Male sex chromosomal complement exacerbates the pathogenicity of Th17 cells in a chronic model of central nervous system autoimmunity

MicroRNAs (miRNAs) released from the activated microglia upon neurotropic virus infection may exacerbate the neuronal damage. Here, we identified let‐7a and let‐7b (let‐7a/b) as one of the essential miRNAs over‐expressed upon Japanese Encephalitis virus (JEV) infection and released in the culture supernatant of the JEV‐infected microglial cells through extracellular vesicles. The let‐7a/b was previously reported to modulate inflammation in microglial cells through Toll‐like receptor 7 (TLR7) pathways; although their role in accelerating JEV pathogenesis remain unexplored. Therefore, we studied the role of let‐7a/b in modulating microglia‐mediated inflammation during JEV infection and investigated the effect of let‐7a/b‐containing exosomes on primary neurons. To this end, we examined let‐7a/b and NOTCH signaling pathway in TLR7 knockdown (KD) mice. We observed that TLR7 KD or inhibition of let‐7a/b suppressed the JEV‐induced NOTCH activation possibly via NF‐κB dependent manner and subsequently, attenuated JEV‐induced TNFα production in microglial cells. Furthermore, exosomes secreted from let‐7a/b over‐expressed microglia when transferred to uninfected mice brain induced caspase activation. Exosomes secreted from virus‐infected or let‐7a/b over‐expressed microglia when co‐incubated with mouse neuronal (Neuro2a) cells or primary cortical neurons also facilitated caspase activation leading to neuronal death. Thus, our results provide evidence for the multifaceted role of let‐7a/b miRNAs in JEV pathogenesis. Let‐7a/b can interact with TLR7 and NOTCH signaling pathway and enhance TNFα release from microglia. On the other hand, the exosomes secreted by JEV‐infected microglia can activate caspases in uninfected neuronal cells which possibly contribute to bystander neuronal death. Cover Image for this issue: doi: .

BackgroundActivated platelets release cytokines/proteins including CXCL4 (PF4), CCL5 and fibrinopeptides, which regulate infection of several pathogenic viruses such as HIV, H1N1 and HCV in human. Since platelet activation is the hallmark of Dengue virus (DV) infection, we investigated the role of platelets in DV replication and also in a closely related Japanese Encephalitis virus (JEV).Methods and findingsMicroscopy and PCR analysis revealed a 4-fold increase in DV replication in primary monocytes or monocytic THP-1 cells in vitro upon incubation with either DV-activated platelets or supernatant from DV-activated platelets. The mass spectrometry based proteomic data from extra-nuclear fraction of above THP-1 lysate showed the crucial association of PF4 with enhanced DV replication. Our cytokine analysis and immunoblot assay showed significant inhibition of IFN-α production in monocytes via p38MAPK-STAT2-IRF9 axis. Blocking PF4 through antibodies or its receptor CXCR3 through inhibitor i.e. AMG487, significantly rescued production of IFN-α resulting in potent inhibition of DV replication in monocytes. Further, flow cytometry and ELISA data showed the direct correlation between elevated plasma PF4 with increased viral NS1 in circulating monocytes in febrile DV patients at day-3 of fever than day-9. Similarly, PF4 also showed direct effects in promoting the JEV replication in monocytes and microglia cells in vitro. The in vitro results were also validated in mice, where AMG487 treatment significantly improved the survival of JEV infected animals. Interpretation: Our study suggests that PF4-CXCR3-IFN axis is a potential target for developing treatment regimen against viral infections including JEV and DV.

Japanese Encephalitis Virus (JEV), a globally important pathogen, belongs to the family Flaviviridae, is transmitted between vertebrate hosts by mosquitoes, principally by Culex tritaeniorhynchus. The E-glycoprotein of the virus mediates its attachment to the host cell receptors. In this study, we cloned and purified JEV E-glycoprotein in pET28a vector using E. coli BL21 (DE3) cells. A pull down assay was performed using plasma membrane fraction of BALB/c mouse brain and E-glycoprotein as a bait protein. 2-Dimensional Gel Electrophoresis based separation of the interacting proteins was analyzed by mass spectrometry. Among all the identified partners of E-glycoprotein, PLVAP (Plasmalemma vesicle associated protein) and GKN3 (Gastrokine3) showed significant up-regulation in both JEV infected mouse brain and neuro2a cells. In-silico studies also predicted significant interaction of these receptors with E-glycoprotein. Additionally, overexperssion and silencing of these receptors resulted in increase and reduction in viral load respectively, suggesting them as two critical cellular receptors governing JEV entry and propagation in neurons. In support, we observed significant expression of PLVAP but not GKN3 in post-mortem autopsied human brain tissue. Our results establish two novel receptor proteins in neurons in case of JEV infection, thus providing potential targets for antiviral research.

RNA viruses are known to modulate host microRNA (miRNA) machinery for their own benefit. Japanese encephalitis virus (JEV), a neurotropic RNA virus, has been reported to manipulate several miRNAs in neurons or microglia. However, no report indicates a complete sketch of the miRNA profile of neural stem/progenitor cells (NSPCs), hence the focus of our current study. We used an miRNA array of 84 miRNAs in uninfected and JEV-infected human neuronal progenitor cells and primary neural precursor cells isolated from aborted fetuses. Severalfold downregulation of hsa-miR-9-5p, hsa-miR-22-3p, hsa-miR-124-3p, and hsa-miR-132-3p was found postinfection in both of the cell types compared to the uninfected cells. Subsequently, we screened for the target genes of these miRNAs and looked for the biological pathways that were significantly regulated by the genes. The target genes involved in two or more pathways were sorted out. Protein-protein interaction (PPI) networks of the miRNA target genes were formed based on their interaction patterns. A binary adjacency matrix for each gene network was prepared. Different modules or communities were identified in those networks by community detection algorithms. Mathematically, we identified the hub genes by analyzing their degree centrality and participation coefficient in the network. The hub genes were classified as either provincial (P < 0.4) or connector (P > 0.4) hubs. We validated the expression of hub genes in both cell line and primary cells through qRT-PCR after JEV infection and respective miR mimic transfection. Taken together, our findings highlight the importance of specific target gene networks of miRNAs affected by JEV infection in NSPCs. IMPORTANCE JEV damages the neural stem/progenitor cell population of the mammalian brain. However, JEV-induced alteration in the miRNA expression pattern of the cell population remains an open question, hence warranting our present study. In this study, we specifically address the downregulation of four miRNAs, and we prepared a protein-protein interaction network of miRNA target genes. We identified two types of hub genes in the PPI network, namely, connector hubs and provincial hubs. These two types of miRNA target hub genes critically influence the participation strength in the networks and thereby significantly impact up- and downregulation in several key biological pathways. Computational analysis of the PPI networks identifies key protein interactions and hubs in those modules, which opens up the possibility of precise identification and classification of host factors for viral infection in NSPCs.