Intestinal epithelial cells are constantly bombarded with pathogenic, cytotoxic, metabolic stresses which trigger apoptotic and necrotic cell death, leading to gut barrier damage, microbial influx and inflammatory responses[15,16,31,32,145,146]. Evidence supporting the notion that gut permeability defects precedes the onset of mucosal inflammation was found in spontaneous enterocolitis models of IL-10-/- and SAMP1/YitC mice[147-149]. Moreover, mucosal inflammation was seen in areas adjacent to epithelium with TJ disruption (loss of endogenous E-cadherin) due to the expression of a dominant negative N-cadherin mutant lacking an extracellular domain in mice. Recent studies using epithelial-specific knockout models provide direct evidence of the cause-and-effect relationship between cell death-dependent epithelial barrier defects and intestinal inflammation. Mice with conditional deletion of caspase-8 or Fas-Associated protein with Death Domain on intestinal epithelial cells spontaneously developed epithelial cell necrosis and inflammatory lesions in the ileum and colon[145,146]. On the other hand, a number of studies have demonstrated that pro-inflammatory cytokines (e.g., IFNγ and TNFα) and phagocytic mediators (e.g., free radicals and proteases) cause tight junctional breakdown and intestinal permeability rise[139,151,152], and thus argue in favor of inflammation as the cause for epithelial barrier disruption. Regardless of the starting point, a feed-forward vicious cycle between barrier dysfunction and inflammatory reaction is crucial for the perpetuation and aggravation of chronic inflammation in intestines.
Several lines of evidence suggest a critical role of dysbiosis in the pathogenesis of inflammatory bowel disease (IBD). In IBD patients, not only the quantity of commensal bacteria in the intestine is reduced (about ten-fold lower than control subjects), but also the diversity of the microbiota is altered[6,153,154]. Reduction of major classes of commensals, Firmicutes and Bacteroidetes, and increase of mucosal adherent bacteria are documented in patients[6,153-155]. Experimental models such as IL-2- or IL-10-deficient mice that spontaneously develop colitis do not develop disease when raised in a germ-free environment[156,157]. In addition, monoassociation with Bacteroides vulgatus or E. coli is sufficient to induce colitis in human leukocyte antigen-B27 transgenic rats. Recent findings that transmission of colitogenic commensal bacteria is able to trigger colitis in the genetically intact recipient mice further strengthen this view. Mice with genetic deficiency in RAG-1 and T-bet displayed dysbiosis and developed spontaneous colonic inflammation that resembles human ulcerative colitis. Interestingly, T-bet-competent wild type pups develop colitis after being crossfostered to female mutant mice, suggesting a communicable nature of this form of colitis by the gut microbiota.
Aberrant bacterial signaling by microbe-associated molecular pattern receptors, e.g., nucleotide-binding oligomerisation domain 2 (NOD2) and toll-like receptors (TLRs), on mucosal cells is incriminated in the development of chronic intestinal inflammation. Mutations in the gene encoding NOD2 were identified in patients with Crohn’s disease[161,162]. NOD2 has been known as a cytosolic innate receptor able to sense peptidoglycan from Gram-positive and -negative bacteria inside enterocytes to trigger RIP2- and nuclear factor kappa B (NF-κB)-mediated pro-inflammatory responses and to induce antimicrobial defensin synthesis[163,164]. Recent studies demonstrated that NOD2-deficient mice display altered microbiota composition, and elevated bacterial load in the feces and terminal ileum compared to their wild-type counterparts[165,166], supporting that NOD2 dysfunctions and its subsequent dysbiosis may result in the breakdown of gut homeostasis and predispose to chronic inflammation.
Accumulating evidence points out that changes in the expression levels of receptors to Gram-negative bacterial LPS in the intestinal mucosa may be involved in the pathogenesis of IBD and colorectal cancer[167-170]. The multi-unit receptor for LPS (CD14/TLR4/MD-2 complex) was originally detected on blood monocytes in the context of the pathogenesis of septic shock[171,172]. It becomes clear now that intestinal epithelial cells and resident macrophages bear a distinct expression pattern of receptors unlike circulating monocytes and peritoneal macrophages. Recent data show that in purified enterocytes isolated from normal human biopsy samples, CD14 protein is constitutively expressed, whereas TLR4 is barely detectable[167-170,173]. Moreover, human intestinal macrophages isolated from normal jejunal specimens do not express innate immune receptors, such as receptors for LPS (CD14), Fcα (CD89), Fcγ (CD64, CD32, CD16), CR3 (CD11b/Cd18) and CR4 (CD11a/CD18). Low TLR4 levels have also been reported in the lamina propria macrophages in comparison to blood monocytes in normal human subjects. It is noteworthy that these intestinal resident macrophages show downregulated LPS-induced production of proinflammatory cytokines, but retain potent phagocytic and bactericidal activities in physiological conditions[174,176]. The distinct characteristics of LPS receptors on enterocytes and mucosal macrophages may reflect its tolerance to the presence of commensal bacteria, which is crucial for limiting unwanted inflammation and for maintaining gut homeostasis.
