Immunogenetics of Infection and Inflammation in the Gastrointestinal 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: Sander Ouburg Druk: 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 INFLAMMATION AND INFECTION IN THE GASTROINTESTINAL AND UROGENITAL TRACTS

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: prof.dr. A.S. Peña prof.dr. C.J.J. Mulder copromotoren: 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

Introduction 11

Aims and Outline 31

Part I 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 63 The role of the bacterial CpG sensing toll-like receptor 9 (TLR9) in Dutch 6 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 83 Combined carriership of TLR9-1237*C and CD14-260*T alleles enhances the risk of developing chronic relapsing pouchitis

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 189 Samenvatting 197

Addendum 207 Curriculum Vitae 208 Curriculum Vitae in English 209 Publications 210 Author affiliations 212 Acknowledgments 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

INFLAMMATION AND INFECTION

IN THE GASTROINTESTINAL AND UROGENITAL TRACTS

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. G 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.

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? 13

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

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.

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 14 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

to the thesis 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 15 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.

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.

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Chlamydia trachomatis Biovar Serovars Host Site of Inflammation Trachoma A, B, Ba, C Human Conjunctivae Urogenital tract (rare) D, Da, E, F, G, Ga, H, I, Ia, J, K Human Urogenital tract Conjunctivae Respiratory tract (rare) LGV L1, L2, L2a, L3 Human Inguinal lymph nodes Urogenital tract Rectum Mouse 1 Mouse Respiratory tract (MoPn agent, currently known as C. muridarum) 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). 17

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.

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”.

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 18 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. 19 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.

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.

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 20 (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).

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 D- isoGln 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 22 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-to- measure’ treatment of individual patients.

23

Introduction

References

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to the thesis

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Introduction

receptor antagonist and anti-IL-1 receptor monoclonal antibody, Kim W. McIntyre, George J. Stepan, Kenneth D. Kolinsky, William R. Benjamin, Joseph M. Plocinski, Kimberlee L. Kaffka, Caroline A. Campen, Richard A. Chizzonite & Patricia L. Kilian, Journal of Experimental Medicine 1991; 173 (4): pp. 931 - 939, PubMed: 1826128 51. Immunoregulation in the gut: success and failures in human disease, I. Monteleone, P. Vavassori, L. Biancone, G. Monteleone & F. Pallone, Gut 2002; 50 (Suppl 3): pp. III60 - III64, PubMed: 11953335 52. Two novel IL-1 family members, IL-1 delta and IL-1 epsilon, function as an antagonist and agonist of NF-kappa B activation through the orphan IL-1 receptor-related protein 2, Reno Debets, Jackie C. Timans, Bernhard Homey, Sandra Zurawski, Theodore R. Sana, Sylvia Lo, Janet Wagner, Gina Edwards, Teresa Clifford, Satish Menon, J. Fernando Bazan & Robert A. Kastelein, Journal of Immunology 2001; 167 (3): pp. 1440 - 1446, PubMed: 11466363 53. 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to the thesis

73. 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 74. Deficient host-bacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn's disease and ulcerative colitis, D. Franchimont, Séverine Vermeire, H. El Housni, M. Pierik, K. Van Steen, T. Gustot, E. Quertinmont, M. Abramowicz, A. van Gossum, J. Deviere & Paul Rutgeerts, Gut 2004; 53 (7): pp. 987 - 992, PubMed: 15194649 75. Human toll-like receptor 4 mutations but not CD14 polymorphisms are associated with an increased risk of gram- negative infections, Doreen M. Agnese, Jacqueline E. Calvano, Sae J. Hahm, Susette M. Coyle, Siobhan A. Corbett, Steve E. Calvano & Stephen F. 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Introduction

94. Sp1 is a critical factor for the monocytic specific expression of human CD14, Dong-Er Zhang, Christopher J. Hetherington, Shencao Tan, Suzan E. Dziennis, David A. Gonzalez, Hui-Min Chen & Daniel G. Tenen, Journal of Biological Chemistry 1994; 269 (15): pp. 11425 - 11434, PubMed: 7512565 95. C(-260)-->T polymorphism in the promoter of the CD14 monocyte receptor gene as a risk factor for myocardial infarction, Jaroslav A. Hubacek, G. Rothe, Jan Pit'ha, Zdena Škodová, Vladimir Stanek, Rudolf Poledne & Gerd Schmitz, Circulation 1999; 99 (25): pp. 3218 - 3220, PubMed: 10385492 96. Interaction of polymorphisms in the CARD15 and CD14 genes in patients with Crohn disease, Wolfram Klein, Andreas Tromm, Thomas Griga, Christian Folwaczny, Michael Hocke, Klaus Eitner, Michaela Marx, Natascha Duerig & Jörg Thomas Epplen, Scandinavian Journal of Gastroenterology 2003; 38 (8): pp. 834 - 836, PubMed: 12940436 97. Ulcerative colitis is associated with a promoter polymorphism of lipopolysaccharide receptor gene, CD14, N. Obana, S. Takahashi, Y. Kinouchi, K. Negoro, S. Takagi, N. Hiwatashi & T. Shimosegawa, Scandinavian Journal of Gastroenterology 2002; 37 (6): pp. 699 - 704, PubMed: 12126249 98. A polymorphism in the CD14 gene is associated with Crohn disease, Wolfram Klein, Andreas Tromm, Thomas Griga, Harald Fricke, Christian Folwaczny, Michael Hocke, Klaus Eitner, Michaela Marx, Natascha Duerig & Jörg Thomas Epplen, Scandinavian Journal of Gastroenterology 2002; 37 (2): pp. 189 - 191, PubMed: 11843056 99. CD14 and TNFa promoter polymorphisms in patients with acute arthritis. Special reference to development of chronic spondyloarthropathy, Heikki Repo, Krista Anttonen, Sami K. Kilpinen, Aarno Palotie, Petri Salven, Arto Orpana & Marjatta Leirisalo-Repo, Scandinavian Journal of Rheumatology 2002; 31 (6): pp. 355 - 361, PubMed: 12492251 100. 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Schall, Dan R. Littman & Nathaniel R. Landau, Nature 1996; 381 (6584): pp. 661 - 666 104. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5, Tatjana Dragic, Virginia Litwin, Graham P. Allaway, Scott R. Martin, Yaoxing Huang, Kirsten A. Nagashima, Charmagne Cayanan, Paul J. Maddon, Richard A. Koup, John P. Moore & William A. Paxton, Nature 1996; 381 (6584): pp. 667 - 673, 28 PubMed: 8649512 105. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene, Michel Samson, Frédérick Libert, Benjamin J. Doranz, Joseph Rucker, Ccorinne Liesnard, Claire-Michèle Farber, Sentob Saragosti, Claudine Lapouméroulie, Jacqueline Cognaux, Christine Forceille, Gaetan Muyldermans, Chris Verhofstede, Guy Burtonboy, Michel Georges, Tsuneo Imai, Shalini Rana, Yanji Yi, Robert J. Smyth, Ronald G. Collman, Robert W. Doms, Gilbert Vassart & Marc Parmentier, Nature 1996; 382 (6593): pp. 722 - 725, PubMed: 8751444 106. Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes, J. Claiborne Stephens, David E. Reich, David B. Goldstein, Hyoung Doo Shin, Michael W. Smith, Mary Carrington, Cheryl Winkler, Gavin A. Huttley, Rando Allikmets, Lynn Schriml, Bernard Gerrard, Michael Malasky, Maria D. Ramos, Susanne Morlot, Maria Tzetis, Carole Oddoux, Francesco S. di Giovine, Georgios Nasioulas, David Chandler, Michael Aseev, Matthew Hanson, Luba Kalaydjieva, Damjan Glavac, Paolo Gasparini, E. Kanavakis, Mireille Claustres, Marios Kambouris, Harry Ostrer, Gordon Duff, Vladislav Baranov, Hiljar Sibul, Andres Metspalu, David Goldman, Nick Martin, David Duffy, Jorg Schmidtke, Xavier Estivill, Stephen J. O'Brien & Michael Dean, American Journal of Human Genetics 1998; 62 (6): pp. 1507 - 1515, PubMed: 9585595 107. Evaluating plague and smallpox as historical selective pressures for the CCR5-Delta 32 HIV-resistance allele, Alison P. Galvani & Montgomery Slatkin, Proceedings of the National Academy of Sciences of the United States of America 2003; 100 (25): pp. 15276 - 15279, PubMed: 14645720 108. The geographic spread of the CCR5 Delta32 HIV-resistance allele, John Novembre, Alison P. Galvani & Montgomery Slatkin, PLoS Biology 2005; 3 (11): pp. e339, PubMed: 16216086 109. The case for selection at CCR5-Delta32, Pardis C. Sabeti, Emily Walsh, Steve F. Schaffner, Patrick Varilly, Ben Fry, Holli B. Hutcheson, Mike Cullen, Tarjei S. Mikkelsen, Jessica Roy, Nick Patterson, Richard Cooper, David Reich, David Altshuler, Stephen O'Brien & Eric S. Lander, PLoS Biology 2005; 3 (11): pp. e378, PubMed: 16248677 110. The Black Death and AIDS: CCR5-Delta32 in genetics and history, S. K. J. Cohn & L. T. Weaver, QJM 2006; 99 (8): pp. 497 - 503, PubMed: 16880184 111. Host Recognition of Bacterial Muramyl Dipeptide Mediated through NOD2. IMPLICATIONS FOR CROHN'S DISEASE, Naohiro Inohara, Yasunori Ogura, Ana Fontalba, Olga Gutierrez, Fernando Pons, Javier Crespo, Koichi Fukase, Seiichi Inamura, Shoichi Kusumoto, Masahito Hashimoto, Simon J. Foster, Anthony P. Moran, Jose L. Fernandez-Luna & Gabriel Núñez, Journal of Biological Chemistry 2003; 278 (8): pp. 5509 - 5512, PubMed: 12514169 112. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease., Yasunori Ogura, Denise K. Bonen, Naohiro Inohara, Dan L. Nicolae, Felicia F. Chen, R. Ramos, H. Britton, T. Moran, R. Karaliuskas,

to the thesis

Richard H. Duerr, J. P. Achkar, Steven R. Brant, T. M. Bayless, B. S. Kirschner, S. B. Hanauer, Gabriel Núñez & Judy H. Cho, Nature 2001; 411 (6837): pp. 603 - 606, PubMed: 11385577 113. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease, Jean-Pierre Hugot, Mathias Chamaillard, Habib Zouali, Suzanne Lesage, Jean-Pierre Cézard, Jacques Belaiche, Sven Almer, Curt Tysk, Colm A. O'Morain, Miquel Gassull, Vibeke Binder, Yigael Finkel, Antoine Cortot, Robert Modigliani, Pierre Laurent-Puig, Corine Gower-Rousseau, Jeanne Macry, Jean-Frédéric Colombel, Mourad Sahbatou & Gilles Thomas, Nature 2001; 411 (6837): pp. 599 - 603, PubMed: 11385576 114. A novel NOD2/CARD15 haplotype conferring risk for Crohn disease in Ashkenazi Jews, Kazuhito Sugimura, Kent D. Taylor, Ying-Chao Lin, Tieu Hang, Dai Wang, Yong-Ming Tang, Nathan Fischel-Ghodsian, Stephan R. Targan, Jerome I. Rotter & Huiying Yang, American Journal of Human Genetics 2003; 72 (3): pp. 509 - 518, PubMed: 12577202 115. Crohn disease: frequency and nature of CARD15 mutations in Ashkenazi and Sephardi/Oriental Jewish families, Turgut Tukel, Adel Shalata, Daniel Present, Daniel Rachmilewitz, Lloyd Mayer, Deniera Grant, Neil Risch & Robert J. Desnick, American Journal of Human Genetics 2004; 74 (4): pp. 623 - 636, PubMed: 15024686 116. Absence of mutation in the NOD2/CARD15 gene among 483 Japanese patients with Crohn's disease, Keiko Yamazaki, Masakazu Takazoe, Torao Tanaka, Toshiki Ichimori & Yusuke Nakamura, Journal of Human Genetics 2002; 47 (9): pp. 469 - 472, PubMed: 12202985 117. Association between insertion mutation in NOD2 gene and Crohn's disease in German and British populations, Jochen Hampe, Andrew Cuthbert, Peter J. P. Croucher, Muddassar M. Mirza, Silvia Mascheretti, Sheila Fisher, Henning Frenzel, Kathy King, Anja Hasselmeyer, Andrew J. S. MacPherson, Stephen Bridger, S. van Deventer, Alastair Forbes, Susanna Nikolaus, John E. Lennard-Jones, Ulrich R. Foelsch, Michael Krawczak, Cathryn Lewis, Stefan Schreiber & Christopher G. Mathew, The Lancet 2001; 357 (9272): pp. 1925 - 1928, PubMed: 11425413 118. Crohn's disease-associated NOD2 variants share a signaling defect in response to lipopolysaccharide and peptidoglycan, Denise K. Bonen, Yasunori Ogura, Dan L. Nicolae, Naohiro Inohara, Lisa Saab, Tsuyoshi Tanabe, Felicia F. Chen, Simon J. Foster, Richard H. Duerr, Steven R. Brant, Judy H. Cho & Gabriel Nuñez, Gastroenterology 2003; 124 (1): pp. 140 - 146, PubMed: 12512038 119. Assay of locus-specific genetic load implicates rare Toll-like receptor 4 mutations in meningococcal susceptibility, Irina Smirnova, Navjiwan Mann, Annemiek Dols, H. H. Derkx, Martin L. Hibberd, Michael Levin & Bruce Beutler, Proceedings of the National Academy of Sciences of the United States of America 2003; 100 (10): pp. 6075 - 6080, PubMed: 12730365

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 T 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.

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. 33

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.

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 T “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.

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. 39

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.

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 metaplasia- I dysplasia-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.

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).

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 42 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 P 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, IL- 10, 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 43 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. 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)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.

Chapter 1

The aim of this study was to identify whether or not polymorphisms in the IL-1B and IL- 1RN 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.

Cases Controls Remarks

N 210 334 44 Age (years) 59 (21-81) 43 (19-92) Males (%) 80.5 52.7 Smokers (%) N/A Data in 71 patients only Never 12.7 Previous 7.0 Current 80.3 Alcohol use (%) N/A Data in 73 patients only Never 37.0 Previous 21.9 Current 41.1 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 in- house 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

IL-1B, IL-1RN, CD14 & TLR4 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.

Cases Controls N = 191 N = 334 TLR4 % % 45 Genotype AA 84.3 86.5 AG 15.7 13.2 GG 0 0.3

Allele A 92.1 93.1 G 7.9 6.9 Table 2: TLR4 genotype and allele frequencies in cases and controls

Cases Controls N = 191 N = 334 CD14 % % Genotype CC 34.0 25.7 CT 44.5 46.1 TT 21.5 28.1

Allele C 56.3 48.8 T 43.7 51.2 Table 3: CD14 genotype and allele frequencies in cases and controls

Chapter 1

Cases Controls N = 191 N = 334 IL-1RN % % Genotype* TT 51.8 61.4 TC 36.6 32.0 CC 11.5 6.6

Allele T 70.2 77.4 C 29.8 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

Cases Controls N = 191 N = 334 IL-1B % % Genotype CC 42.4 42.2 CT 46.6 45.2 TT 11.0 12.6

Allele C 65.7 64.8 T 34.3 35.2 46 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 TLR4 IL-1B IL-1RN Cases Controls Effect p Odds -260 +896 -511 +2018 (%) (%) ratio C>T A>G C>T T>C TT AA 14.8 24.6 Protection 0.007 1.9 TT AA CT TT 1.8 6.1 Protection 0.030 3.3 TC AG CC TT 0.6 4.0 Protection 0.013 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).

IL-1B, IL-1RN, CD14 & TLR4 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 95% C.I. Lower Upper Gender < .001 5.830 3.031 11.214 IL-1RNa .006 2.241 1.266 3.969 TLR4a .119 1.782 .862 3.687 Age < .001 .852 .827 .877 CD14a .251 .718 .407 1.265 IL-1Ba .081 .601 .339 1.065 Table 7: Logistic regression analysis of the IL-1B, IL-1RN, CD14, and TLR4 genotypes in patients suffering from Barrett Oesophagus. aMutation carriers vs. homozygous wildtype 47

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 IL- 1RN+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,

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.

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 48 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.

IL-1B, IL-1RN, CD14 & TLR4 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 OR (95% CI) Remarks Reference Gender (Male) 2.7 (2.2 – 3.4) Ford, 20054 BMI (Obesity) 3.0 (1.6 – 6.7) Bu, 200645 4.0 (1.4 – 11.1) El-Serag, 200546 Ethnicity (Caucasian) 6.0 (3.6 – 10) Ford, 20054 49 Diet (vitamin C) 0.44 (0.2 – 0.98) Veugelers, 200647 Hiatal hernia 5.4 (3.1 – 9.4) García Rodríguez, 200648 Socio–economical 1.6 (1.2 – 2.2) Ford, 20054 status Oesophagitis 1.8 (1.6 – 2.0) Ford, 20054 Drinking p: 0.001 No OR given Conio, 200249 Medication (NSAIDs) 0.40 (0.19 – 0.81) Anderson, 200650 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).

