Immunogenetics of Inflammation and Infection in the Gastointestinal

Transcription

Immunogenetics
of
Infection and Inflammation
in the
G astrointestinal and Urogenital Tracts
Sander Ouburg
IMMUNOGENETICS OF
INFLAMMATION AND INFECTION
IN THE GASTROINTESTINAL AND UROGENITAL TRACTS
The work described in this thesis was performed at the VU University Medical Center, Laboratory of Immunogenetics
(Head: Prof. A.S. Peña, MD, PhD, FRCP, AGAF) of the Department of Pathology (Head: Prof. C.J.L.M. Meijer), in
collaboration with the Department of Gastroenterology (Head: Prof. C.J.J. Mulder, MD, PhD). This thesis was
enrolled in the research line “Immunogenetics of Infectious Diseases” (Project-leader: Dr. S.A. Morré). The work
described in this thesis was financially supported by AstraZeneca Nederland BV.
Publication of this thesis was financially supported by:
AstraZeneca Nederland BV
Greiner Bio-One BV
bioMérieux Benelux BV
Microbiome Ltd.
BMD BioMedical Diagnostics SA – Ani Labsystems Ltd. Oy
Medac GmbH – Oxoid BV
Roche Diagnostics Netherlands
Goffin Meyvis Analytical & Medical Systems
Eric Ouburg
Marga van Putten
Herman & Ria Pleijster
Cover design:
Druk:
Sander Ouburg
Gildeprint Drukkerijen B.V. - www.gildeprint.nl
Lay out:
Sander Ouburg
ISBN-10: 90-9021321-X
ISBN-13: 978-90-9021321-7
© Sander Ouburg, Mijdrecht, Nederland 2006.
All rights reserved. Any part of this publication may be reproduced, stored in a retrieval system, or transmitted in any
form or by any means, mechanical, photocopying, recording, or otherwise, with written permission from the publisher.
VRIJE UNIVERSITEIT
IMMUNOGENETICS OF
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad Doctor aan
de Vrije Universiteit Amsterdam,
op gezag van de rector magnificus
prof.dr. L.M. Bouter,
in het openbaar te verdedigen
ten overstaan van de promotiecommissie
van de faculteit der Geneeskunde
op maandag 18 december 2006 om 10.45 uur
in de aula van de universiteit,
De Boelelaan 1105
door
Sander Ouburg
geboren te Amsterdam
promotoren:
copromotoren:
prof.dr. A.S. Peña
prof.dr. C.J.J. Mulder
dr. S.A. Morré
dr. E.C. Klinkenberg-Knol
I am among those who think that science has great beauty.
A scientist in his laboratory is not only a technician:
he is also a child placed before natural phenomena which
impress him like a fairy tale.
Marie Curie
French (Polish-born) chemist & physicist (1867 - 1934)
Table of Contents
Contents
Part I
Introduction
11
Aims and Outline
31
Inflammation and Infection of the gastrointestinal tract
35
Aims and Outline
37
Chapter 1
41
Polymorphisms in the immune regulatory genes IL-1B & IL-1RN and the
bacterial sensing genes CD14 & TLR4 are associated with Barrett
oesophagus
Chapter 2
53
The toll-like receptor 4 (TLR4) Asp299Gly polymorphism is associated with
colonic localisation of Crohn's disease without a major role for the
Saccharomyces cerevisiae mannan-LBP-CD14-TLR4 pathway
Chapter 3
6
63
The role of the bacterial CpG sensing toll-like receptor 9 (TLR9) in Dutch
Caucasians and Spanish Galicians patients with Crohn’s disease: evidence
for genetic heterogeneity
Chapter 4
71
CD14 and TLR4 gene polymorphisms in Galician patients with Crohn’s
disease: genetic and environmental interactions
Chapter 5
Combined carriership of TLR9-1237*C and CD14-260*T alleles enhances
the risk of developing chronic relapsing pouchitis
83
Table of contents
Part II
Inflammation and Infection of the urogenital tract
97
Aims and Outline
99
Chapter 6
103
The first strong genetic susceptibility marker for Chlamydia trachomatis
infections: The interleukin 1 receptor antagonist IL-1RN +2018 T>C gene
polymorphism
Chapter 7
109
The CD14 functional gene polymorphism –260 C>T is not involved in either
the susceptibility to Chlamydia trachomatis infection or the development of
tubal pathology
Chapter 8
123
Host inflammatory response and development of complications of Chlamydia
trachomatis genital infection in CCR5 deficient mice and subfertile women
with the CCR5delta32 gene deletion
Chapter 9
141
Do host genetic traits in the bacterial sensing system play a role in the
development of Chlamydia trachomatis-associated tubal pathology in
subfertile women?
7
General Discussion
153
Summary
Samenvatting
189
197
Addendum
Curriculum Vitae
Curriculum Vitae in English
Publications
Author affiliations
Acknowledgments
207
208
209
210
212
216
Table of Contents
8
All truths are easy to understand
once they are discovered;
the point is to discover them
Galileo Galilei
Italian astronomer & physicist (1564 - 1642)
IMMUNOGENETICS OF
Sander Ouburg
10
If life gives us rocks,
it's our choice whether
to build a bridge or a wall
Anonymous
Introduction
Partially based on
A candidate gene approach of immune mediators effecting the susceptibility to and severity
of upper gastrointestinal tract diseases in relation to Helicobacter pylori and
Epstein-Barr virus infections
Sander Ouburg, J. Bart A. Crusius, Elly C. Klinkenberg-Knol, Chris J.J. Mulder,
A. Salvador Peña & Servaas A. Morré
European Journal of Gastroenterology and Hepatology 2005; 17 (11): pp. 1213 - 1224
&
The true ligand of the NOD2 receptor is peptidoglycan instead of lipopolysaccharide:
a schematic representation of ligand-receptor interactions and NF-kappa B activation
Servaas A. Morré, Sander Ouburg, Elly C. Klinkenberg-Knol, Chris J.J. Mulder &
A. Salvador Peña
Gastroenterology 2004; 126 (1): pp. 371 - 373
Introduction
12
There is nothing like looking,
if you want to find something.
You certainly usually find something,
if you look,
but it is not always quite the something you were after.
J.R.R. Tolkien
(English writer, 1892 – 1973)
to the thesis
Introduction to the thesis
astrointestinal diseases can be affected by a wide range of pathological
conditions, from simple passing infections to severe chronic inflammation.
The chronic inflammatory conditions can have a severe impact of the patient’s
lifestyle and / or quality of life. Many patients require life long medication, changes in life
style, or in severe cases require surgical intervention. Barrett oesophagus, a condition
predisposing to the development of oesophageal cancer, and Inflammatory Bowel Disease
(IBD) are two of these chronic inflammatory conditions.
G
Chlamydia trachomatis is the most prevalent sexually transmitted bacterium around the
world and the leading cause of bacteria related blindness. Due to its mostly asymptomatic
course of infection, infected persons are at increased risk of spreading the disease and of
developing late complications. Furthermore infected persons are at increased risk of
acquiring other STDs, such as HIV1.
Clear differences in the course of these inflammatory processes and infections have been
described, however the aetiopathogenesis remains currently unknown. In order to be able to
better treat patients and to potentially prevent disease, or at least severe complications,
many questions need to be addressed, including: Why do some people acquire infection
while others exposed to the same pathogen do not? Why do some people develop
complications or more severe inflammations, while others do not? Since inflammation is a
defensive mechanism of the immune system: does regulation of the immune system play a
role in the aetiopathogenesis of these diseases? Does recognition or lack thereof, of
pathogens influence the course of infection?
These are questions that this thesis will try to address.
Contents
1.
Introduction
2.
Gastrointestinal diseases
3.
Urogenital diseases
4.
Genetic variation in the immune system
1. Introduction
The human body remains one of the humankinds biggest scientific puzzles. Although
science progresses at a rapid pace due to the current high rate of technological
development, relatively little is known about the complex mechanisms and biological
interactions that make life possible. One of the big mysteries that remain is the human
immune system. The immune system is very versatile and efficient at dealing with most
pathogens. Unfortunately, the more complex something is, the more difficult it is to
understand and the more easily something can go wrong.
Approximately 5% of the human genome is thought to be structurally variable2. Genetic
variation in genes encoding the immune system may help to create a more flexible immune
13
Introduction
system, however these variations may also result in aberrant immune responses.
In order to gain a better understanding of the immune system, the way it works and the way
it is regulated, we studied mucosal infections in two distinct organ systems, the
gastrointestinal tract and the urogenital tract.
2. Gastrointestinal diseases
2.1 Barrett oesophagus
2.2 Crohn’s Disease
2.3 Pouchitis
The human gastrointestinal tract is a very complex system, interlinking several organs, each
with its own specific biological functions and mechanisms. Apart from being the digestive
system providing the body with essential nutrients, the gastrointestinal tract plays an
important role in the immune system. On the one hand it provides a homeostatic balance
with the intestinal flora3, on the other hand it forms a immune barrier against (food-borne)
pathogens. Due to its complexity, the gastrointestinal system is capable of responding to
changing patterns in both diet and immune stimuli. However the complexity makes the
gastrointestinal tract also vulnerable to factors that may influence key processes that
deregulate the gastrointestinal system.
14
2.1 Barrett oesophagus
Barrett Oesophagus (BO) is an inflammatory condition of the oesophagus and patients have
an increased risk of malignancies. BO is a complication of gastro-oesophageal reflux
disease (GORD), where acid contents of the stomach are regurgitated into the oesophagus.
This exposure to gastric acid leads to tissue damage and changes in the oesophageal
epithelium4. These changes range from metaplasia to dysplasia to carcinoma, and although
the risk of developing high grade dysplasia or adenocarcinoma from BO is small (estimated
annual risk 0.2-2.0%), the risk is 30 – 125 times that of an age matched population5, 6.
Recent studies have implicated the involvement of the host immune system in the
pathogenesis of gastrointestinal tract diseases7. Although comparatively little is known
about the immunogenetics of the gastrointestinal tract diseases, recent studies have shown
clear associations between polymorphisms in genes encoding cytokines and the
development of gastric cancer8, 9. Furthermore, twin studies have reported the role of
genetic factors in the aetiology of reflux disease10, 11 and familial clustering has been
reported in Barrett oesophagus patients12, indicating that a genetic component may be
involved in the development of GORD and Barrett oesophagus.
2.2 Crohn’s Disease
Crohn’s Disease (CD) is one of the two main clinical phenotypes of inflammatory bowel
disease (IBD). It is a chronic inflammation of the small intestine, whereas ulcerative colitis
(UC) is a chronic inflammation of the large intestine.
The prevalence of CD is 10-200 cases per 100.000 persons in Europe and North America,
with the highest incidences in highly urbanised areas. CD most commonly affects the
terminal ileum, caecum, peri-anal areas and the colon. Patches of normal colon between
affected areas, known as “skip lesions” are characteristic of the disease13. Symptoms are
based on the location and the extent of the inflammation, and include amongst others
diarrhoea, fatigue, weight loss, narrowing of the gut and blood loss. Further complications
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include stenosis, fistulae and extraintestinal manifestations, including rheumatoid arthritis,
ankylosing spondylitis and inflammation of eyes, skin, liver, and bones.
The generally accepted idea is that CD is caused by a complex interplay between genetic,
bacterial and environmental factors14. Furthermore, previous studies in families and in
twins have provided evidence for a genetic component in CD15 - 18.
2.3 Pouchitis
Total proctocolecotomy with ileal pouch-anal anastomosis (IPAA) is currently a standard
surgical procedure in patients with ulcerative colitis (UC)19, 20. The procedure preserves the
sphincter functions, while allowing the removal of the diseased colorectal tissue, which is a
vast improvement compared to an ileostomy. Most patients who undergo IPAA surgery for
severe chronic UC achieve excellent functional results.
Unfortunately, circa 30% of the patients who have IPAA surgery will develop an
idiopathic, non-specific inflammation of the ileal reservoir, called “pouchitis”. The
aetiology of pouchitis is diverse and ischemic complications of surgery, faecal stasis,
bacterial overgrowth, dysbiosis, nutritional deficiencies, novel forms of inflammatory
bowel disease (IBD), recurrence of UC in the pouch, missed diagnosis of CD and genetic
susceptibility have all been suggested as causative factors21. Due to the familial
predisposition in patients with IBD and the potential role of intestinal microorganisms in
IBD pathogenesis, it has been suggested that host genes may affect susceptibility to IBD
and pouchitis.
3. Urogenital diseases
The urogenital tract shares many similarities with other mucosal sites, like the
gastrointestinal and respiratory tracts. However, clear differences exist apart from the most
obvious differences in the functions of these organ systems.
The female reproductive tract is a very complex system where many factors, including
hormones, vaginal flora and immune mediators, combine to provide protection against
infection on the one hand, while on the other hand maintaining an environment suitable for
conception22 - 24.
The vagina can be colonised by a variety of commensal microorganisms (e.g. Candida
albicans and Lactobacillus spp.) and pathogens (e.g. Chlamydia, trachomatis, Neisseria
gonorrhoeae, Treponema pallidum, and Trichomonas vaginalis). In healthy premenopausal
women up to 50 different species of microorganism may colonise the vagina at any given
time. These microorganisms are thought to help prevent infection with pathogenic
microorganisms, for instance through biofilm formation, lowering of vaginal pH, or
biosurfactant production25. The precise biological mechanisms that result in protection
remain unclear.
However, colonisation with pathogenic microorganisms does occur and chronic or
persistent infection may ascend up the urogenital tract and may infect other organs,
including the uterus and tubae. Ascending infections may increase the risk of developing
cervical cancer, tubal infertility and ectopic pregnancy, and may pose a risk for the foetus if
the infected woman is pregnant.
Chlamydia spp. are associated with a broad clinical spectrum of human diseases, including
cardiovascular disease, and pulmonary, ocular and urogenital tract infections26 - 29.
15
Introduction
Chlamydia trachomatis is an obligate intracellular pathogen. Its life cycle has two distinct
stages, called elementary bodies (EB) and reticulate bodies (RB), and requires the host cell
for replication (figure 1).
16
Figure 1: General features of C. trachomatis developmental cycle (Abdelrahman, FEMS Microbiology Reviews
2005). Details may vary between different serovars and biovars. TARP (Translocated Actin-Recruiting
Phosphoprotein, CT456), TTSS (Type III Secretion System), MEP (non-MEvalonate Pathway), CPAF (Chlamydial
Protease/proteasomelike Activity Factor).
The elementary body is the extracellular, infectious and metabolically inactive form.
Following uptake into the host cell, the EB-containing phagosome avoids lysosomal
fusion30 and the EB transforms into the intracellular replicative form called a reticulate
body (RB). RBs are non-infectious, metabolically active, and replicate by binary fission.
Chlamydia trachomatis is divided in three biovars (table 1). The trachoma biovar consists
of serovars A – C, causing trachoma, and the serovars D – K, which infect the urogenital
tract. The LGV biovar, consisting of serovars L1 – L3, causes lymphogranuloma venereum
(LGV), currently mostly found in men who have sex with men (MSM)31. And the mouse
biovar, previously known as the mouse pneumonitis (MoPn) agent and now called
C. muridarum, causes respiratory tract infections in mice32.
to the thesis
Chlamydia trachomatis
Biovar
Serovars
Trachoma A, B, Ba, C
Host
Human
D, Da, E, F, G, Ga, H, I, Ia, J, K
Human
LGV
L1, L2, L2a, L3
Human
Mouse
1
(MoPn agent, currently known as
C. muridarum)
Mouse
Site of Inflammation
Conjunctivae
Urogenital tract (rare)
Urogenital tract
Conjunctivae
Respiratory tract (rare)
Inguinal lymph nodes
Urogenital tract
Rectum
Respiratory tract
Table 1: Chlamydia trachomatis serovar classification and tissue tropism
Chlamydia trachomatis infection is the most common sexually transmitted infection in
Europe and the USA. The course of infection is in most women asymptomatic, resulting in
a reduced likelihood of consulting a physician for treatment, an increased risk of
transmission to sexual partners, increased risk of acquiring other STDs1, and an increased
risk of chronic infection and late complications.
Clear differences in the clinical course of Chlamydia infection have been described and are
due to an interaction between environmental (e.g. co-infection), bacterial (e.g. virulence
factors) and host factors (genetic differences between individuals).
4. Genetic variation in the immune system
4.1
Candidate gene approach
4.2
Immune mediators
4.2.1
Interleukin 1 family
4.2.2
Toll Like Receptor family
4.2.3
CD14
4.2.4
CCR5
4.2.5
CARD15/NOD2
4.3
Multiple polymorphisms and multiple genes
4.4
In conclusion
Bacterial infections and genetic influences have been implicated in gastrointestinal and
urogenital diseases. Therefore, the influence of host genetic variations in the immune
system is a very interesting and relevant subject to study in relation to these diseases.
The traditional point of view on susceptibility to infection and the severity of ensuing
disease was based on the environmental and microbial factors. Most research was devoted
to the risk of infection and identification of transmission routes. The microbe based
research focused on analysis of specific strains with potential differences in virulence and
subsequently to the identification of specific genes linked to severe phenotypes of disease.
However, it became clear that the analysis of environmental and microbial factors alone,
could not explain the observed differences in the course of infection.
17
Introduction
Recent studies have provided a wealth of information on the role of host genetics in disease
aetiopathogenesis, and a clear and prominent role for genes encoding immune mediators
has been established. The data on the effects of functional polymorphisms in genes
encoding cytokines, chemokines, and pathogen recognition receptors have provided an
insight into the susceptibility to and severity of infections and disease. It has been shown
that functional polymorphisms occur with different frequencies in diverse ethnic
populations and that these polymorphisms can differently influence aetiopathogenesis in
these populations. Based on these data, it can be concluded that the host (immuno-) genetic
background, combined with environmental and microbial factors contribute to the course
and outcome of infection33 - 36.
Genetic linkage and positional cloning are appropriate for the identification of relatively
high-risk genes, but this approach has not been successful for the identification of genes in
complex forms of polygenic diseases like infectious disease susceptibility and severity37, 38.
Candidate gene studies can detect small to moderate relative risks in the context of
aetiological and genetic heterogeneity by studying the relevance of functional single
nucleotide polymorphisms (SNPs) in genes. The field that studies the host genetic
background in relation to inflammation, infection and disease susceptibility and severity is
called “immunogenetics”.
18
4.1 Candidate gene approach
The candidate gene approach of common SNPs can detect small to moderate relative risks
in the context of aetiological and genetic heterogeneity. Epidemiologic and immunogenetic
approaches consider the detection of an association a crucial step in understanding disease
aetiology, rather than a means to establish causality37. The first step is to identify
potentially relevant genes. The selection of genes is based on a careful consideration of
current knowledge of disease phenotype, expression studies and infectious disease models.
For example, if a certain immune mediator is thought to play a role in disease phenotype,
knockout and knockin mice could establish the importance of this particular gene.
Subsequently, expression profiling of mRNA and protein levels in cases and controls could
further confirm the role of the gene in disease. The next step would be to identify
polymorphisms in this gene, which might influence the function of this gene. Once the
polymorphisms are identified, larger scale studies can be performed39, 40.
When a hypothesis on the influence of potential genes on disease is formed and potentially
functional genetic polymorphisms in the genes are identified, the polymorphisms are
genotyped in a case-control study. If statistically significant associations are found, then
further studies (e.g. expression profiling of mRNA and protein levels) are initiated to
identify the exact biological mechanisms through which the genetic polymorphisms
influence the disease pathogenesis. These studies will also provide information on whether
the polymorphism under study is directly related to the disease pathogenesis or linked to
another polymorphism, which may influence disease. This kind of linkage is referred to as
linkage disequilibrium. Linkage disequilibrium (or ‘allelic association’) describes the
tendency of alleles to be inherited together more often than would be expected under
random inheritance (e.g. the C allele of IL-1B–511 is found in 99.5% of the cases together
with the T allele of IL-1B–31. Under random segregation, the C allele would have been
observed in approximately 50% of the cases with the T allele of IL-1B–31 and
approximately 50% of the cases with the C allele of IL-1B–31). This knowledge may one
to the thesis
day be used to develop diagnostic tests to identify patients at high risk for disease
development or adverse outcome of disease.
4.2 Immune Mediators
Several key immune mediators have been studied in relation to gastrointestinal and
urogenital disease pathogenesis. In this thesis the Interleukin 1 (IL-1) family, Cluster of
Diffentiation 14 (CD14), Toll-Like Receptor 4 (TLR4), TLR9, CC motif chemokine
Receptor 5 (CCR5), Caspase Activation Recruitment Domain 15/Nucleotide
Oligomerization Domain 2 (CARD15/NOD2) will be discussed. These regulatory cytokines
and bacteria sensing receptors are involved in the activation of Nuclear Factor kappa B
(NF-κB), which is of paramount importance in the regulation of the inflammatory response.
4.2.1 Interleukin 1 family
Interleukin 1
The IL-1 family consists of four genes coding for IL-1 Alpha (IL-1α), IL-1 Beta (IL-1β),
IL-1 receptor antagonist (IL-1ra)41 and the IL-1 like protein (IL-1L1)42, 43. IL-1 is involved
in a wide variety of physiologic processes, including the regulation of inflammatory,
metabolic, haematopoietic and immunologic mechanisms. It is produced by macrophages,
neutrophils and endothelial cells.
IL-1β initiates the expression of several genes, coding for lymphokines. It induces natural
killer cells and activates T- and B-cells44, 45. IL-1β is a powerful inhibitor of gastric acid
secretion and upregulates COX2 expression46.
IL-1ra is the natural inhibitor of IL-1β and is produced by the same cell types as IL-1β. For
a 50% inhibition of IL-1, a 10- to 500-fold excess of IL-1ra is required47.
Binding of IL-1 to the IL-1 receptor induces a signal, via NF-κB responsive genes in the
nucleus, leading to cellular responses like the stimulation of prostaglandin E2 (PGE2)
synthesis, up regulation of COX genes46, 48, 49, stimulation of collagenase production and
cytoadherence of leukocytes to endothelial cells50, 51.
Binding of IL-1ra to the IL-1 receptor induces no signal transduction and blocks the
receptor for IL-1 binding, thus effectively inhibiting IL-1 function51. IL-1 homologues have
been described, though their biological roles are yet unknown42, 43, 52.
Polymorphisms of IL-1
Many SNPs have been reported in the IL-1B gene, however three SNPs, at positions –511
(IL-1B–511; rs16944), -31 (IL-1B–31; rs1143627) and +3954 (IL-1B+3954;
rs1143634)53 - 55, are frequently studied. The polymorphisms at positions –511 and –31 are
in 99.5% linkage disequilibrium56 - 58.
The IL-1B–31 SNP is located in a TATA-box and influences DNA-protein interactions56, 59.
Alleles of IL-1B–511 (via linkage disequilibrium with IL-1B–31) and IL-1B+3954 have
been associated with increased IL-1β production.
The IL-1RN (receptor antagonist) gene contains a hexa-allelic 86bp variable number of
tandem repeats (VNTR) at position +2951 (in intron 244, 47, 60, 61; rs2234663). The most
common allele (allele 1) in all populations studied thus far contains 4 repeats of the 86bp
fragment56, 62. The second allele of the VNTR is in near perfect linkage disequilibrium with
the rare allele of the IL-1RN +2018 T>C SNP (rs419598). This polymorphism has been
associated with increased expression of IL-1ra60, 63, 64.
19
Introduction
Variations in allele frequencies were found in different ethnic and geographic populations.
However, allele 1 is the most common in all populations, and alleles 1 and 2 are present in
over 90% of the population65 - 67.
4.2.2 Toll Like Receptor Family
The Toll Like Receptor (TLR) family is a group of transmembrane pathogen-associated
molecular pattern (PAMP) receptors, which recognise several microbial products, including
bacterial cell wall components and DNA68. The family currently consists of ten different
members, each with its own specific ligand. The TLRs use different signalling cascades
generally resulting in NF-κB and AP-1 activation in MyD88 dependent pathways and
activation of type IFNs in TRIF dependent pathways69.
TLR4
Poltorak et al. associated TLR4 with lipopolysaccharide (LPS) recognition in mice70.
Further studies in mice corroborated these data 71, 72. The recognition of LPS is facilitated
through the LPS – LPS binding protein (LBP) – CD14 complex. TLR4 specifically
recognizes the LipidA fraction of LPS73. Functional polymorphisms in the TLR4 gene have
been associated with increased susceptibility to severe bacterial infections and IBD74 and
may predispose to septic shock with Gram-negative microorganisms75, 76.
20
Polymorphisms in TLR4
TLR4 contains two well studied co-segregating SNPs at positions +896 (A>G) and +1196
(C>T), better known as the TLR4 Asp299Gly (rs4986790) and the TLR4 Thr399Ile
(rs4986791), respectively. Both SNPs cause amino acid changes in the extracellular domain
of TLR4 and have been associated with LPS hyporesponsiveness in humans77, 78. However
heterozygous carriage of the TLR4+896 SNP does not affect LPS responsiveness and only
the rare homozygous carriers are less responsive to LPS79, 80. The TLR4+896 SNP has been
associated with increased susceptibility to severe bacterial infections and IBD74 and may
predispose to septic shock with Gram-negative microorganisms75, 76.
TLR9
TLR9 is required for the recognition of CpG motifs, short sequences of unmethylated DNA
predominantly present in bacterial DNA. CpG motifs have immunostimulatory activity by
inducing dendritic cell maturation, B-cell proliferation and production of cytokines,
including interleukin-6 (IL-6) and interleukin-12 (IL-12)81, 82. TLR9 signalling has been
shown to mediate the resolution of intestinal inflammation in experimental colitis83,
suggesting that the release of bacterial DNA from the microflora might favour immune
homeostasis.
Polymorphisms in TLR9
The promoter TLR9 -1237 T>C SNP (TLR9-1237; rs5743836) located on chromosome
3p21.3 is associated with susceptibility to asthma in European Americans, but not in
Hispanic or African Americans84 and the marker D3S1076 in this region shows association
with IBD in a classical TDT test85. The TLR9-1237 T>C and TLR9+2848 G>A SNPs
(rs352140) SNPs are part of a four SNP haplotype. Lazarus et al. only found seven out of
sixteen possible haplotype combinations and the -1237 and +2848 SNPs allow distinction
between the four most common haplotypes84.
to the thesis
4.2.3 CD14
CD14 acts as a co-receptor for TLR4 and confers responsiveness to LPS, a component of
the cell wall of most Gram-negative bacteria. CD14 forms a complex with LPS and the
LPS-binding protein (LBP)86. CD14 exists in a membrane form on monocytes and
neutrophils and in a soluble form in serum87 - 89. Combined with TLR4 this complex induces
NF-κB associated immune responses including the release of a broad spectrum of cytokines
that include tumour necrosis factor alpha (TNF-α), IL-1, IL-6, and IL-8 to initiate immune
response90. Increased expression of CD14 in macrophages has been found in inflammatory
bowel disease (IBD)91
Polymorphisms in CD14
The promotor region of the CD14 gene contains a C>T SNP at position –260 (rs2569190).
This SNP is also known as CD14 -159 C>T. The differences in SNP position are based on
the major transcription start site and the translation start site. According to the current
naming conventions, the SNP position is calculated relative to the translation start site
(ATG; Figure 2)92, 93. The SNP is positioned at -260bp relative to the translation start94 and
this position will be used throughout this thesis.
21
Figure 2: Promotor region of the CD14 gene. Depicted are the -260 SNP, the transcription and translation start
sites, and the distances in basepairs. All nucleotide positions are relative to the ATG start codon.
The -260 C>T SNP affects the binding of transcription factors94 and has been associated
with high levels of soluble CD14 (sCD14) and with lower serum IgE levels90. This SNP has
been associated with myocardial infarction95, IBD96 - 98, atherosclerosis95 and an increased
susceptibility to develop chronic spondyloarthropathy in women99. It has been
demonstrated that carriers of the T allele of this promotor polymorphism have a higher
expression of both membrane bound CD14 (mCD14) and sCD14 and that TNF-α
production is increased in the homozygous CD14 –260 T carriers100, 101. Genetically
determined variation in CD14 serum levels may have functional consequences given the
ability of soluble CD14 to confer pathogen responsiveness to cells such as intestinal,
epithelial and endothelial cells that do not express CD14 on their membranes102.
4.2.4 CCR5
The chemokine receptor CCR5 is a member of the 7-transmemebrane G-protein coupled
receptor superfamily, expressed on monocytes, killer T cells, T helper cells, and dendritic
cells. It plays a role in T cell activation and function, and plays an important role in
infection related immune and inflammatory responses. CCR5 was identified as a coreceptor
for the human HIV-1 virus by Deng et al. and Dragic et al.103, 104.
Introduction
Polymorphisms in CCR5
CCR5 contains several polymorphisms, with the best known being the 32bp deletion
(CCR5Δ32 / CCR5δ32; rs333). This deletion results in a frameshift and a truncated
protein105. The CCR5δ32 polymorphism is present in high frequencies in European
Caucasian and American Caucasian populations, but virtually absent in Asian and African
populations. It was originally postulated that the high frequency in Caucasians was caused
by the evolutionary pressure of the Medieval plague epidemics106, although current
understanding suggests that a disease like smallpox is a more likely candidate107 - 110. The
CCR5δ32 polymorphism is associated with a level of protection against HIV-1 infection
and a slower disease progression in infected patients105, 108. The polymorphisms has further
been related to asthma, and allograft rejection (OMIM, *60137349).
22
4.2.5 CARD15/NOD2
CARD15, also known as NOD2, is an intracellular pattern recognition receptor. It
recognizes the MurNac-L-Ala-D-iso-Gln motif, derived from peptidoglycan (PGN), with a
strong stereoselective recognition, as shown by replacement of L-Ala for D-Ala or DisoGln for L-isoGln73, 111. Although CARD15/NOD2 can mediate the recognition of
muropeptides, the mechanism involved is unclear and remains to be determined. Because
the Leucine Rich Repeats (LRR), involved in the binding of the muropeptide part of PGN,
are required for recognition, muropeptides could interact directly with CARD15/NOD2
through its LRRs or via an as yet to be identified cellular factor(s). PGN is present in both
Gram-positive and Gram-negative bacteria, indicating that CARD15/NOD2 is capable of
recognising a wide variety of intracellular bacteria and thus initiate immune responses
against intracellular bacteria that otherwise may have evaded the immune system73.
Polymorphisms in CARD15/NOD2
Ogura et al. and Hugot et al. identified multiple polymorphisms in the CARD15/NOD2
gene112, 113, associated with CD. Up to 67 polymorphisms have been described in
CARD15/NOD2, however 3 polymorphisms (R702W (also known as SNP8), G908R
(SNP12), and 3020InsC (SNP13)) have consistently been associated with CD. The
incidence of these polymorphisms is variable between different ethnic groups, with high
frequencies of CARD15/NOD2 polymorphisms found in Ashkenazi Jewish populations114 117
.
CARD15/NOD2 SNPs have been associated with functionally deficient responses to LPS
and PGN118.
4.3 Multiple polymorphisms and multiple genes
Multiple polymorphisms in one gene could in an increasing manner influence gene
expression, protein expression or protein function. For instance, several mutations in the
TLR4 gene were studied, but no single variant was significantly associated with
meningococcal sepsis. However, when multiple rare mutants in the TLR4 gene were
combined, an over-representation of these rare mutants was found in the systemic
meningococcal infected patients119. Combinations of multiple polymorphisms across
multiple genes may influence pathogenesis. For example, El-Omar et al. showed that
carriage of multiple proinflammatory polymorphisms conferred greater risk for
to the thesis
development of gastric cancer, with increasing odd’s ratios reaching 27.3 in high-risk
genotypes8.
4.4 In conclusion
The rapid advances in the field of human genetics have offered new opportunities to
investigate the role of various immune mediators in disease susceptibility and severity.
Combining different studies and different research methods provides a valuable insight into
the complex and dynamic host-pathogen interactions. This insight may help to further the
understanding of both gastrointestinal tract and urogenital tract pathophysiology and
immunopathology, and may help identify new research targets and help define potential
therapeutic targets. The knowledge acquired by this approach will result in the development
of improved general medical practices, and perhaps in the future help to establish ‘made-tomeasure’ treatment of individual patients.
23
Introduction
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to the thesis
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29
Introduction
30
The success of tomorrow
is the result of the failures of today
Aims & Outline of this Thesis
Aims & Outline
32
The great tragedy of Science – the slaying of a beautiful hypothesis by an ugly fact.
Thomas H. Huxley
English biologist (1825 - 1895)
of the thesis
Aims & Outline
he gastrointestinal and the urogenital tracts are part of the mucosal immune
system and represent organs of the protective barrier of the organism, such as
the lung and the skin. The exposure to pathogens or abnormal responses to
ubiquitous microorganisms are responsible for the immunopathogenesis of diseases
affecting these organs. The studies presented in this thesis were designed to understand the
immunogenetic contribution to chronic inflammatory responses in diseases affecting the
gastrointestinal and urogenital tract. We choose diseases which are complex in nature but
share similar elements, such as the presence of chronic inflammation and a risk to develop
malignancy.
T
The aim of this thesis is:
First, to assess the role of nucleotide polymorphisms in genes that are involved in the
regulation of the inflammatory response and in the detection of pathogens, in order to
determine their role in the susceptibility to and severity of selected gastrointestinal and
urogenital diseases. The genes selected are important in the regulation of both the innate
and acquired immune responses.
Second, to use the knowledge obtained in these studies to better understand of the
underlying polygenic regulatory and sensory mechanisms involved in gastrointestinal and
urogenital immune responses.
Third, to compare the results of the gene polymorphisms studied in the gastrointestinal and
urogenital tracts, in order to gain a better understanding of the regulation of biological
mechanisms in different sites of the organism to different noxious agents, and ubiquitous
and pathogenic bacteria.
The Discussion reviews the results of Parts I & II in the context of the current literature and
compares the results obtained in the diseases selected in the gastrointestinal and urogenital
tract, in order to gain a deeper insight into the immunogenetics of the immunological
mechanisms underlying infection and inflammation at human mucosal epithelia.
I hope that the generated data and insight will advance the knowledge of inflammation and
immune regulation in the gastrointestinal and urogenital tracts and provide a basis for
further research.
33
Aims & Outline
34
I am passionately curious
Albert Einstein
Part I
Inflammation and Infection
in the
Gastrointestinal Tract
Part I
36
Als je het belang van je onderzoek niet aan je moeder uit kan leggen
is er waarschijnlijk niets mis met je moeder,
wel met je onderzoek
If you can’t explain the importance of your research to your mother
then there is, most likely, nothing wrong with your mother,
but with your research
Aims & Outline Part I
Aims & Outline
38
What a caterpillar calls the end,
the rest of the world calls a butterfly
Part I
Aims & Outline Part I
he incidence of gastro–oesophageal reflux disease (GORD) is on the rise,
increasing the risk of Barrett oesophagus and oesophageal cancer. In this case
“acid” appears to be the major offending factor.
Inflammatory bowel diseases (IBD) – Crohn’s disease and ulcerative colitis – are
considered to represent an abnormal response to the ubiquitous intestinal microflora.
Ulcerative colitis often requires the resection of the whole colon. Ileo-anal anastomosis
with the construction of a reservoir (“pouch”) is the operation of choice but 30% of the
patients develop chronic inflammation in the form of pouchitis.
All of these diseases have a severe impact on quality of life of the patient. Patients with
IBD, often have an associated risk of extraintestinal manifestations of the disease.
T
Part I of this thesis addresses the role of SNPs in genes encoding immune regulatory and
bacteria sensing proteins in the development of chronic inflammatory diseases affecting the
gastrointestinal tract.
Chapter 1 focuses on regulatory cytokine genes associated with the inflammation of the
stomach and reflux disease, as well as genes involved in bacteria sensing receptor genes in
the development of Barrett oesophagus.
Chapters 2 – 4 assess the role of genes in regulatory and pathogen sensing genes in Dutch
Caucasian and Spanish Galician Crohn’s disease patients, in order to gain more
understanding of the heterogeneity observed among CD patients and between different
ethnic populations.
The main question addressed in Chapter 5 is whether these genes predispose to
development of pouchitis and/or cause a more severe course of infection.
39
Aims & Outline
40
Knowing others is intelligence;
knowing yourself is true wisdom.
Mastering others is strength;
mastering yourself is true power
Tao Te Ching
Chapter 1
Polymorphisms in the immune regulatory genes IL-1B & IL-1RN and the bacterial sensing
genes CD14 & TLR4 are associated with Barrett oesophagus
Sander Ouburg, Marieke Emonts, A. Salvador Peña, Agnieszka M. Rygiel, Kausilia K. Krishnadath,
Peter W.M. Hermans, Jacques G.H.M. Bergman, Chris J.J. Mulder, Elly C. Klinkenberg-Knol &
Servaas A. Morré
Chapter 1
Abstract
ntroduction: Patients with Barrett oesophagus are at increased risk for
malignancy. Progression to invasive adenocarcinoma follows the metaplasiadysplasia-carcinoma sequence. The eradication of H. pylori influences gastric
acid production and reflux disease. Therefore, polymorphisms in genes regulating gastric
acid secretion (IL-1), immune regulation (IL-1 family) and bacterial recognition genes
(CD4 and TLR4) may be involved in the susceptibility to develop Barrett oesophagus.
I
Material and methods: 210 Dutch Caucasian Barrett patients and 334 ethnically matched
controls were typed for IL-1B -511, IL-1RN +2018, CD14 -260 and TLR4 +896 SNPs.
Results: The IL-1RN*C allele at position +2018 was significantly increased in Barrett’s
patients compared to controls (p: 0.033; OR: 1.5; 95% CI: 1.1 – 2.1). Homozygotes for
CD14-260 TT were significantly decreased in Barrett’s patients compared to controls
(p: 0.040; OR: 1.7; 95% CI: 1.0 – 2.7) and carriers of CD14-260 TT with TLR4+896 AA
were also significantly decreased in patients (p: 0.007; OR: 1.9; 95% CI: 1.2 – 3.0).
Combined carriage of specific combinations of these four genes confers protection from
Barrett oesophagus. Multivariate logistic regression analysis showed a strong correlation
between IL-1RN+2018 and development of Barrett oesophagus (p: 0.006; OR: 2.2.; 95%
CI: 1.3 – 3.0).
42
Conclusions: IL-1B, IL-1RN, CD14 and TLR4 gene polymorphisms influence susceptibility
to Barrett oesophagus. IL-1RN correlates very strongly in multivariate analysis with Barrett
oesophagus. Combined carriage of specific genotypes in bacteria sensing genes protects
against development of Barrett oesophagus, while carriage of a regulatory carrier trait for
gastric acid secretion and inflammation increases the risk of Barrett oesophagus.
IL-1B, IL-1RN, CD14 & TLR4
Barrett oesophagus
Introduction
atients with Barrett oesophagus are at increased risk for malignancy and
progression to invasive adenocarcinoma is reflected histologically by the
metaplasia-dysplasia-carcinoma sequence. The incidence of development of
high grade dysplasia or adenocarcinoma from Barrett oesophagus is small (estimated
annual risk 0.2-2.0%), however the risk is 30-125 times that of an age matched
population1, 2. The presence of low grade dysplasia and long duration of reflux symptoms
are independent risk factors for development of high grade dysplasia and oesophageal
carcinoma3. The diagnosis of Barrett oesophagus is made endoscopically and confirmed by
histological examination of biopsies.
Decreasing rates of H. pylori infection are reported to coincide with increased rates of
Barrett oesophagus in the Western countries. Other risk factors appear to be age over 40,
male gender, Caucasian race and increased body mass. Protective factors may be high fiber
diets and successful antireflux surgery3 - 6.
Recent studies have implicated the involvement of the host immune system in the
pathogenesis of gastrointestinal tract diseases7. Although comparatively little is known
about the immunogenetics of upper gastrointestinal tract diseases, recent studies have
shown clear associations between polymorphisms in genes encoding cytokines and the
development of gastric cancer8, 9. For instance, carriage of polymorphisms in the IL-1B, IL10, TNF-A and IL-1RN genes increases the risk for non-cardia gastric cancer as compared
to single gene analysis OR: 2.8 (single gene) vs. OR: 27.3 (multiple genes)8. Furthermore,
twin studies have reported the role of genetic factors in the etiology of reflux disease10, 11.
The interleukin 1 (IL-1) gene family is involved in a wide variety of physiologic processes,
including the regulation of inflammatory, metabolic, hematopoietic and immunologic
mechanisms. IL-1β induces lymphokine expression and activates natural killer cells, T
cells, and B cells12. Furthermore, it is a powerful inhibitor of gastric acid secretion and an
up regulator of COX2 expression13. The IL-1 receptor antagonist (IL-1ra) is the natural
inhibitor of IL-1β. Polymorphisms have been described in both genes. The polymorphism
at position -31 in the IL-1B gene is located in a TATA-box and influences DNA-protein
interactions. This polymorphism is associated with decreased IL-1β production14. The
polymorphism at position +2018 in the IL-1RN gene is in linkage disequilibrium with the
IL-1RN 86bp tandem repeat, which is associated with increased IL-1ra production15.
The Toll Like Receptor (TLR) family is a group of pattern recognition receptors, which
recognize several microbial products, including bacterial cell wall components and DNA16.
LPS-binding protein (LBP)17. Combined with TLR4 this complex induces NF-κB
associated immune responses including the release of a broad spectrum of cytokines that
include tumor necrosis factor alpha (TNF-α), IL-1, IL-6, and IL-8, to initiate immune
responses18. The TLR4 gene contains an A to G substitution at position +896, which is
associated with LPS hyporesponsiveness19. The promotor region of the CD14 gene contains
a single nucleotide polymorphism (SNP) at position –260. The -260 C>T genetic variation
affects the binding of transcription factors20 and has been associated with increased levels
of soluble CD14 and membrane-bound CD14. Since H. pylori infection has been described
in relation to the pathogenesis of reflux disease and Barrett oesophagus development,
recognition of H. pylori and the subsequent immune response to its presence might be
important.
P
43
Chapter 1
The aim of this study was to identify whether or not polymorphisms in the IL-1B and IL1RN genes (immune regulation), and in the TLR4 and CD14 genes (bacterial recognition)
influence the susceptibility to and severity of Barrett oesophagus both in single gene and
carrier trait analysis.
Material & Methods
Patients & controls
The study comprised of 210 Dutch Caucasian patients, attending the outpatient departments of
Gastroenterology of the VU University Medical Centre (n=88) and the Academic Medical Centre
(n=122), Amsterdam, The Netherlands. A total of 334 ethnically matched healthy controls were
included. The mean age was 59y (21y-81y) and 43y (19y-92y) in the Barrett and control cohorts
respectively, while 80.5% was male in the Barrett cohort as compared to 52.7% in the control cohort.
