Lack of Interferon (IFN) Regulatory Factor 8 Associated with Restricted IFN-γ Response Augmented Japanese Encephalitis Virus Replication in the Mouse Brain

Abstract

Interferon Regulatory Factor 8 (IRF8), a myeloid lineage transcription factor, emerges as an essential regulator for microglia activation. However, the precise role of IRF8 during Japanese encephalitis virus (JEV) infection in the brain remains elusive. Here we report that JEV infection enhances IRF8 expression in the infected mice brain. Comparative transcriptional profiling of whole-brain RNA analysis and validation by qRT-PCR reveals an impaired IFNγ and related gene expression in Irf8 knockout ( Irf8 -/- ) infected mice. Further, Ifnγ knockout ( Ifnγ -/- ) mice exhibit a reduced level of Irf8. Both Ifnγ -/- and Irf8 -/- mice exhibit significantly reduced levels of activated (CD11b + CD45 hi , CD11b + CD45 lo , Cd68, and CD86 ) and infiltrating immune cells (Ly6C + , CD4, and CD8) in the infected brain as compared to WT mice. However, a higher level of granulocyte cells (Ly6G + ) infiltration is evident in Irf8 -/- mice and the increased concentration of TNFα, IL6, MCP1 levels in the brain. Interestingly, neither Irf8 -/- nor Ifnγ -/- has conferred protection against lethal JEV challenge to mice and exhibits augmentation in JEV replication in the brain. The gain of function of Irf8 by overexpressing functional IRF8 in an IRF8 deficient cell line attenuates viral replication and enhances IFNγ production. Overall, we summarise that in the murine model of JEV encephalitis, IRF8 modulation affects JEV replication. We also evidence that lack of Irf8 affects immune cells abundance in circulation and the infected brain leading to a reduction in IFNγ level and increased viral load in the brain. Importance Microglial cells, the resident macrophages in the brain, play a vital role in Japanese encephalitis virus (JEV) pathogenesis. The deregulated activity of microglia can be lethal for the brain. Therefore, it is crucial to understand the regulators that drive microglia's phenotype changes and induce inflammation in the brain. Interferon regulatory factor 8 (IRF8) is a myeloid lineage transcription factor involved in microglial activation. However, the impact of IRF8 modulation on JEV replication remains elusive. Moreover, the pathways regulated by IRF8 to initiate and amplify pathological neuroinflammation are not well understood. Here, we demonstrated the effect of IRF8 modulation on JEV replication, microglial activation, and immune cells infiltration in the brain.

Neurovirulence of the Australian outbreak Japanese Encephalitis virus genotype 4 is lower compared to genotypes 2 and 3 in mice and human cortical brain organoids

“…To provide additional potential insights into the nature of the cellular infiltrates (e.g. perivascular cuffing and leukocyte infiltrates, Figure 6), immune cell type abundance estimates were obtained from reanalyzed (59) publically available JEV-infected mouse brain RNA-Seq expression data (60) using SpatialDecon (61). The dominant cell types identified were CD4 T cells, NKT cells, monocytes/macrophages, neutrophils, innate lymphoid cells, T regs, and CD8 T cells, with an increase in microglia cells also identified (Supplementary Figure 12).…”

“…A publically available RNA-Seq data (raw read counts) of C57BL/6 brain infection with JEV isolate P20778 (Vellore strain, genotype 3) 52 (GEO accession: GSE154002) was reanalyzed 53 to provide a list of differentially expressed genes (DEGs) for infected brains versus uninfected brains from C57BL/6 mice. GFAP was significantly up-regulated 5.4 fold by infection (Supplementary Table 2, log2FC 2.4), consistent with the GFAP IHC (Figure 2C) and reactive astrogliosis seen herein.…”

Acquisition of Immune Privilege in GBM Tumors: Role of Prostaglandins and Bile Salts

“…IRF4 expression drives Teff > Treg in tumor-infiltrating T cells [ 117 , 118 ] via PRDM1 to generate IL10-expressing Tregs [ 119 ]. IRF8 is a significant controller of microglial and macrophage activation [ 114 , 115 , 120 ]. IRF8 causes an upregulation of SPI1 [ 89 ] and a downregulation of TCF4 [ 121 ] and is critical for microglial expansion and activation [ 120 , 122 ].…”

“…IRF8 is a significant controller of microglial and macrophage activation [ 114 , 115 , 120 ]. IRF8 causes an upregulation of SPI1 [ 89 ] and a downregulation of TCF4 [ 121 ] and is critical for microglial expansion and activation [ 120 , 122 ]. These two expression factors may be used in a cross-correlation analysis to differentiate between Treg and microglial-rich tumors.…”

Characterisation of a Japanese Encephalitis virus genotype 4 isolate from the 2022 Australian outbreak

“…To provide additional potential insights into the nature of the cellular infiltrates (e.g., perivascular cuffing and leukocyte infiltrates, Fig. 6), immune cell type abundance estimates were obtained from reanalyzed 63 publically available JEV-infected mouse brain RNA-Seq expression data 64 using SpatialDecon 65 . The dominant cell types identified were CD4 T cells, NKT cells, monocytes/macrophages, neutrophils, innate lymphoid cells, T regs, and CD8 T cells, with an increase in microglia cells also identified (Supplementary Fig.…”