Chapter 1

Reference s

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Barrett's oesophagus is characterized by a predominantly humoral inflammatory response, Leon M. G. Moons, Johannes G. Kusters, Evelien Bultman, Ernst J. Kuipers, Herman van Dekken, Wendy M. W. Tra, Alex Kleinjan, Jaap Kwekkeboom, Arnoud H. M. van Vliet & Peter D. Siersema, Journal of Pathology 2005; 207 (3): pp. 269 - 276, PubMed: 16177953 27. Inflammation and Cancer II. Role of chronic inflammation and cytokine gene polymorphisms in the pathogenesis of gastrointestinal malignancy, Mairi Macarthur, Georgina L. Hold & Emad M. El-Omar, American Journal of Physiology: Gastrointestinal and Liver Physiology 2004; 286 (4): pp. G515 - G520, PubMed: 15010360 28. Interleukin 1B proinflammatory genotypes protect against gastro-oesophageal reflux disease through induction of corpus atrophy, T. Ando, Emad M. El-Omar, Y. Goto, K. Nobata, O. Watanabe, O. Maeda, K. Ishiguro, M. Minami, N. Hamajima & H. Goto, Gut 2006; 55 (2): pp. 158 - 164, PubMed: 16120761 29. 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Bird, American Journal of Gastroenterology 2005; 100 (5): pp. 1012 - 1018, PubMed: 15842572 33. IL-1 RN polymorphism is not associated with Barrett's oesophagus and oesophageal adenocarcinoma, Leon M. G. Moons, Peter D. Siersema, Ernst J. Kuipers, Arnoud H. M. van Vliet & Johannes G. Kusters, American Journal of Gastroenterology 2005; 100 (12): pp. 2818, PubMed: 16393246 51 34. Helicobacter pylori infection and chronic gastric acid hyposecretion, Emad M. El-Omar, Karin Oien, Adil El- Nujumi, Derek Gillen, Angela Wirz, Stephen Dahill, Craig Williams, Joy E. S. Ardill & Kenneth E. L. McColl, Gastroenterology 1997; 113 (1): pp. 15 - 24, PubMed: 9207257 35. Association study of a functional Toll-like receptor 4 polymorphism with susceptibility to gastric mucosa-associated lymphoid tissue lymphoma, Stephan Hellmig, Wolfgang Fischbach, Maria Elisabeth Goebeler-Kolve, Ulrich Robert Fölsch, Jochen Hampe & Stefan Schreiber, Leukemia and Lymphoma 2005; 46 (6): pp. 869 - 872, PubMed: 16019531 36. Toll-like receptor 4 regulates gastric pit cell responses to Helicobacter pylori infection, Tsukasa Kawahara, Yuki Kuwano, Shigetada Teshima-Kondo, Ttomoko Kawai, Takeshi Nikawa, Kkyoichi Kishi & Kazuhito Rokutan, Journal of Medical Investigation 2001; 48 (3-4): pp. 190 - 197, PubMed: 11694959 37. Gastric mucosal recognition of Helicobacter pylori is independent of Toll-like receptor 4, Fredrik Bäckhed, Bachra Rokbi, Elisabeth Torstensson, Ying Zhao, Christina Nilsson, Delphine Seguin, Staffan Normark, Alison M. J. Buchan & Agneta Richter-Dahlfors, Journal of Infectious Diseases 2003; 187 (5): pp. 829 - 836, PubMed: 12599057 38. Toll-like receptor (TLR) 2 and TLR5, but not TLR4, are required for Helicobacter pylori-induced NF-kappa B activation and chemokine expression by epithelial cells, Michael F. Smith Jr, Anastasia Mitchell, Guolian Li, Song Ding, Ann Marie Fitzmaurice, Kieran Ryan, Sheila Crowe & Joanna B. Goldberg, Journal of Biological Chemistry 2003; 278 (35): pp. 32552 - 32560, PubMed: 12807870 39. Helicobacter pylori lipopolysaccharide from type I, but not type II strains, stimulates apoptosis of cultured gastric mucosal cells, Tsukasa Kawahara, Yuki Kuwano, Shigetada Teshima-Kondo, Toshiro Sugiyama, Tomoko Kawai, Takeshi Nikawa, Kyoichi Kishi & Kazuhito Rokutan, Journal of Medical Investigation 2001; 48 (3-4): pp. 167 - 174, PubMed: 11694956 40. Lipopolysaccharides from Helicobacter pylori can act as antagonists for Toll-like receptor 4, Philipp M. Lepper, Martha Triantafilou, Christian Schumann, E Marion Schneider & Kathy Triantafilou, Cellular Microbiology 2005; 7 (4): pp. 519 - 528, PubMed: 15760452 41. Expression and subcellular distribution of toll-like receptors TLR4, TLR5 and TLR9 on the gastric epithelium in Helicobacter pylori infection, B. Schmaußer, M. Andrulis, S. Endrich, S. K. Lee, C. Josenhans, H. K. Müller- Hermelink & M. Eck, Clinical and Experimental Immunology 2004; 136 (3): pp. 521 - 526, PubMed: 15147355 42. Review article: Helicobacter pylori and gastro-oesophageal reflux disease, Brendan C. Delaney & Kenneth E. L. McColl, Alimentary Pharmacology and Therapeutics 2005; 22 (Suppl 1): pp. 32 - 40, PubMed: 16042657 43. Mechanisms of increased acid secretion after eradication of Helicobacter pylori infection, Emad M. El-Omar, Gut 2006; 55 (2): pp. 144 - 146, PubMed: 16407378

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44. Bacterial biota in reflux oesophagitis and Barrett's oesophagus, Zhiheng Pei, Liying Yang, Richard M. Peek, Steven M. Jr Levine, David T. Pride & Martin J. Blaser, World Journal of Gastroenterology 2005; 11 (46): pp. 7277 - 7283, PubMed: 16437628 45. Body Mass Index is Associated with Barrett Oesophagus and Cardiac Mucosal Metaplasia, Xiangdong Bu, Yanling Ma, Roger Der, Tom Demeester, Leslie Bernstein & Parakrama T. Chandrasoma, Digestive Diseases and Sciences 2006; 51 (9): pp. 1589 - 1594, PubMed: 16927134 46. Abdominal obesity and the risk of Barrett's oesophagus, Hashem B. El-Serag, Peter Kvapil, Joan Hacken-Bitar & Jennifer R. Kramer, American Journal of Gastroenterology 2005; 100 (10): pp. 2151 - 2156, PubMed: 16181362 47. Obesity and lifestyle risk factors for gastroesophageal reflux disease, Barrett oesophagus and oesophageal adenocarcinoma, P. J. Veugelers, G. A. Porter, D. L. Guernsey & A. G. Casson, Diseases of the Oesophagus 2006; 19 (5): pp. 321 - 328, PubMed: 16984526 48. Gastric acid suppression and risk of oesophageal and gastric adenocarcinoma: a nested case-control study in the United Kingdom, Luis A. García Rodríguez, Jesper Lagergren, & Mats Lindblad, Gut 2006; PubMed: 16785284, DOI: 10.1136/gut.2005.086579 [Article submitted / in press] 49. Risk factors for Barrett's oesophagus: a case-control study, Massimo Conio, Rosangela Filiberti, Sabrina Blanchi, Roberto Ferraris, Santino Marchi, Paolo Ravelli, Gabriella Lapertosa, Gaetano Iaquinto, Renato Sablich, Riccardo Gusmaroli, Hugo Aste & Attilio Giacosa, International Journal of Cancer 2002; 97 (2): pp. 225 - 229, PubMed: 11774268 50. Nonsteroidal anti-inflammatory drugs and the oesophageal inflammation-metaplasia-adenocarcinoma sequence, Lesley A. Anderson, Brian T. Johnston, R. G. P. Watson, Seamus J. Murphy, Heather R. Ferguson, Harry Comber, Jim McGuigan, John V. Reynolds & Liam J. Murray, Cancer Research 2006; 66 (9): pp. 4975 - 4982, PubMed: 16651456

52

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 I 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.

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.

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. 54 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 T 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 55 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

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

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. 56 ASCA ELISA: ASCA IgA and IgG were evaluated in commercially available ELISA kits, kindly sponsored by Inova Diagnostics, San Diego, CA24 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.

CD14-260 C>T TLR4+896 A>G 1.1 1.2 2.2 1.1 1.2 2.2 CC CT TT AA AG GG Group Total n % n % n % n % n % n % HC 170 48 28 82 48 40 24 153 90 16 9 1 1 CD 109 33 30 53 49 23 21 89 82 18 17 2 2 UC 101 25 25 42 42 34 34 90 90 10 10 1 1 57 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).

Chapter 2

CD14-260 C>T TLR4+896 A>G 1.1 1.2 2.2 1.1 1.2 2.2 CC CT TT AA AG GG Group Vienna Total n % n % n % n % n % n % CD Total 109 33 30 53 49 23 21 89 82 18 17 2 2 ASCA+ 60 18 30 28 47 14 23 47 78 12 20 1 2 CD A1 96 29 30 48 50 19 20 77 80 18 19 1 1 ASCA+ 53 16 30 26 49 11 21 41 77 12 23 0 0 A2 13 4 31 5 38 4 31 12 92 0 0 1 8 ASCA+ 8 3 38 2 25 3 38 7 88 0 0 1 13 B1 42 13 31 20 48 9 21 35 83 6 14 1 2 ASCA+ 20 6 30 9 45 5 25 16 80 4 20 0 0 B2 44 14 32 21 48 9 20 36 82 7 16 1 2 ASCA+ 24 8 33 10 42 6 25 19 79 4 17 1 4 B3 23 6 26 12 52 5 22 18 78 5 22 0 0 ASCA+ 16 4 25 9 56 3 19 12 75 4 25 0 0 L1 40 13 33 17 43 10 25 35 88 4 10 1 3 ASCA+ 22 7 31 8 36 7 32 18 82 3 14 1 5 L2 22 6 27 12 55 4 18 13 59 8 36 1 5 ASCA+ 9 3 33 5 56 1 11 6 67 3 33 0 0 L3 46 14 30 23 50 9 20 40 87 6 13 0 0 ASCA+ 29 8 28 15 52 6 21 23 79 6 21 0 0 L4 1 0 0 1 100 0 0 1 100 0 0 0 0 58 ASCA+ 0 0 0 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. 59 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.

Chapter 2

References

1. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein, Samuel D. Wright, Robert A. Ramos, Peter S. Tobias, Richard J. Ulevitch & John C. Mathison, Science 1990; 249 (4975): pp. 1431 - 1433, PubMed: 1698311 2. Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex. transfer from CD14 to TLR4 and MD-2, Jean da Silva Correia, Katrin Soldau, Urs Christen, Peter S. Tobias & Richard J. Ulevitch, Journal of Biological Chemistry 2001; 276 (24): pp. 21129 - 21135, PubMed: 11274165 3. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction, Jesse C. Chow, Donna W. Young, Douglas T. Golenbock, William J. Christ & Fabian Gusovsky, Journal of Biological Chemistry 1999; 274 (16): pp. 10689 - 10692, PubMed: 10196138 4. Endocytosed HSP60s use toll-like receptor 2 (TLR2) and TLR4 to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells, Ramunas M. Vabulas, Parviz Ahmad-Nejad, Clarissa da Costa, Thomas Miethke, Carsten J. Kirschning, Hans Häcker & Hermann Wagner, Journal of Biological Chemistry 2001; 276 (33): pp. 31332 - 31339, PubMed: 11402040 5. Learning from discrepancies: CD14 polymorphisms, atopy and the endotoxin switch, Donata Vercelli, Clinical and Experimental Allergy 2003; 33 (2): pp. 153 - 155, PubMed: 12580903 6. A common single nucleotide polymorphism in the CD14 promoter decreases the affinity of Sp protein binding and enhances transcriptional activity, Tricia D. LeVan, John W. Bloom, Thomas J. Bailey, Christopher L. Karp, Marilyn Halonen, Fernando D. Martinez & Donata Vercelli, Journal of Immunology 2001; 167 (10): pp. 5838 - 5844, PubMed: 11698458 7. A Polymorphism* in the 5' flanking region of the CD14 gene is associated with circulating soluble CD14 levels and with total serum immunoglobulin E, Mauro Baldini, I. Carla Lohman, Marilyn Halonen, Robert P. Erickson, Patrick G. Holt & Fernando D. Martinez, American Journal of Respiratory Cell and Molecular Biology 1999; 20 (5): pp. 976 - 983, PubMed: 10226067 8. C(-260)-->T polymorphism in the promoter of the CD14 monocyte receptor gene as a risk factor for myocardial infarction, Jaroslav A. Hubacek, G. Rothe, Jan Pit'ha, Zdena Škodová, Vladimir Stanek, Rudolf Poledne & Gerd Schmitz, Circulation 1999; 99 (25): pp. 3218 - 3220, PubMed: 10385492 9. Ulcerative colitis is associated with a promoter polymorphism of lipopolysaccharide receptor gene, CD14, N. Obana, S. Takahashi, Y. Kinouchi, K. Negoro, S. Takagi, N. Hiwatashi & T. Shimosegawa, Scandinavian Journal of Gastroenterology 2002; 37 (6): pp. 699 - 704, PubMed: 12126249 10. A polymorphism in the CD14 gene is associated with Crohn disease, Wolfram Klein, Andreas Tromm, Thomas Griga, Harald Fricke, Christian Folwaczny, Michael Hocke, Klaus Eitner, Michaela Marx, Natascha Duerig & Jörg Thomas Epplen, Scandinavian Journal of Gastroenterology 2002; 37 (2): pp. 189 - 191, PubMed: 11843056 11. TLR4 mutations are associated with endotoxin hyporesponsiveness in humans, Nancy C. Arbour, Eva Lorenz, 60 Brian C. Schutte, Joseph Zabner, Joel N. Kline, Michael Jones, Kathy Frees, Janet L. Watt & David A. Schwartz, Nature Genetics 2000; 25 (2): pp. 187 - 191, PubMed: 10835634 12. Monocytes Heterozygous for the Asp299Gly and Thr399Ile Mutations in the Toll-like Receptor 4 Gene Show No Deficit in Lipopolysaccharide Signalling, Clett Erridge, John Stewart & Ian R. Poxton, Journal of Experimental Medicine 2003; 197 (12): pp. 1787 - 1791, PubMed: 12796470 13. Heterozygous toll-like receptor 4 polymorphism does not influence lipopolysaccharide-induced cytokine release in human whole blood, Sonja von Aulock, Nicolas W. J. Schröder, Katja Gueinzius, Stephanie Traub, Sebastian Hoffmann, Kathrin Graf, Stefanie Dimmeler, Thomas Hartung, Ralf R. Schumann & Corinna Hermann, Journal of Infectious Diseases 2003; 188 (6): pp. 938 - 943, PubMed: 12964127 14. Antibody to Saccharomyces cerevisiae (bakers' yeast) in Crohn's disease, J. Main, H. McKenzie, G. R. Yeaman, M. A. Kerr, D. Robson, C. R. Pennington & D. Parratt, British Medical Journal 1988; 297 (6656): pp. 1105 - 1106, PubMed: 3143445 15. Evaluation of serological markers to differentiate between ulcerative colitis and Crohn's disease: pANCA, ASCA and agglutinating antibodies to anaerobic coccoid rods, Ronald K. Linskens, Rosalie C. Mallant-Hent, Z. M. Anthonie Groothuismink, Liesbeth E. Bakker-Jonges, Joop P. van de Merwe, Herbert Hooijkaas, B. Mary E. von Blomberg & Stephan G. M. Meuwissen, European Journal of Gastroenterology and Hepatology 2002; 14 (9): pp. 1013 - 1018, PubMed: 12352222 16. Antibody to selected strains of Saccharomyces cerevisiae (baker's and brewer's yeast) and Candida albicans in Crohn's disease, H. McKenzie, J. Main, C. R. Pennington & D. Parratt, Gut 1990; 31 (5): pp. 536 - 538, PubMed: 2190866 17. Antibodies to Saccharomyces cerevisiae in patients with Crohn's disease and their possible pathogenic importance, M. H. Giaffer, A. Clark & C. D. Holdsworth, Gut 1992; 33 (8): pp. 1071 - 1075, PubMed: 1398231 18. Specific antibody response to oligomannosidic epitopes in Crohn's disease, B. Sendid, J. F. Colombel, P. M. Jacquinot, C. Faille, J. Fruit, A. Cortot, D. Lucidarme, D. Camus & D. Poulain, Clinical and Diagnostic Laboratory Immunology 1996; 3 (2): pp. 219 - 226, PubMed: 8991640 19. Cross-reactivity of yeast antigens in human colon and peripheral leukocytes, Nobuhide Oshitani, Fumihiko Hato, Kenichi Suzuki, Yoshinori Sawa, Takayuki Matsumoto, Kiyoshi Maeda, Kazuhide Higuchi, Seiichi Kitagawa & Tetsuo Arakawa, Journal of Pathology 2003; 199 (3): pp. 361 - 367, PubMed: 12579538 20. Marker antibody expression stratifies Crohn's disease into immunologically homogeneous subgroups with distinct clinical characteristics, Eric A. Vasiliauskas, L. Y. Kam, Loren C. Karp, J. Gaiennie, Huiying Yang & Stephan R. Targan, Gut 2000; 47 (4): pp. 487 - 496, PubMed: 10986208 21. Association of antibody responses to microbial antigens and complications of small bowel Crohn's disease, William S. Mow, Eric A. Vasiliauskas, Ying-Chao Lin, Phillip R. Fleshner, Konstantinos A. Papadakis, Kent D. Taylor,

TLR4 Crohn’s Disease Carol J. Landers, Maria T. Abreu-Martin, Jerome I. Rotter, Huiying Yang & Stephan R. Targan, Gastroenterology 2004; 126 (2): pp. 414 - 424, PubMed: 14762777 22. Classification of inflammatory bowel disease, J. E. Lennard-Jones, Scandinavian Journal of Gastroenterology. Supplement 1989; 170 pp. 2 - 6, PubMed: 2617184 23. A simple classification of Crohn's disease: report of the Working Party for the World Congresses of Gastroenterology, Vienna 1998, C. Gasche, J. Scholmerich, J. Brynskov, G. D'Haens, S. B. Hanauer, E. J. Irvine, D. P. Jewell, D. Rachmilewitz, D. B. Sachar, W. J. Sandborn & L. R. Sutherland, Inflammatory Bowel Diseases 2000; 6 (1): pp. 8 - 15, PubMed: 10701144 24. Diagnostic value of anti-Saccharomyces cerevisiae and antineutrophil cytoplasmic autoantibodies in inflammatory bowel disease, Marc Peeters, Sofie Joossens, Severine Vermeire, Robert Vlietinck, Xavier Bossuyt & Paul Rutgeerts, American Journal of Gastroenterology 2001; 96 (3): pp. 730 - 734, PubMed: 11280542 25. Innate immunity and toll-like receptors: clinical implications of basic science research, Maria T. Abreu & Moshe Arditi, Journal of Pediatrics 2004; 144 (4): pp. 421 - 429, PubMed: 15069387 26. CARD15 gene and the classification of Crohn's disease, Laura S. Murillo, J. Bart A. Crusius, Ad A. van Bodegraven, Behrooz Z. Alizadeh & A. Salvador Peña, Immunogenetics 2002; 54 (1): pp. 59 - 61, PubMed: 11976792 27. Genetic and serological markers to identify phenotypic subgroups in a Dutch Crohn' s disease population, Ronald K. Linskens, Rosalie C. Mallant-Hent, Laura S. Murillo, B. Mary E. von Blomberg, Behrooz Z. Alizadeh & A. Salvador Peña, Digestive and Liver Disease 2004; 36 (1): pp. 29 - 34, PubMed: 14971813 28. NOD2/CARD15, TLR4 and CD14 mutations in Scottish and Irish Crohn's disease patients: evidence for genetic heterogeneity within Europe?, Ian D. R. Arnott, E. R. Nimmo, H. E. Drummond, J. Fennell, B. R. K. Smith, E. MacKinlay, J. Morecroft, N. Anderson, D. Kelleher, M. O'Sullivan, R. McManus & Jack Satsangi, Genes & Immunity 2004; 5 (5): pp. 417 - 425, PubMed: 15190267 29. A functional single polymorphism of the TLR4 gene is correlated with Crohn's Disease but not with Ulcerative Colitis, Henri Braat, Marcel Dijkgraaf, Wouter Curvers, Esther Vogels, Ad A. van Bodegraven, Pieter Stokkers, Daan Hommes & Sander J. H. van Deventer, Gastroenterology 2004; 124 pp. A367 30. Deficient host-bacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn's disease and ulcerative colitis, D. Franchimont, Séverine Vermeire, H. El Housni, M. Pierik, K. Van Steen, T. Gustot, E. Quertinmont, M. Abramowicz, A. van Gossum, J. Deviere & Paul Rutgeerts, Gut 2004; 53 (7): pp. 987 - 992, PubMed: 15194649 31. Current concepts of the etiology and pathogenesis of ulcerative colitis and Crohn's disease, R. Balfour Sartor, Gastroenterology Clinics of North America 1995; 24 (3): pp. 475 - 507, PubMed: 8809232 32. Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing Toll-like receptors, Elke Cario, Ian M. Rosenberg, Steven L. Brandwein, Paul L. Beck, Hans Christian Reinecker & Daniel K. Podolsky, Journal of Immunology 2000; 164 (2): pp. 966 - 972, PubMed: 10623846 33. Relevance of the innate immune system, Mónica Peñate & A. Salvador Peña, Revista Española de Enfermedadas 61 Digestivas 2001; 93 (11): pp. 721 - 739, PubMed: 11995372 34. Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism, Catherine Werts, Richard I. Tapping, John C. Mathison, Tsung Hsien Chuang, Vladimir Kravchenko, Isabelle Saint Girons, David A. Haake, Paul J. Godowski, Fumitaka Hayashi, Adrian Ozinsky, David M. Underhill, Carsten J. Kirschning, Hermann Wagner, Alan Aderem, Peter S. Tobias & Richard J. Ulevitch, Nature Immunology 2001; 2 (4): pp. 346 - 352, PubMed: 11276206 35. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease, Jean-Pierre Hugot, Mathias Chamaillard, Habib Zouali, Suzanne Lesage, Jean-Pierre Cézard, Jacques Belaiche, Sven Almer, Curt Tysk, Colm A. O'Morain, Miquel Gassull, Vibeke Binder, Yigael Finkel, Antoine Cortot, Robert Modigliani, Pierre Laurent-Puig, Corine Gower-Rousseau, Jeanne Macry, Jean-Frédéric Colombel, Mourad Sahbatou & Gilles Thomas, Nature 2001; 411 (6837): pp. 599 - 603, PubMed: 11385576 36. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease., Yasunori Ogura, Denise K. Bonen, Naohiro Inohara, Dan L. Nicolae, Felicia F. Chen, R. Ramos, H. Britton, T. Moran, R. Karaliuskas, Richard H. Duerr, J. P. Achkar, Steven R. Brant, T. M. Bayless, B. S. Kirschner, S. B. Hanauer, Gabriel Núñez & Judy H. Cho, Nature 2001; 411 (6837): pp. 603 - 606, PubMed: 11385577 37. Autoimmunity and apoptosis: the Crohn's connection, Bruce Beutler, Immunity 2001; 15 (1): pp. 5 - 14, PubMed: 11485733 38. Innate immune recognition, Charles A. J. Janeway & Ruslan Medzhitov, Annual Review of Immunology 2002; 20 pp. 197 - 216, PubMed: 11861602

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 T 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).

