Increased Expression of Autotaxin / Lysophospholipase D on Intestinal Vessels Involves in Aggravation of Intestinal Damage Through Lymphocytes Migration

Abstract

Background and Aims: Clostridium difficile infection is the major cause of nosocomial antibiotic-associated colitis. C. difficile produces toxin A, a proinflammatory enterotoxin involved in the pathophysiology of C. difficile infection. Cathelicidin is an endogenous antimicrobial peptide that protects the host from infections. Recent findings also support the ability of cathelicidin to modulate innate immunity and alter the development of intestinal inflammation. Here we explored whether toxin Amodifies the expression of cathelicidin inmouse ileum and cultured human colonocytes. We also determined the anti-inflammatory mechanism of cathelicidin in toxin A-mediated intestinal inflammation using a mouse model of enteritis and human primary peripheral blood monocytes. Methods: Ileal loops were prepared in anesthetized mice and injected with either toxin A (10 microg/20g mouse) or saline (control) (n= 6 loops per group). A separate group was treated with cathelicidin (200 microg/mouse) administered into the ileal loops. After 4 hrs, levels of cathelicidin and TNFalpha and mucosal histological changes of ileal loops were evaluated. Results: Toxin A significantly increased cathelicidin mRNA and protein expression (up to 5 fold, P=0.005) in mouse ileum. In human colonic epithelial NCM460 cells, toxin A induced cathelicidin protein (by ~2 fold, P 50%, P 50%, P 80%, P 50%, P<0.001) from human primary peripheral blood monocytes and mouse RAW 264.3 macrophages. This anti-inflammatory pathway was NFkappaB dependent since cathelicidin-induced inhibition of TNFalpha expression was augmented by the NF-kappaB inhibitor CAPE and reversed by the NF-kappaB activator PMA. In addition, cathelicidin (5 microM) reduced toxin A mediated NF-kappaB p65 phosphorylation in human monocytes, indicating that cathelicidin exerts its anti-inflammatory effects by inhibiting NF-kappaB. Conclusion: Toxin A induces cathelicidin mRNA and protein expression inmouse intestine and only protein expression in human colonocytes. Administration of exogenous cathelicidin reduces toxin Amediated intestinal inflammation, suggesting that this approach may be beneficial in controlling C. difficile-associated diarrhea and colitis. Supported by a Pilot and Feasibility Study grant from UCLA-CURE Center, a Career Development Award from Crohn's and Colitis Foundation of America and K01DK084256 from National Institute of Health to HWK.