Intestinal epithelial cells (IEC) interact with a high density of Gram-positive bacteria and are active participants in mucosal immune responses. Recognition of Gram-positive organisms by Toll-like receptor (TLR)2 induces proinflammatory gene expression by diverse cells. We hypothesized that IEC are unresponsive to Gram-positive pathogen-associated molecular patterns and sought to characterize the functional responses of IEC to TLR2-specific ligands. Human colonic epithelial cells isolated by laser capture microscopy and IEC lines (Caco-2, T84, HT-29) were analyzed for expression of TLR2, TLR6, TLR1, and Toll inhibitory protein (Tollip) mRNA by RT-PCR and quantitative real-time PCR. Response to Gram-positive bacterial ligands was measured by NF-κB reporter gene activation and IL-8 secretion. TLR2 protein expression was analyzed by immunofluorescence and flow cytometry. Colonic epithelial cells and lamina propria cells from both uninflamed and inflamed tissue demonstrate low expression of TLR2 mRNA compared with THP-1 monocytes. IECs were unresponsive to TLR2 ligands including the staphylococcal-derived Ags phenol soluble modulin, peptidoglycan, and lipotechoic acid and the mycobacterial-derived Ag soluble tuberculosis factor. Transgenic expression of TLR2 and TLR6 restored responsiveness to phenol soluble modulin and peptidoglycan in IEC. In addition to low levels of TLR2 protein expression, IEC also express high levels of the inhibitory molecule Tollip. We conclude that IEC are broadly unresponsive to TLR2 ligands secondary to deficient expression of TLR2 and TLR6. The relative absence of TLR2 protein expression by IEC and high level of Tollip expression may be important in preventing chronic proinflammatory cytokine secretion in response to commensal Gram-positive bacteria in the gut.

The intestinal epithelium serves as a barrier to the intestinal flora. In response to pathogens, intestinal epithelial cells (IEC) secrete proinflammatory cytokines. To aid in defense against bacteria, IEC also secrete antimicrobial peptides, termed defensins. The aim of our studies was to understand the role of TLR signaling in regulation of β-defensin expression by IEC. The effect of LPS and peptidoglycan on β-defensin-2 expression was examined in IEC lines constitutively or transgenically expressing TLRs. Regulation of β-defensin-2 was assessed using promoter-reporter constructs of the human β-defensin-2 gene. LPS and peptidoglycan stimulated β-defensin-2 promoter activation in a TLR4- and TLR2-dependent manner, respectively. A mutation in the NF-κB or AP-1 site within the β-defensin-2 promoter abrogated this response. In addition, inhibition of Jun kinase prevents up-regulation of β-defensin-2 protein expression in response to LPS. IEC respond to pathogen-associated molecular patterns with expression of the antimicrobial peptide β-defensin-2. This mechanism may protect the intestinal epithelium from pathogen invasion and from potential invaders among the commensal flora.

Enteropathogenic Escherichia coli (EPEC) virulence requires a type III secretion system (TTSS) to deliver effector molecules in host cells. Although the TTSS is crucial to EPEC pathogenesis, its function in EPEC-induced inflammation is not known. The aim of this study was to investigate the role of the TTSS in EPEC-induced inflammation. HT-29 intestinal epithelial cells were infected with wild-type (WT) EPEC or select mutant strains or exposed to corresponding filter-sterilized supernatants (SN), and interleukin-8 (IL-8) secretion was determined by ELISA. EPEC SN stimulated significantly greater IL-8 production than EPEC organisms. Flagellin, as well as a TTSS-independent >50-kDa nonflagellin protein, was found to significantly contribute to this response. Dose-response studies showed that increasing concentrations of WT SN proportionally increased IL-8, whereas increasing multiplicity of infection of EPEC inversely correlated with IL-8 secretion, suggesting that EPEC dampens this host response. Infection with DeltaescN (nonfunctional TTSS) markedly increased IL-8 compared with WT, indicating that a functional TTSS is required for this anti-inflammatory property; complementation of escN restored the attenuated response. Mutation of espB also enhanced the IL-8 response, and complementation returned IL-8 to near WT levels, suggesting involvement of this effector. The anti-inflammatory effect extends to both bacterial and host-derived proinflammatory stimuli, since prior infection with EPEC suppressed the IL-8 response to tumor necrosis factor-alpha, IL-1beta, and enterohemorrhagic E. coli flagellin. These findings indicate that EPEC-induced inflammation is a balance between pro- and anti-inflammatory proteins; extracellular factors, including flagellin and an unidentified TTSS-independent, >50-kDa protein, trigger inflammation while intracellular TTSS-dependent factors, including EspB, attenuate this response.

Shiga toxin (Stx) is implicated in the development of hemorrhagic colitis and hemolytic-uremic syndrome, but early symptoms of enterohemorrhagic Escherichia coli (EHEC) infection such as non-bloody diarrhea may be Stx-independent. In this study, we defined the effects of EHEC, in the absence of Stx, on the intestinal epithelium using a murine model. EHEC colonization of intestines from two groups of antibiotic-free and streptomycin-treated C57Bl/6J mice were characterized and compared. EHEC colonized the cecum and colon more efficiently than the ileum in both groups; however, greater amounts of tissue-associated EHEC were detected in streptomycin-pretreated mice. Imaging of intestinal tissues of mice infected with bioluminescent EHEC further confirmed tight association of the bacteria to the cecum and colon. Greater numbers of EHEC were also cultured from stool of streptomycin-pretreated mice, as compared to those that received no antibiotic. Transmission electron microscopy demonstrated that EHEC infection leads to microvillous effacement of mouse colonocytes. Hematoxylin and eosin staining of colonic tissues of infected mice revealed a slight increase in the number of lamina propria polymorphonuclear leukocytes. Transmucosal electrical resistance, a measure of epithelial barrier function, was reduced in colonic tissues of infected animals. Increased mucosal permeability to 4KDa FITC-Dextran was also observed in colonic tissues of infected mice. Immunofluorescence microscopy revealed that EHEC infection resulted in redistribution of the tight junction proteins occludin and claudin-3 and increased expression of claudin-2 while ZO-1 localization remained unaltered. Quantitative real-time PCR revealed that EHEC altered mRNA transcription of Ocln, Cldn2 and Cldn3. Most notably, claudin-2 expression was significantly increased and correlated with increased intestinal permeability. Our data indicate that C57Bl/6J mice serve as an in vivo model to study the physiological effects of EHEC infection on the intestinal epithelium and suggest that altered transcription of tight junction proteins plays a role in the increase in intestinal permeability.

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