Polymorphism of CD14 and TLR4 genes was identified in subsets of IBD patients[177-183], suggesting that abnormal bacterial LPS signaling may play a role in the pathogenesis. Since both intestinal epithelial cells and lamina propria macrophages express CD14 and TLR4 proteins at variable levels, their changes related to chronic colitis will be discussed in a cell type-specific fashion. Upregulated epithelial TLR4 expression was observed in IBD patients compared to normal subjects[167,168]. A similar increase in TLR4 was found in crypt epithelial cells in DSS-induced mouse colitis models[184,185]. Moreover, CD14 mRNA and protein levels in the intestinal epithelial cells of DSS-induced and spontaneous colitic mice were also higher than those in healthy animals[184,186]. These finding suggest that at the interface with commensal microbes, altered expression of LPS receptor components (CD14 and TLR4) on enterocytes may trigger epithelial-derived proinflammatory signals.
A wide array of differential expression patterns and subcellular location of LPS receptors was seen in different intestinal epithelial cell lines that correlated with their responsiveness to LPS for proinflammatory cytokine synthesis. For example, Caco-2 cells that express cell surface CD14 but have low levels of TLR4 mRNA and proteins, similar to normal human enterocytes, neither activate their NF-κB pathway nor produce IL-8 after LPS challenge[57,68,116,187], showing one of the possible mechanisms for endotoxin tolerance by enterocytes. Transfection of TLR4/MD2 to Caco-2 cells restores the responsiveness to LPS and synergistic activation of NF-κB and IL-8 reporter genes. Moreover, HT29 cells that express membrane-bound CD14 and cytoplasmic TLR4 are responsive to IFNγ for upregulation of intracellular TLR4 levels and the cells are sensitized for LPS-induced IL-8 production[57,116]. Among human intestinal epithelial cell lines that express constitutively high cell surface levels of TLR4, such as SW480 and T84 cells, exposure to LPS stimulates the activation of NF-κB and AP-1 signaling and the production of TNFα and IL-8[57,8,187]. It is clear from in vitro data that induction or heightened expression of individual LPS receptor components on intestinal epithelial cells may overrule their hyporesponsiveness to luminal bacterial LPS as a trigger for proinflammatory signals. Augmented expression of LPS receptors was also noted in lamina propria macrophages in inflamed tissues of IBD patients[168,175,188,189]. Heightened TLR4 expression was localized to intestinal macrophages in biopsy or surgical specimens obtained from both ulcerative colitis and Crohn’s disease patients. In Crohn’s disease patients, recent studies found increased subsets of CD14+ macrophages in comparison to the typical resident macrophages (CD14-CD33+) in the intestinal lamina propria[168,188,189]. The CD14+ population of macrophages exhibit potent antigen-presenting ability to evoke differentiation of Th17 cells and produce large amounts of proinflammatory cytokines (e.g., TNFα and IL-23) that stimulate lamina propria mononuclear cells to synthesize IFNγ in a positive feedback loop. These abnormal CD14+ macrophages may decrease the threshold to mount an inflammatory response upon exposure to low concentrations of LPS and to commensal bacteria, and may amplify the production of proinflammatory cytokines from different cell types through the positive feedback loop of IL-23/IFNγ[189,190].
Other reports indicated that a decrease in IL-10-producing intestinal macrophage subsets (CD11b+F4/80+CD11c-) also plays a role in the development of chronic intestinal inflammation[191,192]. Studies in IL-10-deficient colitis mouse models have demonstrated that bone marrow-derived macrophages from IL-10-/-mice produce large amounts of IL-12 and IL-23 upon stimulation with heat-killed bacterial antigens, whereas those from wild type mice produce high levels of IL-10 but neither IL-12 nor IL-23, which is correlated to the phenomenon where IL-10-/- mice fail to develop spontaneous colitis and intestinal histopathology if reared in germ-free conditions. These findings suggest that commensal microbes or bacterial LPS may stimulate different subsets of macrophages, leading to varied patterns of macrophage-derived cytokine production (IL-10 vs IL-12/IL-23) that determine the progress to immune hyporesponsiveness or development of colitis[118,190]. It remains unknown whether the low baseline levels of Fcα and Fcγ on normal intestinal resident macrophages are also upregulated in IBD patients, which may increase opsonization and phagocytosis for more efficient antigen presenting capability to stimulate long-term immune memory and chronic reactions.
