Transcriptomic analysis unveils bona fide molecular signatures of microglia under conditions of homeostasis and viral encephalitis

Microglia provide a front-line defense against neuroinvasive viral infection, however, determination of their bona fide transcriptional profiles under conditions of health and disease is challenging. Here, we used a combination of experimental tools to delineate the overall transcriptional landscape of microglia during viral infection. By exploiting the ribosomal tagging approach, we developed the concept of enrichment of relevant marker genes by comparing immunoprecipitated RNA with total RNA. Enriched transcripts corresponding to genes expressed in target cells were instrumental in defining bona fide signatures of microglia. With this approach, we generated a comprehensive and accurate transcriptome of microglia at the in situ environment under conditions of health and virus infection. These unified microglial signatures may serve as a benchmark to retrospectively assess ex vivo artefacts from available atlases. Leveraging the microglial translatome, we found enrichment of genes implicated in T-cell activation and cytokine production during the course of VSV infection. These data linked microglia with T-cell re-stimulation and further underscored that microglia shape anti-viral T-cell responses in the brain. Collectively, this study faithfully defines the transcriptional landscape of microglia in steady state and during viral encephalitis and highlights cellular interactions between microglia and T cells that contribute to the control of virus dissemination. Overall design: Mixed gender CX3CR1-CreERT2+/- Rpl22wt/HA mice aged 6 weeks were subcutaneously injected with 4 mg of tamoxifen for two consecutive days. 8 weeks after initial tamoxifen injection, mice weeks were intranasally instilled with 10 µL of VSV. Controls animals received same volume of PBS. Six days post infection the animals were sacrificed by cervical dislocation prior to brain harvesting. Thereafter, brain was homogenized and lysate used to pull down HA-tagged ribosomes and to isolate RNA from “input” and RiboTag-IP fractions for bulk RNA-sequencing.