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Keywords:

  • inflammatory bowel diseases;
  • intestinal epithelial cells;
  • nuclear transcription factor κB (NF-κB);
  • host-derived anti-inflammatory signals;
  • endoplasmic reticulum stress;
  • glucose regulated protein (Grp)-78;
  • interleukin 10;
  • commensal bacteria

Abstract

  1. Top of page
  2. Abstract
  3. RECEPTOR-MEDIATED RECOGNITION OF ENTERIC BACTERIA: IMPLICATIONS FOR THE INITIATION AND REGULATION OF CHRONIC INTESTINAL INFLAMMATION
  4. INTESTINAL EPITHELIUM: A SENSITIVE INTERFACE BETWEEN INNATE AND ADAPTIVE IMMUNE SIGNALS
  5. CONCLUSION
  6. REFERENCES

The genetic predisposition to deregulated mucosal immune responses and the concurrent prevalence of certain environmental triggers in developed countries are strong etiologic factors for the development of inflammatory bowel diseases in human subjects, including Crohn's disease and ulcerative colitis. Numerous clinical and experimental studies have shown that the intestinal microbes are critical for the initiation and progression of chronic intestinal inflammation. Activation of pattern recognition receptor signaling via members of the Toll-like receptor (TLR) and the nucleotide-binding oligomerization domain (NOD)-like families initiates inflammatory defense mechanisms that are required to alert and protect the host. Key inflammatory mechanisms such as nuclear transcription factor κB (NF-κB) activation and endoplasmic reticulum stress responses are controlled by a complex network of pathways that includes intrinsic feedback effectors and is targeted by immunosuppressive cytokines such as interleukin 10 (IL-10) and transforming growth factor (TGF)-β. In the absence or after functional loss of these antiinflammatory feedback signals, physiological defense mechanisms may turn into pathological responses. The data discussed in the present review suggest that disturbances in the homeostasis between bacteria- and host-derived signals at the epithelial cell level lead to a break in the intestinal barrier function and to the development of mucosal immune disorders in genetically susceptible hosts.

(Inflamm Bowel Dis 2007)

Ulcerative colitis (UC) and Crohn's disease (CD), the 2 main idiopathic pathologies of inflammatory bowel diseases (IBDs), are spontaneously relapsing, immunologically mediated disorders of the gastrointestinal tract. Polymorphisms associated with CD have been reported for several genes, including NOD2/(CARD15),1, 2NOD1/(CARD4),3TLR4,4TLR9,5SLC22A4 and SLC22A5,6, 7ABCB1,8, 9ATG16L1,10DLG5,11TNFSF15,12 and IL-23R.13 On the other hand, the low concordance rate for CD and UC in identical twins (≈50% and 10%, respectively) confirm that environmental factors contribute to the disease progression.14 Both CD and UC affect people in approximately equal female/male proportions, with a combined mean frequency of 5–200 cases per 100,000 inhabitants in Europe and North America.15 Since IBDs are to date not curable and the incidence of CD is still increasing in Western societies, it is crucial to add mechanistic insights to the yet-unknown etiology of IBD.

Clinical data and studies in gnotobiotic animal models of experimental colitis have shown that enteric bacteria are crucial for the development of chronic inflammation. In CD patients, diversion of the fecal stream in distal ileal segments improved the disease state.16 However, 1-week perfusion of bypassed ileum with ileostomic effluents triggered immune responsiveness and inflammation.17 Experiments in germ-free animals have shown that there is a bacterial specificity in the ability to induce chronic inflammation. For instance, Bacteroides vulgatus TUSVM40G2-33 was important for carrageenan-induced colitis in guinea pigs18 and triggered colitis in gnotobiotic HLA-B27 transgenic rats.19 However, B. vulgatus strain mpk showed protective effects on the development of experimental colitis induced by E. coli mpk (serogroup H8) in IL-2−/− mice.20 Also, the colonization of germ-free IL-10−/− mice with E. coli and Enterococcus faecalis, but not with B. vulgatus, triggered chronic experimental inflammation.21, 22 The absence of colitis and pathologic immune responses in colonized wildtype mice demonstrates the nonpathogenic nature of these bacterial species and suggests the presence of effective regulatory mechanisms under “controlled or physiologic” intestinal inflammation. To characterize the involvement of certain bacterial groups in IBD, many studies have described intestinal microbiota under chronic inflammation. Beyond the role of Mycobacterium spp. and adherent-invasive E. coli in IBD,23, 24 fecal microbiota of CD patients seem to be less diverse than that of control subjects.25 Also, luminal enteric bacteria possibly colonize the intestinal mucosa more easily under chronic inflammation.26 In UC patients the prevalence of E. faecalis in rectal mucosal samples was higher than in control subjects and was associated with a higher bacterial-surface-specific IgA response (n = 9 in each group).27 Interestingly, besides their involvement in IBD, bacteria may promote beneficial effects. Indeed, several clinical trials and animal studies have shown that E. coli Nissle 191728, 29 and the probiotic mixture VSL#330–34 are of therapeutic relevance.

