Inhibiting Mucosal HIV-1 Transmission by Host Cell RNA Interference

6.项目摘要 粘膜免疫系统在预防HIV-1的传播中起着核心作用，其通过以下方式的组合： 快速，非特异性反应介导的先天免疫细胞，以及通过诱导特异性 获得性免疫细胞介导的反应。粘膜组织中的巨噬细胞和树突状细胞（DC） 表达由病毒蛋白质和核酸触发的受体，导致抗病毒抗体的分泌。 破坏入侵病原体的因子这些专职抗原呈递细胞（APC）也 内吞HIV-1并将病毒肽呈递给与HLA I类和II类相关的应答T细胞 proteins.然而，APC也表达CD4、CCR5和CXCR4受体，并且可以变得富有成效。 感染了HIV-1。此外，DC可以通过与包括DC-SIGN在内的C型凝集素结合来捕获HIV-1 （DC特异性ICAM-抓取非整联蛋白）。HIV感染的巨噬细胞或DC与 抗原呈递过程中的T细胞可导致HIV-1感染T细胞，通过分泌新的 合成病毒穿过这些细胞之间形成的病毒突触（"顺式"感染），或通过转移 未修饰的HIV-1从DC到T细胞（"反式"感染）。直接感染艾滋病毒的T细胞- 1，或者在与感染的APC相互作用后被感染，然后迁移到邻近的淋巴结，导致淋巴结转移。 HIV-1从粘膜部位向外周的传播。这些研究的总体目标是确定 通过阻断CCR5和DC-SIGN的表达可以抑制粘膜HIV-1传播的程度。 粘膜巨噬细胞、DC和T细胞。我们的假设是阻止HIV-1与巨噬细胞结合， DC和T细胞将显著减少这些细胞的感染，以及随后的APC- 介导的应答T细胞的感染。我们建议使用RNA干扰（RNAi）与短干扰 RNA（siRNA）沉默女性粘膜白细胞上CCR5和DC-SIGN的表达 生殖道和胃肠道的免疫缺陷小鼠模型，该模型用 人祖细胞并表达所有主要的人免疫细胞群体。我们的具体目标将 确定在人源化CCR5和DC-SIGN的粘膜组织内沉默CCR5和DC-SIGN的程度。 NOD/SCID/BLT小鼠在阴道内或直肠内激发后导致HIV-1感染的抑制 HIV-1（具体目标1）。这些研究将包括确定siRNA的最佳剂量和给药途径， 以及确定siRNA是否可以抑制无细胞以及细胞相关的HIV-1感染。在 此外，我们还将确定抑制HIV-1在粘膜组织中的感染是否与 受体沉默的程度，因为额外的抗病毒机制可能作为siRNA暴露的函数而发生。 在随后的研究中，我们将确定有助于抑制HIV-1的抗病毒机制 通过测定白细胞上CCR5和DC-SIGN表达沉默的程度， NOD/SCID/BLT小鼠的粘膜组织增强对HIV-1和模型抗原的免疫应答， 破伤风类毒素（具体目标2）。这一目标的研究将量化对HIV-1的获得性免疫应答， 通过测量体液和细胞免疫应答，在siRNA处理的小鼠中检测破伤风类毒素。我们的总目标 是开发新的方法来抑制HIV-1从粘膜部位向外周的传播。 沉默对HIV-1感染重要的细胞受体可以增强其他治疗方法 旨在减少粘膜HIV-1传播，包括疫苗和杀菌剂。

6. Project Abstract The mucosal immune system plays a central role in preventing the transmission of HIV-1 by a combination of rapid, non-specific responses mediated by innate immune cells, as well as by the induction of specific responses mediated by acquired immune cells. Macrophages and dendritic cells (DC) in mucosal tissues express receptors that are triggered by viral proteins and nucleic acids, leading to the secretion of anti-viral factors that destroy the invading pathogen. These professional antigen presenting cells (APC) also endocytose HIV-1 and present viral peptides to responding T cells in association with HLA class I and class II proteins. However, APC also express CD4, CCR5, and CXCR4 receptors and can become productively infected with HIV-1. In addition, DC can capture HIV-1 via viral binding to C type lectins including DC-SIGN (DC-specific ICAM-grabbing non-integrin). Cell-to-cell contact between an HIV-infected macrophage or DC and a T cell during antigen presentation can lead to infection of T cells by HIV-1, either by the secretion of newly- synthesized virus across the viral synapse that forms between these cells ("cis" infection), or by the transfer of unmodified HIV-1 from the DC to the T cell ("trans" infection). T cells that are either directly infected with HIV- 1, or become infected after interaction with infected APC, then migrate to adjacent lymph nodes, leading to the transmission of HIV-1 from mucosal sites to the periphery. The overall goal of these studies is to determine the extent to which mucosal HIV-1 transmission can be inhibited by blocking expression of CCR5 and DC-SIGN on mucosal macrophages, DC and T cells. Our hypothesis is that preventing HIV-1 binding to macrophages, DC and T cells would dramatically reduce infection of these cells, and also the subsequent APC- mediated infection of responding T cells. We propose to use RNA interference (RNAi) with short-interfering RNA (siRNA) to silence expression of CCR5 and DC-SIGN on mucosal leukocytes from the female reproductive and gastrointestinal tracts using an immunodeficient mouse model that is reconstituted with human progenitor cells and expresses all major human immune cell populations. Our specific aims will determine the extent to which silencing CCR5 and DC-SIGN within mucosal tissues of humanized NOD/SCID/BLT mice leads to the inhibition of HIV-1 infection after intravaginal or intrarectal challenge with HIV-1 (specific aim 1). These studies will include defining optimal doses and routes of administration of siRNA, and also determining whether siRNA can inhibit both cell-free as well as cell associated HIV-1 infection. In addition, we will determine whether inhibition of HIV-1 infection across mucosal tissues correlates with the degree of receptor silencing, as additional anti-viral mechanisms may occur as a function of siRNA exposure. In subsequent studies, we will define the anti-viral mechanisms that contribute to the inhibition of HIV-1 infection by determining the extent to which silencing expression of CCR5 and DC-SIGN on leukocytes in mucosal tissues of NOD/SCID/BLT mice enhances immune responses to HIV-1 and to a model antigen, tetanus toxoid (specific aim 2). Studies in this aim will quantify acquired immune responses to HIV-1 and to tetanus toxoid in siRNA-treated mice by measuring humoral and cellular immune responses. Our overall goal is to develop novel approaches to inhibit the transmission of HIV-1 from mucosal sites to the periphery. Silencing cellular receptors important for HIV-1 infection could augment other therapeutic approaches designed to reduce mucosal HIV-1 transmission, including vaccines and microbicides.