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Pathophysiology of Inflammatory Bowel Diseases

If the aetiology of Inflammatory Bowel Diseases is still unclear, it is commonly admitted that they are multifactor pathologies arising from a combination of genetic susceptibilities and environmental factors, which trigger an inappropriate mucosal immune response.

Genetic factors in IBD

The involvement of genetics in IBD has been suspected for almost 70 years from the observation of familial aggregation. The rate of clustering is 2-14% in patients with CD reporting a family history of CD and 5–16% for report of any type of IBD ; 7–11% in patients with UC reporting family history of UC and 8–14% for any type of IBD.24–28
The degree of familial clustering of a pathology (lS) can be expressed as the ratio (or percentage) of the risk of siblings over the reported prevalence in population. Literature reports lS ratio ranging from 15 to 42 for CD 24,25,29,30 and 7–17 for UC.25,30 In comparison with other highly complex disorders, type-1 diabetes has lS equal to 15, type-2 diabetes and schizophrenia have lS < 10, and coeliac disease has lS ratio ranging from 7 to 30.
Large concordance studies in twins have since brought new evidence to confirm the relative importance of inherited and environmental factors in the aetiology of IBD. Theoretically, a pathology that would depend entirely on genetic factors should have a concordance in monozygotic twins (identical twins) close to a 100%, while its concordance in dizygotic (non- identical twins) would decrease to 50%. On the opposite side, a disease entirely free from inherited factors would express similar concordance in both homozygotic and dizygotic twins. Studies in Northern Europe (Sweden 31,32, Denmark 33 and the UK34) found concordance rates between 20 and 50% for CD in identical twins, and lower than 10% for non-identical twins. Concordance rates for UC were lower, ranging 14–19% in monozygotic twins and 0–7% in dizygotic twins.
Orholm et al. furthermore demonstrated that the risk of developing CD for a first-degree relative to CD-patients was 10 times higher than in population, while the risk of UC in UC-relative was 8 times higher.
The identification of susceptibility genes for IBD started with the discovery of mutations in the NOD2 gene located on chromosome 16 in CD patients.35 This gene encodes the NOD2 protein for Nucleotide-binding Oligomerization Domain-containing protein 2, but also known as Caspase Recruitment Domain-containing protein 15 (CARD15) or Inflammatory Bowel Disease protein 1 (IBD1). This protein is involved in the immune response as an intracellular pattern-recognition receptor (PRR) and can sense intracellular bacterial molecules such as the muramyl dipeptide present in the peptidoglycans forming the bacterial cell wall, especially of Gram-positive bacteria.36,37 NOD2 protein can be found in many cell types, including macrophages, monocytes, dendritic cells, B- and T-lymphocytes, and intestinal epithelial cells. NOD2 polymorphisms are associated with increased susceptibility to CD, but not UC. NOD2 mutations are extensively associated with European ancestry CD : about 30% of patients of European decent carry at least one of the three identified polymorphisms, when these variants are rare in patients of African origin and absent in patients of Asian ancestry.38–41 Mutated NOD2 carriers more often present with ileal disease and are more prone to develop strictures and fistulae.
Other than NOD2, autophagy gene mutations and especially that of ATG16L1, have been associated with CD.42 Located on chromosome 2, this gene is expressed in intestinal epithelial cells, antigen-presenting cells, B cells and CD4+/CD8+ T cells. It is involved with two other partners in the formation of a complex necessary for autophagosome formation, which role is essentially in pathogen clearance. Several studies have identified significant associations between the rs2241880 variant of ATG16L1 and CD ; individuals with CD and the ATG16L1 risk variant presented with defective autophagy mechanisms.43–45
Another main autophagy gene has been linked to Crohn’s disease: the IRGM gene located on chromosome 5. This gene is involved in autophagy against intracellular pathogens, such as Toxoplasma gondii, Mycobacterium tuberculosis or Salmonella typhimurium.46–48 The association of single-nucleotide polymorphisms (SNPs) in NOD2, ATG16L1 and IRGM with CD stress how innate immune defects are central features in CD, but not in UC.
As previously demonstrated by twin concordance studies, genetic predispositions seem to have a more modest contribution in ulcerative colitis than in Crohn’s disease. Nevertheless, some susceptibility loci could be identified for UC as well, such as polymorphisms in PTPN2, HERC2 and ECM1I.49,50 The anti-inflammatory cytokine IL-10 has also drawn a lot of attention towards its involvement in intestinal immune regulation and colitis. It was demonstrated that IL-10 deficient mice spontaneously developed enterocolitis.51–53 In parallel, important defects in IL-10 signaling in the lamina propria cells from patients with UC have been described.
Finally some polymorphisms have been associated with both CD and UC, among which the most significant ones were related to the interleukin IL-23R gene. IL-23 is a regulatory cytokine secreted by CD4+ T cells, CD8+ T cells, activated macrophages, dendritic cells, natural killer cells (NK cells) and NKT-cells54, that may be involved in chronic inflammation.55–58 The functional IL-23 is an heterodimeric cytokine, composed of IL12p40 and IL23-specific p19 subunit. IL-23 expression is particularly enriched in the intestine, and it can supress the regulatory T-cell responses in the gut, hence favouring inflammation. Consequently, increased levels on IL-17 have been observed in the lamina propria of UC patients.59
Genetic polymorphisms within the interleukin-23 receptor (IL23R), IL12B and STAT3 have been significantly associated with both CD and UC 60,61, although the mechanisms by which these SNPs interfere with gene function and affect IBD pathogenesis is still unclear.
Overall recent analyses have reported a total of 163 IBD risk loci 62 accounting for up to 71 associations (when only 71 susceptibility loci had been identified in 2012 63). The candidate genes within these loci implicate a broad array of genes, but unsurprisingly many are related to immune response mechanisms, cytokine production and cytokine receptors…etc. A large number – 110 out of 163 – of these loci are associated with both UC and CD, with more or less heterogenous effects. Among the remaining mutations, 30 are identified as CD-specific are 23 as UC-specific. Furthermore, 70% of the loci (113 out of 163) have also been identified in other complex diseases, including immune disorders (e.g. type-1 diabetes, psoriasis and ankylosing spondylitis) and immunodeficiencies leading to severe infections.64,65 Despite the large number of loci identified, it seems they account for 20% only of the genetic susceptibility to IBD.66

