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

项目摘要：炎症性疾病患者肠道艰难梭菌感染(CDI)增加 在过去的十年里，肠道疾病(IBD)导致了重大的医疗负担。治疗方法： 然而，由于目前对基本细胞缺乏了解，这种共病是极其有限的。 CDI中涉及的加重IBD的类型和关键信号蛋白。人们认为，与壁龛细胞一起， Lgr5hi和Lgr5低位肠上皮干细胞(IESCs)控制肠上皮(IEC)谱系修复 在急性粘膜损伤之后。此外，肠道潘氏细胞(PC)或结肠深隐窝的形成 分泌细胞(DC)的小生境细胞受相反的Wnt和Notch信号的差异调节。诱导性 产生这些小生境细胞的IESCs可以减轻IBD背景下的CDI或促进IECS的IESC修复 在IBD。然而，如何维持IESCs以指定进而支持IESC再生的PC和DSC 目前仍不清楚。我们报道了酪氨酸磷酸化(PYSTAT5)的激活促进了IESC 急性回肠炎和结肠炎后再生。重要的是，JAK2或 Stat5a与IEC愈合缺陷、CDI和IBD并发症有关。JAK2 SNP携带者是 与回肠-结肠同步定位的儿童克罗恩病的CDI发生率增加一倍相关 (回结肠炎)。然而，IBD中的JAK2或STAT5A SNPs是否对确定易感性至关重要 并存的CDI尚不清楚。我们目前的数据显示，单一的STAT5A SNP降低了干细胞的存活率。状态5 与对照组相比，耗竭导致严重的伪膜性结肠炎；相比之下，结构性活跃 Stat5a(Ca-pYSTAT5)重新激活Lgr5low IESCs对PC的作用并增加分泌的生态位的产生 各种因素。因此，我们推测，Ca-pYSTAT5调控IESCs产生PC和DCs的壁龛细胞 这反过来支持IESC的再生，并且JAK2-STAT5途径中的SNPs损害了Ca-pYSTAT5， 导致IBD合并CDI。我们将通过解决以下具体目标来检验这一假设。在……里面 目的1，我们将研究JAK2-STAT5基因缺陷对IBD和CDI共病发生率的影响 再生和小生境细胞分化。我们将测试JAK2或STAT5A SNP对从头开始IESC的影响 小龛细胞分化。这些研究将确定为什么CDI经常出现在IBD中，并将提出一种 降低CDI合并IBD患病率的潜在治疗目标。在目标2中，我们将定义机制 其中，Ca-pYSTAT5调控IESC壁龛细胞分化。我们将确定减少状态5的影响 STAT5A基因SNP在小鼠结肠炎易感性中的表达、pYSTAT5水平及敲入 这些研究将测试JAK2-pYSTAT5缺陷与合并CDI的IBD之间的联系。总的来说，我们的 该提案将确定一种肠道血统受限的转录因子，用于IESCs诱导利基细胞，这在 继而保护IESC再生免受IBD的侵袭，并伴有CDI。我们的研究将为 JAK2和STAT5A在IBD和CDI中的风险多态机制。

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.