All patients underwent an upper gastrointestinal (GI) endoscopy and biopsies (duodenum, antrum,
corpus, and Barrett) were taken at regular intervals. Presence of Barrett epithelium and intestinal
metaplasia was confirmed by histology. Patients who underwent multiple GI endoscopies were
classified as suffering from Barrett oesophagus if this condition was diagnosed in at least 1 endoscopy
and confirmed by histology in the biopsy samples. Data was available on alcohol use and smoking for
one-third of the patients for logistic regression (gender distribution was equal to the total population).
Patients were defined as non-user, previous user or current user of alcohol and/or tobacco. The patient
characteristics are presented in table 1.
44
N
Age (years)
Males (%)
Smokers (%)
Cases
Controls
Remarks
210
59 (21-81)
80.5
334
43 (19-92)
52.7
N/A
Data in 71 patients only
N/A
Data in 73 patients only
Never
Previous
Current
12.7
7.0
80.3
Never
Previous
Current
37.0
21.9
41.1
Alcohol use (%)
Table 1: patient characteristics
N/A: Not available
DNA isolation
Peripheral venous blood was collected in 10ml EDTA-tubes and stored at room temperature until the
genomic DNA was extracted from peripheral blood mononuclear cells (PBMC) according to an inhouse DNAzol extraction procedure (Invitrogen, The Netherlands).
Genotyping
All samples were genotyped for the IL-1RN +2018 T>C (rs419598), CD14 -260 C>T (rs2569190),
IL-1B -511 C>T (rs16944) and TLR4 +896 A>G (rs4986790) polymorphisms using PCR-RFLP or
TaqMan analyses.
The detection of the CD14 -260 C>T and TLR4 +896 A>G polymorphisms was performed as
described previously by our group21, 22. The IL-1B -511 C>T was assessed by PCR-RFLP as described
previously by di Giovine et al.23. The IL-1RN+2018 T>C, of which the mutant (C) allele is in linkage
disequilibrium with the second allele of the IL-1RN VNTR (rs2234663), was assessed according
Barrett oesophagus
standard TaqMan protocol in 96 well plates (Greiner Bio-One), using the primers: forward: 5’- CAA
CCA CTC ACC TTC TAA ATT GAC AT -3’, and reverse: 5’- CTG AGT CCT TTT CCT TTT CAG
AAT CT -3’. The probes used were: MGB-AGT ATC CAG CAA CTA GT-FAM for the T allele and
MGB-CAA GTA TCC GGC AAC TA-VIC for the C allele.
Statistical analyses
All groups were tested for Hardy-Weinberg equilibrium to check for Mendelian inheritance.
Statistical analyses were performed using Instat Graphpad and SPSS version 11. Fisher exact and χ2
tests were used to test for differences in allele / genotype / carrier frequencies between the
(sub)groups and p-values <0.05 were considered statistically significant. Logistic regression analysis
was used to assess the association of different genotypes with Barrett Oesophagus, adjusted for age
and gender.
Results
All groups were in Hardy-Weinberg equilibrium, confirming genotype distributions
according to Mendelian inheritance principles.
Single gene analyses
An increased incidence of the IL-1RN*C allele was found in patients with Barrett
oesophagus compared to controls (48.1% vs. 38.6%; p: 0.033; OR: 1.5; 95% CI: 1.1 – 2.1).
The IL-1B -511 alleles were equally distributed among cases and controls. The distribution
of genotypes and allele frequencies in cases and controls is shown in tables 2 - 5.
TLR4
Genotype
AA
AG
GG
Allele
A
G
Cases
N = 191
%
Controls
N = 334
%
84.3
15.7
0
86.5
13.2
0.3
92.1
7.9
93.1
6.9
Table 2: TLR4 genotype and allele frequencies in cases and controls
CD14
Genotype
CC
CT
TT
Allele
C
T
Cases
N = 191
%
Controls
N = 334
%
34.0
44.5
21.5
25.7
46.1
28.1
56.3
43.7
48.8
51.2
Table 3: CD14 genotype and allele frequencies in cases and controls
45
Chapter 1
IL-1RN
Genotype*
TT
TC
CC
Allele
T
C
Cases
N = 191
%
Controls
N = 334
%
51.8
36.6
11.5
61.4
32.0
6.6
70.2
29.8
77.4
22.6
Table 4: IL-1RN genotype and allele frequencies in cases and controls
* vs. controls. 48.1% vs. 38.6%; p: 0.033; OR: 1.5; 95% CI: 1.1 – 2.1. p values calculated using 2 by 2 χ2 test
IL-1B
Genotype
CC
CT
TT
46
Allele
C
T
Cases
N = 191
%
Controls
N = 334
%
42.4
46.6
11.0
42.2
45.2
12.6
65.7
34.3
64.8
35.2
Table 5: IL-1B genotype and allele frequencies in cases and controls
Carrier trait analysis
Carriage of the CD14 -260 TT genotype together with the TLR4 AA genotype is
significantly associated with a decreased risk for Barrett oesophagus compared to controls
(14.8% vs. 24.6% (controls); p: 0.007; OR: 1.9; 95% CI: 1.2 – 3.0) (Table 6).
CD14
-260
C>T
TT
TT
TC
TLR4
+896
A>G
AA
AA
AG
IL-1B
-511
C>T
IL-1RN
+2018
T>C
CT
CC
TT
TT
Cases
(%)
Controls
(%)
Effect
p
Odds
ratio
14.8
1.8
0.6
24.6
6.1
4.0
Protection
Protection
Protection
0.007
0.030
0.013
1.9
3.3
9.1
Table 6: Multiple gene analysis of CD14, TLR4, IL-1B and IL-1RN gene polymorphisms in Dutch Caucasian
Barrett’s oesophagus patients and controls.
Combination of the two carrier traits
Combining the regulatory and bacteria-sensing carrier traits into a larger carrier trait
resulted in significant differences between cases and controls. Homozygous wildtype
carriage of the IL-1B and IL-1RN genes combined with heterozygous carriage of the CD14
and TLR4 genes is significantly decreased in cases compared to controls (0.6% vs. 4.0%; p:
0.013; OR: 9.1; 95% CI: 1.2 – 70.1) (Table 6).
Barrett oesophagus
Carriage of the carrier trait IL-1B -511*CT, IL-1RN +2018*TT, CD14 -260*CT and TLR4
+896*AA is significantly decreased in patients with Barrett oesophagus compared to
controls (1.8% vs. 6.1%; p: 0.030; OR: 3.3; 95% CI: 1.1 – 9.7). The results are summarized
in table 6.
Logistic regression analysis
Logistic regression analysis was used to assess the association of different genotypes with
Barrett oesophagus, adjusted for age and gender. Age, gender and carriage of the IL-1RN
+2018 SNP were significantly associated with Barrett oesophagus, with gender and IL-1RN
being risk factors (table 7). We analyzed the immune regulatory, bacteria sensing, and
combined carrier traits in the logistic regression model, however this did not reach
statistical significance.
p
OR
Gender
< .001
5.830
Lower
3.031
Upper
11.214
IL-1RNa
.006
2.241
1.266
3.969
a
95% C.I.
.119
1.782
.862
3.687
< .001
.852
.827
.877
a
CD14
.251
.718
.407
1.265
IL-1Ba
.081
.601
.339
1.065
TLR4
Age
Table 7: Logistic regression analysis of the IL-1B, IL-1RN, CD14, and TLR4 genotypes in patients suffering from
Barrett Oesophagus.
a
Mutation carriers vs. homozygous wildtype
Discussion
In this study we found on a single gene level that the IL-1RN+2018*C allele was
significantly increased in Barrett patients compared to controls. The carriertrait
CD14-260 TT – TLR4+896 AA was significantly decreased in patients. Combined carriage
of specific combinations of these four genes confers protection against Barrett oesophagus.
Finally, multivariate logistic regression analysis showed a strong correlation between IL1RN+2018 and development of Barrett oesophagus.
The studied SNPs in the IL-1B and IL-1RN genes have shown to be associated with altered
inflammatory responses. Carriage of the IL-1RN*C allele and combined carriage of
IL-1B-511 CT and IL-1RN+2018 CT is associated with risk for Barrett oesophagus.
These results concur with those reported in the literature, namely, IL-1β increases COX2
production which is known to up-regulate Th2 cytokines when associated with oesophageal
carcinogenesis24, 25. Moons et al. have demonstrated that a predominantly humeral immune
response is characteristic for Barrett oesophagus26. Furthermore, an increased inflammatory
response and/or chronic inflammation may result in increased tissue damage and
carcinogenesis27.
In Japanese it was shown that IL-1B genotypes protect against gastro-oesophageal reflux
disease through induction of corpus atrophy28. Even though this population is Japanese,
47
Chapter 1
while our population consists of Dutch Caucasian patients, these results indicate that the
genetic risk factors for the development of Barrett oesophagus could differ between
different ethnicities. The increased expression of CD14 in CD14-260*T carriers might
explain the association with protection against Barrett oesophagus. A better recognition of
an antrum predominant H. pylori infection which is associated with increased acid
production29 may result in a stronger inflammatory response. CD14 stimulates both IL-1
and TNF-α production18. Both cytokines reduce gastric acid production13, 30, 31 and thus
reduce the effect of gastric acid in the oesophagus and so reduce the risk for development
of Barrett oesophagus. The mutant allele of TLR4 has been associated with a reduced LPS
recognition. The mutant TLR4*G allele affects the extracellular domain of the TLR4
receptor thus blunting the response to LPS19. We observed a trend towards risk for Barrett
oesophagus in carriers of this mutant allele.
When the regulatory and bacteria-sensing carrier traits were combined in a larger carrier
trait, significant differences between cases and controls could be observed. Specific carrier
traits of the IL-1B, IL-1RN, CD14, and TLR4 genes were significantly decreased in patients
with Barrett oesophagus when compared to controls. These carrier traits differ from the
regulatory and bacteria-sensing carrier traits, adding to the complexity of the observed
effects.
48
Recent literature has provided conflicting evidence for the role of IL-1 polymorphisms and
H. pylori infections in Barrett oesophagus. For instance, Gough et al. have shown that the
IL-1RN +2018 2.2 genotype was associated with Barrett oesophagus when compared to
oesophagitis32, while Moons and colleagues failed to confirm this association in a similar
population33. In another paper, Moons et al. demonstrated a predominantly humeral
immune response in patients with Barrett oesophagus, which may explain a lack of
association with the Th1 type cytokine IL-1.
Combined carriage of the CD14 TT genotype and the TLR4 AA genotype confers optimal
recognition of H. pylori and thus protection in an antrum predominant infection. In a corpus
predominant infection which is associated with reduced acid production34, this effect may
be reversed and this effect of the location of H. pylori infection should be researched in a
population with clearly defined corpus or antrum gastritis. It should be noted that
conflicting reports have been published on the role of TLR4 in H. pylori infection. Both
positive35, 36 and negative37, 38 associations have been reported. Different H. pylori LPS
types39, 40 and TLR4 expression and subcellular distribution41, have been reported as
possible explanations for these conflicting results.
Equally conflicting reports have been published on the effect of H. pylori on development
of reflux disease and Barrett oesophagus. However, recent findings may offer an
explanation. An antrum predominant gastritis results in increased acid production, while a
corpus predominant (atrophic) gastritis results in reduced gastric acid secretion29, 34. These
differences in infection pattern may explain the differences in reported influences of
H. pylori on reflux disease, since increased acid production has been associated with reflux
disease after eradication of the bacteria42, 43. Unfortunately, we currently do not have data
on H. pylori infection in this cohort, so we can not comment on the influence of H. pylori
on Barrett oesophagus.
Barrett oesophagus
Pei and colleagues have recently described a variety of bacterial species in oesophageal
biopsy specimens in patients with oesophageal reflux related disorders44. These bacteria
might influence development of oesophagitis and Barrett oesophagus. The interaction
between these bacterial species and CD14 – TLR4, and the regulation of the immune
response against these bacteria by IL-1 may influence pathogenesis of Barrett oesophagus.
However, it should be noted that the study of Pei et al., is only a preliminary report and
further studies are required to confirm the results, to elucidate the role of the bacterial
species in oesophageal reflux related disorders, and to elucidate the role of the IL-1B,
IL-1RN, TLR4, and CD14 genes in upper gastrointestinal tract pathogenesis.
Complex mechanisms underlay development of Barrett oesophagus and pathogenic
mechanisms in one disease may be protective against another. An overview of variables
currently associated with Barrett oesophagus is presented in table 8.
Further research is required to elucidate these pathological mechanisms and to gain a better
understanding of Barrett oesophagus. The present results suggest that a combined analysis
of environmental, bacterial and host factors will help to identify patients at high risk for
development of Barrett oesophagus and oesophageal cancer.
Variable
Gender (Male)
BMI (Obesity)
Ethnicity (Caucasian)
Diet (vitamin C)
Hiatal hernia
Socio–economical
status
Oesophagitis
Drinking
Medication (NSAIDs)
OR (95% CI)
2.7 (2.2 – 3.4)
3.0 (1.6 – 6.7)
4.0 (1.4 – 11.1)
6.0 (3.6 – 10)
0.44 (0.2 – 0.98)
5.4 (3.1 – 9.4)
Remarks
1.6 (1.2 – 2.2)
Reference
Ford, 20054
Bu, 200645
El-Serag, 200546
Ford, 20054
Veugelers, 200647
García Rodríguez,
200648
Ford, 20054
1.8 (1.6 – 2.0)
p: 0.001
0.40 (0.19 – 0.81)
Ford, 20054
Conio, 200249
Anderson, 200650
No OR given
Table 8: Overview of current known factors associated with Barrett oesophagus
Acknowledgements
S. Ouburg is supported by a PhD-fellowship from AstraZeneca, the Netherlands. The authors would like to thank
M.L. Laine for excellent statistical assistance and J. Pleijster for outstanding technical assistance.
K.K. Krishnadath is supported by the Dutch Cancer Society (KWF Kankerbestrijding).
49
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Chapter 2
Based on
The toll-like receptor 4 (TLR4) Asp299Gly polymorphism is associated with colonic
localisation of Crohn's disease without a major role for the
Saccharomyces cerevisiae mannan-LBP-CD14-TLR4 pathway
Sander Ouburg*, Rosalie Mallant-Hent*, J. Bart A. Crusius, Ad A. van Bodegraven, Chris J.J. Mulder,
Ronald Linskens, A. Salvador Peña & Servaas A. Morré
* Both authors contributed equally to the manuscript
Gut 2005; 54 (3): pp. 439 – 440
Chapter 2
Abstract
ntroduction: Both genetic and microbial factors seem to play a pivotal role in
the aetiopathogenesis of inflammatory bowel disease (IBD). It has been
suggested that a genetic predisposition leads to a dysregulated intestinal
immune response in part to environmental factors. The recently identified toll-like receptor
(TLR) 4, recognising amongst others LPS (via CD14) and host & bacterial heat shock
proteins, is a potential candidate gene involved in mediating essential immune responses in
the intestinal tract, predisposing patients to IBD in general or to Crohn’s disease (CD), or
ulcerative colitis (UC) specifically. CD patients with high serological levels of anti
Saccharomyces cerevisiae antibodies (ASCA) have more severe disease, as defined by
fibrostenotic and internal perforating disease, and are more likely to require small bowel
surgery.
I
Aim: To assess the frequency of candidate gene polymorphisms in the bacterial agonist
recognizing CD14 and TLR4 genes in the susceptibility to and severity of CD and UC in
Dutch Caucasian IBD patients.
54
Methods: 210 IBD patients (109 CD patients and 101 UC patients) and 170 healthy
controls were included in the study. PCR based RFLP analyses were used to identify the
CD14-260 C>T and TLR4+896 A>G SNPs. Genotype and allele frequency analyses were
performed and the Vienna classification (CD patients) was used to assess potential
association with disease phenotype. Antibodies against Saccharomyces cerevisiae (ASCA)
were measured using an ASCA ELISA.
Results: The frequency of the G allele of TLR4+896 was increased in CD patients
compared to controls (19% vs. 10%; p: 0.049; OR: 2.0). The TT genotype of CD14 -260
showed an increased frequency in UC patients compared to healthy controls (34% vs. 24%;
p: 0.09). Carriage of TLR4+896*G significantly increases the risk for colonic localization
of CD compared to non-colonic localization (41% vs. 13%; p: 0.0047; OR: 4.8; 95% CI: 1.7
- 14).
Conclusion: The association of the TLR4+896 polymorphism with CD suggests a
potentially important role for bacteria in the immunopathogenesis of CD. The association
we demonstrated between TLR4 and CD and colonic localization, is most likely not
strongly based on the S. cerevisiae mannan–LBP–CD14–TLR4 pathway, as we have shown
based on the ASCA data in our group.
TLR4
Crohn’s Disease
Introduction
he acquired immune system and the innate immune system form the host
defence against invading pathogens. The innate immune system is an inborn
defence mechanism and is already present the first time a pathogen is
encountered. The innate immune response does not require prior exposure and it is not
significantly modified by repeated exposures to the same pathogen. The acquired immune
system is a weak defence on the first exposure, however the efficiency and effectiveness of
this defence mechanism are greatly increased with subsequent exposures to the pathogen.
Toll-like receptor 4 (TLR4) and Cluster of differentiation 14 (CD14) are an integral part of
the recognition complex for lipopolysaccharide (LPS), HSP60 and other bacterial agonists.
This complex acts as a signalling receptor for the innate immune system. LPS is a
component of Gram-negative bacterial cell walls. CD14 is present on the membranes of
monocytes and macrophages and recognizes LPS – LPS binding protein (LBP) complexes1.
LPS is transferred from the CD14–LBP complex to a TLR-MD2 membrane receptor
complex2. The subsequent transmembrane signal induces NF-κB activation, resulting in the
activation of NF-κB responsive genes and the release of pro-inflammatory cytokines,
including interleukin 1 (IL-1) and tumour necrosis factor alpha (TNF-α)3. Besides LPS,
other bacterial and host agonists are recognised by TLR4, including both host and bacterial
heat shock protein 60 (HSP60)4.
CD14 mediates sensitive responses to a wide variety of pathogens (bacteria, both gram
negative and gram positive, mycobacteria and possible viruses)5. Studies showed that a low
dose of LPS resulted in a Th2 mediated immune response, while a high bacterial load
induces a Th1 mediated immune response5.
The T allele (mutant) of the CD14-260 SNP enhances transcriptional activity6 and
monocyte CD14 expression7, 8. Carriers of TT genotype have significantly increased serum
levels of soluble CD14 and a higher density of membrane-bound CD14, compared to the
CC and CT genotypes7, 8.
The T allele of the CD14 SNP has been associated with ulcerative colitis (UC) in a
Japanese population9. In a German population, the same mutant allele was increased in
Crohn’s disease (CD), but not in UC10.
The TLR4+896 A>G SNP causes an aspartic acid to glycine substitution in the extracellular
receptor domain of the TLR4 receptor. This SNP has been associated with an “endotoxin
hyporesponsive phenotype”11. However, monocytes of individuals carrying one copy of the
G allele do not exhibit a deficient LPS recognition12 or an altered LPS induced cytokine
release13. For other agonists, including both host and bacterial HSP60/70, the effect of
heterozygous carriage for TLR4+896 has not yet been assessed.
In 1988, Main et al. first described the presence of anti-Saccharomyces cerevisiae
antibodies (ASCA) in patients with CD14. About 65% of CD patients are known to be
positive for ASCA antibodies, while only 0–5% in healthy controls is positive for these
antibodies. The presence of the antibodies is currently used to subtype the disease in
different phenotypes14 - 18. The ASCA test for diagnosing CD has a sensitivity of 72% and a
specificity of 82%15.
The nature of S. cerevisiae antigens supporting the specific antibody response in CD is still
unknown. ASCA are thought to result from a specific antibody response to the S. cerevisiae
cell wall mannan (phosphopeptidomannans). It is not known whether this is a direct
response towards the yeast itself or an epiphenomenon with a similar immunologic
response towards another antigen. It is postulated that the yeast wall cell mannan may
T
55
Chapter 2
mimic a high mannose-containing molecule towards which the antibody is directed
inducing a hypersensitivity reaction during inflammation19. Patients with high serological
levels of ASCA have more severe disease, as defined by fibrostenotic and internal
perforating disease and are more likely to require small bowel surgery20, 21.
The aim of this study was to assess the frequencies of the CD14-260 and TLR4+896 SNPs
to study their influence on IBD (subdivided in UC and CD) susceptibility and severity.
Furthermore, we assessed the potential relevance of the S. cerevisiae mannan–LBP–CD14–
TLR4 pathway using the ASCA status of the patients.
Materials & methods
56
Study population:
Participants were recruited from the outpatient clinic of the department of Gastroenterology of the VU
University Medical Centre, Amsterdam, The Netherlands. The group consisted of 210 IBD patients
(109 CD patients and 101 UC patients). Mean age was 43 years (range 20-84) for CD and 48 (range
22-88) for UC. Seventy percent of CD patients were of female gender compared to 53% in the UC
population. Diagnosis of disease was based on clinical, histopathologic and endoscopic findings
according to the classification of Lennard-Jones22. CD patients were categorised using the Vienna
classification23, which subdivides patients according to age of onset (below 40 years (A1) or older
(A2)), localization of disease (terminal ileum (L1), colon (L2), ileocolonic (L3) or upper
gastrointestinal tract (L4)) and disease behaviour (penetrating (B1), stricturing (B2), non-penetrating /
non-stricturing (B3)) in the disease period from diagnosis until the first surgical procedure.
The ethical committee approved the study. All patients were informed and gave informed consent.
The control group comprised of 170 healthy controls. Both patients and controls were unrelated
Dutch Caucasians.
ASCA ELISA:
ASCA IgA and IgG were evaluated in commercially available ELISA kits, kindly sponsored by Inova
24
Diagnostics, San Diego, CA as described by Linskens et al.15. The antigen consisted of
phosphopeptidomannan (PPM) extracted from Saccharomyces cerevisiae. Results were expressed as
arbitrary units with a cut off positivity of 25U/ml. Sera were considered positive if either IgA or IgG
both were positive. Sera were considered negative if both IgA and IgG ASCA were negative.
Genotyping:
Genomic DNA was isolated from blood samples using a Roche DNA isolation kit (Roche Molecular
Biochemicals, Mannheim, Germany). Both the CD14-260 C>T and TLR4+896 A>G SNPs were
genotyped from genomic DNA, using PCR RFLP.
Genotyping the CD14-260 C>T SNP (rs2569190) was performed with forward primer 5’-TCA CCT
CCC CAC CTC TCT T-3’ and reverse primer 5’-CCT GCA GAA TCC TTC CTG TT-3’. The PCR
program consisted of 94˚C for 5 minutes, followed by 35 cycles of 30 seconds at 94˚C, 30 seconds at
59˚C, and 1 minute at 72˚C. A final step at 72˚C for 7 minutes was followed by 4˚C, in 96 wells PCR
plates (Greiner Bio-One). The 107bp amplificates were digested overnight with HaeIII (Invitrogen,
Paisley, UK). The digestion resulted in two fragments of 83bp and 24bp (C allele) or 107bp (T allele),
respectively.
The TLR4+896 A>G SNP (rs4986790) was genotyped and the oligonucleotides forward 5’-TT ACC
CTT TCA ATA GTC ACA CTC A-3’ and reverse 5’-AGC ATA CTT AGA CTA CCT CCA TG-3’
flanking this region were used as primers. Conditions for the PCR were as follows: 94˚C for 5
minutes followed by 35 cycles of 94˚C for 30 seconds, 55˚C for 30 seconds, 72˚C for 30 seconds and
finally 72˚C for 7 minutes followed by cooling to 4˚C, in 96 wells PCR plates (Greiner Bio-One). The
102bp amplificates were digested overnight with NcoI (Invitrogen, Paisley, UK). The digestion
resulted in two fragments of 80bp and 22bp (G allele) or 102bp (A allele), respectively.
TLR4
Crohn’s Disease
All restriction fragments were analyzed by electrophoresis on 4% agarose gels, stained with ethidium
bromide and photographed under UV-light.
Statistical analyses:
The CD14-260 and TLR4+896 allele and carrier frequencies as well as ASCA status, their
combinations (carrier trait) and carriers of at least one copy of a rare allele were compared between
control and patient groups. The patient group was compared as a whole (IBD) or subdivided in UC
and CD. Furthermore CD patients were subdivided according to the Vienna Classification. Statistical
methods used were the χ2 and Fisher exact test (two-sided) and logistic regression analyses. Odd’s
ratios (OR) and 95% Confidence Intervals (95% CI) were calculated. A p-value <0,05 was considered
significant. Statistical analyses were performed using SPSS 11.0 and Graphpad Instat.
Results
The TT genotype of CD14-260 showed a slightly increased frequency in UC patients
compared to healthy controls (34% vs. 24%; p: 0.09; OR: 1.6; 95% CI: 1.0 – 2.8), see
table 1. No differences were found when CD patients (classified according to the Vienna
Classification) were compared to controls.
1.1
CC
Group Total
170
HC
109
CD
101
UC
n
48
33
25
CD14-260 C>T
1.2
2.2
CT
TT
%
n
%
n
%
28
82
48
40
24
30
53
49
23
21
25
42
42
34
34
1.1
AA
n
153
89
90
TLR4+896 A>G
1.2
2.2
AG
GG
%
n
%
n
%
90
16
9
1
1
82
18
17
2
2
90
10
10
1
1
Table 1: Genotype distribution of TLR4 and CD14 for UC, CD and healthy controls (HC).
The frequency of the G allele of TLR4+896 was significantly increased in CD patients
compared to controls (19% vs. 10%; p: 0.049; OR: 2.0; 95% CI: 1.0 – 4.1).
Disease phenotype was assessed in CD patients using the Vienna Classification. Carriage of
TLR4+896*G significantly increases the risk for pure colonic localization of CD compared
to other localizations (41% vs. 13%; p: 0.0047; OR: 4.8; 95% CI: 1.7 – 14).
We also assessed if ASCA status was correlated with carriage of the TLR4*G allele.
however, there was no difference between TLR4*G allele carriage in the ASCA positive
and ASCA negative patients (22% vs. 14%; p: 0.45) and there was no difference between
TLR4*G allele carriage in the ASCA positive and negative CD patients with colonic
localization (33% vs. 46%; p: 0.67), while the frequency of G allele carriage was identical
to that of CD patients with colonic localization (41%) without correcting for the ASCA
status, see table 2.
Although there was a difference in the frequencies of the TLR4*G allele between the two
age classifications for CD (20% vs. 8%), this was not statistically significant (p: 0.45).
57
Chapter 2
Group
CD
CD
58
Vienna
Total
ASCA+
A1
ASCA+
A2
ASCA+
B1
ASCA+
B2
ASCA+
B3
ASCA+
L1
ASCA+
L2
ASCA+
L3
ASCA+
L4
ASCA+
Total
109
60
96
53
13
8
42
20
44
24
23
16
40
22
22
9
46
29
1
0
CD14-260 C>T
1.1
1.2
2.2
CC
CT
TT
n % n
%
n %
33 30 53
49 23 21
18 30 28
47 14 23
29 30 48
50 19 20
16 30 26
49 11 21
4 31
5
38
4 31
3 38
2
25
3 38
13 31 20
48
9 21
6 30
9
45
5 25
14 32 21
48
9 20
8 33 10
42
6 25
6 26 12
52
5 22
4 25
9
56
3 19
13 33 17
43 10 25
7 31
8
36
7 32
6 27 12
55
4 18
3 33
5
56
1 11
14 30 23
50
9 20
8 28 15
52
6 21
0
0
1 100
0
0
0
0
0
0
0
0
TLR4+896 A>G
1.1
1.2
2.2
AA
AG
GG
n
%
n %
n
%
89
82 18 17
2
2
47
78 12 20
1
2
77
80 18 19
1
1
41
77 12 23
0
0
12
92
0
0
1
8
7
88
0
0
1 13
35
83
6 14
1
2
16
80
4 20
0
0
36
82
7 16
1
2
19
79
4 17
1
4
18
78
5 22
0
0
12
75
4 25
0
0
35
88
4 10
1
3
18
82
3 14
1
5
13
59
8 36
1
5
6
67
3 33
0
0
40
87
6 13
0
0
23
79
6 21
0
0
1 100
0
0
0
0
0
0
0
0
0
0
Table 2: Results of CD14 and TLR4 genotyping for CD in relation to ASCA status.
Discussion
The primary role of TLRs is to recognise bacterial, viral, fungal and other pathogens that
may be deleterious to the host. Recently published phenotype – genotype studies of TLRs
demonstrate that mutations in TLRs may contribute to increased susceptibility to certain
infections or to differences in disease course25. In this study we investigated the effect of
both CD14-260 and TLR4+896 SNPs on the susceptibility to and severity of IBD, showing
that the frequency of the G allele of TLR4 +896 was significantly increased in CD patients
compared to controls. Disease phenotype was assessed in CD patients using the Vienna
classification and we showed that carriage of TLR4+896*G significantly increases the risk
for colonic localization of CD compared to non-colonic localization. These results were not
confounded by the three main CARD15 SNPs as has been published in part by our group
before (Murillo et al., Immunogenetics 2002)26 and Linskens et al. (Dig. Liv. Dis. 2004)27
(data not shown).
Several studies have described the TLR4+896 A>G and CD14-260 C>T SNPs in CD. Klein
and colleagues describe a German population of 361 IBD patients (142 UC and 219 CD)
and found an increased incidence of CD14-260 heterozygous and homozygous mutants in
CD patients compared to healthy controls10.
This association could not be confirmed in our Dutch Caucasian population nor in a
TLR4
Crohn’s Disease
Scottish and Irish population described by Arnott et al.28.
Preliminary data by Braat et al.29 demonstrated an increased risk of suffering from CD in a
Dutch population carrying the TLR SNP confirming our results. Franchimont and
colleagues corroborate the results from Braat et al.30. In contrast to Franchimont and
colleagues we found a clear association between the G allele of TLR4+896 and disease
phenotype (colonic localization). In contrast to the aforementioned studies and results,
Arnott et al. were unable to demonstrate an association between CD and the TLR4 SNPs in
a Scottish and Irish population28.
The association between TLR4 and CD underscores the role of impaired innate immunity in
CD. TLR4 signalling is based on both exogenous (e.g. LPS) and endogenous (e.g. human
HSPs) agonists, and heterozygous carriage of the TLR4+896 A>G does not seem to impair
LPS signaling12. Further agonist identification to elucidate the microorganisms involved in
CD and especially in the colonic localization is essential to get insight in both
pathophysiological and monogenetic aspects of CD. This insight is potentially helpful to
develop strategies for prevention and treatment of CD.
Although there is increasing evidence that IBD results from the combined effects of
environmental agents and host genetic factors, there is still a long way to go in order to
define other genes and the environmental triggers that initiate the disease31. The mucosal
innate immune system in the gastrointestinal tract has a critical function in the defence
against intestinal pathogens. Intestinal epithelial cell lines constitutively express several
functional Toll-like receptors (TLRs) which appear to be key regulators of the innate
response system32. Individual TLRs activate specialized antifungal or antibacterial genes
through the activation of NF-κB family members. Yeasts, for instance, activate TLR233, 34.
Both Hugot and Ogura have emphasized the possibility of a functional connection between
the Leucine Rich Repeats (LRR) in the NOD2 protein35, 36 and the TLRs37, which all
recognise their own conserved molecular patterns38.
In conclusion, the association of the TLR4+896 polymorphism and CD suggest a potentially
important role for bacteria in the immunopathogenesis of CD. The association we
demonstrated between TLR4 and CD and colonic localization, is most likely not strongly
based on the S. cerevisiae mannan-LBP-CD14-TLR4 pathway, as we have shown based on
the ASCA data in our group. Further agonist identification will be helpful in elucidating the
microorganisms involved in CD.
59
Chapter 2
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61
Chapter 2
62
Nothing shocks me. I'm a scientist.
Harrison Ford, as Indiana Jones
US movie actor (1942 - )
Chapter 3
The role of the bacterial CpG sensing toll-like receptor 9 (TLR9) in Dutch
Caucasian and Spanish Galician patients with Crohn’s disease: evidence for genetic
heterogeneity
Sander Ouburg, Manuel Barreiro, J. Bart A. Crusius, Ad A. van Bodegraven,
J. Enrique Dominguez-Muñoz, A. Salvador Peña & Servaas A. Morré
Chapter 3
Abstract
he frequency of the TLR9+2848 AA was significantly decreased in Dutch CD
patients (p: 0.02) and especially in penetrating disease (p: 0.01). In Galicians
the TLR9-1237 CC increased the risk of stricturing disease (p: 0.03) and was
associated with age over 40 years (p: 0.02). The TLR9+2848 AA was associated with
colonic localization in Galician CD patients (p: 0.02).
T
64
TLR9
Crohn’s Disease
Introduction
rohn’s disease (CD) is characterized by chronic intestinal inflammation
resulting from an aberrant or excessive activation of the immune response
generated against ubiquitous bacteria or bacterial products that gain access to
the lamina propria. Genetic predisposition for CD is well established by epidemiological
and genomic-wide linkage studies1, 2. The clear association of CARD15 (NOD2), the
bacterial peptidoglycan receptor, with CD strengthens the dysbiosis theory3 and emphasizes
the relevance of bacterial triggers in host susceptibility. Toll like receptor 9 (TLR9) is
required for the recognition of bacterial CpG motifs, which are short sequences of
unmethylated DNA predominantly present in bacterial DNA. CpG motifs have
immunostimulatory activity by inducing dendritic cell maturation, B cell proliferation and
production of cytokines, including interleukin-6 (IL-6) and interleukin-12 (IL-12)4, 5.
Rachmilewitz et al. have recently described the essential role of TLR9 in mediating the anti
inflammatory effects of probiotics in dextran sodium sulfate induced colitis in mice6.
Subsequently, Török et al.7 described the association of a genetic variation in TLR9 with
CD in German patients. They described an association for the promoter TLR9 –1237 T>C
SNP with CD and Lazarus et al. described an association with susceptibility to develop
asthma in European Americans, but not in Hispanic or African Americans8.
C
Due to the clear differences in genotype distribution described by Lazarus et al.8,
differences between clearly defined ethnically and geographically isolated populations are
expected, especially since differences in CD between the Dutch Caucasian and Galician
populations have been described9.
Here we report the results of a candidate gene based study of TLR9 polymorphisms in over
600 CD patients and controls from Dutch Caucasian and Spanish Galician ethnicity, to
determine if genetic heterogeneity within the TLR9 gene plays a role in the differences in
CD between these distinct populations.
Patients and methods
Two ethnically different patient and control cohorts were recruited. The first cohort was recruited
from the Outpatient Clinic of the Department of Gastroenterology of the VU University Medical
Centre, Amsterdam, The Netherlands. The group consisted of 133 CD patients and 147 unrelated
Dutch Caucasian controls. Diagnosis of disease was based on clinical, histopathologic and endoscopic
findings. CD patients were categorized using the Vienna classification (general patient characteristics
are described elsewhere10). The second cohort consisted of 165 unrelated Galician (NW Spain)
patients with Crohn's disease and 163 healthy unrelated ethnically matched individuals with no family
history of IBD who were recruited from the Department of Gastroenterology at the “Hospital Clínico
Universitario” of Santiago de Compostela (Galicia, Spain).
Genotyping of two TLR9 SNPs – TLR9 –1237 T>C (NCBI SNP CLUSTER ID: rs5743836) and TLR9
+2848 G>A genotyping (NCBI SNP CLUSTER ID: rs352140) – was performed using standard
TaqMan analysis, in standard 96 well plates (Greiner Bio-One). The primers and probes used for
TLR9 -1237 were: forward primer 5’- GGC CTT GGG ATG TGC TGT T-3’ and reverse primer 5’GGT GAC ATG GGA GCA GAG ACA-3’; and dual-labeled fluorogenic hybridization MGB-probes:
CTG CCT GAA AAC T-5’ Fluor Label (FAM, 6-carboxyfluorescein) and CTG GAA ACT CCC C5’ Fluor Label (VIC). The primers and probes used for TLR9+2848 were: forward primer 5’-CCG
65
Chapter 3
GTC TGC AGG TGC TAG AC-3’ and reverse primer 5’-CCA AAG GGC TGG CTG TTG TA-3’;
and dual-labeled fluorogenic hybridization MGB probes: AGC TAC CGC GAC TGG-5’ Fluor Label
(FAM) and AGC TAC CAC GAC TGG A-5’ Fluor Label (VIC).
TLR9 haplotype
The two TLR9 SNPs analyzed were chosen based on the study of Lazarus et al.8 in which a set of four
frequent TLR9 SNPs (allele frequencies of at least 10% in four ethnically different populations,
including the European American population), designated as TLR9 –1486, TLR9 –1237, TLR9 +1174
and TLR9 +2848, were described. Genotyping of both TLR9 –1237 and TLR9 +2848 SNPs allows all
4-locus haplotypes commonly present in the European American population to be distinguished. We
therefore calculated genotype-based haplotypes in our populations.
The TLR9 SNP genotypes, allele, carrier frequencies and genotype-based haplotypes were compared
between the different clinical patient groups and controls, and between the two ethnically different
cohorts. The data were analysed in a multivariate logistic regression model to assess the association of
different genotypes with CD, adjusted for age and gender.
Results
66
The results are displayed in table 1. In Dutch Caucasians the frequency of the AA genotype
of TLR9 +2848 G>A was significantly decreased in CD patients compared to controls (23%
vs. 36%, p: 0.018, OR: 1.9, 95% CI: 1.1 – 3.2). This was not observed in the Spanish
Galicians. In addition, the TLR9 +2848 AA genotype significantly decreased the risk of
penetrating disease (Vienna Classification B3) in CD patients compared to healthy controls
in Dutch Caucasians (11.1% vs. 36%, p: 0.013, OR: 4.5, 95% CI: 1.3 – 15.5) but not in
Spanish Galicians. However, this same genotype is significantly increased in Galician
patients with colonic disease (L2) compared to those without colonic disease (50% vs. 26%,
p: 0.022, OR: 2.9, 95% CI: 1.2 – 6.6).
The TLR9 -1237 CC was significantly increased in Galician patients over 40 years old (A2)
compared to younger patients and in Galician patients with stricturing disease (B2)
compared to non stricturing disease (p: 0.02, OR: 14.7, 95% CI: 1.5 – 146.5 and p: 0.031,
OR: 11.9, 95% CI: 1.2 – 118.4, respectively). Remarkably, there was a significant
difference (p: 0.036, OR: 1.9, 95% CI: 1.1 – 3.3) when comparing the wildtype genotype
GG frequency for the TLR9+2848 between Dutch Caucasians (16%) and Spanish Galicians
(26%).
Haplotype analysis showed that the difference between Duch Caucasian CD patients and
controls for the +2848*A allele is defined in haplotype II. The haplotype frequencies for
haplotypes I and II seem reversed when comparing Dutch Caucasian patients and controls,
while Dutch Caucasian CD patients have identical haplotype frequencies compared to
Galician CD patients and controls. Analyses of the complete haplotypes defined by Lazarus
et al.8 showed that the TLR9 +1174 was always G and could be C or T at TLR9 –1486
(making future analyses of this promotor location an interesting site for future analyses).
Logistic regression analysis confirmed the associations between TLR9 +2848 AA and CD
in Dutch Caucasians, between TLR9 -1237 CC and stricturing disease in Galicians, between
TLR9 -1237 CC and age over 40 years in Galicians, and between TLR9 +2848 AA and
TLR9
Crohn’s Disease
colonic localization in Galicians (data not shown).
Dutch Caucasians
Crohn’s
Healthy
Disease
Controls
n=133
n=147
n
%
n
%
Spanish Galicians
Crohn’s
Healthy
Disease
Controls
n=163
n=165
n
%
n
%
TLR9 –1237 T>C
TT
TC
CC
101
29
3
76
22
2
105
42
0
71
29
0
119
40
4
73
25
3
112
47
6
68
29
4
TLR9 +2848 G >A
GG
GA
AA
27
76
30
20
57
23
23
71
53
16
48
36
43
71
49
27
44
30
42
74
49
26
45
30
TLR9 Haplotypes1
-1237 +2848
I
T
G
II
T
A
III
C
A
IV
C
G
2n
128
103
33
2
%
48
39
12
0.8
2n
115
139
39
3
%
39
47
13
1
2n
154
124
45
3
%
47
38
14
0.9
2n
152
119
53
6
%
46
36
16
2
Table 1: TLR9 genotypes and haplotypes in Dutch Caucasian and Spanish Galician Crohn’s disease patients and
controls
1
TLR9 haplotype 2n definition: haplotypes were based on the TLR9 –1486, TLR9 –1237, TLR9 +1174 and TLR9
+2848 position described by Lazarus et al 8. European American and Hispanic American haplotype definitions
and frequencies: Haplo type I (TTAG) 44 vs. 40%; Haplo type II (CTGA) 39 vs. 56%; Haplo type III (TCGA) 15
vs. 4.2%; Haplo type IV (TCGG) 0 vs. 0%, (CTGG) 0 vs. 0%, (CCGG) 0 vs. 0%, and (TTGA) 2.2 vs. 0%.
Discussion
Török and colleagues found an increased incidence of –1237*C allele carriers in German
CD patients compared to controls, while the +2848 AG genotype was found to be slightly
decreased in CD patients compared to controls7. These associations were partially
confirmed in our populations. Our Dutch population did show a clear decreased frequency
of the +2848 AA genotype in CD patients and specifically in CD patients with penetrating
disease, while the -1237 CC genotype was increased in stricturing disease in Galicians.
When we compared the haplotypes in our Galician population to the haplotypes in the
Hispanic American population described by Lazarus et al.8, we found that the –1237*T /
+2848*G haplotype was expressed in lower frequencies in the Hispanic American
population compared to both our Galician CD and control populations (40% vs. 47% /
46%), while the –1237*T / +2848*A haplotype was expressed in higher frequencies (56%
vs. 38% / 36%). Galicia is a geographically and ethnically rather isolated region, while most
Hispanic populations are mixtures of Spanish, Mexican and Latin American populations.
These differences in population composition may explain the observed haplotype
differences.
67
Chapter 3
The TLR9 gene is located on chromosome 3p21.2 in the vicinity of the marker D3S1076
which shows an association with IBD in a classical TDT test 11. We did find evidence for
an association of the TLR9 SNPs and haplotypes with CD and with penetrating CD (Vienna
classification B3). However these TLR9 SNPs are not known to influence the level of
expression of TLR9 and the association observed might therefore result from linkage
disequilibrium with other polymorphisms in a nearby gene. In a recent study by van Heel et
al. a synergistic interaction between TLR9 and NOD2 was demonstrated, where NOD2
enhances TLR9 signal transduction. Well characterized NOD2 SNPs drastically reduce
TLR9 initiated immune responses12. In a similar study, Netea et al. did not find an
interaction between NOD2 and TLR9, but they did find interaction between NOD2 and
TLR4 and TLR313.
