The junctions of the epithelial barrier
In order to fulfill the intestinal barrier challenge, IECs are held together by three intracellular adhesion complexes, Tight Junctions (TJ), Desmosomes and adherens junctions (AJs) (Figure 6). These cell-cell interconnections, likely formed between two different cell types, are crucial for the paracellular transport and barrier integrity providing mechanical properties to the barrier function (Antoni, Nuding, Wehkamp, & Stange, 2014). An important feature of the intestinal epithelium is the high polarization of the IECs. Due to the asymmetric organization of specific protein along the cell, such as TJs, AJs or ion distribution, the composition between the apical and the basolateral pole differs (Cereijido, Contreras, Shoshani, Flores-Benitez, & Larre, 2008). IECs polarization contributes to intestinal functions like absorption and secretion. Increased intestinal permeability is associated to several GI disease such as inflammatory bowel disease (IBD), mentioned in chapter 4 (Zeissig et al., 2007).
The TJ network is the apical-most junctional complex that delimits the border between apical and basolateral domain. They are composed by four different families of transmembrane proteins including occludin, claudin, tricellulin and junctional adhesion molecules (JAM) (Figure 7). Occludin, claudin, tricellulin are structures proteins with two extracellular loops and two cytoplasmic domains. The intracellular tail of TJs connects with cytosolic scaffold proteins, such as the zonula occludens (ZO) proteins family, which consecutively binds to the actin cytoskeleton. In order to sustain the contractile tension caused by the TJs, the cytoskeleton, is closely interconnected to a peri-junctional ring of myosin II light chain (MLC). The phosphorylation of MLC via myosin light chain kinase (MLCK) induces contraction of the actin-myosin cytoskeleton leading to a TJs opening and leakage across intestinal epithelium (Feighery et al., 2008; Turner et al., 1997). Disruption of TJs integrity leads to a decrease of transepithelial electrical resistance (TEER)-mentioned in chapter 6 section 1-, a manner to measure paracellular flux.
Occludin was the first transmembrane components of TJ to be discovered, in 1993. Occludin is mainly expressed in epithelia and endothelial cells, but its functions are not fully delineated. In vivo and in vitro data have shown that Occludin plays a role in the regulation of the paracellular permeability by maintaining the integrity of TJs network (Al-Sadi et al., 2011). Occludin is an integral membrane protein made up by four domains-two extracellular loops, two cytoplasmic domains, a long carboxyl region and a short N-terminus region. Regulation and localization of Occludin is arranged by phosphorylation on multiples residues sites- tyrosine, serine and threonine. Kinases, such as protein kinase C (PKC) and phosphates, regulate occludin phosphorylation and its localization to TJs. Highly phosphorylated occludin is selectively localized at TJs regions. However, depending on the phosphorylated residues such as Tyrosine phosphorylation, it triggers dissociation of the occludin-ZO1 complex. On the contrary, non-phosphorylated molecules are mainly located at the basolateral membrane (Dörfel & Huber, 2012; Gonzalezmariscal, 2003).
Cytosolic C-terminus domain anchors to several TJ scaffold proteins, such as PDZ-domain containing ZO proteins, which, in turn, link to the actin-cytoskeleton (Figure 7). Intracellular scaffold proteins are localized at the cytoplasmic surfaces of junctional structures to establish specialization and localization of the junctions. Thus cytoplasmic platforms such as PDZ-domain-containing zona occludens proteins (ZO-1, ZO-2 and ZO-3) constitute a bridge between transmembrane proteins and the actin-cytoskeleton to mediate intracellular and extracellular signals (Figure 7). Some TJs can also interact with non-PDZ domains such as cingulin, which connects the junctional membrane protein to ZO-1 (Groschwitz & Hogan, 2009; Lee, 2015; Umeda et al., 2004).
Figure 7. Tight Junction complex. The TJs are the most apical junctional complexes which create a selective permeability between adjacent IECs. It is made up by a branch network of sealing strands including, occludin, claudins, tricellulin and JAMs. Extracted from (Lee, 2015).
