Immune-responsive gene-1: The mitochondrial Key to Th17 Cell Pathogenicity in CNS Autoimmunity

Pathogenic Th17 cells play crucial roles in CNS autoimmune diseases such as multiple sclerosis (MS), but their regulation by endogenous mechanisms remains unknown. Through RNA-seq analysis of primary brain glial cells, we identified immuno-responsive gene 1 (Irg1) as one of the highly upregulated gene under inflammatory conditions. Validation in the spinal cord of animals with experimental autoimmune encephalomyelitis (EAE), an MS model, confirmed elevated Irg1 levels in myeloid, CD4, and B cells in the EAE group raising the concern if Irg1 is detrimental or protective. Irg1 knockout (KO) mice exhibited severe EAE disease, increased mononuclear cell infiltration, and increased levels of triple-positive CD4+ T cells expressing IL17a, GM-CSF, and IFN?. A lack of Irg1 in macrophages elevates Class II expression, promoting the polarization of myelin-primed CD4+ T cells into pathogenic Th17 cells via the NLRP3/IL-1ß axis. Adoptive transfer in Rag-1 KO and single-cell RNA sequencing highlighted the crucial role of Irg1 in shaping pathogenic Th17 cells. Moreover, bone marrow chimeras revealed that immune cells lacking Irg1 maintained pathogenic and inflammatory phenotypes, suggesting its protective role in autoimmune diseases, including MS. Overall design: For the generation of bone marrow chimeras, host (CD45.1) animals were treated with three doses of busulfan (20 mg/ml), and on the fourth day, the host received an i.v. injection of 10 million BM cells [50:50 mixture of CD45.1 (Wt) and CD45.2 (Irg1 KO)]. Using flow cytometry, we sorted CD45.1+ and CD45.2+ cells from BILs isolated via a Percoll gradient from the brain and spinal cord after EAE induction. BILs were stained with primary antibodies against mouse PerCP-Cy5.5-CD45.1 and BV421-CD45.2 (BioLegend) and sorted on a BD FACSAria™ III. Furthermore, the sorted populations were re-run to check the purity of the sorted cells. Next, we used an automated cell counter (Bio-Rad) to determine the viability of CD45+1+ and CD45.2 cells via trypan blue and found that >98% of the cells were viable. Then, RNA was harvested from the sorted cells and processed for RNA sequencing. Three mice per group (WT and Irg1 knockout) were pooled for analysis. Experimental autoimmune encephalomyelitis (EAE) was induced using MOG peptide in combination with Pertussis toxin and monitored for 20 days. Following euthanasia, spinal cords were harvested, and single-cell suspensions were prepared using Accumax enzymatic dissociation followed by Percoll gradient purification. A total of approximately 200,000 cells per group were processed using the Fluent pipeline. Two single-cell RNA-seq libraries per group were generated using the Fluent PIPseq T20 3' capture and lysis kit. The timed pregnant rat was purchased from Charles River Laboratory. The brains of 2–3-day-old female and male pups were used to prepare mixed glial cell cultures. We followed our previously published protocol to prepare primary mixed glial cell cultures (3). After 10 days of culture, the cells were treated with LPS (0.5 µg/ml) + IFN? (20 ng/ml) to induce inflammatory conditions in our study. After 8 h of treatment, the cells were processed for RNA isolation and RNA-seq