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 C 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. 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 65 600 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 C- 5’ Fluor Label (VIC). The primers and probes used for TLR9+2848 were: forward primer 5’-CCG

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

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 66 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 Spanish Galicians Crohn’s Healthy Crohn’s Healthy Disease Controls Disease Controls n=133 n=147 n=163 n=165 n % n % n % n % TLR9 –1237 T>C TT 101 76 105 71 119 73 112 68 TC 29 22 42 29 40 25 47 29 CC 3 2 0 0 4 3 6 4

TLR9 +2848 G >A GG 27 20 23 16 43 27 42 26 GA 76 57 71 48 71 44 74 45 AA 30 23 53 36 49 30 49 30

TLR9 Haplotypes1 2n % 2n % 2n % 2n % -1237 +2848 I T G 128 48 115 39 154 47 152 46 II T A 103 39 139 47 124 38 119 36 III C A 33 12 39 13 45 14 53 16 IV C G 2 0.8 3 1 3 0.9 6 2 Table 1: TLR9 genotypes and haplotypes in Dutch Caucasian and Spanish Galician Crohn’s disease patients and controls 1TLR9 haplotype 2n definition: haplotypes were based on the TLR9 –1486, TLR9 –1237, TLR9 +1174 and TLR9 67 +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.

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.

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 68 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

1. Familial aggregation of inflammatory bowel disease: a population-based study in South Limburg, The Netherlands. 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 2. Genome-wide scanning in inflammatory bowel diseases, J. P. Hugot & G. Thomas, Digestive Diseases 1998; 16 6): pp. 364 - 369, PubMed: 10207223 3. Dysbiosis in inflammatory bowel disease, C. P. Tamboli, C. Neut, P. Desreumaux & J. F. Colombel, Gut 2004; 53 1): pp. 1 - 4, PubMed: 14684564 4. CpG motifs in bacterial DNA trigger direct B-cell activation, Arthur M. Krieg, Ae Kyung Yi, Sara Matson, Thomas J. Waldschmidt, Gail A. Bishop, Rebecca Teasdale, Gary A. Koretzky & Dennis M. Klinman, Nature 1995; 374 6522): pp. 546 - 549, PubMed: 7700380 5. CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma, Dennis M. Klinman, Ae Kyung Yi, Serge L. Beaucage, Jacqueline Conover & Arthur M. Krieg, Proceedings of the National Academy of Sciences of the United States of America 1996; 93 7): pp. 2879 - 2883, PubMed: 8610135 6. Toll-like receptor 9 signaling mediates the anti-inflammatory effects of probiotics in murine experimental colitis, Daniel Rachmilewitz, Kyoko Katakura, Fanny Karmeli, Tomoko Hayashi, Constantin Reinus, Bernard Rudensky, Shizuo Akira, Kiyoshi Takeda, Jongdae Lee, Kenji Takabayashi & Eyal Raz, Gastroenterology 2004; 126 2): pp. 520 - 528, PubMed: 14762789 7. 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 8. 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 9. 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 10. Genetic and serological markers to identify phenotypic subgroups in a Dutch Crohn' s disease population, Ronald K. Linskens, Rosalie C. Mallant-Hent, Laura S. Murillo, B. Mary E. von Blomberg, Behrooz Z. Alizadeh & A. Salvador Peña, Digestive and Liver Disease 2004; 36 1): pp. 29 - 34, PubMed: 14971813 11. Fine mapping of the chromosome 3p susceptibility locus in inflammatory bowel disease, Jochen Hampe, N. J. Lynch, S. Daniels, Stephen Bridger, Andrew J. S. MacPherson, P. Stokkers, Alastair Forbes, John E. Lennard- 69 Jones, Chistopher G. Mathew, M. E. Curran & Stefan Schreiber, Gut 2001; 48 2): pp. 191 - 197, PubMed: 11156639 12. 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 13. 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

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 T 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).

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.

Results: An increased incidence of TLR4*G was observed in cases compared to controls 72 (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 C 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.

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.

The Toll Like Receptor (TLR) family is a group of pattern recognition receptors, which recognise several microbial products, including bacterial cell wall components and DNA11. 73 Poltorak et al. associated TLR4 with lipopolysaccharide (LPS) recognition in mice12. 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).

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.

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. 74 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 Cases Controls N = 165 N = 162 % % TLR4 Genotype AA 93.3 85.7 AG* 6.1 12.9 GG* 0.6 1.4

Allele A 96.4 92.1 G 3.6 7.9

CD14 CD14 Genotype CC 23.0 23.6 CT 50.9 50.7 TT 26.1 25.7

Allele C 48.5 48.9 T 51.5 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

75 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).

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).

Cases Controls N = 119 N = 46 N = 140 N = 22 TLR4 CARD15/NOD2 CARD15/NOD2 CARD15/NOD2 CARD15/NOD2 – + – + (%) (%) (%) (%) Genotype AA 91.6 97.8 85.7 86.4 AG 7.6 2.2 12.9 13.6 GG 0.8 0.0 1.4 0.0

Allele A 95.4 98.9 92.1 93.2 G 4.6 1.1 7.9 6.8 76 Table 2: TLR4 genotype distribution and allele frequencies in relation to CARD15/NOD2 SNPs in cases and controls

Cases Controls N = 119 N = 46 N = 140 N = 22 CD14 CARD15/NOD2 CARD15/NOD2 CARD15/NOD2 CARD15/NOD2 – + – + (%) (%) %) (%) Genotype CC 24.4 19.6 23.6 36.4 CT* 48.7 56.5 50.7 27.3 TT* 26.9 23.9 25.7 36.4

Allele C 48.7 47.8 48.9 50 T 51.3 52.2 51.1 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 95% CI p OR Lower Upper Gender 0.68 0.89 0.54 1.50 Age * 0.01 1.02 1.00 1.04 Origin (rural or urban)* 0.05 0.59 0.35 1.00 Smoking <0.001 0.15 0.90 0.25 SNP8 0.34 1.46 0.67 3.19 SNP12* 0.02 6.55 1.30 33.10 Carriage of: SNP13* 0.009 6.00 1.56 23.07 TLR4* 0.04 0.41 0.18 0.96 CD14 0.88 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 77 origin and carriage of the TLR4 SNP are protective.

Fistulae 95% CI p OR Lower Upper Gender * 0.02 4.08 1.21 13.73 Age 0.27 0.97 0.92 1.02 Origin (rural or urban)* 0.71 0.80 0.24 2.67 Smoking 0.10 3.17 0.79 12.72 SNP8 0.44 0.42 0.05 3.86 SNP12* 0.001 13.30 2.81 63.06 Carriage of: SNP13 1.00 <0.001 <0.001 ∞ TLR4 AA & CD14*T* 0.02 0.27 0.08 0.84 Table 5: Logistic regression analysis. *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.

Chapter 4

Ileal resection 95% CI p OR Lower Upper Gender 0.41 0.74 0.35 1.54 Age 0.10 0.98 0.95 1.01 Origin (rural or urban) 0.29 1.47 0.71 3.04 Smoking 0.28 0.67 0.33 1.39 SNP8 0.37 1.56 0.58 4.28 SNP12 0.07 3.07 0.91 10.42 Carriage of: SNP13* 0.02 4.48 1.31 15.25 TLR4 0.93 1.06 0.25 4.49 CD14 0.20 1.80 0.73 4.45 Table 6: Logistic regression analysis. *Carriage of the SNP13 polymorphism was a risk factor for ileal resection.

Discussion

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 78 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 ileo- coecal 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 79 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.

Chapter 4

References

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Immunocytochemical evidence of Listeria, Escherichia coli, and Streptococcus antigens in Crohn's disease, Ying Liu, Herbert J. van Kruiningen, A. Brian West, Richard W. Cartun, Antoine Cortot & Jean-Frédéric Colombel, Gastroenterology 1995; 108 (5): pp. 1396 - 1404, PubMed: 7729631 11. Toll-like receptors, Kiyoshi Takeda, Tsuneyasu Kaisho & Shizuo Akira, Annual Review of Immunology 2003; 21 pp. 335 - 376, PubMed: 12524386 80 12. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene, Alexander Poltorak, Xiaolong He, Irina Smirnova, Mu-Ya Liu, Christophe van Huffel, Xin Du, Dale Birdwell, Erica Alejos, Maria Silva, Chris Galanos, Marina Freudenberg, Paola Ricciardi-Castagnoli, Betsy Layton & Bruce Beutler, Science 1998; 282 (5396): pp. 2085 - 2088, PubMed: 9851930 13. TLR4: central component of the sole mammalian LPS sensor, Bruce Beutler, Current Opinion in Immunology 2000; 12 (1): pp. 20 - 26, PubMed: 10679411 14. The role of toll-like receptor (TLR) 2 and TLR4 in the host defense against disseminated candidiasis, Mihai G. Netea, Chantal A. A. van der Graaf, Alieke G. Vonk, Ineke Verschueren, Jos W. M. van der Meer & Bart Jan Kullberg, Journal of Infectious Diseases 2002; 185 (10): pp. 1483 - 1489, PubMed: 11992285 15. TLR4 mutations are associated with endotoxin hyporesponsiveness in humans, Nancy C. Arbour, Eva Lorenz, Brian C. Schutte, Joseph Zabner, Joel N. Kline, Michael Jones, Kathy Frees, Janet L. Watt & David A. Schwartz, Nature Genetics 2000; 25 (2): pp. 187 - 191, PubMed: 10835634 16. The role of TLR4 in endotoxin responsiveness in humans, David A. Schwartz, Journal of Endotoxin Research 2001; 7 (5): pp. 389 - 393, PubMed: 11753209 17. Response of human pulmonary epithelial cells to LPS involves toll-like receptor 4 (TLR4)-dependent signaling pathways: Evidence for an intracellular compartmentalization of TLR4., Loïc Guillot, Samir Medjane, Karine Le- Barillec, Viviane Balloy, Claire Danel, Michel Chignard & Mustapha Si-Tahar, Journal of Biological Chemistry 2004; 279 (4): pp. 2712 - 2718, PubMed: 14600154 18. Chlamydial heat shock protein 60 activates macrophages and endothelial cells through Toll-like receptor 4 and MD2 in a MyD88-dependent pathway, Yonca Bulut, Emmanuelle Faure, Lisa Thomas, Hisae Karahashi, Kathrin S. Michelsen, Ozlem Equils, Sandra G. Morrison, Richard P. Morrison & Moshe Arditi, Journal of Immunology 2002; 168 (3): pp. 1435 - 1440, PubMed: 11801686 19. Human heat shock protein 60 stimulates vascular smooth muscle cell proliferation through Toll-like receptors 2 and 4, Rick de Graaf, Geoffrey Kloppenburg, Peter J. H. M. Kitslaar, Cathrien A. Bruggeman & Frank Stassen, Microbes & Infection 2006; 8 (7): pp. 1859 - 1865, PubMed: 16843693 20. Endocytosed HSP60s use toll-like receptor 2 (TLR2) and TLR4 to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells, Ramunas M. Vabulas, Parviz Ahmad-Nejad, Clarissa da Costa, Thomas Miethke, Carsten J. Kirschning, Hans Häcker & Hermann Wagner, Journal of Biological Chemistry 2001; 276 (33): pp. 31332 - 31339, PubMed: 11402040 21. HSP70 as endogenous stimulus of the Toll/interleukin-1 receptor signal pathway, Ramunas M. Vabulas, Parviz Ahmad-Nejad, Sanghamitra Ghose, Carsten J. Kirschning, Rolf D. Issels & Hermann Wagner, Journal of Biological Chemistry 2002; 277 (17): pp. 15107 - 15112, PubMed: 11842086 22. Cutting edge: heat shock protein (HSP) 60 activates the innate immune response: CD14 is an essential receptor for

CD14 & TLR4 Crohn’s Disease HSP60 activation of mononuclear cells, Amir Kol, Andrew H. Lichtman, Robert W. Finberg, Peter Libby & Evelyn A. Kurt-Jones, Journal of Immunology 2000; 164 (1): pp. 13 - 17, PubMed: 10604986 23. A Polymorphism* in the 5' flanking region of the CD14 gene is associated with circulating soluble CD14 levels and with total serum immunoglobulin E, Mauro Baldini, I. Carla Lohman, Marilyn Halonen, Robert P. Erickson, Patrick G. Holt & Fernando D. Martinez, American Journal of Respiratory Cell and Molecular Biology 1999; 20 (5): pp. 976 - 983, PubMed: 10226067 24. Sp1 is a critical factor for the monocytic specific expression of human CD14, Dong-Er Zhang, Christopher J. Hetherington, Shencao Tan, Suzan E. Dziennis, David A. Gonzalez, Hui-Min Chen & Daniel G. Tenen, Journal of Biological Chemistry 1994; 269 (15): pp. 11425 - 11434, PubMed: 7512565 25. Interaction of polymorphisms in the CARD15 and CD14 genes in patients with Crohn disease, Wolfram Klein, Andreas Tromm, Thomas Griga, Christian Folwaczny, Michael Hocke, Klaus Eitner, Michaela Marx, Natascha Duerig & Jörg Thomas Epplen, Scandinavian Journal of Gastroenterology 2003; 38 (8): pp. 834 - 836, PubMed: 12940436 26. 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 27. Synergistic effect of Toll-like receptor 4 and CD14 polymorphisms on the total atherosclerosis burden in patients with peripheral arterial disease, Tryfon Vainas, Frank R. M. Stassen, Cathrien A. Bruggeman, Rob J. T. Welten, Luc H. J. M. van den Akker, Peter J. E. H. Kitslaar, A. Salvador Peña & Servaas A. Morré, Journal of Vascular Surgery 2006; 44 (2): pp. 326 - 332, PubMed: 16890863 28. Frequency of glucocorticoid resistance and dependency in Crohn's disease, P. Munkholm, E. Langholz, M. Davidsen & V. Binder, Gut 1994; 35 (3): pp. 360 - 362, PubMed: 8150347 29. Role of the toll-like receptor 4 Asp299Gly polymorphism in susceptibility to Candida albicans infection, Servaas A. Morré, Laura S. Murillo, Joke Spaargaren, Han S. A. Fennema & A. Salvador Peña, Journal of Infectious Diseases 2002; 186 (9): pp. 1377 - 1379, PubMed: 12402214 30. 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 31. CARD15 gene and the classification of Crohn's disease, Laura S. Murillo, J. Bart A. Crusius, Ad A. van Bodegraven, Behrooz Z. Alizadeh & A. Salvador Peña, Immunogenetics 2002; 54 (1): pp. 59 - 61, PubMed: 11976792 32. Appendectomy is followed by increased risk of Crohn's disease, Roland E. Andersson, Gunnar Olaison, Curt Tysk & Anders Ekbom, Gastroenterology 2003; 124 (1): pp. 40 - 46, PubMed: 12512028 33. A polymorphism in the CD14 gene is associated with Crohn disease, Wolfram Klein, Andreas Tromm, Thomas Griga, Harald Fricke, Christian Folwaczny, Michael Hocke, Klaus Eitner, Michaela Marx, Natascha Duerig & 81 Jörg Thomas Epplen, Scandinavian Journal of Gastroenterology 2002; 37 (2): pp. 189 - 191, PubMed: 11843056 34. Association between polymorphisms in the Toll-like receptor 4, CD14, and CARD15/NOD2 and inflammatory bowel disease in the Greek population, Maria Gazouli, Gerassimos Mantzaris, Athanassios Kotsinas, Panayotis Zacharatos, Efstathios Papalambros, Athanassios Archimandritis, John Ikonomopoulos & Vassilis G. Gorgoulis, World Journal of Gastroenterology 2005; 11 (5): pp. 681 - 685, PubMed: 15655821 35. 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, Gut 2005; 54 (3): pp. 439 - 440, PubMed: 15710998 36. NOD2/CARD15, TLR4 and CD14 mutations in Scottish and Irish Crohn's disease patients: evidence for genetic heterogeneity within Europe?, Ian D. R. Arnott, E. R. Nimmo, H. E. Drummond, J. Fennell, B. R. K. Smith, E. MacKinlay, J. Morecroft, N. Anderson, D. Kelleher, M. O'Sullivan, R. McManus & Jack Satsangi, Genes & Immunity 2004; 5 (5): pp. 417 - 425, PubMed: 15190267 37. A functional single polymorphism of the TLR4 gene is correlated with Crohn's Disease but not with Ulcerative Colitis, Henri Braat, Marcel Dijkgraaf, Wouter Curvers, Esther Vogels, Ad A. van Bodegraven, Pieter Stokkers, Daan Hommes & Sander J. H. van Deventer, Gastroenterology 2004; 124 pp. A367 38. Deficient host-bacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn's disease and ulcerative colitis, D. Franchimont, Séverine Vermeire, H. El Housni, M. Pierik, K. Van Steen, T. Gustot, E. Quertinmont, M. Abramowicz, A. van Gossum, J. Deviere & Paul Rutgeerts, Gut 2004; 53 (7): pp. 987 - 992, PubMed: 15194649 39. NOD2/CARD15 and TNFA, but not IL1B and IL1RN, are associated with Crohn's disease, António Carlos Ferreira, Susana Almeida, Marta Tavares, Paolo Canedo, Fábio Pereira, Gonçalo Regalo, Céu Figueiredo, Eunice Trindade, Raquel Seruca, Fátima Carneiro, Jorge Amil, José Carlos Machado & Fernando Tavarela-Veloso, Inflammatory Bowel Diseases 2005; 11 (4): pp. 331 - 339, PubMed: 15803022 40. Mutations in the NOD2/CARD15 gene in Crohn's disease are associated with ileocecal resection and are a risk factor for reoperation, Carsten Büning, J. Genschel, S. Bühner, S. Krüger, K. Kling, A. Dignass, P. Baier, B. Bochow, J. Ockenga, H. H. J. Schmidt & H. Lochs, Alimentary Pharmacology and Therapeutics 2004; 19 (10): pp. 1073 - 1078, PubMed: 15142196 41. Association of NOD2/CARD15 mutations with previous surgical procedures in Crohn's disease, Manuel Barreiro, Conchita Núñez, J. Enrique Domínguez-Muñoz, Aurelio Lorenzo, F. Barreiro, J. Potel & A. Salvador Peña, Revista Española de Enfermedades Digestivas 2005; 97 (8): pp. 547 - 553, PubMed: 16266221 42. The molecular classification of the clinical manifestations of Crohn's disease, Tariq Ahmad, Alessandro Armuzzi,

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Mike Bunce, Kim Mulcahy-Hawes, Sara E. Marshall, Timothy R. Orchard, Jonathan Crawshaw, Oliver Large, Arjuna de Silva, Julia T. Cook, Martin Barnardo, Sue Cullen, Ken I. Welsh & Derek P. Jewell, Gastroenterology 2002; 122 (4): pp. 854 - 866, PubMed: 11910336 43. The contribution of NOD2 gene mutations to the risk and site of disease in inflammatory bowel disease, Andrew P. Cuthbert, Sheila A. Fisher, Muddassar M. Mirza, Kathy King, Jochen Hampe, Peter J. P. Croucher, Silvia Mascheretti, Jeremy Sanderson, Alastair Forbes, John Mansfield, Stefan Schreiber, Cathryn M. Lewis & Christopher G. Mathew, Gastroenterology 2002; 122 (4): pp. 867 - 874, PubMed: 11910337 44. Card15 gene overexpression in mononuclear and epithelial cells of the inflamed Crohn's disease colon, D. Berrebi, R. Maudinas, J. P. Hugot, M. Chamaillard, F. Chareyre, P. De Lagausie, C. Yang, P. Desreumaux, M. Giovannini, J. P. Cezard, H. Zouali, D. Emilie & M. Peuchmaur, Gut 2003; 52 (6): pp. 840 - 846, PubMed: 12740340 45. NOD2 and Crohn's Disease: Loss or Gain of Function?, Lars Eckmann & Michael Karin, Immunity 2005; 22 (6): pp. 661 - 667, PubMed: 15963781 46. 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 47. 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 48. NOD2 regulation of Toll-like receptor responses and the pathogenesis of Crohn's disease, T. Watanabe, A. Kitani & W. Strober, Gut 2005; 54 (11): pp. 1515 - 1518, PubMed: 16227353

82

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

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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 A 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.