Intestinal epithelial cells (IEC) must adapt to constant changes in their environment by processing both bacterial and host-derived immune signals. This implies that IEC are essential for the maintenance of mucosal homeostasis. Advances in cell-mediated innate and adaptive immune responses have helped to understand the disease pathologies of IBD. Yet little is known about how intestinal bacteria target protective and detrimental signal transduction pathways in genetically susceptible hosts. An emerging paradigm suggests that stress responses or dysfunctions in the endoplasmic reticulum and in mitochondria contribute to the loss of tissue homeostasis and to the development of chronic intestinal inflammation. The current review discusses the interrelated role of innate and adaptive immune signals in the complex network of pro- and antiinflammatory signals at the level of IEC, highlighting novel mechanisms underlying receptor-mediated recognition of enteric bacteria and host-derived feedback mechanisms.

RECEPTOR-MEDIATED RECOGNITION OF ENTERIC BACTERIA: IMPLICATIONS FOR THE INITIATION AND REGULATION OF CHRONIC INTESTINAL INFLAMMATION

  1. Top of page
  2. Abstract
  3. RECEPTOR-MEDIATED RECOGNITION OF ENTERIC BACTERIA: IMPLICATIONS FOR THE INITIATION AND REGULATION OF CHRONIC INTESTINAL INFLAMMATION
  4. INTESTINAL EPITHELIUM: A SENSITIVE INTERFACE BETWEEN INNATE AND ADAPTIVE IMMUNE SIGNALS
  5. CONCLUSION
  6. REFERENCES

The mammalian gastrointestinal tract harbors a very complex microbiota,35 which is important for the development of the immune system.36 A recent study proposed that intestinally derived bacterial signals that are transported to the lactating breast within mononuclear cells may stimulate the neonatal immune system.37 Although a variety of host-derived tolerogenic mechanisms allow a peaceful and productive coexistence of host cells with enteric bacteria, the gut-associated immune system (GALT) remains highly responsive to enteropathogens. This paradox is a pivotal feature of efficient immune tolerance but is broken under chronic intestinal inflammation.38, 39 As one of the first lines of defense against luminal aggressions, IEC play a central role in transducing bacterial stimuli via pattern recognition receptor (PRR)-associated signaling pathways. Taking into account the complexity of the intestinal microbiota, host cells are stimulated by numerous and highly diverse bacterial molecules. These molecules can be recognized by the soluble mannose binding lectin40 and by mindin, an extracellular matrix protein.41 However, a set of well conserved PRR that belong to the Toll-like receptor (TLR) and the nucleotide-binding oligomerization domain (NOD)-like receptor families is essential for recognition of extracellular and intracellular molecules associated with both nonpathogenic and pathogenic bacteria.42 PRR signaling is crucial for the initiation of innate responses, which in turn induce the adaptive immune system by providing adequate costimulatory molecules and cytokines.43

TLR Signaling

TLRs are transmembrane proteins that have an extracellular domain containing leucine-rich repeats (LRR) and an intracellular domain homologous to the IL-1 receptor named Toll/IL-1R (TIR). Ligand-specific binding to TLR promotes distinct signals depending on the interaction of the TIR domain with various adaptor proteins (MyD88, MAL/TIRAP, TRIF/TICAM-1, TRAM/TIRP/TICAM-2). These adaptor proteins target downstream effector systems such as the mitogen-activated kinases (MAPK) and the IκB/NF-κB transcriptional system.44 Recently, Rakoff-Nahoum et al45 showed that the absence of TLR/MyD88-derived signals in IL-10−/− mice prevented the development of experimental colitis at the level of T cell-mediated adaptive immune responses. In contrast, TLR/MyD88-deficient mice were characterized by increased histopathological scores after induction of colitis with dextran sodium sulfate (DSS) when compared with control animals.46 This suggests protective effects of MyD88-dependent signaling cascades at the epithelial cell level and agrees with the fact that TLR4-mutant mice are more sensitive to DSS-induced colitis47, 48 and to allergic responses toward food antigens49 than wildtype mice. Further evidence for protective TLR-mediated effects on experimental colitis was shown by Katakura et al.50 The authors demonstrated that the induction of TLR9 signaling activated interferon regulated factors (IRF1 and 8) and triggered production of protective type I interferons (IFN-α/β) through mechanisms that involve MyD88 and DNA-dependent protein kinase (DNA-PK). Consistently, TLR9−/− mice were more susceptible to colitis induced by a low dose (1.5%) of DSS when compared with wildtype and TLR2−/− mice.51 Primary colonocytes from TLR9−/− mice showed an increase in DNA binding activity of NF-κB, suggesting a pathological role for persistent activation of NF-κB signaling in IEC. Most important, whereas apical stimulation of epithelial cells by TLR9-specific ligands (ISS-ODN) inhibited NF-κB signaling by blocking the degradation of ubiquitinated IκBα, basolateral stimulation led to IκBα degradation and activation of NF-κB.51 These findings show that polarization of innate signals may be crucial for the control of intestinal homeostasis. Interestingly, bacterial DNA from the probiotic mixture VSL#3 had TLR9-mediated beneficial effects on chronic intestinal inflammation in IL-10−/− mice and in mice with colitis induced by DSS and trinitrobenzene sulfonic acid (TNBS).34 The data presented above support the hypothesis that the loss of PRR signaling may incapacitate the host to mount an appropriate innate response leading to deregulated adaptive immune responses.52