Environmental factors in IBD

The imperfect concordance observed in monozygotic twins studies suggests an important role for environmental factors and lifestyle in Inflammatory Bowel Disease. These data are further supported by several epidemiologic observations :
– First, the existence of north-south, east-west and urban-rural gradients in incidence rates and prevalence of IBD is observed worldwide,67
– Ethnic groups previously less affected by IBD such as Asians and Hispanics,68 nowadays exhibit increased incidence rates that correlate with their change in lifestyle towards westernization,
– Increased incidence rates in immigrants from low incidence regions that moved in countries with a high incidence, within a single generation timespan.69,70
Many environmental factors have been pointed out at the moment : less women breastfeeding71, improved domestic hygiene and sanitation72, quality of drinkable tap water73, air pollution exposure74, western diet75 high in sugars and polyunsaturated fats but low in fibres… But no strict cause-consequence relation could be demonstrated, only correlations. Only two environmental factors have been clearly identified up to date : tobacco consumption and appendicectomy.
Current cigarette smoking at time of disease has been shown to be protective in ulcerative colitis against non-smoking.76,77 UC patients who are smokers tend to have a milder course of disease, and disease activity tends to increase in patients that quit smoking78 while ex-smokers have more chances of developing UC.77 Surprisingly, smoking has totally the opposite effect on Crohn’s disease, where it tends to increase both the risks of relapse and of surgical intervention.79–81
Early appendicectomy before the age of 20 years old for acute appendicitis has been shown to be protective against UC.82,83 In the case of Crohn’s Disease, many different opinions have been expressed in the literature, but the overall trend seems to the support the theory that appendicectomy increases the risk of developing CD.84,85