Like Occludin, the transmembrane protein Claudins also form the core of the TJs and control for ion selectivity and permeability. To date, 24 Claudins have been identified and their functions differ depending on their tissue-specific expression. Like Occludin, some Claudins are regulated and localized to TJs via phosphorylation. The two extracellular loops, hemophilic and/or heterophilic, interconnect with neighbouring cells, establishing a selective permeability and ion channel-forming. The cytosolic C-terminus tail anchors Claudins to the actin-cytoskeleton via scaffold proteins including ZO-1, ZO-2 and ZO-3 (Itoh et al., 1999). Some of the pores forming claudins reduce intestinal permeability whereas others alter paracellular selection. Claudins-1, – 5 and -7, fulfill the formation of the barrier function decreasing permeability (Günzel, 2017). On the other hand, claudin-2, -15 and -16 possess the “leaking” phenotype, increasing transepithelial resistance (TEER)(Furuse, Furuse, Sasaki, & Tsukita, 2001; Overgaard, Daugherty, Mitchell, & Koval, 2011).
Located in the tricellulin TJ, the newest discovered TJs tetraspanin protein, tricellulin (marvelD2) and marvelD3, together with marvelD1 (occludin) are members of the TJ-associated Marvel protein family (TAMPs). These three proteins share a conserved MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain that contributes to epithelial function and TJs regulation (Oda, Otani, Ikenouchi, & Furuse, 2014; Riazuddin et al., 2006).
Junctional adhesion molecules
Junctional adhesion molecules (JAM) is the last component of the TJs complex. The JAM family including mostly A and B subtypes belongs to the immunoglobulin superfamily which comprises two IgG-like fold extracellular domains and one cytoplasmic tails. Two homophilic and heterophilic extracellular domains, from two different JAM, are required to stabilize cell-cell junctions to regulate the cellular function and paracellular permeability. Besides homeostatic functions, JAMs are required for cell migration and proliferation (Nava et al., 2011). Intracellular C-terminus domain interacts with the scaffolding and cytoplasmic proteins , such as ZO-1, which in turn, links to the actin cytoskeleton (Campbell, Maiers, & DeMali, 2017; Monteiro et al., 2013).
Underneath the tight junction, adherens junction, also termed zonula adherens, form the major lateral cell-cell adhesion belt connecting transmembrane proteins, intracellular adaptor proteins and actin filaments. The AJs consist in two adhesive proteins units; the most commune AJs structure is formed by cadherin-catenin (Figure 8).
Figure 8. Structural model of Adherens Junctions. The most studied AJ complex is the cadherin-catenin network. The main function is to maintain the physical cohesion between IECs. Extracted from (Perry, Lins, Lobie, & Mitchell, 2010).
Classical epithelial (E)-cadherins anchor to the catenin complex – α-, β-, and p120 catenin. Through the armadillo repeats, the N-terminus domain of p120 catenin and β-catenin (also termed plakoglobin) binds directly to the intracellular domain of E-cadherins. β-catenin C-terminus domain, in turn, binds to α-catenin which links to the cytoskeleton. α -catenin has the ability to bridge the filaments of actin with E-cadherin (Figure 8). The extracellular domain of E-cadherins interacts with neighbouring cells that mediates cell-cell adhesion (Groschwitz & Hogan, 2009; Niessen, 2007). Together, these AJs complexes provide a strong mechanical connection required to maintain the integrity of the epithelial barrier (Niessen, 2007).
The intracellular junction desmosomes are essential for mediating strong cell-cell cohesion and for maintaining a mechanical seal between cells. Thus, they are abundant in the epidermis and myocardium tissues due to their exposure to repetitive mechanical forces. Placed in the basal side of IECs, desmosomes are intracellular junctions with an extracellular tail that anchors to neighbouring desmosomes, and a cytoplasmic domain that anchors to cytoskeleton-associated proteins. Altogether, the desmosome complex forms a network that provides mechanical strength to the intestine named scaffold complex, and it consists in three units: two intracellular components and one cell to cell (Figure 9)(Hatzfeld, Keil, & Magin, 2017). Intracellularly, actin bind to the desmosomal adhesion molecules by the linkage of intermediate filaments (keratins). Thus, intermediate protein linkage is mediated by desmoplakin (linker 1) and the armadillo proteins plakoglobin and plakophilin (linker 2). To sum up, intermediate filaments bind to linker 1. Linker 1, in turn, binds to the linker 2 that binds to the desmosomal cadherins complex, conforming integrity and plasticity to the epithelium (Garrod and Chidgey 2007).