NOD Signaling

There are to date more than 20 members of the NOD-like receptor family. Nevertheless, NOD signaling pathways are still largely unknown. The current knowledge is that intracellular NOD ligands, including the NOD2 (CARD15) ligand muramyl dipeptide (MDP) and the NOD1 (CARD4) ligand γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), trigger NF-κB activation through protein–protein interaction of the caspase recruitment domain (CARD) with the serine-threonine kinase Rip2 (RICK or CARDIAK).53 Additional adaptor proteins such as GRIM-1954 and Erbin55 may be required to trigger to activation of NF-κB and the antibacterial function of intracellular NOD proteins.56 The main NOD2 gene variations associated with chronic inflammation are the missense mutations R702W and G908R and the frameshift mutation L1007fsinsC, with a prevalence of 25%–35% in CD patients having European but not Asian or African-American ancestors.57 Flagellin has been identified as a disease-relevant antigen and an increased antibody response toward CBir1 flagellin occurs in 50% of CD patients.58, 59 The anti-CBir-1 antibody response was maximal in CD patients with 1 of the 3 dominant NOD2 mutations.60 Similarly, the expression of antimicrobial peptides in Paneth cells was maximally reduced in CD patients bearing a mutation in the NOD2 gene.61 These findings suggest that PRR-associated genetic polymorphisms combined with microbial disorders lead to the development of chronic intestinal inflammation. NOD2 mutant (NOD22939iC) mice harbor a homolog of the most common CD susceptibility allele, 3020insC, which encodes a truncated protein lacking the last 33 amino acids.62 These mice were characterized by elevated NF-κB activation in response to MDP and by a more efficient processing and secretion of IL-1β, suggesting gain-of-function mechanisms for certain NOD2 variations. In contrast, MDP suppressed TLR2-induced IL-12 production in wildtype but not in NOD2−/− mice after stimulation with peptidoglycan (PGN). This suggests loss-of-function mechanisms, i.e., loss of NOD-associated negative feedback signals toward TLR-mediated Th1 immune activation.63 In addition, the complete lack of NOD2 in NOD2−/− mice did not lead to increased NF-κB activity and increased susceptibility to DSS-induced colitis.56, 64 None of the animal models with NOD2 variations develop chronic intestinal inflammation spontaneously. Moreover, NOD2 mutations also occur in healthy Caucasian subjects.65 This suggests that additional mechanisms are required to manifest the pathological effects of NOD2 mutations.

An essential role of PRR signaling is to alert and protect the host. However, PRR-mediated signals are also involved in chronic inflammation. An important question is to what extent NOD and TLR-mediated signals contribute to the early and late phases of chronic intestinal inflammation. Also, it is intriguing that commensal bacteria signal through the same PRR pathways as pathogenic bacteria. To understand how the innate immune system specifically responds to various bacteria, efforts must be put into the analysis of: 1) bacterial cell wall structures and novel colitogenic factors, 2) (sub)cellular distribution and expression levels of PRR in tissues, and 3) negative regulators of PRR signaling.

INTESTINAL EPITHELIUM: A SENSITIVE INTERFACE BETWEEN INNATE AND ADAPTIVE IMMUNE SIGNALS

  1. Top of page
  2. Abstract
  3. RECEPTOR-MEDIATED RECOGNITION OF ENTERIC BACTERIA: IMPLICATIONS FOR THE INITIATION AND REGULATION OF CHRONIC INTESTINAL INFLAMMATION
  4. INTESTINAL EPITHELIUM: A SENSITIVE INTERFACE BETWEEN INNATE AND ADAPTIVE IMMUNE SIGNALS
  5. CONCLUSION
  6. REFERENCES

The intestinal epithelium is a highly selective barrier between the luminal environment and lamina propria immune cells. IEC constitutively express, or can be induced to express, costimulatory molecules66 and components of the human major histocompatibility complex (MHC),67, 68 TLR and NOD2 proteins,69, 70 inflammatory and chemoattractive cytokines,71 as well as antimicrobial peptides.72, 73 Importantly, most of these molecules are at least in part transcriptionally regulated by the transcription factor NF-κB.74 The intestinal epithelium is considered a constitutive component of the mucosal immune system. Indeed, IEC contribute to the initiation and regulation of innate and adaptive defense mechanisms by directly interacting with lamina propria dendritic cells (DC), lamina propria lymphocytes (LPL), intraepithelial lymphocytes (IEL), as well as mediators of the immune and the enteric nerve system.75–77 Specific attention has been focused on the interaction between IEC and DC as a mechanism to polarize colitogenic T cell responses toward Th1/Th13 and Th2 effector functions in CD and UC, respectively.78–80