Identification of all aspects of the TLR9 signalling pathway, including the aforementioned
potential enhancing role of NOD2, is essential to both elucidate the role of microorganisms
in CD, and especially specific phenotypes of CD, and to gain insight into both the
pathophysiological and immunogenetic aspects of CD. The findings reported here
underscore the role of innate immunity in CD.
68
In conclusion, using a candidate gene approach we showed that the TLR9+2848 AA was
associated with a decreased risk of developing of CD and of penetrating CD in Dutch
Caucasian patients, but in Spanish Galicians the TLR9-1237 CC increased the risk of
stricturing CD, suggesting that these markers might play different roles in the susceptibility
to CD in populations from different ethnic backgrounds. Our findings need to be confirmed
and functional studies are required for a better understanding of the biological role of TLR9
in CD.
Acknowledgements
S. Ouburg is supported by a PhD-fellowship from AstraZeneca, the Netherlands. S. A. Morré is supported by
Tramedico BV, the Netherlands, the Falk Foundation, Germany and the Department of Internal Medicine of the
VU University Medical Centre, the Netherlands. We thank Jolein Pleijster for superb technical assistance in the
genotyping of TLR9 polymorphisms and database management. Part of this work was financed by a grant from the
Gastrostart foundation (grant code 10-2003), The Netherlands.
TLR9
Crohn’s Disease
References
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3.
4.
5.
6.
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8.
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10.
11.
12.
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The South Limburg IBD Study Group, M. G. Russel, C. J. Pastoor, K. M. Janssen, C. T. van Deursen, J. W.
Muris, E. H. van Wijlick & R. W. Stockbrugger, Scandinavian Journal of Gastroenterology. Supplement 1997; 223
pp. 88 - 91, PubMed: 9200312
Genome-wide scanning in inflammatory bowel diseases, J. P. Hugot & G. Thomas, Digestive Diseases 1998; 16 6):
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Krieg, Proceedings of the National Academy of Sciences of the United States of America 1996; 93 7): pp. 2879 2883, PubMed: 8610135
126 2): pp. 520 - 528, PubMed: 14762789
Crohn's disease is associated with a toll-like receptor-9 polymorphism, Helga Paula Török, Jürgen Glas, Laurian
Tonenchi, Guenter Bruennler, Matthias Folwaczny & Christian Folwaczny, Gastroenterology 2004; 127 1): pp.
365 - 366, PubMed: 15236225
Single-nucleotide polymorphisms in the Toll-like receptor 9 gene TLR9): frequencies, pairwise linkage disequilibrium,
and haplotypes in three U.S. ethnic groups and exploratory case-control disease association studies, Ross Lazarus,
Walter T. Klimecki, Benjamin A. Raby, Donata Vercelli, Lyle J. Palmer, David J. Kwiatkowski, Edwin K.
Silverman, Fernando Martinez & Scott T. Weiss, Genomics 2003; 81 1): pp. 85 - 91, PubMed: 12573264
CARD15 mutations in patients with Crohn's disease in a homogeneous Spanish population, Concepción Núñez,
Manuel Barreiro, J. Enrique Dominguez-Muñoz, Aurelio Lorenzo, Carlos Zapata & A. Salvador Peña,
American Journal of Gastroenterology 2004; 99 3): pp. 450 - 456, PubMed: 15056084
Salvador Peña, Digestive and Liver Disease 2004; 36 1): pp. 29 - 34, PubMed: 14971813
Lynch, S. Daniels, Stephen Bridger, Andrew J. S. MacPherson, P. Stokkers, Alastair Forbes, John E. LennardJones, Chistopher G. Mathew, M. E. Curran & Stefan Schreiber, Gut 2001; 48 2): pp. 191 - 197, PubMed:
11156639
Synergy between TLR9 and NOD2 innate immune responses is lost in genetic Crohn's disease, David A. van Heel,
Subrata Ghosh, Karen A. Hunt, C. G. Mathew, A. Forbes, Derek P. Jewell & Raymond J. Playford, Gut 2005;
54 11): pp. 1553 - 1557, PubMed: 15928043
Nucleotide-binding oligomerization domain-2 modulates specific TLR pathways for the induction of cytokine release,
Mihai G. Netea, Gerben Ferwerda, Dirk J. de Jong, Trees Jansen, Liesbeth Jacobs, Matthijs Kramer, Ton H. J.
Naber, Joost P. H. Drenth, Stephen E. Girardin, Bart Jan Kullberg, Gosse J. Adema & Jos W. M. van der
Meer, Journal of Immunology 2005; 174 10): pp. 6518 - 6523, PubMed: 15879155
69
Chapter 3
70
The most exciting phrase to hear in science,
the one that heralds new discoveries,
is not 'Eureka!' (I found it!)
but 'That's funny ...'
Isaac Asimov
US science fiction novelist & scholar (1920 - 1992)
Chapter 4
CD14 and TLR4 gene polymorphisms in Galician patients with Crohn’s disease:
genetic and environmental interactions
Sander Ouburg*, Manuel Barreiro de Acosta*, A. Salvador Peña, Aurelio Lorenzo,
J. Enrique Domínguez-Muñoz & Servaas A. Morré
* Both authors contributed equally to the manuscript
Chapter 4
Abstract
oll-like receptor 4 (TLR4) acts as the transducing subunit of the LPS receptor
complex in the detection of Gram-negative pathogens. CD14 is a co-receptor of
TLR4, and it is essential for recognition of LPS and the subsequent signal
transduction that involves activation of NFκB. The recently described Asp299Gly
polymorphism in the TLR4 gene has been associated with a decreased sensitivity to LPS,
while the CD14-260 SNP has been associated with increased receptor density and higher
serum levels of soluble CD14. We have previously described the association of
CARD15/NOD2 in Galicians patients with CD. We hypothesize that the TLR4 and CD14
SNPs may, either alone or in a multi gene combination with each other or CARD15/NOD2,
influence the susceptibility to or severity of Crohn’s disease (CD).
T
Patients and methods: To test this hypothesis, we studied 165 consecutive patients with
CD (95 female and 70 male, mean age 36 years, age range 17 – 76) and 163 healthy,
unrelated controls, matched by ethnicity, sex and age, and with no family history of
inflammatory bowel disease. All cases and controls were originally from and current
residents of Galicia (NW of Spain). A blood sample was obtained from all cases and
controls, and all were genotyped for the TLR4+896 and the CD14-260 SNPs. Previously
obtained data for CARD15/NOD2 SNP8, SNP12, and SNP13 were included in the analyses.
Associations were expressed as odds ratios (OR) with 95% confidence interval (CI). Data
were analyzed by the χ2 test and the Fisher’s exact test, whenever appropriate.
72
Results: An increased incidence of TLR4*G was observed in cases compared to controls
(p: 0.0298, OR: 2.3, 95% CI: 1.1 – 5.0), while CD14*T was equally distributed between
cases and controls. Carriage of CD14*T appeared to protect against the development of
fistulae (p: 0.045, OR: 2.8, 95% CI: 1.4 – 5.8). Carriage of the CD14-260*T with the TLR4
wildtype was significantly associated with protection against fistulae (p: 0.03, OR: 3.2,
95% CI: 1.2 – 8.4). Corrections for alcohol use or smoking did not alter the results.
Increased incidence of combined carriage of the CD14*T with CARD15/NOD2 SNPs was
observed in cases compared to controls (p: 0.0007, OR: 3.1, 95% CI: 1.6 – 5.9). Addition of
the TLR4 wildtype to the combination resulted in an increased OR (p: 0.0003, OR: 3.5,
95% CI: 1.7 – 7.0). The CD14*T – CARD15/NOD2 SNP combination was significantly
increased in fistulae patients (p: 0.004, OR: 3.2, 95% CI: 1.5 – 6.8). TLR4 wildtype carriage
combined with CD14 and CARD15/NOD2 SNPs increased the risk for ileal resection
(p: 0.002, OR: 3.4, 95% CI: 1.6 – 7.3). The same genotype combination was significantly
increased in steroid resistant patients compared to non resistant patients (p: 0.04, OR: 2.5,
95% CI: 1.0 – 6.2).
Multivariate regression analysis showed that smoking, rural origin and the TLR4*G were
protective against CD, while age and SNP12 and SNP13 were risk factors. Carriage of
SNP12 was a risk factor for fistulae and CD14*T – TLR4 AA was protective. SNP13 was a
significant risk factor for ileal resection.
Conclusions: The TLR4+896 and CD14-260 SNPs influence the pathogenesis of CD also
in combination with the CARD15/NOD2 SNP8 / SNP12 / SNP13, providing further
evidence for the complex genetic contributions underlying CD.
CD14 & TLR4
Crohn’s Disease
Introduction
rohn’s disease (CD) is a chronic inflammatory condition of the
gastrointestinal tract. The prevalence is 10 – 200 cases per 100.000 persons in
Europe and North America, with highest incidences in highly urbanised
areas. CD most commonly affects the terminal ileum, coecum, peri-anal areas and the
colon. Patches of normal colon between affected areas, known as “skip lesions” are
characteristic of the disease1. Symptoms are based on the location and the extent of the
inflammation, and include amongst others diarrhoea, fatigue, weight loss, narrowing of the
gut and blood loss. Further complications include stenosis, fistulae and extraintestinal
manifestations, including rheumatoid arthritis / ankylosing spondylitis and inflammation of
eyes, skin, liver, and bones.
C
The generally accepted idea is that CD is caused by an complex interplay between genetic,
bacterial and environmental factors2. Previous studies in twins have provided evidence for a
genetic component in CD3, 4. CARD15/NOD2 single nucleotide polymorphisms (SNPs)
have been associated with CD5, 6 and have been demonstrated to be deficient in signalling in
response to PGN7, 8, however the CARD15/NOD2 SNPs alone do not explain the
concordance of CD as shown in a Swedish monozygotic twin study9. Since bacteria have
been associated with CD and since CARD15/NOD2 recognizes bacterial components10, one
might hypothesise that other bacterial recognition receptors may also influence the
susceptibility to or severity of CD.
recognise several microbial products, including bacterial cell wall components and DNA11.
Further studies in mice corroborated these data13, 14, while studies in humans demonstrated
associations between TLR4 mutations and LPS hyporesponsiveness15 - 17. TLR4 acts as the
transducing subunit of the LPS receptor complex in the detection of Gram-negative
pathogens. CD14 is a co-receptor of TLR4, and it is essential for recognition of LPS and
the subsequent signal transduction that involves activation of NF-κB. The TLR4 – CD14
receptor complex is also involved in the recognition of bacterial and human HSPs18 - 22. The
CD14-260 promotor SNP has been associated with increased CD14 receptor density and
increased serum levels of soluble CD1423, 24. Furthermore, the CD14-260 SNP has recently
been associated with CD in combination with CARD15/NOD2 SNPs25. CARD15/NOD2,
which has been associated to CD, also plays a role in regulating NF-κB activation by
interacting with LPS. Klein et al. demonstrated that carriage of the CD14-260 SNP in
combination with any of the CARD15/NOD2 SNPs significantly increases the risk of
developing CD in a German population. We have previously demonstrated that
CARD15/NOD2 mutations are associated with susceptibility and severity of CD in this
Galician population26 and we have extended our analyses to include the pathogen
recognition receptors, CD14 and TLR4.
We hypothesize that the TLR4 and CD14 SNPs may, either alone (as we have previously
shown in atherosclerosis patients27) or in a multi gene combination with each other or
CARD15/NOD2, influence the susceptibility to and severity of Crohn’s disease (CD).
73
Chapter 4
Material & Methods
Patients
We studied 165 consecutive patients with CD (95 female and 70 male, mean age 36 years, age range
17-76) and 163 healthy, unrelated controls, matched by ethnicity, sex and age, and with no family
history of inflammatory bowel disease. All cases and controls were from and residents in Galicia
(NW of Spain). Data concerning steroid dependency, origin of the patient (rural or urban), family
history of CD, CD related surgery, extraintestinal manifestations, Vienna classification, and smoking,
were collected for patients and controls, where appropriate.
Patient definitions
Steroid-dependency was defined as a relapse within 30 days after the end of steroid treatment or after
at least two attempts of tapering the steroid dose within the last 12 months.
Steroid-resistant patients were defined as those who did not respond to steroid therapy (minimum 50
mg of prednisolone) for more than seven days28. A positive family history of CD was defined as
presence of at least one first degree relative suffering from the disease. A retrospective study of the
patients regarding previous CD related surgery was performed. The analysis was also stratified
according to the two most recent procedures performed on these patients, namely, ileal resection and
treatment of fistulae (mainly perianal). The history of appendectomy as either previous to the
development of the disease or at the time of the diagnosis was also analyzed. Extraintestinal
manifestations were defined as chronic inflammatory conditions that involve other organ systems and
included articular, skin, eye and liver diseases. Smokers include all patients who were smokers at the
moment of the blood samples.
74
Genetic analyses
A blood sample was obtained from all cases and controls, and all were genotyped for the TLR4+896
A>G (D299G; rs4986790) and the CD14-260 C>T (rs2569190) SNPs.
Previously we typed for CARD15/NOD2 SNP8 (+2209 A>T; R702W; rs2066844), SNP12 (+2722
G>T; G908R; rs2066845), and SNP13 (+3020 InsC; L1007P; rs2066847) SNPs which we used in this
manuscript in the multigene and interaction analyses. The aforementioned SNPs were analysed by
PCR-RFLP as described previously6, 29 - 31.
Statistics
Data were analyzed by the χ2 test and the Fisher’s exact test, where appropriate. The data were
analysed in a multivariate logistic regression model to assess the association of different genotypes
with CD, adjusted for age, gender, smoking, and place of origin.
Results
CD14 and TLR4 single gene analysis
Carriage of the TLR4+896 SNP was increased in controls compared to patients (14.2% vs.
6.7%; p: 0.0298, OR: 2.3, 95% CI: 1.1 – 5.0), while CD14 SNPs were equally distributed
amongst cases and controls (see Table 1).
CD14 and TLR4 SNPs in relation to patient variables
Carriage of the CD14-260*T allele was significantly increased in non-fistulae patients as
compared to patients with fistulae (79.5% vs. 57.9%; p: 0.045, OR: 2.8, 95% CI: 1.4 – 5.8).
When corrections were made for gender or smoking, no significant associations were
observed, nor when corrections were made for both gender and smoking.
CD14 & TLR4
Crohn’s Disease
TLR4
CD14
Cases
N = 165
%
Controls
N = 162
%
Genotype
AA
AG*
GG*
93.3
6.1
0.6
85.7
12.9
1.4
Allele
A
G
96.4
3.6
92.1
7.9
CD14
Genotype
CC
CT
TT
23.0
50.9
26.1
23.6
50.7
25.7
Allele
C
T
48.5
51.5
48.9
51.1
Table 1: TLR4 genotype and allele frequencies in cases and controls
* Cases vs. controls: p: 0.0298, OR: 2.3, 95% CI: 1.1 – 5.0
Multiple gene analysis
Interaction between CD14 and TLR4 SNPs
An increased frequency of TLR4 AA with CD14*T was observed in younger patients
(Vienna A1) compared to patients above 40 years (Vienna A2). This did not reach
statistical significance although a trend was observed (p: 0.06, OR: 4.0, 95% CI: 1.0 – 11.9)
Interaction between CD14 and TLR4 SNPs in relation to patient variables
Combined carriage of the CD14-260 SNP with the TLR4 wildtype was significantly
increased in patients without fistulae compared to those with fistulae (p: 0.03, OR: 3.2, 95%
CI: 1.2 – 8.4). Corrections for smoking or gender did not result in statistically significant
associations.
Interaction between CD14 & TLR4 and CARD15/NOD2
We analysed carriage of the CD14-260 and the TLR4+896 SNPs in combination with
carriage of one or more CARD15/NOD2 SNPs (Tables 2 and 3). Carriage of CD14*T
combined with one or more CARD15/NOD2 SNPs was significantly increased in cases
compared to controls (22.4% vs. 8.6%; p: 0.0007, OR: 3.1, 95% CI: 1.6 – 5.9). Carriage of
CD14*T combined with the CARD15/NOD2 SNPs and homozygous carriage of the
TLR4+896 wildtype was significantly increased in cases compared to controls, with an
increased Odds ratio when compared to the analysis without TLR4 correction (21.8% vs.
7.4%; p: 0.0003, OR: 3.5, 95% CI: 1.7 – 7.0).
75
Chapter 4
Interaction between CD14 & TLR4 and CARD15/NOD2 in relation to patient variables
Carriage of CD14*T combined with one or more CARD15/NOD2 SNPs was significantly
increased in patients who had an ileal resection compared to those who did not have an ileal
resection (37.0% vs. 15.8%; p: 0.004, OR: 3.2, 95% CI: 1.5 – 6.8).
TLR4 wildtype carriage combined with CD14 and CARD15/NOD2 SNPs slightly increased
the risk for ileal resection (p: 0.002, OR: 3.4, 95% CI: 1.6 – 7.3). Combined carriage of the
CD14 SNP, one or more CARD15/NOD2 SNPs and the TLR4 wildtype was significantly
increased in steroid resistant patients compared to non resistant patients (37.0% vs. 19.6%;,
p: 0.04, OR: 2.5, 95% CI: 1.0 – 6.2).
TLR4
76
Cases
N = 119
N = 46
CARD15/NOD2 CARD15/NOD2
–
+
(%)
(%)
Controls
N = 140
N = 22
–
+
(%)
(%)
Genotype
AA
AG
GG
91.6
7.6
0.8
97.8
2.2
0.0
85.7
12.9
1.4
86.4
13.6
0.0
Allele
A
G
95.4
4.6
98.9
1.1
92.1
7.9
93.2
6.8
Table 2: TLR4 genotype distribution and allele frequencies in relation to CARD15/NOD2 SNPs in cases and
controls
CD14
Cases
N = 119
N = 46
–
+
(%)
(%)
Controls
N = 140
N = 22
–
+
%)
(%)
Genotype
CC
CT*
TT*
24.4
48.7
26.9
19.6
56.5
23.9
23.6
50.7
25.7
36.4
27.3
36.4
Allele
C
T
48.7
51.3
47.8
52.2
48.9
51.1
50
50
Table 3: CD14 genotype distribution and allele frequencies in relation to CARD15/NOD2 SNPs in cases and
controls
* Cases vs. controls: p: 0.0007, OR: 3.1, 95% CI: 1.6 – 5.9
CD14 & TLR4
Crohn’s Disease
Multivariate logistic regression
We entered age, gender, origin of the patient (rural or urban area), smoking, and carriage of
the SNPs into a multivariate logistic regression model. Age, smoking, origin and carriage of
the SNP12, SNP13 and TLR4 SNPs were interdependently associated with development of
CD. Smoking, rural origin and carriage of the TLR4 SNP were protective while age and
carriage of the SNP12 and SNP13 polymorphisms were risk factors (Table 4).
Carriage of the SNP12 and the CD14 polymorphism combined with the TLR4 wildtype
were associated with development of fistulae. SNP12 was a risk factor, while the
CD14 – TLR4 combination was protective against fistulae (Table 5). SNP13 appeared as a
significant risk factor in the ileal resection model (Table 6).
CD
Gender
Age
Origin
Smoking
*
(rural or urban)*
Carriage of:
SNP8
SNP12*
SNP13*
TLR4*
CD14
p
0.68
0.01
0.05
<0.001
0.34
0.02
0.009
0.04
0.88
OR
0.89
1.02
0.59
0.15
1.46
6.55
6.00
0.41
0.96
95% CI
Lower
Upper
0.54
1.50
1.00
1.04
0.35
1.00
0.90
0.25
0.67
3.19
1.30
33.10
1.56
23.07
0.18
0.96
0.53
1.73
Table 4: Logistic regression analysis.
*Age, and carriage of the SNP12 and SNP13 polymorphisms increase the risk of development of CD, while rural
origin and carriage of the TLR4 SNP are protective.
Fistulae
Gender
Age
Origin
Smoking
Carriage of:
*
(rural or urban)*
SNP8
SNP12*
SNP13
TLR4 AA & CD14*T*
p
0.02
0.27
0.71
0.10
0.44
0.001
1.00
0.02
OR
4.08
0.97
0.80
3.17
0.42
13.30
<0.001
0.27
95% CI
Lower
Upper
1.21
13.73
0.92
1.02
0.24
2.67
0.79
12.72
0.05
3.86
2.81
63.06
<0.001
∞
0.08
0.84
*Gender, and carriage of the SNP12 polymorphism increased the risk of fistulae, while carriage of the TLR4
wildtype combined with carriage of the CD14 SNP protected against fistulae.
77
Chapter 4
Ileal resection
Gender
Age
Origin
Smoking
Carriage of:
(rural or urban)
SNP8
SNP12
SNP13*
TLR4
CD14
p
0.41
0.10
0.29
0.28
0.37
0.07
0.02
0.93
0.20
OR
0.74
0.98
1.47
0.67
1.56
3.07
4.48
1.06
1.80
95% CI
Lower
Upper
0.35
1.54
0.95
1.01
0.71
3.04
0.33
1.39
0.58
4.28
0.91
10.42
1.31
15.25
0.25
4.49
0.73
4.45
*Carriage of the SNP13 polymorphism was a risk factor for ileal resection.
Discussion
78
Our results show that carriage of CD14-260*T was associated with protection against
development of fistulae, especially in smokers. Carriage of the TLR4+896 A allele was
significantly reduced in CD patients, indicating a protective effect of this SNP. The
CD14*T appeared protective against development of fistulae. Our data showed that
combined carriage of CD14, TLR4, and CARD15/NOD2 SNPs increased the risk of
development of CD and the requirement of ileal resection. Multivariate analysis showed
that age, smoking, rural origin, and carriage of the SNP12, SNP13, and TLR4 SNPs affect
the susceptibility to CD. SNP12 was a risk factor for fistulae, while CD14*T – TLR4 AA
appeared protective. SNP13 carriage was a risk factor for the requirement of ileal resection.
The CD14 -260 SNP has been associated with increased CD14 receptor density and
increased sCD14 level in serum23, 24. Increased recognition of intestinal bacteria may lead to
a stronger or aberrant immune response against these bacteria and thus may result in tissue
damage. CD14 is not the only factor in this process indicated by the fact that carriage of the
CD14 SNP alone does not significantly associate with development of CD. However, in
combination with CARD15/NOD2 SNPs, CD14 is a risk factor for both CD and ileal
resection. In contrast, CD14*T does appear to protect against fistulae. A recent study
demonstrated an increased risk of development of CD after appendectomy32. Further
studies are required to elucidate if and how carriage of the SNP presented in this study may
affect the development of CD after appendectomy.
The TLR4 SNP has been associated with LPS hyporesponsiveness15 - 17. A reduced immune
response to intestinal bacteria may result in a longer duration of infection, but with less
tissue damage and associated pathogenesis. This is reflected in our results showing that
TLR4*G protects against development of CD and in the fact that carriage of the wildtype in
combination with the CD14 SNP associates with pathogenesis.
Several studies have provided evidence for in involvement of TLR4 and CD14 in CD.
Klein et al. described increased incidence of the CD14 SNP in a German CD population
CD14 & TLR4
Crohn’s Disease
compared to controls33. This result was corroborated by the work of Gazouli et al. who
found an increased incidence of the CD14 SNP in a Greek CD population34. We did not
observe an increased incidence of the CD14 SNP alone in the CD patients in this Galician
population nor in a Dutch population studied by our group35, however combined carriage of
CD14 with CARD15/NOD2 SNPs was associated with risk for CD. Arnott and colleagues
did not find associations between CD and the CD14 and TLR4 SNPs in either Scottish or
Irish populations36. Other studies associate the TLR4+896 SNP with Crohn’s
disease34, 35, 37, 38, while our Galician populations shows a protective effect for this SNP. The
populations described by Gazouli and Arnott, and our Galician population are rather
isolated populations with little admixture. These data clearly demonstrate the genetic
heterogeneity in CD and underscore the complexity of this disease.
In a recent publication Ferreira et al. described an increased incidence of ileal or ileocolonic
disease in CD patients carrying the CARD15/NOD2 SNPs39. These patients were also at
higher risk for abdominal surgery. Büning et al. demonstrated an increased risk for ileocoecal resection and reoperation in CD patients carrying the CARD15/NOD2 SNPs40.
Although we did not observe a significant association between the CARD15/NOD2 SNPs
and disease location, our findings that CARD15/NOD2 SNPs alone41 and combined with the
CD14*T and TLR4 wildtype increase the risk of ileal resection are in concordance with the
results of Ferreira and Büning.
CD is a complex disease where the interplay between different factors in the host immune
system, the intestinal flora and other yet unknown factors influence the aetiopathogenesis.
CARD15/NOD2 is well known to be associated with CD, however 60 – 70% of CD
patients do not carry the CARD15/NOD2 SNPs while these SNPs are common in the
healthy population42, 43. CARD15/NOD2 SNPs are thought to inhibit signalling7, while at
the same time increased expression of CARD15/NOD2 and NF-κB is found in CD
patients44, 45. These results fuel the “loss-of-function” vs. “gain-of-function” in
CARD15/NOD2 SNPs discussion45. CD14 and TLR4 are also potent NF-κB inducers and
might explain the increase in NF-κB expression. The studies by Netea et al. and van Heel et
al. add to the complexity of this problem by showing that CARD15/NOD2 is capable of
enhancing the responses generated by several (but not all) TLRs46 - 48.
In conclusion, our results shed a light on the complex puzzle that is the pathogenesis of CD
and underscores the need for further research into the underlying biological mechanisms
that contribiute to the development of CD.
79
Chapter 4
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Chapter 5
Combined carriership of TLR9-1237*C and CD14-260*T alleles enhances the risk of
developing chronic relapsing pouchitis
Karen M. Lammers, Sander Ouburg, Servaas A. Morré, J. Bart A. Crusius, Paolo Gionchetti,
Fernando Rizzello, Claudia Morselli, Elisabetta Caramelli, Roberto Conte, Gilberto Poggioli,
Massimo Campieri & A. Salvador Peña
World Journal of Gastroenterology 2005; 11 (46): pp. 7323 – 7329
Chapter 5
Abstract
im: About 40% of the patients with ileal-pouch anal anastomosis (IPAA) for
ulcerative colitis develops pouchitis. Given the important role of enteric bacteria
in the pathogenesis of pouchitis, host genetic variation in innate immunity genes
may underly susceptibility to pouchitis onset or severity. To investigate single nucleotide
polymorphisms (SNPs) in genes involved in bacterial recognition and the susceptibility to
develop pouchitis or pouchitis severity.
A
Materials and Methods: Analyses of CD14 –260 C>T, CARD15/NOD2 3020insC, Tolllike receptor (TLR)4 +896 A>G, TLR9 –1237 T>C, TLR9 +2848 G>A and IRAKM +22148
G>A SNPs were performed in 157 IPAA patients (79 patients who did not develop
pouchitis, 43 infrequent pouchitis patients, 35 chronic relapsing pouchitis patients) and 224
Italian Caucasian healthy controls.
Results: No significant differences were found in SNP frequencies between controls and
IPAA patients. However, a significant difference in carriership frequency of the TLR9
–1237*C allele was found between the infrequent pouchitis and chronic relapsing pouchitis
groups (p: 0.028; OR: 3.2, 95% CI: 1.2 – 8.6). This allele uniquely represents a 4-locus
TLR9 haplotype comprising both studied TLR9 SNPs in Caucasians. Carrier trait analysis
revealed an enhanced combined carriership of the alleles TLR9 –1237*C and CD14 –260*T
in the chronic relapsing pouchitis vs. infrequent pouchitis group (p: 0.018; OR: 4.1, 95%
CI: 1.4 – 12.3).
84
Conclusion: We found no evidence that the SNPs studied predispose to the need for IPAA
surgery. The significant increase of the combined carriership of the CD14 –260*T and
TLR9 –1237*C alleles in the chronic relapsing pouchitis group suggests that these markers
identify a subgroup of IPAA patients with a risk to develop chronic or refractory pouchitis.
CD14, TLR4, TLR9, CARD15/NOD2 & IRAK-M
Pouchitis
Introduction
atients with ulcerative colitis may need surgery for their disease and
proctocolectomy with ileal-pouch anal anastomosis (IPAA) is the surgical
procedure of choice for the management of these patients1, 2. Most patients
undergoing IPAA for severe colitis or chronic continuous disease achieve good functional
results, but some patients develop a pouchitis, a non-specific idiopathic inflammation of the
ileal reservoir. Frequency rates of pouchitis are highly variable, ranging from 10 – 59%
depending on the length of follow up and the diagnostic criteria used3. Although the origin
of pouchitis remains unknown, genetic and immunological factors are likely to be involved
in addition to an ileal mucosa that needs to adapt to its new role as reservoir4. This is
illustrated by the fact that pouchitis occurs almost exclusively in patients with IPAA for
ulcerative colitis and not in patients with IPAA for familial adenomatous polyposis, a
hereditary non-inflammatory disease of the colon with high risk for developing colon
cancer. An important role for luminal bacteria in the development of pouchitis is
underscored by various reports on bacterial overgrowth and dysbiosis in pouchitis5 and is
further confirmed by efficacy of antibiotic and probiotic therapy6, 7.
P
Pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), are essential
components of the innate immune system as recognition of microbial products occurs via
PRRs that are expressed by innate effector cells. Microbial recognition results in a rapid
and efficient immune response against invading microorganisms8.
Given the role of luminal bacteria in driving the inflammatory response in pouchitis,
identification and functional characterization of polymorphisms in innate immunity genes
may provide insight in a possible genetically determined susceptibility to develop pouchitis
and/or to suffer from chronic relapsing pouchitis9.
CD14 is part of the endotoxin/lipopolysaccharide (LPS) receptor complex8 and is, in
conjunction with TLR4 and with TLR210, important in the recognition of LPS a membrane
glycolipid on Gram-negative bacteria. CD14 may also recognize cell membrane
components of Gram-positive, mycobacteria and viruses11 - 15. CD14 exists in membrane
form on monocytes and neutrophils, and in a soluble form in serum16 - 18. The SNP at
position –260 C>T (also known as CD14–159 C>T) in the promoter region of the CD14
gene (located on chromosome 5q31) has been associated with enhanced transcriptional
activity19 and with significantly higher CD14 serum levels20. Increased expression of CD14
on macrophages has been found in inflammatory bowel disease (IBD)21. An association of
the CD14 –260 C>T gene polymorphism with IBD22, 23 and with atherosclerosis24 has been
described. Genetically determined variation in CD14 serum levels may have functional
consequences given the ability of soluble CD14 to confer pathogen responsiveness to cells
that do not express CD14 on their membrane such as intestinal epithelial and endothelial
cells25.
The TLR4 gene is located on chromosome 9q32-q33. The TLR4 +896 A>G SNP affects the
leucine-rich repeat domain of TLR4 and is associated with hyporesponsiveness to LPS26,
with increased susceptibility to severe bacterial infections and to develop IBD27, and may
predispose to develop septic shock with Gram-negative microorganisms28, 29.
TLR9 is required for the recognition of CpG motifs, short sequences of unmethylated DNA
predominantly present in bacterial DNA. CpG motifs have immunostimulatory activity by
inducing dendritic cell maturation, B cell proliferation and production of cytokines,
85
Chapter 5
86
including interleukin-6 (IL-6) and interleukin-12 (IL-12)30, 31. TLR9 signaling has been
shown to mediate the resolution of intestinal inflammation in experimental colitis32,
suggesting that the release of bacterial DNA from the microflora might favor immune
homeostasis. The promoter TLR9 –1237 T>C SNP located on chromosome 3p21.3 has been
associated with susceptibility to develop asthma in European Americans, but not in
Hispanic or African Americans33 and the marker D3S1076 in this region shows association
with IBD in a classical TDT test34. Török and colleagues35 studied the TLR9 –1237 T>C
and TLR9 +2848 G>A SNPs in German patients with Crohn’s disease, ulcerative colitis and
healthy blood controls and reported an association of the allele TLR9 –1237*C carrier status
with Crohn’s disease compared to controls. TLR9 +2848 G>A allele frequencies were not
different between the study groups.
CARD15/NOD2 is a cytoplasmatic bacterial PRR expressed in monocytes and intestinal
epithelial cells and mediates responsiveness of muramyl dipeptide derived from
peptidoglycan36, 37. The CARD15/NOD2 gene is located on chromosome 16q12. Three
major polymorphisms in this gene (R702W, G908R, and and the L1007 frameshift
mutation) have been associated with susceptibility to develop Crohn’s disease, possibly as a
result of a defective response against muramyl dipeptide derived from peptidoglycan38.
Recently, an increased frequency of the L1007 frameshift mutation has been observed in
Italian patients with ulcerative colitis when compared to controls39.
The intracellular domains of TLRs are homologous to the interleukin-1 receptor (IL-1R)
type I intracellular domain and use a common pathway of intracellular signaling with
shared components including the protein kinase IL-1R-associated kinase1 (IRAK1) and
IRAK-M, recently reported to be a negative regulator of TLR signaling. Increased cytokine
production was measured in IRAK-M-/- macrophages after TLR/IL-1 stimulation and
bacterial challenge, and endotoxin tolerance was reduced in these cells. Furthermore,
IRAK-M-/- mice had increased inflammatory responses to bacterial infection and
developed intestinal inflammation. These data suggest that IRAK-M has a regulatory
function in TLR/IL-1R signaling and in innate immune homeostasis40. The IRAKM (or
IRAK3) gene is located at chromosome 12q14.2 within the IBD2 region linked with and
associated to ulcerative colitis41, 42. Genetic variation in the IRAKM gene may be involved
in the development of chronic intestinal inflammation. For this reason, we chose to analyze
a non-synonymous SNP in exon 5 resulting in an Ile/Val substitution.
A candidate genes approach, which is based on determination of frequencies of functional
SNPs, can be used to investigate the relevance of genes to the susceptibility to and severity
of disease. Carrier trait analysis investigates combinations of SNPs and allows studying the
implication of different SNPs in disease susceptibility and severity as a result of their
synergistic action.
The aim of this study was to study whether SNPs in innate immunity genes contribute to
the susceptibility to develop pouchitis and/or to severity of pouchitis. We chose candidate
genes of interest CD14, TLR4, TLR9, NOD2/CARD15, and IRAKM for their involvement in
bacterial recognition and intracellular signaling pathways.
Pouchitis
Materials and Methods
Patients
Hundred and fifty-seven unrelated patients with IPAA for ulcerative colitis and 224 healthy blood
donors were studied. All individuals were Italian Caucasians. Consent was obtained and the local
ethics committee approved the protocol. Demographic and clinical information is described in
Table 1. IPAA patients were subdivided into three test groups according to the pouchitis pattern. One
group consisted of IPAA patients who never developed pouchitis, one group consisted of IPAA
patients who had up to two episodes of pouchitis during the duration of IPAA (infrequent pouchitis),
and a third group consisted of IPAA patients who developed three or more episodes of pouchitis
(chronic relapsing pouchitis).
DNA isolation
Venous blood (5-10 ml) was drawn and genomic DNA was isolated using standard protocols. The
CD14, TLR4, TLR9, CARD15/NOD2 and IRAKM gene polymorphisms in these groups were
analyzed. 5-100 ng genomic DNA was used for each genotyping.
Analysis of gene polymorphisms
Polymerase chain reaction (PCR) for RFLP analyses was performed on a thermal cycler GeneAmp
9700 (Perkin-Elmer Cetus, Norwalk, CT, USA). Digested fragments were analyzed on a 4% agarose
gel, except for the IRAKM SNP analyzed on a 2% a agarose gel, and visualized with an UVilluminator after ethidium bromide staining. SNPs were analyzed with the TaqMan assay (Applied
Biosystems, Foster City, CA). MGB TaqMan probes and primer pairs were designed with Primer
Express software (version 2.0). TaqMan thermocycling consisted of an initial step at 50°C for 2 min
and denaturation at 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 15 s and
annealing/extension at 60°C for 1 min, in 96 well plates (Greiner Bio-One). We used the ABI Prism
7000 Sequence Detector (Applied Biosystems) for data acquisition.
Genotyping
CD14 –260C>T genotyping (NCBI SNP CLUSTER ID: rs2569190) was performed by PCR. Primers
used were: forward primer 5’-TCA CCT CCC CAC CTC TCT T-3’ and reverse primer 5’-CCT GCA
GAA TCC TTC CTG TT-3’. The PCR conditions were initial denaturation at 95°C for 5 min,
followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 59°C for 30 s and extension at
72°C for 1 min. The cycling was followed a final step at 72°C for 5 min and cooling to 4°C. The PCR
reactions were performed in 96 well plates (Greiner Bio-One). The 107 bp amplicons were digested
overnight with HaeIII (New England Biolabs, UK). Digestion resulted in two fragments of 83 bp and
24 bp (C allele) or 107 bp (T allele), respectively.
Genotyping of the TLR4 +896 A>G SNP (NCBI SNP CLUSTER ID: rs4986790) was performed with
forward primer 5’-TTT ACC CTT TCA ATA GTC ACA CTC A-3’ and reverse primer 5’-AGC ATA
CTT AGA CTA CTA CCT CCA TG-3’. PCR conditions were: initial denaturation at 94°C for 5 min
followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at
72°C for 30 s. The cycling was followed a final extension step at 72°C for 5 min was followed by
cooling to 4°C. The PCR reactions were performed in 96 well plates (Greiner Bio-One). The 102 bp
amplicons were digested overnight with NcoI (New England Biolabs, UK). Digestion resulted in two
fragments of 80 bp and 22 bp (G allele) or 102 bp (A allele), respectively.
CARD15/NOD2 3020InsC (CARD15 L1007fs ) (NCBI SNP CLUSTER ID: rs2066847) genotyping
was performed with forward primer 5’-GGC AGA AGC CCT CCT GCA GGG CC-3’ and reverse
primer 5’-CCT CAA AAT TCT GCC ATT CC-3’. PCR conditions were: initial denaturation at 94°C
for 5 min followed by 35 cycles of denaturation at 94°C for 30s, annealing at 59°C for 30 s and
extension at 72°C for 45 s. The cycling was followed a final extension step at 72°C for 5 min was
followed by cooling to 4°C. The PCR reactions were performed in 96 well plates (Greiner Bio-One).
87
Chapter 5
The 150 bp amplicons were digested overnight with ApaI. Digestion resulted in a fragment of 150 bp
(no insertion) or 128 bp and 22 (insertion C), respectively.
TLR9 –1237 T>C (NCBI SNP CLUSTER ID: rs5743836) genotyping was performed with TaqMan
method. Primers used were: forward primer 5’- GGC CTT GGG ATG TGC TGT T-3’ and reverse
primer 5’- GGT GAC ATG GGA GCA GAG ACA-3’. Dual-labeled fluorogenic hybridization MGBprobes used were: CTGCCTGAAAACT 5’ Fluor Label (FAM, 6-carboxyfluorescein) and
CTGGAAACTCCCC 5’ Fluor Label (VIC).
TLR9 +2848 G>A genotyping (NCBI SNP CLUSTER ID: rs352140) was performed with TaqMan
method. Primers used were: forward primer 5’-CCG GTC TGC AGG TGC TAG AC-3’ and reverse
primer 5’-CCA AAG GGC TGG CTG TTG TA-3’. Dual-labeled fluorogenic hybridization MGB
probes used were: AGCTACCGCGACTGG 5’ Fluor Label (FAM) and AGCTACCACGACTGGA
5’ Fluor Label (VIC).
Genotyping the IRAKM +22148 G>A exon 5 SNP (NCBI SNP CLUSTER ID: rs1152888) was
performed by performing PCR with forward primer 5´- AGT GGA AC T GAT GTC CTG TGA
CAG -3´ and reverse primer 5´- GCA ACA CAT TGA CCT AAT GAC CAG -3´.
The PCR program consisted of 95°C for 5 min, followed by 35 cycles of 50 s at 95°C, 50 s at 60°C,
and 150 s at 72°C and a final step at 72°C for 5 min was followed by 4°C. Digestion overnight with
RsaI (Invitrogen Life Technologies) of the 505 bp amplicons resulted in two fragments of 188 bp +
317 bp (allele G) or 505 bp (allele A).
88
Statistical analysis
Hardy-Weinberg equilibrium was determined in healthy controls to assess Mendelian inheritance.
Comparisons of the genotypes between control and different groups of IPAA patients were performed
by Fisher’s exact or χ2 two-tailed tests where appropriate. Carrier trait analysis was performed to
determine whether combinations of SNPs were acting synergistically on the risk to develop pouchitis
or to predispose to chronic relapsing pouchitis. Adjusted Odd’s ratio (OR) and 95% confidence
intervals (95% CI) were calculated. Uncorrected P values <0.05 were considered statistically
significant.
Results
Characteristics of patients and control groups
Demographic features of IPAA patients and healthy controls are shown in Table 1. No
statistical differences between the IPAA group and healthy controls were found for the
variables analyzed including gender and age.
Total number (n)
Gender M/F
Mean age in yr (SD)
Range
Median
IPAA group
157
88/69
42.9 (11.8)
17 – 73
41
Healthy controls
224
118/106
45.8 (12.8)
21 – 77
45.5
Table 1: Demographic features of IPAA patients and healthy controls
Clinical characteristics of the patients with IPAA for ulcerative colitis are summarized in
table 2. Information on the pattern of pouchitis within the group of IPAA patients for
Pouchitis
ulcerative colitis, that is patients who did not develop pouchitis, patients who had
infrequent pouchitis, and patients who suffered from chronic relapsing pouchitis,
respectively, is shown in Table 2. No statistically significant differences were found
between controls and the IPAA groups for gender and age. No statistically significant
differences were found between the IPAA groups and between infrequent pouchitis and
chronic relapsing pouchitis groups for the duration of pouch and the first episode of
pouchitis after IPAA surgery, respectively.
IPAA group
Age (yr) at diagnosis ulcerative colitis
Time (yr) from diagnosis ulcerative colitis to IPAA surgery
Time from IPAA surgery to first episode of pouchitis
Total pouchitis group (n=78)
Infrequent pouchitis (≤2 episodes, n=43)
Chronic relapsing pouchitis (≥3 episodes, n=35)
Pattern of pouchitis
Duration of pouch (yr)
No episodes of pouchitis (n=79)
Infrequent pouchitis (≤2 episodes, n=43)
Chronic relapsing pouchitis (≥3 episodes, n=35)
Mean (SD)
29.7 (11.7)
6.3 (5.5)
Median
27
5
Range
8 – 61
0 – 34
2.8 (3.1)
3.7 (3.3)
2 (2.6)
2
3
1
0 – 12
0 – 12
0 – 12
Mean (SD)
6.3 (4.1)
7.6 (3.8)
7.9 (3.6)
Median
5
7
7
Range
0 – 14
1 – 16
2 – 14
Table 2: Clinical data IPAA patients
Genotyping
The genotype frequencies in the control group were in Hardy-Weinberg equilibrium for the
CD14, TLR4, TLR9, CARD15/NOD2 and IRAKM gene polymorphisms analyzed. Genotype
frequencies of these polymorphisms are described in Table 3. No significant differences in
allele-, genotype- or carrier frequencies of the gene polymorphisms studied between the
healthy control group and IPAA patients were found.
