Interrogating the role of reactive astrocytes during Toxoplasma-induced brain inflammation - PROJECT SUMMARY Toxoplasma gondii is a common protozoan parasite that forms a lifelong infection in the brain. Despite chronic infection, Toxoplasma only causes clinical pathology in the immune compromised and therefore triggers an effective and well-balanced immune response in the brain. T. gondii replication and innate immunity are strongly regulated by CNS resident astrocytes. Astrocytes ordinarily execute vital processes for proper brain function and reprogram into reactive astrocytes (RAs) during brain inflammation. The beneficial or detrimental roles of RAs in brain inflammation and neurotoxicity has remained debated and largely unknown. During Toxoplasma infection, astrocytes limit parasite proliferation in a STAT1-depend- ent manner, produce multiple cytokines and chemokines to attract immune cells to the brain, and modify extracellular concentrations of neurotransmitters to regulate the inflammatory environment of the tissue Like many immune cells, astrocytes are increasingly recognized as falling into distinct functional subsets but unlike immune cells our ability to manipulate RAs or study their function in a disease- or infection- specific manner has been limited. Here we propose to characterize, track and selectively manipulate RAs to determine their diversity and functional roles in the immune response during chronic Toxoplasma in- fection. We have generated a novel tamoxifen-inducible Cre knock-in mouse line driven by the lipocalin 2 (Lcn2) promoter (Lcn2CreERT2). By crossing to the Ai9 TdTomato reporter line, we have visualized RAs in various pathological states including systemic inflammation and T. gondii infection. Traditional astro- cyte promoters will be used in combination with Lcn2CreERT2 to selectively target RAs for genetic manipu- lation at any point during progression of infection, while leaving healthy astrocytes, neurons, and other glial cell types intact, transforming our understanding of astrocyte function and underlying inflammatory mechanisms during Toxoplasma infection. Three aims are proposed: In Aim1 infection-induced astrocyte subsets will be identified and characterized over the course of infection using flow cytometry and scRNAseq analysis. In Aim2 we will track the resolution of RAs in the context of high and low inflammatory environments following infection and in Aim3 we will manipulate RA gene expression to determine the beneficial and detrimental roles of RAs during chronic Toxoplasma infection. The rationale for the pro- posed research is that determining the development, function and responsiveness of astrocyte subsets during chronic infection will provide new knowledge on the long-term consequences of Toxoplasma in- fection and the basis of a working inflammatory response in the brain.