Materials and Methods: Analyses of CD14 –260 C>T, CARD15/NOD2 3020insC, Toll- like 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 P 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.

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 85 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,

Chapter 5

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 86 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.

CD14, TLR4, TLR9, CARD15/NOD2 & IRAK-M 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 UV- illuminator 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. 87 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).

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 MGB- probes 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).

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 88 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.

IPAA group Healthy controls Total number (n) 157 224 Gender M/F 88/69 118/106 Mean age in yr (SD) 42.9 (11.8) 45.8 (12.8) Range 17 – 73 21 – 77 Median 41 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

CD14, TLR4, TLR9, CARD15/NOD2 & IRAK-M 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 Mean (SD) Median Range Age (yr) at diagnosis ulcerative colitis 29.7 (11.7) 27 8 – 61 Time (yr) from diagnosis ulcerative colitis to IPAA surgery 6.3 (5.5) 5 0 – 34 Time from IPAA surgery to first episode of pouchitis Total pouchitis group (n=78) 2.8 (3.1) 2 0 – 12 Infrequent pouchitis (≤2 episodes, n=43) 3.7 (3.3) 3 0 – 12 Chronic relapsing pouchitis (≥3 episodes, n=35) 2 (2.6) 1 0 – 12

Pattern of pouchitis Duration of pouch (yr) Mean (SD) Median Range No episodes of pouchitis (n=79) 6.3 (4.1) 5 0 – 14 Infrequent pouchitis (≤2 episodes, n=43) 7.6 (3.8) 7 1 – 16 Chronic relapsing pouchitis (≥3 episodes, n=35) 7.9 (3.6) 7 2 – 14 Table 2: Clinical data IPAA patients

Genotyping The genotype frequencies in the control group were in Hardy-Weinberg equilibrium for the 89 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

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.

Carrier trait analysis 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.

Polymorphisms Genotype Controls No pouchitis Infrequent Chronic pouchitis relapsing pouchitis n=224 % n=79 % n=43 % n=35 % CD14 –260 C>T CC 55 24.6 24 30.4 11 25.6 7 20.0 CT 102 45.5 34 43.0 21 48.8 22 62.9 TT 67 29.9 21 26.6 11 25.6 6 17.1

CARD15 3020InsC WT/WT 218 97.3 77 97.5 43 100.0 34 97.1 WT/InsC 6 2.7 2 2.5 0 0.0 1 2.9 InsC/InsC 0 0.0 0 0.0 0 0.0 0 0.0 90 TLR4 +896 A>G AA 208 92.9 73 92.4 38 88.4 33 94.3 AG 16 7.1 5 6.3 5 11.6 2 5.7 GG 0 0.0 1 1.3 0 0.0 0 0.0

TLR9 –1237 T>C TT 158 70.5 52 65.8 34 79.1 19 54.3 TC 60 26.8 24 30.4 9 20.9 14 40.0 CC 6 2.7 3 3.8 0 0.0 2 5.7

TLR9 +2848 G>A GG 40 17.9 20 25.3 9 20.9 6 17.1 GA 104 46.4 38 48.1 22 51.2 16 45.7 AA 80 35.7 21 26.6 12 27.9 13 37.2

IRAKM +22148G>A GG 181 80.8 64 81.0 33 76.7 28 80.0 GA 42 18.8 14 17.7 7 16.3 7 20.0 AA 1 0.4 1 1.3 3 7.0 0 0.0 Table 3: Genotypes of the CD14, CARD15, TLR4, TLR9 and IRAKM polymorphisms in subgroups of patients with IPAA and controls

CD14, TLR4, TLR9, CARD15/NOD2 & IRAK-M Pouchitis TLR9 TLR9 Haplotype Controls No pouchitis Infrequent Chronic −1237 +2848 pouchitis relapsing pouchitis 2n=448 % 2n=158 % 2n=86 % 2n=70 % T G I 181 40 51 32 40 47 28 40 T A II 195 44 77 49 37 43 24 34 C A III 69 15 29 18 9 101 18 261 C G IV 3 1 1 1 0 0 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 91 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 no- pouchitis 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

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.

If this is true, carriership of allele TLR9 –1237*C would become an important predictive 92 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.

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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 C uneventful course of infection, even when not treated. However, untreated infections may result in pelvic inflammatory disease, ectopic pregnancy and tubal infertility.

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 101 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.

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. C 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.

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 A 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. 105

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%)

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.

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References

1. Genetic epidemiology: strengths, weaknesses, and opportunities, David Sharp, The Lancet 2005; 366 (9489): pp. 880, PubMed: 16154001 2. Key concepts in genetic epidemiology, Paul R. Burton, Martin D. Tobin & John L. Hopper, The Lancet 2005; 366 (9489): pp. 941 - 951, PubMed: 16154023 3. 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 4. Human leukocyte antigen and cytokine gene variants as predictors of recurrent Chlamydia trachomatis infection in high- risk 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 5. 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 6. 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 7. 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 8. 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

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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

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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 O 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.

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.

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 110 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 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 111 including HLA-DQ and interleukin 10 (IL-10) have been associated with Chlamydia infection11.

The Toll Like Receptor (TLR) family is a group of pattern recognition receptors, which recognise several microbial products, including bacterial cell wall components and DNA12. Poltorak et al. associated TLR4 with lipopolysaccharide (LPS) recognition in mice13. 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. 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) (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

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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 Susceptibility to and severity of C. trachomatis infections 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 Subfertility cohort n 576 253 CT DNA (LCx) + 184 – 392 CT IgG + 217 – 359 CT IgG (MIF) + (>=32) 41 – (<32) 212 Coinfections – 401 + 175 C. albicans 160 N. gonorrhoeae 7 113 T. vaginalis 6 H. simplex virus 1 2 H. simplex virus 2 5 Symptoms – 335 + 221 Vulvovaginal discharge 157 Abdominal pain 81 Dysuria 58 Bleeding during / after coïtus 25 Age Average 23.6y 30y Range 15 – 41y 19 – 39y Median 23y 31y Tubal Pathology + 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 Susceptibility to and severity of C. trachomatis infections 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 115 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.

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CD14 –260 C>T 1.1 (CC) 1.2 (CT) 2.2 (TT) Total n % n % 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+ 42 12 28.6% 24 57.1% 6 14.3% Symp 56 14 25.0% 31 55.4% 11 19.6% CT IgG+ LAP+ 17 4 23.5% 10 58.8% 3 17.6% LCx– (CT DNA–) 82 28 34.1% 38 46.3% 16 19.5% MO+ 29 12 41.4% 11 37.9% 6 20.7% Symp 43 16 37.2% 14 32.6% 13 30.2% LAP+ 17 5 29.4% 8 47.1% 4 23.5% Total 359 96 26.7% 185 51.5% 78 21.7% LCx+ (CT DNA+) 49 15 30.6% 23 46.9% 11 22.4% MO+ 16 3 18.8% 10 62.5% 3 18.8% Symp 19 5 26.3% 11 57.9% 3 15.8% CT IgG– LAP+ 10 3 30.0% 6 60.0% 1 10.0% LCx– (CT DNA–) 310 81 26.1% 162 52.3% 67 21.6% MO+ 88 20 22.7% 53 60.2% 15 17.0% Symp 103 26 25.2% 51 49.5% 26 25.2% 116 LAP+ 37 6 16.2% 20 54.1% 11 29.7% Healthy Controls 170 48 28.2% 82 48.2% 40 23.5% 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

CD14 –260 C>T 1.1 (CC) 1.2 (CT) 2.2 (TT) Total n % n % n % Total 253 70 27.7% 124 49.0% 59 23.3% TP+ 50 14 28.0% 24 48.0% 12 24.0% TP– 203 56 27.6% 100 49.3% 47 23.2% CT IgG+ TP+ 28 9 32.1% 15 53.6% 4 14.3% CT IgG+ TP– 13 4 30.8% 6 46.2% 3 23.1% Healthy Controls 170 48 28.2% 82 48.2% 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 Susceptibility to and severity of C. trachomatis infections

Figure 2: CD14 genotype distribution in the STD, subfertility and control cohorts. 117 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

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.

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 118 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 Susceptibility to and severity of C. trachomatis infections 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.

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References

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Serotypes of Chlamydia trachomatis and risk for development of cervical squamous cell carcinoma, Tarja Anttila, Pekka Saikku, Pentti Koskela, Aini Bloigu, Joakim Dillner, Irma Ikaheimo, Egil Jellum, Matti Lehtinen, Per Lenner, Timo Hakulinen, Ale Närvänen, Eero Pukkala, Steinar Thoresen, Linda Youngman & Jorma Paavonen, Journal of the American Medical Association 2001; 285 (1): pp. 47 - 51, PubMed: 11150108 9. Analysis of genetic heterogeneity in Chlamydia trachomatis clinical isolates of serovars D, E and F by Amplified Fragment length Polymorphism, Servaas A. Morré, Jacobus M. Ossewaarde, Paul H. M. Savelkoul, Jeroen Stoof, Chris J. L. M. Meijer & Adriaan J. C. van den Brule, Journal of Clinical Microbiology 2000; 38 (9): pp. 3463 - 3466, PubMed: 10970405 10. Interrelationship between polymorphisms of incA, fusogenic properties of Chlamydia trachomatis strains, and clinical manifestations in patients in The Netherlands, Yvonne Pannekoek, Joke Spaargaren, Ankie A. J. Langerak, Judith Merks, Servaas A. Morré & Arie van der Ende, Journal of Clinical Microbiology 2005; 43 (5): pp. 2441 - 2443, PubMed: 15872278 120 11. HLA DQ alleles and interleukin-10 polymorphism associated with Chlamydia trachomatis-related tubal factor infertility: a case-control study, Anne H. Kinnunen, Helja Marja Surcel, Matti Lehtinen, J. Karhukorpi, Aila Tiitinen, M. Halttunen, A. Bloigu, Richard P. Morrison, Riitta Karttunen & Jorma Paavonen, Human Reproduction 2002; 17 (8): pp. 2073 - 2078, PubMed: 12151439 12. Toll-like receptors, Kiyoshi Takeda, Tsuneyasu Kaisho & Shizuo Akira, Annual Review of Immunology 2003; 21 pp. 335 - 376, PubMed: 12524386 13. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene, Alexander Poltorak, Xiaolong He, Irina Smirnova, Mu-Ya Liu, Christophe van Huffel, Xin Du, Dale Birdwell, Erica Alejos, Maria Silva, Chris Galanos, Marina Freudenberg, Paola Ricciardi-Castagnoli, Betsy Layton & Bruce Beutler, Science 1998; 282 (5396): pp. 2085 - 2088, PubMed: 9851930 14. Tlr4: central component of the sole mammalian LPS sensor, Bruce Beutler, Current Opinion in Immunology 2000; 12 (1): pp. 20 - 26, PubMed: 10679411 15. The role of toll-like receptor (TLR) 2 and TLR4 in the host defense against disseminated candidiasis, Mihai G. Netea, Chantal A. A. van der Graaf, Alieke G. Vonk, Ineke Verschueren, Jos W. M. van der Meer & Bart Jan Kullberg, Journal of Infectious Diseases 2002; 185 (10): pp. 1483 - 1489, PubMed: 11992285 16. TLR4 mutations are associated with endotoxin hyporesponsiveness in humans, Nancy C. Arbour, Eva Lorenz, Brian C. Schutte, Joseph Zabner, Joel N. 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A Polymorphism* in the 5' flanking region of the CD14 gene is associated with circulating soluble CD14 levels and with total serum immunoglobulin E, Mauro Baldini, I. Carla Lohman, Marilyn Halonen, Robert P. Erickson, Patrick G. Holt & Fernando D. Martinez, American Journal of Respiratory Cell and Molecular Biology 1999; 20 (5): pp. 976 - 983, PubMed: 10226067 21. Sp1 is a critical factor for the monocytic specific expression of human CD14, Dong-Er Zhang, Christopher J. Hetherington, Shencao Tan, Suzan E. Dziennis, David A. Gonzalez, Hui-Min Chen & Daniel G. Tenen, Journal

CD14 Susceptibility to and severity of C. trachomatis infections of Biological Chemistry 1994; 269 (15): pp. 11425 - 11434, PubMed: 7512565 22. C(-260)-->T polymorphism in the promoter of the CD14 monocyte receptor gene as a risk factor for myocardial infarction, Jaroslav A. Hubacek, G. Rothe, Jan Pit'ha, Zdena Škodová, Vladimir Stanek, Rudolf Poledne & Gerd Schmitz, Circulation 1999; 99 (25): pp. 3218 - 3220, PubMed: 10385492 23. Interaction of polymorphisms in the CARD15 and CD14 genes in patients with Crohn disease, Wolfram Klein, Andreas Tromm, Thomas Griga, Christian Folwaczny, Michael Hocke, Klaus Eitner, Michaela Marx, Natascha Duerig & Jörg Thomas Epplen, Scandinavian Journal of Gastroenterology 2003; 38 (8): pp. 834 - 836, PubMed: 12940436 24. CD14 and TNFa promoter polymorphisms in patients with acute arthritis. Special reference to development of chronic spondyloarthropathy, Heikki Repo, Krista Anttonen, Sami K. Kilpinen, Aarno Palotie, Petri Salven, Arto Orpana & Marjatta Leirisalo-Repo, Scandinavian Journal of Rheumatology 2002; 31 (6): pp. 355 - 361, PubMed: 12492251 25. A CD14 promoter polymorphism is associated with CD14 expression and Chlamydia-stimulated TNFalpha production, Hock-Liew Eng, Chiou-Huey Wang, Chih-Hung Chen, M. H. Chou, C. T. Cheng & Tsun-Mei Lin, Genes & Immunity 2004; 5 (5): pp. 426 - 430, PubMed: 15164100 26. CD14 promoter polymorphism -159C>T is associated with susceptibility to chronic Chlamydia pneumoniae infection in peripheral blood monocytes, J. Rupp, W. Goepel, E. Kramme, J. Jahn, W. Solbach & Matthias Maass, Genes & Immunity 2004; 5 (5): pp. 435 - 438, PubMed: 15175649 27. Comparison between the LCx probe system and the COBAS AMPLICOR system for detection of Chlamydia trachomatis and Neisseria gonorrhoeae infections in patients attending a clinic for treatment of sexually transmitted diseases in Amsterdam, The Netherlands, Gerard J. J. van Doornum, L M. Schouls, A. S. Pijl, Irina Cairo, M. Buimer & Sylvia M. Bruisten, Journal of Clinical Microbiology 2001; 39 (3): pp. 829 - 835, PubMed: 11230391 28. Diagnosing genital ulcer disease in a clinic for sexually transmitted diseases in Amsterdam, The Netherlands, Sylvia M. Bruisten, Irina Cairo, Han Fennema, A. Pijl, M. Buimer, Paul G. H. Peerbooms, E. van Dyck, Adam Meijer, Jacobus M. Ossewaarde & Gerard J. J. van Doornum, Journal of Clinical Microbiology 2001; 39 (2): pp. 601 - 605, PubMed: 11158114 29. Amplified fragment length polymorphism fingerprinting for identification of a core group of Neisseria gonorrhoeae transmitters in the population attending a clinic for treatment of sexually transmitted diseases in Amsterdam, The Netherlands, Joke Spaargaren, J. Stoof, Han Fennema, Roel A. Coutinho & Paul H. Savelkoul, Journal of Clinical Microbiology 2001; 39 (6): pp. 2335 - 2337, PubMed: 11376085 30. Manual of Clinical Microbiology, Editors: Patrick R. Murray, Ellen Jo Baron, Michael A. Pfaller, Fred C. Tenover & Robert H. Yolken, American Society for Microbiology Press, Washington D.C., USA 1995, ISBN: 1-55581-086-1 31. Host and pathogen interaction during vaginal infection by Trichomonas vaginalis and Mycoplasma hominis or Ureaplasma urealyticum, Cindy van der Schee, Hans J. F. Sluiters, Willem I. van der Meijden, Patricia van Beek, Paul G. H. Peerbooms, Henri Verbrugh & Alex van Belkum, Journal of Microbiological Methods 2001; 45 (1): pp. 61 - 67, PubMed: 11295198 121 32. Performance of five serological chlamydia antibody tests in subfertile women, Jolande A. Land, Anna P. Gijsen, A. G. H. Kessels, Marlea E. P. Slobbe & Cathrien A. Bruggeman, Human Reproduction 2003; 18 (12): pp. 2621 - 2627, PubMed: 14645182 33. How to use Chlamydia antibody testing in subfertility patients, Jolande A. Land, Johannes L. H. Evers & Valère J. Goossens, Human Reproduction 1998; 13 (4): pp. 1094 - 1098, PubMed: 9619578 34. Comparison of three commercially available peptide-based immunoglobulin G (IgG) and IgA assays to microimmunofluorescence assay for detection of Chlamydia trachomatis antibodies, Servaas A. Morré, Christian Munk, Kenneth Persson, Susanne Krüger-Kjaer, Rogier van Dijk, Chris J. L. M. Meijer & Adriaan J. C. van den Brule, Journal of Clinical Microbiology 2002; 40 (2): pp. 584 - 587, PubMed: 11825974 35. The inflammatory cytokine response to Chlamydia trachomatis infection is endotoxin mediated, Robin R. Ingalls, Peter A. Rice, Nilofer Qureshi, Kuni Takayama, Juey Shin Lin & Douglas T. Golenbock, Infection and Immunity 1995; 63 (8): pp. 3125 - 3130, PubMed: 7542638 36. Endotoxic activity and chemical structure of lipopolysaccharides from Chlamydia trachomatis serotypes E and L2 and Chlamydophila psittaci 6BC, Holger Heine, Sven Müller-Loennies, Lore Brade, Buko Lindner & Helmut Brade, European Journal of Biochemistry 2003; 270 (3): pp. 440 - 450, PubMed: 12542694 37. Lipopolysaccharide interaction with cell surface Toll-like receptor 4-MD-2: higher affinity than that with MD-2 or CD14, Sachiko Akashi, Shin-ichiroh Saitoh, Yasutaka Wakabayashi, Takane Kikuchi, Noriaki Takamura, Yoshinori Nagai, Yutaka Kusumoto, Koichi Fukase, Shoichi Kusumoto, Yoshiyuki Adachi, Atsushi Kosugi & Kensuke Miyake, Journal of Experimental Medicine 2003; 198 (7): pp. 1035 - 1042, PubMed: 14517279 38. Monocytes can phagocytose Gram-negative bacteria by a CD14-dependent mechanism, Uwe Grunwald, Xiaolong Fan, Robert S. Jack, Grefachew Workalemahu, Axel Kallies, Felix Stelter & Christine Schütt, Journal of Immunology 1996; 157 (9): pp. 4119 - 4125, PubMed: 8892647 39. Response to Neisseria gonorrhoeae by cervicovaginal epithelial cells occurs in the absence of toll-like receptor 4- mediated signaling, Raina N. Fichorova, Amanda O. Cronin, Egil Lien, Deborah J. Anderson & Robin R. Ingalls, Journal of Immunology 2002; 168 (5): pp. 2424 - 2432, PubMed: 11859134 40. Differential expression of Toll-like receptors 2 and 4 in tissues of the human female reproductive tract, Patricia A. Pioli, Eyal Amiel, Todd M. Schaefer, John E. Connolly, Charles R. Wira & Paul M. Guyre, Infection and Immunity 2004; 72 (10): pp. 5799 - 5806, PubMed: 15385480 41. Characterization of Toll-like receptors in the female reproductive tract in humans, A. Fazeli, C. Bruce & D. O. Anumba, Human Reproduction 2005; 20 (5): pp. 1372 - 1378, PubMed: 15695310 42. Cutting edge: naturally occurring soluble form of mouse Toll-like receptor 4 inhibits lipopolysaccharide signaling, Ken-Ichiro Iwami, Tetsuya Matsuguchi, Akio Masuda, Takeshi Kikuchi, Tipayaratn Musikacharoen &