Neither were significant differences in allele-, genotype- or carrier frequencies of the gene
polymorphisms between the three subgroups of IPAA patients apparent, except for
carriership of allele TLR9 –1237*C, which was more frequent in patients with chronic
relapsing pouchitis (45.7%) as compared to those with infrequent pouchitis (20.9%) (p:
0.028; OR: 3.2, 95% CI: 1.2 – 8.6). When comparing the combined groups of infrequent
pouchitis and chronic relapsing pouchitis (i.e. total pouchitis group) with the patients
without pouchitis, carriership of this allele was not significantly different (p: 0.87, OR: 1.1,
95% CI: 0.6 – 2.1).
TLR9 haplotype
The two TLR9 SNPs analyzed were chosen based on the study of Lazarus et al.33 in which a
set of four frequent TLR9 SNPs (allele frequencies in four ethnically different populations,
among others the European American population, at least 10%) designated as TLR9 –1486,
TLR9 –1237, TLR9 +1174 and TLR9 +2848, was described. Genotyping of both TLR9 –
1237 and TLR9 +2848 SNPs allows to distinguish all 4-locus haplotypes commonly present
in the European American population. We therefore calculated haplotypic genotypes in the
Italian Caucasian population (Table 4). The haplotype frequencies in the healthy control
group were identical to the European American population reported by Lazarus et al.
Haplotype III was more frequent in chronic relapsing pouchitis as compared to infrequent
89
Chapter 5
pouchitis (p: 0.018; OR: 3.0, 95% CI: 1.2 – 7.1). This haplotype however, did not show
nucleotides uniquely present (tag SNPs) on position –1486 (allele T present in haplotypes I
and III) or on position +1174 (allele G present in haplotypes II and III), indicating that
allele TLR9 –1237*C provides the strongest association.
To investigate if SNPs in different genes act synergistically on disease susceptibility and/or
disease severity, a carrier trait analysis with the associated TLR9 allele was performed.
Simultaneous carriership of alleles TLR9 –1237*C and CD14 −260*T was more frequent in
patients with chronic relapsing pouchitis as compared to those with infrequent pouchitis: p:
0.018; OR: 4.1; 95% CI: 1.4 – 12.3) which is more significant as compared to the analysis
of allele TLR9 –1237*C alone (p: 0.028; OR: 3.2, 95% CI: 1.2 – 8.6). No other significant
carrier traits were observed.
90
Polymorphisms
Genotype
CD14 –260 C>T
CC
CT
TT
CARD15 3020InsC
Controls
No pouchitis
Infrequent
pouchitis
Chronic
relapsing
pouchitis
n=35
%
7
20.0
22
62.9
6
17.1
n=224
55
102
67
%
24.6
45.5
29.9
n=79
24
34
21
%
30.4
43.0
26.6
n=43
11
21
11
%
25.6
48.8
25.6
WT/WT
WT/InsC
InsC/InsC
218
6
0
97.3
2.7
0.0
77
2
0
97.5
2.5
0.0
43
0
0
100.0
0.0
0.0
34
1
0
97.1
2.9
0.0
TLR4 +896 A>G
AA
AG
GG
208
16
0
92.9
7.1
0.0
73
5
1
92.4
6.3
1.3
38
5
0
88.4
11.6
0.0
33
2
0
94.3
5.7
0.0
TLR9 –1237 T>C
TT
TC
CC
158
60
6
70.5
26.8
2.7
52
24
3
65.8
30.4
3.8
34
9
0
79.1
20.9
0.0
19
14
2
54.3
40.0
5.7
TLR9 +2848 G>A
GG
GA
AA
40
104
80
17.9
46.4
35.7
20
38
21
25.3
48.1
26.6
9
22
12
20.9
51.2
27.9
6
16
13
17.1
45.7
37.2
IRAKM +22148G>A GG
GA
AA
181
42
1
80.8
18.8
0.4
64
14
1
81.0
17.7
1.3
33
7
3
76.7
16.3
7.0
28
7
0
80.0
20.0
0.0
Table 3: Genotypes of the CD14, CARD15, TLR4, TLR9 and IRAKM polymorphisms in subgroups of patients
with IPAA and controls
Pouchitis
TLR9
−1237
TLR9
+2848
T
T
C
C
G
A
A
G
Haplotype
I
II
III
IV
Controls
2n=448
181
195
69
3
%
40
44
15
1
No pouchitis
2n=158
51
77
29
1
%
32
49
18
1
Infrequent
pouchitis
2n=86
40
37
9
0
%
47
43
101
0
Chronic
relapsing
pouchitis
2n=70
%
28
40
24
34
18
261
0
0
Table 4: Frequencies of the TLR9 haplotypes formed by the –1237 T>C and +2848 G>A SNPs
1
p: 0.018; OR: 3.0, 95% CI: 1.2 – 7.1
Discussion
There is convincing evidence that enteric bacteria have a role in driving the inflammatory
response in inflammatory bowel diseases (IBD), and further, that genetic factors contribute
not only to the pathogenesis but also to the course and extent of these disorders. Given
these premises, we investigated whether polymorphisms in the following genes encoding
for proteins that are involved in innate immunity, TLR4 +896 A>G, TLR9 +2848 G>A,
TLR9 –1237 T>C, CD14 –260 C>T, CARD15/NOD2 3020insC and IRAKM +22148 G>A,
are associated with development of pouchitis, with disease frequency or severity.
Analysis of the three subgroups of IPAA patients, i.e. patients who never developed
pouchitis, patients with infrequent pouchitis and patients with a chronic refractory form of
pouchitis, revealed a positive association of allele TLR9 –1237*C with the risk to develop
chronic refractory pouchitis once these patients develop pouchitis. Haplotype analysis
showed that out of the four SNPs defining TLR9 haplotypes this allele was uniquely
responsible for this finding. Subsequently, we investigated whether interactions of allele
TLR9 –1237*C with SNPs in the other candidate genes might strengthen this association.
Carrier trait analysis revealed that an even stronger association is apparent with the
combination of alleles TLR9 –1237*C and CD14 –260*T. These data suggest that this
combination of alleles might be a valuable genetic marker to identify a clinical subgroup of
IPAA patients with an enhanced risk to develop a chronic relapsing form of pouchitis, in
contrast to alleles of the SNPs TLR4 +896 A>G, TLR9 +2848 G>A, CARD15/NOD2
3020insC and IRAKM +22148 G>A.
It can not be excluded that the group of patients that did not develop pouchitis consists of a
mixture of patients who will never develop pouchitis on the one hand and patients who will
proceed to the infrequent or chronic relapsing pouchitis group on the other hand. This could
explain why we did not detect an association of allele TLR9 –1237*C between the nopouchitis and the chronic relapsing pouchitis group. It should be noted that no significant
differences were found in the mean duration of IPAA between the three groups.
At present it is unknown what may be the effect of the TLR9 –1237 T>C SNP on the level
of expression of TLR9 given its location in the far promoter region where no DNA-binding
site for known transcription factors is apparent. The association observed might therefore
result from linkage disequilibrium with another polymorphism(s) in a nearby gene.
The mechanism underlying increased risk to develop a chronic relapsing form of pouchitis
91
Chapter 5
by a combined carriership of alleles TLR9 –1237*C and CD14 –260*T might be a
dysfunction in bacterial recognition or a lack of an adequate immune response to bacterial
challenge. This could start at the level of the plasmacytoid dendritic cell which has a central
role in bacterial recognition, selectively expresses TLR9 and may bind soluble CD14
facilitating reactivity towards a broad array of bacterial components43, or at the level of the
intestinal epithelium, and soluble CD14 might confer epithelial cell responsiveness25.
The regulatory role of dendritic cells is of particular importance in the intestine where the
mucosal immune system is in close association with the external environment44; dendritic
cells sample bacterial products either indirectly via M cells or directly by reaching between
epithelial cells into the gut lumen45. In this perspective, it is noteworthy to mention a recent
article that reported a lack of immature blood dendritic cells, which possibly migrate to the
gut, in IBD patients with active disease46.
Recently, carriership of the TLR9 –1237*C allele has been associated with Crohn’s
disease35. Ileal involvement is present in about 60% of patients with Crohn’s disease. Since
the pouch is an ileal reservoir that is more vulnerable to the continuous contact with high
bacterial titers it could be hypothesized that carriership of the allele TLR9 –1237*C (with or
without CD14 –260*T) is associated with an impaired immune response at the level of the
ileal tissue. Possibly at the level of enterocytes and, in particular, paneth cells which are
located in the crypts and are central in host defence to luminal bacteria by releasing
antimicrobial substances47, 48.
92
If this is true, carriership of allele TLR9 –1237*C would become an important predictive
marker to enhanced risk to develop a refractory chronic form of pouchitis and eventual
pouch failure.
SNPs in different genes might work synergistically and might constitute a small to
moderate relative risk to develop disease. Although the observations we describe in this
article are based on relatively small numbers of patients, it should be realized that this study
represents one of the largest series available.
In conclusion, our data suggest that the alleles TLR9 –1237*C and CD14 –260*T
synergistically enhance the risk to develop chronic relapsing pouchitis and eventually
pouch failure in ulcerative colitis patients who need surgical intervention. Larger studies are
required to determine whether this allelic combination could become a valuable predictive
marker and functional studies on the biological role of TLR9 and CD14 in pouchitis are
warranted.
Acknowledgements
We are indebted to Italian patients and healthy controls for their participation in this study. S.A. Morré is
supported by Tramedico BV, the Netherlands, the Falk Foundation, Germany, The Foundation of
Immunogenetics, The Netherlands, and the Department of Internal Medicine of the VU University Medical Centre,
the Netherlands. We thank Jolein Pleijster and Roel Heijmans for excellent technical assistance in the genotyping.
Pouchitis
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95
Chapter 5
96
The road to success is lined with many tempting parking spaces
Part II
Inflammation and Infection
in the
Urogenital Tract
Part II
98
There is a theory which states that if ever anybody discovers exactly what the Universe is for and why it is here, it
will instantly disappear and be replaced by something even more bizarre and inexplicable.
There is another theory which states that this has already happened.
Douglas Adams
English humorist & science fiction novelist (1952 - 2001)
Aims & Outline Part II
Aims & Outline
100
As an adolescent I aspired to lasting fame, I craved factual certainty, and I thirsted for a meaningful vision of
human life – so I became a scientist.
This is like becoming an archbishop so you can meet girls.
M. Cartmill
Part II
Aims & Outline Part II
hlamydia trachomatis is the most prevalent sexually transmitted bacterium
worldwide. In most cases, infected patients undergo an asymptomatic and
uneventful course of infection, even when not treated. However, untreated
infections may result in pelvic inflammatory disease, ectopic pregnancy and tubal
infertility.
C
Part II of this thesis aims to assess the role of the genes involved in innate and acquired
immune response, described in Part I in the susceptibility to and severity of C. trachomatis
infections. Four chapters have been devoted to this end.
Chapter 6 addresses the question: Does the interleukin 1 receptor antagonist affect
susceptibility to C. trachomatis infection?
Since it is known that the CD14-TLR4 complex is able to detect Chlamydia, Chapter 7
focuses on the role of these two pathogen receptors in the development of tubal pathology,
a late complication of C. trachomatis infection.
In Chapter 8, both murine and human studies are used in a translational model to gain a
better understanding of the role CCR5 plays in primary and secondary C. trachomatis
infections and the development of late complications.
Chapter 9 aims to determine the risk associated with SNPs in different innate immunity
genes in order to identify genetic traits that may result in an aberrant immune response
following Chlamydia infection, resulting in tubal pathology.
101
Aims & Outline
102
We must not forget that when radium was discovered no one knew that it would prove useful in hospitals. The
work was one of pure science. And this is a proof that scientific work must not be considered from the point of
view of the direct usefulness of it. It must be done for itself, for the beauty of science, and then there is always the
chance that a scientific discovery may become like the radium, a benefit for humanity.
Marie Curie,
Lecture at Vassar College, Poughkeepsie, New York
Chapter 6
The first strong genetic susceptibility marker for Chlamydia trachomatis infections:
The interleukin 1 receptor antagonist IL-1RN +2018 T>C gene polymorphisms
Joke Spaargaren, Sander Ouburg, Han S.A. Fennema, A. Salvador Peña, Servaas A. Morré
Chapter 6
Abstract
hlamydia trachomatis infection is the most prevalent sexually transmitted
disease with a variable course of infection in different infected patients.
Currently, no clear relationship has been identified between bacterial factors
and susceptibility to Chlamydial infection. Evidence for the influence of host genetic
factors on C. trachomatis pathogenesis is just emerging.
The IL-1RN +2018 T>C polymorphism is strongly reduced (p: 0.0005, OR: 1.5) in women
with a urogenital C. trachomatis infection and may indicate a potential therapeutic target.
C
104
IL-1RN
Susceptibility to C. trachomatis infection
Introduction
bout half of all deaths and diseases are associated with preventable behaviours
and avoidable exposures, which would seem to leave the remainder attributable to
genetic influences. Confirming this almost certainly oversimplified subtraction
and filling in the details that will be of practical clinical value, is the task that genetic
epidemiology is trying to address1. For reasons mainly of statistical power and recruitment
of ever larger cohorts, genetic epidemiology is moving away from linkage studies based on
families to allelic association studies based on unrelated individuals. The biology
underpinning genetic epidemiology offers a potential useful way to study environmental
determinants including infectious agents in disease without residual confounding2.
Chlamydia trachomatis is the most common sexually transmitted bacterial infection. The
course of C. trachomatis infection is quite different in different individuals: Only part of the
exposed women get infected and only part of those infected develops more severe disease,
such as pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility.
Although environmental, bacterial and host factors are known to be involved in disease
course, no clear bacterial virulence factors have been identified in relation to disease
susceptibility and the first relevance of genetic variation in host factors have been published
recently3, 4. For instance, the CD14 -260 C>T polymorphism is not associated with
C. trachomatis infection or it’s late complications3, while specific HLA and IL-10
genotypes have been associated with C. trachomatis infection and late sequelae4, and twin
studies showed a 40% genetic predisposition5. The IL-1 receptor antagonist (IL-1ra) is the
natural inhibitor of the proinflammatory cytokine IL-1 and plays an important role in the
regulation of inflammation, infection, and immune responses. We studied the IL-1RN+2018
T>C polymorphism in relation to the susceptibility to C. trachomatis infections.
A
Participants and methods
1213 Dutch Caucasian women visiting the STD outpatient clinic in Amsterdam, The Netherlands
were included in this study. The cohort (as described previously3) consisted of 748 C. trachomatis
DNA positive women and 465 C. trachomatis DNA negative women.
Analyses for the presence of Candida albicans, Neisseria gonorrhoeae, Trichomonas vaginalis, and
Herpes simplex virus type 1/2 were included in our analyses since infections with these
microorganisms may result in symptoms similar to a C. trachomatis infection. Peripheral venous
blood was collected for the detection of anti C. trachomatis IgG antibodies (Medac Diagnostika
mbH). The functional IL-1RN+2018 T>C (rs419598), of which the mutant (C) allele is in linkage
disequilibrium with the second allele of the IL-1RN VNTR (rs2234663), was assessed according
standard TaqMan protocol, in 96 well TaqMan plates (Greiner Bio-One), using the primers: forward:
5’- CAA CCA CTC ACC TTC TAA ATT GAC AT -3’, and reverse: 5’- CTG AGT CCT TTT CCT
TTT CAG AAT CT -3’. The MGB probes used were: AGT ATC CAG CAA CTA GT-FAM for the T
allele and CAA GTA TCC GGC AAC TA-VIC for the C allele. Fisher’s exact and χ2 tests were used
when appropriate. A p-value <0.05 was considered significant.
Results
The frequency of the IL-1RN*C allele was significantly decreased in C. trachomatis DNA
positive women (39%) compared to C. trachomatis DNA negative women (50%)
105
Chapter 6
(p: 0.0005, OR: 1.5, 95% Confidence interval: 1.2 – 1.9). When C. trachomatis serology
was introduced in the analyses, similar results were found. The IL-1RN*C allele was
significantly reduced in C. trachomatis DNA positive / C. trachomatis IgG positive women
(41%) when compared to C. trachomatis DNA negative / C. trachomatis IgG negative
women (52%) (p: 0.0144, OR: 1.6, 95% confidence interval: 1.1 – 2.3). The results are
summarized in figure 1. Introduction of coinfection status or symptomatology in the
analyses did not alter the results.
106
Figure 1: IL-1RN genotype distribution.
Depicted on the left is the distribution in the cohort, divided in C. trachomatis (CT) DNA positive and
C. trachomatis DNA negative. Depicted on the right is the IL-1RN genotype distribution for the samples with
C. trachomatis serology. The bars represent the IL-1RN distribution between C. trachomatis DNA positive and
C. trachomatis DNA negative women. The differences in colours represent the relative distribution of IgG
positivity and negativity, with each bar representing 100%. The figures next to the bars represent the relative
percentage of IgG positive women in that group.
Comment
We have shown that the carriage of the IL-1RN*C allele is significantly reduced in women
with a C. trachomatis infection. This allele has previously been associated with increased
expression of IL-1ra and an anti-inflammatory immune response. Previous studies have
shown that the carriage of the IL-1RN*C allele is associated with protection against
infection related pre-term birth6. In contrast, foetal carriage of IL-1RN*C has been
associated with increased intra-amniotic levels of IL-1β and induction of an intra-uterine
inflammatory response may predispose to pre-term birth in foetuses carrying this SNP7.
A previous study of our group did not show an association between IL-1RN and tubal
pathology, a late complication of C. trachomatis infection8. This indicates that genetic
IL-1RN
Susceptibility to C. trachomatis infection
variation in the IL-1RN gene might protect against primary C. trachomatis infections but
may not have a strong influence on the development of late complications, reflecting
complex underlying pathogenic mechanisms, and indicating the potential existence of other
factors that influence the risk of tubal pathology. From these data it might be hypothesized
that stimulation of IL-1ra might be used as a potential treatment in patients infected with
C. trachomatis. Stimulation of IL-1ra may reduce infection related pre-term births6,
however the study of Witkin et al. appears to contradict this7, indicating that conclusions
about IL-1ra in pregnancy can not be easily drawn.
Although further studies are required, the identified defects in different molecular
signalling pathways will provide extensive insight in the individual differences in the
human immunopathogenesis of C. trachomatis disease, knowledge which will be translated
to improve infectious diseases and health care issues.
107
Chapter 6
References
1.
2.
3.
4.
5.
6.
7.
8.
108
Genetic epidemiology: strengths, weaknesses, and opportunities, David Sharp, The Lancet 2005; 366 (9489): pp. 880,
PubMed: 16154001
Key concepts in genetic epidemiology, Paul R. Burton, Martin D. Tobin & John L. Hopper, The Lancet 2005; 366
(9489): pp. 941 - 951, PubMed: 16154023
The CD14 functional gene polymorphism –260 C>T is not involved in either the susceptibility to Chlamydia trachomatis
infection or the development of tubal pathology, Sander Ouburg, Joke Spaargaren, Janneke E. den Hartog, Jolande
A. Land, Han S. A. Fennema, Jolein Pleijster, A. Salvador Peña, Servaas A. Morré & ICTI consortium, BMC
Infectious Diseases 2005; 5 (1): pp. 114, PubMed: 16368002
Human leukocyte antigen and cytokine gene variants as predictors of recurrent Chlamydia trachomatis infection in highrisk adolescents, Chengbin Wang, Jianming Tang, William M. Geisler, Peggy A. Crowley-Nowick, Craig M.
Wilson & Richard A. Kaslow, Journal of Infectious Diseases 2005; 191 (7): pp. 1084 - 1092, PubMed: 15747244
Lymphoproliferative responses to C. trachomatis EBS in a Gambian Twin population; Estimating the role of host genetic
factors, Robin L. Bailey, Amanda Fowler, Rosanna Peeling, David Mabey, Hilton Whittle & Annette Jepson,
Chlamydial Infections. Proceedings of the Ninth International Symposium on Human Chlamydial Infections. Napa,
California, USA. June 21 - 26, 1998 1998; pp. 474 - 477
Polymorphism in intron 2 of the interleukin-1 receptor antagonist gene, local midtrimester cytokine response to vaginal
flora, and subsequent preterm birth, Mehmet R. Genc, Andrew B. Onderdonk, Santosh Vardhana, Mary L. Delaney,
Errol R. Norwitz, Ruth E. Tuomala, Lilly Rose Paraskevas & Steven S. Witkin, American Journal of Obstetrics and
Gynecology 2004; 191 (4): pp. 1324 - 1330, PubMed: 15507961
Polymorphism in intron 2 of the fetal interleukin-1 receptor antagonist genotype influences midtrimester amniotic fluid
concentrations of interleukin-1beta and interleukin-1 receptor antagonist and pregnancy outcome, Steven S. Witkin,
Santosh Vardhana, Melissa Yih, Kunihiko Doh, Ann Marie Bongiovanni & Stefan Gerber, American Journal of
Obstetrics and Gynecology 2003; 189 (5): pp. 1413 - 1417, PubMed: 14634579
Interleukin-1B (IL-1B) and interleukin-1 receptor antagonist (IL-1RN) gene polymorphisms are not associated with tubal
pathology and Chlamydia trachomatis-related tubal factor subfertility, Laura S. Murillo, Jolande A. Land, Jolein
Pleijster, Cathrien A. Bruggeman, A. Salvador Peña & Servaas A. Morré, Human Reproduction 2003; 18 (11): pp.
2309 - 2314, PubMed: 14585879
Chapter 7
The CD14 functional gene polymorphism –260 C>T is not involved in either the
susceptibility to Chlamydia trachomatis infection or the development of tubal pathology
Sander Ouburg, Joke Spaargaren, Janneke E. den Hartog, Jolande A. Land, Han S.A. Fennema,
Jolein Pleijster, A. Salvador Peña, Servaas A. Morré & the ICTI consortium
BMC Infectious Diseases 2005; 5 (1): pp. 114
Chapter 7
Abstract
bjectives: The functional polymorphism -260 C>T in the LPS sensing TLR4
co-receptor CD14 gene enhances the transcriptional activity and results in a
higher CD14 receptor density. Individuals carrying the TT genotype also
have significantly higher serum levels of soluble CD14. The T allele of this polymorphism
has recently been linked to Chlamydia pneumoniae infection. We investigated the role of
the CD14 –260 C>T polymorphism in the susceptibility to and severity (defined as
subfertility and/or tubal pathology) of C. trachomatis infection in Dutch Caucasian women.
O
Methods: The different CD14 –260 C>T genotypes were assessed by PCR-based RFLP
analysis in three cohorts: 1) A cohort (n=576) of women attending a STD clinic, 2) a cohort
(n=253) of women with subfertility, and 3) an ethnically matched control cohort (n=170).
The following variables were used in the analysis: In cohort 1 the CT-DNA status, CT IgG
serology status, self-reported symptoms and in cohort 2, the CT IgG serology status and the
tubal status at laparoscopy.
110
Results: In the control cohort the CC, CT and TT genotype distribution was: 28.2%, 48.2%,
and 23.5% respectively. No differences were found in the overall prevalence of CD14 –260
genotypes (28.1%, 50.7%, and 21.2%) in cohort 1 when compared to the control cohort.
Also no differences were observed in women with or without CT-DNA, with or without
serological CT responses, with or without symptoms, or in combinations of these three
variables. In subfertile women with tubal pathology (cohort 2, n=50) the genotype
distribution was 28.0%, 48.0%, and 24.0% and in subfertile women without tubal
pathology (n=203), 27.6%, 49.3% and 23.2%. The genotype distribution was unchanged
when CT IgG status was introduced in the analyses.
Conclusions: The CD14 –260 C>T genotype distributions were identical in all three
cohorts, showing that this polymorphism is not involved in the susceptibility to or severity
of sequelae of C. trachomatis infection.
CD14
Susceptibility to and severity of C. trachomatis infections
Introduction
hlamydia species are related to a broad clinical spectrum of human disease
including Chlamydia pneumoniae in lung and cardiovascular disease,
C. psittaci in pulmonary emphysema and psittacosis, and C. trachomatis in
ocular and urogenital infections1 - 3.
C. trachomatis is the most prevalent sexually transmitted disease in Europe and the USA.
Due to the mostly asymptomatic course of infection, these women will most likely not be
treated resulting in an enhanced risk for the development of late complications, which
include pelvic inflammatory disease (PID), ectopic pregnancy and tubal infertility.
The female reproductive tract is a very complex system where many factors, including
hormones, vaginal flora and immune mediators, combine to provide protection on the one
hand, while on the other hand maintaining an environment suitable for conception4. Clear
differences in the clinical course of infection have been described and are due to an
interaction between environmental (e.g. co-infection), bacterial (e.g. virulence factors) and
host factors (genetic differences between individuals). In previous studies no clear
associations have been demonstrated between C. trachomatis serotype, C. trachomatis
genotype, and the course of C. trachomatis infection5, 6, although differences in cytotoxicity
for different serovars have been described7 and an association between C. trachomatis
serovar G and cervical squamous cell carcinoma has been suggested8. In addition, virulence
gene expression studies, and genomic comparisons of strains, isolated from clearly
symptomatic or asymptomatic infected persons, revealed no strong role for the
C. trachomatis bacterium in relation to the course of infection9, 10.
A limited number of studies have recently demonstrated the influence of host genetic
factors on the susceptibility to and the severity of C. trachomatis infection. Host factors
including HLA-DQ and interleukin 10 (IL-10) have been associated with Chlamydia
infection11.
C
recognise several microbial products, including bacterial cell wall components and DNA12.
Further studies in mice corroborated these data 14, 15, while studies in human demonstrated
associations between TLR4 mutations and LPS hyporesponsiveness16. We did not observe
an association between the TLR4 Asp299Gly polymorphism in patients with tubal
pathology although the study population was relatively small17. The lack of association can
be explained by recent publications showing that heterozygous carriage of the TLR4
Asp299Gly mutation does not affect LPS responsiveness and that only the rare
homozygous carriers are less responsive to LPS18.
LPS-binding protein (LBP) (figure 1)19. Combined with TLR4 this complex induces NF-κB
associated immune responses including the release of a broad spectrum of cytokines that
include tumour necrosis factor alpha (TNF-α), IL-1, IL-6, and IL-8 to initiate immune
response20.
The promotor region of the CD14 gene contains a single nucleotide polymorphism (SNP) at
position –260. The -260 C>T genetic variation affects the binding of transcription factors21
and has been associated with levels of sCD14 and inversely associated with serum IgE
levels20. This SNP has been associated with myocardial infarction22, Crohn’s disease23 and
111
Chapter 7
an increased susceptibility to develop chronic spondyloarthropathy in women24.
112
Figure 1: CD14 localisation.
Panel A: Membrane-bound CD14 (mCD14) complexed with TLR4 and the LBP – LPS complex. Panel B: Soluble
CD14 (sCD14). Abbreviations: TLR: Toll-Like Receptor; LBP: LPS Binding Protein; LPS: Lipopolysaccharide;
NF-κB: Nuclear Factor κ B.
Eng et al. demonstrated that carriers of the T allele of this promotor polymorphism have a
higher expression of both mCD14 and sCD14 and that TNFα production is increased in the
homozygous CD14 –260*T carriers when stimulated with either C. pneumoniae or
C. trachomatis25. In a recent article, Rupp and colleagues described an association between
the mutant allele and an increased susceptibility to chronic C. pneumoniae infection in
coronary artery disease patients26. Since the CD14 –260 C>T is functional25 and is
associated with C. pneumoniae infection26, one could hypothesize that in Chlamydia
trachomatis infection this polymorphism could influence the susceptibility to and severity
of this most prevalent sexually transmitted bacterium which is associated with female
infertility.
Therefore, we investigated the role of the CD14 –260 C>T polymorphism in the
susceptibility to and severity (defined as subfertility and/or tubal pathology) of
C. trachomatis infection in Dutch Caucasian women. A cohort of women attending a STD
clinic was used to assess the susceptibility to C. trachomatis infection, taking into account
both C. trachomatis DNA and C. trachomatis IgG detection, symptoms and coinfections. A
cohort of subfertile women with or without clinically well-defined tubal pathology was
used to assess the role of CD14 in the severity of sequelae of C. trachomatis infection.
CD14
Material and Methods
Patient populations:
STD cohort
Women of Dutch Caucasian (DC) origin (n=576), under the age of 33 (range 14 to 33 years; median
22 years) and visiting the STD outpatient clinic in Amsterdam, The Netherlands, were included in this
study (collection period: July 2001 – December 2004) (Table 1). All 576 women were consecutively
included as the first part of a large prospective study. For every CT-DNA positive woman two
consecutive CT-DNA negative controls were included in the study. The women were asked to sign an
informed consent and to fill out a questionnaire, regarding their complaints at that moment, varying
from increased discharge, having bloody discharge during and/or after coïtus, recent abdominal pain
(not gastrointestinal or menses related) and/or dysuria. A cervical swab was taken for the detection of
C. trachomatis DNA (CT-DNA) by PCR (COBAS AMPLICOR; Hoffman – La Roche, Basel,
Switzerland)27.
STD cohort
n
CT DNA (LCx)
CT IgG
CT IgG (MIF)
Coinfections
576
184
392
217
359
+
–
+
–
+ (>=32)
– (<32)
–
+
41
212
C. albicans
N. gonorrhoeae
T. vaginalis
H. simplex virus 1
H. simplex virus 2
Symptoms
–
+
Vulvovaginal discharge
Abdominal pain
Dysuria
Bleeding during / after coïtus
Age
Tubal Pathology
Average
Range
Median
+
–
Subfertility
cohort
253
401
175
160
7
6
2
5
335
221
157
81
58
25
23.6y
15 – 41y
23y
113
30y
19 – 39y
31y
50
203
Table 1: Patient characteristics in the STD and subfertility cohorts.
Abbreviations: CT: C. trachomatis; STD: sexually transmitted disease; TP: tubal pathology
Peripheral venous blood was collected for the analysis of IgG antibodies against C. trachomatis (CT)
(Medac Diagnostika mbH, Hamburg, Germany). A titre of ≥ 1:50 was considered positive. Samples
with grey zone values, e.g. cut off ± 10%, were repeated and considered positive when the result was
positive or again within the grey zone. Infections with the microorganisms: Candida albicans,
Neisseria gonorrhoeae, Trichomonas vaginalis, Herpes simplex virus 1 / 2, may result in symptoms
similar to CT infection. Infection status for these microorganisms was recorded. HSV 1 / 2 was
detected according the methods described by Bruisten et al.28. N. gonorrhoeae was detected according
methods described by Spaargaren et al.29. T. vaginalis was cultured on Trichosel medium according
Chapter 7
standard procedures30 and detection of T. vaginalis was according the methods described by van der
Schee et al.31. C. albicans was cultured on Chrom agar and detection of C. albicans was performed
according standard procedures30.
Subfertility cohort
The study was performed in 253 consecutive Dutch Caucasian women who visited the department of
Obstetrics and Gynaecology of the Academisch Ziekenhuis Maastricht, The Netherlands, between
December 1990 and November 2000 because of subfertility32. In these women a laparoscopy with
tubal testing had been performed as part of their fertility work-up. Preoperatively blood was drawn
from all patients for Chlamydia IgG antibody testing (CAT), and spare sera were cryopreserved.
Two independent investigators, who were unaware of the CAT results, scored the laparoscopy reports
to assess the grade of tubal pathology. Tubal pathology was defined as extensive peri-adnexal
adhesions and/or distal occlusion of at least one tube at laparoscopy33. Subfertile women who had no
peri-adnexal adhesions and had patent tubes at laparoscopy served as negative controls. Based on
these criteria, 50 women had tubal pathology and 203 women served as controls.
IgG antibodies to C. trachomatis were detected with a species-specific MIF test (AniLabSystems,
Finland), as described previously32, with comparable sensitivity and specificity as compared to the
IgG ELISA from Medac used for the STD cohort 34. A positive C. trachomatis IgG MIF test was
defined as a titre ≥1:32. Findings at laparoscopy were correlated with the MIF test results. Based on
the MIF test, 41 women were found to be CT IgG positive, while 212 were CT IgG negative. Of the
CT IgG positive women 28 (68.8%) had tubal pathology, while 22 women (10.4%) of the CT IgG
negative women had tubal pathology.
Healthy controls
A healthy Dutch Caucasian control group (n=170) was included to assess the general frequency of the
CD14 -260 genotypes in the Dutch Caucasian population.
114
Immunogenetic analyses
DNA Extraction
STD cohort
Eukaryotic DNA from PBMC was isolated using the isopropanol isolation method. In short: 100μl
PBMC in PBS were added to 600μl L6 (Nuclisens Lysisbuffer, BioMerieux, Boxtel, The
Netherlands) and 1μl glycogen (Roche Molecular Diagnostics, Almere, The Netherlands). The
samples were incubated for 30 minutes at 65°C and left to cool at RT. An equal volume of cold
(-20°C) isopropanol was added to the samples. The samples were then centrifuged (20 min at
20.000G). The supernatant was discarded and the pellets were washed twice in 75% EtOH. The
pellets were dissolved in T10 overnight (O/N) at 4°C and then stored at –20°C until further analysis.
Subfertility cohort
Genomic DNA was extracted out of the cryopreserved sera using High Pure PCR Template
Preparation Kit (HPPTP kit) according to the manufacturer’s instructions (Roche Molecular
Biochemicals, Mannheim, Germany).
Healthy controls
Blood was collected in EDTA-tubes and stored at room temperature until the genomic DNA was
extracted from peripheral blood leukocytes (PBMC) according to an in-house DNAzol (Invitrogen,
The Netherlands) isolation procedure.
CD14 –260C>T gene polymorphism
The C>T substitution in the proximal CD14 promoter GC box at position –260 from the translation
start site (NCBI SNP CLUSTER ID: rs2569190) results in a HaeIII restriction site. We developed a
CD14
PCR assay using the primers, 5’ TCA CCT CCC CAC CTC TCT T 3’ (sense) and 5’ CCT GCA GAA
TCC TTC CTG TT 3’ (antisense) (Invitrogen Life Technologies, Breda, The Netherlands), flanking
this restriction site. Amplification was performed using a thermal cycler Perkin-Elmer 9700 (Applied
Biosystems, Forter City, CA, USA), in standard 96 well plates (Greiner Bio-one). The parameters
were an initial denaturation at 95°C for 5 min, followed by 35 cycles: denaturation at 95°C for 30 s,
annealing at 59°C for 30 s, and elongation at 72°C for 1 min. The final elongation was at 72°C for 7
min followed for a cooling to 4°C. The 107-bp fragments were digested overnight at 37°C with
HaeIII (Invitrogen, The Netherlands) resulting in fragments that either were cut in two fragments of
83-bp and 24-bp (allele C) or were not restricted (T allele). These fragments were analyzed by
electrophoresis on 4% low melting agarose gels (Tebu-Bio, The Netherlands) stained with ethidium
bromide.
Statistical analyses
All groups were tested for Hardy-Weinberg equilibrium to check for Mendelian inheritance.
Statistical analyses were performed using Instat Graphpad and SPSS version 11 (SPSS Inc., Chicago,
IL, USA). Fisher exact and χ2 tests were used to test for differences in CD14 allele / genotype /
carrier frequencies between the (sub)groups and p-values <0.05 were considered statistically
significant.
Results
All genotype distributions assessed were in Hardy-Weinberg Equilibrium. The CD14 -260
C>T SNP was assessed in the STD, subfertility and control cohorts.
CD14 –260 in the susceptibility to C. trachomatis infection
To determine the effects of CD14 –260 C>T on the susceptibility to C. trachomatis
infection, the prevalence of CD14 –260 C>T genotypes were assessed in the STD cohort
(table 2). The overall genotype distribution was 28.1% CC, 50.7% CT, 21.2% TT. This
distribution was comparable to the healthy controls (figure 2). The distribution was 28.8%
CC, 50.0% CT, 21.2% TT in CT DNA positive women, while in CT DNA negative women
the distribution was 27.8% CC, 51.0% CT, 21.2% TT. In women with or without
serological CT responses the distribution was 30.4% CC, 49.3% CT, 20.3% TT and 26.7%
CC, 51.5% CT, 21.7% TT, respectively. No differences could be observed in women with
or without symptoms. Coinfection with other microorganisms or combinations of these four
variables (CT DNA, CT serology, symptoms and microorganisms) did not introduce
statistically significant differences or trends in CD14 genotype distributions.
CD14 -260 in the severity of sequelae of C. trachomatis infection
The effect of CD14 –260 C>T on the severity of sequelae of C. trachomatis infection was
assessed in a cohort of subfertile women with clinically well-defined tubal pathology. The
overall genotype distribution in the cohort was 27.7% CC, 49.0% CT and 23.3% TT
(figure 2). The genotype distribution in women with tubal pathology was similar to the
distribution in women without tubal pathology (28.0% CC, 48.0% CT, 24.0% TT and
27.6% CC, 49.3% CT, 23.2% TT respectively) and to the distribution in the healthy
controls (table 3). Introduction of CT IgG serology, with special attention to C. trachomatis
positive women who did develop tubal pathology as compared to those who did not
develop tubal pathology, did not alter the observed genotype distribution.
115
Chapter 7
Total
1.1 (CC)
n
%
2.2 (TT)
n
%
Total
217
66
30.4%
107
49.3%
44
20.3%
LCx+ (CT DNA+)
135
38
28.1%
69
51.1%
28
20.7%
MO+
Symp
42
56
12
14
28.6%
25.0%
24
31
57.1%
55.4%
6
11
14.3%
19.6%
LAP+
17
4
23.5%
10
58.8%
3
17.6%
82
28
34.1%
38
46.3%
16
19.5%
29
43
17
12
16
5
41.4%
37.2%
29.4%
11
14
8
37.9%
32.6%
47.1%
6
13
4
20.7%
30.2%
23.5%
Total
LCx+ (CT DNA+)
MO+
Symp
CT IgG–
LAP+
LCx– (CT DNA–)
MO+
Symp
LAP+
359
49
16
19
10
310
88
103
37
96
15
3
5
3
81
20
26
6
26.7%
30.6%
18.8%
26.3%
30.0%
26.1%
22.7%
25.2%
16.2%
185
23
10
11
6
162
53
51
20
51.5%
46.9%
62.5%
57.9%
60.0%
52.3%
60.2%
49.5%
54.1%
78
11
3
3
1
67
15
26
11
21.7%
22.4%
18.8%
15.8%
10.0%
21.6%
17.0%
25.2%
29.7%
Healthy Controls
170
48
28.2%
82
48.2%
40
23.5%
CT IgG+
LCx– (CT DNA–)
MO+
Symp
LAP+
116
CD14 –260 C>T
1.2 (CT)
n
%
Table 2: CD14 genotype distribution in the Dutch Caucasian STD cohort.
C. trachomatis IgG positive and negative patients, divided in CT DNA (LCx) positive and negative and subdivided
in coinfection with other microorganisms (N. gonorrhoeae, T. vaginalis, C. albicans,and H. simplex virus 1 & 2),
symptoms (vulvovaginal discharge, abdominal pain, dysuria, bleeding during / after coitus) and lower abdominal
pain. Abbreviations: CT: C. trachomatis; MO+: microorganism positive; LAP+: lower abdominal pain positive;
Symp: symptoms positive
Total
TP+
TP–
CT IgG+ TP+
CT IgG+ TP–
Healthy Controls
Total
253
50
203
28
13
170
1.1 (CC)
n
%
70
27.7%
14
28.0%
56
27.6%
9
32.1%
4
30.8%
48
28.2%
CD14 –260 C>T
1.2 (CT)
n
%
124
49.0%
24
48.0%
100
49.3%
15
53.6%
6
46.2%
82
48.2%
2.2 (TT)
n
%
59
23.3%
12
24.0%
47
23.2%
4
14.3%
3
23.1%
40
23.5%
Table 3: CD14 genotype distribution in the Dutch Caucasian subfertility cohort.
Distribution in the total cohort and subdivided in women with or without tubal pathology, and C. trachomatis IgG
positive women with or without tubal pathology. C. trachomatis positivity defined as a titre ≥ 1:32 (MIF).
Abbreviations: TP: tubal pathology; CT: Chlamydia trachomatis.
CD14
Figure 2: CD14 genotype distribution in the STD, subfertility and control cohorts.
Abbreviations: STD: sexually transmitted disease; HC: healthy controls
Discussion
We did not find an association between the functional upregulating CD14 –260 C>T
polymorphism and the susceptibility to or subsequent severity of sequelae of
C. trachomatis infection, as assessed in the STD and subfertility populations (figure 2).
However, these results do not exclude that a still unknown CD14 expression decreasing
SNP may influence the course of C. trachomatis infection.
Recent studies have shown that Chlamydia LPS is capable of inducing an inflammatory
response through CD1435, 36, although the potency to induce an inflammatory response was
100 – 1000 times less when compared to the responses induced by S. minnesota,
N. gonorrhoeae35 and the enterobacteria S. enterica and E. coli36. Heine et al. demonstrated
that the CD14 associated inflammatory response was TLR4 but not TLR2 mediated36.
These results are corroborated by studies showing the role of the CD14–TLR4–MD2
complex in intracellular signalling by LPS13, 37 and studies showing the dependency on
CD14 of phagocytosis of Gram negative bacteria38.
The absence of an association between CD14 and susceptibility to C. trachomatis infection
might be explained by the compartmentalisation of TLR4. The differential expression of
TLR4 has been described in immortalised cell-lines derived from the female urogenital
117
Chapter 7
tract39 and recently demonstrated in cells isolated from patients by Pioli40 and Fazeli41.
TLRs 1 – 6 were found to be expressed in the epithelia of the female urogenital tract. TLR2
and TLR4 were the only Toll like receptors with a clear differential expression. Low
expression in the lower urogenital tract and high expression in the upper genital tract40, 41.
The expression remained similar in all subjects irrespective of age or status of the
reproductive cycle41. It is hypothesized that through this expression pattern TLR4
modulates immunological tolerance in the lower genital tract and induces host defence
against ascending infection in the upper genital tract41. In the upper genital tract, Fazeli and
colleagues found TLR4 positive vacuole like structures that seemed to be secreted from
endocervical glands41. A secretory form of TLR4 has been described in mice, where the
soluble TLR4 appears to inhibit LPS mediated signals, while at the same time sTLR4
mRNA is upregulated by LPS42. This may represent a feedback mechanism to prevent
excessive responses to LPS in the endocervix, which can be seen as a boundary between the
lower and upper genital tract. Further evidence for the regulation of immune responses to
LPS by TLR4 is provided by the study of Harju et al., who demonstrated the intrauterine
expression of TLR4 and endotoxin responsiveness in mice in the perinatal period43. mCD14
is expressed on human endometrial stromal cells but not on endometrial epithelial gland
cells. The epithelial cells are dependent on sCD14 for LPS recognition44. Soluble CD14 is
present in the cervical mucosa and may be present in the endometrium45.