IBD and microbiota : a complex interplay

There is abundant data supporting the intricate link between IBD and microbiota. Antibiotics have for instance been reported to induce remission in active CD patients and to play a role in the prevention of post-operative recurrence in CD.86 16S rRNA sequencing in children has demonstrated differences in microbiota between IBD and non-IBD paediatric patients.87
Microbiota is intended as the microorganisms inhabiting a defined ecological niche. The intestinal microbiota encompasses bacteria, archaea, fungi and viruses, all of which make up diverse ecosystems within us and live in symbiosis with their host. It is commonly admitted that the human gut contains up to 1014 bacteria, an estimate 10-fold more than the number of eukaryotic cells in our organism.88 The intestinal microbiota begins to establish at birth and during early infancy, when the GI tract is colonised by microorganisms from diverse taxa among which Bifidobacterium, Lactobacillus and Enterobacteriaceae, that are acquired from many sources such as the mother’s vaginal microbiota (if the infant is born naturally and not by C-section), skin and faecal microbiota of parents, siblings, classmates or breast milk.89 The microbiota is then shaped by host genetics, microbial exposure, diet, medical treatments and other environmental pressures, to reach its final structure around the age of three years old.90 Gut microbes are adapted to a certain type of lifestyle such as a diet 91, and extreme diet changes may result in wide alterations of the microbiota composition.92

Healthy or normal gut microbiota

The definition of a healthy microbiota remains unclear and elusive despite the enormous progresses achieved along the past decades. This imprecision is largely imputable to culture-based difficulties to identify and grow the anaerobic bacteria that make up the majority of our gut microbiota, but also to the new sequencing methods which, if they brought numerous answers, have unveiled a bacterial complexity and diversity we were previously unaware of.
The knowledge of the human gut microbiota was for long limited to the bacterial strains that could be cultivated in the laboratory. The development of the first anaerobic techniques – pioneered by Hungate and Freter93,94 – opened the field to the discovery of a large number of micro-organisms. The dominant cultivable genera then identified were the following:
Bacteroides, Eubacterium, Peptostreptococcus, Ruminococcus, Bifidobacterium, Fusobacterium and Clostridium (Table 4).
Based solely on the known and cultivable micro-organisms, it was believed for several decades that the human microbiota held only 400 species, among which 25 to 40 species made up the dominant microbiota of each individual.
The advent of new high throughput sequencing methods, bioinformatics, and the improvement of anaerobic culture broadened our knowledge of the gut microbiota. The Human Microbiome Project was one of the first large-scale studies to provide thorough data on the human microbiome. It was observed that a healthy human gut is likely to be comprised of more than a 1000 bacterial species and as much as 1011 bacteria per gram of colonic content.97 In contrast a taxonomic approach could only identify 70-100 known species.
Following this project, several groups attempted to set up a definition of a “core” human microbiome. Li and co-workers compared the microbial diversity of different body sites ; the vaginal microbiota revealed to be the least diverse, while oral and skin were the most complex ones, closely followed by the gut microbiota.98 Similarly, the group of B. A. Méthe analysed phyla abundance over the body sites by exploiting the data from the Human Microbiome
Project.97 Its conclusions joined that of Li : stool samples mainly contained representatives of phyla Bacteroidetes, Firmicutes, and Proteobacteria. Results may however differ based on the analytic technique used for sequencing (16S rRNA versus Whole Genome Sequencing).
