MicroRNAs in Epithelial Innate Immunity to C. parvum

描述(申请人提供)：为了更好地了解上皮细胞对病原体的先天免疫反应的分子机制，我们将研究微小隐孢子虫对胆管细胞(胆管内的上皮细胞)的先天免疫反应。微小隐孢子虫是一种NIAID B类优先病原体，可导致人类肠道和胆道疾病。利用我们建立的人胆道隐孢子虫病体外模型，我们已经证明：(I)两个Toll样受体(TLR2和TLR4)及其相关的细胞内信号通路(核因子-kappaB激活)在胆管细胞识别和诱导先天性免疫反应(如释放细胞因子/趋化因子)中起核心作用；(Ii)微小隐孢子虫感染改变 胆管细胞表达特定的内源性microRNAs(MiRNAs)，这是一类新发现的在转录后基因调控中重要的小调节RNAs；以及(Iii)TLR/NF-kappaB信号参与了微小毛滴虫诱导的胆管细胞miRNA的表达。因此，我们将检验这一中心假设，即上皮细胞对微小弧菌感染的天然免疫反应涉及TLR介导的病原体识别以及随后miRNA介导的转录后基因调控的改变。在我们的三个综合的特定目标中，我们将检验以下假设：(I)宿主细胞TLRs的微小激活改变胆管细胞内源性miRNAs的表达；(Ii)微小的宿主细胞诱导的胆管细胞miRNA的表达是通过核转录因子NF-kappaB的TLR激活而介导的；以及(Iii)c.微小的宿主细胞诱导的miRNA表达的改变影响相关的转录后基因调控，并有助于胆管细胞的先天免疫反应。该应用的创新方面包括miRNA分析的新方法，人类胆道隐孢子虫病的体外模型，以及新概念(通过转录因子调节miRNA的表达，以及在上皮先天免疫中miRNA介导的转录后基因调节)。这些研究将解决与上皮对B类病原体的先天免疫有关的一个基本问题；具体地说，miRNAs在TLR介导的胆管细胞对微小弧菌的先天免疫反应中起什么作用？这一新概念很可能与先天免疫和获得性免疫有关，我们的结果也可能为设计和实施新的微生物感染治疗策略提供合理的基础。

DESCRIPTION (provided by applicant): With a long-term goal to better understand the molecular mechanisms of epithelial innate immune responses to pathogens, we will investigate innate immunity in cholangiocytes (epithelial cells lining the biliary tract) in response to Cryptosporidium parvum, an NIAID Category B Priority Pathogen that causes both intestinal and biliary disease in humans. Using an in vitro model of human biliary cryptosporidiosis established by us, we have demonstrated that: (i) two Toll-like receptors (TLRs), TLR2 and TLR4, and an associated intracellular signaling pathway (NF-kappaB activation) play a central role in C. parvum recognition and induction of innate immune responses (e.g., release of cytokines/ chemokines) in cholangiocytes; (ii) C. parvum infection alters cholangiocyte expression of specific endogenous microRNAs (miRNAs), a newly identified class of small regulatory RNAs important in post-transcriptional gene regulation; and (iii) TLR/NF-kappaB signals are involved in C. parvum-induced cholangiocyte miRNA expression. Thus, we will test the CENTRAL HYPOTHESIS that epithelial innate immunity in response to C. parvum infection involves TLR-mediated pathogen recognition and subsequent alteration of miRNA-mediated post-transcriptional gene regulation. In our three integrated SPECIFIC AIMS, we will test the hypotheses that: (i) C. parvum activation of host-cell TLRs alters cholangiocyte expression of endogenous miRNAs; (ii) C. parvum-induced miRNA expression in cholangiocytes is mediated by TLR activation of the nuclear transcription factor, NF-kappaB; and (iii) C. parvum-induced alterations in miRNA expression influence associated post-transcriptional gene regulation and contribute to cholangiocyte innate immune responses. Innovative aspects of the application include novel methodologies for miRNA analysis, an in vitro model of human biliary cryptosporidiosis, and new concepts (regulation of miRNA expression by transcription factors and miRNA-mediated post-transcriptional gene regulation in epithelial innate immunity). These studies will address a fundamental question related to epithelial innate immunity to a Category B Pathogen; specifically, what is the role of miRNAs in TLRmediated cholangiocyte innate immune responses to C. parvum? This NEW CONCEPT is likely relevant to innate and adaptive immunity in general and our results could also provide a rational basis for the design and implementation of new therapeutic strategies for microbial infection.