Kyoko Katakura

Maintenance of colonic homeostasis by distinctive apical TLR9 signalling in intestinal epithelial cells

The mechanisms by which commensal bacteria suppress inflammatory signalling in the gut are still unclear. Here, we present a cellular mechanism whereby the polarity of intestinal epithelial cells (IECs) has a major role in colonic homeostasis. TLR9 activation through apical and basolateral surface domains have distinct transcriptional responses, evident by NF-κB activation and cDNA microarray analysis. Whereas basolateral TLR9 signals IκBα degradation and activation of the NF-κB pathway, apical TLR9 stimulation invokes a unique response in which ubiquitinated IκB accumulates in the cytoplasm preventing NF-κB activation. Furthermore, apical TLR9 stimulation confers intracellular tolerance to subsequent TLR challenges. IECs in TLR9-deficient mice, when compared with wild-type and TLR2-deficient mice, display a lower NF-κB activation threshold and these mice are highly susceptible to experimental colitis. Our data provide a case for organ-specific innate immunity in which TLR expression in polarized IECs has uniquely evolved to maintain colonic homeostasis and regulate tolerance and inflammation.

Toll-like receptor 9–induced type I IFN protects mice from experimental colitis

Experimental colitis is mediated by inflammatory or dysregulated immune responses to microbial factors of the gastrointestinal tract. In this study we observed that administration of Toll-like receptor 9 (TLR9) agonists suppressed the severity of experimental colitis in RAG1-/- but not in SCID mice. This differential responsiveness between phenotypically similar but genetically distinct animals was related to a partial blockade in TLR9 signaling and defective production of type I IFN (i.e., IFN-alpha/beta) in SCID mice upon TLR9 stimulation. The addition of neutralization antibodies against type I IFN abolished the antiinflammatory effects induced by TLR9 agonists, whereas the administration of recombinant IFN-beta mimicked the antiinflammatory effects induced by TLR9 agonists in this model. Furthermore, mice deficient in the IFN-alpha/beta receptor exhibited more severe colitis than wild-type mice did upon induction of experimental colitis. These results indicate that TLR9-triggered type I IFN has antiinflammatory functions in colitis. They also underscore the important protective role of type I IFN in intestinal homeostasis and suggest that strategies to modulate innate immunity may be of therapeutic value for the treatment of intestinal inflammatory conditions.

TLR4 signaling in effector CD4+ T cells regulates TCR activation and experimental colitis in mice

TLRs sense various microbial products. Their function has been best characterized in DCs and macrophages, where they act as important mediators of innate immunity. TLR4 is also expressed on CD4+ T cells, but its physiological function on these cells remains unknown. Here, we have shown that TLR4 triggering on CD4+ T cells affects their phenotype and their ability to provoke intestinal inflammation. In a model of spontaneous colitis, Il10-/-Tlr4-/- mice displayed accelerated development of disease, with signs of overt colitis as early as 8 weeks of age, when compared with Il10-/- and Il10-/-Tlr9-/- mice, which did not develop colitis by 8 months. Similar results were obtained in a second model of colitis in which transfer of naive Il10-/-Tlr4-/- CD4+ T cells into Rag1-/- recipients sufficient for both IL-10 and TLR4 induced more aggressive colitis than the transfer of naive Il10-/- CD4+ T cells. Mechanistically, LPS stimulation of TLR4-bearing CD4+ T cells inhibited ERK1/2 activation upon subsequent TCR stimulation via the induction of MAPK phosphatase 3 (MKP-3). Our data therefore reveal a tonic inhibitory role for TLR4 signaling on subsequent TCR-dependent CD4+ T cell responses.

S1707 Conventional Dendritic Cells Regulate the Outcome of Colonic Inflammation Independently of T Cells

We explored the physiological role of conventional dendritic cells (cDCs) in acute colitis induced by a single cycle of dextran sodium sulfate administration. Depending on their mode of activation and independently of T cells, cDCs can enhance or attenuate the severity of dextran sodium sulfate-induced colitis. The latter beneficial effect was achieved, in part, by IFN-1 induced by Toll-like receptor 9-activated cDCs. IFN-1 inhibits colonic inflammation by regulating neutrophil and monocyte trafficking to the inflamed colon and restraining the inflammatory products of tissue macrophages. These data highlight a novel role of cDCs in the regulation of other innate immune cells and position them as major players in acute colonic inflammation.

S1731 Tolerance of Toll-Like Receptor 4 Mediated Signaling Pathway Protects Mice from Experimental Colitis

Background:The basis of inflammatory bowel disease (IBD) is multi-factorial involving susceptibility genes, and immune and environmental factors. There has been a rapid increase in the prevalence of IBD in industrialized countries. The hygiene hypothesis proposes that the present clean surroundings in less exposure to bacteria are involved in development of this disease. Toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS), has a phenomenon termed LPS tolerance such as an initial exposure to LPS induce a transient state of hyporesponsiveness to a subsequent challenge with LPS. Based on In Vitro studies, the mechanisms of this phenomenon are gradually becoming clear, although that has not been formally proven in inflammatory disease models In Vivo. Therefore we hypothesized that repeated LPS administration could protect colonic inflammation of mouse experimental colitis model. Methods:Murine colitis was induced to Balb/c mice by oral administration of 7% dextran sulphate sodium (DSS) with or without daily intraperitoneal administration of LPS. Colitis was evaluated by a macroscopic disease activity index, myeloperoxidase (MPO) activity in colon and histological score. Cytokine mRNA expressions in colon were also measured by RT-PCR. To confirm the phenomenon of LPS tolerance, we generated mouse conventional bone marrow derived dendritic cells (BMDC), and these were preincubated with or without LPS and restimulated with LPS after 24 hours from first stimulation. Cytokine productions in the culture supernatant were measured by ELISA, and mRNA expressions of the cells were evaluated by RT-PCR. Furthermore, we investigated negative regulators of LPS tolerance and expression of them in model of experimental colitis. Results:Administration of LPS significantly suppressed colonic inflammation of DSS-induced colitis, although there were no difference in mRNA expression of IL-12p40, IL-6, TNF-α, IL-10, and TLR4. TLR4 in BMDC was activated with LPS and induced TNF-α production. After second stimulation with LPS, TNF-α and IL-6 production were reduced which followed previous reports. IRAKM mRNA expression, which had participation in LPS tolerance, was upregulated in the LPS treated BMDC and mice colon in comparison to the control mice. Conclusion:LPS tolerance could protect mice from DSS-induced colitis, and IRAK-M had participation in LPS tolerance of TLR4 signaling pathway. Taken together, these observations suggested that loss of exposure to LPS may be one of the pathogenesis of IBD.