Charkes Parkos

IL-36g Promotes Intestinal Inflammation Via Intestinal Macrophage/DC Activation: P-199

vehicle from day 0 to 6. Mice were sacrificed on day 7 and colon tissue sections were analyzed for colitis severity, disease activity index (DAI), Ki-67 staining and for myeloperoxidase (MPO) activity. Levels of SP-333 in blood plasma were analyzed by LC-MS/MS method (LLOQ: 1 ng/mL). Colon explant cultures were used to assess production of inflammatory cytokines. Effects of SP-333 on activation of NF-kappa B and on production of cytokines were also measured in T84 colon carcinoma cells. RESULTS: Treatment with SP-333 (0.05 mg/kg) was as effective as 5-ASA (100 mg/ kg) in ameliorating colitis, suppressing DAI, reducing MPO activity and normalizing Ki-67 staining. The pharmacokinetics study in DSS-colitic mice indicated that systemic absorption of orally administered SP-333 was minimal. In T84 cells, treatment with SP-333 reduced levels of p-NF-kappaB-p65 in the nuclear fraction and increased levels of cytosolic I-kappa B without affecting I-kappa kinase (IKK). Treatment with 8-Br-cGMP, a positive control, exhibited similar inhibition in NFkappa B activation. SP-333 also suppressed LPS-stimulated production of IL-8, IL17a, IL-23 and TNF-alpha in T-84 cells. CONCLUSION(S): Oral treatment with SP-333 ameliorated colitis in mice possibly via activation of GC-C signaling to inhibit NF-kappa B activation and to suppress production of pro-inflammatory cytokines (Fig 1). This is the first ever study suggesting that the anti-inflammatory effect of GC-C agonists might be through inhibition of NF-kappa B activation and it opens a novel avenue for development of GC-C agonists as a new class of orally delivered, mucosally active, drug candidates for treatment of IBD. SP-333 is entering clinical development for the treatment of mild to moderate ulcerative colitis. 1. Shailubhai K, et al., Cancer Res. 60: 5151-5157, 2000. 2. Shailubhai K, et al., Am J Gastroenterol.106(S1):S455, 2011

Claudin-7 in Colonic Intestinal Epithelial Cell Differentiation and IBD: P-211 YI

was induced by exposure to IFNc (100U/mL, 24h) and IFNcþTNFa (500ng each, 24h, ip) respectively, and analyzed by flow cytometry and immunofluorescence. The effects of neutrophil-IEC interactions, and specifically of ICAM-1 engagement by antibody crosslinking on the regulation of epithelial barrier function was examined in T84 IECs by measuring changes in transepithelial resistance (TER) and permeability to 4kDa FITC-Dextran, and by using an intestinal loop model from anesthetized mice, measuring absorption of FITC-dextran from the intestinal lumen into the circulation. RESULTS: We confirmed upregulation of ICAM-1 expression in IFNc treated T84 cells, in the intestinal epithelium of patients with IBD, and further showed induction in ICAM-1 expression in-vivo in murine small intestine pre-treated with IFNc and TNFa. In cultured IECs, expression of ICAM-1 enhanced neutrophil adhesion to the apical epithelial membrane. The majority ( 60%) of apically adhered neutrophil that completed TEM were found to exhibit luminal crawling behavior, which was significantly enhanced (>33%) when T84 cells were pre-exposed to IFNc. Neutrophil interactions with apical epithelial membrane of IFNc pretreated IECs also resulted in enhanced neutrophil survival as evident by decreased apoptosis. The effects on neutrophil crawling and apoptosis were ICAM-1-dependent and were reversed by treatment with an anti-ICAM-1 function-blocking antibody. Furthermore, neutrophil interactions with the apical epithelial membrane resulted in time-dependent decrease in TER. This decrease was prevented by inhibition of neutrophil adhesion and crawling using an anti-CD11b/CD18 function-blocking antibody, suggesting that neutrophil induced effects on barrier function were due to direct engagement of ligands on epithelial surface. Experiments modeling neutrophil apical engagement using antibody-mediated crosslinking of cell surface ICAM-1 in cultured IECs and in-vivo, using murine intestinal loop model confirmed a direct role for ICAM-1 engagement in regulating epithelial barrier function. Antibody crosslinking of ICAM-1 resulted in time-dependent decrease in TER (up to 30%) and increased flux of FITC-dextran ( 2.5-fold at 4 hours) in-vitro and increased flux of FITC-dextran ( 1.6-fold) in-vivo. ICAM-1 mediated changes in barrier function were accompanied by actin rearrangement and dependent on MLCK. CONCLUSION(S): These studies suggest that neutrophil engagement of apically expressed ICAM-1during IBD results in signaling events that can inhibit neutrophil clearance and alter barrier. Such interactions may contribute to further recruitment of inflammatory cells and impede resolution of inflammation. Thus, targeting ICAM-1 in the intestinal epithelium may provide new/improved therapeutic approaches for treating IBD.

The Fucosyltransferase 3 Driven Expression of Sialyl Lewis A on the Intestinal Epithelium Regulates PMN Clearance During Inflammation: P-206 YI

IBD pathogenesis. While epidemiological evidence demonstrates vitamin D-deficiency is highly prevalent in IBD and vitamin D status is inversely associated with disease severity, the mechanistic impact(s) of vitamin D deficiency remains unclear. Vitamin D hormone activity is mediated by the vitamin D receptor (VDR). Previous studies using IL-10(-/-)/VDR(-/-) mice concluded that VDR signaling in immune cells inhibits colonic inflammation and colitis development. In addition, our investigations of VDR(-/-) mice suggested that the epithelial VDR attenuates experimental colitis by protecting the mucosal epithelial barrier and that colonic epithelial VDR levels were markedly reduced in the biopsies from patients with IBD. Aims: To identify the anti-colitic role of the colonic epithelial vs. immune VDR signaling in the development of experimental colitis. METHODS: Transgenic (Tg) mice were generated in which villin promoter was used to target Flag-tagged human (h)VDR to intestinal epithelial cells (IEC). Transgenic-VDR(-/-) (Tg-KO) mice were also produced through breeding in which only the IECs were reconstituted with hVDR in the VDR(-/-) background. Tg, Tg-KO, VDR(-/-) and wild-type (WT) mice were studied in parallel using TNBSand DSScolitis models. RESULTS: In both TNBS and DSS models, Tg mice were highly resistant to colitis induction compared to WT mice, manifested by lower clinical and histological scores and marked reduction in colonic epithelial damage and inflammatory cytokine production. Colonic epithelial permeability was well preserved in Tg mice. VDR(-/-) mice developed the most severe colitis that led to high mortality in both models, whereas Tg-KO mice were highly resistant to colitis with almost no mortality. Mechanistically, VDR overexpression attenuated TNBSor DSS-induced IEC apoptosis, with marked suppression of PUMA induction and caspase 3 activation. In vitro and in vivo data demonstrated that VDR signaling inhibited PUMA expression by blocking NF-kB activation. CONCLUSION(S): These results demonstrate that intestinal epithelial VDR signaling inhibits colonic inflammation by protecting the integrity of the mucosal epithelial barrier. The observation that the hVDR transgene rescues VDR-null mice from developing severe colitis despite a VDR-null immune system demonstrates that the anticolitic activity of the epithelial VDR is independent of the immune VDR actions.