Mucus layer of the intestine
The mucus layer covers the whole epithelium of the GI tract keeping harmful antigens away from the epithelial monolayer. The small intestine presents a single and discontinuous mucus layer whereas the large bowel consists in double and well defined mucus layer. Due to the high number of bacteria living in symbiosis in the colon, the mucus layer is essential to avoid the contacts of pathogens with the intestinal mucosa (Figure 10). Moreover, the lubrication ability of the mucus is crucial to improve intestinal transit (In et al., 2016).
Figure 10. Schematic model of the mucus organization in the small intestine and colon. Two layers of adherent mucus are present in the large bowel. Goblet cell-derived mucin are mostly present in the colon. Adapted from (M. E. V. Johansson, Larsson, & Hansson, 2011)
Mucins, the main protein of the mucus, are stored and secreted, by goblet cells – described at chapter 1 section 2.B –. Mucins are subdivided in two groups: the secreted mucins and the transmembrane mucins. The secreted mucin (MUC2, MUC5 and MUC6) form long polymers and are tissue-specifically expressed. On the other hand, transmembrane mucins (MUC1, MUC3, MUC4, MUC12, MUC13, MUC16, and MUC17) are adherent to the apical cell surface of IECs (Malin E V Johansson, Sjövall, & Hansson, 2013). These cell-membrane-associated mucins belong to the inner mucus layer of the distal colon and form an important element of the glycocalyx –mentioned in chapter 1 section 2.B – and epithelium protection (Malin E V Johansson et al., 2011; Thornton, 2004).
Mucin proteins are translocated into the endoplasmic reticulum (ER) where they are folded and form disulfide-bonded dimers. Thereafter, mucins PTS domains (tandem repeats) rich in proline (P), threonine (T), and serine (S) become densely O-glycosylated in the Golgi apparatus. This highly glycosylated mucin domains have a high capacity to bind water that contributes to the gel-forming properties of the mucus (Ijssennagger, van der Meer, & van Mil, 2016).
The mucus layer of the distal colon consists in two different mucus coats including an inner layer firmly adherent to the epithelium and an outer loose mucus layer (Figure 10) (Atuma, Strugala, Allen, & Holm, 2001; M. E. V. Johansson et al., 2011; Malin E V Johansson et al., 2011). The inner firm mucus layer, free of bacteria is transformed into another mucus layer expended in volume. The loose outer mucus layer is in contact with bacteria and can be degraded by their enzymes. Bacteria strains can bind to the outer mucus thanks to the abundant glycans present in MUC2 mucin. The major component and the skeleton of the mucus layer is the gel-forming mucin MUC2 (Thornton, 2004).
Other important peptide produced by goblet cells that regulates the physical barrier and stabilizes mucin polymers are trefoil-factor 3 (TFF3) and Resistin-like molecule-β (RLMβ)(table 1) (Peterson & Artis, 2014). RELMβ belongs to the resistin-like molecules including four members:
RELM-α, -β, -γ and resistin. Unlike the other proteins of the family, RELMβ is tightly produced by goblet cells and secreted apically into the lumen content. Although its function is not fully defined, RELMβ is induced upon bacterial colonization and promotes MUC2 secretion and inflammation control by the stimulation of Th2 cytokines. Within the inflammatory area, RELMβ regulates macrophages and T cells resident in the gut (Artis et al., 2004; McVay et al., 2006). On the other hand, TFF3, also named intestinal trefoil factor, is predominantly expressed by goblet cell-derived peptide in the small intestine and colon, and it is abundantly secreted into the lumen surface. TFF3, belongs to the trefoil peptide family which includes the gastric peptides pS2 (also named TFF1) and spasmolytic polypeptide (also called TFF2), all of them are involved in gastrointestinal epithelial restitution (Sands & Podolsky, 1996). TFF3, is involved not just in the structural integrity of the mucus but also provides epithelial healing, IECs migration and turnover as well as resistance to apoptosis (Taupin, Kinoshita, & Podolsky, 2000).
Within the mucus layer, the apical IECs surface is covered with secretory IgA (sIgA). They serve as a first line of defence and display antibacterial properties protecting the gut mucosa from enteric toxins and harmful microorganisms. SIgA is capable of controlling the inflammation and regulating immune response to commensal microbiota, pathogens and antigens by a system known as immune exclusion (Mantis, Rol, & Corthésy, 2011). SIgA blocks the access of the bacteria to epithelial receptors, retaining the pathogen within the mucus and facilitating its expulsion by peristaltic movements of the intestine (a J. Macpherson et al. 2008). They shape the intestinal microflora by re-transporting antigens across the mucosa barrier to dendritic cells, subsets in GALT, which promotes pro-inflammatory signals associated with uptake of pathogens.
