Human Beta-Defensin-1 in HSV-1 Innate Immunity

描述（由申请人提供）：由单纯疱疹病毒-1 （HSV-1）引起的病毒感染，通常是免疫系统受损的结果。先天免疫系统是预防这些病原体的第一道防线，由许多可溶性因子和细胞类型组成，包括树突状细胞（DC）和自然杀伤细胞。浆细胞样DC （plasmacytoid DC， PDC）是DC的一种，它通过产生干扰素- γ在先天免疫功能中起重要作用。由包膜病毒如HSV-1诱导的IFN。最近有研究表明，PDC在诱导Th1对流感和HSV-1的免疫中发挥作用，并在小鼠感染模型中被招募到淋巴结以刺激抗病毒细胞毒性t细胞。在艾滋病患者中，循环中PDC的缺乏以及CD4 t淋巴细胞的耗竭与机会性感染（包括病毒感染）的发生有关。除IFN外，PDC在HSV刺激下产生趋化因子和肿瘤坏死因子-a。我们证明PDC还能产生人β -防御素-1 (hBD-1)，这是一种抗菌肽，对转染CCR6受体的细胞具有抗病毒活性和趋化特性。我们假设，针对HSV-1的抗病毒免疫的第一步是在PDC等免疫细胞中产生hBD-1。我们的初步数据显示，HSV-1刺激后2小时内，PDC中hBD-1 mRNA和肽水平升高。由于PDC在病毒感染中具有潜在的重要性，因此在确定PDC在临床疾病患者中的存在之前，有必要首先了解病毒诱导PDC中hBD-1的性质和机制。我们假设HSV-1通过刺激toll样受体9和激活转录因子从而增加hBD-1基因的转录，直接诱导PDC中相应数量的hBD-1。本研究拟探索HSV-1诱导PDC中hBD-1 mRNA和肽的初始反应途径，建立一种量化hBD-1在这一罕见DC亚群中诱导的方法，确定HSV-1诱导hBD-1的转录激活途径。在第一个目标中，我们将确定HSV-1是否通过直接途径刺激hBD-1，或者细胞因子和趋化因子是否通过间接机制影响诱导。我们还将通过对病毒和hBD-1的共定位研究来研究HSV-1和PDC的相互作用，以及使用氯喹治疗PBMC时hBD-1是否需要在病毒进入时进行内体酸化。为了开始解决hBD-1的生物学功能，将进行趋化性研究。我们将研究tlr2、7和9信号，干扰素调节因子的诱导和hBD-1启动子区域转录因子结合位点的鉴定。这项资助的数据将提供不同于hBD-2的防御素调节新机制的新信息，并增强对PDC生物学功能的认识，并使未来的体内研究成为可能，设计模拟HSV-1感染或临床研究的动物研究。单纯疱疹病毒-1 （HSV-1）是免疫功能低下患者（如接受化疗、器官移植、免疫缺陷和艾滋病毒感染者）严重且经常持续的机会性感染的原因。抵抗这种病毒和其他病毒病原体的第一道防线是先天免疫系统。先天免疫的一个组成部分是产生抗生素肽，如防御素。我们研究的长期目标是了解-防御素在对抗病毒的先天免疫防御中的作用，以便根据我们的发现开发出新的治疗方法。

DESCRIPTION (provided by applicant): Viral infections caused by Herpes Simplex virus-1 (HSV-1), often occur as a result of a compromised immune system. The innate immune system is the first line of defense against prevention of these pathogens and consists of numerous soluble factors and cell types, including dendritic cells (DC) and natural killer cells. One type of DC, the plasmacytoid DC (PDC) is important in innate immune function through the production of interferon-? (IFN) induced by enveloped viruses like HSV-1. PDC have recently been shown to play a role in induction of Th1 immunity against influenza and HSV-1 and are recruited in mouse infection models to lymph nodes to stimulate antiviral cytotoxic T-cells. In AIDS patients, the lack of PDC in the circulation, along with depletion of CD4 T-lymphocytes is associated with the development of opportunistic infections, including viral infections. In addition to IFN, PDC produce chemokines and tumor necrosis factor-a upon HSV stimulation. We demonstrated that PDC also produce human beta-defensin-1 (hBD-1), an antimicrobial peptide that has antiviral activity and chemotactic properties towards cells transfected with CCR6 receptor. We hypothesize that an initial step in anti-viral immunity against HSV-1 is the production of hBD-1 in immune cells such as PDC. Our preliminary data show that HSV-1 induces increased hBD-1 mRNA and peptide in PDC within 2 hours of HSV-1 stimulation. Since PDC have potential importance in viral infections, it is essential to first understand the nature and mechanism of the viral induction of hBD-1 in PDC before determining its presence in clinical disease in patients. We hypothesize that HSV-1 directly induces relevant amounts of hBD-1 in PDC via stimulation of Toll-like receptor 9 and activation of transcription factors subsequently increasing hBD-1 gene transcription. This study proposes to explore the initial response pathway of HSV-1 induced hBD-1 mRNA and peptide in PDC, to develop a method to quantify hBD-1 induction in this rare DC subset, and to determine the transcriptional activation pathway of hBD-1 induction by HSV-1. In the first aim we will determine whether hBD-1 is stimulated by HSV-1 via a direct pathway or whether cytokines and chemokines influence the induction via an indirect mechanism. We will also study HSV-1 and PDC interactions using co- localization studies of virus and hBD-1 and whether hBD-1 requires endosomal acidification upon viral entry using chloroquine treatment of PBMC. To begin to address the biological function of hBD-1, chemotaxis studies will be done. We will examine TLR 2, 7 and 9 signaling, induction of interferon regulatory factors and identification of binding sites of transcription factors in the hBD-1 promoter region. The data from this grant will yield new information on a new mechanism for ¿-defensin regulation, different from hBD-2, and enhance the knowledge of the biological function of PDC and also enable future studies in vivo, designing animal studies that model HSV-1 infection or clinical studies. Herpes Simplex Virus-1 (HSV-1) is the cause of severe and often persistent opportunistic infections in immunocompromised patients, such as those undergoing chemotherapy, organ transplantation, and those with immunodeficiencies and HIV. The first line of defense against this and other viral pathogens is the innate immune system. One component of innate immunity is the production of antibiotic peptides such as ¿- defensins. The long-range goal of our research is to understand the role of ¿-defensins in the innate immune defense against viruses so that novel therapies can be developed from our findings.