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Yasunobu Yoshikai, Journal of Immunology 2000; 165 (12): pp. 6682 - 6686, PubMed: 11120784 43. Expression of toll-like receptor 4 and endotoxin responsiveness in mice during perinatal period, Kirsi Harju, Marja Ojaniemi, Samuli Rounioja, Virpi Glumoff, Reija Paananen, Reetta Vuolteenaho & Mikko Hallman, Pediatric Research 2005; 57 (5 Pt 1): pp. 644 - 648, PubMed: 15718365 44. TLR4-dependent recognition of lipopolysaccharide by epithelial cells requires sCD14, Fredrik Bäckhed, Lisa Meijer, Staffan Normark & Agneta Richter-Dahlfors, Cellular Microbiology 2002; 4 (8): pp. 493 - 501, PubMed: 12174084 45. Correlation between human immunodeficiency virus type 1 RNA levels in the female genital tract and immune activation associated with ulceration of the cervix, Stephen D. Lawn, Shambavi Subbarao, Thomas C. Wright Jr, Tammy Evans-Strickfaden, Tedd V. Ellerbrock, Jeffrey L. Lennox, Salvatore T. Butera & Clyde E. Hart, Journal of Infectious Diseases 2000; 181 (6): pp. 1950 - 1956, PubMed: 10837174 46. Binding of lipopeptide to CD14 induces physical proximity of CD14, TLR2 and TLR1, Maria Manukyan, Kathy Triantafilou, Martha Triantafilou, Alan Mackie, Nadra Nilsen, Terje Espevik, Karl-Heinz Wiesmüller, Artur J. Ulmer & Holger Heine, European Journal of Immunology 2005; 35 (3): pp. 911 - 921, PubMed: 15714590 47. Non-LPS components of Chlamydia pneumoniae stimulate cytokine production through Toll-like receptor 2- dependent pathways, Mihai G. Netea, Bart Jan Kullberg, Jochem M. D. Galama, Anton F. H. Stalenhoef, Charles A. Dinarello & Jos W. M. van der Meer, European Journal of Immunology 2002; 32 (4): pp. 1188 - 1195, PubMed: 11932927 48. Bacterial lipopolysaccharide induces transforming growth factor beta and hepatocyte growth factor through toll-like receptor 2 in cultured human colon cancer cells, T. Yoshioka, Y. Morimoto, H. Iwagaki, H. Itoh, S. Saito, N. Kobayashi, T. Yagi & N. Tanaka, Journal of International Medical Research 2001; 29 (5): pp. 409 - 420, PubMed: 11725828 49. Toll-like receptor-2, but not Toll-like receptor-4, is essential for development of oviduct pathology in chlamydial genital tract infection, Toni Darville, Joshua M. O'Neill, C. W. Andrews Jr, Uma M. Nagarajan, Lynn Stahl & David M. Ojcius, Journal of Immunology 2003; 171 (11): pp. 6187 - 6197, PubMed: 14634135 50. Lipopolysaccharides of Bacteroides fragilis, Chlamydia trachomatis and Pseudomonas aeruginosa signal via Toll- like receptor 2, Clett Erridge, Alison Pridmore, Adrian Eley, John Stewart & Ian R. Poxton, Journal of Medical Microbiology 2004; 53 (Pt 8): pp. 735 - 740, PubMed: 15272059 51. Lipopolysaccharide recognition: CD14, TLRs and the LPS-activation cluster, Martha Triantafilou & Kathy Triantafilou, Trends in Immunology 2002; 23 (6): pp. 301 - 304, PubMed: 12072369 52. Description of the ICTI consortium: an integrated approach to the study of Chlamydia trachomatis infection, Servaas A. Morré, Joke Spaargaren, Jacobus M. Ossewaarde, Jolande A. Land, Caroline J. Bax, P. Joep Dörr, Paul M. Oostvogel, Daisy Vanrompay, Paul H. M. Savelkoul, Yvonne Pannekoek, Jan E. A. M. van Bergen, Han S. A. Fennema, Henry J. C. de Vries, J. Bart Crusius, A. Salvador Peña, James I. Ito Jr & Joseph M. Lyons, Drugs of Today 2006; 42 (Suppl A): pp. 107 - 114, PubMed: 16683050 122

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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

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Abstract

cell immunity protects against diseases caused by the obligate intracellular bacterium Chlamydia trachomatis. Incidentally, host inflammatory response T 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 genitally- infected 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) 124 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.

CCR5 Susceptibility to and severity of C. trachomatis infections Introduction

he genus Chlamydia comprises obligate intracellular, gram negative-like bacteria that cause numerous oculo-genital and respiratory infections in T 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.

There is urgency to develop intervention and prevention measures to control Chlamydial 125 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

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

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 126 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 Susceptibility to and severity of C. trachomatis infections o 37 C 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 anti-C. trachomatis IgG antibodies, as described previously47, 48. A positive C. trachomatis IgG MIF 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 Bio- 127 One), with the sense primer CCR5-d32S: 5’ CAA AAA GAA GGT CTT CAT TAC ACC 3’ and anti- sense 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

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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.

6 IFU/ml) 10 4 Wild type 2 CCR5KO 0

Chlamydia (log Chlamydia 6 122127 128 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 long- term 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 Susceptibility to and severity of C. trachomatis infections 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

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100 W/T noninfected 80 CCR5 KO noninfected 60 W/T % pregnant 40 CCR5KO

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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.

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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

10000

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2000 IFN-gamma (pg/ml) IFN-gamma 130 0 CCR5 KO WT Controls infected infected noninfected Figure 3a

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CCR5 Susceptibility to and severity of C. trachomatis infections

12

10 8 6 4 2 Fold Enhandement Fold Enhandement (Wildtype/CCR5KO) 0 IFN-gamma TNF-alpha Cytokines 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 131 (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

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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

1600 1400 1200 1000 CCR5KO 800 Wild type 600

IP-10 (pg/ml) IP-10 400 200 0 7 142128 Time (days post infection)

Figure 4a

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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 Susceptibility to and severity of C. trachomatis infections

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.

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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, 134 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 CCR5- related specific Th1 and inflammatory responses in the pathogenesis of infectious tubal pathologies.

CCR5 Susceptibility to and severity of C. trachomatis infections

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 anti- microbials 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. 135

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.

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Gray, Alberto Mantovani & Francesco Sinigaglia, Journal of Experimental Medicine 1998; 187 (1): pp. 129 - 134, PubMed: 9419219 37. CCR5 is characteristic of Th1 lymphocytes, Pius Loetscher, Mariagrazia Uguccioni, Lorenza Bordoli, Marco Baggiolini, Bernhard Moser, Carlo Chizzolini & Jean-Michel Dayer, Nature 1998; 391 (6665): pp. 344 - 345, PubMed: 9450746 38. Chemokines as regulators of T cell differentiation, Sanjiv A. Luther & Jason G. Cyster, Nature Immunology 2001; 2 (2): pp. 102 - 107, PubMed: 11175801 39. Impaired T cell function in RANTES-deficient mice, Yasuhiko Makino, Donald N. Cook, Oliver Smithies, Olivia Y. 137 Hwang, Eric G. Neilson, Laurence A. Turka, Hiroshi Sato, Andrew D. Wells & Theodore M. Danoff, Clinical Immunology 2002; 102 (3): pp. 302 - 309, PubMed: 11890717 40. The chemokine system: redundancy for robust outputs, Alberto Mantovani, Immunology Today 1999; 20 (6): pp. 254 - 257, PubMed: 10354549 41. Cutting edge: Role of C-C chemokine receptor 5 in organ-specific and innate immunity to Cryptococcus neoformans, Gary B. Huffnagle, Lisa K. McNeil, Roderick A. McDonald, Juneann W. Murphy, Galen B. Toews, Nobuya Maeda & William A. Kuziel, Journal of Immunology 1999; 163 (9): pp. 4642 - 4646, PubMed: 10528159 42. CCR5 deficiency decreases susceptibility to experimental cerebral malaria, Elodie Belnoue, Michele Kayibanda, Jean Christophe Deschemin, Mireille Viguier, Matthias Mack, William A. Kuziel & Laurent Renia, Blood 2003; 101 (11): pp. 4253 - 4259, PubMed: 12560237 43. CCR5 mediates specific migration of Toxoplasma gondii-primed CD8 lymphocytes to inflammatory intestinal epithelial cells, Souphalone Luangsay, Lloyd H. Kasper, Nicolas Rachinel, Laurie A. Minns, Franck J. Mennechet, Alain Vandewalle & Dominique Buzoni-Gatel, Gastroenterology 2003; 125 (2): pp. 491 - 500, PubMed: 12891552 44. Humoral immune response to chlamydial genital infection of mice with the agent of mouse pneumonitis, Kyle H. Ramsey, Wilbert J. th & Roger G. Rank, Infection and Immunity 1989; 57 (8): pp. 2441 - 2446, PubMed: 2744854 45. Intravaginal inoculation of mice with the Chlamydia trachomatis mouse pneumonitis biovar results in infertility, Luis M. de la Maza, Sukumar Pal, Ali Khamesipour & Ellena M. Peterson, Infection and Immunity 1994; 62 (5): pp. 2094 - 2097, PubMed: 8168974 46. 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 47. The role of chlamydia genus-specific and species-specific IgG antibody testing in predicting tubal disease in subfertile women, Janneke E. den Hartog, Jolande A. Land, Frank R. M. Stassen, Marlea E. P. Slobbe-van Drunen, A. G. H. Kessels & Cathrien A. Bruggeman, Human Reproduction 2004; 19 (6): pp. 1380 - 1384, PubMed: 15105400 48. Performance of five serological chlamydia antibody tests in subfertile women, Jolande A. Land, Anna P. Gijsen, A. G. H. Kessels, Marlea E. P. Slobbe & Cathrien A. Bruggeman, Human Reproduction 2003; 18 (12): pp. 2621 - 2627, PubMed: 14645182 49. Suppression of endogenous IL-10 gene expression in dendritic cells enhances antigen presentation for specific Th1 induction: potential for cellular vaccine development, Joseph U. Igietseme, Godwin A. Ananaba, Jacqueline Bolier, Samera Bowers, Terri Moore, Tesfaye Belay, Francis O. Eko, Deborah Lyn & Carolyn M. Black, Journal of Immunology 2000; 164 (8): pp. 4212 - 4219, PubMed: 10754317

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50. IFN-gamma knockout mice show Th2-associated delayed-type hypersensitivity and the inflammatory cells fail to localize and control chlamydial infection, Shuhe Wang, Yijun Fan, Robert C. Brunham & Xi Yang, European Journal of Immunology 1999; 29 (11): pp. 3782 - 3792, PubMed: 10556835 51. The intercellular adhesion molecule type-1 is required for rapid activation of T helper type 1 lymphocytes that control early acute phase of genital chlamydial infection in mice, Joseph U. Igietseme, Godwin A. Ananaba, Jacqueline Bolier, Samera Bowers, Terri Moore, Tesfaye Belay, Deborah Lyn & Carolyn M. Black, Immunology 1999; 98 (4): pp. 510 - 519, PubMed: 10594682 52. Fc receptor-mediated antibody regulation of T cell immunity against intracellular pathogens, Terri Moore, Charles O. Ekworomadu, Francis O. Eko, LuCinda MacMillan, Kiantra Ramey, Godwin A. Ananaba, John W. Patrickson, Periakaruppan R. Nagappan, Deborah Lyn, Carolyn M. Black & Joseph U. Igietseme, Journal of Infectious Diseases 2003; 188 (4): pp. 617 - 624, PubMed: 12898452 53. Chlamydia trachomatis serovar E isolates from patients with different clinical manifestations have similar courses of infection in a murine model: host factors as major determinants of C trachomatis mediated pathogenesis, Joseph M. Lyons, James I. Ito Jr & Servaas A. Morré, Journal of Clinical Pathology 2004; 57 (6): pp. 657 - 659, PubMed: 15166277 54. Impact of host genetics on susceptibility to human Chlamydia trachomatis disease, Olaimatu S. M. Mahdi, British Journal of Biomedical Science 2002; 59 (2): pp. 128 - 132, PubMed: 12113404 55. The persistence of chlamydial inclusions in clinically quiescent trachoma, O. E. Babalola & S. D. Bage, West African Journal of Medicine 1992; 11 (1): pp. 55 - 61, PubMed: 1322165 56. Severe disease in children with trachoma is associated with persistent Chlamydia trachomatis infection, L. D. Bobo, N. Novak, B. Munoz, Y. H. Hsieh, T. C. Quinn & S. West, Journal of Infectious Diseases 1997; 176 (6): pp. 1524 - 1530, PubMed: 9395364 57. Factors influencing the induction of infertility in a mouse model of Chlamydia trachomatis ascending genital tract infection, Sukumar Pal, W. Hui, Ellena M. Peterson & Luis M. de la Maza, Journal of Medical Microbiology 1998; 47 (7): pp. 599 - 605, PubMed: 9839564 58. Susceptibility of mice to vaginal infection with Chlamydia trachomatis mouse pneumonitis is dependent on the age of the animal, Sukumar Pal, Ellena M. Peterson & Luis M. de la Maza, Infection and Immunity 2001; 69 (8): pp. 5203 - 5206, PubMed: 11447208 59. Epidemiological and genetic correlates of incident Chlamydia trachomatis infection in North American adolescents, William M. Geisler, Jianming Tang, Chengbin Wang, Craig M. Wilson & Richard A. Kaslow, Journal of Infectious Diseases 2004; 190 (10): pp. 1723 - 1729, PubMed: 15499525 60. Scarring trachoma is associated with polymorphism in the tumor necrosis factor alpha (TNF-alpha) gene promoter and with elevated TNF-alpha levels in tear fluid, David J. Conway, Martin J. Holland, Robin L. Bailey, Alison E. Campbell, Olaimatu S. Mahdi, Richard Jennings, Ephraim Mbena & David C. W. Mabey, Infection and Immunity 1997; 65 (3): pp. 1003 - 1006, PubMed: 9038309 138 61. Association of Chlamydia trachomatis heat-shock protein 60 antibody and HLA class II DQ alleles, Lakshmi K. Gaur, Rosanna W. Peeling, M. Cheang, J. Kimani, Job J. Bwayo, F. Plummer & Robert C. Brunham, Journal of Infectious Diseases 1999; 180 (1): pp. 234 - 237, PubMed: 10353888 62. Risk factors for Chlamydia trachomatis pelvic inflammatory disease among sex workers in Nairobi, Kenya, J. Kimani, I. W. Maclean, Job J. Bwayo, K. MacDonald, J. Oyugi, G. M. Maitha, Rosanna W. Peeling, M. Cheang, N. J. Nagelkerke, F. A. Plummer & Robert C. Brunham, Journal of Infectious Diseases 1996; 173 (6): pp. 1437 - 1444, PubMed: 8648217 63. Chlamydia trachomatis heat shock protein-60 induced interferon-gamma and interleukin-10 production in infertile women, Anne H. Kinnunen, Helja Marja Surcel, M. Halttunen, Aila Tiitinen, Richard P. Morrison, Sandra G. Morrison, Pentti Koskela, Matti Lehtinen & Jorma Paavonen, Clinical and Experimental Immunology 2003; 131 (2): pp. 299 - 303, PubMed: 12562392 64. The role of a mutant CCR5 allele in HIV-1 transmission and disease progression, Yaoxing Huang, William A. Paxton, Stevan M. Wolinsky, Avidan U. Neumann, Linqi Zhang, Tian He, Stanley Kang, Daniel Ceradini, Zhanqun Jin, Karina Yazdanbakhsh, Kevin Kunstman, Daniel Erickson, Elizabeth Dragon, Nathaniel R. Landau, John Phair, David D. Ho & Richard A. Koup, Nature Medicine 1996; 2 (11): pp. 1240 - 1243, PubMed: 8898752 65. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE Study, Michael Dean, Mary Carrington, Cheryl Winkler, Gavin A. Huttley, Michael W. Smith, Rando Allikmets, James J. Goedert, Susan P. Buchbinder, Eric Vittinghoff, Edward Gomperts, Sharyne Donfield, David Vlahov, Richard Kaslow, Alfred Saah, Charles Rinaldo, Roger Detels & Stephen J. O'Brien, Science 1996; 273 (5283): pp. 1856 - 1862, PubMed: 8791590 66. The CCR5-{Delta}32 mutation: impact on disease outcome in individuals with hepatitis C infection from a single source, C. Goulding, R. McManus, A. Murphy, G. Macdonald, S. Barrett, J. Crowe, J. Hegarty, S. McKiernan & D. Kelleher, Gut 2005; 54 (8): pp. 1157 - 1161, PubMed: 15863470 67. Identification of T-cell stimulatory antigens of Chlamydia trachomatis using synovial fluid-derived T-cell clones, A. B. Hassell, D. J. Reynolds, M. Deacon, J. S. Gaston & J. H. Pearce, Immunology 1993; 79 (4): pp. 513 - 519, PubMed: 7691730 68. [Use of an experimental Chlamydia trachomatis salpingitis model for evaluating the effectiveness of antibiotics and anti- inflammatory agents on fertility], P. Verhoest, J. Orfila & E. Bissac, Journal de Gynecologie, Obstetrique et Biologie de la Reproduction 1997; 26 (3): pp. 309 - 314, PubMed: 9265054 69. Dexamethasone in the complex treatment of Chlamydial conjunctivitis, Y. F. Maichuk, Revue Internationale du Trachome et de Pathologie Oculaire Tropicale et Subtropicale et de Sante Publique 1991; 68 pp. 83 - 93, PubMed: 1669654

CCR5 Susceptibility to and severity of C. trachomatis infections 70. Does addition of anti-inflammatory agents to antimicrobial therapy reduce infertility after murine chlamydial salpingitis?, D. V. Landers, M. L. Sung, K. Bottles & Julius Schachter, Sexually Transmitted Diseases 1993; 20 (3): pp. 121 - 125, PubMed: 8511704 71. Clindamycin and ibuprofen effects on Chlamydial salpingitis in mice, J. D. Blanco, R. M. Patterson, I. Ramzy & T. Turner, Sexually Transmitted Diseases 1989; 16 (4): pp. 192 - 194, PubMed: 2595517 72. Effects of doxycycline and antiinflammatory agents on experimentally induced chlamydial upper genital tract infection in female macaques, Dorothy L. Patton, Y. C. Sweeney, N. J. Bohannon, A. M. Clark, James P. Hughes, A. Cappuccio, Lee Ann Campbell & Walter E. Stamm, Journal of Infectious Diseases 1997; 175 (3): pp. 648 - 654, PubMed: 9041337 73. Aspirin inhibits Chlamydia pneumoniae-induced NF-kappa B activation, cyclo-oxygenase-2 expression and prostaglandin E2 synthesis and attenuates Chlamydial growth, Hiroshi Yoneda, Koshiro Miura, Hiroshi Matsushima, Kazuro Sugi, Tomoyuki Murakami, Kazunobu Ouchi, Katsuhiro Yamashita, Haruhide Itoh, Teruko Nakazawa, Michiyasu Suzuki & Mutsunori Shirai, Journal of Medical Microbiology 2003; 52 (Pt 5): pp. 409 - 415, PubMed: 12721317

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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

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Abstract

ackground: In women, Chlamydia (C.) trachomatis upper genital tract infection can cause distal tubal damage and occlusion, increasing the risk of tubal factor B 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.