To maintain gut homeostasis the intestinal epithelium integrates numerous signals from both enteric bacteria and immune cells (Fig. 1). Since the break in intestinal epithelial barrier function precedes the onset of chronic immune-mediated histopathology in IBD patients and animal models of IBD, the loss of epithelial cell homeostasis seems to be critical for the development of chronic intestinal inflammation.81, 82

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Figure 1. The central role of intestinal epithelial cells in IBD. The development of chronic intestinal inflammation is a complex and long-term process that involves intestinal bacteria, host genetic background, immune signals, and environmental factors. The intestinal epithelium integrates numerous bacterial and host stimuli and is thereby essential for the regulation of innate and adaptive inflammatory immune signals. The figure shows stimuli associated with epithelial cell homeostasis or chronic inflammation. Protective (blue) and colitogenic (red) mechanisms are associated with bacterial and host-derived factors. CpG DNA, unmethylated CpG motif oligonucleotides; EF-Tu, elongation factor Tu associated with the cellular surface of L. johnsonii; IFN, interferon; IL, interleukin; TGF, transforming growth factor; Th, T helper; Tr, T regulatory.

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Crosstalk Between Bacteria and Epithelial Cells: NF-κB Signal Transduction at the Crossroad Between Host Defense and Chronic Inflammation

TLR expression and NF-κB activity seem to be increased in lamina propria macrophages and in the intestinal epithelium under chronic intestinal inflammation.83, 84 Moreover, the local administration of antisense oligonucleotides to the p65 unit of NF-κB abrogated colitis in TNBS-treated mice.85 These data suggest that the NF-κB transcription factor system plays a central role in the activation of proinflammatory genes. On the other hand, the inhibition of NF-κB activity with pharmacological inhibitors during the resolution phase of carrageenin-induced inflammation had adverse effects on the host.86 Consistently, the activation of NF-κB promotes cellular restitution of the wounded epithelium.87 Also, a mouse model characterized by IKKβ-deficiency in IEC was more sensitive to ischemia-reperfusion-induced epithelial cell apoptosis and showed loss of mucosal integrity, likely due to the failure of IκB kinase (IKK) to activate a protective NF-κB-dependent gene program.88 Thus, the activation of NF-κB signaling during the course of inflammation possibly has a dual function. In this context, we propose that the acute and transient activation of NF-κB in the intestinal epithelium may be protective for the host, while sustained and uncontrolled NF-κB activation contributes to chronic inflammation.

We have shown that monoassociation of germ-free wildtype mice with E. faecalis induced transient TLR2-mediated NF-κB activation (RelA Ser536 phosphorylation) and proinflammatory gene expression in the intestinal epithelium at an early stage of bacterial colonization (3 days).89 The transient induction of NF-κB signaling preceded any histological evidence of colitis and was associated with decreased TLR2 protein expression. At a late stage of bacterial colonization (14 weeks), IEC isolated from IL-10−/− mice showed persistently active TLR/NF-κB signaling in association with clinical and histological signs of intestinal inflammation. These results support the idea that PRR signaling in IEC contribute to the immune surveillance of enteric bacteria during early stages of host colonization.90

Also, we have shown that B. vulgatus triggered TLR4 signaling to induce NF-κB activation and proinflammatory gene expression in epithelial cell lines and in the native intestinal epithelium.91–93 Immunostaining of tissue sections confined the induction of RelA phosphorylation to the epithelium, with no induction in underlying lamina propria immune cells, suggesting a compartmentalized activation of NF-κB in the gut mucosa. In addition, Hornef et al94 showed that lipopolysaccharide (LPS) from E. coli K12 D31m4 were internalized by murine IEC and stimulated the IκB/NF-κB system via intracellular located TLR-4, supporting the concept that nonpathogenic Gram-negative bacteria can activate proinflammatory signals in the gut epithelium. Consistent with these findings, Lotz et al95 found that the intestinal epithelium of mice acquires postnatal endotoxin tolerance in response to TLR-induced signals through mechanisms that involve the selective ubiquitin-mediated degradation of IRAK-1 via the proteasome complex. In contrast, Steinhoff and colleagues96 revealed a pathophysiological and proinflammatory role for proteasome-mediated degradation of NF-κB p105 and IκBα in CD and UC patients, supporting a dual function of NF-κB in the development of inflammation.

Interestingly, Bifidobacterium lactis BB12 targeted the TLR2 signaling cascade in primary and IEC lines, suggesting that probiotic bacteria also trigger innate responses in the gut epithelium. Similar to the association of wildtype rats or mice with B. vulgatus and E. faecalis, colonization of Fisher 344 rats with B. lactis BB12 induced transient NF-κB activation and proinflammatory gene expression in the native epithelium.97 In addition, we found that the colonization of reconstituted lactobacilli-free (RLF) mice with Lactobacillus reuteri trigged a transient activation of an NF-κB-dependent proinflammatory gene program (Hoffmann, Tannock, and Haller, unpubl.). The fact that colitogenic and probiotic bacteria signal through the same PRR systems to initially trigger proinflammatory signaling cascades underlines the concept that normal hosts develop hard-wired mechanisms to inhibit persistent immune activation of IEC.

