West Nile virus (WNV) is a neurotropic flavivirus that can cause significant neurological disease. Mouse models of WNV infection demonstrate that a proinflammatory environment is induced within the central nervous system (CNS) after WNV infection, leading to entry of activated peripheral immune cells. We utilized ex vivo spinal cord slice cultures (SCSC) to demonstrate that anti-inflammatory mechanisms may also play a role in WNV-induced pathology and/or recovery. Microglia are a type of macrophage that function as resident CNS immune cells. Similar to mouse models, infection of SCSC with WNV induces the upregulation of proinflammatory genes and proteins that are associated with microglial activation, including the microglial activation marker Iba1 and CC motif chemokines CCL2, CCL3, and CCL5. This suggests that microglia assume a proinflammatory phenotype in response to WNV infection similar to the proinflammatory (M1) activation that can be displayed by other macrophages. We now show that the WNV-induced expression of these and other proinflammatory genes was significantly decreased in the presence of minocycline, which has antineuroinflammatory properties, including the ability to inhibit proinflammatory microglial responses. Minocycline also caused a significant increase in the expression of anti-inflammatory genes associated with alternative anti-inflammatory (M2) macrophage activation, including interleukin 4 (IL-4), IL-13, and FIZZ1. Minocycline-dependent alterations to M1/M2 gene expression were associated with a significant increase in survival of neurons, microglia, and astrocytes in WNV-infected slices and markedly decreased levels of inducible nitric oxide synthase (iNOS). These results demonstrate that an anti-inflammatory environment induced by minocycline reduces viral cytotoxicity during WNV infection in ex vivo CNS tissue.

IMPORTANCE West Nile virus (WNV) causes substantial morbidity and mortality, with no specific therapeutic treatments available. Antiviral inflammatory responses are a crucial component of WNV pathology, and understanding how they are regulated is important for tailoring effective treatments. Proinflammatory responses during WNV infection have been extensively studied, but anti-inflammatory responses (and their potential protective and reparative capabilities) following WNV infection have not been investigated. Minocycline induced the expression of genes associated with the anti-inflammatory (M2) activation of CNS macrophages (microglia) in WNV-infected SCSC while inhibiting the expression of genes associated with proinflammatory (M1) macrophage activation and was protective for multiple CNS cell types, indicating its potential use as a therapeutic reagent. This ex vivo culture system can uniquely address the ability of CNS parenchymal cells (neurons, astrocytes, and microglia) to respond to minocycline and to modulate the inflammatory environment and cytotoxicity in response to WNV infection without peripheral immune cell involvement.

西尼罗河病毒（WNV）是一种神经营养性黄病毒，可以引起严重的神经系统疾病。WNV感染的小鼠模型表明，WNV感染后在中枢神经系统（CNS）内诱导了促炎环境，导致活化的外周免疫细胞进入。我们离体利用脊髓切片培养物（SCSC）证明抗炎机制也可能在WNV诱导的病理和/或恢复中起作用。小胶质细胞是一种巨噬细胞，可作为常驻中枢神经系统免疫细胞。与小鼠模型相似，用WNV感染SCSC会诱导与小胶质细胞激活相关的促炎基因和蛋白质的上调，包括小胶质细胞激活标记Iba1和CC基序趋化因子CCL2，CCL3和CCL5。这表明小胶质细胞假定对WNV感染具有促炎表型，类似于其他巨噬细胞可以显示的促炎（M1）激活。我们现在显示，在米诺环素存在的情况下，WNV诱导的这些和其他促炎基因的表达显着降低，而米诺环素具有抗神经炎特性，包括抑制促炎性小胶质细胞反应的能力。米诺环素还引起与替代性抗炎（M2）巨噬细胞激活相关的抗炎基因表达的显着增加，包括白介素4（IL-4），IL-13和FIZZ1。M1 / M2基因表达的米诺环素依赖性改变与WNV感染切片中神经元，小胶质细胞和星形胶质细胞的存活率显着增加以及诱导型一氧化氮合酶（iNOS）水平显着降低有关。这些结果表明，米诺环素诱导的抗炎环境降低了WNV感染期间的病毒细胞毒性。米诺环素还引起与替代性抗炎（M2）巨噬细胞激活相关的抗炎基因表达的显着增加，包括白介素4（IL-4），IL-13和FIZZ1。M1 / M2基因表达的米诺环素依赖性改变与WNV感染切片中神经元，小胶质细胞和星形胶质细胞的存活率显着增加以及诱导型一氧化氮合酶（iNOS）水平显着降低有关。这些结果表明，米诺环素诱导的抗炎环境降低了WNV感染期间的病毒细胞毒性。米诺环素还引起与替代性抗炎（M2）巨噬细胞激活相关的抗炎基因表达的显着增加，包括白介素4（IL-4），IL-13和FIZZ1。M1 / M2基因表达的米诺环素依赖性改变与WNV感染切片中神经元，小胶质细胞和星形胶质细胞的存活率显着增加以及诱导型一氧化氮合酶（iNOS）水平显着降低有关。这些结果表明，米诺环素诱导的抗炎环境降低了WNV感染期间的病毒细胞毒性。WNV感染切片中的星形胶质细胞和星形胶质细胞，诱导型一氧化氮合酶（iNOS）的水平明显降低。这些结果表明，米诺环素诱导的抗炎环境降低了WNV感染期间的病毒细胞毒性。WNV感染切片中的星形胶质细胞和星形胶质细胞，诱导型一氧化氮合酶（iNOS）的水平明显降低。这些结果表明，米诺环素诱导的抗炎环境降低了WNV感染期间的病毒细胞毒性。离体中枢神经系统组织。

重要性西尼罗河病毒（WNV）导致大量发病和死亡，没有可用的特殊治疗方法。抗病毒炎症反应是WNV病理学的重要组成部分，了解其调控方式对于制定有效的治疗方法非常重要。已对WNV感染期间的促炎反应进行了广泛研究，但尚未研究WNV感染后的抗炎反应（及其潜在的保护和修复能力）。Minocycline诱导WNV感染的SCSC中CNS巨噬细胞（小胶质细胞）抗炎（M2）激活相关基因的表达，同时抑制与促炎（M1）巨噬细胞激活相关的基因表达，并保护多种CNS细胞类型，表明其可能用作治疗试剂。离体培养系统可以独特地解决中枢神经系统实质细胞（神经元，星形胶质细胞和小胶质细胞）对米诺环素作出反应并调节炎性环境和细胞毒性的能力，以响应WNV感染而无需外周免疫细胞参与的能力。