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型干扰素产生细胞，被认为是“专业的干扰素产生细胞”。在HIV感染者中，PDC在数量和功能上都存在缺陷，这种功能障碍与发展为机会性感染密切相关。除了证据表明PDC在HIV感染中具有重要的保护作用外，过度热情的PDC干扰素-α反应也与免疫激活和HIV的发病有关。本应用中的实验是基于我们观察到的病毒携带者外周血PDC不仅在数量上耗尽，而且在功能上也发生了改变。自上次竞争性提交这项资助以来，我们的研究表明，这种数量和功能障碍是由几个因素造成的，包括一些循环中的PDC的凋亡，激活和潜在的外周招募，以及外周PDC被新移居的骨髓细胞取代。此外，我们还发现，与未感染的细胞相比，PDC优先从感染病毒的活细胞中摄取细胞膜和细胞质，随后产生干扰素-α，并在这种“蚕食”反应中成熟。然而，在与HIV感染的细胞相互作用时，这一过程被颠覆，PDC与HIV感染的细胞融合。我们提出了三个具体目标来跟进这些观察：在第一个具体目标中，我们将调查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干扰素-a的产生和PDC：艾滋病毒感染细胞的相互作用，无论是作为无细胞病毒还是作为有病毒感染的细胞。这些病毒包括早期传播的病毒以及从CHAVI获得的慢性期病毒。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来确定PDC：T细胞在体内和淋巴组织中是否发生融合：首先，我们将确定是否有证据表明艾滋病毒感染患者的外周血中存在PDC：T细胞融合；其次，我们将确定在扁桃体悬浮细胞和扁桃体器官培养中是否发生PDC：T细胞融合；第三，我们将与加州大学洛杉矶分校的Christel Uittenbogaart博士合作，确定在感染HIV的HIS小鼠中是否发生PDC：T细胞融合。在最终的特定目标中，我们将研究PDC不同表型亚群在HIV感染者和非HIV感染者中的功能意义，并将研究HIV感染者骨髓中的PDC。总之，本申请中提出的研究有望为人类PDC的生物学及其在HIV-1感染中功能障碍的机制提供有价值的新信息。 公共卫生相关性：浆细胞样树突状细胞(PDC)是HIV-1先天免疫反应的重要组成部分。在这个持续的更新应用中，我们将跟踪我们对HIV感染者中PDC异常激活以及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的发病机制。

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.