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传播方面发挥着核心作用，它通过以下几种途径相结合 由先天免疫细胞介导的快速、非特异性反应，以及通过诱导特异性 由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 表达由病毒蛋白质和核酸触发的受体，导致抗病毒物质的分泌 破坏入侵病原体的因素。这些专业的抗原提呈细胞(APC)也 内吞HIV-1并将病毒多肽递呈给与人类白细胞抗原I和II类相关的反应性T细胞 蛋白质。然而，APC也表达CD4、CCR5和CXCR4受体，并且可以高效地 感染了HIV-1病毒。此外，DC可以通过病毒结合包括DC-SIGN在内的C型凝集素来捕获HIV-1 (DC特异性ICAM-抓取非整合素)。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T细胞感染HIV-1，可能是通过分泌新的- 通过这些细胞之间形成的病毒突触合成的病毒(“顺式”感染)，或通过转移 未经修饰的HIV-1从DC转移到T细胞(“反式”感染)。T细胞要么直接感染HIV- 1，或与受感染的APC相互作用后感染，然后迁移到邻近的淋巴结，导致 HIV-1从粘膜部位传播到外周。这些研究的总体目标是确定 阻断CCR5和DC-Sign ON的表达可在多大程度上抑制HIV-1的粘膜传播 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细胞将极大地减少对这些细胞的感染，以及随后的APC- 介导反应性T细胞的感染。我们建议使用RNA干扰(RNAi)和短干扰 RNA(SiRNA)沉默雌性粘膜白细胞CCR5和DC-SIGN的表达 用免疫缺陷小鼠模型重建生殖道和胃肠道 人类祖细胞，并表达人类所有主要免疫细胞群。我们的具体目标将是 人胃粘膜组织中CCR5和DC-SIGN沉默程度的测定 NOD/SCID/BLT小鼠经阴道或直肠内给药后抑制HIV-1感染 艾滋病毒-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是否既能抑制无细胞感染又能抑制细胞相关的HIV-1感染。在……里面 此外，我们将确定抑制跨越粘膜组织的HIV-1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 通过检测CCR5和DC-SIGN在白细胞上沉默表达的程度来确定 NOD/SCID/BLT小鼠的粘膜组织增强了对HIV-1和模型抗原的免疫应答， 破伤风类毒素(特效靶2)。这一目标的研究将量化对艾滋病毒-1的获得性免疫反应，并 通过测量体液和细胞免疫反应来检测siRNA处理的小鼠的破伤风类毒素。我们的总目标是 是开发新的方法来抑制艾滋病毒-1从粘膜部位向外围传播。 沉默对HIV-1感染至关重要的细胞受体可能会增加其他治疗方法 旨在减少艾滋病毒-1粘膜传播，包括疫苗和杀微生物剂。

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.