Host-derived Signals Regulate Proinflammatory Mechanisms at the Epithelial Cell Level

Intrinsic Regulatory Mechanisms

As seen above, an intricate network of kinases, adapter proteins, and scaffolding proteins transmit TLR signals to various effector systems, including the NF-κB signaling machinery. Among other molecules, A20,98 IRAK-M,99 ST2,100 TIR8,101 TOLLIP,102 and TRIAD3A103 are intrinsic proteins involved in the negative regulation of TLR-mediated gene expression. Toll/IL-1R 8 (TIR8), also known as single immunoglobulin IL-1R-related receptor (SIGIRR), has been shown to directly interfere with the IL-1/TLR cascade by blocking the interaction between TRAF6 and IRAK1.101 When compared with wildtype mice, TIR8−/− mice were more susceptible to DSS-induced intestinal inflammation.104 The authors also showed that TIR8 is expressed in both IEC and DC. Another important intrinsic negative regulator of TLR-mediated signals is the Toll-interacting protein (TOLLIP). In IEC, TOLLIP impaired NF-κB and AP-1 transcriptional activity via association with TLR4 and TLR2 and via suppression of LPS-induced IRAK phosphorylation.105 Also, TLR hyporesponsiveness to bacterial ligands was associated with the induction of TOLLIP in IEC.106

Peroxisome proliferator-activated receptor (PPAR)-γ is a member of the steroid receptor superfamily and is involved in various cellular functions, e.g., differentiation, apoptosis, lipid metabolism, and antiinflammatory responses.107 Although PPAR-γ is expressed in multiple tissues, the highest expression is found in the adipose tissue and in the colonic epithelium.108 PPAR-γ was identified as a susceptibility gene in both the SAMP1/YitFc mouse model of experimental ileitis and in CD patients.109 In the colonic epithelium of UC patients and in DSS-treated mice, PPAR-γ expression was substantially reduced.110, 111 Consistently, PPAR-γ+/- mice were more susceptible to TNBS-induced colitis when compared to wildtype mice.112 The fact that TLR4−/− mice failed to induce PPAR-γ expression in IEC after the colonization with enteric bacteria110 supports the idea that PPAR-γ is implicated in the negative regulation of bacteria-meditated PRR signaling in the intestinal epithelium. Indeed, Bacteroides thetaiotaomicron triggered PPAR-γ-mediated nuclear export of transcriptionally active RelA and abolished Salmonella enteriditis-induced inflammatory effects in IEC cultures.113 This suggests that commensal enteric bacteria play an important role in intestinal homeostasis.

Phosphorylation and dephosphorylation of transcription factors regulates their DNA binding properties and their transactivating potential. Protein complexes containing both kinases and phosphoprotein phosphatases (PP) seem to be important in maintaining the phosphorylation state of intracellular substrates. Protein serine/threonine phosphatases, such as PP1, PP2A, PP2B, and PP2C, are involved in the regulation of signaling pathways including the NF-κB cascade.114 We have shown that the PPAR-γ-specific ligand 15-deoxy-Δ12,14-prostaglandin J2 triggered PP2A activity in IEC, thereby inducing dephosphorylation of B. vulgatus-induced phospho-RelA and, as a consequence, inhibited NF-κB-dependent gene expression.93 Although monoassociation of Fisher 344 rats with B. vulgatus induced PPAR-γ nuclear expression in the native epithelium, the antiinflammatory effect of 15-deoxy-D12,14-prostaglandin J2 in IEC was not dependent on the presence of PPAR-γ.

An interrelated role of MyD88-dependent and prostaglandin-mediated signals was shown to be required for maintenance of intestinal epithelial cell proliferation during DSS-induced injury.115 Prostaglandin-endoperoxide synthase 2 deficient mice (Ptgs2−/−) exhibited an inhibition of epithelial cell proliferation and cellular organization within rectal crypts. The exogenous application of prostaglandin E2 derivatives (16,16-dimethyl-PGE2) rescued the pathologic phenotype in both MyD88−/− and Ptgs2−/− mice. The authors proposed that Myd88 and prostaglandin signaling pathways interact through a mechanism that requires proper cellular mobilization within the crypt niche. These results suggest that intercellular communication and host-derived mediators interfere with the complex signaling network involved in acute and chronic inflammatory processes, thus balancing the fine-tuned mechanisms underlying tissue injury and healing.

Cytokine-mediated Regulatory Mechanisms

In addition to intrinsic regulatory mechanisms, host-derived immune signals are critical for epithelial cell homeostasis. Although many pathways are likely involved in the regulation of innate and adaptive immunity in the intestine, the immunosuppressive mediators IL-10 and TGF-β are highly relevant to IBD.

