Role of Innate Immunity and Stat3 Activation in Colitis Mouse Model

描述(由申请人提供)： 炎症性肠病(IBD)，即克罗恩病或溃疡性结肠炎，据估计影响着140万美国人，表现为胃肠道的慢性炎症，发病率很高，有时还会出现危及生命的并发症。IBD被认为是由于遗传易感个体对结肠菌群调节失调的粘膜免疫(先天和适应性)反应所致。脆弱类杆菌是一种常见的寄生于结肠的人类共生菌。脆弱芽孢杆菌的一个亚群，产肠毒素脆弱杆菌(ETBF)，与炎症性腹泻疾病、IBD和结直肠癌有关。我们的ETBF小鼠模型数据显示，ETBF的定植导致了促炎反应，在C57BL/6小鼠中表现出由信号转导和转录激活因子3(STAT3)促进的以Th17(白介素17分泌)CD4+T细胞为主的浸润。在口服接种ETBF后的几个小时内，激活的STAT3和结肠炎的存在是明显的。然而，到目前为止，还没有关于先天性免疫系统在ETBF结肠炎中的作用的研究。最近在一种不同的感染模型中证实了IL-17的偏斜，该模型涉及核苷酸结合的寡聚化结构域包含(NOD)天然免疫途径的下游产物。NOD2基因突变与克罗恩病有很强的相关性。因此，这一数据支持先天免疫因子和信号转导与转录激活因子3(STAT3)在ETBF小鼠结肠炎模型中发挥重要作用，为深入了解炎症性肠病的发病机制提供了依据。本研究的目的是探讨先天性免疫信号通路，特别是NOD/RIP2和Toll样受体(TLR)/MyD88在ETBF诱导的结肠炎模型中的作用。我们将检验这样的假设，即MyD88-/-和RIP2-/-小鼠将表现出ETBF诱导的结肠炎和STAT3激活的增强。为此，提出了以下特定目标：特定目的1：利用不同的转基因小鼠评估NOD/RIP2和TLR/MyD88先天免疫通路在ETBF诱导的结肠炎反应中的作用；特定目的2：确定STAT3激活在ETBF诱导的NOD/RIP2和TLR/MyD88缺陷小鼠中的作用；特定目标3：通过建立和利用骨髓来源的嵌合移植，确定TLR/MyD88和NOD/RIP2缺陷在造血和非造血细胞来源的细胞中对STAT3激活的作用和ETBF诱导的结肠炎的发病机制。该项目将使用ETBF诱导的结肠炎小鼠(C57BL/6)模型以及各种免疫学分析、基因敲除小鼠、骨髓移植、免疫组织化学、流式细胞术、细胞因子和RNA分析。这项研究的意义在于，它将使人们能够深入了解与IBD相关的结肠细菌的失调免疫反应，这是一种发病率很高的疾病，并希望对这种流行疾病有新的治疗见解。 公共卫生相关性： 本研究探讨先天免疫系统，特别是TLR和NOD受体信号通路，以及信号转导和转录激活因子3(STAT3)在细菌诱导的结肠炎小鼠模型中的作用。从这项研究中获得的信息将使人们能够深入了解与IBD相关的免疫反应失调，IBD是一种发病率很高的疾病。

DESCRIPTION (provided by applicant): Inflammatory bowel disease (IBD), Crohn's disease or ulcerative colitis, affects an estimated 1.4 million Americans and is manifested by chronic inflammation of the gastrointestinal tract with significant morbidity and, at times, life-threatening complications. IBD is proposed to result from a dysregulated mucosal immune (innate and adaptive) response to the colonic flora in genetically susceptible individuals. Bacteroides fragilis is a common human commensal residing in the colon. A subset of B. fragilis, enterotoxigenic B. fragilis (ETBF), has been associated with inflammatory diarrheal disease, IBD, and colorectal cancer. Our ETBF murine model data shows that colonization with ETBF leads to a proinflammatory response exhibiting a predominant Th17 (interleukin 17-secreting) CD4+ T cell infiltrate promoted by signal transducer and activator of transcription-3 (Stat3) in C57BL/6 mice. The presence of activated Stat3 as well as colitis is evident within hours of orogastric inoculation with ETBF. However, there have been no investigations to date regarding the role of the innate immune system in ETBF colitis. IL-17 skewing has been recently demonstrated in a different infectious model involving downstream products of the Nucleotide-binding oligomerization domain containing (NOD) innate immune pathway. There is a strong association of NOD2 gene mutations and Crohn's disease. Hence, this data supports that innate immune factors and signal transducer and activator of transcription 3 (Stat3) plays a major role in the ETBF mouse model of colitis providing insight into the genesis of Inflammatory Bowel Disease. The objective of this research project is to address the role of the innate immune signaling pathways, in particular the NOD/Rip2 and toll like receptor (TLR)/MyD88, in the ETBF-induced colitis model. We will test the hypothesis that MyD88-/- and Rip2-/- mice will exhibit enhanced ETBF-induced colitis and Stat3 activation. To that end, the following Specific Aims are proposed: Specific Aim 1: To evaluate the role of NOD/Rip2 and TLR/MyD88 innate immune pathways in response of ETBF induced colitis using various genetically manipulated mice; Specific Aim 2: To define the role of Stat3 activation in the pathogenesis of ETBF-induced colitis in the NOD/Rip2- and TLR/MyD88- deficient mice; Specific Aim 3: To determine the role of TLR/MyD88 and NOD/Rip2 deficiency in hematopoietic versus non-hematopoietic derived cells on Stat3 activation and the pathogenesis of ETBF-induced colitis by creating and utilizing chimeric mice derived via bone marrow (BM) transplantation. The project will use the murine (C57BL/6) model of ETBF-induced colitis and various immunologic assays, knock-out mice, bone marrow transplantation, immunohistochemistry, flow cytometery, cytokine and RNA analysis. The significance of this research is that it will allow insight into the dysregulated immune response to colonic bacteria associated with IBD, a disorder with substantial morbidity, with hopes for new therapeutic insights to this prevalent disease. PUBLIC HEALTH RELEVANCE: This study examines the role of innate immune system, specifically TLR and NOD receptor signaling pathways, and the role of signal transducer and activator of transcription 3 (Stat3) in a bacteria induced colitis mouse model. Information obtained from this study will allow insight into the dysregulated immune response associated with IBD, a disorder with substantial morbidity.