REGULATION OF NICHE CELL DIFFERENTIATION TO SUSTAIN INTESTINAL STEM CELL REGENERATION AGAINST GUT INFLAMMATION

项目总结：肠道艰难梭菌感染（CDI）在炎症性疾病患者中增加 在过去的十年中，肠道疾病（IBD），导致了重大的医疗保健负担。治疗方法 然而，由于目前缺乏对基本细胞的了解， 参与CDI的类型和关键信号蛋白，加剧IBD。据信，沿着与龛细胞， Lgr5hi和Lgr5low肠上皮干细胞（IESC）控制肠上皮（IEC）谱系修复 急性粘膜损伤后。此外，肠潘氏细胞（PC）或结肠深隐窝的形成 分泌细胞（DCS）小生境细胞通过相反的Wnt和Notch信号传导而被差异调节。诱导 产生这些小生境细胞的IESC可以减轻IBD背景下的CDI或促进IESC修复IEC 在IBD。然而，如何维持IESC以指定PC和DSC，进而维持IESC再生， 仍不清楚我们报道了酪氨酸磷酸化（pYSTAT 5）的激活促进IESC 急性回肠炎和结肠炎后的再生。重要的是，JAK2或JAK3基因中的单核苷酸多态性（SNP）是一个重要的基因。 STAT5A与IEC愈合缺陷、CDI和IBD并发症有关。JAK2 SNP载体是 与儿童克罗恩病患者的CDI发生率高两倍相关，伴回肠-结肠同步定位 （回肠结肠炎）。然而，IBD中JAK2或STAT5A SNP是否对确定IBD的易感性至关重要， 共病CDI未知。我们目前的数据表明，单个STAT5A SNP会降低干细胞存活率。STAT5 与对照组相比，消耗导致严重的伪膜性结肠炎;相反，组成性活性 STAT5A（Ca-pYSTAT5）重新激活Lgr5low IESC向PC并增加分泌型小生境的产生 因素因此，我们假设Ca-pYSTAT5调节IESC产生PC和DCS小生境细胞， 这反过来又维持IESC再生，JAK2-STAT 5通路中的SNP损害Ca-pYSTAT 5， 导致IBD和CDI并存我们将通过解决以下具体目标来检验这一假设。在 目的1，我们将研究JAK2-STAT5基因缺陷对IBD和CDI合并症发生率、IESC 再生和小生境细胞分化。我们将检测JAK2或STAT5A SNP对新生IESC的影响， 小生境细胞分化这些研究将确定为什么CDI经常发生在IBD中，并将建议 潜在的治疗靶点，以降低IBD的CDI患病率。在目标2中，我们将定义 Ca-pYSTAT5通过其调控IESC小生境细胞分化。我们将确定减少Stat5的影响 表达、pYSTAT5水平和小鼠中STAT5A SNP的敲入对结肠炎对CDI的易感性的影响。 这些研究将测试有缺陷的JAK2-pYSTAT5和IBD与CDI共病之间的联系。总体而言，我们 一项提案将确定IESC诱导小生境细胞的肠道谱系限制性转录因子， 反过来保护IESC再生免受IBD与CDI共病的影响。我们的研究将提供关键的见解， JAK2和STAT5A风险多态性在IBD和CDI背景下的机制。

Project Summary: Intestinal Clostridium difficile infection (CDI) has increased in patients with inflammatory bowel disease (IBD) over the past decade, resulting in a major healthcare burden. Therapeutic approaches for this comorbidity are extremely limited, however, due to the current lack of understanding of the essential cell types and key signaling proteins involved in CDI that exacerbate IBD. It is believed that along with niche cells, Lgr5hi and Lgr5low intestinal epithelial stem cells (IESCs) control intestinal epithelial (IEC) lineage repair following acute mucosal injuries. Further, the formation of intestinal Paneth cell (PC) or colonic deep crypt secretory cell (DCS) niche cells are differentially regulated by opposing Wnt and Notch signaling. Inducing IESCs to give rise to these niche cells could mitigate CDI in the setting of IBD or promote IESC repair of IECs in IBD. However, how IESCs are sustained to specify PCs and DSCs that in turn sustain IESC regeneration remains unclear. We reported that the activation of Tyrosine phosphorylated (pYSTAT5) promoted IESC regeneration following acute ileitis and colitis. Importantly, single nucleotide polymorphisms (SNPs) in JAK2 or STAT5A have been linked to IEC healing defects, CDI, and IBD complications. JAK2 SNP carriage is associated with two-fold higher rates of CDI in pediatric Crohn’s disease with synchronous ileal-colonic location (ileocolitis). However, whether JAK2 or STAT5A SNPs in IBD are critical to determine the susceptibility to comorbid CDI is not known. Our current data show that a single STAT5A SNP reduces stem cell survival. Stat5 depletion leads to a severe pseudomembranous colitis compared to controls; in contrast, constitutively active STAT5A (Ca-pYSTAT5) reactivates Lgr5low IESCs toward PCs and increases production of secreted niche factors. Therefore, we hypothesize that Ca-pYSTAT5 regulates IESCs to give rise to PC and DCS niche cells that in turn sustain IESC regeneration and that the SNPs in the JAK2-STAT5 pathway impair Ca-pYSTAT5, leading to IBD with comorbid CDI. We will test this hypothesis by addressing the following Specific Aims. In Aim 1, we will examine the effects of genetic defects in JAK2-STAT5 on IBD and CDI comorbidity rates, IESC regeneration, and niche cell differentiation. We will test the effects of JAK2 or STAT5A SNP on de novo IESC niche cell differentiation. These studies will determine why CDI frequently occurs in IBD and will suggest a potential therapeutic target to reduce the prevalence of CDI with IBD. In Aim 2, we will define the mechanisms by which Ca-pYSTAT5 regulates IESC niche cell differentiation. We will determine the effects of reduced Stat5 expression, pYSTAT5 level and knock-in of the STAT5A SNP in mice on the susceptibility of colitis to CDI. These studies will test the link between defective JAK2-pYSTAT5 and IBD comorbid with CDI. Collectively, our proposal will identify an intestinal lineage-restricted transcription factor for IESCs to induce niche cells, which in turn protect IESC regeneration against IBD with comorbid CDI. Our studies will provide critical insights into mechanisms of JAK2 and STAT5A risk polymorphisms in the context of IBD and CDI.