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的重要辅助因素，与增加病毒暴露和破坏治疗依从性的危险行为有关。但与行为因素无关，可卡因似乎增加了艾滋病毒的复制，加速了艾滋病的发展。我们假设发生这种情况是因为可卡因促进了HIV对关键宿主防御的颠覆。HIV本身是如何破坏免疫防御的？一种途径涉及树突状细胞（dc），树突状细胞通常捕获病原体并在溶酶体中降解它们。HIV可以通过与DC-SIGN和细胞骨架蛋白络合绕过这一途径，转运到核内体而不是溶酶体，因此它不会被降解。内核体相关病毒通过dc通过免疫突触大量呈递到CD4+ t细胞。HIV还可以通过改变新的Robo受体来破坏完整的淋巴内皮（LE）屏障，这些受体通常发出信号来稳定细胞骨架。机器人的失调会增加内皮细胞的渗透性，这会促进病毒传播到血液中。类似的HIV效应可能发生在高内皮小静脉（HEVs）中，它是免疫细胞在淋巴结运输的“看门人”。研究表明，可卡因可以特异性地改变dc和内皮细胞的细胞骨架和信号通路，这可能对HIV有益。本提案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破坏宿主防御。我们将在BLT人源化小鼠体内和体外模拟它们的作用，并探索靶向新型Robo受体如何对抗可卡因引发的增强HIV病理生物学的有害变化。我们的初步研究支持这一策略：Robo信号保护LE免受HIV高渗透性，并且在dc中抑制病毒向t细胞的传播。具体的创新点包括：(1)专注于改变细胞骨架装置，作为艾滋病毒和可卡因可能解除免疫反应的独特方式；(2)利用纯化的LEs和hev群体，它们在限制HIV方面的作用尚未很好地表征；(3)利用新发现的Slit/Robo家族配体和受体拮抗可卡因介导的HIV对免疫防御的破坏；(4)在体内HIV-BLT小鼠模型中应用前沿方法翻译体外观察结果。具体目标是：(1)进一步表征艾滋病毒和可卡因在dc中的分子效应，增强病毒向t细胞的传播；(2)表征HIV和可卡因导致的LE和HEV屏障的分子变化，并进一步评估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相互作用的模型；(4)在BLT人源化小鼠体内评估Robo信号如何拮抗可卡因增强的HIV病理生物学。通过产生这种新知识，我们希望为预防或控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.