Diverse subpopulations of reactive mouse astrocytes following chronic Toxoplasma infection.

Astrocytes provide physical and metabolic support for neurons, regulate the blood brain barrier, and react to injury, infection, and disease. When astrocytes become reactive, the regulation of the inflammatory state, the forms it takes, and its longevity are all poorly understood. Several models of acute inflammation have revealed astrocyte subpopulations that go beyond a two-activation state model, instead encompassing distinct functional subsets. However, how reactive astrocyte (RA) subsets evolve over time during chronic inflammatory disease or infection have been technically difficult to address. Here we use a prolific human pathogen, Toxoplasma gondii, that causes lifelong infection in the brain alongside a novel Lcn2CreERT2 reporter mouse line to examine astrocyte subsets during chronic inflammation. Single cell RNA sequencing revealed diverse astrocyte populations during chronic inflammation. Furthermore, we observed transcriptionally unique Lcn2CreERT2+ RAs, some of which changed over time in a manner suggestive of potential resolution and overall plasticity of RAs. In addition to an immune regulating Lcn2CreERT2+ astrocyte population enriched with gene transcripts encoding chemokines Ccl5, Cxcl9, Cxcl10 and receptors Ccr7 and Il7r, a specific set of Lcn2CreERT2+ astrocytes highly expressed transthyretin (Ttr), a secreted carrier protein involved in glycolytic enzyme activation and potential vasculature regulation and angiogenesis. These findings provide novel information about the evolution and diversity of reactive astrocyte subtypes and functional signatures at different stages of infection and an undocumented role for transthyretin expressing astrocytes in immune regulation at the central nervous system (CNS) vasculature. Overall design: We performed scRNAseq on the mixed samples of whole brain taken from brain mononuclear cells collected following 14, 28, and 42 days of Toxoplasma infection and obtained 21,941 high quality cells. We repeated this utilizing ACSA2 for each time point to get 3,736 high quality ACSA2 astrocytes. Lastly, we performed scRNAseq on Lcn2CreErt2;Ai9 transgenic mice following 14 and 42 days of Toxoplasma infection to collect Lipocalin (Lcn2) expressing cells obtaining 5,875 high quality Lcn2+ cells. A tamoxifen rested time point was included as well, and mice were infected with the chronic 42 day infection regimen and collected 2 weeks later to assay any changes to previously labeled chronic Lcn2 astrocytes.