EphB2-mediated ephrin-B reverse signaling on microglia drives an antiviral but also inflammatory response in HIV

HIV Associated Neurocognitive Disorder (HAND) is a condition behaviorally characterized by cognitive and neurological impairments, and pathologically characterized by neuroinflammation and a loss of synaptic integrity and function. Although therapeutics exist to increase the lifespan of people living with HIV, they are not effective at preventing HIV induced neuronal damage and the prevalence of HAND remains virtually unchanged. We observed an increase in expression of ephrin-B/EphB in post-mortem CNS specimen of people who lived with HIV and brain pathology. Given the previously recognized impact of EphB2 on inflammation in the periphery, the functional role of EphB2-mediated ephrin-B reverse signaling on microglia was assessed. A a pro-inflammatory and antiviral mRNA expression profile was observed. The stimulation produced condirioned media, including secreted inflammatory factors, that were sufficient to induce non-cell contact-dependent neurotoxicity. Finally, knockdown of microglial ephrin-B1, EphB2 binding partner, resulted in a partial alleviation of the microglial pro-inflammatory signature and subsequent neurotoxicity. In this study, we show that elevated EphB2, and its reverse signaling through ephrin-B1 in microglia, can drive neuroinflammation and toxicity suggesting this pathway can mediate neurotoxicity in neuroHIV. Furthermore, we propose that this neuroinflammatory mechanism may play a role in promoting other neurodegenerative diseases. HMC3 cells were transfected with ephrin-B1 siRNA or non-targeting, negative control siRNA for 48 hours prior to treatment with pre-clustered EphB2-Fc or Control-Fc for 24 hours. Pre-clustering was accomplished by incubation for 1 hour on ice, mixing every 15 minutes. Following 24 hour EphB2-Fc or Control-Fc treatment, HMC3 cells were lysed for RNA isolation using Qiagen's RLT Buffer with 1% beta-mercaptoethanol. Isolated RNA was quality-controlled and sequenced.