118
Combining the aforementioned studies with the knowledge that CD14 can signal through
TLR4, it might be hypothesized that the absence of an association between the CD14 –260
SNP and the susceptibility to C. trachomatis infection might be due to the low expression
or absence of TLR4 in the lower urogenital tract. In the upper genital tract, strict regulation
of immune responses to LPS by TLR4 may inhibit CD14 signalling through TLR442, 43, thus
limiting the influence of CD14 on the development of tubal pathology.
However, this hypothesis does not take into account the ability of CD14 to signal through
TLR246, nor does it take into account that the study of Netea et al. which demonstrated that
non-LPS components of Chlamydia pneumoniae can stimulate cytokine production through
TLR2 dependent, CD14 independent pathways47 and that a similar mechanism may exist
and stimulate C. trachomatis induced cytokine production in urogenital infections.
Since TLR2 is involved in Chlamydia-induced TGF-beta, an anti-inflammatory cytokine
with an important role in fibrosis, and thus very likely in post-infection tubal pathology, it
might explain why CD14 polymorphisms may not severely impact the development of
tubal pathology48.
Darville et al. have demonstrated that TLR2 is an important mediator of innate immune
responses in C. trachomatis infection in mice and plays an important role in early
production of immune mediators and development of tubal pathology49, 50. In a recent
publication by Pitz et al. it was shown that C. pneumoniae is capable of activating
endothelial cells by TLR2 as initial extracellular C. pneumoniae receptor, whereas NOD1
was shown to be a potent intracellular immune receptor for C. pneumoniae in endothelial
cells. Further research may extend these results to C. trachomatis infections. Overall, the
recognition of bacterial LPS involves a complex system of multiple receptors and a
complex orchestration of protein-protein interactions51.
CD14
Conclusions
Our study showed that the functional up-regulating CD14 -260 C>T SNP did neither
influence the susceptibility to nor the severity of late sequelae of Chlamydia trachomatis
infection. However, this does not exclude a prominent role for CD14 in the course of an
active C. trachomatis infection and not yet described CD14 expression decreasing SNPs
may affect the course of C. trachomatis infection profoundly. Further studies on the
immunogenetics of C. trachomatis infection will provide more insight in the clear
differences in the clinical course that this microorganism induces in individuals and lead to
potential vaccine candidates.
Acknowledgements
Sander Ouburg is an AstraZeneca Nederland BV fellow.
Servaas A. Morré is supported by the Department of Internal Medicine of the VU University Medical Centre, the
Netherlands.
The authors are indebted to Prof. Cathrien Bruggeman, head of the department of Medical Microbiology,
Academisch Ziekenhuis Maastricht, Maastricht, The Netherlands, for the serological testing of the subfertility
cohort.
The ICTI consortium (Integrated approach to Chlamydia trachomatis Infections52) provides a broad specialized
network for the multidisciplinary studies described.
119
Chapter 7
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Chapter 8
Host inflammatory response and development of complications of Chlamydia trachomatis
genital infection in CCR5 deficient mice and subfertile women with the
CCR5δ32 gene deletion
Erika L. Barr, Sander Ouburg, Joseph U. Igietseme, Servaas A. Morré, Edith Okwandu, Francis O. Eko,
Godwin Ifere, Tesfaye Belay, Qing He, Deborah Lyn, Gift Nwankwo, James Lillard, Carolyn M. Black &
Godwin A. Ananaba
Journal of Microbiology, Immunology & Infection 2005; 38 (4): pp. 244 – 254
Chapter 8
Abstract
cell immunity protects against diseases caused by the obligate intracellular
bacterium Chlamydia trachomatis. Incidentally, host inflammatory response
that includes T cells appears to also contribute to the pathogenesis of
Chlamydial diseases such as trachoma and tubal factor infertility (TFI). Therefore,
designing effective prevention strategies require a delineation of immune processes
responsible for pathology and those mediating immunity, and defining the immunogenetic
factors predisposing to complication development. The chemokine receptor CCR5 is crucial
for T cell activation and function since its deficiency causes suppression of T cell response.
We investigated the hypothesis that the clearance of genital Chlamydial infection in CCR5
deficient mice could be delayed in the short-term; however, a beneficial effect could
include protection against inflammation-related complications such as TFI. In a
translational study in humans, we investigated the effect of functional CCR5δ32 gene
deletion on the risk of developing tubal pathology in Dutch Caucasian women with
immunological evidence (i.e. IgG responses) of Chlamydial infection. When genitallyinfected wild-type (WT) and CCR5 knockout (CCR5KO) mice were evaluated for
microbiological shedding of Chlamydiae, there was a greater intensity of infection and
delayed resolution in the knockout mice. However, compared to WT mice, the fertility of
infected CCR5KO mice (measured by pregnancy rate) was only mildly affected on the
short-term and unaffected on the long-term (70 versus 30% reduction in the short-term, and
50 vs. 0% on the long-term, respectively). Immunobiologic analysis revealed that the
diminished capacity of CCR5KO to control acute Chlamydial infection correlated with the
relatively low chemokine (IP-10 and RANTES) and cytokine (mainly IFN-γ and TNF-α)
expression corresponding to a poor early Th1 response. However, the reduced incidence of
complications in the CCR5KO mice appears to correlate with the low activity of long-term
inflammatory mediators. Besides, the translational studies in humans revealed that among
patients with positive anti-Chlamydial IgG responses, tubal pathology correlated with a low
incidence of CCR5δ32 deletion (7%), while women without tubal pathology had higher
incidence of the CCR5δ32 deletion (31%), as compared to controls (19%). Thus, in mice
and humans the inflammation associated with CCR5 function may predispose to
development of complications of Chlamydial infection, such as TFI.
T
124
CCR5
Introduction
he genus Chlamydia comprises obligate intracellular, gram negative-like
bacteria that cause numerous oculo-genital and respiratory infections in
humans, animals and birds. Trachoma, caused by C. trachomatis serovars A, B,
Ba and C, is the world’s most common preventable blinding disease, and of epidemic
proportion in several developing nations, including Africa, South East Asia, and the Middle
East, with an estimated 150 million people affected, of whom six million are irreversibly
blinded or severely visually impaired1. Genital infection by the different genovars of
C. trachomatis constitutes the most common bacterial STD in the United States and several
other industrialized nations, including the United Kingdom, Germany, Japan, and France.
Pelvic inflammatory disease (PID) and tubal factor infertility (TFI) are major complications
of genital infection, occurring respectively in approximately 40 and 10 percent of untreated
infections, respectively, and constituting an enormous morbidity and socioeconomic burden
of chlamydial infections2 - 5. The Lymphogranuloma venereum (LGV) infections are
invasive and often ulcerative with lymphatic tissue involvement (e.g. inguinal bubo)6, 7,
which are endemic in certain developing nations including parts of Africa, Asia, South
America, and the Caribbean8. The recent epidemic outbreak of LGV disease among men
who sex with men in Europe is attracting considerable public health attention in many
countries9. Ulcerative STDs in general are a major risk factor for HIV acquisition.
Moreover, reports suggesting that genital Chlamydial infection may be on the rise10, 11, and
could predispose to HIV-related AIDS12 - 18 and human papilloma virus-associated cervical
dysplasia, have heightened these concerns19.
T
There is urgency to develop intervention and prevention measures to control Chlamydial
infections in the human population. However, a better understanding of the pathobiology of
the disease is crucial for efforts to design preventive measures including the use of targeted
immunomodulators and selective anti-inflammatory agents to control the onset of
pathologies, and the application of effective vaccines as prophylaxis. Although T cell
immunity is crucial for Chlamydial control20 - 22, clinicopathologic and experimental studies
have suggested that the pathogenesis of complications such as trachoma and TFI is due to
the deleterious host inflammatory immune response against the infectious agent. Thus,
studies defining the key elements of protective immunity against Chlamydia, and
establishing the parameters for vaccine selection and evaluation, are often confounded by
the complexities in Chlamydia biology, serovariation, infection manifestations and
induction of paradoxical immune effectors that can be both protective and pathologic23 - 27.
This double-edge effect poses a dilemma to the effort to dissect the role of host immune
response on pathogenesis and immunity. In this respect, various inflammatory chemokines
and cytokines and strains of mice with differential susceptibilities to Chlamydial infection
have been evaluated to define some of the immunopathogenic factors responsible for
Chlamydial disease28 - 34. Among other findings, TLR2 deficiency caused decreased
secretion of specific inflammatory cytokines, and a significant reduction in oviduct and
mesosalpinx pathology even without affecting the course of the infection in mice30. Also,
cytokines and molecules indicative of T cell activation were upregulated in active
trachoma35. These observations and others would suggest that there are key
immunobiological pathways of T cell activation with limited redundancies that often
complicate such studies. CCR5 is a member of the 7-transmembrane, G protein-coupled
receptor superfamily, functioning as an important chemokine receptor that is preferentially
125
Chapter 8
expressed on certain leukocytes (monocytes, CTLs CD4 Th1 and dendritic cells), and
binding specific chemokines (e.g. RANTES, MIP-1α and MIP-1β) that activate and induce
Th1-like cells36 - 40. As a crucial receptor involved in T cell activation and function, a
deficiency of CCR5 is associated with a suppression of T cell induction and leukocyte
migration under certain infectious and non-infectious conditions41 - 43, suggesting that it
plays a role in both infection-related immune and inflammatory processes. The effect of a
targeted suppression of the critical specific T cell response on both immune-mediated
microbial clearance and the development of complications of Chlamydial infection is
largely unknown. We investigated the hypothesis that the suppression of T cell response
against genital Chlamydial infection in CCR5 deficient mice could delay the clearance of
the pathogen in the short-term, but it could also confer a beneficial effect by protecting the
animals from complications such as TFI. In addition, in a translational study in humans, we
investigated if the functional 32bp deletion in the CC-chemokine receptor 5 gene
(CCR5δ32) had an effect on the risk of developing tubal pathology in women with
serological evidence (IgG responses) of C. trachomatis infection. The results supported the
working hypothesis and could pave the way for a more detailed analysis and definition of
the specific host-related immune effectors and immunopathologic processes underlying the
pathogenesis of Chlamydial disease.
Materials and Methods
126
Animals: Knockout (KO) and Wild-type mice
Female chemokine receptor-5 (CCR5) knockout (CCR5KO or CCR5-/-) and the wild-type (WT)
control (i.e. CCR5+/+) mice, on (C57BL/6J) background, 5-8 weeks old, were obtained from The
Jackson Laboratory, Bar Harbor, MA. All animals were fed with food and water ad libitum, and
maintained in Laminar flow racks under pathogen-free conditions of 12-hour light and 12-hour
darkness.
Chlamydia stocks and antigens.
Stocks of Chlamydia muridarum (the C. trachomatis agent of mouse pneumonitis or MoPn) used for
infections were prepared by propagating elementary bodies (EBs) in McCoy or HeLa cells, according
to standard procedures44. Chlamydial stock titers were expressed as inclusion-forming units per
milliliter (IFU/ml). Chlamydial antigens were prepared by growing the agent in HeLa cells and
purifying EBs over renografin gradients, followed by inactivation under ultraviolet (UV) light for 3
hours.
Animal Infection
Groups of CCR5KO and control mice were intravaginally infected with 106 IFU of MoPn. The status
of the infection was monitored by periodic cervico-vaginal swabbing of individual animal and
isolation of Chlamydiae in tissue culture44. Experiments were repeated 2 times to give 10-12 mice per
group.
Chlamydia-induced cell activation, cytokine and chemokine secretion by leukocytes from
Chlamydial-infected CCR5KO WT control mice
The profile of cytokines and chemokines secreted by leukocytes from Chlamydial-infected CCR5KO
and wild type mice was compared by measuring the levels of specific cytokines and chemokines that
are released following in vitro restimulation of the total splenic cells with UV-inactivated
Chlamydiae. CCR5KO and control mice were genitally infected with MoPn as previously described
above and at various times post-infection (7, 14, 21 and 28 days) splenic cells were isolated from
infected mice and 2x10^5 cells were stimulated with 10 μg/ml of Chlamydial antigen and incubated at
CCR5
37oC in 5% CO2 incubators for 120 hr. At the end of the incubation period, the supernatants were
collected and assayed for the Th1 cytokine IFN-γ, and the Th1 chemokines IP-10 and RANTES,
using a quantitative ELISA (CytoscreenTM Immunoassay Kit; BioSource) according to the supplier’s
instructions. The concentration of the cytokine and chemokines in each sample was obtained by
extrapolation from a standard calibration curve generated simultaneously. Data were calculated as the
mean values (± S.D.) of triplicate cultures for each experiment. The results were derived from at least
3 independent experiments.
Animal fertility studies
Animals were infected with 106 IFU/mouse with MoPn. Two weeks and five weeks post infection
groups of animals were mated with male counterparts by placing one female to one male for 19 days,
and subsequently observed and weighed daily for 19 days to determine pregnancy, as previously
described45. The numbers of pregnant mice in the different groups were enumerated after 19 days in
each case.
CCR5δ32 gene deletion in women with subfertility
The study cohort included 256 Dutch Caucasian women who presented with subfertility at the
Research Institute Growth and Development (GROW) and the Department of Obstetrics and
Gynaecology, Academisch Ziekenhuis Maastricht, The Netherlands). This subfertility cohort has been
described elsewhere46. Tubal pathology was defined as extensive periadnexal adhesions and/or distal
occlusions of one or both tubes, and 50 women had severe tubal pathology based on these criteria.
Chlamydial antibodies were assessed by indirect microimmunofluorescence (MIF) test for
47, 48
. A positive C. trachomatis IgG MIF
anti-C. trachomatis IgG antibodies, as described previously
test was defined as a titer ≥ 1:32. A healthy Dutch Caucasian control group (n = 145) was included to
assess the general frequency of the CCR5δ32 genotypes in the Dutch Caucasian population. Genomic
DNA was extracted from blood using the MagNaPure LC isolator according to the manufacturer’s
instructions (Roche Molecular Biochemicals, Mannheim, Germany). The human CCR5 gene δ32
deletion was determined by polymerase chain reaction (PCR) in standard 96 well plates (Greiner BioOne), with the sense primer CCR5-d32S: 5’ CAA AAA GAA GGT CTT CAT TAC ACC 3’ and antisense primer CCR5-d32AS: 5’ CCT GTG CCT CTT CTT CTC ATT TCG 3’ under the following
PCR conditions: 5 min at 94°C, followed by 35 cycles of 60 sec at 94°C, 60 sec at 55°C, and 60 sec at
72°C, and the cycling programme was followed by 7 min at 72°C and finally stored at 4°C (Perkin
Elmer PE9700). The PCR product was electrophoresed on 3% agarose and the following three
fragment patterns were identified: wild type CCR5 gene (1.1) 189bp, homozygote CCR5 mutant gene
(2.2) of 157bp, and the heterozygote CCR5 gene (1.2) 189bp + 157bp.
Statistical Analysis
The levels of cytokines in samples from different experiments were analyzed and compared by
performing a one- or two-tailed t test, and the relationship between different experimental groupings
was assessed by analysis of variance (ANOVA). Minimal statistical significance was judged at
P< 0.05. The χ2-test or Fisher exact test was used for comparison of CCR5δ32 genotype frequencies
between patient (sub) groups and/or controls. SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA)
was used for statistical analysis.
Results
High intensity of infection and delayed clearance of genital Chlamydial infection in
CCR5KO mice
CCR5 is a crucial chemokine receptor that supports the activation and induction of specific
T cells during infection- and non-infection inflammatory processes. We investigated the
effect of CCR5 deficiency on the ability of mice to control and clear genital Chlamydial
127
Chapter 8
Chlamydia (log 10 IFU/ml)
infection. Figure 1 shows results from studies that compared the course of genital
Chlamydial infection in CCR5KO and control (wild type, WT) mice. The data revealed that
within the first week of infection, there was no difference in the level of infectivity of
CCR5KO and WT mice. However, by the second and forth weeks, the ability of CCR5KO
mice to control the infection was compromised, with a higher intensity of infection revealed
by the isolation of higher Chlamydiae from the mice. By the fifth week, all WT mice had
cleared the infection but the CCR5KO mice remained infected (0.0 versus Log10 3.0
IFU/ml, respectively). The results suggested that the deficiency of CCR5 could have
adversely affected the ability the mice to elicit the required T cell response that is known to
clear Chlamydiae in mice20.
128
6
4
Wild type
2
CCR5KO
0
6
12
21
27
Days post infection
Figure 1: Female CCR5-/- and the control CCR5+/+ mice were intravaginally infected with 106 IFU of MoPn. The
status of the infection was monitored by periodic cervico-vaginal swabbing of individual animal and isolation of
Chlamydiae in tissue culture44. Experiments were repeated 2 times to give 10-12 mice per group.
Protection of CCR5 deficient mice from certain complications of Chlamydial infection
The effect of the diminished capacity of CCR5KO mice to clear genital Chlamydial
infection on the infertility that is commonly associated with a genital infection45 was
studied. Infected mice were mated at two and five weeks after the initial infection, and the
fertility was assessed by the number of pregnancies recorded. The mating at different time
periods was targeted at evaluating the short- and long-term effect of the infection on
fertility, since the WT mice cleared their infection at this latter time. Interesting, at 2 weeks
post genital infection, WT mice exhibited a significantly lower fertility (with < 40%
pregnancy rate) than CCR5KO mice (> 70%; p > 0.021) (Figure 2a). Furthermore, at 5
weeks post genital infection, all the CCR5KO mice exhibited 100% fertility whereas the
control mice scored approximately 50% (Figure 2b). These results suggested that the
immunocompetence of the host is possibly a relevant factor in the development of the longterm complication of Chlamydial infection such as infertility. To test this proposition, we
evaluated the likely immune correlates of this inverse relationship between the ability to
clear Chlamydial infection and the development of complications in the context of CCR5
integrity.
CCR5
Immunological correlates of clearance of infection and protection from disease
The direct immunobiologic impact of CCR5 deficiency includes the limitation of T cell
activation and reduction or elimination of recruitment of leukocytes to inflammatory sites
of infection41 - 43. Since these processes are mediated by chemokines and cytokines, their
production by WT and CCR5KO leukocytes that are exposed to Chlamydiae was evaluated.
When splenic cells containing T cells and other leukocytes were exposed to Chlamydiae,
the antigen-specific IFN-γ and TNF-α response was expectedly elevated in the cells from
the WT mice (Figure 3a).
150
% pregnant
100
W/T noninfected
CCR5 KO noninfected
50
W/T
CCR5 KO
0
Figure 2a
129
% pregnant
120
100
80
W/T noninfected
CCR5 KO noninfected
60
40
W/T
CCR5KO
20
0
Figure 2B
Figure 2: Animals were infected with 106 IFU/ mouse with MoPn. Two (Fig 2a) or 5 (Fig 2b) weeks post infection,
groups of animals were mated with males, and subsequently observed for 19 days to determine pregnancy, as
previously described45. The numbers of pregnant mice in the different groups were enumerated after 19 days in
each case. Experiments were repeated 3 times with 6 mice per experiment. W/T = wild-type control mice.
Chapter 8
The results presented in Figure 3a also revealed that the level of antigen-specific IFN-γ
secreted by leukocytes from CCR5KO mice was not statistically different from the levels
secreted by leukocytes from non-infected mice, indicating that that CCR5 is required for
adequate activation of Th1 response against Chlamydia.
When the levels of the inflammatory chemokines RANTES and IP-10 secreted by
Chlamydial exposed leukocytes from infected CCR5KO and WT mice were compared, it
was also found that the knockout mice exhibited a diminished capacity (Figures 3 and 4),
suggesting the deficiency of CCR5 results in a compromised Th1 response. Figure 3c
expresses the data in Figures 3a and 3b as fold enhancement of WT response over
CCR5KO response to emphasize the significance of Th1 suppression due to CCR5
deficiency.
12000
IFN-gamma (pg/ml)
10000
8000
6000
4000
2000
130
0
CCR5 KO
infected
WT
infected
Controls
noninfected
Figure 3a
1200
TNF-alpha (pg/ml)
1000
Figure 3b
800
600
400
200
0
CCR5 KO
infected
WT
infected
Control
noninfected
CCR5
Fold Enhandement
(Wildtype/CCR5KO)
12
10
8
6
4
2
0
IFN-gamma
Cytokines
TNF-alpha
Figure 3c
Figure 3: CCR5KO and control mice were genitally infected with MoPn and at 2 wk post-infection splenic cells
were isolated from infected mice and 2x10^5 cells were stimulated with 10 μg/ml of Chlamydial antigen for 120
hr. The supernatants were collected assayed for IFN-γ and TNF-α, as described in the Materials and Methods
section. Data were calculated as the mean values (± S.D.) of triplicate cultures for each experiment. The results
were derived from at least 3 independent experiments. Figure 3a (IFN-γ). Figure 3b (TNF-α). Figure 3c (Fold
enhancement of WT response over CCR5KO response from data in Figures 3a and 3b).
Inverse relation between CCR5δ32 gene deletion and development of tubal pathology
among women with evidence of C. trachomatis infection.
In a translational study involving subfertile Dutch Caucasian women, we investigated by
PCR method whether a functional 32bp deletion in the CC-chemokine receptor 5 gene
(CCR5δ32) had an effect on the risk of developing tubal pathology in women with
serological evidence of C. trachomatis infection based on IgG responses. Results revealed
that in the control women (n=145) the CCR5δ32 wild-type genotype (1.1) was present in
81%, the heterozygous genotype 1.2 in 18%, and the 2.2 homozygous mutant genotype in
1% (with a total of 19% being carrier of the mutant allele). Also, the subfertility cohort
(n=256) had frequencies of the wild-type, heterozygous, and homozygous mutant
genotypes (1.1 = 80%, 1.2 = 19.5% and 2.2 = 0.5%, respectively) essentially identical to the
control women. Therefore, there was no difference in the frequency of the CCR5δ32 gene
deletion among subfertile (20%) and control (19%) women.
However, in women with laparoscopically confirmed tubal pathology (n=50), cariership of
the δ32 deletion was lowered to 14%, while in women without tubal pathology (n=206)
cariership of the δ32 deletion remained at the normal (21%). This suggested that the
incidence of tubal pathology was lower in women carrying the CCR5 mutation. Since only
a proportion of the women with proven C. trachomatis infection (MIF IgG responses)
actually develop late complications such as tubal pathology, we wondered whether CCR5
mediated inflammatory response has a role in the development of Chlamydial-associated
complications. Therefore, to directly investigate the role of CCR5 status on the incidence of
Chlamydial-associated TFI, and we hypothesized that as in mice, a functional deficiency in
CCR5 gene will moderate tubal pathology in Chlamydial infected women. To test this
hypothesis, we compared the frequency of δ32 deletion in women with C. trachomatis IgG
responses who developed tubal pathology (TP+CT+, n=28) to women with C. trachomatis
IgG responses without tubal pathology (TP-CT+, n=13)). The results revealed that there
131
Chapter 8
was a significant difference the TP+CT+ women with only 7% carriership of the 32bp
deletion in the CCR5 gene and the TP-CT+ women with 31% carriership (OR 5.8)
(Figure 5 presents a summary of these results).
IP-10 (pg/ml)
IP-10
1600
1400
1200
1000
800
600
400
200
0
CCR5KO
Wild type
7
14
21
28
Time (days post infection)
Figure 4a
RANTES
132
RANTES (pg/ml)
500
400
300
CCR5KO
200
wild type
100
0
7
14
21
28
Time (days post infection)
Figure 4b
Figure 4: CCR5KO and control mice were genitally infected with MoPn and at various times post-infection (7, 14,
21 and 28 days) splenic cells were isolated from infected mice and 2x10^5 cells were stimulated with 10 μg/ml of
Chlamydial antigen for 120 hr. The supernatants were IP-10 and RANTES, using a quantitative ELISA as
described in the Materials and Methods section. Data were calculated as the mean values (± S.D.) of triplicate
cultures for each experiment. The results were derived from at least 3 independent experiments. Figure 4a (IP-10).
Figure 4b (RANTES).
CCR5
133
Figure 5: Analysis of the CCR5δ32 carriership (genotypes 1.2 + 2.2) in Dutch Caucasian controls (DC) and
women with subfertility (Sub). Subfertily women with laparoscopically confirmed tubal pathology (TP+) were
compared to those without tubal pathology (TP-). Also, CCR5δ32 carriership was compared in women with
C. trachomatis (CT) IgG responses, with or without tubal pathology (TP+ versus TP-).
Discussion
The ability of Chlamydia to induce both protective and immunopathogenic immune
responses poses a phenomenal challenge to research efforts to define the conditions
favoring either response, and vaccine design research to skew the responses along the
protective pathway. A detailed knowledge of the conditions engendering pathogenesis
could lead to the design of targeted immunopharmacologic strategies to avert pathologies.
In this respect, the recent findings from an IL-10 deficient dendritic cell-based cellular
vaccine20, 49 suggested that a fast and vigorous Th1 response after an infection will rapidly
arrest Chlamydial replication, clear the infection, eliminate residual antigens and prevent
the establishment of a latent infection. On the other hand, it was suggested that an
inadequate or suboptimal Th1 response delays clearance of the pathogen, leading to the
establishment of a latent or persistent infection, which fuels a low-grade chronic immune
response that causes tissue damage. This proposition is supported by several experimental
and clinical findings relating to the cytokine and leukocyte responses that are associated
with the onset of Chlamydial diseases31, 50 - 52.
Chapter 8
134
The microbiologic and host factors governing the development of complications of
Chlamydial disease have been inadequately defined. The increasing experimental and
clinical evidence that certain host genetic factors are crucial for the manifestation of
specific disease phenotypes in certain individuals has led to the proposition that host
genetic and immunologic or inflammatory processes are relevant for the development of
complications of Chlamydial disease53 - 55. In this respect, clinical evidence from a sibling
cohort study in a trachoma-hyperendemic setting indicated that persistence and inadequate
clearance of Chlamydiae (possibly due to a delayed or inadequate Th1 response) were
hallmarks of individuals who suffered severe trachoma56. Also, different isolates of
C. trachomatis causing symptomatic and asymptomatic infections in women exhibited
similar growth characteristics in vivo and in vitro53, suggesting that the clinical outcome
was mostly host dependent. In addition, history of prior infection, age, and the hormonal
status relating to the phase of estrous cycle and HLA alleles were significant factors in the
development of PID and infertility following genital Chlamydial infection57 - 62.
Furthermore, various inflammatory chemokines and cytokines and strains of mice with
differential susceptibilities to Chlamydial infection have been analyzed to define some of
the immunopathogenic factors responsible for Chlamydial disease28 - 34, 63. Among other
findings, TLR2 deficiency caused decreased secretion of specific inflammatory cytokines,
and a significant reduction in oviduct and mesosalpinx pathology even without affecting the
course of the infection in mice30. The lack of manifestation of the effect of TLR2 deficiency
on the clearance of Chlamydial infection was probably due to the natural redundancy in the
TLR signaling system, such that the absence of TLR2 was duly compensated by other
pathways. However, the reduction in tissue pathology could be associated with the
suppression of inflammatory response caused by the inhibition of TLR signaling. Also,
cytokines and molecules indicative of T cell activation were upregulated in active
trachoma35, and animals that exhibit a high incidence of ascending genital Chlamydial
infection tend to secrete a higher TNF-α in response to an infection28, 29. These observations
strongly support the involvement of host inflammatory response in the pathogenesis of the
complication of Chlamydial infection.
We have experimentally demonstrated in this study that CCR5-related inflammatory
response is crucial for the development of TFI following genital Chlamydial infection. In
translational immunogenetic and pathobiological clinical studies in humans, functional
defect in CCR5 also appears to moderate the development of tubal pathologies associated
with genital Chlamydial infection in women. These data are corroborated by previous
propositions that certain host factors are relevant for the development of the complications
of Chlamydial infection. Specifically, the 32-base pair deletion in the CCR5 gene, which
results in a truncated protein with impaired signal-transduction capacity, was associated
with resistance to human immunodeficiency virus type 1 (HIV-1) infection64, 65. Recently, it
has been suggested that heterozygosity for CCR5δ32 was also associated with spontaneous
hepatitis C viral clearance and with significantly lower hepatic inflammatory scores66. Our
experimental studies indicated that a deficiency of specific anti-Chlamydial Th1 response
led to a suppression of Th1 response and delayed clearance of genital Chlamydial infection.
However, CCR5KO mice were protected from the complication of the genital infection
relating to infertility. In addition, C. trachomatis-exposed women with CCR5δ32 deletions
appear to be protected from tubal pathology as well, suggesting a crucial role for CCR5related specific Th1 and inflammatory responses in the pathogenesis of infectious tubal
pathologies.
CCR5
Perhaps this study represents the first concurrent demonstration of a strong causal
relationship between CCR5-related specific Th1 and inflammatory response and
development of complications of genital Chlamydial infection in both animal models and
humans. The implications include the fact that although Th1 response is crucial for
Chlamydial control, there are host conditions that could skew the response toward
pathology. Such conditions may include the involvement of immunopathogenic Chlamydial
antigens26 and vaccine design effort may focus on defining the existence of clonotypic T
cells that recognize such antigens or develop additional strategies to eliminate them from
promising vaccine candidates. T cell clones reactive against specific Chlamydial antigens
have been isolated from the synovial fluids of patients suffering Chlamydial-induced
reactive arthritis67. In addition, since an early and relatively robust T cell response is
protective against subsequent development of complications of Chlamydial infection20, 28,
these results from the CCR5KO system may suggest that the lack of an early T cell
activation caused the delay in resolution of the infection; however, the persisting
suppression of T cell activation prevented the chronic host inflammatory response that
induces pathologies. Therefore, an early treatment of Chlamydial infection with antimicrobials followed by specifically targeted anti-inflammatory agents may hold promise as
a strategy for preventing the complications of Chlamydial infection. Finally, it is pertinent
to mention that although previous studies along this proposition yielded conflicting
results68 - 72, the selective use of specifically targeted anti-inflammatory agents in
combination with antibiotics could prove useful in the management of Chlamydial
infections to avert pathology. In fact, inflammatory processes induced by several species of
Chlamydia could be suppressed by a select non-steroidal anti-inflammatory drugs,
including aspirin and indomethacin73.
Aknowledgements
This research was supported by PHS grants (5P60MD000525, AI41231, GM08247, GM08248, and RR03034)
from the National Institutes of Health and the Center for Disease Control and Prevention (CDC). The authors with
to thank Jolande A. Land, MD, PhD (Research Institute Growth and Development (GROW) and Department of
Obstetrics and Gynaecology, Academisch Ziekenhuis Maastricht, The Netherlands) for providing the subfertility
cohort and patient characteristics, and Prof. A Salvador Peña, MD, PhD, FRCP, Head of the Laboratory of
Immunogenetics, Vrije Universiteit Medical Centre, Amsterdam The Netherlands, for fostering the excellent
setting for the immunogenetic studies and for valuable discussion. Sander Ouburg is an AstraZeneca Nederland
BV fellow.
135
Chapter 8
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139
Chapter 8
140
There is one thing even more vital to science than intelligent methods;
and that is, the sincere desire to find out the truth, whatever it may be.
Charles Sanders Pierce
Chapter 9
Do host genetic traits in the bacterial sensing system play a role in the development of
Chlamydia trachomatis-associated tubal pathology in subfertile women?
Janneke E. den Hartog, Sander Ouburg, Jolande A. Land, Joseph M. Lyons, James I. Ito,
A. Salvador Peña & Servaas A. Morré
BMC Infectious Diseases 2006; 6 (1): pp. 122
Chapter 9
Abstract
ackground: In women, Chlamydia (C.) trachomatis upper genital tract infection
can cause distal tubal damage and occlusion, increasing the risk of tubal factor
subfertility and ectopic pregnancy. Variations, like single nucleotide
polymorphisms (SNPs), in immunologically important host genes are assumed to play a
role in the course and outcome of a C. trachomatis infection. We studied whether genetic
traits (carrying multiple SNPs in different genes) in the bacterial sensing system are
associated with an aberrant immune response and subsequently with tubal pathology
following a C. trachomatis infection. The genes studied all encode for pattern recognition
receptors (PRRs) involved in sensing bacterial components.
B
Methods: Of 227 subfertile women, serum was available for C. trachomatis IgG antibody
testing and genotyping (common versus rare allele) of the PRR genes TLR9, TLR4, CD14
and CARD15/NOD2. In all women, a laparoscopy was performed to assess the grade of
tubal pathology. Tubal pathology was defined as extensive peri-adnexal adhesions and/or
distal occlusion of at least one tube.
Results: Following a C. trachomatis infection (i.e. C. trachomatis IgG positive), subfertile
women carrying two or more SNPs in C. trachomatis PRR genes were at increased risk of
tubal pathology compared to women carrying less than two SNPs (73% vs. 33% risk). The
differences were not statistically significant (p: 0.15), but a trend was observed.
142
Conclusions: Carrying multiple SNPs in C. trachomatis PRR genes tends to result in an
aberrant immune response and a higher risk of tubal pathology following a C. trachomatis
infection. Larger studies are needed to confirm our preliminary findings.
CD14, TLR4, TLR9, CARD15/NOD2
Severity of C. trachomatis infections
Background
large variation exists in the individual response to a Chlamydia (C.) trachomatis
infection. Some women clear a C. trachomatis infection adequately without
developing tissue damage, whereas others get a persistent infection which may
ascend to the upper genital tract, increasing the risk of tubal damage and subfertility. The
susceptibility, course and outcome of infectious diseases are determined by environmental
factors, virulence factors of the pathogen and host factors.
A
Immunogenetic studies evaluate the role of genetic variations in immunologically important
host genes as determinants of the susceptibility, course and outcome of infectious diseases.
Among these variations are single nucleotide polymorphisms (SNPs), in which one
nucleotide has been substituted, inserted or deleted. This may lead to synthesis of a
potentially aberrant protein, or to up- or downregulation of the normal protein, and
subsequently to an aberrant immune response, increasing the risk of late sequelae of
infectious diseases (e.g. tubal pathology following a C. trachomatis infection).
In the present study, we have evaluated SNPs in genes encoding for pattern recognition
receptors (PRRs). PRRs are present on or in circulating cells of the innate immune system
(e.g. macrophages) and local cells (e.g. epithelial cells of the upper genital tract). PRRs are
involved in the bacterial sensing pathways of the innate immune system by recognizing the
so-called pathogen-associated molecular patterns (PAMPs), which are pathogen-specific
cell wall components or intracellular components. Since different PRRs recognize different
PAMPs, pathogen recognition and initiation of the immune response is a complex and
flexible system.
Carrying a SNP in a single PRR may not result in a large effect on disease severity, since
other PRRs may compensate for the partial loss of function in a specific pathogen
recognition route. Subsequently, SNPs in only one PRR may not play a significant role as
risk factors for the development of C. trachomatis-associated tubal pathology, as shown for
the PRR toll-like receptor (TLR) 41 and its co-receptor cluster of differentiation (CD) 142.
However, carrying multiple SNPs in one gene or in multiple genes (in so-called carrier
traits) may be associated with an increased risk of tubal pathology. Smirnova et al. (2003)3
have found that combinations of TLR4 variants are markedly more common in patients with
meningococcal infections, whereas single variants are not over-represented in those
patients. In studies on gastrointestinal malignancies, it has been concluded that carrying
multiple pro-inflammatory polymorphisms is associated with an increased risk of gastric
cancer4, 5. Furthermore, studies on the relationship between caspase recruitment domain
(CARD) 15/nucleotide oligomerisation domain (NOD) 2 genetic variants, of which SNP8,
SNP12 and SNP13 are most studied, and Crohn’s disease have shown that compound
heterozygous subjects (carriers of two different genetic variants, e.g. SNP12 genotype 1.2
and SNP13 genotype 1.2) have a higher risk of Crohn’s disease as compared to
homozygous subjects (carriers of the same genetic variant on both chromosomes, e.g.
SNP12 genotype 2.2)6, 7.
Analogous to these findings, we hypothesized that carrying multiple genetic variations in
multiple PRRs (in a so-called carrier trait) may increase the risk of C. trachomatisassociated tubal pathology in subfertile women. According to their biological function
143
Chapter 9
(recognition of C. trachomatis PAMPs: see Table 1), four PRRs were selected: TLR9,
TLR4, CD14 and CARD15/NOD2. Five relatively common SNPs, which are assumed to
influence the receptor function, in these four PRR genes were studied in this carrier trait
analysis (see Table 1).
Methods
144
Study population
The study was performed in women who visited the Academic Hospital Maastricht between
December 1990 and November 2000 because of subfertility. In all patients blood was drawn at their
initial visit for a Chlamydia IgG antibody test (CAT). All spare sera were cryopreserved. Only
patients who had undergone a laparoscopy and tubal testing as part of their fertility work-up were
included in the present study. Since the prevalence of SNPs may depend on ethnical background, only
Dutch Caucasian women were included. Patients who had undergone previous pelvic surgery (except
for an uneventful appendectomy or Caesarean section) were excluded. In the Netherlands, for
retrospective analysis of anonymized patient data and stored sera no ethical committee approval is
required. In the fertility clinic of the Academic Hospital Maastricht, all couples are informed at intake
about possible use of their anonymized data and stored sera for research purposes, and a “no objection
procedure” is followed. Only patients having not objected participated in the present study.
Two independent investigators, who were unaware of the CAT results, scored 259 successive
laparoscopy reports to assess the grade of tubal pathology. Tubal pathology was defined as extensive
peri-adnexal adhesions and/or distal occlusion of at least one tube8. In case of disagreement,
consensus was reached by consultation.
Of the 259 women who underwent a laparoscopy, 43 (17%) had tubal pathology (according to the
above-mentioned definition) and 184 (71%) had no tubal pathology (no peri-adnexal adhesions and
patent tubes), and these 227 women participated in the present study. Thirty-two women (12%) had
minor or non-C. trachomatis-related abnormalities (any peri-adnexal adhesions and/or proximal
occlusion of at least one tube) and were excluded.
C. trachomatis IgG antibody testing
IgG antibodies to C. trachomatis were detected using the species-specific Chlamydia pneumoniae
IgG micro-immunofluorescence (MIF) test (AniLabsystems, Finland), as described previously9. This
species-specific test is able to detect IgG antibodies to both C. pneumoniae and C. trachomatis (using
an antigen derived from a C. trachomatis LGV strain, serovar L2). We have previously studied the
test performances of five commercially available C. trachomatis IgG tests, including the
C. trachomatis IgG spot in the C. pneumoniae MIF (AniLabsystems)10. In our hands, the
C. trachomatis IgG titre obtained by the C. pneumoniae MIF (AniLabsystems) had the best predictive
value for tubal factor subfertility10. Therefore, we have used this test in the present study. The cut-off
titre used for a positive test was 32.
Immunogenetic analysis
For the immunogenetic analyses, genomic DNA was extracted from the cryopreserved serum samples
using either the MagNaPure LC isolator according to the manufacturers’ instructions (Roche
Molecular Biochemicals, Germany) or the High Pure PCR Template Preparation (HPPTP) Kit
according to the manufacturers’ instructions (Roche Molecular Biochemicals, Germany). Both
techniques provide enough DNA for reproducible genetic analyses. Genotyping was performed using
a polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP)-assay or
TaqMan-assay in standard 96 well plates (Greiner Bio-One) as described previously2, 11 - 13. The SNPs
studied are summarized in Table 1. Chromosomal locations and further information on the genes
studied are: TLR9 chromosomal location 3p21.3, TLR9 –1237 T>C rs5743836 and TLR9 +2848 G>A
rs352140; TLR4 chromosomal location 9q32-q33, TLR4 +896 A>G rs4986790; CD14 chromosomal
location 5q31.1, CD14 –260 C>T rs25691909; CARD15/NOD2 chromosomal location 16q21,
CARD15/NOD2 Leu1007fsinsC (SNP13) rs2066847. For ethnically-matched background genotyping,
genomic DNA was extracted from whole blood of 97 healthy Dutch Caucasian employees of the VU
University Medical Center. They gave written informed consent for use of their anonymized sera to
serve as control sera for genetic research purposes.
PRR
TLR9
TLR4
CD14
CARD15/NOD2
PAMP
CpG-rich motifs
LPS and HSP
LPS and HSP (co-receptor of TLR4)
Peptidoglycans
SNP
-1237 T>C and +2848 G>A
+896 A>G
-260 C>T
Leu1007fsinsC (SNP13)
Table 1: The pattern recognition receptors (PRRs), which recognize C. trachomatis pathogen-associated
molecular patterns (PAMPs), and the single nucleotide polymorphisms (SNPs) studied
TLR = toll-like receptor; CD = cluster of differentiation; CARD = caspase recruitment domain; NOD =
nucleotide oligomerisation domain; CpG = cytosine-phosphate-guanine; LPS = lipopolysaccharide; HSP = heat
shock protein; T = thymine; C = cytosine; G = guanine; A = adenine; Leu = leucine-rich repeat domain; fsins =
frameshift insertion.
Statistical methods
The genotype distribution was tested for Hardy-Weinberg equilibrium to assess Mendelian
inheritance. Fisher’s exact or χ2 tests were used to compare the single genotypes between
C. trachomatis IgG-positive and IgG-negative subfertile women with and without tubal pathology and
the healthy control group. Subsequently, the single genotypes were used to define carrier traits. The
carrier traits were tested in χ2 and trend analyses. P < 0.05 was considered statistically significant.
Results
Of all 227 subfertile women participating in the present study, 43 (19%) had tubal
pathology, whereas 184 (81%) did not have tubal pathology. C. trachomatis IgG antibodies
were present in 39 women, of whom 26 (67%) had tubal pathology and 13 (33%) did not
have tubal pathology. C. trachomatis IgG antibodies were absent in 188 women, of whom
17 (9%) had tubal pathology and 171 (91%) did not have tubal pathology.
For all genes studied, the genotype distribution was in Hardy-Weinberg equilibrium in the
subfertile women and the ethnically-matched control group.
The genotype distribution did not differ between subfertile women, the C. trachomatis IgGpositive subgroup of subfertile women and the healthy control group (Table 2), indicating
that the subfertile women participating in the study reflect an average Dutch-Caucasian
population regarding the genotype distribution.
Single gene analysis
The risk of tubal pathology was assessed in all subfertile women and in the C. trachomatis
IgG-positive subgroup in relation to the genotype of TLR9, TLR4, CD14 and
CARD15/NOD2 (Table 2; Figure 1). An increasing risk of tubal pathology was observed
across the genotypes in all genes except CD14. Carrying SNPs in these genes increased the
risk of tubal pathology (on average almost 20%). These differences did not reach statistical
significance. These single genotypes were used to define carrier traits.
