The Spinal Cord as a CNS Latent Reservoir for Replication Competent SIV

项目摘要 艾滋病毒感染脊髓和大脑中的巨噬细胞，即使有，也经常导致临床症状。 抗逆转录病毒治疗（ART）。在脊髓和脑中，巨噬细胞（MΦs）可能作为长期的细胞功能， 潜伏的艾滋病毒的储存库。如果ART停止，有复制能力的HIV可以从这些潜伏的宿主中出现。 SIV在急性感染期间在脊髓和脑中以相同的水平复制;然而， 在整个感染过程中，包括潜伏期和潜伏期， 与急性感染相反，病毒动力学在急性感染中有显著不同。 这些不同的中枢神经系统隔室后停止ART和跟踪SIV反弹。SIV RNA很容易被检测到 在停止ART后的第一周内，脊髓中的SIV含量增加;相反，大多数SIV感染的动物没有 在大脑中检测到SIV RNA。脊髓和大脑之间的反弹复制的差异可能导致 来自这些不同CNS隔室中MΦ和星形胶质细胞的独特细胞免疫应答。大多数SIV- 接受ART的感染猕猴在脊髓或脑、CSF或血浆中没有可检测到的SIV RNA。 尽管如此，前病毒DNA和免疫反应在脊髓和大脑中仍然存在。有趣的是， 持续免疫反应的差异：在脊髓中，GFAP和CCL 2的表达升高， 表明持续的星形胶质细胞活化;在脑中，升高的CD 68和TNFα水平表明持续的脑 MΦ激活。这一建议的中心前提是脊髓MΦ作为一个独特的SIV水库， 停止ART后迅速重新激活的CNS。脊髓中的细胞神经免疫反应与 在停止ART后的潜伏期和再激活期间从大脑中释放。特别是， 脊髓MΦ和星形胶质细胞的免疫反应本质上不同于脑反应。具体目标1是 确定脊髓MΦ是否构成CNS中独特的SIV储库， 1）急性SIV感染期间的CNS细胞群，2）ART延长的潜伏期，和3） 具体目标2将确定脊髓和大脑中的神经炎症反应， 1)急性SIV感染，2）ART潜伏期延长，和3）停止ART后病毒反弹。 将使用nCounter免疫调节试验进行分析;单细胞RNA-seq基因表达 对分离自脊髓和脑的单细胞（包括MΦ和星形胶质细胞）进行的分析将 补体靶向基因分析。具体目标3是确定是否耗尽脊髓和脑 通过用CSF 1 R抑制剂PLX 3397治疗，在潜伏期期间和停止ART后持续的MΦ改变 在SIV从潜伏期反弹期间CNS SIV复制。MΦ耗竭将补充SA 1对CNS的研究 MΦ作为潜伏性储库及SIV潜伏期和期间脊髓基因表达谱的SA 2研究 反弹，以推进我们对脊髓MΦ在HIV潜伏期中所起作用的理解。

PROJECT SUMMARY HIV infects macrophages in the spinal cord and brain, often leading to clinical symptoms even with antiretroviral treatment (ART). In spinal cord and brain, macrophages (MΦs) may serve as long-term cellular reservoirs of latent HIV. Replication competent HIV can emerge from these latent reservoirs if ART is stopped. SIV replicates in both spinal cord and brain at equivalent levels during acute infection; however, the specific cellular targets of SIV in the CNS have not been classified throughout infection, including during latency and rebound after interrupting ART. In contrast with acute infection, viral dynamics are significantly different in these distinct CNS compartments after stopping ART and tracking SIV rebound. SIV RNA is readily detectable in spinal cord in the first weeks after stopping ART; in contrast, most SIV-infected animals do not have detectable SIV RNA in the brain. The disparity in rebound replication between spinal cord and brain may result from unique cellular immune responses by MΦ and astrocytes in these distinct CNS compartments. Most SIV- infected macaques receiving ART have no detectable SIV RNA in spinal cord or brain, CSF, or plasma. Proviral DNA and immune responses persist nonetheless in both spinal cord and brain. Intriguingly, the nature of the sustained immune response differs: in the spinal cord, elevated expression of both GFAP and CCL2 point to sustained astrocyte activation; in the brain, elevated CD68 and TNFα levels indicate persistent brain MΦ activation. The central premise of this proposal is that spinal cord MΦs serve as a distinct SIV reservoir in the CNS that reactivates rapidly after stopping ART. Cellular neuroimmune responses in the spinal cord differ from the brain during latency and reactivation after stopping ART. In particular, coordinate regulation of spinal cord MΦ and astrocyte immune responses intrinsically differ from brain responses. Specific Aim 1 is to determine whether spinal cord MΦs constitute a unique SIV reservoir in the CNS by identifying SIV-infected CNS cell populations during 1) acute SIV infection, 2) prolonged latency with ART, and 3) viral rebound after stopping ART. Specific Aim 2 will identify the neuroinflammatory responses in the spinal cord and brain during 1) acute SIV infection, 2) prolonged latency on ART, and 3) viral rebound after stopping ART. Gene expression profiling will be performed using nCounter immunoregulatory assays; single cell RNA-seq gene expression profiling performed on single cells isolated from spinal cord and brain including MΦs and astrocytes will complement targeted gene profiling. Specific Aim 3 is to determine whether depleting spinal cord and brain MΦs during latency and continuing after stopping ART by treatment with the CSF1R inhibitor PLX3397 alters CNS SIV replication during SIV rebound from latency. MΦ depletion will complement SA1 studies on CNS MΦs as latent reservoirs and SA2 studies on spinal cord gene expression profiles in latency and during SIV rebound to advance our understanding of the role that spinal cord MΦs play in HIV latency.