Dysregulation of enterocytic apoptosis, proliferation and tumorigenesis
The abnormal TLR4 overexpression on enterocytes and intestinal macrophages in IBD patients suggests that bacterial LPS stimulation may initiate mucosal-derived proinflammatory signals in the pathogenesis of chronic colitis. Based on this theory, a number of laboratories investigated the possibility that targeted deficiency of TLR4 signaling might decrease gut inflammation. Unexpectedly, mice with spontaneous mutation or targeted knock-out of TLR4 and MyD88 displayed poorer colitis scores and lower survival rates in DSS models[114,193,194]. Besides the heightened mucosal inflammatory responses, the lack of TLR4 signaling also resulted in other abnormalities, such as elevated epithelial cell apoptosis, decreased crypt cell proliferation, and impaired epithelial restitution accompanied with more severe mucosal ulceration in the DSS-induced colitis model[114,193,194]. The findings in these TLR4-/- and MyD88-/- mice were similar to those with commensal bacteria depletion in DSS-induced colonic injury, whereby more extensive denudation of the surface epithelium results in ulceration or erosion of mucosa accompanied by pronounced compensatory crypt proliferation. These novel observations point out that presence of commensal bacteria and LPS-mediated TLR4 signaling may also be involved in epithelial cell survival that is critical in maintaining epithelial barrier integrity in physiological conditions and recovery to gut homeostasis in diseased states.
Many studies have shown that a lack of NF-κB sig-naling leads to increased epithelial apoptosis and impaired epithelial restitution after DSS challenge in colitis development[114,193-196]. Mice with epithelial-specific deficiency of IKKγ/NEMO develop spontaneous chronic intestinal inflammation associated with increased epithelial apoptosis and bacterial translocation. Targeted ablation of IKKβ in intestinal epithelial cells also resulted in severe cell apoptosis upon radiation or ischemic challenge, further supporting a universal role of IKKβ for cell survival against various types of stresses. Another study also showed that enterocyte-specific knockout of Raf-1 leads to NF-κB inactivation that is responsible for increased epithelial apoptosis and impaired epithelial proliferation and regeneration after oral DSS challenge. Taken together, the aforementioned studies indicated that epithelial-derived TLR4/NF-κB pathways are involved in anti-apoptotic events.
From a physiological point of view, LPS signaling in the normally tolerant gut epithelial cells may serve as a warning system to the underlying immune cells while trying to promote epithelial restitution and maintain epithelial barrier functions via multiple pathways for proinflammatory, anti-apoptotic and proliferative effects. Short-term epithelial TLR4/NF-κB signaling is crucial for preventing pathogenic epithelial cell death and epithelial barrier disruption, which may help limit the exposure of the immune cells to bacterial antigens and toxins that could cause full-blown reactions. On the other hand, a chronic epithelial-derived LPS signaling may shift the normal cell cycle into tumorigenic phenotypes in the long run.
A strong link between inflammation and cancer formation was suggested by the higher incidence of gastric and colorectal cancer in patients with early onset of IBD[199,200]. Accumulating evidence indicates that TLR4 expression in intestinal epithelial cells is upregulated in patients with colorectal cancer[169,170], suggesting that altered expression pattern and malformed signals of epithelial LPS receptor components may also play crucial roles in tumorigenesis. Aberrant reactions to bacterial LPS by CD14/TLR4 may induce an imbalance of apoptosis and proliferation, resulting in cancer formation. Recent data showed that TLR4-/- and MyD88-/- mice failed to develop colitis-associated and carcinogen-induced colorectal tumors[201-204]. TLR4 may be responsible for upregulated production of cyclooxygenases and activation of epidermal growth factor receptors which may contribute to cancer formation[193,201]. A recent study pointed out that MyD88-dependent signaling controls the expression of several key modifier genes of intestinal tumorigenesis and has a critical role in both spontaneous and carcinogen-induced tumor development.
Mice with epithelial-specific IKKβ deficiency had a lower incidence of tumor formation, partly due to increased levels of epithelial apoptosis, compared to wild type animals after injection with azoxymethane (AOM) followed by treatment with DSS. It is noteworthy that deletion of IKKβ in myeloid cells led to smaller tumor size, but no change of tumor incidence compared to wild type mice after AOM-DSS challenge. These findings suggest that IKKβ in different cell types contributes to tumorigenesis via variable cellular functions, of which epithelial-specific IKKβ promotes tumor formation by conferring resistance to cell apoptotic pathways, whereas IKKβ signals in myeloid cells are involved in boosting epithelial cell cycle progression and cell division. Therefore, it is important to identify the different types of mucosal cells (enterocytes or macrophages) responding to LPS when explaining the pathogenesis of intestinal inflammation and colorectal cancer formation.
In summary, LPS/TLR4-mediated signals which are normally downregulated in the gut epithelium are now linked with various pathological phenomena and disease states such as chronic inflammation, anti-apoptosis, hyperproliferation and tumorigenesis in the gastrointestinal tract.