145
Chapter 9
1.1
n
TLR9 –1237 T>C
TLR9 +2848 G>A
TLR4 +896 A>Ga
CD14 –260 C>Tb
CARD15/NOD2
Leu1007fsinsC
(SNP13)
All subfertile women
CT+ subfertile women
Control group
Control group
Control group
Control group
Control group
155 (68%)
26 (67%)
66 (68%)
45 (20%)
6 (15%)
15 (15%)
200 (88%)
33 (85%)
87 (90%)
60 (26%)
12 (31%)
26 (27%)
211 (93%)
37 (95%)
95 (98%)
1.2 and 2.2
Risk of TP
20%
62%
–
18%
50%
–
19%
64%
–
17%
67%
–
18%
65%
–
n
72 (32%)
13 (33%)
31 (32%)
182 (80%)
33 (85%)
82 (85%)
27 (12%)
6 (15%)
10 (10%)
167 (74%)
27 (69%)
71 (73%)
16 (7%)
2 (5%)
2 (2%)
Risk of TP
17%
77%
–
19%
70%
–
22%
83%
–
20%
67%
–
25%
100%
–
Table 2: The risk of tubal pathology (TP) in relation to the genotype of the single genes studied
All subfertile women: n = 227, of whom 19% has tubal pathology (TP).
CT + (C. trachomatis IgG-positive) subfertile women: n = 39, of whom 67% has TP.
Control group: n = 97 ethnically-matched healthy employees of the VU University Medical Center.
1.1 = normal genotype (homozygous for the common allele); 1.2 = heterozygous SNP carrier (one common allele
and one rare allele); 2.2 = homozygous SNP carrier (homozygous for the rare allele).
a
Adapted from Morré et al., 20031
b
Adapted from Ouburg et al., 20052
146
Figure 1; The risk of tubal pathology (TP) in C. trachomatis IgG-positive subfertile women in relation to the
genotype of the single pattern recognition receptor genes.
a
Adapted from Morré et al., 20031
b
Adapted from Ouburg et al., 20052
The SNPs in the single genes were combined in carrier traits. The risk of tubal pathology
was assessed in C. trachomatis IgG-positive and IgG-negative subfertile women in relation
to the number of SNPs. Carrying two or more SNPs did not influence the risk of tubal
pathology in C. trachomatis IgG-negative women as compared to C. trachomatis IgGnegative women carrying less than two SNPs (9% vs. 8% risk respectively; Figure 2).
However, carrying multiple SNPs doubled the risk of tubal pathology in C. trachomatis
IgG-positive women as compared to C. trachomatis IgG-positive women with less than two
SNPs (73% vs. 33% risk respectively; Figure 2). These differences did not reach statistical
significance (p: 0.15) but a clear trend was observed.
Figure 2: The risk of tubal pathology (TP) in C. trachomatis IgG-positive (CT+) and IgG-negative (CT-) subfertile
women in relation to carrying five single nucleotide polymorphisms (SNPs) in four pattern recognition receptor
genes.
Discussion
Over the last decade, immunogenetic studies have provided insight in the pathogenesis of
and susceptibility to infectious diseases. So far, the role of SNPs in immunologically
relevant genes has been established in numerous diseases, e.g. sexually transmitted
infections14 - 17 and inflammatory bowel diseases (Crohn’s disease and ulcerative
colitis)17, 18. In this study, we analyzed the role of single SNPs and multiple SNPs in
multiple genes (in a so-called carrier trait) as risk factors of C. trachomatis-related tubal
pathology and we confirmed our hypothesis that a carrier trait based on genes in bacterial
sensing pathways had a stronger association with the risk of tubal pathology than a single
gene analysis.
Recent studies have shown the value of genetic traits in complex diseases. Carrying
multiple SNPs in the same gene, or multiple SNPs in multiple genes, has been associated
with an increased risk of infectious diseases and malignancies3, 4, 6, 7. Analogous to these
findings, we hypothesized that the disregulation of the immune response by the presence of
multiple SNPs may lead to an even higher risk of tubal pathology following a
C. trachomatis infection as compared to carrying a single SNP.
In this study, we investigated the role of five SNPs in four genes which are assumed to play
a role in the recognition of C. trachomatis (see Table 1). An adequate recognition of
C. trachomatis by PRRs is the first step in the immune response. Recent studies have
147
Chapter 9
shown that TLR1 – 9 are expressed in the human female genital tract. TLR4 and its coreceptor CD14 are predominantly expressed in the fallopian tubes, where they may play an
important role in the innate host defence mechanism against ascending C. trachomatis
infections19 - 21. Regarding CARD15/NOD2, it is not clear whether it plays a role in the
C. trachomatis recognition in the genital tract, although NOD proteins have been shown to
be involved in the intracellular sensing of C. pneumoniae in endothelial cells22.
Our data show a doubling of the risk of tubal pathology in C. trachomatis IgG-positive
women carrying two or more SNPs as compared to C. trachomatis IgG-positive women
carrying less than two SNPs (73% vs. 33% risk). The differences did not reach statistical
significance (p: 0.15) due to the small sample size (227 subfertile women in total, including
39 C. trachomatis IgG-positive subfertile women, of whom 67% has tubal pathology). If
the association found in this pilot study would be confirmed in a larger cohort, a 50%
increase in sample size (to 341 subfertile women) would result in a significant difference
(p: 0.047) between C. trachomatis IgG-positive subfertile women carrying two or more
SNPs versus C. trachomatis IgG-positive subfertile women carrying less than two SNPs
regarding the risk of tubal pathology. A 100% increase in sample size (to 454 subfertile
women) would result in a strong association (p: 0.016; OR: 5.3) between carrying two or
more SNPs and an increased risk of tubal pathology. Increasing the sample size twofold
would not be possible however in our setting in a reasonable time frame. Although the
present study was performed in a large fertility clinic and patients were included during a
ten-year period, the number of affected women remained small, due to the low prevalence
of IgG-positivity in combination with both carrying multiple SNPs and tubal pathology.
148
From our results it can be concluded that an adequate recognition of the pathogen at the site
of infection seems to be a relevant step in the immune response, and that carrying multiple
SNPs in multiple C. trachomatis PRR genes tends to increase the risk of an aberrant
immune response and tubal pathology. To draw significant conclusions, our hypothesis
should be retested in further studies using a larger cohort.
As expected, a difference in risk of tubal pathology between C. trachomatis IgG-positive
women and C. trachomatis IgG-negative women was observed. In previous studies, the
presence of C. trachomatis IgG antibodies, an indicator of a previous C. trachomatis
infection, has been associated with tubal pathology23. Our data show that carrying multiple
SNPs in bacterial sensing pathways and a previous C. trachomatis infection synergistically
enhance the risk of tubal pathology, while carrying these SNPs does not influence the risk
of tubal pathology in the absence of a previous C. trachomatis infection (Figure 2).
Conclusion
We hypothesized that carrier traits (i.e. carrying multiple SNPs in multiple genes) that
likely result in an aberrant immune response are associated with tubal pathology following
a C. trachomatis infection. In 227 subfertile women, we studied five variations in four
genes encoding for pattern recognition receptors, which recognize several pathogenassociated molecular patterns of C. trachomatis. The presence of two or more SNPs tends
to correlate with an increased risk of tubal pathology following a C. trachomatis infection
as compared to a lower number of SNPs. Further studies in a larger cohort are needed to
confirm our preliminary findings. An adequate recognition of C. trachomatis by receptors
in the genital tract seems to be a relevant step in the immune response, and may play a role
in protecting the host against the development of late sequelae following a C. trachomatis
infection.
Acknowledgements
Sander Ouburg is an AstraZeneca Nederland BV fellow.
Servaas A. Morré is supported by the Foundation of Immunogenetics, The Netherlands.
The authors acknowledge Jolein Pleijster, of the Laboratory of Immunogenetics of the VU University Medical
Center, for excellent technical assistance, and Gert Grauls, of the Department of Medical Microbiology of the
Academic Hospital Maastricht, for laboratory assistance.
The ICTI consortium (Integrated approach to the study of Chlamydia trachomatis Infections) provides a broad
specialized network for the multidisciplinary studies described24. The EpiGenChlamydia consortium
(www.EpiGenChlamydia.eu) is a European Framework Programme 6 (FP6) financially supported Co-ordination
Action (CA) in functional genomics research, entitled: Contribution of molecular epidemiology and host–pathogen
genomics to understand Chlamydia trachomatis disease.
Part of the data on the single gene analysis has previously been published by our research group1, 2. For the
purpose of the present study, these data have been completed, adapted to the current format and (with accurate
references) used in the Results section as a basis for the carrier trait analysis. Part of the data has been presented as
poster presentations at the 16th Biennial Meeting of the International Society for Sexually Transmitted Diseases
Research (ISSTDR; Amsterdam, The Netherlands, 10-13 July 2005), the 11th International Symposium on Human
Chlamydial Infections (ISHCI; Niagara-on-the-Lake, Canada, 18-23 June 2006) and the 22nd Annual Meeting of
the European Society of Human Reproduction and Embryology (ESHRE; Prague, Czech Republic, 18-21 June
2006).
149
Chapter 9
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151
Chapter 9
152
Amoebas at the start
Were not complex;
They tore themselves apart
And started Sex.
Arthur Guiterman
Discussion
Discussion
C
hronic inflammation and infection play an important role in determining the
prognosis of diseases affecting the mucosal barrier of the body.
In the first part of this thesis, we concentrated on understanding the role of Immunogenetics
in chronic inflammatory diseases affecting the oesophagus (Barrett oesophagus), the small
intestine (Crohn’s disease) and the large intestine (Crohn’s disease, ulcerative colitis and
pouchitis).
In the second part, we studied the role of Immunogenetics in determining the susceptibility
to and severity of Chlamydia trachomatis infections.
The gastrointestinal and urogenital tracts share the immunology of the “mucosas” and
genetic, environmental, bacterial and behavioural factors play a role in the development of
disease, and in the short- and long-term prognosis. The diseases are therefore multifactorial
and, as shown in this thesis, polygenic. Technical limitations prompted the selection of a
few candidate genes, but recent technical advances in genetics will enable the simultaneous
assessment of multiple genes in the same sample.
154
Part I
Part I
he first part of the thesis focuses on the role of selected genes involved in the
regulation of innate and acquired immunity in relation to gastrointestinal
inflammation and infection. Polymorphisms in these genes may affect the
susceptibility to and / or severity of the gastrointestinal diseases described. The genes
studied in Part I of this thesis and their respective polymorphisms are presented in table 1.
T
Polymorphism
IL-1B
IL-1RN
TLR4
-511 C>T
+2018 T>C
+896 A>G
CD14
-260 C>T
—
rs2569190
TLR9
-1237 T>C
+2848 G>A
rs5743836
rs352140
CARD15/NOD2
+2209 A>T
(SNP8)
+2722 G>T
(SNP12)
+3020 insC
(SNP13)
+22148 G>A
—
Pro545Pro
(P545P)
Arg702Trp
(R702W)
Gly908Arg
(G908R)
Leu1007Pro
(L1007P)
Val147Ile (V147I)
—
—
—
IRAK-M
(IRAK3)
Multigene
analysis
Amino Acid
substitution
—
Ala#bAla (A#bA)
Asp299Gly
(D299G)
dbSNPa
Gene
rs16944
rs419598
rs4986790
rs2066844
Disease
studied
Barrett
Barrett
Crohn
UC
Barrett
Crohn
UC
UC
(Pouchitis)
Crohn
UC
(Pouchitis)
Crohn
Ch.
1
1
1
2
2
1
2,4
2
5
3
5
4
rs2066845
rs2066847
rs1152888
UC
(Pouchitis)
Barrett
Crohn
UC
(Pouchitis)
5
1
4
5
Table 1: Genes and polymorphisms studied in some chronic inflammatory diseases of the gastrointestinal tract.
The rs numbers of the dbSNP database and the chapters (Ch.) in the thesis where the results are discussed are
shown.
a
Available from1: http://www.ncbi.nlm.nih.gov/SNP/
b
Depending on the isoform: amino acid 57 (isoform 1), 60 (isoform 2), 39 (isoform 3), or 23 (isoform 4)
IL-1β and IL-1ra
The IL-1B-511 C>T and IL-1RN+2018 T>C polymorphisms have previously been
associated with an anti-inflammatory profile. Our results demonstrate that carriage of the
IL-1RN+2018 T>C polymorphism confers risk for the development of Barrett oesophagus
(Chapter 1). Our results are similar to those of Gough et al., who demonstrated that
155
Discussion
carriage of IL-1RN TT is associated with risk for Barrett oesophagus2. In contrast, IL-1β
has been associated with an increased expression of COX2, which is known to up regulate
Th2 cytokines when associated with oesophageal carcinogenesis3, 4. A predominantly
humoral immune response is characteristic for Barrett oesophagus5 and an increased
inflammatory response or chronic inflammation may result in tissue damage and
carcinogenesis6. Furthermore, it has been shown in a Japanese population that carriage of
IL-1B genotypes are protective against gastro-oesophageal reflux disease (GORD)7.
Barrett oesophagus is a gastric acid induced inflammation of the oesophagus. The risk
observed may be due, not only to direct influence of IL-1ra on the gastric acid induced
inflammation of the oesophagus, but also indirectly on the possible presence of H. pylori8 10
, although conflicting reports have been published on the influence of H. pylori in the
development of reflux disease and Barrett oesophagus11 - 13. The influence of IL-1B and
IL-1RN SNPs on Helicobacter infection and pathogenesis has been extensively studied and
described14. Microorganisms infecting the oesophagus may play a role in the development
of Barrett oesophagus, however studies are still preliminary15 and no data is currently
available on the influence of IL-1B and IL-1RN SNPs in relation to oesophageal infection. It
should be noted that conflicting results have been published in the literature on the effect of
the IL-1RN SNPs on the expression levels of IL-1ra16 - 21. These inconsistencies can in part
be explained by different methodologies, differences in IL-1β / IL-1ra balance,
environmental factors, and differences in expression between different ethnic populations.
156
Increased levels of IL-1ra may reduce the immune response and thus protect against tissue
damage. This may be protective in Barrett oesophagus, however high levels of IL-1ra
inhibit IL-1β, which is known to be a potent inhibitor of gastric acid22, 23. High levels of
gastric acid in reflux disease are a risk factor for Barrett oesophagus. From the differences
observed in the literature and in our results it can be concluded that IL-1B and IL-1RN
SNPs influence the susceptibility to Barrett oesophagus. The differences also show that
IL-1β and IL-1ra are not the only factors in this disease model and that more factors have to
be taken into account in order to explain the observed clinical phenotype.
TLR4
The results of TLR4 genotyping in Dutch (Chapter 2) and Galician (Chapter 4) CD
patients demonstrate that carriage of the mutant allele of TLR4 +896 confers risk in Dutch
Caucasian patients, while conferring protection in Spanish Galician patients.
The role of TLR4 in a diversity of human infections and diseases has been extensively
researched, and its role in gastrointestinal diseases is no exception, however the literature is
divided on the role of TLR4 in gastrointestinal diseases. Carriage of the TLR4 mutation is
associated with gastric H. pylori infection according to some publications24, 25 and not
associated according to others26, 27. Arnott and colleagues did not find an association
between TLR4 SNPs and CD in Scottish and Irish populations28, while in other populations
carriage of TLR4 SNPs is a risk factor for development of CD29 - 31. Studies in murine
models of colitis have demonstrated that TLR4 exerts a protective effect in the intestine, by
regulating the immune response to epithelial injury and limiting bacterial translocation32, 33.
Absence of TLR4 in TLR4 KO mice exacerbates mucosal damage in experimental colitis34.
The underlying mechanism could be (in part) TLR4 mediated COX2 expression. In an
Part I
acute intestinal injury (i.e. intestinal inflammation) TLR4 upregulates COX2 and COX2
upregulates PGE2, resulting in increased proliferation and decreased apoptosis of the
epithelium. Chronic COX2 upregulation through TLR4 may, in contrast, result in an
increased risk for COX2 associated colon cancer development35. A non functional TLR4
may thus lead to decreased tissue proliferation and repair, and increased apoptosis.
Translating the murine data to the human model indicates that carriage of the TLR4 SNP,
which has been associated with LPS hyporesponsiveness36, 37, could result in an imbalance
in intestinal homeostasis, increased risk of CD and potentially the promotion of disease
pathogenesis32, 35. Although the murine model does offer an explanation for the observed
risk for development of CD in carriers of the TLR4 SNP, as observed in the Dutch and
Greek populations30, 31, 38, it does not explain the lack of association between TLR4 and CD
in the Scottish, Irish28 and the Galician populations (Chapter 4). TLR expression studies in
the intestine show that low levels of TLR4 are expressed at the apical membrane of
intestinal epithelial cells39, 40, and that TLR4 expression is upregulated in IBD patients39. In
contrast, Naik et al. found an absence of TLR4 expression in children with IBD41.
These data provide evidence for genetic heterogeneity between the populations and are
indicative that more factors are involved in the aetiopathogenesis of CD. Environmental
factors, differences in TLR expression in different populations or perhaps age groups,
related biological mechanisms (e.g. COX2 expression), selection bias or differences in
selection criteria should be taken into consideration when comparing the different results
from these publications, as well as possible differences in the composition of intestinal
flora, which may influence the results, as was shown for H. pylori infection42.
The role of TLR4 may be mediated not through direct recognition of the microorganisms,
but also through recognition of proteins related to cell damage and cell death such as
HSP60 and HSP7043, 44. Regulation of TLR expression can influence pathogenesis and the
regulation of tolerance in the intestine is expertly reviewed by Cario and Podolsky45. The
mechanisms they present for intestinal “TOLLerance” vs. “inTOLLerance” can easily be
translated to other diseases where TLRs might be involved. Furthermore, recent
publications indicate that not only that regulation may play an important role, but also
TLR4 – CD14 – MD2 stoichiometry.
CD14
We observed a protective effect for the development of fistulae in carriers of the CD14 -260
SNP in the Galician CD population. Furthermore, an increased carriage of this SNP was
observed in male patients who had not had an appendectomy prior to or at the time of CD
diagnosis (Chapter 4). No apparent differences were observed in the carriage of CD14
genotypes between pouchitis patients and healthy controls (Chapter 5).
The CD14 –260 SNP affects the binding of transcription factors46 and has been associated
with increased levels of sCD14 and inversely associated with serum IgE levels47. This SNP
has been associated with myocardial infarction48, Crohn’s disease49 and an increased
susceptibility to develop chronic spondyloarthropathy in women50.
157
Discussion
For almost every reported association between CD14 and a disease, a lack of association
has been reported. This is especially true for IBD and cardiovascular diseases, where both
positive30, 48, 51 - 56 and negative associations28, 57 - 63 have been reported for the CD14 -260
SNP.
Apart from differences in methodologies, ethnic differences, and differences in disease
aetiopathogenesis between the reported studies, it should be noted that the biological nature
of the CD14 signalling pathway may contribute to the heterogeneity in the observed results.
CD14 has a soluble (sCD14) and a membrane bound (mCD14) form64, 65.
Griga et al. have shown that the CD14 -260 TT genotype reduces mCD14 expression on
monocytes and increases serum sCD14 in patients with inactive Crohn’s disease66, while
this genotype did not influence levels of mCD14 and sCD14 in healthy controls. This
confirms previous findings that the TT genotype increases serum sCD1447 and suggests that
CD14 may play a role in the aetiopathogenesis of CD.
The sCD14 and mCD14 forms are incapable of signal transduction and require another
receptor for signalling. CD14 complexes with TLR4 or with TLR2 to confer responsiveness
lipopolysaccharides (LPS) and lipopeptides, respectively67 - 69. Disruption of the complex
formation or a non-functional TLR2/4 may thus influence CD14 signalling. The observed
associations may thus be due to other factors in the signalling pathway.
158
Increased expression of TLR4 is observed in the inflamed intestine39 (reviewed earlier in
this discussion) and it has been shown that TLR2 may influence intestinal inflammation,
potentially in the presence of CARD15/NOD2 mutations70 - 72.
Differences in LPS structure influences recognition and signalling efficiency, as has already
been demonstrated for H. pylori LPS recognition by TLR427, 42, 73. The LipA subfraction of
LPS is the epitope recognised by TLR474. Penta-acylated LipA inhibits a pro-inflammatory
immune response, while hexa-acylated LipA results in TLR4 activation and subsequent
immune activation73, 75. A similar effect of hypo-acylated LipA on LPS recognition by
CD14 may have far reaching implications for immune recognition. It has already been
reported that structural differences in sCD14 are required for optimal recognition of
peptidoglycan (PGN) and LPS by sCD1476.
The results seem to suggest that in the analysis of CD14 SNPs other factors should be taken
into account such as LPS subtypes, and bacterial load77, but also expression of TLR2, TLR4
and CARD15/NOD2 and their respective polymorphisms. The analysis of multiple genes in
relation to disease aetiopathogenesis is reviewed later in this discussion.
TLR9
The TLR9 -1237 T>C and +2848 G>A SNPs influence susceptibility and severity of
Crohn’s Disease in both the Dutch Caucasian and Spanish Galician populations
(Chapter 3). In the Dutch Caucasian population associations with susceptibility for CD and
protection against penetrating disease were observed, while in Spanish Galicians
associations with higher age, colonic localisation and stricturing disease were observed.
Part I
The TLR9 -1237*C allele is significantly associated with chronic relapsing pouchitis in
Italian Caucasians, who underwent IPAA surgery (Chapter 5). Combining the TLR9 SNPs
into a haplotype, as described by Lazarus et al., resulted in a significant association
between haplotype III and chronic relapsing pouchitis.
Murine studies revealed that TLR9 is capable of inhibiting hepatitis B replication and of
reducing experimental colitis, through induction of type I interferons78 - 81. Furthermore,
murine studies point to a potential role of TLR9 in autoimmunity82. TLR9 is involved in H.
pylori induced overexpression of COX2 and results in the invasion and angiogenesis
observed in gastric carcinogenesis83, 84.
The TLR9 SNPs were first described by Lazarus et al., who found an increased risk for
asthma in European Americans harbouring the -1237*C allele85. Török et al. associated
increased carriage of the -1237*C allele with Crohn’s disease, while the +2848 AG
genotype was slightly reduced in Crohn’s partients86.
The TLR9 SNPs can be subdivided in 4 common haplotypes, shown in table 2.
Haplotype
I
II
III
IV
-1486 T>C
T
C
T
T
C
C
T
TLR9
-1237 T>C +1174 G>A
T
A
T
G
C
G
C
G
T
G
C
G
T
G
+2848 G>A
G
A
A
G
G
G
A
Comments
Rare
Rare
Rare
Rare
Table 2: TLR9 haplotypes as described by Lazarus et al.85. The SNPs at positions -1237 and +2848 can be used
to differentiate between the 4 most common haplotypes.
Carriage of the C allele at position -1237 appears to confer risk for CD in Germans, for
asthma in European Americans85, for older age of onset and a stricturing phenotype in
Galician CD patients (Chapter 3), and for relapsing pouchitis (Chapter 5). Carriage of the
A allele at position +2848 confers protection against development of CD and penetrating
disease in Dutch Caucasians, but confers risk for colonic localisation of CD in Galicians
(Chapter 3).
The possibility exists that the TLR9 SNPs do not influence the function or expression of the
protein, however the TLR9 SNPs may be linked to nearby functional polymorphisms. The
TLR9 gene is located on chromosome 3p21.2 in the vicinity of the marker D3S1076
showing association with IBD in a classical TDT test87, which may explain the associations
with gastrointestinal diseases. CARD15/NOD2 mutations, well known for increased
susceptibility for IBD, have recently been shown to abolish NOD2 – TLR9 synergy,
resulting in a reduced immune response after TLR9 stimulation88. Netea et al. failed to
replicate these findings, but suggest an interaction between CARD15/NOD2 and TLR2,
TLR3, and TLR489. Due to overlap in TLR signalling pathways, it may be hypothesised
that CARD15/NOD2 SNPs may affect multiple TLRs. Carriage of these CARD15/NOD2
SNPs may have a profound influence on TLR signalling and thus explain the heterogeneity
159
Discussion
in TLR9 associations.
TLR9 is an intracellular receptor, detecting bacterial DNA in endosomes90 - 92 and may
recognise DNA from necrotic cells93. Therefore proteins that mediate the uptake of
bacterial DNA may critically control the efficiency of TLR9 signalling, although TLR9
may itself be involved in the uptake of bacterial DNA92, 94, 95.
TLR9 is capable of recognising CpG DNA of different bacterial species, however the TLR9
activity differs depending on the DNA of these different species. High concentrations of
bacterial DNA are required and higher genomic frequencies of [CG] dinucleotides result in
increased activation of TLR996.
Furthermore, Lundberg et al. have shown that the ratio between immunostimulatory and
immunoinhibitory sequences (figure 1) influences the effect of CpG DNA stimulation.
Their research has produced a formula, where a positive outcome represents a stimulatory
effect and a negative outcome represents an inhibitory effect, with the greater the difference
between the sequence types determining the strength of the effect97.
160
Figure 1: Immunostimulatory and inhibitory sequences. Figure based on Lundberg et al., 200397
Analysis of the microbial species at the site of infection (i.e. specific species, number of
microbes present, number of CpG motifs, and the ratio of stimulatory and inhibitory motifs)
together with regulation of DNA uptake and presence of CARD15/NOD2 mutations may
provide more information on the role of TLR9 in gastrointestinal disease. Expression
studies as well as stimulation studies in cells with specific TLR9 genotypes will elucidate
whether or not the TLR9 SNPs affect TLR9 expression and / or function.
CARD15/NOD2
CARD15/NOD2 SNPs have previously been associated with susceptibility to Crohn’s
disease98, 99. Rahman et al. referred to CARD15/NOD2 as a pleiotropic autoimmune gene,
since it confers susceptibility to Crohn’s disease, Blau syndrome, and psoriatic arthritis100.
Of all mutations in the CARD15/NOD2 gene, 93% are reported to be located in the distal
third of the gene, which contains the bacterial recognition domain. This confirms the gene
dosage effect that is observed in Crohn’s disease, meaning that patients carrying two or
more mutations are characterised by a younger age at onset, a more stricturing phenotype,
and a less frequent colonic localisation, compared to patients without CARD15/NOD2
Part I
mutations101. Furthermore, carriage of two of these common SNPs means that there will be
no normally functioning CARD15/NOD2 protein, since the 3 common SNPs are carried on
separate ancestral haplotypes99, 102. Increased carriage of CARD15/NOD2 SNPs and
increased incidence of Crohn’s disease is observed in Jewish population, especially
Ashkenazi Jews103 - 105. The three main CARD15/NOD2 SNPs SNP8, SNP12, and SNP13
are virtually absent in Asian populations106, 107, while a low incidence of these SNPs has
been reported in northern European populations108.
The CARD15/NOD2 SNP12 and SNP13 SNPs predispose to development of CD in Spanish
Galicians, although the association is not as strong as in Castilian109 and Dutch patients.
SNP12 is a risk factor for development of fistulae110, especially in female patients. SNP13
appears to be a risk factor for ileal resection (Chapter 4).
Both SNP8 and SNP12 result in non-synonymous amino acid changes, although a
functional effect of these changes has not yet been described. The C insertion in SNP13
results in a frameshift and a premature stop codon98. The last part of the protein is most
likely involved in the regulation of cellular localisation, since the truncated protein fails to
locate to the membrane111. Activation of NF-κB by CARD15/NOD2 is impaired through
failure of membrane localisation and impaired peptidoglycan (PGN) recognition111, 112.
Leung et al. have studied the biological activity of truncated isoforms of
CARD15/NOD2113. They suggest that the terminal regions of the RNA transcript are
alternatively spliced, resulting in eight putative CARD15/NOD2 variants. Expression of the
full-length and spliced protein variants changes when cells are stimulated with bacterial
products. Some isoforms are unresponsive to peptidoglycan, although they do not
antagonise the activity of the wildtype protein. Leung et al. suggest that alternative splicing
of the CARD15/NOD2 transcripts is a potential mechanism for regulation of bacteriasensing through CARD15/NOD2113. Rosenstiel et al. have shown that a short isoform of
CARD15/NOD2, NOD2-S, inhibits CARD15/NOD2 – RIP2 induced activation of NF-κB,
thus confirming the role of CARD15/NOD2 isoforms in the regulation of CARD15/NOD2
induced inflammatory responses114.
CARD15/NOD2 is expressed on intestinal epithelial cells and macrophages111, 115. It is
upregulated by proinflammatory cytokines115 and is involved in the regulation of
bactericidal peptides, that may have a gatekeeper function against the intestinal flora116.
A defective CARD15/NOD2 protein may thus result in a defective regulation of
antibacterial peptides and a reduced epithelial immune defence117. The gut barrier function
is known to be impaired in IBD patients118. Recent evidence suggests that the SNP13
polymorphism may be an important factor in the impairment of the intestinal barrier
function, although it should be noted that bacterial products and local inflammatory
responses may also facilitate barrier breakdown119.
Familial, twin, and epidemiological studies have clearly shown that CD clearly has an
genetic component120 - 125, however CARD15/NOD2 SNPs alone do not explain the
concordance of CD as shown in a Swedish monozygotic twin study126. Only 30 – 40% of
CD patients carry one or more of the three main CARD15/NOD2 SNPs101, 127 - 131 and
homozygous carriers of CARD15/NOD2 SNPs have a less than 10% likelihood of
developing CD. Although some residual evidence of linkage is observed between CD and
the IBD1 locus after corrections for the predisposing SNPs have been made132, 133, these
161
Discussion
results indicate that more factors are involved in the development of CD. Analysis of
multiple genes in relations to susceptibility and severity of CD will be discussed later in this
chapter, however recent literature has provided evidence for the involvement of
CARD15/NOD2 in the regulation of gastrointestinal immune responses. CARD15/NOD2
synergises with TLRs. Synergy with TLR2 – 4, and TLR9 has been reported and carriage of
CARD15/NOD2 SNPs attenuates TLR mediated immune responses88, 89.
The number of biological processes in which CARD15/NOD2 is involved, clearly shows
the importance of this protein in microorganism detection, as well as in regulation of
immune responses and intestinal homeostasis. However, the same diversity of biological
processes makes identification of the biological mechanisms through which
CARD15/NOD2 affects disease susceptibility and severity rather difficult, especially since
CARD15/NOD2 is not the only causative factor.
IRAK-M
162
IRAK-M has recently been identified as a negative regulator of TLR and IL-1R signalling
and it has been suggested that IRAK-M has a regulatory function in innate immune
homeostasis, for instance in intestinal inflammation134. The IRAK-M (or IRAK3) gene is
located at chromosome 12q14.2 within the IBD2 region linked to and associated with
ulcerative colitis and asthma135 - 137. Therefore it may be hypothesized that genetic variation
in the IRAK-M gene may be involved in the development of chronic intestinal
inflammation. We did not observe any differences in the genotype frequencies of the
IRAK-M +22148 G>A SNP between cases and controls. Currently only one other
publication has studied this SNP in relation to disease. Nakashima et al. studied 19 SNPs in
the IRAK-M gene in relation to asthma in a Japanese population138. They did not observe
significant differences in the frequencies of this SNP between cases and controls. Although
this SNP results in an amino acids substitution at position 147 (V>I), the SNP is located
outside the two major functional domains of IRAK-M, the death domain and the kinase
domain. They did not observe strong linkage disequilibrium between this SNP and the other
SNPs in their study.
These results suggest that the IRAK-M +22148 G>A SNP has no effect in pouchitis and in
asthma. However, little data is currently available on the effect of this SNP and more
research is required to confirm these results.
Multigene approach
Carriage of a polymorphism in a gene may affect disease pathogenesis. As discussed
before, the carriage of CARD15/NOD2 SNP13 increases the risk of developing CD.
Carriage of multiple polymorphisms across multiple genes may have a more profound
effect, especially when biological effect of the polymorphisms is additive. For instance, the
TLR4+896 SNP has been associated with atherosclerotic disease139, while the CD14-260
SNP is associated with myocardial infarction48. Combined carriage of these two SNPs has
been associated with extensive and clinically relevant atherosclerotic disease139, indicating
that the combined carriage of these two SNPs has a clinically more severe impact.
Part I
In the gastrointestinal tract, we observed that combined carriage of the IL-1B -511 and
IL-1RN SNPs, combined carriage of the CD14 -260 SNP and TLR4 wildtype, and combined
carriage of specific genotype in these four genes increases the risk of Barrett oesophagus
(Chapter 1). Combined carriage of CD14 -260 with one or more CARD15/NOD2 SNPs
increases the risk of developing CD, and of a disease phenotype requiring ileal resection in
CD patients.
Carriage of the CD14 SNP combined with the CARD15/NOD2 SNP12 increase the risk of
fistulae (Chapter 4). Combined carriage of TLR9 and CD14 SNPs is a risk factor for
chronic relapsing pouchitis, as shown in Chapter 5.
El-Omar et al. and Machado et al. have previously demonstrated that an increasing number
of polymorphisms in IL-1B, IL-1RN, IL-10, and TNF-A progressively increase the risk of
atrophic gastritis and gastric cancer140 - 142.
Mendoza and colleagues assessed the combination of CARD15/NOD2 SNPs and SNPs in
the anti-inflammatory gene IL-10 in Spanish CD patients. The combination of any
CARD15/NOD2 SNP with the IL-10 -1082*G allele predisposes to ileocolonic disease,
while CARD15/NOD2 SNPs combined with the IL-10G14 microsatellite is associated with
an history of appendectomy and smoking habits at the time of CD diagnosis143, 144.
An increased incidence of the CD14 -260*T allele combined with one or more
CARD15/NOD2 SNPs was observed in German CD patients49. This was confirmed in a
Greek population, where carriage of either the TLR4+896 SNP or the CD14-260 SNP
combined with one or more CARD15/NOD2 SNPs significantly increased risk for the
development of CD30. In a recent publication by Brand and colleagues an interesting
additional insight is provided, namely that CD patients carrying the TLR4 SNP combined
with the CARD15/NOD2 wildtype had an increased risk of stricturing disease, while
penetrating disease was more prevalent in patients carrying the TLR4 wildtype with one or
more CARD15/NOD2 SNPs145.
163
Discussion
Part II
T
he second part of this thesis describes candidate genes involved in the innate
and acquired immunity to urogenital tract infection.
Polymorphisms in these genes may affect the susceptibility to and / or severity of diseases
of the urogenital tract and in particular Chlamydia trachomatis infection. The genes studied
in part II of this thesis and their respective polymorphisms are presented in table 3.
Gene
Polymorphism
IL-1B
IL-1RN
-511 C>T
+2018 T>C
TLR4
+896 A>G
CD14
-260 C>T
Amino Acid
substitution
—
Ala#bAla
(A#bA)
Asp299Gly
(D299G)
—
CCR5
δ32 (32bp
deletion)
+3020 insC
(SNP13)
—
Premature
stop codon
Leu1007Pro
(L1007P)
—
CARD15/NOD2
164
Multigene
analysis
dbSNPa
rs16944
rs419598
Chlamydia
trachomatis
Susceptibility
Susceptibility
rs4986790
Severity
9
rs2569190
7
9
8
rs2066847
Susceptibility
/ Severity
Severity
Susceptibility
/ Severity
Severity
—
Severity
9
rs333
Chapter
6
6
9
Table 3: Genes and polymorphisms studied in urogenital Chlamydia infection. The rs numbers of the dbSNP
database and the chapters in the thesis where the results are discussed are shown.
a
Available from1: http://www.ncbi.nlm.nih.gov/SNP/
b
Depending on the isoform: amino acid 57 (isoform 1), 60 (isoform 2), 39 (isoform 3), or 23 (isoform 4)
IL-1β and IL-1ra
The IL-1B-511 C>T and IL-1RN+2018 T>C polymorphisms have previously been
associated with an anti-inflammatory profile. In Chlamydial infection of the urogenital tract
(Chapter 6) we observed that carriage of the IL-1RN+2018 SNP is reduced in women with
a C. trachomatis infection. In a previous study by our group, it was shown that neither
IL-1B nor IL-1RN SNPs influence the risk of development of tubal factor infertility, a late
complication of C. trachomatis infection146. Genc and colleagues have shown that the
IL-1RN SNP is associated with protection against bacteria related pre-term birth147, while
Witkin et al. showed that carriage of the IL-1RN SNP is associated with increased intraamniotic levels of IL-1β and hypothesize that the IL-RN SNP may associate with pre-term
birth only in an IL-1β inducing event148. Care should be taken when drawing conclusions
on the influence of IL-1 on disease in pregnant women, since different effects have been
described for different pathogens and ethnicities149 - 151, and the IL-1 family is involved in
the maternal–foetal interaction152.
Part II
Infection with C. trachomatis is likely directly influenced by the IL-1RN SNP.
Furthermore, carriage of this SNP has been associated with protection against Ureaplasma
urealyticum infection by van der Schee et al.153 and Doh et al.149, while Barton et al.
associated this SNP with risk for U. urealyticum infection and elevated vaginal IL-1ra
levels149, 150.
LPS, present in the cell wall of most gram negative bacteria, induces increased IL-1β
expression in uterine macrophages, and the expression is enhanced by estradiol. IL-1β
induces antimicrobial beta-defensin 2 secretion by uterine epithelial cells154. However,
estradiol also downregulates IL-1R1, thus effectively limiting the proinflammatory immune
response155. This downregulation may be essential in dampening the immune response
during ovulation and pregnancy155.
As mentioned before, conflicting results have been published in the literature on the effect
of the IL-1RN SNPs on the expression levels of IL-1ra16 - 21. Different pathogens (e.g.
U. urealyticum, M. hominis149), differences between ethnicities, environmental factors,
hormonal influences and different methodologies, may all contribute to the observed
diversity of the published results.
High levels of IL-1ra during pregnancy reduce immune responsiveness and thus preventing
an immune response that would result in harm to the foetus. Limiting the immune response
may explain the protection against bacteria related pre-term birth147. The biological
mechanisms explaining how an anti-inflammatory response may protect against Chlamydia
infection remain unclear. One might hypothesize that a reduced immune response results in
a slightly longer duration of infection but also in a better adaptive immune response. Upon
reinfection the immune system is better primed against C. trachomatis, resulting in
protection and faster resolution of infection.
From our results and those published in the literature, it can be concluded that IL-1B and
IL-1RN SNPs influence the susceptibility to Chlamydia infection. These results also show
that IL-1β and IL-1ra are not the only factors in the evolution of Chlamydia infection and
that more factors have to be taken into account in order to explain the observed clinical
phenotype.
TLR4
In the urogenital tract, carriage of the TLR4 SNP appears to increase the risk of developing
tubal pathology in subfertile women (Chapter 9; not significant). TLR4 is differentially
expressed in the urogenital tract, with low expression in the lower tract and high expression
in the upper urogenital tract156 - 158. This differential expression allows an immunological
tolerance for commensal microorganisms in the lower urogenital tract159, while maintaining
an immunological barrier in the upper urogenital tract156. Decreased responsiveness to LPS
in the lower genital tract may result in an increased risk of ascending and chronic
Chlamydia infection and thus in increased tissue damage, resulting tubal pathology and
reduced fertility. It should be noted that the literature also reports absence of TLR4
expression in the oviduct160, 161. O’Connel et al. report that recognition of C. trachomatis is
mainly mediated by intracellular expression of TLR2 and that TLR4 has no or minimal
effect on the immune response against C. trachomatis, measured by IL-8 production.
165
Discussion
Direct recognition of microorganisms by TLR4 may not be the mechanism through which
TLR4 exerts the observed effect, but it may also exacerbate immune reactions in response
to recognition of proteins related to cell damage and cell death such as human HSP60 and
HSP7043, 44, and Chlamydial HSP60162. Rallabhandi and colleagues present an interesting
model on how polymorphism induced structural changes in TLR4 may alter responsiveness
to Chlamydial HSP60163, showing that TLR4 – CD14 – MD2 stoichiometry affects ligand –
receptor interaction. Recent findings in Candida bloodstream infections show that carriers
of the TLR4 +896, together with the co-segregating TLR4 +1196 C>T (Thr399Ile;
rs4986791) SNP, are at increased risk for Candida infections and that this may be due to
increased production of IL-10164. TLR4 induces expression of a Th1 cytokine profile and
defective TLR4 signalling may result in a more Th2 type cytokine profile165. IL-10 is a well
known anti-inflammatory cytokine and upregulation of this cytokine may increase
susceptibility to microbial infection, however it may also protect against an aberrant
immune response.
It is most likely that in C. trachomatis infection, a combination of the aforementioned
mechanisms plays a role in the susceptibility and severity of Chlamydial infection,
indicating the need for more research.
CD14
166
No significant differences in the frequencies of the CD14-260 SNP between patients and
controls were observed in the susceptibility or severity of C. trachomatis infections
(Chapter 7 & 9). CD14 requires TLR4 or TLR2 for signal transduction67 - 69. TLR4 is
differentially expressed in the female urogenital tract156 - 158. This differential expression
pattern may offer a possible explanation for the observed lack of association with
susceptibility to Chlamydia trachomatis infection, while the strict regulation of immune
responses against LPS in the upper genital tract may explain the lack of associations in
tubal pathology166, 167.
A recent study reports that CD14 is not expressed on oviductal epithelial cells and stromal
fibroblasts, suggesting that CD14 may not play a role in the immunity of the human
fallopian tubes160. However, CD14 may also signal through TLR2, and TLR2 does play a
role in C. trachomatis recognition and C. trachomatis induced tubal pathology161, 168.
Differences in the structural requirements for optimal recognition of PGN and LPS has
been observed76, and molecular structures of LPS may influence recognition and signalling
efficiency, as reviewed previously in this discussion..
Similar to the observations in the gastrointestinal tract, these results seem to suggest that
multiple factors should be taken into account in the analysis of CD14 SNPs in relation to
infection and inflammation.
Part II
TLR9
Carriage of the TLR9 SNPs (TLR9 -1237 T>C and +2848 G>A) increases the risk of
development of tubal pathology in subfertile women, especially in C. trachomatis positive
women (Chapter 9). In a murine study performed by our group, we observed that TLR9
had no effect on primary Chlamydia infection, however upon reinfection a significantly
reduced duration of infection was observed in TLR9 KO mice compared to wildtype
controls169. Furthermore, when studying carriage of the TLR9 SNPs in subfertile women it
was observed that carriage of TLR9 haplotype I was significantly increased in women with
serological responses against C. trachomatis who did develop tubal pathology, compared to
those women who did not develop tubal pathology. The reverse was observed in haplotype
III, indicating that carriage of these specific haplotypes influence development of tubal
pathology in the presence of a C. trachomatis infection169.
Murine studies have shown that TLR9 is involved in Hepatitis B infection and autoimmune
disease (reviewed previously in this discussion). Innate immunity against Plasmodium
falciparum (malaria) is activated through TLR9 in both humans and mice170 - 172. Recent
evidence has implicated TLR9 activation in the induction of HIV replication and
subsequent high viral loads in HIV infected patients173, 174.
The TLR9 SNPs can be subdivided in 4 common haplotypes, as shown in table 2.
