A TLR3-STAT3-miR-155 axis and astrocyte-myeloid crosstalk in viral encephalitis

摘要 嗜神经性病毒感染期间中枢神经系统(CNS)内稳态的维持是至关重要的 为了宿主的生存。中枢神经系统炎症的特征是白细胞重新聚集到大脑和 激活驻留的中枢神经系统细胞，包括星形胶质细胞。星形胶质细胞在病毒感染过程中的体内关键功能 脑炎的定义并不明确。Toll样受体3(TLR3)是一种干扰素诱导的双链RNA传感器 它能感觉到病毒感染。虽然众所周知，星形胶质细胞表达功能性的TLR3，但它在反应中的作用 对病毒RNA感染中枢神经系统仍知之甚少。转录因子和转录因子之间的相互作用 MicroRNAs及其上游模式识别受体在病毒性脑炎中的作用尚未得到广泛研究 学习。MicroRNA-155(miR-155)在TLR反应的微调中起关键作用，并发挥两种积极作用 炎症过程中的负性调节。我们和其他人已经确定miR-155是最多的- 用合成的dsRNA激动剂Poly(I：C)刺激TLR3后，巨噬细胞表达miRNAs。在 CNS、miR-155在神经胶质细胞中也有表达。然而，miR-155在中枢神经系统中的内在作用尚未在 活着。信号转导和转录激活子(STAT)蛋白及其负调控因子， 细胞因子信号转导抑制因子(SOCS)蛋白家族在调节多种细胞因子和 增长因素。最近，在miR-155的启动子中发现了两个STAT3结合位点。此外， MIR-155靶向SOCS1和SOCS3抑制其对JAK/STAT信号的负调控作用 路径。冠状病毒(CoV)是一种包膜、阳性、单链、多腺化的RNA病毒。 小鼠肝炎病毒JHM(MHV-JHM)是一种嗜中枢神经系统的毒株，可引起致命性脑炎，与 与加剧的趋化因子相关的非保护性、增强的巨噬细胞和中性粒细胞的渗透 Th1应答，无Th17参与。我们已经鉴定出一种依赖TLR3的miR-155/STAT3调节性基因 促进加重的、有害的神经炎和血脑屏障(BBB)破坏的环路 对MHV-JHM致命感染的反应。有趣的是，TLR3不需要TRIF，这是已知的唯一TLR3适配器- 体内和体外对STAT3的依赖激活和miR-155的诱导。我们已经鉴定出星形胶质细胞 炎症单核细胞作为推动STAT3激活的细胞来源渗入大脑，加剧了STAT3的激活。我们的 这些发现促使我们研究了以下假设：目的1 MHV-JHM在星形胶质细胞和 巨噬细胞通过TRIF非依赖、TLR3依赖的非正规轴诱导STAT3激活 受其接头TRIF的负调控；目的2细胞类型特异性的STAT3和miR-155的遗传消融 在星形胶质细胞或髓系细胞中的表达将减少有害的神经炎症并提高宿主的存活率 在致命的MHV-JHM感染后，AIM 3TLR3通过早期和持续的方式促进血脑屏障的破坏 星形胶质细胞中miR-155表达上调，星形胶质细胞可向脑内皮细胞输送外切体-miR155 影响AJPs、TJPs和/或MMPs的表达(在AIM 3中有待鉴定)。

ABSTRACT The maintenance of homeostasis in the central nervous system (CNS) during neurotropic viral infections is critical for host survival. Inflammation of the CNS is characterized by the recruitment of leukocytes to the brain and the activation of resident CNS cells, including astrocytes. The pivotal in vivo function of astrocytes during viral encephalitis is poorly defined. Toll like receptor 3 (TLR3) is an interferon-inducing double stranded RNA sensor that senses viral infections. Although it is well known that astrocytes express functional TLR3, its role in response to viral RNA infections of the CNS remains poorly understood. The interplay between transcription factors and microRNAs, and their upstream pattern recognition receptors during viral encephalitis has not been extensively studied. MicroRNA-155 (miR-155) has a key role of in the fine-tuning of TLR responses, and exerts both positive and negative regulation during inflammation. We and others have identified miR-155 as one of the most over- expressed miRNAs after stimulation of TLR3 with the synthetic dsRNA agonist Poly (I:C) in macrophages. In the CNS, miR-155 is also expressed in glial cells. However, a CNS-intrinsic role of miR-155 has yet to be defined in vivo. Signal transducers and activators of transcription (STAT) proteins and their negative regulators, the suppressors of cytokine signaling (SOCS) protein family play central roles in regulating many cytokines and growth factors. Recently, two STAT3 binding sites have been identified in the promoter of miR-155. Furthermore, miR-155 targets SOCS1 and SOCS3 suppressing their negative regulatory effect on JAK/STAT signaling pathway. Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded, polyadenylated RNA viruses. Mouse hepatitis virus JHM (MHV-JHM) is a CNS-tropic strain that induces fatal encephalitis, associated with non-protective, enhanced macrophage and neutrophil infiltration that correlates with exacerbated chemokine and Th1 response and no Th17 involvement. We have identified a TLR3-dependent, miR-155 /STAT3 regulatory loop that promotes exacerbated, detrimental neuroinflammation and blood brain barrier (BBB) disruption in response to MHV-JHM fatal infection. Intriguingly, TRIF, the only TLR3 adaptor known, is not required for TLR3- dependent activation of STAT3 and induction of miR-155 both in vitro and in vivo. We have identified astrocytes and inflammatory monocytes infiltrating the brain as the cell sources driving exacerbated STAT3 activation. Our findings prompted us to investigate the following hypotheses: AIM 1 MHV-JHM infection in astrocytes and macrophages induces STAT3 activation through a TRIF-independent, TLR3-dependent non-canonical axis that is negatively regulated by its adaptor TRIF; AIM 2 Cell type specific genetic ablation of STAT3 and miR-155 expression in astrocytes or myeloid cells will reduce detrimental neuroinflammation and increase host survival after fatal MHV-JHM infection; and AIM 3 TLR3 contributes to BBB disruption through early and sustained upregulation of miR-155 expression in astrocytes, which deliver exosome-miR155 to brain endothelial cells affecting the expression of (to be identified in AIM 3) AJPs, TJPs, and/or MMPs.