EphB2-mediated ephrin-B reverse signaling on microglia drives an anti-viral, but inflammatory and neurotoxic response associated with HIV

Background: Pathological inflammation with a loss of synaptic integrity and function has been implicated in HIV Associated Neurocognitive Disorders (HAND). Although therapeutics exist to increase the lifespan of people living with HIV (PLWH), they are not effective at preventing neuroinflammation and HIV induced neuronal damage persists. In this study, we investigate the ephrin-B/EphB axis, which regulates inflammation, in post-mortem brain specimen of PLWH and experimental models in order to assess its potential role in HIV induced neuroinflammation. Methods: We analyze mRNA samples of post-mortem brain specimen of PLWH and uninfected controls obtained from the National NeuroAIDS Tissue Consortium (NNTC) and, for comparison, of a transgenic mouse model of neuroHIV using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Follow-up experiments employ mouse brain tissue and in vitro models, including immortalized human microglia, human induced pluripotent stem cell (iPSC)-derived mixed neuroglial cell cultures, cellular and molecular interference, functional and multiplex assays, immunofluorescence and mRNA sequencing to examine the role of the ephrin-B/EphB axis in neuroinflammation and the associated neurotoxicity. Results: Using qRT-PCR we find increased expression of EphB2 in post-mortem brain of PLWH, and detect a correlation with pro-viral DNA, viral RNA and an inverse correlation with abstract executive function and verbal fluency. Increased expression of ephrin-B/EphB at mRNA and protein level is also observed in brains of a transgenic mouse model of neuroHIV suggesting the upregulation can be driven, at least in part, by expression of viral gp120 envelope protein and a type I interferon, IFNβ. Additionally, we find induction of ephrin-B1 expression in microglia following activation by IFNβ. Given the previously reported impact of EphB2 on inflammation in the periphery, the functional role of EphB2-mediated ephrin-B reverse signaling on microglia is assessed for a pro-inflammatory and anti-viral signature. We find that EphB2 treated microglia secrete inflammatory and anti-viral factors but also exert contact-independent neurotoxicity. Finally, knockdown of microglial ephrin-B1, an EphB2 binding partner, shows a partial alleviation of the microglial pro-inflammatory signature and neurotoxicity. Conclusion: Our study suggests that elevated EphB2, and its reverse signaling through ephrin-B1 in microglia contribute to neuroinflammation and neurotoxicity in neuroHIV.