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. Overall design: 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.

HIV相关神经认知障碍（HIV Associated Neurocognitive Disorder, HAND）是一类以认知与神经功能损害为行为学特征，以神经炎症、突触完整性与功能丧失为病理学特征的疾病。尽管现有治疗手段可延长HIV感染者的生存期，但无法有效阻断HIV诱导的神经元损伤，HAND的患病率几乎未发生改变。我们在死于HIV感染且伴脑部病理改变的个体的死后中枢神经系统（Central Nervous System, CNS）标本中，观察到ephrin-B/EphB的表达上调。鉴于此前已明确EphB2对外周炎症的调控作用，本研究评估了EphB2介导的ephrin-B反向信号在小胶质细胞中的功能角色。结果观测到促炎与抗病毒的mRNA表达谱。该刺激诱导产生的条件培养基（包含分泌型炎症因子）足以引发非细胞接触依赖性的神经毒性。最后，敲低小胶质细胞的EphB2结合蛋白ephrin-B1后，可部分缓解小胶质细胞的促炎特征，并减轻后续的神经毒性。本研究证实，EphB2表达升高及其通过小胶质细胞内ephrin-B1介导的反向信号，可驱动神经炎症与神经毒性，提示该通路可介导神经HIV中的神经毒性。此外，我们提出该神经炎症机制可能在其他神经退行性疾病的发生发展中发挥作用。整体实验设计：将HMC3细胞用ephrin-B1小干扰RNA（small interfering RNA, siRNA）或非靶向阴性对照siRNA转染48小时，随后用预聚集的EphB2-Fc或对照-Fc处理24小时。预聚集操作通过冰上孵育1小时、每15分钟混匀一次完成。经24小时EphB2-Fc或对照-Fc处理后，使用Qiagen公司的含1%β-巯基乙醇的RLT缓冲液裂解HMC3细胞以提取RNA。对提取的RNA进行质量质控后进行测序。