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.

Conclusions: Carrying multiple SNPs in C. trachomatis PRR genes tends to result in an 142 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 A 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.

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. 143

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. trachomatis- associated tubal pathology in subfertile women. According to their biological function

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

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 144 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

CD14, TLR4, TLR9, CARD15/NOD2 Severity of C. trachomatis infections 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 PAMP SNP TLR9 CpG-rich motifs -1237 T>C and +2848 G>A TLR4 LPS and HSP +896 A>G CD14 LPS and HSP (co-receptor of TLR4) -260 C>T CARD15/NOD2 Peptidoglycans 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 145

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 IgG- positive 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.

Chapter 9

1.1 1.2 and 2.2

n Risk of TP n Risk of TP TLR9 –1237 T>C All subfertile women 155 (68%) 20% 72 (32%) 17% CT+ subfertile women 26 (67%) 62% 13 (33%) 77% Control group 66 (68%) – 31 (32%) – TLR9 +2848 G>A All subfertile women 45 (20%) 18% 182 (80%) 19% CT+ subfertile women 6 (15%) 50% 33 (85%) 70% Control group 15 (15%) – 82 (85%) – TLR4 +896 A>Ga All subfertile women 200 (88%) 19% 27 (12%) 22% CT+ subfertile women 33 (85%) 64% 6 (15%) 83% Control group 87 (90%) – 10 (10%) – CD14 –260 C>Tb All subfertile women 60 (26%) 17% 167 (74%) 20% CT+ subfertile women 12 (31%) 67% 27 (69%) 67% Control group 26 (27%) – 71 (73%) – CARD15/NOD2 All subfertile women 211 (93%) 18% 16 (7%) 25% Leu1007fsinsC CT+ subfertile women 37 (95%) 65% 2 (5%) 100% (SNP13) Control group 95 (98%) – 2 (2%) – 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

CD14, TLR4, TLR9, CARD15/NOD2 Severity of C. trachomatis infections Carrier trait analysis 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 IgG- negative 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. 147

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

Chapter 9

shown that TLR1 – 9 are expressed in the human female genital tract. TLR4 and its co- receptor 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 pathogen- associated 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

CD14, TLR4, TLR9, CARD15/NOD2 Severity of C. trachomatis infections 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 149 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).

Chapter 9

References

1. The role that the functional Asp299Gly polymorphism in the toll-like receptor-4 gene plays in susceptibility to Chlamydia trachomatis-associated tubal infertility, Servaas A. Morré, Laura S. Murillo, Cathrien A. Bruggeman & A. Salvador Peña, Journal of Infectious Diseases 2003; 187 (2): pp. 341 - 342, PubMed: 12552467 2. 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 3. Assay of locus-specific genetic load implicates rare Toll-like receptor 4 mutations in meningococcal susceptibility, Irina Smirnova, Navjiwan Mann, Annemiek Dols, H. H. Derkx, Martin L. Hibberd, Michael Levin & Bruce Beutler, Proceedings of the National Academy of Sciences of the United States of America 2003; 100 (10): pp. 6075 - 6080, PubMed: 12730365 4. Increased risk of noncardia gastric cancer associated with proinflammatory cytokine gene polymorphisms, Emad M. El- Omar, Charles S. Rabkin, Marilie D. Gammon, Thomas L. Vaughan, Harvey A. Risch, Janet B. Schoenberg, Janet L. Stanford, Susan T. Mayne, James Goedert, William J. Blot, Joseph F. Fraumeni Jr & Wong-Ho Chow, Gastroenterology 2003; 124 (5): pp. 1193 - 1201, PubMed: 12730860 5. A proinflammatory genetic profile increases the risk for chronic atrophic gastritis and gastric carcinoma, José Carlos Machado, Céu Figueiredo, Paolo Canedo, Paul Pharoah, Ralph Carvalho, Sérgio Nabais, Catarina Castro Alves, Maria Luisa Campos, Leen-Jan van Doorn, Carlos Caldas, Raquel Seruca, Fátima Carneiro & Manuel Sobrinho- Simões, Gastroenterology 2003; 125 (2): pp. 364 - 371, PubMed: 12891537 6. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease, Jean-Pierre Hugot, Mathias Chamaillard, Habib Zouali, Suzanne Lesage, Jean-Pierre Cézard, Jacques Belaiche, Sven Almer, Curt Tysk, Colm A. O'Morain, Miquel Gassull, Vibeke Binder, Yigael Finkel, Antoine Cortot, Robert Modigliani, Pierre Laurent-Puig, Corine Gower-Rousseau, Jeanne Macry, Jean-Frédéric Colombel, Mourad Sahbatou & Gilles Thomas, Nature 2001; 411 (6837): pp. 599 - 603, PubMed: 11385576 7. NOD2/CARD15 gene polymorphisms in Crohn's disease: a genotype- phenotype analysis, Denis Heresbach, Véronique Gicquel-Douabin, Brigitte Birebent, Pierre-Nicolas D'halluin, Nathalie Heresbach-Le Berre, Stéphane Dreano, Laurent Siproudhis, Alain Dabadie, Michel Gosselin, Jean Mosser, Gilbert Semana, Jean-François Bretagne & Jacqueline Yaouanq, European Journal of Gastroenterology and Hepatology 2004; 16 (1): pp. 55 - 62, PubMed: 15095853 8. How to use Chlamydia antibody testing in subfertility patients, Jolande A. Land, Johannes L. H. Evers & Valère J. Goossens, Human Reproduction 1998; 13 (4): pp. 1094 - 1098, PubMed: 9619578 9. The role of Chlamydia genus-specific and species-specific IgG antibody testing in predicting tubal disease in subfertile women, Janneke E. den Hartog, Jolande A. Land, Frank R. M. Stassen, Marlea E. P. Slobbe-van Drunen, A. G. H. 150 Kessels & Cathrien A. Bruggeman, Human Reproduction 2004; 19 (6): pp. 1380 - 1384, PubMed: 15105400 10. Performance of five serological chlamydia antibody tests in subfertile women, Jolande A. Land, Anna P. Gijsen, A. G. H. Kessels, Marlea E. P. Slobbe & Cathrien A. Bruggeman, Human Reproduction 2003; 18 (12): pp. 2621 - 2627, PubMed: 14645182 11. Role of the toll-like receptor 4 Asp299Gly polymorphism in susceptibility to Candida albicans infection, Servaas A. Morré, Laura S. Murillo, Joke Spaargaren, Han S. A. Fennema & A. Salvador Peña, Journal of Infectious Diseases 2002; 186 (9): pp. 1377 - 1379, PubMed: 12402214 12. CARD15 gene and the classification of Crohn's disease, Laura S. Murillo, J. Bart A. Crusius, Ad A. van Bodegraven, Behrooz Z. Alizadeh & A. Salvador Peña, Immunogenetics 2002; 54 (1): pp. 59 - 61, PubMed: 11976792 13. 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 Gionchett, Fernando Rizzello, Claudia Morselli, Elisabetta Caramelli, R. Conte, G. Poggioli, Massimo Campieri & A. Salvador Peña, World Journal of Gastroenterology 2005; 11 (46): pp. 7323 - 7329, PubMed: 16437636 14. Relationship between Ureaplasma urealyticum vaginal colonization and polymorphism in the interleukin-1 receptor antagonist gene, Jan Jeremias, Paulo Giraldo, Shane Durrant, Ayrton Ribeiro-Filho & Steven S. Witkin, Journal of Infectious Diseases 1999; 180 (3): pp. 912 - 914, PubMed: 10438393 15. HLA DQ alleles and interleukin-10 polymorphism associated with Chlamydia trachomatis-related tubal factor infertility: a case-control study, Anne H. Kinnunen, Helja Marja Surcel, Matti Lehtinen, J. Karhukorpi, Aila Tiitinen, M. Halttunen, A. Bloigu, Richard P. Morrison, Riitta Karttunen & Jorma Paavonen, Human Reproduction 2002; 17 (8): pp. 2073 - 2078, PubMed: 12151439 16. Human leukocyte antigen and cytokine gene variants as predictors of recurrent Chlamydia trachomatis infection in high- risk 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 17. Deficient host-bacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn's disease and ulcerative colitis, D. Franchimont, Séverine Vermeire, H. El Housni, Marie Pierik, Kristel Van Steen, T. Gustot, E. Quertinmont, M. Abramowicz, André van Gossum, J. Deviere & Paul Rutgeerts, Gut 2004; 53 (7): pp. 987 - 992, PubMed: 15194649 18. Radiological sacroiliitis, a hallmark of spondylitis, is linked with CARD15 gene polymorphisms in patients with Crohn's disease, Harald Peeters, Bart Vander Cruyssen, D. Laukens, P. Coucke, D. Marichal, M. Van Den Berghe, C. Cuvelier, E. Remaut, H. Mielants, F. De Keyser & M. D. Vos, Annals of the Rheumatic Diseases 2004; 63 (9): pp. 1131 - 1134, PubMed: 15308523 19. Differential expression of Toll-like receptors 2 and 4 in tissues of the human female reproductive tract, Patricia A. Pioli, Eyal Amiel, Todd M. Schaefer, John E. Connolly, Charles R. Wira & Paul M. Guyre, Infection and Immunity 2004;

CD14, TLR4, TLR9, CARD15/NOD2 Severity of C. trachomatis infections 72 (10): pp. 5799 - 5806, PubMed: 15385480 20. Toll-like receptor (TLR) expression and TLR-mediated cytokine/chemokine production by human uterine epithelial cells, Todd M. Schaefer, Kristin Desouza, John V. Fahey, Kenneth W. Beagley & Charles R. Wira, Immunology 2004; 112 (3): pp. 428 - 436, PubMed: 15196211 21. Characterization of Toll-like receptors in the female reproductive tract in humans, A. Fazeli, C. Bruce & D. O. Anumba, Human Reproduction 2005; 20 (5): pp. 1372 - 1378, PubMed: 15695310 22. Nod1-mediated endothelial cell activation by Chlamydophila pneumoniae, Bastian Opitz, Stefanie Förster, Andreas C. Hocke, Matthias Maass, Bernd Schmeck, Stefan Hippenstiel, Norbert Suttorp & Matthias Krüll, Circulation Research 2005; 96 (3): pp. 319 - 326, PubMed: 15653568 23. Chlamydial serology in infertile women by immunofluorescence, R. Punnonen, P. Terho, V. Nikkanen & O. Meurman, Fertility and Sterility 1979; 31 (6): pp. 656 - 659, PubMed: 376359 24. Description of the ICTI consortium: an integrated approach to the study of Chlamydia trachomatis infection, Servaas A. Morré, Joke Spaargaren, Jacobus M. Ossewaarde, Jolande A. Land, Caroline J. Bax, P. Joep Dörr, Paul M. Oostvogel, Daisy Vanrompay, Paul H. M. Savelkoul, Yvonne Pannekoek, Jan E. A. M. van Bergen, Han S. A. Fennema, Henry J. C. de Vries, J. Bart Crusius, A. Salvador Peña, James I. Ito Jr & Joseph M. Lyons, Drugs of Today 2006; 42 (Suppl A): pp. 107 - 114, PubMed: 16683050

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152

Amoebas at the start Were not complex; They tore themselves apart And started Sex.

Arthur Guiterman

Discussion

Discussion

hronic inflammation and infection play an important role in determining the prognosis of diseases affecting the mucosal barrier of the body. C 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 T 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.

Gene Polymorphism Amino Acid dbSNPa Disease Ch. substitution studied IL-1B -511 C>T — rs16944 Barrett 1 IL-1RN +2018 T>C Ala#bAla (A#bA) rs419598 1 TLR4 +896 A>G Asp299Gly rs4986790 Barrett 1 (D299G) Crohn 2 UC 2 CD14 -260 C>T — rs2569190 Barrett 1 Crohn 2,4 UC 2 UC 5 (Pouchitis) TLR9 -1237 T>C — rs5743836 Crohn 3 +2848 G>A Pro545Pro rs352140 UC 5 (P545P) (Pouchitis) CARD15/NOD2 +2209 A>T Arg702Trp rs2066844 Crohn 4 (SNP8) (R702W) 155 +2722 G>T Gly908Arg rs2066845 (SNP12) (G908R) +3020 insC Leu1007Pro rs2066847 (SNP13) (L1007P) IRAK-M +22148 G>A Val147Ile (V147I) rs1152888 UC 5 (IRAK3) (Pouchitis) Multigene — — — Barrett 1 analysis Crohn 4 UC 5 (Pouchitis) 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. aAvailable from1: http://www.ncbi.nlm.nih.gov/SNP/ bDepending 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

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.

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 156 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. 157 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.

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.

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. 158 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.

TLR9 Haplotype -1486 T>C -1237 T>C +1174 G>A +2848 G>A Comments I T T A G II C T G A III T C G A IV T C G G Rare C T G G Rare 159 C C G G Rare T T G A 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

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 161 of the CARD15/NOD2 transcripts is a potential mechanism for regulation of bacteria- sensing 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

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

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 162 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 163 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.

Discussion

Part II

he second part of this thesis describes candidate genes involved in the innate and acquired immunity to urogenital tract infection. T 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 Amino Acid dbSNPa Chlamydia Chapter substitution trachomatis IL-1B -511 C>T — rs16944 Susceptibility 6 IL-1RN +2018 T>C Ala#bAla rs419598 Susceptibility 6 (A#bA) TLR4 +896 A>G Asp299Gly rs4986790 Severity 9 (D299G) CD14 -260 C>T — rs2569190 Susceptibility 7 / Severity Severity 9 CCR5 δ32 (32bp Premature rs333 Susceptibility 8 deletion) stop codon / Severity CARD15/NOD2 +3020 insC Leu1007Pro rs2066847 Severity 9 (SNP13) (L1007P) Multigene — — — Severity 9 164 analysis 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. aAvailable from1: http://www.ncbi.nlm.nih.gov/SNP/ bDepending 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 intra- amniotic 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 165 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.

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

No significant differences in the frequencies of the CD14-260 SNP between patients and 166 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 167 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 IFN- α 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

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.

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 168 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 169 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.

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 T with a function in immune regulation or pathogen recognition were studied using a candidate gene approach to elucidate their influence on infection and inflammation.

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.

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 170 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 43, 44, 162 HSP70 . 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 171 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.

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.

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 172 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).

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 176 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 high- throughput 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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Discussion

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Discussion

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Human oviductal stromal fibroblasts, but not oviductal epithelial cells, express Toll-like receptor 4: the site-specific mucosal immunity of the human fallopian tube against bacterial infection, Hiroko Itoh, Kaei Nasu, Masakazu Nishida, Harunobu Matsumoto, Akitoshi Yuge & Hisashi Narahara, American Journal of Reproductive

