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

摘要 在嗜神经病毒感染期间，维持中枢神经系统（CNS）的稳态至关重要 宿主的生存。CNS的炎症的特征在于白细胞向脑的募集和白细胞向神经元的转移。 活化驻留的CNS细胞，包括星形胶质细胞。星形胶质细胞在病毒感染中的关键作用 脑炎的定义不明确。Toll样受体3（TLR 3）是一种干扰素诱导的双链RNA传感器 能感知病毒感染尽管众所周知星形胶质细胞表达功能性TLR 3，但其在应答中的作用 CNS的病毒RNA感染仍然知之甚少。转录因子和转录因子之间的相互作用 病毒性脑炎中的microRNA及其上游模式识别受体尚未被广泛研究。 研究了microRNA-155（miR-155）在TLR反应的微调中具有关键作用，并发挥积极的免疫调节作用。 和炎症过程中的负调节。我们和其他人已经确定miR-155是最多的一个- 在巨噬细胞中用合成的dsRNA激动剂Poly（I：C）刺激TLR 3后表达miRNA。在 在CNS中，miR-155也在神经胶质细胞中表达。然而，miR-155在CNS中的内在作用尚未确定。 vivo.信号转导和转录激活因子（STAT）蛋白及其负调节因子， 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中起着重要作用， 生长因子最近，已经在miR-155的启动子中鉴定了两个STAT 3结合位点。此外，委员会认为， miR-155靶向SOCS 1和SOCS 3，抑制其对JAK/STAT信号转导的负调节作用 通路冠状病毒（CoV）是有包膜的、正义的、单链的、多腺苷酸化的RNA病毒。 小鼠肝炎病毒JHM（MHV-JHM）是一种中枢神经系统嗜性毒株，可引起致命性脑炎，与 非保护性的、增强的巨噬细胞和中性粒细胞浸润，与趋化因子恶化相关 和Th 1应答，没有Th 17参与。我们已经鉴定了TLR 3依赖的miR-155 /STAT 3调控的 环，促进恶化，有害的神经炎症和血脑屏障（BBB）破坏， 对MHV-JHM致命感染的反应。有趣的是，TRIF，唯一已知的TLR 3适配器，对于TLR 3- STAT 3的依赖性活化和miR-155的诱导。我们已经鉴定出星形胶质细胞 以及浸润脑的炎性单核细胞作为驱动STAT 3活化加剧的细胞源。我们 这些发现促使我们研究以下假设：星形胶质细胞中的AIM 1 MHV-JHM感染， 巨噬细胞通过TRIF非依赖性、TLR 3依赖性非典型轴诱导STAT 3活化， 受其衔接子TRIF负调控; AIM 2 STAT 3和miR-155的细胞类型特异性基因消融 在星形胶质细胞或骨髓细胞中的表达将减少有害的神经炎症并增加宿主存活 在致死性MHV-JHM感染后; AIM 3 TLR 3通过早期和持续的免疫抑制作用促进BBB破坏。 星形胶质细胞中miR-155表达上调，其将外泌体-miR 155递送至脑内皮细胞 影响AJPs、TJPs和/或MMP的表达（在AIM 3中鉴定）。

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.