Viral encephalitis is characterized by a series of immunological reactions that can control virus infection in the brain, but dysregulated responses may cause excessive inflammation and brain damage. Microglia are brain-resident myeloid cells that are specialized on surveillance of the local environment and in case of viral brain infection they contribute to control of the infection and restriction of viral dissemination. Here, we report that after exposure to neurotropic vesicular stomatitis virus (VSV), murine in vitro microglia cultures showed rapid upregulation of a broad range of pro-inflammatory and antiviral genes, which were stably expressed over the entire 8 hours infection period. Additionally, a set of immunomodulatory genes was upregulated between 6 and 8 hours post infection. In in vitro microglia, the induction of several immune response pathways including cytokine responses was dependent on mitochondrial antiviral-signaling protein (MAVS). Consequently, in Mavs-deficient microglia control of virus infection failed as indicated by augmented accumulation of viral transcripts. Thus, in the analyzed in vitro system, MAVS signaling is critically required to achieve full microglia activation and to mediate profound antiviral effects. In Mavs-deficient mice, intranasal VSV instillation caused higher disease severity than in WT mice and the virus disseminated beyond the olfactory bulb and entered more distal brain regions. Virus spread to inner regions of the olfactory bulb, i.e., the granular cell layer, correlated with infiltration of highly inflammatory non-microglial myeloid cells into the olfactory bulb. Furthermore, increased cytokine levels were detected in the nasal cavity, the olfactory bulb and in other brain regions. Thus, microglial MAVS signaling is critically needed for virus sensing, full microglia activation, and for orchestration of protective immunity in the virus-infected CNS.
Overall design: Brains from C57BL/6 and B6.STOCK-Mavs (tm1Tsc) (Mavs-/-) newborns were dissected at day 3 post birth, meninges and blood vessels were removed using a stereomicroscope. Next, the brains were minced and digested with 0.1% trypsin and 0.25% DNAse, single cell suspensions were seeded in pre-coated poly-L-lysine flasks in DMEM with 1% penicillin/streptomycin supplemented with 10% FCS. Medium was changed on day 1 and 5. After 10 days of mixed glial culture, microglia were harvested by shaking the flasks (125 rpm) for 40 min at 37°C. Microglial cells were re-plated at a density of 2.0 x 105 cells per well in 24 well plates. The cultured microglia were inoculated at a MOI of 0.5 of VSV diluted in DMEM medium for 1 hr at 37°C in 5% CO2. At 2, 4, 6 and 8 h post infection, the supernatants were removed and the adhering cells were then harvested and lysed by TRIzol. RNA were extracted from cell lysates and sequenced on Illumina NovaSeq-6000 with 50 bp paired-end reads configuration.
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