Multimodal profiling of microglia during HIV infection and substance use disorder

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Early CNS entry of HIV fosters its expression in perivascular macrophages and microglia; but, whether HIV expression in these cells is associated with persistent neuropsychiatric damage is unclear. Non-HIV-related comorbidities such as substance use disorders can worsen neuropsychiatric deficits. Stimulants such as cocaine have been reported to alter HIV replication dynamics in macrophages and microglia and change their inflammatory state, and may contribute to neuropsychiatric deficits in PWH. A recent preprint provides the only evidence that frequent cocaine use is associated with larger HIV latent reservoir size in CD4+ T cells. Low- level/residual inflammation has been shown to foster a persistent HIV reservoir in CD4+ T cells. However, a major knowledge gap is whether cocaine exposure supports long-term HIV expression in macrophages and microglia and whether inflammation mediates this effect. A clear understanding of cocaine’s impact on HIV expression in complex multicellular contexts is a prerequisite for the identification of novel targets for effective ART regimens, adjunctive neuroprotective therapies, and latency reversal strategies. Our overarching hypothesis is that long- term HIV expression in macrophages and microglia contributes to neuropsychiatric damage which is exacerbated by cocaine. Addressing these key questions requires methods that can determine the identity of cells that harbor HIV DNA and RNA and simultaneously link the presence of HIV DNA and RNA to functional cellular outcomes via the transcriptome in the same cell. Proteogenomic approaches that combine DNA and RNA sequencing with the analysis of surface marker expression to identify cell populations provide insight into the expression of HIV DNA and RNA in specific CD4+ T cell subtypes. However, significant caveats hinder their applicability in CNS cells. We will develop a novel molecular, high-resolution, single-cell level method we call HIV integrated proviral DNA (HID)/single-nuclear RNASeq (HID-Seq). In this combined approach, dCas9-Tn5 tagmentation amplifies proviral DNA integrated in the host genome, and single-nuclear RNASeq uses validated primer libraries to detect specific HIV RNA species along with the detection of all host-cell RNA species in the same cell. HIV proviral DNA, HIV RNA, and host-cell RNA are molecularly barcoded using split-sequencing, which tracks DNA and RNA products to individual cells. In the R61 phase (Aim 1), we will develop HID-Seq for multilevel indexing of HIV DNA and RNA expression while simultaneously examining the host cell transcriptome in vitro. In the R33 phase (Aim 2), we will use HID-Seq to determine the impact of cocaine on HIV DNA and RNA expression in human induced pluripotent stem cell-derived microglia and macrophages in vitro (Aim 2a) and in the peripheral blood mononuclear cells and CNS cells obtained from autopsy specimens of PWH (Aim 2b-c). Bioinformatic analyses will (i) confirm the identity and characteristics of cells harboring HIV DNA and RNA in the CNS of PWH with history of cocaine use and (ii) determine how cocaine use alters the associations between HIV DNA and RNA and host-cell transcriptomic profile.