The human mucosa faces an enormous density of bacteria, antigens, and pathogens in the gut lumen. The epithelial cell layer has the task of selectively blocking the passage of toxins and pathogens into the mucosa, while ensuring resorption of food and fluid. Defects in the tightly regulated barrier function of the mucosa and the epithelium can lead to the influx of antigens into the intestinal wall. Recognition and clearance of these pathogens are critical to the survival of the organism. Pattern recognition receptors (PRRs), which include members of the intracellular NOD protein family, recognize molecular patterns of various pathogens (PAMPs).1, 2 One member, NOD2, has been linked to susceptibility to Crohn's disease (CD), an inflammatory bowel disease (IBD).3, 4
The ligand of NOD2 is the bacterial wall component muramyldipeptide (MDP). After MDP binding to a leucine-rich repeat (LRR) domain, NOD2-mediated signaling normally results in activation of the transcription factor nuclear factor kappa B (NF-κB) via a kinase called RICK. Activation and nuclear translocation of the NF-κB signaling pathway has different consequences. On the one hand, NF-κB induces numerous proinflammatory genes that play a role in the regulation of innate and adaptive immune responses,5 e.g., the controlled expression of immune modulators such as IL-2, IL-6, IL-8, GM-CSF, or chemokines and their receptors, enzymes (iNOS, MMP-9), or cell-surface adhesion molecules (E-selectins).6 On the other hand, expression of antiapoptotic genes like FLIPL, TRAF1, or members of the Bcl-2 family is induced via NF-κB signaling.7 For the 3 frequent NOD2 variants, so-called single nucleotide polymorphisms (SNPs) 8, 12, and 13, there exist conflicting data regarding their functionality. The “loss of function” theory is supported by the observation that a defect of NOD2 variants to activate defense mechanisms results in an increased bacterial translocation, at least in animal models.8–11 On the other hand, mice with SNP13 mutation showed an excessive IL-1β production upon bacterial challenge, rather supporting the “gain of function” scenario.12 Most of the data on NOD2 function have been derived in cell lines overexpressing the wildtype (WT) protein and its variants or in different mouse models. However, it is obvious that patients have to be studied to understand the impact of NOD2 variants in human disease.
To date, the etiology of CD is not yet clear. However, recent years have brought new and important insights. Besides genetic and environmental factors, the luminal flora seems to be involved in the pathogenesis of chronic intestinal inflammation.13 In CD patients the mucosal barrier may become leaky, leading to uncontrolled uptake of antigens and proinflammatory molecules, including luminal bacteria and bacterial products from the gut lumen.13 An increased permeability in patients (and their relatives) with CD was shown 20 years ago.14, 15
Cell–cell contacts between intestinal epithelial cells for obvious reasons play an important role in the barrier function of the epithelium. The junctional complex is composed of different adhesion proteins, forming apical tight junctions and subjacent adherens junctions. Three prominent tight junction proteins or protein families are occludin, the claudins, and the Zonula occludens (ZO) proteins. Occludin was shown to be a central mediator of tight junction formation as occludin-deficient embryonic stem cells were unable to form functional tight junctions.16 In addition, overexpression of mutated occludin variants changed barrier capacities.17–19 The claudin family is composed of 24 transmembrane proteins. The most prominent members claudin-1, -2, and -4 regulate the “tightness” and specific ion selectivity of the epithelium.20–22 ZO-1 acts as a linker between the actin cytoskeleton and other tight junction-associated proteins and regulates paracellular permeability.23–28 The adherens junction protein family prominently includes cadherins and catenins that fulfil anchoring functions.29, 30
Our hypothesis was that in CD patients luminal bacteria might reach the lamina propria via modified cell–cell contacts followed by a dysregulated immune response. In NOD2 variant patients a lack of detection of invaded bacteria and bacterial material could be followed by impaired clearance of those bacterial products and subsequent activation of proinflammatory pathways. Therefore, the aim of the study was to establish a link between NOD2-mediated signaling and bacterial translocation in CD patients.
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- PATIENTS AND METHODS
To avoid the invasion of bacteria or specific pathogens from the intestinal lumen into the body the healthy mucosa possesses local protection mechanisms like mucus production, synthesis of antimicrobial peptides, intraluminal IgA-secretion, or gut motility.34, 35 In CD these protective factors are impaired. The intestinal barrier becomes “leaky,” resulting in an invasion of luminal bacteria.
Therefore, in this study we asked 1) whether the transepithelial passage of bacteria is associated with the NOD2 genotype of the patients, 2) whether modified cell–cell contacts may be the route for translocation, and 3) what consequence bacterial stimulation after mucosal translocation has on activation of NOD2-associated proinflammatory signaling pathways.
