ABSTRACT Persistent immune activation is the defining feature of HIV-1 infection in vivo and a driver of progression to end- stage AIDS. Systemic immune activation in people living with HIV (PWH) has been hypothesized to account for higher incidence of chronic inflammatory diseases, including HIV-associated neurocognitive disorders (HAND). Importantly, numerous studies have demonstrated that populations living with opioid-use disorder (OUD) have a higher occurrence of comorbid HIV infection, suggesting both potential socio-economic and neurocognitive influence. This comorbidity of OUD and HIV has also been shown to increase neuroinflammation and exacerbate the progression of HIV infection and HAND. The proposal will primarily focus on macrophages and CNS-resident innate immune cell, microglia, which both serve as the primary virus reservoirs in the CNS. While some studies have investigated the interactive effects of OUD and HAND in myeloid cells of non-human origin or transgenic rodent models, the molecular mechanisms linking the two conditions in primary human microglia is still largely unknown. Hence, we believe, this proposal fulfills a critical unmet need by establishing primary human iPSC- derived microglia cultures to systematically interrogate the effect of HIV infection and opioids on inflammasome activation. This application will attempt to delineate the molecular mechanisms by which HIV-1 infection activates inflammasome activation in microglia and how opioid exposure degrades auto-inhibitory mechanisms that restrain spontaneous activation of the inflammasome, and is responsive to the FOA, “Targeting Inflammasomes in Substance Abuse and HIV (RFA-DA-24-003)”. We will utilize detailed molecular and mechanistic analysis to identify the microglia-intrinsic pathway that primes and activates NLRP1 inflammasome upon co-exposure to HIV and opiates. Specifically, we will determine if HIV-1 intron-containing RNA (icRNA) expression and binding of HIV-1 icRNA by NLRP1 in microglia induces inflammasome activation. We will next determine if opioid exposure enhances expression of HIV icRNA in microglia and promotes a cellular danger state to erode negative regulatory mechanisms that prevent spurious NLRP1 inflammasome activation in microglia. We postulate that these proposed experiments will identify a microglia-intrinsic mechanism of NLRP1 inflammasome activation driven by HIV icRNA expression and opioid exposure that fuels neuroinflammation and HAND. Importantly, results from these studies will inform future therapeutic development to prevent HIV and opiate-induced neuroinflammation and prevent development of HAND in HIV+ substance use population.

PROJECT NARRATIVE Continued opioid abuse and HIV infection contribute to exacerbated neuroinflammation and neurocognitive impairments. The proposed project aims to define molecular mechanisms of opiate and HIV-induced NLRP1 inflammasome activation in iPSC-derived primary human microglia. These in vitro model systems will be leveraged to identify host pathways to prevent microgliosis and the resulting neuroinflammation, which are critical to preventing the neuropathogenesis of in HIV+ OUD+ patients.