P045 Direct effects of infliximab (IFX) on intestinal mucosa: Exploring mechanisms of mucosal healing

Abstract

BACKGROUND Mucosal healing is one of the biggest issue on the management of IBD. Although clinical use of these drugs in achieving mucosal healing is a reality, the mechanisms of it are still not clear. Aim of our study is to identify the effect of IFX on intestinal epithelial cells in order to induce mucosal healing, by exploring whether IFX is really acting at intestinal mucosal level and whether IFX acts on «wound healing» process of intestinal epithelial cells, using In Vitro and In Vivo approach. METHODS Human intestinal mucosal biopsies from active IBD patients were cultured for 48 h with or without IFX (50 ng/ml), and supernatants were assessed for cytokines content and histology performed. Effect of IFX was explored in C57BL/6 mice exposed for 7 days to 2,5 % of DSS. IFX was administered iv (5 mg/Kg) at day 5 or by daily enema (300 mg/200 ml) for 3 days starting at day 5. At day 9 mice were sacrificed and histology was taken. A scratch assay was performed on CT26 and Caco 2 cell line monolayer. Precice scratches were obtained by seading cells in micro-chamber formed by culture inserts. Cells were exposed for 24 h to IFX (50 ng/ml) and/or TNF-α (100 U/ml), or to supernatants of PBMC or fibroblast treated or not for 24 h with IFX and/ or TNFα. Seriated pictures were taken for 24 h following the stimulation and analized by computer software. RESULTS Human biopsies exposed to IFX showed a decrease in TNFa content, as well as of innate and adaptive immunity cytokines. Furthermore at histology IFX-treated biopsies showed a a decreased immune cells infiltration. In Vivo, IFX iv or given as enema ameliorated the severity of colitis in mice, by lowering the disease activity index (DAI) and the loss of body weight. At day 9 a reduction of intestinal inflammatory infiltrate was observed in treated mice. At scratch assay, TNF-a slightly decreased the healing process, while IFX alone or in combination did not. TNF-a exposed PBMC supernatants decreased epithelial healing; also TNF-a fibroblast supernatants slowed the healing process but adding IFX the healing process was restored. CONCLUSIONS Taken together our preliminary observations suggest that IFX acts locally at mucosa levels, as shown by the decrease in inflammatory infiltrate and cytokines contents in human biopsies and the effect on murine colitis when given intrarectally. The effect of IFX is only partially dependent on direct effect on epithelial cells. IFX ameliorates epithelial cell healing by blocking the detrimental capacity of TNF-a pre-treated fibroblast on intestinal epithelial cells. More data are necessary to unravel molecular pathways of these observations. Understanding mechanisms of action of IFX may clarify mechanisms of loss of responses to it as well as new targets for potential new therapies.