Antimicrobial peptides (AMPs) belong to the innate immune defence and play a homeostatic key role maintaining the composition of the commensal flora and intestinal homeostasis. AMPs are found in the most exposed areas of the body to microbes, such as skin, eyes, oral mucosa, lung or intestinal mucosa. AMPs are produced by Paneth cells, goblet cells and enterocytes. This defence peptides possess a wide-spectrum of antibacterial properties towards pathogens and microbiota, both Gram-positive and Gram-negative (Dupont et al., 2015). Besides killing bacteria, some AMPs possess non-antimicrobial functions such as immune modulator. These peptides reinforce the total immune response by a range of mechanisms: as a chemoattractant to recruit immune cells, pro-inflammatory cytokines or as a Toll-Like Receptor ligand –mentioned in chapter 2. section 3.2 A- (Islam et al., 2001).
AMPs are usually cationic, not longer than 50 amino acids, and positively charged to prevent the diffusion of the peptide into the lumen. These features are ideal to trap the peptide into the mucus layer and to target the negatively charged bacteria surface (Hancock & Diamond, 2000; Zasloff, 2002). Depending on the peptide, the bactericidal mechanism of action differs. The peptide kill bacteria by (a) disruption of membrane integrity, (b) inhibiting DNA or RNA synthesis or (c) targeting specific intracellular molecules (Figure 11) (Bahar & Ren, 2013).
Figure 11. Biological function of antimicrobial peptides. AMPs act not just as a bacteria killer but also modulate host immune response. Bactericidal AMPs bind to the bacterial membrane by electrostatic interaction and kill them by disrupting their membrane or by inhibiting crucial intracellular functions. Immunoregulatory AMPs recruit or activate immunocytes by chemoattraction or by acting as a TLR ligand that leads to the activation of pro-inflammatory downstream signaling pathways. Adapted from (Zhang & Gallo, 2016).
Defensins and cathelicidins are the two major mammalian antimicrobial peptides.
Defensins are cationic peptides with three intramolecular disulphide bonds. To date, three subclasses of defensins have been described (α, β and θ) (table 2). Although the α-, and β-defensins have been identified in humans (O’Neil et al. 1999), θ-defensinse has been characterized from primates’ leukocytes (M E Selsted, 2004). Six human α-defensins have been characterized. Up to 4 different α-defensins are expressed by neutrophil, known as a human neutrophil peptide-1 (HNP1, HNP2, HNP3 and HNP4). Human defensine-5 and -6 (HD-5, HD-6) are tissue-specific only expressed by Paneth cells in the small intestine. Compared to α-defensins, four different β-defensins have been identified (hBD1-4) in human. In mice, it has been described 6 different α-defensins, named cryptdins (crypt defensins) and up to 45 different β-defensins gens (Michael E Selsted & Ouellette, 2005). They are quite abundant within the GI tract, especially in the colon, mainly expressed by epithelial cells. While hBD1 is constitutively expressed along the small and large colon, hBD2, -3, and -4 are induced by pro-inflammatory or pathogen stimuli, through PPR-activated signals – mentioned in chapter 2 section 3.2 A- which, in turn, activate transcription factor nuclear factor kappa-B (NF-κB) (O’Neil et al., 1999).
Secreted at the surface of the colonic crypts, like defensins, cathelicidins are another dominant class of AMPs. Although about 30 subfamilies of cathelicidin have been identified in mammalian, only LL-37/hCAP18 and CRAMP have been found in human and mice, respectively (Dürr, Sudheendra, & Ramamoorthy, 2006). These peptides carry a large spectrum of bactericidal activity against Gram-negative and -positive bacteria. Stored in neutrophils, macrophages and epithelial cells as secretory granules, cathelicidins are released upon leukocyte activation (Kościuczuk et al., 2012; Zanetti, 2005).
Paneth cells are the main source of AMPs in the small intestine, and beside α-defensins, they secret phospholipase A2 (sPLA2), RegIII, and lysozyme C. AMPs are regulated and stored as inactive peptides in secretory granules. Triggered by bacteria among other stimulus, the granules are release on the lumen and are cleaved by trypsin or matrix metalloproteinase to generate the peptide mature form (Tollin et al., 2003; Wang, 2014).
