Signaling pathway of TNF-alpha production and Clostridium difficile infection (CD

描述（由申请人提供）：这是NIDDK在艰难梭菌感染（CDI）领域的K01申请。我的长期职业目标是利用我和其他人通过工作获得的知识来制定预防和治疗CDI和其他肠道病原体相关疾病的措施。本研究的目的是阐明艰难梭菌毒素介导的TNF-¿产生的信号通路，并通过靶向阻断TNF-¿的产生来开发一种新的治疗CDI的方法。将实现三个具体目标。特异性目的1：根据我们的假设，肠道树突状细胞（dc）和巨噬细胞是细菌感染期间主要的TNF-¿产生者，确定体内对艰难梭菌毒素反应中主要产生TNF-¿的免疫细胞亚群。特异性目的2：研究导致巨噬细胞毒素介导的TNF-产生的信号事件。特异性目标3：评估阻断TNF-产生作为原发性和复发性CDI肠道炎症的辅助治疗。在小鼠回肠环模型和小鼠艰难梭菌感染中，通过免疫荧光染色和免疫组织化学鉴定产生TNF-的免疫细胞。为了更精确地评估DC和巨噬细胞在TNF-产生中的作用，将使用DC或巨噬细胞耗尽的小鼠。对于特定目的2，将采用多种方法，包括siRNA敲除，Western-blot分析，RT-PCR来确定小Rho GTPases和双特异性磷酸酶（DUSPs）在艰难梭菌毒素诱导的TNF-¿产生中的作用。在特定目标3中，一种新的葡聚糖颗粒（GP）依赖性siRNA递送系统将被用于评估阻断TNF-产生作为CDI肠道炎症的辅助治疗。

DESCRIPTION (provided by applicant): This is a K01 application from NIDDK in the field of Clostridium difficile infection (CDI). My long- term career objective is to utilize knowledge acquired through my work and others to develop measures of prevention and therapy against CDI and other enteric pathogens-associated diseases. The object of this study is to elucidate the signaling pathway of C. difficile toxin- mediated TNF-¿ production and develop a novel therapy against CDI by targeted blocking TNF- ¿ production. Three specific aims will be pursued. Specific Aim 1: Identify subsets of immune cells that are major producer of TNF-¿ in response to C. difficile toxins in vivo, based on our hypothesis that intestinal dendritic cells (DCs) and macrophages are major TNF-¿ producer during the bacterial infection. Specific Aim 2: Investigate the signaling events that lead to toxin-mediated TNF-¿ production in macrophages. Specific Aim 3: Evaluate blocking TNF-¿ production as an adjunctive therapy against intestinal inflammation in both primary and recurrent CDI. For specific aim1, TNF-¿ producing immune cells will be identified by immuofluorescence staining and immunohistochemistry in mouse ileal loop model and C. difficile infection in mice. To more precisely assess the roles of DCs and macrophages in TNF-¿ production, DC- or macrophage- depleted mice will be used. For specific aim 2, multiple approaches including siRNA knockdown, Western-blot analysis, RT-PCR will be performed to identify the involvement of small Rho GTPases and dual specificity phosphatases (DUSPs) in C. difficile toxin-induced TNF-¿ production. In specific aim 3, a novel glucan particle (GP)- dependent siRNA delivery system specifically targeting macrophages and DCs will be employed to evaluate blocking TNF-¿ production as an adjunctive therapy against intestinal inflammation in CDI.