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
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.
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.
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.