Haplotype I (-1486T, -1237T, +1174A, +2848G) is associated with an increased risk for
development of tubal pathology in C. trachomatis positive women169, while haplotype III (1486T, -1237C, +1174G, +2848A) is associated with a reduced risk for developing tubal
pathology in C. trachomatis infected subfertile women169. The TLR9-1486*C allele confers
risk for intrauterine growth retardation in P. falciparum infected women175. While
Mockenhaupt et al. tested the -1237 SNP, they did not observe significant differences
between cases and controls175. The -1486*C allele is present in the very common haplotype
II, together with -1237*T and +2848*A.
No consistent pattern can be observed in the results of the different studies and currently no
functional effect of the SNPs on the functions or expression of TLR9 has been described
that would help explain the observed associations. It is important to note that two major
differences between mice and humans may influence the translational value of data
obtained in the murine model to human infections: 1) humans and mice recognise slightly
different CpG motifs although stimulatory indexes for any given motif are around 60 – 80%
between the two species, indicating that oligodeoxynucleotides (ODNs) containing
unmethylated CpG motifs may induce different responses in the two species and 2) in mice
plasmacytotoid dendritic cells (PDCs), myeloid DCs, macrophages and B cells respond to
CpG DNA, while in humans only PDCs and B cells respond to CpG DNA169, 176.
Berghöfer et al. have shown that the TLR9 genotypes and haplotypes do not alter PDC IFNα production in response to stimulation with CpG-ODNs177, however Rothenfusser and
Krug have shown that different CpG motifs result in different responses178, 179. Influence of
the TLR9 SNPs may be dependent on the type of CpG-ODN encountered by TLR9.
Other factors may influence the role of TLR9 in urogenital disease, including microbial
factors, the type and number of CpG motifs, regulation of TLR9 and DNA uptake, and the
167
Discussion
presence of CARD15/NOD2 SNPs, as was reviewed previously.
CCR5
The CCR5δ32 polymorphism protects against development of late complications of
C. trachomatis infection, as shown by our results in both the murine and human infection
models (Chapter 8). Previous studies in mice have demonstrated that a rapid upregulation
of CCR5 expression follows synchronously with C. trachomatis infection180. It should be
noted that CCR5 expression in the female reproductive tract fluctuates during the menstrual
cycle as a function of sex hormone levels181, and that this fluctuating expression could have
an impact on the actual effect of the CCR5δ32 polymorphism in C. trachomatis infection
and the development of tubal pathology.
CCR5 is best known as the HIV-1 receptor and most research into CCR5 is devoted to HIV
infection. CCR5 has been associated with HIV in the female urogenital tract182 and to
colonic inflammation in HIV infected patients183, underscoring the presence and importance
of CCR5 in both gastrointestinal and urogenital diseases.
168
In previous studies it was shown that an early, robust T cell response confers protection
against the development of late complications following C. trachomatis infection,184, 185 and
the results from the CCR5 KO mice suggest that the lack of T cell activation is responsible
for the observed delay in clearance of Chlamydia infection. However, the reduced
activation of T cells may prevent a prolonged immune response that would contribute to
more severe upper tract pathology. The results in the murine model and human infection
offer a compelling explanation for the role of CCR5 in Chlamydia pathogenesis.
CARD15/NOD2
An increased risk for the development of tubal pathology was observed in subfertile women
carrying CARD15/NOD2 SNP13, especially in subfertile women with a positive C.
trachomatis IgG serology (Chapter 9).
CARD15/NOD2 is a well known risk factor for the development of Crohn’s Disease, as
discussed in the previous part of this discussion, and for other diseases such as Blau
syndrome and psoriatic arthritis100.
CARD15/NOD2 enhances the signals transduced by a variety of TLRs and carriage of the
CARD15/NOD2 SNPs has been shown to attenuate signal transduction, resulting in a
reduced inflammatory response88, 89. This also indicates that CARD15/NOD2 may
potentially regulate the homeostasis with commensal microorganisms. From these data it
can be concluded that CARD15/NOD2 is involved in a variety of pathways and that
CARD15/NOD2 SNPs may have a significant impact on aetiopathogenesis. This has been
demonstrated in Crohn’s disease, but also in other diseases such as Graft versus Host
Disease (GVHD) and adverse outcome following allogeneic stem cell transplantation
(SCT)186, 187. The effect on the occurrence of GVHD is dependent on the side from which
the CARD15/NOD2 SNPs came. Carriage of the SNPs on the recipient side results in a
Part II
systemic inflammatory reaction caused by the adoptive immune system and is similar to the
inflammation observed in IBD. Carriage of the SNPs on the donor side will results in an
immune response by the donor derived cells only. The CARD15/NOD2 SNPs may reduce
NF-κB activation and thus the donor cell derived immune response187. The deleterious
effect in SCT is thought to result from an altered immune response of the innate immune
system, resulting in increased mortality due to pneumonia induced respiratory failure186.
Further research is required to elucidate the exact role of CARD15/NOD2 in C. trachomatis
pathogenesis and we should be aware of the many biological functions of CARD15/NOD2,
but our data does provide evidence that CARD15/NOD2 may play a role in C. trachomatis
induced tubal pathology.
Multigene approach
In the urogenital tract it was shown that carriage of two or more SNPs in the TLR9, TLR4,
CD14 and CARD15/NOD2 genes almost doubles the risk of developing tubal pathology in
C. trachomatis positive women, compared to C. trachomatis positive women, who carried
less than two SNPs in these genes (Chapter 9). These results show multiple SNPs may
influence disease susceptibility and severity. The results in the subfertile women clearly
show the interaction between the different risk factors. Carriage of multiple SNPs in C.
trachomatis negative women does not predispose to development of tubal pathology.
However, when women are infected with C. trachomatis, the presence of these SNPs is a
risk factor. These results suggest that suboptimal or non existent recognition of the
Chlamydia infection may result in a chronic/persistent infection and increased tissue
damage. The SNPs in CARD15/NOD2 abolish CARD15/NOD2 mediated NF-κB activation
in response to PGN111, 112 and attenuate TLR signalling88, 89, thus effectively reducing the
immune response. TLR4 inactivation results in reduced COX2 mediated tissue proliferation
and repair35, thus effectively maintaining tissue damage and opportunities for bacteria
and/or bacterial components to persist in the mucosa.
169
Discussion
Part III
The immunology of the gastrointestinal and urogenital mucosa and
the interaction of molecules encoded by genes studied in this thesis
wo models of mucosal immunology are presented in this thesis, chronic
inflammation of the gastrointestinal tract and urogenital tract infection. Proteins
with a function in immune regulation or pathogen recognition were studied
using a candidate gene approach to elucidate their influence on infection and inflammation.
T
IL-1β and IL-1ra
Barrett oesophagus (chronic inflammation) and Chlamydia infection are affected by SNPs
in the IL-1RN gene. The biological mechanisms appear to be distinct. Barrett oesophagus
may be affected directed by the (dys)regulation of the immune response, or indirectly by
the influence on gastric acid production, on COX2 expression, and possibly by infection
with microorganisms in the oesophagus and the stomach.
On the other hand, Chlamydia infection is more likely influenced by the impact of IL-1
SNPs on the regulation of the immune system.
170
The literature appears divided on the exact influence of IL-1B and IL-1RN SNPs on
aetiopathogenesis of many diseases. Our results indicate that these SNPs influence
susceptibility to Barrett oesophagus and Chlamydia infection, but that IL-1β and IL-1ra are
not the only factors and that more factors have to be taken into account in order to explain
the observed clinical phenotypes.
TLR4
Carriage of the TLR4 +896 A>G SNP confers risk for both Crohn’s disease and tubal
pathology, keeping in mind that both diseases are multifactorial and results regarding TLR4
should always be viewed in relation to other genes and predisposing factors. The effect of
TLR4 may be mediated through direct recognition of microorganisms, but also indirectly,
through the sensing of markers of cell damage and death, such as HSP60 and
HSP7043, 44, 162. TLR4 mediated induction of COX2 results in PGE2 induced tissue
proliferation and apoptosis inhibition35. A defective TLR4 may thus in effect increase
inflammation related tissue damage due to lack of tissue proliferation and inhibition of
apoptosis. Differential expression of TLR4 has been reported on a cellular level188 and on a
macroscopic level156 - 158, 189. This compartmentalisation prevents immune responses in
compartments where a homeostasis with local microbial flora is beneficial for the host,
while maintaining pathogen free compartments. Regulation of TLRs45, synergy with
CARD15/NOD289, and structural requirements73, 75, 163 add another level of complexity,
which may potentially explain the observed diversity of the clinical courses of diseases in
which these genes play a role.
Part III
The interaction between the aforementioned biological mechanisms plays a role in the
selected gastrointestinal and urogenital diseases studied in this thesis, and further research
is required to elucidate how the combined biological pathways contribute to susceptibility
and severity of disease.
CD14
Carriage of the CD14 polymorphism is associated with the development of fistulae and
protection against the need of appendectomy in Galician patients. We did not observe an
effect of this polymorphism in C. trachomatis infection and complications. CD14 has been
associated with a variety of diseases, however for almost every positive association a
negative association has been reported. The reported differences may due to differences in
methodologies, ethnicities, and disease aetiopathogenesis, as well as signalling through
TLR4 or TLR2.
Our results and published studies suggest a potential role of CD14 in gastrointestinal
pathogenesis. The results in the urogenital tract indicate that CD14 is not involved in the
susceptibility to C. trachomatis infection or the development of tubal pathology. This does
not exclude a possible role for CD14 in the course of C. trachomatis infection that may
become apparent when yet to be described CD14 expression decreasing SNPs are analyzed.
TLR9
TLR9 SNPs influence susceptibility and severity of Crohn’s Disease, pouchitis and
Chlamydia related tubal pathology, as shown by the results presented in this thesis and from
previous work169.
The results described in the literature do not show a consistent pattern and no functional
effect of these SNPs is currently known. The SNPs might be linked to other functional
polymorphisms. The TLR9 gene is located in a region linked to IBD87, which may explain
the associations with Crohn’s disease, however it does not readily provide an explanation
for the observed effect in urogenital disease. Other factors have been shown to affect TLR9
signalling, including synergy with CARD15/NOD288, differences in CpG motifs96, 97, and
direct regulation of TLR992, 94, 95. Further studies are required to elucidate the functional
effects of the TLR9 SNPs or whether these SNPs are linked other functional SNPs.
CCR5
Using a translational model, we have shown that carriage of the CCR5 polymorphism
protects against development of tubal pathology. CCR5 is best known as the HIV-1
receptor and most research into CCR5 is devoted to HIV infection. CCR5 has been
associated with HIV in the female urogenital tract182 and to colonic inflammation in HIV
infected patients183, underscoring the presence and importance of CCR5 in both
gastrointestinal and urogenital diseases.
171
Discussion
Hampe et al. postulated that it is unlikely that CCR5 is a major susceptibility locus in IBD87
and this was corroborated by the study of Martin et al. who did not observe differences in
CCR5δ32 frequencies between cases and controls190. However, several publications have
shown that CCR5 is involved in a variety of gastrointestinal diseases. Increased expression
of CCR5 was found in H. pylori infected children191. Increased carriage of the δ32
polymorphism was observed in hepatitis C patients192, while decreased incidence of the
polymorphism was observed in primary sclerosing cholangitis (PSC)193. Increased
expression of CCR5 was observed in Peyer’s patches and in non caseating granulomas in
CD patients194, 195.
172
In the gastrointestinal tract, one might hypothesize that the early robust immune response
may be part of the inflammatory response observed in CD and may be caused by the
increased expression of CCR5 in the inflamed tissue194, 195. Carriage of the δ32
polymorphism reduces the inflammatory response against pathogens, which may explain
the increased frequency of this polymorphism observed in hepatitis C patients192. PSC is
caused by inflammation and scarring of the bile ducts196. Although the cause is yet
unknown, severe scarring can theoretically be seen as a late complication of the
inflammation. A reduced immune response, as seen in the murine model, may result in less
scarring, explaining the observed protective effect of CCR5δ32 in PSC193. However, a
strong association between CCR5δ32 and PSC has been described197, contradicting the
protective effect described by Henckaerts et al.193. Unfortunately, in complex diseases the
cause is often multifactorial and the pathogenesis is never so easily explained. Hampe87 and
Martin190 have postulated that CCR5 plays no role in CD, although one might hypothesize
that CCR5 may not influence the development of CD, but may influence the severity of
CD198.
The concordant results in the murine model and human disease offer a compelling
explanation for the role of CCR5 in Chlamydia pathogenesis, however further research is
required to elucidate whether we can translate the results to gastrointestinal diseases.
CARD15/NOD2
CARD15/NOD2 SNPs influence the susceptibility and severity of Crohn’s disease, as
confirmed in this thesis (Chapter 4) and predispose to the development of tubal pathology
in subfertile women, as shown for the first time in this thesis (Chapter 9). As discussed
previously, CARD15/NOD2 is involved in a wide variety of biological processes, including
PGN recognition, synergy with TLRs, and regulation immune responses and intestinal
homeostasis.
These results confirm the importance of CARD15/NOD2 in gastrointestinal diseases and
potentially in urogenital infection, adding more value to the title “pleiotropic autoimmune
gene” as proposed by Rahman et al.100.
Part III
IRAK-M
Little data is available on the influence of IRAK-M SNPs in susceptibility to or severity of
disease. The IRAK-M +22148 G>A SNP does not predispose to pouchitis (this thesis) or
asthma138. IRAK-M is a negative regulator of TLR and IL-1R signalling, thus suggesting a
possible role in innate immune homeostasis. Future studies may elucidate the potential role
of IRAK-M in other gastrointestinal and urogenital diseases.
Multiple genes
Combined carriage of multiple SNPs in the immune regulatory and bacteria sensing genes
affect the susceptibility to and prognosis of both gastrointestinal and urogenital tract
inflammation and infection. If the functional effect of the SNPs is additive, then the
observed effect may be more profound than expected based on the observed effect in single
SNP carriers.
173
Discussion
The results presented and discussed in this thesis, clearly show that an adequate recognition
of microorganisms at the site of infection or colonisation is essential for immune regulation,
homeostasis with commensal flora, and defence against pathogens. Strict regulation of
inflammatory responses allows homeostasis with the ubiquitous flora, while maintaining an
effective immune barrier. Polymorphisms in genes encoding proteins involved in regulatory
and pathogen recognition mechanisms can have a profound effect on infection and / or
inflammation, especially when multiple polymorphisms are present in genes in the same
signalling pathway or in the same functional class (e.g. pathogen recognition receptors).
A graphical representation of the interactions between the genes studied in this thesis and
(part of) the signalling cascade associated with these proteins is given in figure 2.
174
Figure 2: Graphical representation of interactions between IL-1β, IL-1ra, CD14, TLR4, TLR5, CARD15/NOD2,
CCR5, and IRAK-M (IRAK3), resulting in regulation of NF-κB expression and subsequent immune activation.
Nota bene: Interactions presented in the pathway diagram are described in the literature, however these
interactions do not necessarily represent the actual or complete biological pathways. Diagram made with
PathwayStudio 4.0.1199, 200.
Part IV
Although figure 2 represents a small part of the complex biological pathways involved in
the regulation of the immune response, it is clear that, even though there is a redundancy in
signalling routes, dysregulation of key proteins in this pathway can have far reaching
impact.
Future research
Immunogenetic analyses are currently employed for disease profiling and identification of
patients at risk for adverse outcomes of disease.
Preliminary data, in part published at the DDW 2006, describe an IBDChip developed by
Progenika, which screens 61 SNPs in 40 genes, selected for their potential impact on IBD.
Preliminary studies suggest, that this chip is able to discriminate with a very high
specificity between fistulising and inflammatory phenotypes in Crohn’s disease patients,
and to identify a significant proportion of ulcerative colitis patients requiring colectomy or
presenting with extraintestinal manifestations201.
175
Figure 3: The complex nature of the diseases studied in this thesis justify a future integrated approach in order to
understand their aetiopathogenesis. Host factors (e.g. genetic variation and immunologic factors), environmental
factors (e.g. diet, coinfections with other microorganisms, medication, behaviour), and factors of the
microorganism(s) of interest (e.g. virulence factors of C. trachomatis (e.g. IncA and OMP1) in the C. trachomatis
studies) influence the observed differences in the clinical course of infection. The different factors can also
interact with eachother, e.g. infection with other microorganisms may increase the susceptibility to C. trachomatis
infection.
Figure modified from Morré et al.203
The laboratory of Immunogenetics, in collaboration with several European centres, has
Discussion
recently (2006) obtained a European Framework Programme 6 (FP6) grant (Acronym:
IBDChip) focussed on the further development of the IBDChip. It is to be hoped, that this
new tool will help to advance the understanding of genetics of complex diseases.
Preliminary data presented at the ISSTDR meeting in 2005, showed that all subfertile
women with the TLR4*G allele, a positive C. trachomatis IgG serology and a positive
Chlamydial HSP60 serology have tubal pathology (laparoscopy as gold standard).
Screening of women for the presence of TLR4 mutations, Chlamydia IgG and Chlamydial
HSP60 serology can be used to define women at increased risk for tubal pathology,
reducing the need for invasive screening methods, such as laparoscopy202.
The laboratory of Immunogenetics, in collaboration with several European centres, has
recently (2006) obtained a FP6 grant focussed on the Immunogenetics of Chlamydia
trachomatis (Acronym: EpiGenChlamydia).
176
The gastrointestinal and the urogenital tract share striking similarities. Both are mucosa
lined complex systems, continuously exposed to microorganisms. A part of these
microorganisms are commensal and beneficial to the host, colonising the epithelia which
otherwise might be colonised by more pathogenic microorganisms. In both tracts a
homeostasis with these commensals has to be maintained while at the same time
maintaining an effective immunity against pathogens. A careful regulation of the immune
response is therefore required. As shown in this thesis and in other published studies,
altered regulation of the immune response may result in a more rapid initial clearance of
infection, but with a coincidental increase in tissue damage and/or an increased risk of
chronic infection and slower but progressive pathology.
The inflammatory processes and infections studied in this thesis are multifactorial, meaning
that multiple factors are involved in the aetiopathogenesis of the inflammatory responses
and diseases. These factors consist of environmental factors (e.g. diet, smoking, alcohol
(ab)use, medication), microbial factors (e.g. virulence factors) and host factors (e.g. host
genetic variations). Each factor on its own does not cause disease, however a combination
of factors affects susceptibility to and/or severity of disease (figure 3). The precise
combination of factors determines the course of disease and the combination of factors
differs between patients, resulting in a diverse presentation of diseases. An integrated
approach and new research tools are required to advance knowledge in this complex field.
Analysis of the different factors in relation to disease will help the scientific community
gain insight in the susceptibility and severity of disease. New technologies, like highthroughput genotyping and micro-array analysis, will enable researchers to screen ever
larger quantities of potential risk genotypes. The explosive increase in information
exchanges through the internet has enabled researchers to broaden their view, to analyse
their data in relation to world-wide generated research results and to disseminate their
findings to a broad international audience. New computational technologies will help in the
management and interpretation of the data, as well as in integrating data visually in a
graphical format (e.g. Pathway analysis with PathwayStudio). The knowledge can then be
used to further define new research topics and help identify potential targets for therapeutic
intervention.
Part IV
We should, however, also be cautious. In a series of point and counterpoint articles in the
International Journal of Epidemiology, scientists have raised doubts about current research
methods204 - 212. We should keep in mind that we have identified a few pieces of an
unknown shape that should fit in an invisible puzzle of unknown size. The task of solving
that puzzle is daunting, but also challenging. It will take a lot of determination and
innovation to identify the underlying biological mechanisms. But most of all, it will take a
collaborative effort across all scientific fields, combining all scientific knowledge and
expertise, to gain true insight into the puzzle that is the aetiopathogenesis of complex
multifactorial human diseases213.
From my own personal point of view, I am confident that science and scientists alike will
evolve to be able to solve this puzzle and that the generated knowledge will result in the
improved diagnosis and treatment of patients. In the future, made-to-measure treatment
might be a possibility, although it may take some time before a device comparable to a Star
Trek medical tricorder is standard in every doctor’s toolkit.
177
Discussion
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CC-type chemokine receptor 5-Delta32 mutation protects against primary sclerosing cholangitis, Liesbet
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Summary
Summary
190
DNA was the first three–dimensional Xerox machine.
Kenneth Boulding
"Energy and the Environment," Beasts, Ballads, and Bouldingisms, 1976
of the thesis
Summary
n this thesis an overview of our research into the genetic variations that affect
susceptibility to and severity of infections and diseases is given. The study of
these genetic variations has provided insights into the biological mechanisms
that influence gastrointestinal and urogenital diseases. This knowledge will help to identify
patients at increased risk for more severe pathology, and, in due time, lead to potential
therapeutic targets that might lead to made-to-measure treatment of individual patients.
I
The summary highlights the results outlined in this thesis.
Gastrointestinal tract
In Chapter 1 it was shown that IL-1β, IL-1ra, CD14 and TLR4 influence susceptibility to
Barrett oesophagus. The occurrence of the C allele of IL-1RN+2018 was significantly
greater in patients with Barrett oesophagus compared to controls (p: 0.033, OR: 1.5), while
the carriage of the TLR4+896*G allele showed a trend towards an increased risk for Barrett
oesophagus (not significant).
Combining the immunoregulatory and bacteria-sensing carrier traits, resulted in significant
differences between cases and controls. Homozygous wildtype carriage of the IL-1B and
IL-1RN genes combined with heterozygous carriage of the CD14 and TLR4 genes was
significantly decreased in cases compared to controls (p: 0.013; OR: 9.1). Carriage of the
specific carrier trait IL-1B-511*CT, IL-1RN+2018*TT, CD14-260*CT and TLR4+896*AA
is significantly decreased in patients with Barrett oesophagus compared to controls
(p: 0.030; OR: 3.3).
We studied the TLR4, CD14, TLR9, and CARD15/NOD2 genes in relation to the
development of Crohn’s Disease (Chapters 2 – 4). TLR4 and CD14 SNP carriage was
studied in a cohort of Dutch Caucasian IBD patients (Chapter 2). Carrier frequency of the
CD14 SNP was compared between IBD patients and healthy controls and only a slightly
increased frequency was observed in carriage of the CD14 -260 TT genotype in ulcerative
colitis (UC) patients compared to controls. No differences were observed when the CD
patients were subdivided according to the Vienna Classification.
Carriage of the TLR4+896*G was found to be significantly increased in CD patients
compared to controls (p: 0.049; OR: 2.0; 95% CI: 1.0 – 4.1). The patients were subdivided
according the Vienna Classification to assess the role of TLR4 in the severity of CD. An
increased carriage of the TLR4*G allele was found in patients with a colonic localisation
(Vienna classification L2) compared to other localisations (p: 0.0047; OR: 4.8; 95% CI: 1.7
– 14). No difference was observed in TLR4*G allele carriage between ASCA positive and
ASCA negative CD patients and no difference was observed in the frequency of the
TLR4*G allele in ASCA positive and ASCA negative CD patients with colonic disease.
The carrier frequency of the G allele remained similar to the CD patients without colonic
localisation. The data were not confounded by the three main CARD15/NOD2 SNPs1, 2.
In collaboration with the University Hospital in Santiago de Compostela in Spain, we
studied the role of TLR9 polymorphisms in the development of CD in a homogeneous
Spanish Galician population and a Dutch Caucasian population (Chapter 3). Carriage of
the TLR9 +2848 AA genotype confers protection against development of CD in the Dutch
191
Summary
192
Caucasian population (p: 0.018, OR: 1.9, 95% CI: 1.1 – 3.2), but not in the Spanish
Galician population. The same genotype is significantly increased in Spanish Galician CD
patients with colonic disease (Vienna classification L2) compared to patients with noncolonic CD (p: 0.022, OR: 2.9, 95% CI: 1.2 – 6.6). The TLR9 -1237 CC was significantly
increased in Galician patients over 40 years old (Vienna classification A2) compared to
younger patients and in Galician patients with stricturing disease (Vienna classification B2)
compared to non stricturing disease (p: 0.02, OR: 14.7, 95% CI: 1.5 – 146.5 and p: 0.031,
OR: 11.9, 95% CI: 1.2 – 118.4, respectively). Multivariate regression analysis confirmed
the observed associations in the Dutch Caucasian and Spanish Galician populations.
The same homogeneous Spanish Galician population was used for the analysis for TLR4,
CD14 and CARD15/NOD2 polymorphisms in relation to the development of CD (Chapter
4).
Carriage of the TLR4+896 SNP was increased in controls compared to patients (p: 0.0298,
OR: 2.3, 95% CI: 1.1 – 5.0), while the CD14 SNP was equally distributed amongst cases
and controls. Carriage of the CD14-260 T allele was significantly increased in non-fistulae
patients as compared to patients with fistulae (p: 0.045, OR: 2.8, 95% CI: 1.4 – 5.8). When
corrections were made for gender or smoking, no significant associations were observed,
nor when corrections were made for both gender and smoking.
Since the CD14-260 and TLR4+896 SNPs have seemingly opposing effects on bacterial
recognition, analyses were made based on the combination of wildtype TLR4 combined
with carriage of the CD14 mutant allele. An increased frequency of TLR4 AA with
CD14*T was observed in younger patients (Vienna A1) compared to patients over 40 years
old (Vienna A2). This did not reach statistical significance although a trend was observed
(p: 0.06, OR: 4.0, 95% CI: 1.0 – 11.9). Combined carriage of the CD14-260 SNP with the
TLR4 wildtype was significantly increased in patients without fistulae compared to those
with fistulae (p: 0.03, OR: 3.2, 95% CI: 1.2 – 8.4). Corrections for smoking or gender did
not result in statistically significant associations. We analysed carriage of the CD14-260
and the TLR4+896 SNPs in combination with carriage of one or more CARD15/NOD2
SNPs. Carriage of CD14*T combined with one or more CARD15/NOD2 SNPs was
significantly increased in cases compared to controls (p: 0.0007, OR: 3.1, 95% CI: 1.6 –
5.9). Carriage of CD14*T combined with the CARD15/NOD2 SNPs and homozygous
carriage of the TLR4+896 wildtype was significantly increased in cases compared to
controls, with an increased Odds ratio when compared to the analysis without TLR4
correction (p: 0.0003, OR: 3.5, 95% CI: 1.7 – 7.0). Carriage of CD14*T combined with one
or more CARD15/NOD2 SNPs was significantly increased in patients who had an ileal
resection compared to those who did not have an ileal resection (p: 0.004, OR: 3.2, 95% CI:
1.5 – 6.8).
TLR4 wildtype carriage combined with CD14 and CARD15/NOD2 SNPs slightly increased
the risk for ileal resection (p: 0.002, OR: 3.4, 95% CI: 1.6 – 7.3). Combined carriage of the
CD14 SNP, one or more CARD15/NOD2 SNPs and the TLR4 wildtype was significantly
increased in steroid resistant patients compared to non resistant patients (p: 0.04, OR: 2.5,
95% CI: 1.0 – 6.2).
We entered age, gender, origin of the patient (rural or urban area), smoking, and carriage of
the SNPs into a multivariate logistic regression model. Age, smoking, origin and carriage of
the SNP12, SNP13 and TLR4 SNPs are interdependently associated with development of
CD. Smoking, rural origin and carriage of the TLR4 SNP are protective while age and
carriage of the SNP12 and SNP13 polymorphisms are risk factors.
Carriage of the SNP12 and the CD14 polymorphism combined with the TLR4 wildtype
of the thesis
were associated with development of fistulae. SNP12 is a risk factor, while the CD14 –
TLR4 combination is protective against fistulae. SNP13 was shown to be a significant risk
factor for ileal resection.
The results of the Crohn’s disease studies (Chapters 2 – 4) are summarised in table 1.
SNP
TLR4 +896*G
CD14 -260*C
TLR9 +2848 AA
Population
Dutch Caucasian
Spanish Galician
Spanish Galician
Dutch Caucasian
Spanish Galician
Effect
Increased risk for CD
Protection against CD
Protection against fistulae
Increased risk for colonic disease
(L2)
Associated with age above 40 (A2)
Increased risk for stricturing disease
(B2)
Ch.
2
4
3
3
TLR9 -1237 CC
Spanish Galician
Multigene analysis:
TLR4 +896 AA + CD14-260*T
CD14 -260*T + any
CARD15/NOD2 SNP
Spanish Galician
Spanish Galician
Increased risk for CD
4
4
Spanish Galician
Increased in ileal resection patients
Strengthens aforementioned
associations
4
CD14 -260*T + any
CARD15/NOD2 SNP + TLR4
+896 AA
3
Increased in steroid resistant patients
Multivariate logistic regression:
TLR9 +2848 AA
Dutch Caucasian
Spanish Galician
TLR9 -1237 CC
Spanish Galician
Smoking, origin (rural or urban)
and TLR4 +896*G
Age, and carriage of SNP12 and
SNP13
Gender and SNP12
TLR4+896 AA + CD14-260*T
SNP13
Spanish Galician
Increased risk for colonic disease
(L2)
Associated with age over 40 years
Increased risk for structuring disease
(B2)
193
3
3
4
Increased risk of CD
Increased risk of fistulae
Increased in ileal resection patients
Table 1: Overview of the results in Crohn’s disease (Chapters 2 – 4)
The final chapter of Part I discussed pouchitis. We observed no significant differences in
allele, genotype or carrier frequencies of the gene polymorphisms in the CD14, TLR4,
TLR9, CARD15/NOD2 and IRAKM genes between the healthy control group and IPAA
patients. We did observe a significantly increased frequency of the TLR9 –1237*C allele in
patients with chronic relapsing pouchitis compared to those with infrequent pouchitis
(p: 0.028; OR: 3.2)
We analysed the TLR9 SNPs in haplotypes, according to the publication by Lazarus and
colleagues3. The haplotype frequencies in the healthy control group were identical to the
Summary
European American population reported by Lazarus et al.3. Haplotype III was more
frequent in chronic relapsing pouchitis as compared to infrequent pouchitis (p: 0.018; OR:
3.0). This haplotype however, did not show nucleotides uniquely present (tag SNPs) on
position –1486 (allele T present in haplotypes I and III) or on position +1174 (allele G
present in haplotypes II and III), indicating that allele TLR9 –1237*C provides the strongest
association. Synergistic interaction between different bacteria sensing genes was assessed
in a carrier trait analysis. Combined carriage of TLR9 –1237*C and CD14−260*T was
significantly increased in patients with chronic relapsing pouchitis as compared to those
with infrequent pouchitis (p: 0.018; OR: 4.1). This combination strengthens the association
found with TLR9-1237*C alone in patients with chronic relapsing pouchitis.
Urogenital tract
In a STD susceptibility model (Chapter 6), we observed a significantly decreased carriage
of IL-1RN+2018*C in C. trachomatis DNA positive women compared to C. trachomatis
DNA negative women (p: 0.0005, OR: 1.5). Introduction of C. trachomatis serology status
into the analyses resulted in similar results. IL-1RN+2018*C was significantly reduced in
C. trachomatis DNA positive / C. trachomatis IgG positive women compared to
C. trachomatis DNA negative / C. trachomatis IgG negative women (p: 0.014, OR: 1.6).
Introduction of coinfection status or symptoms into the analyses did not alter the
aforementioned associations.
194
The role of CD14 in the susceptibility to and severity of C. trachomatis infection was
reported in Chapter 7. The susceptibility was modelled in a STD cohort. The results
showed a similar distribution of CD14-260 genotypes between cases and controls. No
differences could be observed when C. trachomatis IgG serology, coinfections with other
microorganisms, symptoms, or when any combination of all the variables were introduced
into the analyses.
The severity of sequelae of C. trachomatis infection was assessed in subfertile women with
clinically well-defined tubal pathology. The CD14 genotype distribution in women who
developed tubal pathology was comparable to women who did not develop tubal pathology,
and to the genotype distribution in healthy controls. Introduction of C. trachomatis IgG
serology, with special attention to C. trachomatis positive women who developed tubal
pathology as compared to those who did not develop tubal pathology, did not alter the
observed genotype distribution.
Using a translational model we assessed the role of CCR5 in development of late
complications after Chlamydia infection (Chapter 8). In the mouse CCR5KO model, it was
observed that the ability of CCR5KO mice to control the infection was compromised. The
CCR5KO mice suffered from longer and more intense infections compared to their
wildtype counterparts. The mice were mated at different time points to assess the effect of
the infection on fertility, simulating short- and long-term effects of the infection. Wildtype
mice mated two weeks post genital infection had a less than 40% pregnancy rate, while the
CCR5KO mice had a more than 70% pregnancy rate (p: 0.0021). At five weeks post genital
infection the pregnancy rate was 100% in the KO mice, while the wildtype remained at a
50% pregnancy level. These data suggest that immunocompetence plays a significant role
of the thesis
in development of late complications after Chlamydia infection. To test this theory, levels
of cytokines and chemokines were measured in murine leukocytes. Both IFN-γ and TNF-α
were elevated in wildtype mice, while the levels of these cytokines in CCR5KO mice were
similar to uninfected mice. The CCR5KO mice also displayed a reduced capacity for
production of the pro-inflammatory chemokines RANTES and IP-10, suggesting a
compromised Th1 immune response.
To translate these results to humans, a cohort of clinically well-defined women with and
without tubal pathology were genotyped for the CCR5δ35 polymorphism. A decreased
incidence of tubal pathology was observed in women carrying the CCR5δ32 polymorphism
compared to women not carrying the polymorphism. Since Chlamydia infection is an
important risk factor for development of tubal pathology, we compared the carriage of this
polymorphism between women with and without tubal pathology, with a proven serological
response against Chlamydia trachomatis. We found a significant reduction in carriage of
CCR5δ32 in C. trachomatis IgG positive women who developed tubal pathology compared
to C. trachomatis IgG positive women who did not develop tubal pathology.
The study reported in the last chapter in part II was designed to assess the multi-gene
approach to subfertility and tubal pathology in relation to C. trachomatis infection. The
incidence of mutations in the TLR9, TLR4, CD14 and CARD15/NOD2 genes was
determined in a cohort of subfertile women, and in a specific subgroup of subfertile women
with positive C. trachomatis IgG serology. An average 20% increase in risk for tubal
pathology was observed for the TLR4, TLR9 and CARD15/NOD2 genes, although the
differences did not reach statistical significance. Carrying two or more SNPs in the four
studied genes did not influence the risk of tubal pathology in C. trachomatis IgG negative
women, compared to C. trachomatis IgG negative women carrying less than two SNPs in
these genes. However, in women with a positive C. trachomatis IgG serology, carriage of
two or more SNPs doubles the risk of development of tubal pathology compared to women
carrying less than two SNPs.
In conclusion, the genetic results associated with the selected diseases of the
gastrointestinal and urogenital tracts clearly show that polymorphisms in genes encoding
regulatory and pathogen sensing proteins influence both the susceptibility to and severity of
infection and inflammation. Furthermore, gene – gene, gene – pathogen, and gene –
environment interactions may have a more profound effect. The complex nature of both
tracts should be taken into account when interpreting results and comparing the results of
different studies.
195
Summary
References
1.
2.
3.
196
Single-nucleotide polymorphisms in the Toll-like receptor 9 gene (TLR9): frequencies, pairwise linkage disequilibrium,
Silverman, Fernando Martinez & Scott T. Weiss, Genomics 2003; 81 (1): pp. 85 - 91, PubMed: 12573264
Samenvatting
Samenvatting
198
Success has a thousand fathers,
While failure is always an orphan
van dit proefschrift
Samenvatting
it proefschrift geeft een overzicht van het onderzoek naar genetische variaties,
die van invloed zijn op de vatbaarheid voor en de ernst van beloop van infectie
en ontsteking. Het bestuderen van deze genetische variaties heeft nieuwe
inzichten gegeven in de biologische mechanismen, die ten grondslag liggen aan
gastrointesintale en urogenitale ziektebeelden. De vergaarde kennis zal als hulpmiddel
dienen bij het identificeren van patiënten met een verhoogd risico op een ernstig
ziektebeloop, en, in de toekomst, leiden tot potentiële therapeutische methoden, die kunnen
leiden tot een op maat gemaakte behandeling van individuele patiënten.
D
Deze samenvatting belicht de resultaten, die beschreven zijn in dit proefschrift.
Het maag – darm kanaal
Hoofdstuk 1 laat zien dat IL-1β, IL-1ra, CD14 en TLR4 van invloed zijn op de
ontwikkeling van Barrett oesophagus (een chronische slokdarm ontsteking).
Het C allel op positie +2018 van IL-1RN kwam significant vaker voor bij Barrett patiënten
dan bij controles (p: 0.033, OR: 1.5), terwijl dragerschap voor het TLR4 +896*G allel een
trend vertoonde voor een verhoogd risico (niet significant).
Significante verschillen werden gevonden tussen patiënten en gezonde controles toen de
immuun regulerende en bacterie herkennende carrier traits werden gecombineerd.
Homozygoot dragerschap voor IL-1B en IL-1RN gecombineerd met heterozygoot
dragerschap voor de CD14 en TLR4 genen komt significant minder vaak voor bij patiënten
dan bij gezonde controles (p: 0.013; OR: 9.1). Dragerschap van de specifieke carrier trait
IL-1B-511*CT, IL-1RN+2018*TT, CD14-260*CT en TLR4+896*AA komt significant
minder vaak voor bij Barrett patiënten dan bij controles (p: 0.030; OR: 3.3).
We hebben de TLR4, CD14, TLR9 en CARD15/NOD2 genen bestudeerd in de ontwikkeling
van de ziekte van Crohn (Hoofdstuk 2 – 4).
We hebben gekeken naar dragerschap van de TLR4 en CD14 polymorfismen in een cohort
van Caucasische Nederlandse patiënten met een chronische darm ontsteking (Inflammatory
Bowel Disease; IBD). De frequentie van het CD14 polymorfisme is vergeleken tussen
Crohn patiënten en controles, en er werd alleen een lichte verhoging in het voorkomen van
het CD14 -260 TT genotype waargenomen bij Colitis Ulcerosa patiënten ten opzichte van
de controles. Wij vonden geen verschillen bij de Crohn patiënten, ook niet na
onderverdeling van de patiënten volgens de Vienna Classificatie.
Dragerschap van het TLR4 +896*G allel was significant verhoogd bij Crohn patiënten ten
opzichte van de controles (p: 0.049; OR: 2.0; 95% CI: 1.0 – 4.1). Om de invloed van TLR4
op de ernst van het ziektebeloop te kunnen bepalen zijn de patiënten onderverdeeld volgens
de Vienna Classificatie. Patienten met Crohn van de dikke darm (Vienna Classificatie L2)
blijken vaker drager te zijn van het TLR4*G allel in vergelijking tot Crohn patiënten van
wie andere delen van de darmen aangedaan zijn (p: 0.0047; OR: 4.8; 95% CI: 1.7 – 14).
We hebben geen verschillen waargenomen in het voorkomen van het TLR4 +896*G allel
tussen ASCA positieve en ASCA negatieve Crohn patiënten, of tussen ASCA positieve en
ASCA negatieve patiënten met Crohn van de dikke darm. De data werden niet beïnvloed
199
Samenvatting
door CARD15/NOD2 polymorfismen1, 2.
In samenwerking met het universitair ziekenhuis in Santiago de Compostela in Spanje
hebben we gekeken naar de rol van TLR9 in de ontwikkeling van de ziekte van Crohn in
een Spaans Galicische en een Caucasich Nederlandse populatie (Hoofdstuk 3).
Dragerschap van het TLR9 +2848 AA genotype werkt beschermend tegen de ziekte van
Crohn in de Caucasisch Nederlandse populatie (p: 0.018, OR: 1.9, 95% CI: 1.1 – 3.2), maar
niet in de Spaans Galicische populatie. Dit genotype komt significant vaker voor bij Spaans
Galicische Crohn patiënten van wie de dikke darm is aangedaan (Vienna Classificatie L2)
in vergelijking met patiënten van wie andere delen van de darm zijn aangedaan (p: 0.022,
OR: 2.9, 95% CI: 1.2 – 6.6). Het TLR9 -1237 CC genotype komt vaker voor bij Galicische
patiënten bij wie de ziekte zich pas naar het 40e levensjaar openbaart (Vienna Classificatie
A2) dan bij jongere Crohn patiënten, en bij Galicische patiënten met een darm vernauwend
phenotype (Vienna Classificatie B2) in vergelijking tot een niet vernauwend phenotype
(respectievelijk, p: 0.02, OR: 14.7, 95% CI: 1.5 – 146.5 en p: 0.031, OR: 11.9, 95% CI:
1.2 – 118.4). Multivariate regressie analyses bevestigden de waargenomen associaties in
beide populaties.
200
Dezelfde Spaans Galicische populatie hebben we gebruikt voor het analyseren van TLR4,
CD14 en CARD15/NOD2 polymorfismen in de ziekte van Crohn (Hoofdstuk 4).
Het TLR4 +896 polymorfisme komt vaker voor bij gezonde controles dan bij Crohn
patiënten (p: 0.0298, OR: 2.3, 95% CI: 1.1 – 5.0), terwijl het CD14 polymorfisme gelijk
verdeeld was tussen de patiënten en controles.
Dragerschap van het CD14 -260*T allel komt significant vaker voor bij patiënten zonder
fistels in vergelijking tot patiënten met fistels (p: 0.045, OR: 2.8, 95% CI: 1.4 – 5.8). De
analyses zijn gecorrigeerd voor geslacht en roken, maar dit leverde geen significante
verschillen op.
Aangezien de CD14-260 en TLR4+896 polymorfismen tegenstrijdige effecten hebben op
het herkennen van bacteriën, hebben we analyses uitgevoerd met de combinatie van TLR4
wildtype en dragerschap van het CD14 mutante allel. We vonden een verhoogde frequentie
van TLR4 AA en CD14*T in jonge patiënten (Vienna A1) vergeleken met patiënten ouder
dan 40 jaar (Vienna A2). Wij namen een trend waar die geen significantie bereikte (p: 0.06,
OR: 4.0, 95% CI: 1.0 – 11.9). Dragerschap van het CD14 polymorfisme, gecombineerd met
het TLR4 wildtype komt significant vaker voor in patiënten zonder fistels in vergelijking
met patiënten met fistels (p: 0.03, OR: 3.2, 95% CI: 1.2 – 8.4). Correcties voor roken en/of
geslacht hadden geen invloed op de associaties.