Discussion

Immunology 2006; 56 (2): pp. 91 - 101, PubMed: 16836611 161. Toll-like receptor-2, but not Toll-like receptor-4, is essential for development of oviduct pathology in chlamydial genital tract infection, Toni Darville, Joshua M. O'Neill, C. W. Andrews Jr, Uma M. Nagarajan, Lynn Stahl & David M. Ojcius, Journal of Immunology 2003; 171 (11): pp. 6187 - 6197, PubMed: 14634135 162. Heat shock protein 60 from Chlamydia pneumoniae elicits an unusual set of inflammatory responses via Toll-like receptor 2 and 4 in vivo, Clarissa U. Prazeres da Costa, Nina Wantia, Carsten J. Kirschning, Dirk H. Busch, Nuria Rodriguez, Hermann Wagner & Thomas Miethke, European Journal of Immunology 2004; 34 (10): pp. 2874 - 2884, PubMed: 15368304 163. Analysis of TLR4 polymorphic variants: new insights into TLR4/MD-2/CD14 stoichiometry, structure, and signaling, Prasad Rallabhandi, Jessica Bell, Marina S. Boukhvalova, Andrei Medvedev, Eva Lorenz, Moshe Arditi, Val G. Hemming, Jorge C. G. Blanco, David M. Segal & Stefanie N. Vogel, Journal of Immunology 2006; 177 (1): pp. 322 - 332, PubMed: 16785528 164. Toll-like receptor 4 Asp299Gly/Thr399Ile polymorphisms are a risk factor for Candida bloodstream infection, Chantal A. A. van der Graaf, Mihai G. Netea, Servaas A. Morré, Martin Den Heijer, Paul E. Verweij, Jos W. M. van der Meer & Bart Jan Kullberg, European Cytokine Network 2006; 17 (1): pp. 29 - 34, PubMed: 16613760 165. Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells, Mihai G. Netea, Roger Sutmuller, Corinna Hermann, Chantal A. A. van der Graaf, Jos W. M. van der Meer, Johan H. van Krieken, Thomas Hartung, Gosse Adema & Bart Jan Kullberg, Journal of Immunology 2004; 172 (6): pp. 3712 - 3718, PubMed: 15004175 166. Cutting edge: naturally occurring soluble form of mouse Toll-like receptor 4 inhibits lipopolysaccharide signaling, Ken-Ichiro Iwami, Tetsuya Matsuguchi, Akio Masuda, Takeshi Kikuchi, Tipayaratn Musikacharoen & Yasunobu Yoshikai, Journal of Immunology 2000; 165 (12): pp. 6682 - 6686, PubMed: 11120784 167. Expression of toll-like receptor 4 and endotoxin responsiveness in mice during perinatal period, Kirsi Harju, Marja Ojaniemi, Samuli Rounioja, Virpi Glumoff, Reija Paananen, Reetta Vuolteenaho & Mikko Hallman, Pediatric Research 2005; 57 (5 Pt 1): pp. 644 - 648, PubMed: 15718365 168. Lipopolysaccharides of Bacteroides fragilis, Chlamydia trachomatis and Pseudomonas aeruginosa signal via Toll- like receptor 2, Clett Erridge, Alison Pridmore, Adrian Eley, John Stewart & Ian R. Poxton, Journal of Medical Microbiology 2004; 53 (Pt 8): pp. 735 - 740, PubMed: 15272059 169. An integrated Approach to the Study of Chlamydia trachomatis Infection of the Female Genital Tract, Thesis by: Joseph M. Lyons, VU University Medical Centre, Amsterdam, The Netherlands, 2004 170. Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin, Cevayir Coban, Ken J. Ishii, Taro Kawai, Hiroaki Hemmi, Shintaro Sato, Satoshi Uematsu, Masahiro Yamamoto, Osamu Takeuchi, Sawako Itagaki, Nirbhay Kumar, Toshihiro Horii & Shizuo Akira, Journal of Experimental Medicine 2005; 201 (1): pp. 19 - 25, PubMed: 15630134 171. Cutting edge: the acquisition of TLR tolerance during malaria infection impacts T cell activation, James A. Perry, 186 Christine S. Olver, Robert C. Burnett & Anne C. Avery, Journal of Immunology 2005; 174 (10): pp. 5921 - 5925, PubMed: 15879082 172. Malaria blood stage parasites activate human plasmacytoid dendritic cells and murine dendritic cells through a Toll- like receptor 9-dependent pathway, Sathit Pichyangkul, Kosol Yongvanitchit, Utaiwan Kum-arb, Hiroaki Hemmi, Shizuo Akira, Arthur M. Krieg, D. Gray Heppner, V. Ann Stewart, Hitoshi Hasegawa, Sornchai Looareesuwan, G. Dennis Shanks & R. Scott Miller, Journal of Immunology 2004; 172 (8): pp. 4926 - 4933, PubMed: 15067072 173. Was induction of HIV-1 through TLR9?, Sudhir Agrawal & R. Russell Martin, Journal of Immunology 2003; 171 (4): pp. 1621 - 1622, PubMed: 12902456 174. Toll-like receptor 2 (TLR2) and TLR9 signaling results in HIV-long terminal repeat trans-activation and HIV replication in HIV-1 transgenic mouse spleen cells: implications of simultaneous activation of TLRs on HIV replication, Ozlem Equils, Marco L. Schito, Hiase Karahashi, Zeynep Madak, Ayse Yarali, Kathrin S. Michelsen, Alan Sher & Moshe Arditi, Journal of Immunology 2003; 170 (10): pp. 5159 - 5164, PubMed: 12734363 175. Common polymorphisms of toll-like receptors 4 and 9 are associated with the clinical manifestation of malaria during pregnancy, Frank P. Mockenhaupt, Lutz Hamann, Christiane von Gaertner, George Bedu-Addo, Cordula von Kleinsorgen, Ralf R. Schumann & Ulrich Bienzle, Journal of Infectious Diseases 2006; 194 (2): pp. 184 - 188, PubMed: 16779724 176. Toll-like receptor function and signaling, Tsuneyasu Kaisho & Shizuo Akira, Journal of Allergy and Clinical Immunology 2006; 117 (5): pp. 979 - 987, PubMed: 16675322 177. Common human Toll-like receptor 9 polymorphisms and haplotypes: association with atopy and functional relevance, B. Berghöfer, T. Frommer, I. R. König, A. Ziegler, T. Chakraborty, G. Bein & Holger Hackstein, Clinical and Experimental Allergy 2005; 35 (9): pp. 1147 - 1154, PubMed: 16164440 178. Plasmacytoid dendritic cells: the key to CpG, Simon Rothenfusser, Evelyn Tuma, Stefan Endres & Gunther Hartmann, Human Immunology 2002; 63 (12): pp. 1111 - 1119, PubMed: 12480254 179. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells, Anne Krug, Simon Rothenfusser, Veit Hornung, Bernd Jahrsdörfer, Susan Blackwell, Zuhair K. Ballas, Stefan Endres, Arthur M. Krieg & Gunther Hartmann, European Journal of Immunology 2001; 31 (7): pp. 2154 - 2163, PubMed: 11449369 180. Chemokine and chemokine receptor dynamics during genital chlamydial infection, Tesfaye Belay, Francis O. Eko, Godwin A. Ananaba, Samera Bowers, Terri Moore, Deborah Lyn & Joseph U. Igietseme, Infection and Immunity 2002; 70 (2): pp. 844 - 850, PubMed: 11796619 181. Human immunodeficiency virus receptor and coreceptor expression on human uterine epithelial cells: regulation of

expression during the menstrual cycle and implications for human immunodeficiency virus infection, Grant R. Yeaman, Alexandra L. Howell, Sally Weldon, Douglas J. Demian, Jane E. Collins, Denise M. O'Connell, Susana N. Asin, Charles R. Wira & Michael W. Fanger, Immunology 2003; 109 (1): pp. 137 - 146, PubMed: 12709027 182. HIV-1 infection of the female reproductive tract, Alexandra L. Howell, Susana N. Asin, Grant R. Yeaman & Charles R. Wira, Current HIV/AIDS Reports 2005; 2 (1): pp. 35 - 38, PubMed: 16091247 183. Human immunodeficiency virus type 1 infection is associated with significant mucosal inflammation characterized by increased expression of CCR5, CXCR4, and beta-chemokines, Jenny Olsson, Michael Poles, Anna-Lena Spetz, Julie Elliott, Lance Hultin, Janis Giorgi, Jan Andersson & Peter Anton, Journal of Infectious Diseases 2000; 182 (6): pp. 1672 - 1677, PubMed: 11069233 184. Contemporary approaches to designing and evaluating vaccines against Chlamydia, Joseph U. Igietseme, Francis O. Eko & Carolyn M. Black, Expert Review of Vaccines 2003; 2 (1): pp. 129 - 146, PubMed: 12901604 185. Early local cytokine profiles in strains of mice with different outcomes from Chlamydial genital tract infection, Toni Darville, Charles W. Andrews Jr, James D. Sikes, Patrick L. Fraley & Roger G. Rank, Infection and Immunity 2001; 69 (6): pp. 3556 - 3561, PubMed: 11349013 186. Effect of NOD2/CARD15 variants in T-cell depleted allogeneic stem cell transplantation, Miquel Granell, Alvaro Urbano-Ispizua, Juan Ignacio Aróstegui, Francesc Fernández-Avilés, Carmen Martínez, Montserrat Rovira, Josefa Rius, Susana Plaza, Anna Gaya, Alfons Navarro, Carme Talarn, Enric Carreras, Mariano Monzó, Emili Montserrat & Jordi Yagüe, Haematologica 2006; 91 (10): pp. 1372 - 1376, PubMed: 17018387 187. Mutations in innate immune system NOD2/CARD15 and TLR4 (Thr399Ile) genes influence the risk for severe acute graft-versus-host disease in patients who underwent an allogeneic transplantation, Ahmet H. Elmaagacli, Michael Koldehoff, Heidrun Hindahl, Nina K. Steckel, Rudolf Trenschel, Rudolf Peceny, Hellmut Ottinger, Peter Michael Rath, Rudolf Stefan Ross, Michael Roggendorf, Hans Grosse-Wilde & Dietrich W. Beelen, Transplantation 2006; 81 (2): pp. 247 - 254, PubMed: 16436969 188. Expression and subcellular distribution of toll-like receptors TLR4, TLR5 and TLR9 on the gastric epithelium in Helicobacter pylori infection, B. Schmaußer, M. Andrulis, S. Endrich, S. K. Lee, C. Josenhans, H. K. Müller- Hermelink & M. Eck, Clinical and Experimental Immunology 2004; 136 (3): pp. 521 - 526, PubMed: 15147355 189. Strategic compartmentalization of Toll-like receptor 4 in the mouse gut, Cesar F. Ortega-Cava, Shunji Ishihara, Mohammad A. K. Rumi, Kousaku Kawashima, Norihisa Ishimura, Hideaki Kazumori, Jun Udagawa, Yasunori Kadowaki & Yoshikazu Kinoshita, Journal of Immunology 2003; 170 (8): pp. 3977 - 3985, PubMed: 12682225 190. Delta 32 mutation of the chemokine-receptor 5 gene in inflammatory bowel disease, K. Martin, M. Heinzlmann, R. Borchers, M. Mack, K. Loeschke & C. Folwaczny, Clinical Immunology 2001; 98 (1): pp. 18 - 22, PubMed: 11141322 191. Chemokine Receptor 5 Expression in Gastric Mucosa of Helicobacter pylori-Infected and Noninfected Children, S. Krauss-Etschmann, E. Sammler, S. Koletzko, N. Konstantopoulos, D. Aust, B. Gebert, B. Luckow, D. 187 Reinhardt & D. J. Schendel, Clinical and Diagnostic Laboratory Immunology 2003; 10 (1): pp. 22 - 29, PubMed: 12522035 192. Frequency of the HIV-protective CC chemokine receptor 5-Delta32/Delta32 genotype is increased in hepatitis C, Rainer P. Woitas, Golo Ahlenstiel, Agathe Iwan, Jürgen K. Rockstroh, Hans H. Brackmann, Bernd Kupfer, Bertfried Matz, Ruth Offergeld, Tilman Sauerbruch & Ulrich Spengler, Gastroenterology 2002; 122 (7): pp. 1721 - 1728, PubMed: 12055576 193. CC-type chemokine receptor 5-Delta32 mutation protects against primary sclerosing cholangitis, Liesbet Henckaerts, Johan Fevery, Werner Van Steenbergen, Chris Verslype, Paul Yap, Frederik Nevens, Tania Roskams, Louis Libbrecht, Paul Rutgeerts & Severine Vermeire, Inflammatory Bowel Diseases 2006; 12 (4): pp. 272 - 277, PubMed: 16633049 194. Accumulation of CCR5+ T cells around RANTES+ granulomas in Crohn's disease: a pivotal site of Th1-shifted immune response?, Motoji Oki, Haruo Ohtani, Yoshitaka Kinouchi, Eiichi Sato, Shiro Nakamura, Takayuki Matsumoto, Hiroshi Nagura, Osamu Yoshie & Tooru Shimosegawa, Laboratory Investigation 2005; 85 (1): pp. 137 - 145, PubMed: 15492753 195. Polarized production of T-helper cell type 1 cells in Peyer's patches in Crohn's disease, Takahiro Kudo, Satoru Nagata, Yo Aoyagi, Ryuyo Suzuki, Hironori Matsuda, Yoshikazu Ohtsuka, Toshiaki Shimizu, Ko Okumura & Yuichiro Yamashiro, Digestion 2004; 70 (4): pp. 214 - 225, PubMed: 15627769 196. Sclerosing cholangitis, George R. MacFaul & Roger W. Chapman, Current Opinion in Gastroenterology 2006; 22 (3): pp. 288 - 293, PubMed: 16550044 197. CCR5-Delta32 mutation is strongly associated with primary sclerosing cholangitis, R. Eri, J. R. Jonsson, N. Pandeya, D. M. Purdie, A. D. Clouston, N. Martin, D. Duffy, E. E. Powell, J. Fawcett, T. H. J. Florin & Graham L. Radford-Smith, Genes & Immunity 2004; 5 (6): pp. 444 - 450, PubMed: 15215889 198. Polymorphism of CC chemokine receptors CCR2 and CCR5 in Crohn's disease, Hans Herfarth, Beatrix Pollok- Kopp, Michael Göke, Andreas Press & Martin Oppermann, Immunology Letters 2001; 77 (2): pp. 113 - 117, PubMed: 11377705 199. Pathway studio -- the analysis and navigation of molecular networks, Alexander Nikitin, Sergei Egorov, Nikolai Daraselia & Ilya Mazo, Bioinformatics. 2003; 19 (16): pp. 2155 - 2157, PubMed: 14594725 200. Estimation and testing of genotype and haplotype effects in case-control studies: comparison of weighted regression and multiple imputation procedures, Heather J. Cordell, Genetic Epidemiology 2006; 30 (3): pp. 259 - 275, PubMed: 16496312 201. IBDChip: A new strategy to predict clinical course and development of complications in patients with inflammatory bowel disease (IBD), M. Sans, M. Artieda, T. Diego, C. Cara, L. Rodrigo, M. Barreiro, M. T. Arroyo, M.

Discussion

Aceituno, M. Alvarez-Lobos, J. Yagüe, J. I. Arostegui, L. Quintó, L. Simón, J. M. Piqué, A. Martínez & J. Panés, Gastroenterology 2006; 130 (4): pp. A52 - A53 202. Analysis of genetic variation in the LPS sensing TLR4 gene in relation to tubal pathology and Chlamydial serology status, Janneke E. den Hartog, Sander Ouburg, Jolein Pleijster, A. Salvador Peña, Jolande A. Land & Servaas A. Morré, ISSTDR, 10-13 July 2005, Amsterdam, The Netherlands 2006; 203. Description of the ICTI consortium: an integrated approach to the study of Chlamydia trachomatis infection, Servaas A. Morré, Joke Spaargaren, Jacobus M. Ossewaarde, Jolande A. Land, Caroline J. Bax, P. Joep Dörr, Paul M. Oostvogel, Daisy Vanrompay, Paul H. M. Savelkoul, Yvonne Pannekoek, Jan E. A. M. van Bergen, Han S. A. Fennema, Henry J. C. de Vries, J. Bart Crusius, A. Salvador Peña, James I. Ito Jr & Joseph M. Lyons, Drugs of Today 2006; 42 (Suppl A): pp. 107 - 114, PubMed: 16683050 204. The future of modern epidemiology: genetics, methods, and history, Shah Ebrahim, International Journal of Epidemiology 2006; 35 (3): pp. 511 - 512 205. Everything should be made as simple as possible but not simpler, Rodolfo Saracci, International Journal of Epidemiology 2006; 35 (3): pp. 513 - 514, PubMed: 16723369 206. Dissecting complex disease: the quest for the Philosopher's Stone?, Anne V. Buchanan, Kenneth M. Weiss & Stephanie M. Fullerton, International Journal of Epidemiology 2006; 35 (3): pp. 562 - 571 207. Commentary: grading the credibility of molecular evidence for complex diseases, John P. A. Ioannidis, International Journal of Epidemiology 2006; 35 (3): pp. 572 - 578, PubMed: 16540537 208. Commentary: the Human Genome: philosopher's stone or magic wand?, Robert C. Millikan, International Journal of Epidemiology 2006; 35 (3): pp. 578 - 581, PubMed: 16540534 209. Commentary: complex disease--responding to the challenge, David Coggon, International Journal of Epidemiology 2006; 35 (3): pp. 581 - 583, PubMed: 16540536 210. Commentary: rethinking epidemiology, Douglas L. Weed, International Journal of Epidemiology 2006; 35 (3): pp. 583 - 586, PubMed: 16540538 211. Commentary: what can epidemiology accomplish?, Sharon Schwartz & Ezra Susser, International Journal of Epidemiology 2006; 35 (3): pp. 587 - 590, PubMed: 16540535 212. On stones, wands, and promises, Anne V. Buchanan, Kenneth M. Weiss & Stephanie M. Fullerton, International Journal of Epidemiology 2006; 35 (3): pp. 593 - 596, PubMed: 16540532 213. Genomics and medicine. Dissecting human disease in the postgenomic era, Leena Peltonen & Victor A. McKusick, Science 2001; 291 (5507): pp. 1224 - 1229, PubMed: 11233446

188

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 I 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.

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 191 (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

Summary

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 non- colonic 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 192 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 Population Effect Ch. TLR4 +896*G Dutch Caucasian Increased risk for CD 2 Spanish Galician Protection against CD 4 CD14 -260*C Spanish Galician Protection against fistulae 3 TLR9 +2848 AA Dutch Caucasian Protection against CD 3 Spanish Galician Increased risk for colonic disease (L2) TLR9 -1237 CC Spanish Galician Associated with age above 40 (A2) 3 Increased risk for stricturing disease (B2)

Multigene analysis: TLR4 +896 AA + CD14-260*T Spanish Galician Protection against fistulae 4 CD14 -260*T + any Spanish Galician Increased risk for CD 4 CARD15/NOD2 SNP Increased in ileal resection patients CD14 -260*T + any Spanish Galician Strengthens aforementioned 4 CARD15/NOD2 SNP + TLR4 associations +896 AA Increased in steroid resistant patients 193 Multivariate logistic regression: TLR9 +2848 AA Dutch Caucasian Protection against CD 3 Spanish Galician Increased risk for colonic disease (L2) TLR9 -1237 CC Spanish Galician Associated with age over 40 years 3 Increased risk for structuring disease (B2) Smoking, origin (rural or urban) Spanish Galician Protection against CD 4 and TLR4 +896*G Age, and carriage of SNP12 and Increased risk of CD SNP13 Gender and SNP12 Increased risk of fistulae TLR4+896 AA + CD14-260*T Protection against fistulae SNP13 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 195 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.

Summary

References

1. Genetic and serological markers to identify phenotypic subgroups in a Dutch Crohn' s disease population, Ronald K. Linskens, Rosalie C. Mallant-Hent, Laura S. Murillo, B. Mary E. von Blomberg, Behrooz Z. Alizadeh & A. Salvador Peña, Digestive and Liver Disease 2004; 36 (1): pp. 29 - 34, PubMed: 14971813 2. CARD15 gene and the classification of Crohn's disease, Laura S. Murillo, J. Bart A. Crusius, Ad A. van Bodegraven, Behrooz Z. Alizadeh & A. Salvador Peña, Immunogenetics 2002; 54 (1): pp. 59 - 61, PubMed: 11976792 3. 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

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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 D 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.

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 199 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

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.

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 200 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,

van dit proefschrift

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 201 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.

Samenvatting

Polymorfisme Populatie Effect Hfdst. TLR4 +896*G Cauc. Nederlands Verhoogd risico op Crohn 2 Sp. Galicisch Verlaagd risico op Crohn 4 CD14 -260*C Sp. Galicisch Verlaagd risico op fistels 3 TLR9 +2848 AA Cauc. Nederlands Verlaagd risico op Crohn 3 Sp. Galicisch Verhoogd risico op Crohn van de dike darm (L2) TLR9 -1237 CC Sp. Galicisch Geassocieerd met leeftijd boven 3 40 jaar (A2) Verhoogd risico op een vernauwend phenotype (B2)

Analyse van meerdere genen: TLR4 +896 AA + CD14-260*T Sp. Galicisch Verlaagd risico op fistels 4 CD14 -260*T + any Sp. Galicisch Verhoogd risico op Crohn 4 CARD15/NOD2 SNP Verhoogd in patiënten met een ileum resectie CD14 -260*T + any Sp. Galicisch Versterkt de bovengenoemde 4 CARD15/NOD2 SNP + TLR4 associaties +896 AA Komt vaker voor bij patiënten met steroïden resistentie

Multivariate logistische regressie: 202 TLR9 +2848 AA Cauc. Nederlands Verlaagd risico op Crohn 3 Sp. Galicisch Verhoogd risico op Crohn van de dike darm (L2) TLR9 -1237 CC Sp. Galicisch Geassocieerd met leeftijd boven 3 40 jaar (A2) Verhoogd risico op een vernauwend phenotype (B2) Smoking, origin (rural or urban) Sp. Galicisch Verlaagd risico op Crohn 4 and TLR4 +896*G Age, and carriage of SNP12 and Verhoogd risico op Crohn SNP13 Gender and SNP12 Verhoogd risico op fistels TLR4+896 AA + CD14-260*T Verlaagd risico op fistels SNP13 Verhoogd in patiënten met een ileum resectie Tabel 1: Overzicht van de resultaten van de studies naar de ziekte van Crohn (Hoofdstukken 2 – 4)

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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. 203

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

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.

van dit proefschrift

References

1. Genetic and serological markers to identify phenotypic subgroups in a Dutch Crohn' s disease population, Ronald K. Linskens, Rosalie C. Mallant-Hent, Laura S. Murillo, B. Mary E. von Blomberg, Behrooz Z. Alizadeh & A. Salvador Peña, Digestive and Liver Disease 2004; 36 (1): pp. 29 - 34, PubMed: 14971813 2. CARD15 gene and the classification of Crohn's disease, Laura S. Murillo, J. Bart A. Crusius, Ad A. van Bodegraven, Behrooz Z. Alizadeh & A. Salvador Peña, Immunogenetics 2002; 54 (1): pp. 59 - 61, PubMed: 11976792 3. 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

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

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 208 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. 209 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.