From these analyses emerged a definition of the common core of the distal gut microbiota, that is predominantly composed of phyla Bacteroidetes and Firmicutes, closely followed by Actinobacteria (Collinsella, Bifidobacteria, Atopobium), Verrucomicrobia (Akkermansia) and Proteobacteria.99–101 The Bacteroidetes phylum consists of Gram-negative bacteria ; its most important representatives in the gut ecosystem are genera Bacteroides and Prevotella. On the opposite Firmicutes are Gram-positive bacteria, largely represented by orders Lactobacillales and Clostridiales (Eubacterium, Ruminococcus, Clostridium, Faecalibacterium, Butyrivibrio).
Recent studies have not only described a phylogenetic core100 (Table 5), but also a metagenomic core102, of the human gut microbiota composed of some sixty bacterial species from the main previous phyla.
Less than 0.1% of the gut microbiota is made of overt pathogens (e.g. Campylobacter jejuni, Salmonella enterica, and Vibrio cholera), but opportunistic pathogens such as Bacteroides thetaiotamicron or Bacteroides fragilis have also been identified in an analysis of the Human Microbiome Project by Huttenhower et al.103 These were respectively detected in 46% and 16% of stool samples. Escherichia coli accounted for 0.1% or more of the microbiota in only 15% of stools.
The proportion of each phylum is individual specific, but it is possible to cluster people according to species and functional composition of their microbiome. Arumugam and co-workers classified healthy adult into three groups that appeared to be independent of nationality and gender.104 They suggested a classification of individuals over three preferred ecological configurations of the gut microbiota called “enterotypes” : Enterotypes 1 contain a high proportion of genera Bacteroides, Enterotypes 2 have a high proportion of Prevotella, and Enterotypes 3 show a high proportion of Ruminococcus. The concept was confirmed by other studies105,106, and similar clusters were observed in other mammals such as pigs and mice.107,108 This definition of enterotypes was recently completed by Vandeputte et al, who reported Ruminococcus enterotype to consistently present a high cell count, while they could describe two subtypes of the Bacteroides enterotype based on the raw cell count of donors (low cell count / high cell count).109
Most bacteria of the human body are located in the gastro-intestinal tract, and 70% of them inhabit the colon.110 The composition and density of the human gut microbiota changes along the length of the GI tract from oesophagus and stomach to small intestine, large intestine and colon with the variations of pH, chemical, nutritional and immunological gradients. These variations have been demonstrated by comparing the microbial composition found in upper GI tract analysed by biopsies and the composition found in stool samples that would better mirror the lower tract microbiota.97,111 The bacterial composition of the microbiota in the small intestine increases in diversity and complexity from proximal to distal direction (from duodenum to jejunum and ileum) and reaches its climax in the large intestine ; the small intestine is characterised by high levels of oxygen and antimicrobial agents combined to a short transit time, conditions that limit bacterial growth and favour the rapidly-growing, facultative anaerobes with high adhesion ability to mucus and/or epithelium.91 Microbiological data is scarce for this region of the GI tract (hard to routinely sample), but it appears that genus Streptococcus is dominant in duodenum and jejunum.112,113 On the opposite, ecological colonic conditions allow the development of a dense and rich bacterial community, most of which are anaerobes and thrive on the undigested complex carbohydrates (the fibres).114 The dominant microbiota in the large intestine includes Firmicutes and Bacteroidetes.
In addition to longitudinal variations, our microbiome also varies axially from lumen to mucosal surfaces.101,115 Studies have shown that the phylum Firmicutes was largely predominant in the mucosa-associated microbiota (sampled by biopsy) compared to the luminal one (sampled by stool analysis), where Bacteroidetes would dominate.101,116,117

