Novel Strategies to Antagonize Cocaine-Enhanced HIV Pathobiology

描述(由申请人提供)：可卡因是艾滋病毒流行病学中的一个重要辅助因素，与增加病毒暴露并破坏坚持治疗的危险行为有关。但与行为因素无关，可卡因似乎会增加艾滋病毒的复制，并加速向艾滋病的进展。我们假设这种情况的发生是因为可卡因促进了艾滋病毒对关键宿主防御的破坏。艾滋病毒本身是如何破坏免疫防御的？一条途径涉及树突状细胞(DC)，通常捕获病原体并在溶酶体中将其降解。HIV可以通过与DC-SIGN和细胞骨架蛋白络合来绕过这一途径，运输到内体而不是溶酶体，因此它不会被降解。然后，树突状细胞通过免疫突触将内小体相关病毒丰富地递送给CD4T细胞。HIV还可以通过改变新的Robo受体来破坏完整的淋巴管内皮(LE)屏障，Robo受体通常是稳定细胞骨架的信号。ROBO失调会增加血管内皮细胞的通透性，从而促进病毒传播到血液中。类似的HIV效应也可能发生在高内皮微静脉(HEV)中，HEV是免疫细胞在淋巴结中运输的“守门人”。研究表明，可卡因可以特异性地改变DC和内皮细胞中的细胞骨架和信号通路，从而使艾滋病毒受益。这项提议的总体目标是更深入地了解艾滋病毒和可卡因如何在分子水平上合作颠覆宿主防御。我们将在体外和体内建立它们在BLT人源化小鼠中的作用模型，并探索靶向新的Robo受体如何对抗可卡因引发的有害变化，这些变化增强了艾滋病毒的病理生物学。我们的初步研究支持这一策略：ROBO信号保护LE免受艾滋病毒的高通透性，并在DC中抑制病毒向T细胞的传播。具体创新点包括：(1)专注于细胞骨架器官的变化，作为艾滋病毒和可卡因解除免疫反应的独特方式；(2)利用LES和HEV的纯化种群，其在限制艾滋病毒中的作用尚不清楚；(3)利用新发现的Sit/Robo家族的配体和受体来对抗可卡因促进的艾滋病毒对免疫防御的颠覆；(4)在体内HIV-BLT小鼠模型中应用尖端方法来转化体外观察结果。具体目的是：(1)进一步鉴定艾滋病毒和可卡因对DC的分子作用，从而促进病毒向T细胞的传播；(2)表征艾滋病毒和可卡因引起的LE和HEV屏障的分子变化，进一步评估Robo信号的保护作用；(3)在HIV和可卡因存在的情况下，体外建立DC和T细胞与LE和HEV的相互作用模型；(4)在体内评估Robo信号如何拮抗可卡因增强的艾滋病毒病理生物学。通过产生这一新知识，我们希望为预防或遏制可卡因使用者感染艾滋病毒的创新战略提供基础。

DESCRIPTION (provided by applicant): Cocaine is a prominent cofactor in the epidemiology of HIV, associated with risky behavior that increases exposure to virus and undermines adherence to treatment. But independent of behavioral factors, cocaine appears to increase HIV replication and accelerate progression to AIDS. We hypothesize this occurs because cocaine facilitates HIV subversion of key host defenses. How does HIV itself subvert immune defenses? One pathway involves dendritic cells (DCs) that normally capture pathogens and degrade them in lysosomes. HIV can bypass this pathway by complexing with DC-SIGN and cytoskeletal proteins, trafficking to endosomes rather than lysosomes, so it is not degraded. Endosome-associated virus is then richly presented by DCs to CD4+ T-cells via the immune synapse. HIV can also subvert an intact lymphatic endothelial (LE) barrier through altering novel Robo receptors which normally signal to stabilize the cytoskeleton. Robo dysregulation increases endothelial permeability, which would promote virus dissemination into the bloodstream. Similar HIV effects may occur in high endothelial venules (HEVs), the "gatekeepers" for immune cell trafficking in lymph nodes. Studies show cocaine can specifically alter cytoskeletal and signaling pathways in DCs and endothelium that would work to the benefit of HIV. The overall objective of this proposal is to gain a deeper understanding of how HIV and cocaine partner on a molecular level to subvert host defenses. We will model their effects both in vitro and in vivo in the BLT humanized mouse, and explore how targeting novel Robo receptors may oppose the deleterious changes triggered by cocaine that enhance HIV pathobiology. Our initial studies support this strategy: Robo signaling protected LE from HIV hyperpermeability and in DCs inhibited virus transmission to T-cells. Specific points of innovation include: (1) focus on alterations of the cytoskeletal apparatus as a unique way HIV and cocaine may disarm immune responses; (2) utilize purified populations of LEs and HEVs, whose roles in limiting HIV are not yet well characterized; (3) exploit the newly identified ligands and receptors of the Slit/Robo family in antagonizing cocaine-facilitated HIV subversion of immune defense; (4) apply cutting-edge methodology in the in vivo HIV-BLT mouse model to translate in vitro observations. The specific aims are: (1) further characterize molecular effects of HIV and cocaine in DCs that enhance virus transmission to T-cells; (2) characterize molecular changes in LE and HEV barriers due to HIV and cocaine, and further assess protective effects of Robo signaling; (3) model in vitro DC and T-cell interactions with LE and HEV in the presence of HIV and cocaine; (4) assess in vivo in the BLT humanized mouse how Robo signaling may antagonize cocaine-enhanced HIV pathobiology. By generating this new knowledge, we hope to provide the foundation for innovative strategies to prevent or contain HIV infection in users of cocaine.