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

描述(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在工作中获得的知识，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艰难梭菌毒素介导的肿瘤坏死因子产生的信号通路，并通过靶向阻断肿瘤坏死因子的产生来开发一种新的治疗CDI的方法。将追求三个具体目标。具体目标1：基于我们的假设，即肠道树突状细胞(DC)和巨噬细胞是细菌感染过程中主要产生肿瘤坏死因子的细胞，在体内识别免疫细胞亚群，这些细胞是艰难梭菌毒素反应中主要产生肿瘤坏死因子的细胞。特定目标2：研究导致巨噬细胞产生毒素介导的肿瘤坏死因子的信号事件。具体目标3：评价阻断肿瘤坏死因子的产生作为一种辅助治疗在初次和复发CDI中对抗肠道炎症的作用。通过免疫荧光染色和免疫组织化学方法，在小鼠回肠回肠弯曲模型和艰难梭菌感染小鼠模型中，对特定的AIM 1，产生肿瘤坏死因子的免疫细胞进行鉴定。为了更准确地评估DC和巨噬细胞在产生肿瘤坏死因子中的作用，将使用DC或巨噬细胞耗尽的小鼠。针对特定的目的2，将采用多种方法，包括siRNA敲除、Western-印迹分析、RT-PCR来确定小Rho GTP酶和双特异性磷酸酶(DUSP)在艰难梭菌毒素诱导的肿瘤坏死因子-β的产生中的作用。在具体目标3中，将使用一种新的依赖葡聚糖颗粒(GP)的针对巨噬细胞和DC的siRNA递送系统来评估阻断TNF-β的产生作为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.