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Role of the microbiota

The gut microbiota is a beneficial “organ” to its host due to its multiple implications in metabolism, epithelial integrity, antimicrobial protection and maturation of the immune system.
Bacteria provide the host with nutrients and vitamins and some of their microbial metabolites are strongly involved in our metabolism and regulation of the glucose and lipid homeostasis. Colonic bacteria for instance, can degrade complex carbohydrates otherwise undigested to generate short-chain fatty acids (SCFAs) which are saturated aliphatic acids consisting of one to six carbons, among which acetate, propionate and butyrate are the most abundant.118,119 These are absorbed by intestinal epithelial cells and participate in the regulation of cellular processes and metabolism, e.g. gene expression, proliferation, differentiation, and apoptosis.120–122 Gut bacteria can also synthesize essential vitamins : vitamin B12 is produced by lactic acid bacteria123,124, folate by Bifidobacteria125, but also indoles, vitamin K, biotin, riboflavin, thiamine…etc. Non-reabsorbed bile acids are metabolized by colonic bacteria into secondary biliary acids.126 These many examples of bacterial components are mostly recognised by human G-protein-coupled receptors (GPCRs), resulting in hormone secretion by enteroendocrine cells.127–130
Several microbial species have been identified for their role in epithelial integrity, among which A. muciniphila, L. plantarum, R. gnavus, F. prausnitzii and B. thetaiotaomicron.131–136 These could modulate epithelial and mucus properties, as well as their turnover.
Right beneath the intestinal epithelial cells (IECs) lining the gut mucosa, is located the most extensive human lymphoid compartment : the gut-associated lymphoid tissue (GALT). The GALT is rich in immune cells associated with innate and adaptive immunity. It is involved in maintaining the homeostasis at the intestinal interface between host and microbiota, by differentiating commensal micro-organisms from their pathogen counterparts.137,138 The intestinal bacteria participate to teach the GALT via their constant interactions with dendritic cells (DCs) and IECs through recognition of pathogen-associated molecular patterns (PAMPs) by innate immune receptors named PPRs for Pattern Recognition Receptors (a group of receptors including NOD and TLRs). PAMPs can include lipopolysaccharides (LPS) from Gram-negative bacteria and other molecular patterns such as peptidoglycans and flagellin ; these can for instance trigger the onset of low-grade intestinal inflammation and insulin resistance in obese or diabetic patients.139–141 It has notably been shown that bacteria are required for maturation of the immune system. Segmented filamentous bacteria (SFB), a class of commensal bacteria, have several effects : they are required for Th17 cells differentiation in the lamina propria142, and stimulate the postnatal maturation of the GALT, as well as other immune-stimulatory properties.122 Bacteroides fragilis are needed for the expansion of CD4+ T cells and their conversion into regulatory T cells via secretion of polysaccharide A143,144 ; germ-free animals otherwise lack expansion of the CD4+ population. The physical colonization of the gut epithelium by commensal bacteria is furthermore protective against pathogens for they compete for attachment sites and nutrients, and can also secrete antimicrobial agents.
Finally, the gut microbiota has the property to stimulate its host for production of native antimicrobial substances : cathelicidins, C-type lectins, defensins and IgAs.

Table of contents :