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 Peña & Servaas A. Morré, Gut 2005; 54 (3): pp. 439 - 440, PubMed: 15710998 (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. 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 210 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, BMC Infectious Diseases 2005; 5 (1): pp. 114, PubMed: 16368002 (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

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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)

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uthors & Affiliations

A

Name, Surname(s) Chapter Affiliation

Ananaba, Godwin A. 8 Clark Atlanta University, Atlanta, GA, USA Barr, Erika L. 8 Clark Atlanta University, Atlanta, GA, USA Barreiro de Acosta, Manuel 3, 4 Dept. of Gastroenterology, University Hospital, Santiago de Compostela, Spain Belay, Tesfaye 8 Clark Atlanta University, 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 2, 3 Dept. of Gastroenterology, VU University Medical Centre, 212 Amsterdam, The Netherlands 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, University of Bologna, Bologna, Italy Crusius, J. Bart A. Intro, 2, 3, 5 Laboratory of Immunogenetics, Dept. of Pathology, VU University Medical Centre, Amsterdam, The Netherlands Dominguez-Muñoz, J. Enrique 3, 4 Dept. of Gastroenterology, University Hospital, Santiago de Compostela, Spain 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. 6, 7 STI Outpatient Clinic, Cluster of Infectious Diseases, Municipal Health Service, Amsterdam, Netherlands

Authors & Affiliations

Name, Surname(s) Chapter Affiliation

Gionchetti, Paolo 5 Department of Internal Medicine and Gastroenterology, Policlinico S. Orsola, University of Bologna, 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, Maastricht, The Netherlands He, Qing 8 Center for Disease Control and Prevention, Atlanta, GA, USA Morehouse School of Medicine, 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 Morehouse School of Medicine, Atlanta, GA, USA Igietseme, Joseph U. 8 213 Center for Disease Control and Prevention, Atlanta, GA, USA Morehouse School of Medicine, 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 Dept. of Gastroenterology, VU University Medical Centre, Amsterdam, The Netherlands Krishnadath, Kausilia K. 1 Dept of Gastroenterology and Hepatology, Academical Medical Centre, Amsterdam, The Netherlands Land, Jolande A. 7, 9 Department of Obstetrics and Gynaecology, Research Institute Growth and Development (GROW), Academisch Ziekenhuis Maastricht, Maastricht, The Netherlands Maastricht University, Maastricht, The Netherlands Department of Obstetrics and Gynecology, University Medical Center Groningen, Groningen, The Netherlands Lammers, Karen M. 5 Department of Internal Medicine and Gastroenterology, Policlinico S. Orsola, University of Bologna, Bologna, Italy

Addendum

Name, Surname(s) Chapter Affiliation

Lillard, James 8 Morehouse School of Medicine, Atlanta, GA, USA Linskens, Ronald K. 2 Dept. of Gastroenterology, VU University Medical Centre, Amsterdam, The Netherlands Lorenzo, Aurelio 3, 4 Dept. of Gastroenterology, University Hospital, Santiago de Compostela, Spain Lyn, Deborah 8 Morehouse School of Medicine, 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 Clark Atlanta University, Atlanta, GA, USA Mallant-Hent, Rosalie 2 Dept. of Gastroenterology, VU University Medical Centre, Amsterdam, The Netherlands Morré, Servaas A. Intro, 1 – 9 Laboratory of Immunogenetics, Dept. of Pathology & Dept. of Internal Medicine, VU University Medical Centre, 214 Amsterdam, The Netherlands Dept. of Medical Microbiology, Academic Hospital Maastricht, Maastricht, The Netherlands Morselli, Claudia 5 Department of Internal Medicine and Gastroenterology, Policlinico S. Orsola, University of Bologna, Bologna, Italy Mulder, Chris J.J. Intro, 1, 2 Dept. of Gastroenterology, VU University Medical Centre, Amsterdam, The Netherlands Okwandu, Edith 8 Clark Atlanta University, Atlanta, GA, USA Ouburg, Sander Intro, 1 – 9 Laboratory of Immunogenetics, Dept. of Pathology & Dept. of Gastroenterology, VU University Medical Centre, Amsterdam, The Netherlands Peña, A. Salvador Intro, 1 – 7, 9 Laboratory of Immunogenetics, Dept. of Pathology & Dept. of Gastroenterology, VU University Medical Centre, Amsterdam, The Netherlands Pleijster, Jolein 7 Laboratory of Immunogenetics, Dept. of Pathology, VU University Medical Centre, Amsterdam, The Netherlands

Authors & Affiliations

Name, Surname(s) Chapter Affiliation

Poggioli, Gilberto 5 Department of Surgery and organ transplantation, Policlinic S. Orsola, University of Bologna, Bologna, Italy Rizzello, Fernando 5 Department of Internal Medicine and Gastroenterology, Policlinico S. Orsola, University of Bologna, Bologna, Italy Rygiel, Agnieszka M. 1 Laboratory of Experimental Internal Medicine, Academical Medical Centre, Amsterdam, The Netherlands Spaargaren, Joke 6, 7 Laboratorium Microbiologie Twentse Achterhoek (LABMICTA). Enschede, The Netherlands

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INTERNATIONAL JOURNAL OF GRATITUDE AND ACKNOWLEDGEMENTS, DECEMBER 2006, pp. 1 – 5 Vol. 1, No. 1 2006-1218/00/$3E.48+0 Copyright © 2006, International Society of G&A. All rights reserved. Received October 23, 2006 – Accepted December 18, 2006

Becoming a scientist: an acknowledgment 1, 2, 3, # SANDER OUBURG, PhC, ABD 1Laboratory of Immunogenetics, dept. of Pathology & dept. of Gastroenterology, VU University Medical Centre, Amsterdam, The Netherlands 2Institute of Gratitude, Mijdrecht, The Netherlands 3Secretary 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.

Introduction Apart from citation, which is not usually considered to As defined in Wikipedia, the free encyclopaedia be an acknowledgment, acknowledgment of conceptual [http://en.wikipedia.org/wiki/Acknowledgment_%28cr support is widely considered to be the most important eative_arts%29] in the creative arts and scientific for identifying intellectual debt. Some literature, an acknowledgment (also spelled acknowledgments of financial support, on the other acknowledgement) is an expression of gratitude for hand, may simply be legal formalities imposed by the assistance in creating the work presented. Receiving granting institution. However, for writing a thesis, credit by way of acknowledgment rather than acknowledgements to those who are not involved in the authorship indicates that the person or organization did conceptual support of the thesis are as important as not have a direct hand in producing the work in those who are. 216 question, but may have contributed funding, criticism, or encouragement to the author(s). Various schemes Aim of the current manuscript is to identify those exist for classifying acknowledgments; Giles & persons and groups eligible for acknowledgments using Councill1 proposed the six categories, presented in the Systematic Acknowledger Index (SAI), and to table 1. subsequently define appropriate acknowledgements.

Acknowledgeable support 1 Moral support 2 Financial support 3 Editorial support 4 Presentational support 5 Instrumental/technical support 6 Conceptual support, or Peer Interactive Communication (PIC) Table 1: Acknowledgment scheme according to Giles & Councill1

Material & Methods 7) humorous support. Since humour can be an important factor in a PhD students health and sanity. A total of 49 acknowledgeable persons were collected during the PhD study (January 2003 – December The cohort was subdivided according to the following 2006). criteria: Selection of the study participants: Participants were Promotors and co-promotors: Those involved in the selected according to the criteria of Giles and supervision of the study. Councill1, with the addition of one group: Promotion committee: Those critically reading and commenting on the thesis. # Corresponding Author: Sander Ouburg, Laboratory of Generosity: Those providing the financial support Immunogenetics, Dept. of Pathology, VU University Medical during the PhD study. Centre, De Boelelaan 1117. Postbus 7057, 1007 MB Colleagues: Those working in collaboration with the Amsterdam, The Netherlands. Tel.: +31 - (0)20 - 4448417. PhD student. Fax: +31 - (0)20-4448418. [email protected].

Authors & Affiliations

Inspirational persons: Those who have left a lasting symposia, making sure that whenever we were impression. somewhere together I was introduced to everyone of Fellow PhD students: Those who are working on their importance (at least two or three or four times ☺). You respective PhD studies introduced me to grants and management work, Students: Those who had their internships at the experiences a PhD student rarely encounters and which laboratory or in collaboration with the laboratory and will help me in my future career. Although working who have had support or supervision of the PhD with you can be demanding, it is surprising, full of student experiences and we have had a lot of fun. You have Family & Friends: Those directly or indirectly related helped me all the way to obtaining my PhD and for that to the PhD student & those considered being friends of I will always be grateful. the PHD student. Paranimphs: Those courageous enough to walk down Promotion Committee: Six experts from the that long aisle with me towards that big stage, where Netherlands and abroad – Michael Ward, Joseph my defence will be held. Lyons, Jolande Land, Chris Meijer, Gerrit Meijer, and Miscellaneous: Microbial: Those microbes directly or Paul Savelkoul – have read and evaluated this thesis. I indirectly related to the observed clinical phenotype; would like to thank them for their time and efforts, and Donors: All those persons – patients and healthy their judgment, given in such a short time period. controls – who have voluntarily donated the bodily materials used in the studies. Generosity: The work that has been presented in this LFF: Loveable Furry Friends. thesis and the publication of this thesis would not have Jolein: A small but very significant group consisting of been possible without financial support. Jolein Pleijster AstraZeneca Nederland BV, personified by Andrea Sellink and José van den Berg, has provided me with Statistical analyses: the opportunity to do my PhD study, attend courses and Groups and subgroups were compared by any meetings, and publish this thesis. Andrea and José, you statistical method available (Abacus, version 1.0; have always been very kind to me and you have helped www.asianideas.com). Corrections for multiple testing me realise this dream. For this I will always be were considered unnecessary. A p value <0.05 was grateful. considered significant. Greiner Bio-One, personified by Jeroen Molenaar, has provided most of the high quality laboratory plastics Results used in the experiments described in this thesis and has 217 Promotors and co-promotors: Four people have generously contributed to the publication of this thesis. dedicated a considerable amount of their precious time Jeroen, thank you for all your efforts and the fun to guide me during these years. Each of them an during your visits. honoured expert in his/her respective field of expertise. My parents, Eric and Marga, and my parents-in-law, Salvador, It is hard to find a more kind and modest Herman and Ria, have, apart from the moral and person than you. Your knowledge is only surpassed by emotional support, generously contributed to the your kindness. You were always hard at work, but publication of this thesis. always seemed to find time for everybody to help I would like to thank the CWI for the unemployment them, and to give kind words and advice. Thank you fee I have received since May 2006. for your time and guidance. I would like to thank all sponsors who have contributed Chris, you came in charge of the department of to this thesis, after the writing of this manuscript and Gastroenterology soon after I had started my PhD. who do not feature in this acknowledgement. Your guidance was as everything you do: straightforward and always with your goal in mind. Colleagues: Bart, the walking encyclopedia. Your Thank you for all your help and advice. knowledge of the literature is formidable. Although Elly, it has been a pleasure working with you. If ever your directions could sometimes be a little cryptic: data was needed on a patient, your meticulously kept “You need that article by ‘die dunne en die Griek’ (the records were a blessing. I wish I would have had more thin man and the Greek)”, a search through your time to learn from you, because I think you would have archive resulted in the discovery of the article by Johan broadened my horizon considerably. Thank you for den Dunnen and Stylianos Antonarakis. Thanks for all everything you have done for me. the help. Servaas, it will be hard to find the proper words for Roel and Dion, you both provided the fun on the you. As my daily supervisor, you could sometimes be laboratory. I very much enjoyed working with you my proverbial pain in the posterior region. Either for both. the work you gave me or for kicking the said posterior Marja Laine, paljon kiitoksia for your tine and helping region when I was getting lazy ☺. Your drive and me with the statistical analyses. scientific passion always made sure that there was Jolande land, thank you for allowing us to use the more than enough work to do, but you also created subfertility cohort, for your useful comments and opportunities for me to go to courses, meetings and critical reading of the manuscripts and taking place in

Addendum

the promotion committee. Pa, Moe, Jeroen, Judith, Oma, Wim, Janet, Jolein, Henry de Vries, thank you for all your efforts for the Herman, Ria, Roland, Mirjam, and Helena, you all Chlamydia studies, presented in the second part of my have been interested in the work I have been doing in thesis. the past years and even though sometimes it seemed Marieke Emonts and Adri van der Zande, thank you for mysterious and sorcery to you, you have always the collection of samples and data, the useful supported me in my work. Thank you all for discussions and help with the manuscripts. everything.

Inspirational persons: Sometimes one is lucky to Paranimphs: Roel and Helena, thank you for all the meet a person who inspires you. I have been fortunate help in this last stretch of my PhD study. to have met three such inspirational persons. Michael Ward, Joseph Igietseme and Joseph Lyons, you have Miscellaneous: Donors: None of the research shown me that great wisdom and intelligence can be presented in this thesis was possible without all those wrapped in kindness and friendliness. In our meetings anonymous patients and healthy controls who allowed you have inspired me and I have learned much. us to use their bodily materials for research. Microbial: Joseph Lyons: Joe, you have read my thesis in a very The author would like to thank Chlamydia trachomatis short time and supplied me with a torrent of useful for its ubiquitous lifestyle and for creating such comments and suggestions, which all have added value differences in clinical phenotypes, which has been and improvements to my thesis. When you’re visiting keeping scientists around the world busy for years. I in December we’ll have some oliebollen. would like to thank Helicobacter pylori for its Michael, I have very much enjoyed our tour of interesting infection pattern and keeping the discussion Amsterdam and visiting the Star Pilot. Talisker is on its influence on upper gastrointestinal tract waiting to meet us in December. pathogenesis alive. I would like to thank the commensal flora and ubiquitous microbes for their Fellow PhD students: presence and for making the scientific puzzle a little Joke Spaargaren, your thesis is already at the printer at more complex and interesting. the time of the preparation of this manuscript. I wish you all the luck with the defence of your thesis on LFF: Pets are a source of joy and unconditional love. December 12. You were my supervisor at the public Malina, Remi, Hammie, and Czarnutka have been no health laboratory and afterwards we have together exception. 218 continued working on Chlamydia. Thank you for all you have done for me. Jolein: The smallest group, but with the most Janneke den Hartog, I have always enjoyed working significant impact. Joleintje, you have loved, with you. I wish you all the luck with your thesis supported, and helped me through all these years. Even defence in 2007. I will help you wherever I can, to in this last period when I was locked up in my attic for repay you for all you have done for me. And remember days on end, I could always count on you. This hectic what you called the “Amsterdamse instelling”: Positive PhD fellow period has finally come to an end and we thinking gets you through everything. can look forward to the hectic period of preparations Agnieszka Rygiel, serdecznie dziękuję for all the help for our marriage on April 17, 2007 ☺ ♥. with the Barrett cohort. Erica Barr, I very much enjoyed meeting you at the Dediscere human est. To forget is human. There is a ISHCI in Niagara-on-the-Lake. I am certain that you good statistical chance that I have forgotten to mention will excel at your thesis defence. Hopefully, we’ll meet someone in this manuscript. If you did not find your again in the future, no longer ABD, but PhDs! ☺ name in this manuscript, please be aware that it is not Arnold Catsburg, your time as PhD student is just intentional. You have my gratitude. starting, but I am confident you will succeed. Thank you for your help with the Chlamydia studies. An overview of the person distribution in the different Vitaly Smelov, спасибо for your kindness and удачи subgroups is presented in table 2. There was a with your freshly started PhD study. significant overlap between the groups, i.e. persons who were placed in a group according to the selection Students: A great Roman philosopher once said: criteria had an increased risk of also belonging to “Homines dum docent discunt”. People learn by another selection group, compared to those persons teaching (Lucius Annaeus Seneca, Epistulae morales who did not fit the selection criteria (p: 0.0000001, OR: ad Lucilium 7, 8). Liselotte Kornman, Erik van den 20.3, 95% Confidence Interval: 2.01 – 2.02). 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. Discussion Using a systematic approach, we have identified 49 Family & Friends: Family and friends are always persons with acknowledgeable contributions to the important and one of the greatest sources of support. studied thesis. We observed a statistical increased risk

Authors & Affiliations

Group Number % Promotors and Co-promotors 4 8.16 Promotion Committee 6 12.24 Generosity 8 16.33 Colleagues 8 16.33 Inspirational persons 3 6.12 Fellow PhD students 6 12.24 Students 3 6.12 Family & Friends 13 26.53 Paranimphs 2 4.08 Miscellaneous N/A – LFF 4 8.16 Jolein 1 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 known as the linguistic style, or in a physical the selection criteria, compared to those not fitting the acknowledgment style. An example of the linguistic selection criteria. From these results we can conclude style has been presented in the results section of this that a PhD study and a thesis are indeed multi-person manuscript. approaches. Examples of the most common forms of the physical None of the work presented in the thesis would have style are presented in figure 1. been possible without the collaborative efforts of the all persons mentioned in this manuscript. The tri-morphic stages of the liquid kind of acknowledgment differs significantly from the di- The results of the subgroup analyses indicate that there morphic lifecycle of Chlamydia and the mono-morphic is a significant need for a better subgroup definition. lifestyle of other bacteria, including Helicobacter We are currently researching the possibilities for more pylori. Persons receiving the liquid kind of physical stringent subgroup definitions. Our current proposal, acknowledgment (figure 1A) are at increased risk of available at www.specialunionforconsistentkindness developing late complications, including a hang-over2. andubiquitouspoliteness.nl, results in the identification The second most common type of physical of 49 individual subgroups without significant overlap. acknowledgment is the hidden type (figure 1B). This 219 After a thorough review of the literature, we can type uses a cloaking technique to obscure the contents conclude that we are the first to report these findings of from the receiver and to induce curiosity in the this thesis. 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 box- like 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.

Acknowledgements Sander Ouburg was supported by a PhD fellowship

Figure 1: Physical style of acknowledgments. Panel A: from AstraZeneca Nederland BV, from January 2003 Physical acknowledgment of the liquid kind. Please note the to April 2006. The authors would like to acknowledge tri-morphic appearance, when encountered in its natural several websites – including http://www.elite.net/ environment. Panel B: Physical acknowledgment of the hidden ~runner/jennifers/thankyou.htm, http://www.anglik.net/ kind. This type is known to employ cloaking technology polish_phrases.htm, and http://www.proz.com/kudoz/ commonly known as gift wrapping 57077#136882 – for the non-Dutch / non-English phrases used in the manuscript (and I sure hope the Acknowledgements have two distinct styles. They can translations are correct ☺). be presented as written or verbal acknowledgments, The general concept for this manuscript has been

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. 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 2. http://www.jellinek.nl/ 3. 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

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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.

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