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K 01申请。我的长期职业目标是利用我的工作和其他人获得的知识，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C.艰难梭菌毒素介导的TNF-α产生，并通过靶向阻断TNF-α产生来开发针对CDI的新疗法。将追求三个具体目标。具体目标1：识别免疫细胞亚群，它们是响应C.艰难梭菌毒素在体内，基于我们的假设，肠树突状细胞（DC）和巨噬细胞是主要的TNF-α生产者在细菌感染。具体目标2：研究导致巨噬细胞中毒素介导的TNF-α产生的信号传导事件。具体目标3：评价阻断TNF-α产生作为原发性和复发性CDI中肠道炎症的一种预防性治疗。对于特异性aim 1，将通过免疫荧光染色和免疫组织化学在小鼠回肠袢模型和C.小鼠艰难梭菌感染。为了更精确地评估DC和巨噬细胞在TNF-α产生中的作用，将使用DC或巨噬细胞耗尽的小鼠。针对具体目标2，将采用多种方法，包括siRNA敲除、Western-blot分析、RT-PCR来鉴定小Rho GTP酶和双特异性磷酸酶（DUSPs）在C.艰难梭菌毒素诱导的TNF-α产生。在具体目标3中，将采用特异性靶向巨噬细胞和DC的新型葡聚糖颗粒（GP）依赖性siRNA递送系统来评估阻断TNF-α产生作为针对CDI中肠道炎症的预防性疗法。

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.