AMPs constitute a shield towards commensal microbiota. Dysregulation of peptide production changes the composition of commensal microbiota and disrupts intestinal homeostasis. Failure of AMPs expression and secretion is associated with human disease such as obesity or Irritable Bowel Syndrome (Zhang & Gallo, 2016).
Immunity of the colonic mucosa
In order to protect the GI tract from viruses, bacteria, fungi, or parasites, the intestinal mucosa needs to recognize and respond to foreign organism and harmful substances. To identify and protect the GI tract from those, the intestinal mucosa is equipped with several weapons. IECs express various recognition molecules such as the pattern recognition receptor (PRR). These receptors recognized a broad spectrum of bacteria or microorganism structures, known as a pathogen/microbial associated molecular patterns (PAMPs or MAMPs). PRRs are mostly constitutively expressed in innate immune cells including dendritic cells (DC), macrophages, neutrophils and also in IECs. However, these receptors can also be induced by harmful stimuli. To date, PPRs are classified in four subclasses including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs) and DNA receptors (cytosolic sensors for DNA)(Loo & Gale, 2011). TLRs and NLRs are the two major classes.
Table of contents :
Chapter 1. Biology of the intestine
1. General overview of the gastrointestinal tract
2. Biology of the intestine
2.1 Histology of the intestine
2.2 Cellular organization of the intestine
2.3 The five different cell types
A) Enterocytes and colonocytes
B) Goblet cells
C) Paneth cells
D) Enteroendocrine cells
E) Other minor cells
Chapter 2. Central role of the colonic barrier
1. The main role of intestinal mucosa
2. Physical barrier
2.1 The junctions of the epithelial barrier
A) Tight Junctions
iv) Junctional adhesion molecules
B) Adherens junctions
2.2 Mucus layer of the intestine
3. Bio-chemical barrier
3.1 Antimicrobial peptides
3.2 Immunity of the colonic mucosa
A) Toll-like receptors
B) NOD-like receptors
D) Gut-associated lymphoid tissue
Chapter 3. Inflammatory Bowel disease
1. General introduction
2. Crohn’s disease
3. Ulcerative colitis
4.1 Incidence and prevalence
4.2 Age and gender disparity
4.3 Geographical distribution
5.1 Genetic susceptibility
5.2 Environmental risk factors
D) The hygienic hypothesis
E) Lifestyle: Stress and exercise
6.1 Pharmaceutical treatment
A) Anti-inflammatory drugs
C) Antibiotics and probiotics
D) Biological agents
6.3 Faecal Microbiota transplantation
Chapter 4. Pathogenesis of Inflammatory Bowel Disease
1. Defective physical and biochemical mucosa barrier
1. 1 Impaired Epithelial Junctions
1.2 Defective mucus layer
1.3 Defective antimicrobial factors
2. Immunology factors
3. The role of microbiota
3.1 Commensal microbiota
3.2 Microbiota in IBD
4. Genetic polymorphism involve in IBD pathophysiology
A) NOD2 mechanism of action
B) NOD2 in CD
A) Basal autophagy
B) Autophagy Risk Variants in IBD
C) NOD2 and the link with autophagy
4.3 Endoplasmic reticulum Stress
A) Endoplasmic reticulum stress and cellular homeostasis
B) Endoplasmic Reticulum Stress in IBD
Chapter 5. Proteases in the gut and IBD
1. Proteases in the gut
2. Proteases and their inhibitors
2.1 Matrix Metalloproteinases
2.2 Serine proteases
2.3 Cysteine proteases
3. Mechanism of action of proteases
4. Inflammatory Bowel Disease meet proteases
4.1 Epithelial metalloproteases and their inhibitors in IBD
4.2 Epithelial serine proteases and their inhibitors in IBD
4.3 Epithelial cysteine proteases
5. Protease-based treatments to target IBD
Chapter 6. Our intestinal in vitro models
1. Epithelial cell model
2. Caco-2 cells model
3. HT29-mtx cell model
AIMS AND HYPOTESIS
1. Project 1. The relationship between activation of NOD2 and trypsin-like proteolytic activity secretion in intestinal epithelial cells.
2. Project 2. Autophagy signaling pathway and the release of trypsin-like proteases in intestinal epithelial cells.
3. Project 3. Endoplasmic reticulum stress boosts trypsin activity and release by enterocytes and alters barrier function
a. Paper publication
b. Extra caco-2 results
c. HT29mtx results
GENERAL DISCUSSION AND CONCLUSIONS