1.1. General description of IBD
1.2. Pathophysiology of Inflammatory Bowel Diseases
1.2.1. Genetic factors in IBD
1.2.2. Environmental factors in IBD
1.2.3. IBD and microbiota : a complex interplay Healthy or normal gut microbiota Role of the microbiota Characterising the intestinal dysbiosis in Inflammatory Bowel Diseases
2.1. The Quorum Sensing
2.1.1. Discovery and main characteristics of the Quorum Sensing General mechanism(s) of the bacterial QS The Quorum System of Vibrio fischeri : a paradigm for LuxI/LuxR-type quorum sensing systems
2.1.2. Overview of QS signaling molecules Type-1 autoinducers (AI-1) Type-2 autoinducers (AI-2) Type 3 autoinducers (AI-3) Other signaling molecules in Quorum Sensing Cholerae autoinducer (CAI) 3-hydroxy palmitic acid methyl ester (3OH-PAME) The Pseudomonas Quinolone Signal (PQS) The Diketopiperazines (DKP) The g-butyrolactone Auto-inducing peptides (AIP) Summary table of presented QS molecules
2.1.3. Interspecies Communication and Signaling
2.2. Interkingdom Signaling : Effects of N-Acyl Homoserine Lactones on Eukaryotic cells
2.2.1. AHL penetration into cells
2.2.2. Apoptosis
2.2.3. Immunomodulatory effects and inflammation 3oxoC12-HSL is a chemoattractant per se for neutrophils AHL effects on immune cell functions Inflammatory properties on epithelia
2.2.4. Morphological changes in cells Tight and adherent junctions of the gut epithelium Cell migration and wound healing
2.2.5. Quorum quenching
2.2.6. AHL receptors
2.3. State of the art in our laboratory
2.3.1. Are there AHLs in the intestinal ecosystem?
2.3.2. Objectives of the project
1.1. Cell culture
1.1.1. The Caco-2/TC7 cell line
1.1.2. The Raw 264.7 cell line
1.1.3. The bacterial reporter strain E. coli pSB1075
1.1.4. Bactericidal assay
1.2. Evaluation of the biological activity of molecules in mammal cells
1.2.1. Caco-2/TC7 stimulation with cytokines
1.2.2. Raw 264.7 stimulation with LPS and IFN-g
1.2.3. Measurement of protein concentration in cell lysate
1.2.4. Human cytokines quantification by ELISA
1.2.5. Quantification of murine cytokines
1.2.6. Cytotoxicity assay
1.3. RNA extraction, Reverse transcription and Quantitative PCR
1.4. Biological activity of molecules on bacterial reporter strain E. coli pSB1075
1.5. Immunofluorescence experiments
1.5.1. Fixed-cells imaging
1.5.2. Live-cell imaging
1.6. Reactive Oxygen Species production in Caco-2/TC7 cells
1.7. Statistical analysis
2.1. Evaluation of AHL half-life in cell culture medium at 20°C
2.2. Cell preparation for assessment of kinetics of AHL entry in cells
2.3. AHL detection by tandem HPLC-MS/MS
3.1. Convention for atom numbering in N-acyl homoserine lactones and their analogues
3.2. Experimental procedures for synthesis and physicochemical characterization of natural AHLs,
intermediates and non-natural analogues
3.3. Synthetic procedures for the preparation of AHL probes
4.1. Electrochemistry and cyclic voltammetry
4.2. Spectrometry
4.3. Molecule characterisation (NMR, MS and HRMS)
1.1. The saturated 3oxoC12-HSL
1.2. The unsaturated 3oxoC12:2-HSL
2.1. Results on our biological models
2.1.1. Establishment of stimulation protocols
2.1.2. Natural AHLs can modulate the secretion of pro-inflammatory cytokines in Caco-2/TC7 and Raw 264.7 cell lines Caco-2/TC7 and Raw 264.7 cells have different tolerances towards AHLs in range 1-100 μM AHLs have a bi-modal action over IL-8 secretion by Caco-2/TC7 cells Timing and chronology in AHL treatments AHLs have mild effects on IL-6 secretion by Raw 264.7 cells Other pro-inflammatory cytokines : IL-1ß and TNF-a
2.1.3. No mRNA modulation is observed in Caco-2/TC7 cells submitted to AHLs
2.1.4. AHLs and secretion of anti-inflammatory cytokines
2.1.5. AHLs and bacteria
2.1.6. Discussion
2.2. Stability & metabolism of AHLs
2.2.1. Degradation of AHLs in biological media AHLs are degraded into two major by-products pH-dependent stability in biological medium Biological study of tetramic acid from the 3oxoC12-HSL
2.2.2. Enzymatic degradation and extracellular metabolism of AHLs PON mRNA expression in the studied cell lines Influence of the inhibition of Paraoxonases on AHL effects
2.2.3. Intracellular AHL metabolism : entry and cellular fate
2.2.4. Hydrolysed AHLs and bacteria
2.2.5. Discussion
2.3. Insight into the AHL mechanism of action : cell imaging and receptor investigation
2.3.1. Mapping AHLs in epithelial intestinal cells Use of a commercially available fluorescent molecule Development and evaluation of new molecules Development of a clickable AHL The nitrobenzofurazan: a smaller fluorescent probe Design of potentially fixable naphthalimide-based fluorescent probes Conclusions on cell-imaging
2.3.2. Design of a biotin-tagged AHL
2.4. AHL, Reactive oxygen species and iron
1.1. Natural N-Acyl-Homoserine Lactones
1.2. Introduction to AHLs non-natural analogues
2.1. Compared results on mammalian cell lines
2.1.1. Importance of the keto group
2.1.2. Importance of the acyl chain Shorter acyl chains Longer acyl chains Discussion on acyl chain effects
2.1.3. Importance of the cyclic headgroup Chirality and AHL activity Thiolactones The aminocyclohexanol series Aromatic headgroups The methoxy-anilide analogues The amino-chlorophenol analogue Miscellaneous : diverse compounds
2.1.4. Discussion on AHL modifications
2.2. Further investigation on two hit candidates
2.3. Development of a dimeric AHL
2.4. Results on bacteria strains
2.4.1. Results on LasR bioassay Ketone effects Influence of the acyl chain length Substitution of the headgroup
2.4.2. Bactericidal properties of selected compounds
2.4.3. Investigation of inhibitors
2.4.4. Discussion on the bacterial properties of the analogue library


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