Septins in intestinal fibrosis

摘要 在他们的病程中，超过一半的克罗恩病（CD）患者发展成纤维化诱导的肠道炎症。 梗阻，最终需要手术。没有特定的抗纤维化治疗。尽管取得了进展 的抗炎治疗狭窄的发病率仍然很高，这表明炎症- 独立的机制在疾病的进展中至关重要。介导纤维化的主要效应细胞 是由多种促纤维化生长因子如转化生长因子激活的肌成纤维细胞 （TGF）-β 1。这种激活导致细胞外基质（ECM）的加速分泌和细胞外基质（ECM）的重塑。 肌动球蛋白细胞骨架Septins是研究不足的细胞骨架蛋白，调节分泌和肌动球蛋白- 依赖细胞功能。没有关于Septin细胞骨架在肠道中的作用和调节的数据。 存在纤维化。我们的初步数据显示septins 2，6，7，8，9，10，11在人中的高表达 肠组织中，septin 7是最主要的同种型，在CD中上调。药理学或 Septin细胞骨架的遗传破坏抑制了ECM产生（胶原蛋白）中TGF-β1依赖性的增加 I和纤连蛋白）和永生化和原代人肌/成纤维细胞中的迁移。初步证据表明 这是转录后调节的。隔蛋白调节改善实验鼠纤维化。因此，我们 我建议调查的假设，隔蛋白细胞骨架的重塑是一个驱动器的肠 纤维化和靶向隔蛋白细胞骨架是一种新的方法来治疗纤维狭窄克罗恩病 疾病这一假设将通过三个具体目标进行检验：目标1。Septin改变的表征 在IBD患者的组织样品中的表达和分布。这包括开发高分辨率的 人肠道组织和原代人肠道肌成纤维细胞中septin表达谱图， 包括产生用于狭窄CD和对照的第一个全厚度单细胞RNA测序肠道图谱。 目标2. Septin依赖性调控促纤维化肌成纤维细胞的作用及机制研究 activation.我们将检测septin的破坏或过度表达是否调节TGF-β1信号传导，细胞内囊泡的表达， 贩运或翻译组和转录后调节网络使用的功能丧失和增益- 功能方法AIM3.靶向隔蛋白改善体内肠纤维化的功能探索。我们将 使用药理学和遗传学方法在体内调节septins，并在两个细胞中诱导实验性纤维化。 不同的动物模型我们将暂时控制删除中央分隔符7之前（预防）， 在特异性地在Col I阳性细胞中诱导（逆转）实验性肠纤维化后。如果成功，这 一项提案将挑战纯粹免疫驱动的ECM沉积驱动狭窄的范式 并为预防或治疗狭窄相关的新机制提供概念验证 CD患者肠梗阻。

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.