Powrie et al116 provided experimental evidence for the importance of IL-10 and TGF-β by using severe combined immunodeficient (SCID) and recombination activating gene-deficient (RAG−/−) mice. The adoptive transfer of CD4+ CD45RBhigh T cells from congenic donor mice into T and B cell-deficient SCID and RAG−/− mice triggered experimental colitis. The development of chronic inflammation was associated with the production of high amounts of the proinflammatory Th1 mediator interferon-γ (IFN-γ). In contrast, the adoptive transfer of CD4+ CD45RBlow T cells had protective effects in the recipient SCID and RAG−/− host. These protective effects have been associated with the presence of IL-10 and TGF-β.116, 117 Accordingly, IL-10−/− mice develop immune-mediated colitis under specific pathogen-free (SPF) conditions, but are disease-free under germ-free conditions.22, 118 Thus, in the absence of host-derived immune regulators, bacterial antigens seem to drive chronic inflammatory processes. The protective role of IL-10 in TNBS-induced experimental colitis was indirectly mediated through induction of TGF-β secretion in lamina propria T cells, suggesting an interrelated role of IL-10 and TGF-β.119 TGF-β1-deficient mice spontaneously develop colitis120 and the overexpression of TGF-β1 in lamina propria immune cells inhibited Th1-mediated TNBS-induced colitis.121 Of importance to understand the biological function of TGF-β at the epithelial cell level, the lack of TGF-β signaling in tissue-specific transgenic mice that express a dominant-negative TGF-β receptor in the intestinal epithelium triggered colitis under SPF conditions.122 These mice were also more responsive to DSS-induced intestinal inflammation.

TGF-β1 mediates its biological effect through activation of various signaling cascades, including the Smad and MAPK pathways.123 In B. vulgatus-monoassociated wildtype Fisher 344 rats and in E. faecalis-monoassociated wildtype SvEv129 mice, we have shown that nuclear RelA phosphorylation was followed by the induction of Smad2 phosphorylation in epithelial cells isolated from intestinal tissue sections at early but not late stages of bacterial colonization.89, 91–93 Interestingly, TGF-β-activated Smad signaling induced rapid TLR2 degradation89 and blocked CBP/p300-mediated histone phosphorylation in epithelial cells,92 leading to the inhibition of proinflammatory gene expression. Additional evidence for the importance of proteasome-mediated TLR degradation as a strategy of the host to control pattern recognition receptor signaling has been recently shown by Chuang and Ulevitch.103 The authors demonstrated that the intrinsic RING finger protein TRIAD3 enhanced ubiquitination and proteolytic degradation of TLR4 and TLR9 but not TLR2 due to its E3 ubiquitin-protein ligase activity. Thus, negative feedback regulators may have distinct specificities for the different TLR subsets. Importantly, TGF-β1-induced Smad2 signaling was absent in IEC isolated from IL-10−/− mice monoassociated with E. faecalis.89 Thus, in the absence of the activated TGF-β/Smad cascade in the intestinal epithelium, bacteria-mediated TLR signaling may lead to chronic intestinal inflammation. Altogether, these results show that NF-κB signals are present in the intestinal epithelium after bacterial colonization but that host-derived feedback mechanisms inhibit bacterial-driven proinflammatory mechanisms. An attractive hypothesis is that transient NF-κB induction in IEC triggers protective IL-10-mediated TGF-β responses. Finally, the lack of TGF-β-activated Smad signaling in lamina propria T cells of IBD patients due to overexpression of the inhibitor Smad7 was associated with disease progression.124, 125 Hence, even if immunosuppressive signals are present in diseased tissues, their intracellular blockade may lead to chronic intestinal inflammation.

Endoplasmic Reticulum Stress Responses and Chronic Intestinal Inflammation: Inhibitory Mechanisms of IL-10

In mammalian cells the endoplasmic reticulum (ER) is essential for cholesterol production, for calcium homeostasis, and for the transit of correctly folded proteins to the extracellular space, the plasma membrane, and the exo- and endocytic compartments. Adverse environmental and metabolic conditions activate ER stress responses, including the unfolded protein response (UPR), the ER overload response (EOR), the ER-associated degradation (ERAD), and the sterol regulatory response.126, 127 ER stress can be induced by changes in calcium homeostasis or redox status, elevated protein synthesis, and expression of unfolded or misfolded proteins, energy deficiency and glucose depravation, altered protein glycosylation, cholesterol depletion, and microbial infections. To react against protein accumulation in the ER, molecular mechanisms underlying UPR, EOR, and ERAD lead to translational attenuation, enhanced expression of ER chaperones, and induction of protein degradation. However, upon failure of these adaptive mechanisms, prolonged ER stress results in cell death via mitochondria-dependent and -independent apoptotic pathways.128, 129