We hebben dragerschap van de CD14 en TLR4 polymorfismen bekeken in combinatie met
dragerschap van een of meerdere CARD15/NOD2 polymorfismen. Gecombineerd
dragerschap van de CD14*T allel met een of meerdere CARD15/NOD2 polymorfismen is
een risico factor voor het ontwikkelen van Crohn (p: 0.03, OR: 3.2, 95% CI: 1.2 – 8.4). Als
aan deze combinatie het TLR4 wildtype wordt toegevoegd in de analyses, dan levert dit een
sterkere associatie op met de ziekte van Crohn in vergelijking tot de analyses zonder
correctie voor het TLR4 wildtype (p: 0.0003, OR: 3.5, 95% CI: 1.7 – 7.0). Gecombineerd
dragerschap van de CD14 en CARD15/NOD2 polymorfismen komt significant vaker voor
bij patiënten, die een ileum resectie hebben gehad, in vergelijking tot patiënten, die geen
ileum resectie hebben gehad (p: 0.004, OR: 3.2, 95% CI: 1.5 – 6.8). Dit effect wordt licht
versterkt als de analyses worden gecorrigeerd voor het TLR4 wildtype (p: 0.002, OR: 3.4,
95% CI: 1.6 – 7.3).
De combinatie van de CD14 en CARD15/NOD2 polymorfismen en het TLR4 wildtype
komt significant vaker voor bij patiënten met een steroïden resistentie in vergelijking tot
patiënten zonder steroïden resistentie.
We hebben leeftijd, geslacht, oorsprong van de patiënt (platteland of stedelijk gebied),
roken, en dragerschap voor de diverse polymorfismen getest in een multivariaat logistisch
regressie model. Leeftijd, roken, oorsprong van de patiënt, en dragerschap van de SNP12 en
SNP13 polymorfismen is afhankelijk van elkaar geassocieerd met de ontwikkeling van de
ziekte van Crohn. Roken, oorsprong op het platteland, en dragerschap van het TLR4
polymorfisme zijn beschermende factoren, terwijl SNP12 en SNP13 risico factoren zijn.
SNP12 samen met het CD14 polymorfisme en het TLR4 wildtype zijn geassocieerd met de
ontwikkeling van fistels. SNP12 is een risico factor, terwijl de CD14 – TLR4 combinatie
beschermend werkt. SNP13 is een significante risico factor voor de noodzaak van een
ileum resectie.
De resultaten van de studies naar de ziekte van Crohn (Hoofdstukken 2 – 4) zijn
weergeven in tabel 1.
In het laatste hoofdstuk van Deel I hebben we pouchitis besproken. We vonden geen
verschillen in allel, genotype en dragerschap frequenties van de polymorfismen in de
CD14, TLR4, TLR9, CARD15/NOD2 en IRAKM genen tussen de gezonde controles en
IPAA patiënten. We vonden wel een verhoogd voorkomen van het TLR9 –1237*C allel bij
patiënten met chronisch terugkerende pouchitis in vergelijking met patiënten met
infrequente pouchitis (p: 0.028; OR: 3.2).
We hebben de TLR9 polymorfismen in haplotypen geanalyseerd zoals beschreven door
Lazarus en collega’s3. De haplotype frequenties kwamen overeen met de frequenties in de
Europees Amerikaanse populatie beschreven door Lazarus3. Haplotype III kwam vaker
voor bij patiënten met chronisch terugkerende pouchitis vergeleken met patiënten met
infrequente pouchitis (p: 0.018; OR: 3.0). Dit haplotype heeft geen unieke polymorfismen
(zgn. tag SNPs) op posities -1486 (T allel in haplotype I en III) en +1174 (G allel in
haplotypen II en III), hetgeen aangeeft dat het TLR9 –1237*C allel de sterkste associatie
heeft. De synergistische interactie tussen bacterie herkennende genen is geanalyseerd met
behulp van carrier trait analyses. Gecombineerd dragerschap voor CD14-260*T en TLR9
–1237*C komt significant vaker voor bij patiënten met chronisch terugkerende pouchitis in
vergelijking tot patiënten met infrequente pouchitis (p: 0.018; OR: 4.1). Deze combinatie
versterkt de associatie die gevonden is tussen TLR9–1237*C en patiënten met chronisch
terugkerende pouchitis.
201
Samenvatting
Polymorfisme
TLR4 +896*G
CD14 -260*C
TLR9 +2848 AA
Populatie
Cauc. Nederlands
Sp. Galicisch
Sp. Galicisch
Cauc. Nederlands
Sp. Galicisch
TLR9 -1237 CC
Sp. Galicisch
Analyse van meerdere genen:
TLR4 +896 AA + CD14-260*T
CD14 -260*T + any
CARD15/NOD2 SNP
Sp. Galicisch
Sp. Galicisch
CD14 -260*T + any
CARD15/NOD2 SNP + TLR4
+896 AA
Sp. Galicisch
Effect
Verhoogd risico op Crohn
Verlaagd risico op Crohn
Verlaagd risico op fistels
Verhoogd risico op Crohn van
de dike darm (L2)
Geassocieerd met leeftijd boven
40 jaar (A2)
Verhoogd risico op een
vernauwend phenotype (B2)
Verhoogd in patiënten met een
ileum resectie
Versterkt de bovengenoemde
associaties
Hfdst.
2
4
3
3
3
4
4
4
Komt vaker voor bij patiënten
met steroïden resistentie
202
Multivariate logistische
regressie:
TLR9 +2848 AA
Cauc. Nederlands
Sp. Galicisch
TLR9 -1237 CC
Sp. Galicisch
Smoking, origin (rural or urban)
and TLR4 +896*G
Age, and carriage of SNP12 and
SNP13
Gender and SNP12
TLR4+896 AA + CD14-260*T
SNP13
Sp. Galicisch
Verhoogd risico op Crohn van
de dike darm (L2)
Geassocieerd met leeftijd boven
40 jaar (A2)
Verhoogd risico op een
vernauwend phenotype (B2)
Verhoogd risico op fistels
Verhoogd in patiënten met een
ileum resectie
Tabel 1: Overzicht van de resultaten van de studies naar de ziekte van Crohn (Hoofdstukken 2 – 4)
3
3
4
Het urogenitale kanaal
In een studie naar de vatbaarheid voor SOA’s (Hoofdstuk 6) hebben wij een verlaagde
frequentie waargenomen van het IL-1RN+2018*C allel bij vrouwen met een positieve test
voor C. trachomatis DNA, in vergelijking tot vrouwen met een negatieve test voor
C. trachomatis DNA (p: 0.0005, OR: 1.5). Het meenemen van de serologische status voor
C. trachomatis in de analyses had geen effect op de uitkomst van de analyses. The IL-1RN
+2018*C allel komt significant minder vaak voor bij vrouwen met een positieve test voor
C. trachomatis DNA en een positieve C. trachomatis IgG serologie, in vergelijking tot
vrouwen met een negatieve test voor C. trachomatis DNA en een negatieve C. trachomatis
IgG serologie (p: 0.014, OR: 1.6). Infecties met andere microorganismen of symptomen
hadden geen effect op gevonden associaties.
Hoofdstuk 7 beschrijft de rol van CD14 in de vatbaarheid voor en ernst van beloop van
Chlamydia trachomatis infecties. De vatbaarheid voor infecties hebben we bekeken in een
populatie van vrouwen die een SOA poli bezochten. De verdeling van CD14 genotypen
was vergelijkbaar tussen patiënten en gezonde controles. C. trachomatis IgG serologie,
infecties met andere microorganismen, symptomen, of welke combinatie van factoren dan
ook, was van invloed op de resultaten.
De ernst van late complicaties van C. trachomatis infecties hebben we bekeken in een
groep vrouwen met een verminderde vruchtbaarheid, met klinisch goed gedefinieerde tuba
pathologie. De verdeling van de CD14 genotypes was vergelijkbaar tussen vrouwen met
tuba pathologie, vrouwen zonder tuba pathologie, en gezonde controles. Correcties voor
C. trachomatis IgG serologie hadden geen effect op de analyses.
Om de rol van CCR5 in het ontwikkelen van late complicaties na C. trachomatis infecties
te kunnen bestuderen hebben we een translationeel model gebruikt (Hoofdstuk 8). CCR5
knock out muizen blijken minder goed in staat om een infectie te beheersen. Deze muizen
hadden langduriger en ernstiger infecties dan hun wildtype tegenhangers. Om het effect te
bepalen van infectie op de vruchtbaarheid lieten we de muizen op verschillende tijdspunten
paren, waarmee korte en lange termijn effecten van Chlamydia infecties werden nagebootst.
Het aantal zwangerschappen in de wildtype muizen daalde tot onder de 40% als ze paarden
twee weken na de infectie, terwijl bij de CCR5KO muizen meer dan 70% zwanger raakte
(p: 0.0021). Vijf weken na de infectie was het aantal zwangerschappen 100% bij de
CCR5KO muizen, terwijl dit bij de wildtype muizen slechts 50% was. Uit deze data valt af
te leiden dat immunocompetentie een rol speelt bij de ontwikkeling van complicaties na
C. trachomatis infecties. Om deze theorie te toetsen, hebben wij cytokine en chemokine
niveaus gemeten in witte bloedcellen van deze muizen. Zowel IFN-γ als TNF-α waren
verhoogd in de wildtype muizen, terwijl de niveaus van deze cytokinen in de CCR5KO
muizen gelijk was aan de niveaus in niet geïnfecteerde muizen. De CCR5 muizen waren
minder goed in staat tot de productie van de pro-inflammatoire cytokines RANTES en
IP-10, hetgeen duidt op een verminderde Th1 immuun respons.
Om deze resultaten te kunnen vertalen naar mensen, hebben we het CCR5δ35
polymorfisme getypeerd in een groep van klinisch goed gedefinieerde vrouwen met en
zonder tuba pathologie. Tuba pathologie komt minder vaak voor bij vrouwen, die drager
203
Samenvatting
zijn van het CCR5δ35 polymorfisme. Aangezien bekend is dat Chlamydia infectie een
belangrijke risico factor is voor het krijgen van tuba pathologie, hebben we dragerschap
voor dit polymorfisme vergeleken tussen vrouwen met en zonder tuba pathologie, met een
serologische respons tegen Chlamydia trachomatis. We vonden een verlaagde frequentie
van het CCR5δ32 polymorfisme bij vrouwen met een positieve C. trachomatis IgG
serologie met tuba pathologie in vergelijking tot C. trachomatis IgG positieve vrouwen
zonder tuba pathologie.
De studie, die beschreven is in het laatste hoofdstuk van Deel II, had als doel meerdere
genen tegelijk te analyseren in de ontwikkeling van verminderde vruchtbaarheid en tuba
pathologie na C. trachomatis infectie. We hebben gekeken naar het voorkomen van
polymorfismen in de TLR9, TLR4, CD14 en CARD15/NOD2 genen in een groep van
vrouwen met verminderde vruchtbaarheid en in een specifieke subgroep van vrouwen met
verminderde vruchtbaarheid met een positieve C. trachomatis IgG serologie. Het risico
voor tuba pathology nam met gemiddeld 20% toe voor dragers van de TLR4, TLR9 en
CARD15/NOD2 polymorfismen, alhoewel dit niet statistisch significant werd. Dragerschap
van twee of meer polymorfismen in de vier bestudeerde genen had geen invloed op de
ontwikkeling van tuba pathologie in vrouwen met een negatieve C. trachomatis IgG
serologie. Echter bij vrouwen met een positieve C. trachomatis IgG serologie, verdubbelt
dragerschap van twee of meer polymorfismen in deze vier genen het risico op tuba
pathologie in vergelijking tot vrouwen met een positieve C. trachomatis IgG serologie, die
minder dan twee polymorfismen hebben in deze vier genen.
204
Samenvattend kunnen we stellen, dat de resultaten van de genetische studies naar de
geselecteerde ziektebeelden van het maag – darm kanaal en het urogenitale kanaal
aantonen, dat polymorfismen in genen, die coderen voor immuun regulerende en pathogeen
herkennende eiwitten, van invloed zijn op de vatbaarheid voor en ernst van beloop van
infecties en ontstekingen. Gen – gen, gen – pathogeen, en gen – omgeving interacties
kunnen een nog sterker effect hebben. Bij het analyseren van de resultaten en het
vergelijken van de resultaten van verschillende studies, moet rekening gehouden worden
met de complexe samenstelling van beide orgaansytemen.
References
1.
2.
3.
Single-nucleotide polymorphisms in the Toll-like receptor 9 gene (TLR9): frequencies, pairwise linkage disequilibrium,
Silverman, Fernando Martinez & Scott T. Weiss, Genomics 2003; 81 (1): pp. 85 - 91, PubMed: 12573264
205
Samenvatting
206
If a messy desk is proof of a messy mind,
Then what can I say of an empty desk?
Addendum
207
Addendum
urriculum Vitae
C
208
Sander Ouburg werd geboren op 20 December 1976 in Amsterdam. Na zijn eindexamen op
het Vossius Gymnasium in Amsterdam in 1996, besloot hij zijn fascinatie voor de medische
en genetische kant van de biologie om te zetten in een studie Medische Biologie aan de
Universiteit van Amsterdam (UvA). Tijdens deze studie legde hij zich toe op microbiële
infecties, immunologie en genetica. Hij liep stage op de afdeling Celbiologie van het AMC
in Amsterdam, waar hij zich bezig hield met de regulatie van IL-12Rβ2 door IL-13. Zijn
tweede stage liep hij op het streeklaboratorium van de GG&GD in Amsterdam, waar hij
zich richtte op de rol van IL-1B en IL-1RN polymorfismen in Chlamydia trachomatis
infecties. In augustus 2002 studeerde hij af aan de UvA, waarna hij tot eind 2002
meewerkte aan het implementeren van een kwaliteit systeem op het streeklaboratorium van
de GG&GD. Dankzij de financiële steun van AstraZeneca (verkregen door Elly
Klinkenberg-Knol) kon hij in Januari 2003 beginnen aan zijn promotie onderzoek op de
afdeling Gastroenterologie en op het laboratorium voor Immunogenetica aan de Vrije
Universiteit in Amsterdam. Het laboratorium was samen met de ACTA nauw betrokken bij
het InfoBioMed project, een Europees consortium met als doel het bevorderen van Bio
Medische Informatica (BMI), en intergatie en samenwerking binnen Europa
(www.infobiomed.org). InfoBioMed heeft in 2005 de “1st InfoBioMed Training
Challenge” georganiseerd. Deze training challenge had als doel de uitwisseling van ideeën
en de dialoog tussen verschillende disciplines te bevorderen. Na een strenge selectie
procedure was Sander een van de tien Europese AIOs, die geselecteerd werden uit alle
aanmeldingen voor deelname.
Daarnaast heeft AstraZeneca hem in staat gesteld verschillende cursussen te volgen op hun
kantoor in Zoetermeer, waaronder statistiek en wetenschappelijk schrijven in het Engels.
Al sinds zijn stage op het streeklaboratorium wordt Sander begeleidt door Servaas Morré,
zijn co-promotor.
Het resultaat van zijn promotie onderzoek ligt voor U in de vorm van dit proefschrift, dat
hij zal verdedigen op 18 December 2006.
Curriculum Vitae
urriculum Vitae in English
C
Sander Ouburg was born on December 20 1976, in Amsterdam. After graduating from high
school (Vossius Gymnasium) in Amsterdam, he decided to turn his fascination for the
medical and genetic side of biology into a study of Medical Biology at the University of
Amsterdam (UvA). During his study he specialised in microbial infections, immunology
and genetics. His first internship was at the department of Cell biology of the AMC in
Amsterdam, where he worked on the regulation of IL-12Rβ2 by IL-13. His second
internship was at the public health laboratory of the Municipal Health Service (GG&GD) in
Amsterdam, where he focussed on the role of IL-1B and IL-1RN polymorphisms in
Chlamydia trachomatis infections. After graduating from the university in August 2002, he
helped in the implementation of a quality control system at the public health laboratory
until the end of 2002. With the financial support of AstraZenenca (granted to Elly
Klinkenberg-Knol), he was able to start his PhD study at the department of
Gastroenterology and the Laboratory of Immunogenetics of the VU university in
Amsterdam in January 2003. The laboratory of Immunogenetics collaborated closely with
the ACTA in the InfoBioMed project, a European consortium aiming to promote Bio
Medical Informatics (BMI) and integrative collaboration within Europe
(www.infobiomed.org). InfoBioMed organised the “1st InfoBioMed Training Challenge” in
September 2005. The goal of this training challenge was to promote the exchange of views
and the dialogue between disciplines. After a strict selection procedure, Sander was one of
the ten European PhD students selected out of all applications for participation and invited
to participate in the training challenge.
AstraZeneca has enabled him to take part in PhD courses at their office in Zoetermeer,
including statistics and scientific writing in English.
Ever since his internship at the public health laboratory Sander has been supervised and
guided by Servaas Morré, his co-promotor
The result of his PhD study lies before you in the form of this thesis, which he will defend
on December 18, 2006.
209
Addendum
ublications
P
1.
The true ligand of the NOD2 receptor is peptidoglycan instead of lipopolysaccharide: a
schematic representation of ligand-receptor interactions and NF-kappa B activation,
Servaas A. Morré, Sander Ouburg, Elly C. Klinkenberg-Knol, Chris J. J. Mulder & A.
Salvador Peña, Gastroenterology 2004; 126 (1): pp. 371 - 373, PubMed: 14753217
(Introduction)
2.
The toll-like receptor 4 (TLR4) Asp299Gly polymorphism is associated with colonic
localisation of Crohn's disease without a major role for the Saccharomyces cerevisiae
mannan-LBP-CD14-TLR4 pathway, Sander Ouburg, Rosalie Mallant-Hent, J. Bart A.
Crusius, Ad A. van Bodegraven, Chris J. J. Mulder, Ronald Linskens, A. Salvador
(Chapter 2)
3.
Host inflammatory response and development of complications of Chlamydia trachomatis
genital infection in CCR5-deficient mice and subfertile women with the CCR5delta32 gene
deletion, Erika L. Barr, Sander Ouburg, Joseph U. Igietseme, Servaas A. Morré, Edith
Okwandu, Francis O. Eko, Godwin Ifere, Tesfaye Belay, Qing He, Deborah Lyn, Gift
Nwankwo, James Lillard, Carolyn M. Black & Godwin A. Ananaba, Journal of
Microbiology, Immunology & Infection 2005; 38 (4): pp. 244 - 254, PubMed: 16118671
(Chapter 8)
4.
210
A candidate gene approach of immune mediators effecting the susceptibility to and severity
of upper gastrointestinal tract diseases in relation to Helicobacter pylori and Epstein-Barr
virus infections, Sander Ouburg, J. Bart A. Crusius, Elly C. Klinkenberg-Knol, Chris J.
J. Mulder, A. Salvador Peña & Servaas A. Morré, European Journal of Gastroenterology
and Hepatology 2005; 17 (11): pp. 1213 - 1224, PubMed: 16215434
(Introduction)
5.
Combined carriership of TLR9-1237C and CD14-260T alleles enhances the risk of
developing chronic relapsing pouchitis, Karen M. Lammers, Sander Ouburg, Servaas A.
Morré, J. Bart Crusius, Paolo Gionchetti, Fernando Rizzello, Claudia Morselli,
Elisabetta Caramelli, Roberto Conte, Gilberto Poggioli, Massimo Campieri & A.
Salvador Peña, World Journal of Gastroenterology 2005; 11 (46): pp. 7323 - 7329,
PubMed: 16437636
(Chapter 5)
6.
The CD14 functional gene polymorphism –260 C>T is not involved in either the
susceptibility to Chlamydia trachomatis infection or the development of tubal pathology,
Sander Ouburg, Joke Spaargaren, Janneke E. den Hartog, Jolande A. Land, Han S. A.
Fennema, Jolein Pleijster, A. Salvador Peña, Servaas A. Morré & ICTI consortium,
(Chapter 7)
7.
Do host genetic traits in the bacterial sensing system play a role in the development of
Chlamydia trachomatis-associated tubal pathology in subfertile women?, Janneke E. den
Hartog, Sander Ouburg, Jolande A. Land, Joseph M. Lyons, Jim I. Ito, A. Salvador
Peña & Servaas A. Morré, BMC Infectious Diseases 2006; 6 (1): pp. 122, PubMed:
16859562
(Chapter 9)
Publications
ubmitted or in preparation
S
1.
Polymorphisms in the immune regulatory genes IL-1B & IL-1RN and the bacterial sensing
genes CD14 & TLR4 are associated with Barrett oesophagus, Sander Ouburg, Marieke
Emonts, A. Salvador Peña, Agnieszka M. Rygiel, Kausilia K. Krishnadath, Peter W.M.
Hermans, Jacques G.H.M. Bergman, Chris J.J. Mulder, Elly C. Klinkenberg-Knol &
Servaas A. Morré
(Chapter 1)
2.
The role of the bacterial CpG sensing toll-like receptor 9 (TLR9) in Dutch Caucasian and
Spanish Galician patients with Crohn’s disease: evidence for genetic heterogeneity, Sander
Ouburg, Manuel Barreiro, J. Bart A. Crusius, Ad A. van Bodegraven, J. Enrique
Dominguez-Muñoz, A. Salvador Peña & Servaas A. Morré
(Chapter 3)
3.
CD14 and TLR4 gene polymorphisms in Galician patients with Crohn’s disease: genetic
and environmental interactions, Sander Ouburg, Manuel Barreiro de Acosta, A.
Salvador Peña, Aurelio Lorenzo, J. Enrique Domínguez-Muñoz & Servaas A. Morré
(Chapter 4)
4.
The first strong genetic susceptibility marker for Chlamydia trachomatis infections: The
interleukin 1 receptor antagonist IL-1RN +2018 T>C gene polymorphisms, Joke
Spaargaren, Sander Ouburg, Han S.A. Fennema, A. Salvador Peña, Servaas A. Morré
(Chapter 6)
211
Addendum
uthors & Affiliations
A
Name, Surname(s)
Chapter
Affiliation
Ananaba, Godwin A.
8
Clark Atlanta University,
Atlanta, GA, USA
Barr, Erika L.
8
Atlanta, GA, USA
Barreiro de Acosta, Manuel
3, 4
Dept. of Gastroenterology, University Hospital,
Santiago de Compostela, Spain
Belay, Tesfaye
8
Atlanta, GA, USA
Bergman, Jacques G.H.M.
1
Dept. of Gastroenterology and Hepatology, Academical Medical Centre,
Amsterdam, The Netherlands
Black, Carolyn M.
8
Center for Disease Control and Prevention,
Atlanta, GA, USA
Bodegraven, Ad A. van
212
2, 3
Dept. of Gastroenterology, VU University Medical Centre,
Campieri, Massimo
5
Department of Internal Medicine and Gastroenterology, Policlinico S. Orsola,
University of Bologna,
Bologna, Italy
Caramelli, Elisabetta
5
Institute of Histology and General Embryology, University of Bologna,
Bologna, Italy
Conte, Roberto
5
Department of Immunohaematology and blood transfusion, Policlinico S. Orsola,
Bologna, Italy
Crusius, J. Bart A.
Intro, 2, 3, 5
Laboratory of Immunogenetics, Dept. of Pathology, VU University Medical Centre,
Dominguez-Muñoz, J. Enrique
3, 4
Eko, Francis O.
8
Morehouse School of Medicine,
Atlanta, GA, USA
Emonts, Marieke
1
Laboratory of Paediatrics, Erasmus MC,
Rotterdam, The Netherlands
Fennema, Johan S.A.
STI Outpatient Clinic, Cluster of Infectious Diseases, Municipal Health Service,
Amsterdam, Netherlands
6, 7
Authors & Affiliations
Name, Surname(s)
Chapter
Affiliation
Gionchetti, Paolo
5
Bologna, Italy
Hartog, Janneke E. den
7, 9
Department of Obstetrics and Gynaecology, Research Institute Growth and
Development (GROW), Academisch Ziekenhuis Maastricht,
Maastricht, The Netherlands
Maastricht University,
He, Qing
8
Atlanta, GA, USA
Atlanta, GA, USA
Hermans, Peter W.M.
1
Dept. of Paediatrics, University Medical Center St. Radboud,
Nijmegen, The Netherlands
ICTI Consortium,. The
7
The ICTI consortium (Integrated approach to the study of Chlamydia trachomatis
Infections) provides a broad specialized network for the multidisciplinary studies
described
Ifere, Godwin
8
Atlanta, GA, USA
Igietseme, Joseph U.
8
Atlanta, GA, USA
Atlanta, GA, USA
Ito, James I.
9
Department of Infectious Diseases, City of Hope National Medical Center and
Beckman Research Institute,
Duarte, CA,USA
Klinkenberg-Knol, Elly C.
Intro, 1
Krishnadath, Kausilia K.
1
Dept of Gastroenterology and Hepatology, Academical Medical Centre,
Land, Jolande A.
7, 9
Department of Obstetrics and Gynaecology, Research Institute Growth and
Development (GROW), Academisch Ziekenhuis Maastricht,
Maastricht University,
Department of Obstetrics and Gynecology, University Medical Center Groningen,
Groningen, The Netherlands
Lammers, Karen M.
Bologna, Italy
5
213
Addendum
Name, Surname(s)
Chapter
Affiliation
Lillard, James
8
Atlanta, GA, USA
Linskens, Ronald K.
2
Lorenzo, Aurelio
3, 4
Lyn, Deborah
8
Atlanta, GA, USA
Lyons, Joseph M.
9
Department of Infectious Diseases, City of Hope National Medical Center and
Beckman Research Institute,
Duarte, CA,USA
Nwankwo, Gift
8
Atlanta, GA, USA
Mallant-Hent, Rosalie
2
Morré, Servaas A.
214
Intro, 1 – 9
Laboratory of Immunogenetics, Dept. of Pathology &
Dept. of Internal Medicine, VU University Medical Centre,
Dept. of Medical Microbiology, Academic Hospital Maastricht,
Morselli, Claudia
5
Bologna, Italy
Mulder, Chris J.J.
Intro, 1, 2
Okwandu, Edith
8
Atlanta, GA, USA
Ouburg, Sander
Intro, 1 – 9
Peña, A. Salvador
Intro, 1 – 7, 9
Pleijster, Jolein
Laboratory of Immunogenetics, Dept. of Pathology, VU University Medical Centre,
7
Name, Surname(s)
Chapter
Affiliation
Poggioli, Gilberto
5
Department of Surgery and organ transplantation, Policlinic S. Orsola,
Bologna, Italy
Rizzello, Fernando
5
Bologna, Italy
Rygiel, Agnieszka M.
1
Laboratory of Experimental Internal Medicine, Academical Medical Centre,
Spaargaren, Joke
6, 7
Laboratorium Microbiologie Twentse Achterhoek (LABMICTA).
Enschede, The Netherlands
215
Addendum
INTERNATIONAL JOURNAL OF GRATITUDE AND ACKNOWLEDGEMENTS, DECEMBER 2006, pp. 1 – 5
2006-1218/00/$3E.48+0
Copyright © 2006, International Society of G&A. All rights reserved.
Received October 23, 2006 – Accepted December 18, 2006
Vol. 1, No. 1
Becoming a scientist: an acknowledgment
SANDER OUBURG, PhC, ABD1, 2, 3, #
1
Laboratory of Immunogenetics, dept. of Pathology & dept. of Gastroenterology,
VU University Medical Centre, Amsterdam, The Netherlands
2
Institute of Gratitude, Mijdrecht, The Netherlands
3
Secretary of the Special Union for Consistent Kindness and Ubiquitous Politeness
Abstract
Credits or acknowledgments are an important part of any major achievement. The aim of the current manuscript is
to identify those persons and groups eligible for acknowledgments using the Systematic Acknowledger Index
(SAI), and to subsequently define appropriate acknowledgements.
This study identified and acknowledges forty-nine persons, divided in 12 groups. We concluded that the writing of
a thesis requires a multi-person approach.
216
Introduction
As defined in Wikipedia, the free encyclopaedia
[http://en.wikipedia.org/wiki/Acknowledgment_%28cr
eative_arts%29] in the creative arts and scientific
literature, an acknowledgment (also spelled
acknowledgement) is an expression of gratitude for
assistance in creating the work presented. Receiving
credit by way of acknowledgment rather than
authorship indicates that the person or organization did
not have a direct hand in producing the work in
question, but may have contributed funding, criticism,
or encouragement to the author(s). Various schemes
exist for classifying acknowledgments; Giles &
Councill1 proposed the six categories, presented in
table 1.
1
2
3
4
5
6
Apart from citation, which is not usually considered to
be an acknowledgment, acknowledgment of conceptual
support is widely considered to be the most important
for
identifying
intellectual
debt.
Some
acknowledgments of financial support, on the other
hand, may simply be legal formalities imposed by the
granting institution. However, for writing a thesis,
acknowledgements to those who are not involved in the
conceptual support of the thesis are as important as
those who are.
Aim of the current manuscript is to identify those
persons and groups eligible for acknowledgments using
the Systematic Acknowledger Index (SAI), and to
subsequently define appropriate acknowledgements.
Acknowledgeable support
Moral support
Financial support
Editorial support
Presentational support
Instrumental/technical support
Conceptual support, or Peer Interactive Communication (PIC)
Table 1: Acknowledgment scheme according to Giles & Councill1
Material & Methods
A total of 49 acknowledgeable persons were collected
during the PhD study (January 2003 – December
2006).
Selection of the study participants: Participants were
selected according to the criteria of Giles and
Councill1, with the addition of one group:
# Corresponding Author: Sander Ouburg, Laboratory of
Immunogenetics, Dept. of Pathology, VU University Medical
Centre, De Boelelaan 1117. Postbus 7057, 1007 MB
Amsterdam, The Netherlands. Tel.: +31 - (0)20 - 4448417.
Fax: +31 - (0)20-4448418. [email protected].
7) humorous support. Since humour can be an
important factor in a PhD students health and sanity.
The cohort was subdivided according to the following
criteria:
Promotors and co-promotors: Those involved in the
supervision of the study.
Promotion committee: Those critically reading and
commenting on the thesis.
Generosity: Those providing the financial support
during the PhD study.
Colleagues: Those working in collaboration with the
PhD student.
Inspirational persons: Those who have left a lasting
impression.
Fellow PhD students: Those who are working on their
respective PhD studies
Students: Those who had their internships at the
laboratory or in collaboration with the laboratory and
who have had support or supervision of the PhD
student
Family & Friends: Those directly or indirectly related
to the PhD student & those considered being friends of
the PHD student.
Paranimphs: Those courageous enough to walk down
that long aisle with me towards that big stage, where
my defence will be held.
Miscellaneous: Microbial: Those microbes directly or
indirectly related to the observed clinical phenotype;
Donors: All those persons – patients and healthy
controls – who have voluntarily donated the bodily
materials used in the studies.
LFF: Loveable Furry Friends.
Jolein: A small but very significant group consisting of
Jolein Pleijster
Statistical analyses:
Groups and subgroups were compared by any
statistical method available (Abacus, version 1.0;
www.asianideas.com). Corrections for multiple testing
were considered unnecessary. A p value <0.05 was
considered significant.
Results
Promotors and co-promotors: Four people have
dedicated a considerable amount of their precious time
to guide me during these years. Each of them an
honoured expert in his/her respective field of expertise.
Salvador, It is hard to find a more kind and modest
person than you. Your knowledge is only surpassed by
your kindness. You were always hard at work, but
always seemed to find time for everybody to help
them, and to give kind words and advice. Thank you
for your time and guidance.
Chris, you came in charge of the department of
Gastroenterology soon after I had started my PhD.
Your guidance was as everything you do:
straightforward and always with your goal in mind.
Thank you for all your help and advice.
Elly, it has been a pleasure working with you. If ever
data was needed on a patient, your meticulously kept
records were a blessing. I wish I would have had more
time to learn from you, because I think you would have
broadened my horizon considerably. Thank you for
everything you have done for me.
Servaas, it will be hard to find the proper words for
you. As my daily supervisor, you could sometimes be
my proverbial pain in the posterior region. Either for
the work you gave me or for kicking the said posterior
region when I was getting lazy ☺. Your drive and
scientific passion always made sure that there was
more than enough work to do, but you also created
opportunities for me to go to courses, meetings and
symposia, making sure that whenever we were
somewhere together I was introduced to everyone of
importance (at least two or three or four times ☺). You
introduced me to grants and management work,
experiences a PhD student rarely encounters and which
will help me in my future career. Although working
with you can be demanding, it is surprising, full of
experiences and we have had a lot of fun. You have
helped me all the way to obtaining my PhD and for that
I will always be grateful.
Promotion Committee: Six experts from the
Netherlands and abroad – Michael Ward, Joseph
Lyons, Jolande Land, Chris Meijer, Gerrit Meijer, and
Paul Savelkoul – have read and evaluated this thesis. I
would like to thank them for their time and efforts, and
their judgment, given in such a short time period.
Generosity: The work that has been presented in this
thesis and the publication of this thesis would not have
been possible without financial support.
AstraZeneca Nederland BV, personified by Andrea
Sellink and José van den Berg, has provided me with
the opportunity to do my PhD study, attend courses and
meetings, and publish this thesis. Andrea and José, you
have always been very kind to me and you have helped
me realise this dream. For this I will always be
grateful.
Greiner Bio-One, personified by Jeroen Molenaar, has
provided most of the high quality laboratory plastics
used in the experiments described in this thesis and has
generously contributed to the publication of this thesis.
Jeroen, thank you for all your efforts and the fun
during your visits.
My parents, Eric and Marga, and my parents-in-law,
Herman and Ria, have, apart from the moral and
emotional support, generously contributed to the
publication of this thesis.
I would like to thank the CWI for the unemployment
fee I have received since May 2006.
I would like to thank all sponsors who have contributed
to this thesis, after the writing of this manuscript and
who do not feature in this acknowledgement.
Colleagues: Bart, the walking encyclopedia. Your
knowledge of the literature is formidable. Although
your directions could sometimes be a little cryptic:
“You need that article by ‘die dunne en die Griek’ (the
thin man and the Greek)”, a search through your
archive resulted in the discovery of the article by Johan
den Dunnen and Stylianos Antonarakis. Thanks for all
the help.
Roel and Dion, you both provided the fun on the
laboratory. I very much enjoyed working with you
both.
Marja Laine, paljon kiitoksia for your tine and helping
me with the statistical analyses.
Jolande land, thank you for allowing us to use the
subfertility cohort, for your useful comments and
critical reading of the manuscripts and taking place in
217
Addendum
218
the promotion committee.
Henry de Vries, thank you for all your efforts for the
Chlamydia studies, presented in the second part of my
thesis.
Marieke Emonts and Adri van der Zande, thank you for
the collection of samples and data, the useful
discussions and help with the manuscripts.
Pa, Moe, Jeroen, Judith, Oma, Wim, Janet, Jolein,
Herman, Ria, Roland, Mirjam, and Helena, you all
have been interested in the work I have been doing in
the past years and even though sometimes it seemed
mysterious and sorcery to you, you have always
supported me in my work. Thank you all for
everything.
Inspirational persons: Sometimes one is lucky to
meet a person who inspires you. I have been fortunate
to have met three such inspirational persons. Michael
Ward, Joseph Igietseme and Joseph Lyons, you have
shown me that great wisdom and intelligence can be
wrapped in kindness and friendliness. In our meetings
you have inspired me and I have learned much.
Joseph Lyons: Joe, you have read my thesis in a very
short time and supplied me with a torrent of useful
comments and suggestions, which all have added value
and improvements to my thesis. When you’re visiting
in December we’ll have some oliebollen.
Michael, I have very much enjoyed our tour of
Amsterdam and visiting the Star Pilot. Talisker is
waiting to meet us in December.
Paranimphs: Roel and Helena, thank you for all the
help in this last stretch of my PhD study.
Fellow PhD students:
Joke Spaargaren, your thesis is already at the printer at
the time of the preparation of this manuscript. I wish
you all the luck with the defence of your thesis on
December 12. You were my supervisor at the public
health laboratory and afterwards we have together
continued working on Chlamydia. Thank you for all
you have done for me.
Janneke den Hartog, I have always enjoyed working
with you. I wish you all the luck with your thesis
defence in 2007. I will help you wherever I can, to
repay you for all you have done for me. And remember
what you called the “Amsterdamse instelling”: Positive
thinking gets you through everything.
Agnieszka Rygiel, serdecznie dziękuję for all the help
with the Barrett cohort.
Erica Barr, I very much enjoyed meeting you at the
ISHCI in Niagara-on-the-Lake. I am certain that you
will excel at your thesis defence. Hopefully, we’ll meet
again in the future, no longer ABD, but PhDs! ☺
Arnold Catsburg, your time as PhD student is just
starting, but I am confident you will succeed. Thank
you for your help with the Chlamydia studies.
Vitaly Smelov, спасибо for your kindness and удачи
with your freshly started PhD study.
Students: A great Roman philosopher once said:
“Homines dum docent discunt”. People learn by
teaching (Lucius Annaeus Seneca, Epistulae morales
ad Lucilium 7, 8). Liselotte Kornman, Erik van den
Akker, and Arisja Mauritz, I hope I have been able to
teach you as much as I have learned from teaching you.
I have enjoyed working with you all.
Family & Friends: Family and friends are always
important and one of the greatest sources of support.
Miscellaneous: Donors: None of the research
presented in this thesis was possible without all those
anonymous patients and healthy controls who allowed
us to use their bodily materials for research. Microbial:
The author would like to thank Chlamydia trachomatis
for its ubiquitous lifestyle and for creating such
differences in clinical phenotypes, which has been
keeping scientists around the world busy for years. I
would like to thank Helicobacter pylori for its
interesting infection pattern and keeping the discussion
on its influence on upper gastrointestinal tract
pathogenesis alive. I would like to thank the
commensal flora and ubiquitous microbes for their
presence and for making the scientific puzzle a little
more complex and interesting.
LFF: Pets are a source of joy and unconditional love.
Malina, Remi, Hammie, and Czarnutka have been no
exception.
Jolein: The smallest group, but with the most
significant impact. Joleintje, you have loved,
supported, and helped me through all these years. Even
in this last period when I was locked up in my attic for
days on end, I could always count on you. This hectic
PhD fellow period has finally come to an end and we
can look forward to the hectic period of preparations
for our marriage on April 17, 2007 ☺ ♥.
Dediscere human est. To forget is human. There is a
good statistical chance that I have forgotten to mention
someone in this manuscript. If you did not find your
name in this manuscript, please be aware that it is not
intentional. You have my gratitude.
An overview of the person distribution in the different
subgroups is presented in table 2. There was a
significant overlap between the groups, i.e. persons
who were placed in a group according to the selection
criteria had an increased risk of also belonging to
another selection group, compared to those persons
who did not fit the selection criteria (p: 0.0000001, OR:
20.3, 95% Confidence Interval: 2.01 – 2.02).
Discussion
Using a systematic approach, we have identified 49
persons with acknowledgeable contributions to the
studied thesis. We observed a statistical increased risk
Group
Promotors and Co-promotors
Promotion Committee
Generosity
Colleagues
Inspirational persons
Fellow PhD students
Students
Family & Friends
Paranimphs
Miscellaneous
LFF
Jolein
Number
4
6
8
8
3
6
3
13
2
N/A
4
1
%
8.16
12.24
16.33
16.33
6.12
12.24
6.12
26.53
4.08
–
8.16
2.04
Table 2: Person distribution of the acknowledgeable persons in the different subgroups. N/A: Not available.
of belonging to multiple subgroups in persons fitting
the selection criteria, compared to those not fitting the
selection criteria. From these results we can conclude
that a PhD study and a thesis are indeed multi-person
approaches.
None of the work presented in the thesis would have
been possible without the collaborative efforts of the
all persons mentioned in this manuscript.
The results of the subgroup analyses indicate that there
is a significant need for a better subgroup definition.
We are currently researching the possibilities for more
stringent subgroup definitions. Our current proposal,
available at www.specialunionforconsistentkindness
andubiquitouspoliteness.nl, results in the identification
of 49 individual subgroups without significant overlap.
After a thorough review of the literature, we can
conclude that we are the first to report these findings of
this thesis.
known as the linguistic style, or in a physical
acknowledgment style. An example of the linguistic
style has been presented in the results section of this
manuscript.
Examples of the most common forms of the physical
style are presented in figure 1.
The tri-morphic stages of the liquid kind of
acknowledgment differs significantly from the dimorphic lifecycle of Chlamydia and the mono-morphic
lifestyle of other bacteria, including Helicobacter
pylori. Persons receiving the liquid kind of physical
acknowledgment (figure 1A) are at increased risk of
developing late complications, including a hang-over2.
The second most common type of physical
acknowledgment is the hidden type (figure 1B). This
type uses a cloaking technique to obscure the contents
from the receiver and to induce curiosity in the
receiver. The tentacle–like structure at the top is
intended to lure the receiver to open the
acknowledgment at that end, but may also be intended
to test the knotical prowess of the receiver. The boxlike shape and the use of cloaking technology lead us
to believe that this technology might be obtained from
the Borg. This is currently being researched.
In conclusion, we have identified a large number of
acknowledgeable
persons
and
groups.
Acknowledgements can be presented in different
forms. Further research in PhD studies is required to
confirm our results.
Figure 1: Physical style of acknowledgments. Panel A:
Physical acknowledgment of the liquid kind. Please note the
tri-morphic appearance, when encountered in its natural
environment. Panel B: Physical acknowledgment of the hidden
kind. This type is known to employ cloaking technology
commonly known as gift wrapping
Acknowledgements have two distinct styles. They can
be presented as written or verbal acknowledgments,
Acknowledgements
Sander Ouburg was supported by a PhD fellowship
from AstraZeneca Nederland BV, from January 2003
to April 2006. The authors would like to acknowledge
several websites – including http://www.elite.net/
~runner/jennifers/thankyou.htm, http://www.anglik.net/
polish_phrases.htm, and http://www.proz.com/kudoz/
57077#136882 – for the non-Dutch / non-English
phrases used in the manuscript (and I sure hope the
translations are correct ☺).
The general concept for this manuscript has been
219
Addendum
adapted from an article by Servi Stevens and
Antoinette Brink3, written for the wedding cook book
of S.A. Morré and C.J.M. Böhmer. The manuscript is
available upon request from the corresponding author
of the current manuscript.
The authors would like to thank dr. Danser Obrugu and
dr. Revassa Romér for critically reading the manuscript
and providing useful comments. The authors are
indebted to Felis Malina for refraining from walking on
my keyboard during the writing of this article.
References
1.
2.
3.
220
Who gets acknowledged: measuring scientific
contributions through automatic acknowledgment
indexing, C. Lee Giles & Isaac G. Councill,
Proceedings of the National Academy of Sciences
of the United States of America 2004; 101 (51):
pp. 17599 – 17604, PubMed:: 15601767
http://www.jellinek.nl/
Home-made pasta versus commercially available
pasta: qualitative, culinary and nutritional
aspects, Servi J.C. Stevens & Antoinette A.T.P.
Brink, Journal of Culinary Methods 2000; 1 (1):
pp. 1 – 2
221
To achieve great things,
Two things are needed:
A plan
And not quite enough time
Leonard Bernstein
I will hereby acknowledge the request of my co-promotor, dr. Servaas Morré, not to have
more pages in my thesis than he had in his.
222
223
224

Immunogenetics of Inflammation and Infection in the Gastointestinal

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