Septins in intestinal fibrosis

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ABSTRACT Over their disease course more than half of Crohn’s disease (CD) patients develop fibrosis-induced intestinal obstruction and ultimately require surgery. No specific anti-fibrotic therapies are available. Despite advances of anti-inflammatory therapies the incidence of strictures remains high, suggesting that inflammation- independent mechanisms are crucial in the progression of the disease. The main effector cell mediating fibrosis is the myofibroblast that is activated by multiple pro-fibrotic growth factors, such as transforming growth factor (TGF)-1. Such activation results in accelerated secretion of extracellular matrix (ECM) and remodeling of the actomyosin cytoskeleton. Septins are understudied cytoskeletal proteins that regulate secretory and actomyosin- dependent cellular functions. No data on the roles and regulation of the septin cytoskeleton in intestinal fibrosis exists. Our preliminary data shows a high gene expression of septins 2, 6, 7, 8, 9, 10, 11 in human intestinal tissues with septin 7 as the most predominant isoform, which is upregulated in CD. Pharmacologic or genetic disruption of the septin cytoskeleton inhibited TGF-β1-dependent increase in ECM production (Collagen I & fibronectin) and migration in immortalized and primary human myo/fibroblasts. Preliminary evidence suggests this is post-transcriptionally regulated. Septin modulation improved experimental murine fibrosis. We hence propose to investigate the hypothesis that remodeling of the septin cytoskeleton is a driver of intestinal fibrosis and targeting the septin cytoskeleton is a novel approach to therapy of fibrostenosing Crohn’s disease. This hypothesis will be tested by three specific aims: AIM1. Characterization of alterations in septin expression and distribution in tissue samples of IBD patients. This includes development of a high-resolution map of septin expression profiles in human intestinal tissues and primary human intestinal myofibroblasts, including generation of the first full thickness single cell RNA sequencing gut atlas for stricturing CD and controls. AIM2. Investigation of the roles and mechanisms of septin dependent regulation of pro-fibrotic myofibroblast activation. We will test if septin disruption or overexpression modulates TGF-β1-signaling, intracellular vesicular trafficking or the translatome and post-transcriptionally regulated networks using a loss-of-function and gain-of- function approach. AIM3. Functional exploration if targeting septins ameliorates intestinal fibrosis in vivo. We will modulate septins in vivo using a pharmacologic and genetic approach and induce experimental fibrosis in two different animal models. We will temporally control the deletion of the central septin 7 prior to (prevention) and after induction (reversal) of experimental intestinal fibrosis specifically in Col I positive cells. If successful, this proposal will challenge the paradigm of purely immune-driven ECM deposition driving stricture formation and provide proof-of-concept for a novel mechanism to prevent or treat stricture associated intestinal obstruction in CD patients.