NLRP3 inflammasome-related microglial pyroptosis in EcoHIV infected mice
HIV-associated neurocognitive disorders (HAND) have become a major clinical concern, particularly among the aging HIV-1-seropositive population, which is generally characterized by persistent viral reservoirs and a lower level of chronic inflammation. NLRP3 inflammasome activation exhibits its unique role in the progression of many chronic inflammatory diseases. Furthermore, pyroptosis, an inflammatory form of programmed cell death, has been implicated in numerous neurological diseases. However, the mechanisms linking EcoHIV infection, microglial pyroptosis, and NLRP3 inflammasome activation remain incompletely understood. In this study, EcoHIV was retro-orbitally injected into C57BL/6J wild-type mice and analyzed at 14-, 30-, 60-, and 90-days post-infection to establish a NeuroHIV model. Additionally, in vitro, BV2 microglial cell line was infected with EcoHIV and treated with MCC950, an inhibitor of the NLRP3 inflammasome, for three days. Pyroptosis marker GSDMD, NLRP3 inflammasome components, Caspase-1 (a marker of inflammasome activation), HLA-DR (an immune activation marker), Programmed-death 1 (PD-1, an immune checkpoint molecule), and Ki67 (a cellular proliferation marker) were assessed by immunofluorescence staining. Results showed that EcoHIV-infected mice showed a peak in NLRP3 expression at 14 days post-infection, compared with controls, followed by a modest decline at 30 days, while GSDMD expression increased progressively across 14 and 30 days. These findings demonstrate dynamic changes in microglial pyroptosis and NLRP3 inflammasome activation over the course of EcoHIV infection. In vitro, EcoHIV-infected BV2 cells exhibited significantly increased EcoHIV-eGFP fluorescence compared with controls, confirming the utility of BV2 cells as an in vitro model of microglial EcoHIV infection. Expression levels of GSDMD and NLRP3 were elevated following infection, indicating enhanced pyroptosis and neuroinflammation. Treatment with MCC950 significantly reduced the expression of GSDMD, NLRP3, HLA-DR, PD-1, and Ki67, suggesting that inhibition of NLRP3 inflammasome activity suppresses both pyroptosis and microglial activation and proliferation. Together, elucidating the interplay between microglial pyroptosis and NLRP3 inflammasome activation may provide new insights into the pathogenesis and potential therapeutic strategies for NeuroHIV in the aging HIV-1-seropositive population.