CCR2 Inhibition Reduces Neurotoxic Microglia Activation Phenotype After Japanese Encephalitis Viral Infection.

Swati Singh,Gajendra Singh,Alok Kumar,Swasti Tiwari

doi:10.3389/fncel.2020.00230

Abstract

Controlling the proinflammatory response of microglia by targeting chemokines (C-C motif) receptor 2 (CCR2) could be an important therapeutic approach for Japanese encephalitis virus (JEV) infection. Here, through JEV infection to BV2 microglia and young BALB/c mice, we investigated that CCR2 is highly upregulated after JEV infection and plays a key role in determining microglia activation phenotype and associated with neurotoxic proinflammatory mediators of TNF-α and IFNγ. In addition, we found JEV infection to BV2 microglia causes an increase in microglial proliferation and cell body area at day 1 and day 3. Using the agonist molecule of CCR2 inhibition; RS102895, significantly reduces microglia reactive phenotype and nitric oxide production. Further, to define the role of CCR2 in functional responses of microglia and their activation phenotype, we performed in vitro cell scratch functional assay and ImageJ analysis. When compared with control, microglia cells showed a significant increase in elongated or rod-like activated phenotype in JEV-infected cells at 24 h post-infection and CCR2 inhibition significantly reduced the elongated activation phenotype induced by JEV infection, suggesting that CCR2 acts as a critical regulator for microglia activation phenotype after JEV infection. We found that JEV-infected mice treated with RS102895 had less microglia activation and reduced mRNA expression of CCR2 and proinflammatory mediators such as IFN-γ in cortical tissue. Collectively, our data indicate that CCR2 drives reactive phenotype of microglia and its inhibition reduces microglia activation and neurotoxic proinflammatory mediators after JEV infection.

Highlights

Both experimental and clinical studies demonstrated that uncontrolled neuroinflammatory responses of central nervous system in Japanese encephalitis virus (JEV) infection is a major contributor to cell death and neurological dysfunction (Chen et al, 2012)
We found that post-JEV infection, CCR2 expression is significantly increased and associated with reactive phenotype of microglia and subsequent production of proinflammatory mediators of TNF-α and IFNγ
Our results demonstrated that CCR2 inhibition is an important target for controlling reactive microglia morphology and neuroinflammatory response of JEV infection

Summary

Introduction

Both experimental and clinical studies demonstrated that uncontrolled neuroinflammatory responses of central nervous system in Japanese encephalitis virus (JEV) infection is a major contributor to cell death and neurological dysfunction (Chen et al, 2012). JEV infection induced a neuroinflammatory response including microglia activation that further found engaged in release of proinflammatory mediators (e.g., TNF-α, IFN-γ) along with production of reactive oxygen species that contribute to neuronal cell death in bystander fashion (Das et al, 2008; Thongtan et al, 2010; Chen et al, 2012). In some studies, of microglia migration dynamics, alteration in their morphology including activation from resting phenotype, and adaptation of phagocytic morphologies are observed in association with their neuroinflammatory responses and neuronal cell death after JEV infection (Kreutzberg, 1996; Ghoshal et al, 2007; Kettenmann et al, 2011; Sips et al, 2012). The aims of the current study are: (1) to test the hypothesis that CCR2 activation is associated with proinflammatory response of microglia, and (2) to determine that systemic administration of CCR2 antagonist molecule RS102895 reduces proinflammatory response of microglia in JEV-infected mice

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Conclusion