The glucose-regulated protein (grp)-78 (also referred to as immunoglobulin heavy chain-binding protein, BiP) is an ER chaperone that plays a central role in the UPR. Accumulation of mis- or unfolded proteins in the ER triggers grp-78 liberation from the ER transmembrane protein PERK (PKR-like ER-associated kinase), ATF6 (activating transcription factor 6), and IRE1 (inositol requiring enzyme 1) (Fig. 2A). PERK dimerization and the consequent phosphorylation of eukaryotic initiation factor (eIF)2α lead to translation attenuation and to ATF4-mediated regulation of gene expression, including expression of grp-78 and of the transcription factor CHOP (C/EBP homologous protein, also known as growth arrest and DNA damage 153, GADD153). CHOP subsequently activates the transcription of GADD34, which catalyzes dephosphorylation of P-eIF2α. CHOP and GADD34 expression seem to be particularly important for ER stress regulation and ER-stress-dependent apoptosis.128 After release from the ER, ATF6 migrates to the Golgi apparatus where it is cleaved by site 1 and site 2 proteases. Cleaved-ATF6 translocates to the nucleus and regulates gene expression, such as expression of ER chaperones, CHOP, and the transcription factor XBP1 (X-box binding protein). Finally, the endoribonuclease activity of IRE1 dimers catalyzes the cytoplasmic activation of XBP1 via splicing of a 26-nucleotide-long intronic sequence. Interestingly, ER stress responses seem to be linked to NF-κB activation through mechanisms that involve IRE1 and TNF receptor-associated factor (TRAF) 2, changes in Ca2+ levels, and production of reactive oxygen species (ROS).127, 130 Also, ER stress has been associated with type I and type II diabetes and with neurodegenerative diseases.131, 132 However, despite the fact that IRE1β-deficient mice showed elevated grp-78 levels in the colon and were more sensitive to DSS-induced inflammation,133 knowledge of ER stress responses in chronic intestinal inflammation is scant.

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Figure 2. Endoplasmic reticulum stress signaling. (A) Recruitment of the chaperone grp-78 to mis- or unfolded proteins regulates signaling cascades mediated by the transmembrane protein PERK, ATF6, and IRE-1. The subsequent eIF2 phosphorylation, ATF6 cleavage, and XBP1 splicing lead to changes in gene expression. (B) In the context of chronic intestinal inflammation, ER-stress-associated signals may be important for inflammatory and apoptotic responses and for energy homeostasis. For instance, grp-78 is involved in TNF-induced NF-κB signaling. The antiinflammatory cytokine IL-10 was found to inhibit TNF-induced grp-78 recruitment into the IKK complex and ATF6 nuclear translocation in a p38-dependent manner (the latter is not shown in the figure). AP, activator protein; ARE, antioxidant responsive element; ATF, activating transcription factor; CHOP, C/EBP-homologous protein; ERAD, ER-associated degradation; eIF2α, α-subunit of eukaryotic translation initiation factor 2; ER, endoplasmic reticulum; ERSE, ER stress response element; GADD, growth arrest and DNA damage; Grp, glucose-regulated protein; IL, interleukin; IRE, inositol requiring enzyme; NRF, NF-E2-related factor; p58IPK, 58 kDa-inhibitor of protein kinase; PERK, PKR-like ER-associated kinase; S1P and S2P, site 1 and site 2 proteases; TNF, tumor necrosis factor; TRAF, TNF-receptor associated factor; UPRE, unfolded protein response element; XBP, x-box binding protein.

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Based on proteomic analysis, we found that grp-78 expression was increased in primary IEC from both E. faecalis-monoassociated IL-10−/− mice and inflamed tissues from CD and UC patients.134 Interestingly, cytoplasmic TNF signaling was modulated by recruitment of grp-78 into the IKK complex (Fig. 2B). Consistently, knock-down of grp-78 using small interfering (si) RNA prevented TNF-induced NF-κB RelA phosphorylation, supporting the hypothesis that grp-78 association with the IKK/NF-κB signalsome facilitates the activation of proinflammatory cascades. Since TNF triggers ROS-dependent ER stress135 independent of grp-78 resynthesis,136 the appearance of grp-78 in the IKK complex may reflect TNF-induced ER stress responses via redistribution of grp-78 from the ER lumen into the cytoplasmic space. This agrees with previous data showing that ER stress inducers trigger the redistribution of grp-78 from the ER lumen. Grp-78 may either migrate to the cytoplasm137 or act as a transmembrane protein.138

IL-10 signals through JAK1/STAT3 and p38-MAPK-mediated pathways to trigger antiinflammatory mechanisms dependent on suppressor of cytokine signaling (SOCS)139, 140 and heme oxygenase (HO)-1.141 Although the molecular understanding of IL-10 signaling in IEC is still unclear, we found that IL-10-receptor-reconstituted IEC regain IL-10-mediated p38 phosphorylation, suggesting a direct protective role for IL-10-mediated p38 signaling at the epithelial cell level.134 We showed that the p38 MAPK signaling cascade is activated in primary IEC from E. faecalis-monoassociated wildtype but not IL-10−/− mice. Since IL-10-mediated p38 signaling blocked ER stress responses in IEC via inhibition of the nuclear recruitment of ATF-6 to the grp-78 promoter, we propose that IL-10 may protect the intestinal epithelium by regulating ER stress signaling. Although the host benefits from the ER stress response program at early times to restore normal ER function, sustained ER stress may contribute to the development of epithelial cell dysfunctions and chronic intestinal inflammation. Time and spatial resolution of the various ER signal transduction pathways are required to further specify the pathological mechanisms of ER stress response under conditions of chronic intestinal inflammation.

