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P090. Direct effect of infliximab on intestinal mucosa sustains mucosal healing: Exploring new mechanisms of action

V. Petito1, L. Lopetuso1, E. Stigliano2, V. Arena2, S. Bibbo1, A. Amato3, G. Cammarota1, A. Papa1,4, A. Sgambato2, A. Gasbarrini1, F. Scaldaferri1, 1Catholic University of Sacred Heart, Internal Medicine and Gastroenterology, Rome, Italy, 2Catholic University of Sacred Heart, Pathology, Rome, Italy, 3Catholic University of Sacred Heart, Anesthesiology, Rome, Italy, 4Catholic Universisty, Internal Medicine and Gastroenterology Unit, Complesso Integrato Columbus, Rome, Italy

Background

Infliximab (IFX) has been shown to induce measurable changes in peripheral blood and within intestinal mucosa, through several mechanisms of actions involving immune and non-immune cells, leading to mucosal healing in IBD patients. Despite a clear clinical efficacy, it is still not clear whether IFX really acts directly on intestinal mucosa and how it influences intestinal mucosa healing process.

Aim of this study was to assess whether IFX acts directly on intestinal mucosa and how it contributes to mucosal healing.

Methods

The direct effect of IFX was assessed on human colonic mucosa biopsies, taken from IFX naïve IBD patients in active state of disease. Biopsies were washed, weighted and incubated for 12 hours in 24-well plate, with or without IFX (50 microg/ml). Supernatants were assessed for multiplex cytokines content (Bio-Plex®, BIO-RAD) and cultured biopsies were included for histological evaluation and then stained for CD68, CD3, E-cadherin and phospho-ERK. Apoptosis was evaluated by the terminal deoxynucleotidyl transferase (dUTP) nick end labeling assay (TUNEL). To explore wound/healing process and epithelial cell migration, a scratch assay was performed using Caco2 cells. Briefly, a scratch on a confluent monolayer of CaCo2 cells was applied and migration of cells was measured at regular intervals, in presence of IFX (50 microg/ml) and/or TNF-a (25 ng/ml). CaCo2 cells were also exposed for 24 hours to supernatants obtained from peripheral blood mononuclear cell (PBMC) or human intestinal fibroblast (HIFs) treated with TNF-a (25 ng/ml) and IFX (100 µg/ml) alone or in association for 24 hours.

Results

IFX-treated biopsies displayed reduced inflammatory infiltrate, reduced counts of CD68 and CD3 positive cells, with an increased apoptosis, together with a higher expression of E-cadherin and phospho-ERK expression on enterocytes. IFX exposed biopsies showed a lower content in their supernatants of TNF-a, IL-17, IL-6 and IL-8, with a higher content in INF-g, FGF and IL-1 beta. Biopsies from active IBD patients exposed to IFX, furthermore, expressed a well preserved histology appearance compared to untreated biopsies. At scratch assay, IFX ameliorated wound healing in Caco2 cells alone or exposed to PBMC and HIF supernatants, even in presence of TNF-a.

Conclusion

IFX directly acts on intestinal mucosa, by lowering inflammatory infiltrate, reducing inflammatory cytokines and increasing regenerative cytokines, by preserving enterocytes vitality and by sustaining mucosal healing.