Plasmacytoid Dendritic Cells in HIV pathogenesis

描述（申请人提供）：浆细胞样树突状细胞（pDC）是体内最有效的I型干扰素（IFN）产生细胞，被认为是“专业IFN产生细胞”。pDC在HIV感染个体中变得在数量和功能上都有缺陷，并且这种功能障碍与机会性感染的进展密切相关。除了pDC在HIV感染中的重要保护作用的证据之外，过度热心的pDC IFN-α应答也与免疫活化和HIV发病机制相关。本申请中的实验基于我们的观察，即病毒血症HIV感染个体中的外周血pDC不仅在数量上耗尽，而且在功能上也改变。我们的研究表明，自上次竞争提交该授权以来，这种数量和功能障碍是由几个因素造成的，包括一些循环pDC的凋亡、活化和潜在的外周募集，以及来自骨髓的新迁移细胞替代外周pDC。此外，我们已经发现pDC相对于未感染的细胞优先从活的病毒感染的细胞中摄取膜和细胞质片段，随后产生IFN-α并响应于这种“蚕食”而成熟。然而，在与HIV感染的细胞相互作用时，这一过程被破坏，并且pDC与HIV感染的细胞融合。我们提出了三个具体的目标来跟踪这些观察结果：在第一个具体的目标中，我们将研究pDC与HIV感染细胞融合的要求和后果。我们将确定不同的病毒株如何影响IFN-α的产生和pDC：HIV感染的细胞相互作用，无论是作为无细胞病毒还是作为病毒感染的细胞。这些病毒中包括早期传播病毒以及从CHAVI获得的慢性阶段病毒。成像和传统的流式细胞术将是我们实验方法的核心。在第二个目标中，我们将使用三种不同的方法确定pDC：T细胞融合是否在体内和淋巴组织中发生：首先，我们将确定HIV感染患者的外周血中是否存在pDC：T细胞融合的证据;其次，我们将确定pDC：HIV感染的T细胞融合是否发生在扁桃体悬浮细胞和扁桃体器官培养物中;第三，与UCLA的Christel Uittenbogaart博士合作，我们将确定pDC：T细胞融合是否发生在HIV感染的HIS小鼠中。在最终的具体目标中，我们将研究不同表型的pDC亚群在有和没有HIV感染的个体中的功能意义，并将研究HIV感染个体骨髓中的pDC。总之，本申请中提出的研究预计将提供有关人pDC生物学及其在HIV-1感染中功能障碍的潜在机制的有价值的新信息。 公共卫生相关性：浆细胞样树突状细胞（pDC）是对HIV-1的先天免疫应答的重要组成部分。在这个持续的更新申请中，我们将继续关注我们对HIV感染个体中异常pDC激活以及pDC与HIV感染细胞融合的有趣观察，以确定这些因素如何影响HIV发病机制。

DESCRIPTION (provided by applicant): Plasmacytoid dendritic cells (pDC) are the most potent type I interferon (IFN)- producing cells in the body and are considered to be the "professional IFN producing cells". pDC become both numerically and functionally deficient in HIV-infected individuals, and this dysfunction is strongly correlated with progression to opportunistic infections. In addition to evidence for important protective roles of the pDC in HIV infection, an overzealous pDC IFN-a response has also been associated with immune activation and HIV pathogenesis. The experiments in this application are based on our observations that peripheral blood pDC in viremic HIV-infected-individuals are not only numerically depleted, but are also functionally altered. Our studies since the last competing submission of this grant indicate that this numerical and functional dysfunction is contributed to by several factors including apoptosis of some circulating pDC, activation and potentially recruitment out of the periphery, as well as replacement of peripheral pDC with newly emigrated cells from the bone marrow. In addition, we have discovered that pDC preferentially take-up pieces of membrane and cytoplasm from live, virus-infected cells vs. uninfected cells and subsequently produce IFN-a and mature in response to this "nibbling". Upon interaction with HIV-infected cells, however, this process is subverted, and pDC fuse with the HIV-infected cells. We propose three specific aims to follow-up on these observations: In the first specific aim, we will investigate the requirements for, and consequences of, pDC fusion with HIV-infected cells. We will determine how different viral strains affect IFN-a production and pDC:HIV-infected cell interactions, both as cell-free viruses and as virus-infected cells. Included in these viruses will be early transmitted viruses as well as chronic-stage viruses to be obtained from CHAVI. Imaging and traditional flow cytometry will be central to our experimental approaches. In the second aim, we will determine whether pDC:T cell fusion occurs in vivo and in lymphoid tissues using three distinct approaches: first we will determine whether there is evidence of pDC:T cell fusion in peripheral blood of HIV-infected patients; second, we will determine whether pDC:HIV-infected T cell fusion occurs in tonsil suspension cells and tonsil organ culture; third, in collaboration with Dr. Christel Uittenbogaart of UCLA, we will determine whether pDC:T cell fusion occurs in HIV-infected HIS mice. In the final specific aim, we will investigate the functional significance of the different phenotypic subpopulations of pDC in individuals with and without HIV infection and will investigate pDC in the bone marrow of HIV-infected individuals. Together, the studies proposed in this application are anticipated to provide valuable new information on the biology of human pDC and the mechanisms underlying their dysfunction in HIV-1 infection. PUBLIC HEALTH RELEVANCE: Plasmacytoid dendritic (pDC) cells are an important component of the innate immune response to HIV-1. In this continuing renewal application, we will follow-up on our interesting observations of aberrant pDC activation in HIV-infected individuals and pDC fusion with HIV-infected cells to determine how these factors impact HIV pathogenesis.