It has been suggested that IBD is characterized by energy-deficiency and alteration of oxidative metabolism in epithelial cells.142, 143 In addition, hypoxia and microvascular dysfunction contribute to disease pathology in the chronically inflamed gut.144, 145 In HeLa cells and human diploid fibroblasts, hypoxia triggered PERK-mediated inhibition of the translational machinery.146, 147 Also, hypoxia-inducible factor-1 (HIF-1) regulates a number of barrier protective genes,148, 149 and mutated and functionally inactive HIF-1 in the intestinal epithelium triggered TNBS-induced colitis.150 Grp-78 shares ≈60% homology with the heat shock protein (hsp)70 and, like all hsp70 family members, binds ATP. Since protein folding in the oxidizing ER environment requires energy, the depletion of cellular ATP inhibits protein folding and, thereby, unfolded intermediates become irreversibly trapped in low energy states, contributing to ER stress responses.151, 152 Taken together, these data point at a link between hypoxia, ATP depletion, and ER stress responses at the epithelial cell level under chronic inflammation.

CONCLUSION

  1. Top of page
  2. Abstract
  3. RECEPTOR-MEDIATED RECOGNITION OF ENTERIC BACTERIA: IMPLICATIONS FOR THE INITIATION AND REGULATION OF CHRONIC INTESTINAL INFLAMMATION
  4. INTESTINAL EPITHELIUM: A SENSITIVE INTERFACE BETWEEN INNATE AND ADAPTIVE IMMUNE SIGNALS
  5. CONCLUSION
  6. REFERENCES

Host-derived feedback mechanisms control epithelial cell responses toward enteric bacteria under normal conditions, but deregulation of these protective immune signals is associated with the loss of epithelial cell homeostasis and with chronic activation of proinflammatory immune mechanisms.153 Figure 3 highlights the complex network of signaling cascades involved in the regulation of inflammatory responses at the level of IEC. This network includes bacteria-, immune-, and cell-stress-mediated signals. The use of 2-dimensional protein separation techniques coupled with peptide mass fingerprinting by mass spectrometry allows us to determine changes in protein expression under chronic intestinal inflammation.134, 154 Thereby, we identified 3 major functional clusters that include cytoskeletal proteins and proteins involved in energy and protein metabolism,155 supporting the hypothesis that energy deficiency and cellular stress responses contribute to the development of IBD. It seems now important to characterize the specific contribution of these various proteins in the maintenance of epithelial cell homeostasis.

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Figure 3. Molecular pathways underlying inflammatory and cell stress responses in the intestinal epithelium. Intestinal epithelial cells adapt to their environment through induction and regulation of a complex network of inflammatory and protective immune and cell stress signals. The development of chronic inflammation is characterized by deregulation of these adaptive responses. Red and blue boxes show defense and regulatory effectors, respectively. The molecules depicted are: A20, cytoplasmic zinc-finger protein also referred to as TNFAIP3; AMPK, AMP-activated protein kinase; ATP, adenosine triphosphate; DNA-PK, DNA-dependent protein kinase; Grp-78, glucose regulated protein 78; HIF-1, hypoxia-inducible factor 1; IFN, interferon; IKK, IκB kinase; IL, interleukin; IRAK, IL-1-R-associated kinase; IRF, interferon regulated factor; MAPK, mitogen-activated kinases; MyD88s, spliced variant of myeloid differentiation protein 88; NF-κB, nuclear factor κB; NOD, nucleotide-binding oligomerization domain; PPAR, peroxisome proliferator-activated receptor; PG, prostaglandin; Ptgs, prostaglandin-endoperoxide synthase; PP, phosphoprotein phosphatase; ROS, reactive oxygen species; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; TGF, transforming growth factor; TOLLIP, Toll-interacting protein; TLR, Toll-like receptor; TNF, tumor necrosis factor; TRAF, TNF-receptor associated factor.

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REFERENCES

  1. Top of page
  2. Abstract
  3. RECEPTOR-MEDIATED RECOGNITION OF ENTERIC BACTERIA: IMPLICATIONS FOR THE INITIATION AND REGULATION OF CHRONIC INTESTINAL INFLAMMATION
  4. INTESTINAL EPITHELIUM: A SENSITIVE INTERFACE BETWEEN INNATE AND ADAPTIVE IMMUNE SIGNALS
  5. CONCLUSION
  6. REFERENCES