OP38 Modulation of the septin cytoskeleton ameliorates intestinal fibrogenesis
Naydenov, N.(1);Le, T.H.N.(1);Lechuga Villarauz, S.(1);Elias, M.(1);West, G.(1);Czarnecki, D.(1);Plesec, T.(1);Ivanov, A.(1);Rieder, F.(2)*;
(1)Cleveland Clinic, Inflammation and Immunity, Cleveland, United States;(2)Cleveland Clinic, Department of Gastroenterology- Hepatology and Nutrition, Cleveland, United States;
Background
Fibrosis-induced intestinal strictures leading to obstruction are a common and serious complication of Crohn’s disease (CD) with currently no available specific anti-fibrotic therapies. Cytoskeletal dynamics in mesenchymal cells are associated with organ fibrogenesis. Septin filaments are a novel cytoskeletal element, but the functional role of septins in regulating intestinal inflammation and fibrosis remain unknown. We examined the involvement of the septin cytoskeleton in modulating fibrogenic signaling in intestinal fibrogenesis in vitro and in vivo.
Methods
Expression of different septins in human intestinal resection tissues, primary human intestinal myofibroblasts (HIMF) and immortalized colonic myofibroblast (CCD 18co cells) were examined by real-time PCR and immunoblotting. Functional inhibition of the septin cytoskeleton was achieved using siRNA for septin 7 and the cell-permeable small molecule forchlorfenuron (FCF). Migration and proliferation of HIMF were examined by wound healing and thymidine incorporation assays, respectively. Levels of secreted cytokines were measured using cytometric bead array. Roles of septins in the development intestinal fibrosis in vivo was studied using a chronic DSS colitis model in mice with administration of FCF or vehicle.
Results
All septin genes were expressed in intestinal tissues with a predominant expression of septin 7. Pro-fibrotic transforming growth factor-β1 (TGFβ1) triggered HIMF activation manifested by the increased production of ECM proteins such as fibronectin and collagen 1 and enhanced expression of the myofibroblast markers α-smooth muscle actin, L-caldesmon and tropomyosin. Septin 7 knockdown resulted in loss of other septin proteins thereby disrupting the entire septin cytoskeleton. Either pharmacological, or genetic disruption of the septin cytoskeleton reversed the effect of TGFβ1 by attenuating production of ECM proteins and reducing expression of myofibroblast markers. Interestingly, these effects were only found on protein level and were not due to changes in gene expression, suggesting inhibition of their expression at a post-transcriptional level. Moreover, pharmacological or genetic disruptions of the septin cytoskeleton resulted in reduced migration, and attenuated cytokine secretion by activated myofibroblasts. FCF administration did not affect intestinal inflammation, but decreased the fibrosis score during chronic DSS colitis in mice.
Conclusion
Our data highlights the septin cytoskeleton as a novel regulator of intestinal myofibroblast activation and function. This for the first time implicates the cytoskeleton as an active participant in intestinal fibrosis and as a potential novel target for the development of anti-fibrotic therapies in IBD patients.