MicroRNAs in Epithelial Innate Immunity to C. parvum

描述（由申请方提供）：长期目标是更好地了解上皮对病原体的先天免疫应答的分子机制，我们将研究胆管细胞（胆道内衬上皮细胞）对隐孢子虫（一种NIAID B类优先病原体，可引起人类肠道和胆道疾病）的先天免疫应答。利用我们建立的人胆源性隐孢子虫病体外模型，我们已经证明：（i）两个Toll样受体（TLR），TLR 2和TLR 4，以及相关的细胞内信号通路（NF-κ B激活）在C.细小病毒识别和先天免疫应答的诱导（例如，释放细胞因子/趋化因子）;（ii）C.细小病毒感染改变 胆管细胞表达特异性内源性microRNA（miRNAs），一种新发现的在转录后基因调控中重要的小调节RNA;和（iii）TLR/NF-κ B信号参与C. parvum诱导的胆管细胞miRNA表达。因此，我们将测试中枢假设，即上皮先天免疫应答C。细小病毒感染涉及TLR介导的病原体识别和随后的miRNA介导的转录后基因调控的改变。在我们的三个综合具体目标中，我们将检验以下假设：（i）C。宿主细胞TLR的微小激活改变了胆管细胞内源性miRNA的表达;（ii）C.在胆管细胞中，细小病毒诱导的miRNA表达由核转录因子NF-κ B的TLR激活介导;和（iii）C.细小病毒诱导的miRNA表达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性免疫反应。该申请的创新方面包括miRNA分析的新方法、人胆汁隐孢子虫病的体外模型和新概念（转录因子对miRNA表达的调节和上皮先天免疫中miRNA介导的转录后基因调节）。这些研究将解决一个与上皮细胞对B类病原体的先天免疫相关的基本问题;具体地说，miRNA在TLR介导的胆管细胞对C类病原体的先天免疫应答中的作用是什么。parvum？这一新概念可能与一般的先天性和适应性免疫有关，我们的结果也可以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.