Department of Medicine, Neurovirology Laboratory, University of Minnesota Medical School, 3-107 Microbiology Research Facility, 689 23rd Ave. S.E, Minneapolis, MN, 55455, USA.
J Neuroinflammation. 2018 Mar 5;15(1):66. doi: 10.1186/s12974-018-1107-7.
Peripheral neuropathy is currently the most common neurological complication in HIV-infected individuals, occurring in 35-50% of patients undergoing combination anti-retroviral therapy. Data have shown that distal symmetric polyneuropathy develops in mice by 6 weeks following infection with the LP-BM5 retrovirus mixture. Previous work from our laboratory has demonstrated that glial cells modulate antiviral T-cell effector responses through the programmed death (PD)-1: PD-L1 pathway, thereby limiting the deleterious consequences of unrestrained neuroinflammation.
Using the MouseMet electronic von Frey system, we assessed hind-paw mechanical hypersensitivity in LP-BM5-infected wild-type (WT) and PD-1 KO animals. Using multi-color flow cytometry, we quantitatively assessed cellular infiltration and microglial activation. Using real-time RT-PCR, we assessed viral load, expression of IFN-γ, iNOS, and MHC class II. Using western blotting, we measured protein nitrosylation within the lumbar spinal cord (LSC) and dorsal root ganglion (DRG). Histochemical staining was performed to analyze the presence of CD3, ionized calcium binding adaptor molecule (Iba)-1, MHCII, nitrotyrosine, isolectin B4 (IB4) binding, and neurofilament 200 (NF200). Statistical analyses were carried out using graphpad prism.
Hind-paw mechanical hypersensitivity observed in LP-BM5-infected animals was associated with significantly increased lymphocyte infiltration into the spinal cord and DRG. We also observed elevated expression of IFN-γ (in LSC and DRG) and MHC II (on resident microglia in LSC). We detected elevated levels of 3-nitrotyrosine within the LSC and DRG of LP-BM5-infected animals, an indicator of nitric oxide (NO)-induced protein damage. Moreover, we observed 3-nitrotyrosine in both small (IB4) and large (NF200) DRG sensory neurons. Additionally, infected PD-1 KO animals displayed significantly greater mechanical hypersensitivity than WT or uninfected mice at 4 weeks post-infection (p.i.). Accelerated onset of hind-paw hypersensitivity in PD-1 KO animals was associated with significantly increased infiltration of CD4 and CD8 T lymphocytes, macrophages, and microglial activation at early time points. Importantly, we also observed elevated levels of 3-nitrotyrosine and iNOS in infected PD-1 KO animals when compared with WT animals.
Results reported here connect peripheral immune cell infiltration and reactive gliosis with nitrosative damage. These data may help elucidate how retroviral infection-induced neuroinflammatory networks contribute to nerve damage and neuropathic pain.
周围神经病变是目前 HIV 感染者最常见的神经并发症，在接受联合抗逆转录病毒治疗的患者中发生率为 35-50%。数据显示，LP-BM5 逆转录病毒混合物感染后 6 周，小鼠会发生远端对称性多神经病。我们实验室的先前工作表明，通过程序性死亡 (PD)-1: PD-L1 途径，神经胶质细胞调节抗病毒 T 细胞效应反应，从而限制神经炎症失控的有害后果。
使用 MouseMet 电子 von Frey 系统，我们评估了 LP-BM5 感染的野生型 (WT) 和 PD-1 KO 动物的后爪机械性超敏反应。使用多色流式细胞术，我们定量评估细胞浸润和小胶质细胞激活。使用实时 RT-PCR，我们评估病毒载量、IFN-γ、iNOS 和 MHC Ⅱ类的表达。使用 Western blot，我们测量了腰椎脊髓 (LSC) 和背根神经节 (DRG) 中的蛋白硝化。进行组织化学染色以分析 CD3、离子钙结合接头分子 (Iba)-1、MHCII、硝基酪氨酸、异硫氰酸荧光素 B4 (IB4) 结合和神经丝 200 (NF200) 的存在。使用 GraphPad Prism 进行统计分析。
LP-BM5 感染动物观察到的后爪机械性超敏反应与脊髓和 DRG 中淋巴细胞浸润显著增加有关。我们还观察到 IFN-γ (在 LSC 和 DRG 中) 和 MHC II (在 LSC 中的常驻小胶质细胞上) 的表达升高。我们在 LP-BM5 感染动物的 LSC 和 DRG 中检测到 3-硝基酪氨酸水平升高，这是一氧化氮 (NO) 诱导的蛋白损伤的指标。此外，我们还在小 (IB4) 和大 (NF200) DRG 感觉神经元中观察到 3-硝基酪氨酸。此外，与 WT 或未感染的小鼠相比，感染 PD-1 KO 动物在感染后 4 周时表现出明显更大的机械性超敏反应 (p.i.)。PD-1 KO 动物后爪超敏反应的加速发作与 CD4 和 CD8 T 淋巴细胞、巨噬细胞和小胶质细胞激活的早期显著增加有关。重要的是，与 WT 动物相比，我们还在感染的 PD-1 KO 动物中观察到 3-硝基酪氨酸和 iNOS 水平升高。
这里报告的结果将外周免疫细胞浸润和反应性神经胶质与硝化损伤联系起来。这些数据可能有助于阐明逆转录病毒感染诱导的神经炎症网络如何导致神经损伤和神经病理性疼痛。
