Viral Recognition by plasmacytoid dendritic cell

描述(申请人提供)：浆细胞样树突状细胞(PDCs)是一种关键的天然免疫细胞，能分泌高水平的I型干扰素(IFN)来应对各种病毒。I型干扰素系统在针对病毒感染的早期先天免疫反应中起着关键作用。病原体检测的主要途径之一是通过模式识别受体(PRRs)识别微生物上发现的进化保守的病原体相关分子模式(PAMPs)，如Toll样受体(TLRs)。通过TLRs识别病原体可诱导树突状细胞的激活，并启动清除和保护特定病原体所必需的适应性免疫反应。病毒既可被胞液传感器识别，也可被内体传感器识别。PDC几乎完全利用内体TLR进行病毒识别。在以前的研究中，我们分别证明了TLR9和TLR7在双链DNA病毒、单纯疱疹病毒(HSV)和单链(Ss)RNA病毒、流感和水泡性口炎病毒(VSV)反应中激活和分泌IFN1所必需的。PDC通过TLR7和TLR9识别这两种病毒需要完整的内吞途径。此外，在R01计划的第一个资助期内得到支持的研究表明，pDC利用自噬，这是一种高度保守的途径，将胞浆物质运送到内溶酶体，识别单链RNA病毒的胞液病毒复制中间产物。这些结果提供了TLR识别病毒和I型干扰素诱导途径之间的关键联系，并提示这些和其他TLR在病毒识别和免疫反应中的重要性。然而，在我们对pDC生物学的理解中，主要的空白涉及到内体TLR如何在细胞内运输到适当的隔室，从那里它们可以发出信号来诱导I型IFN和促炎细胞因子。在这项提案中，我们在初步研究部分提供了证据，证明了适配蛋白3(AP-3)复合体、磷脂酰肌醇5激酶和ATG5在TLRs的细胞内运输和信号传递中的作用。基于这些数据，我们描述了系统的方法来研究TLR9在pDC中天然识别的分子和细胞机制。在第一个目标中，我们将确定AP-3复合体介导内体TLRs在pDC中转运的机制，并定义干扰素-内体的细胞生物学。在第二个目标中，我们建议确定pDC中肌醇磷脂介导的信号机制。在最终目标中，我们将整合我们在三条不同途径中的所有发现，并询问自噬与AP-3和磷脂酰肌醇依赖途径介导的TLR贩运之间的可能联系。通过对pDC生物学的基本了解，这些研究将有助于为设计针对病毒疾病的免疫干预提供重要基础。

DESCRIPTION (provided by applicant): Plasmacytoid dendritic cells (pDCs) are critical innate immune cells that secrete high levels of type I interferons (IFNs) in response to a variety of viruses. Type I IFN system plays a pivotal role in the early innate immune responses against virus infection. One of the major pathways of pathogen detection involves the recognition of evolutionarily conserved pathogen associated molecular patterns (PAMPs) found on microbes via pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs). Recognition of pathogens via TLRs induces activation of DCs and the initiation of the adaptive immune responses necessary for clearance of and protection from a given pathogen. Viruses are recognized by both cytosolic sensors and endosomal sensors. The pDCs almost exclusively utilize the endosomal TLRs for viral recognition. In previous studies we demonstrated the requirement of TLR9 and TLR7 in the activation and IFN1 secretion in response to double stranded (ds) DNA viruses, herpes simplex viruses (HSV), and single stranded (ss) RNA viruses, influenza and vesicular stomatitis virus (VSV), respectively. The recognition of both types of viruses through TLR7 and TLR9 by the pDCs requires an intact endocytosis pathway. In addition, research supported during the first funding period of this R01 program demonstrated that pDCs utilize autophagy, a highly conserved pathway of delivering cytosolic materials to the endolysosomes, for recognition of cytosolic viral replication intermediates of ssRNA viruses. These results provided a critical link between TLR recognition of viruses and the type I IFN induction pathways, and suggested the importance of these and other TLRs in viral recognition and immune responses. However, major gaps in our understanding of the biology of pDCs relate to how the endosomal TLRs traffic within the cells to reach the appropriate compartment from which they can signal to induce type I IFNs and pro-inflammatory cytokines. In this proposal, we provide evidence in the Preliminary Studies section that demonstrate the role of adaptor protein 3 (AP-3) complex, phosphatidyl inositol 5 kinase, and Atg5 in intracellular trafficking and signaling by TLRs. Based on these data, we describe systematic approaches to examine the molecular and cellular mechanisms of innate recognition by TLR9 in pDCs. In the first Aim, we will determine the mechanism by which AP-3 complex mediates trafficking of endosomal TLRs in pDCs and define the cell biology of IFN- endosomes. In the second Aim, we propose to identify the phosphoinositide-mediated signaling mechanism in pDCs. In the final Aim, we will integrate all of our findings in the three separate pathways and interrogate a possible link between autophagy and TLR trafficking mediated by AP-3 and phosphoinositide-dependent pathways. Through basic understanding of the biology of pDCs, these studies will help to provide important foundation with which to design immunological interventions against viral diseases.