In the last years several studies focused on the fecal microflora in CD patients and changes in the biodiversity were found.36–38 Investigations regarding the adherens or potential translocation of luminal bacteria are yet limited due to their unspecificity and a lack of quantitative methods.39, 40 Similar to other groups,41, 42 we found that the intestinal mucus layer of CD patients is highly colonized with bacteria, whereas in the mucus layer of normal individuals almost no adherent bacteria were detectable. The abnormal penetration of bacteria into the mucus during CD may be facilitated by a biochemical modification of the mucin layer, a lack of defensins,43 as well as bacterial enzymes that are able to degrade mucin oligosaccharides.44 This is followed by a direct contact between the epithelium and the aggressive microflora, which is normally absent.37 Using FISH technology we effectively detected mucus-associated bacteria. However, in contrast to other reports we could not identify intraepithelial/mucosal bacteria. To obtain more information about potentially translocated bacteria, endotoxin found in the outer membrane of various gram-negative bacteria was localized by immunohistochemical staining. We demonstrate here that accumulation of endotoxin in the intestinal tissue of CD patients reflects the NOD2 genotype: Patients with heterozygous SNP8 or SNP13 variants revealed much stronger endotoxin staining than patients with SNP12 or the WT variant. NOD2 as an MDP sensor protein mediates activation of NF-κB. In this context various in vitro and in vivo experiments proved that NOD2 mutations lead to a loss of MDP recognition.9–11 The NOD2 genotype and the amount of bacterial translocation are likely not directly linked. However, an intracellular accumulation of bacterial products may subsequently lead secondarily to an increased inflammatory reaction. In vitro studies indicated an induction of NOD2 protein synthesis in response to tumor necrosis factor (TNF) stimulation of epithelial cells.45 Similarly, in this study we show that HEK293T-cells are sensitized to bacterial stimuli solely by overexpressing NOD2, as indicated by an increased secretion of IL-8.
Epithelial paracellular permeability as well as barrier function are maintained by tight and adherens junctions.46 In IBD an increase in permeability of the tight junctions mediated by proinflammatory cytokines has been observed. We show here that cell–cell contacts in the intestine of CD patients are morphologically unchanged, but their protein composition is altered. The expression of the tight junction proteins claudin-1, -2, occludin, and ZO-1 was increased in inflamed tissue of CD patients versus controls, claudin-4 was unchanged, and the expression of the adhesion proteins E-cadherin and β-catenin was clearly reduced in the tissue of CD patients.
Claudin-1 and claudin-2 have sealing functions in the epithelial barrier. A change in their expression levels affects the intestinal barrier. An increased expression of claudin-1 was also found in colorectal tumors.47 An induction of claudin-2, as also shown here in CD-associated tissue, was demonstrated to result in the formation of leaky pores20, 48 and an increased permeability. Furthermore, the increased detection of these 2 proteins and their ability to recruit and activate pro-matrix-metallo-proteinase (MMP)-2 or -947, 49, 50 may result in increased proteolysis of extracellular matrix proteins causing tissue remodeling and degradation. Claudin-4 mainly has regulatory functions for transcellular Na+ permeability and no sealing functions.22 Numerous studies showed that occludin plays a major role within the tight junctions. Overexpression of mutated occludin variants resulted in a reduced barrier function of the tight junction.17–19 With respect to this, the finding that occludin was increased in the intestinal tissue of CD patients is a further indicator for the reduced tightness of cell–cell contacts in CD. ZO-1 interacts with the transcription factor ZONAB for regulation of epithelial differentiation and morphogenesis.51 An increased amount of ZO-1 in the tissue may influence the communication between the nucleus and the tight junctions, leading to changes in epithelial differentiation and cell division. In addition, the reduced expression of adherens junction-associated E-cadherin and β-catenin may support the paraepithelial passage of potential noxes.
In summary, these data clearly demonstrate changes in the composition of junctional complex proteins in inflamed CD epithelium that may result in a dysregulated barrier function and loss of cell–cell contacts smooth the way for luminal bacteria to pass the epithelial barrier.
Furthermore, we show that translocated bacterial components are able to activate NF-κB in a NOD2 genotype-specific manner. The effects of NOD2 mutations on intestinal inflammation in CD are still controversial. There exist both “loss” and “gain” of function theories.9–12 In this study we demonstrated in vitro that overexpression of NOD2 in cells expressing the WT gene and remarkably also in cells producing the SNP13 variant activates the NF-κB signaling pathway (IL-8 secretion) without any bacterial stimulus. MDP stimulation increased IL-8 secretion levels in cells that were transfected with the WT NOD2 gene, whereas in cells that produce the truncated SNP13 variant this inducibility was lost. These data correlate with observations from Hugot et al,3 who achieved the same data using lipopolysaccharide (LPS). Our results point out parallels to the regulation of extracellular Toll-like receptors (TLRs) in IEC. Similarly to TLR4,52 NOD2 is only weakly expressed in unstimulated IEC that can be induced by proinflammatory cytokines.53 Due to the demonstrated induction of NOD2 (WT) by bacterial stimulation this protein may additionally fulfil regulatory functions in the gut.
In the intestinal tissue of CD patients an increased activation of NF-κB was found that was further increased in patients with NOD2 variants. NF-κB not only plays a role in inflammatory reactions but also in the regulation of epithelial integrity and intestinal homeostasis.7 In vivo studies showed that an IEC-specific inhibition of NF-κB through NEMO deficiency causes a severe chronic intestinal inflammation that results from epithelial cell apoptosis, reduced expression of antimicrobial peptides, and bacterial translocation.7 In this regard the weakly detected NF-κB activation in the controls reflects a necessity of the intestine to sustain homeostasis. Mainly, a primary defect in innate immunity is presumed that induces proliferation of bacteria and secondarily a NOD2-independent inflammatory reaction via adaptive effector T cells in the host tissue.9 Despite many controversial studies and hypotheses, NOD2 seems to play a key role in the immune defense of the gut against bacterial infections. Mutations in NOD2 advance a predisposition for CD through defect regulation of immune reactions to commensal and/or pathogenic bacteria.
This study for the first time shows that the extent of bacterial translocation occurring in CD is associated with the NOD2 genotype of the patients. Therefore, in patients with NOD2 polymorphism(s) a higher mucosal bacterial translocation additionally triggers intestinal inflammation.