Role of RNA modification in innate immune cell development and function

Invariant natural killer T (iNKT) cells are a specialized subset of T lymphocytes with innate-like properties. They recognize glycolipid antigens presented by the nonclassical MHC class I molecule CD1d. iNKT cells are a keycomponent of the liver-resident immune cells and contribute to the pathogenesis and progression of various liverdiseases such as autoimmune hepatitis. Unraveling the mechanisms that regulate iNKT cell development andfunction could provide therapeutic options for treating liver diseases. N6-methyladenosine (m6A) is the mostprevalent eukaryotic RNA modification and regulates the stability and translation of mRNAs. The stability of m6A-modified mRNA is primarily controlled by the m6A reader protein YTHDF2. Recent studies from our group andothers have demonstrated that YTHDF2 plays a critical role in regulating innate immune cell functions. Howeverits role in iNKT cells and iNKT cell-mediated liver disease remains poorly understood. Our strong preliminarydata indicate that YTHDF2 is indispensable for iNKT cell homeostasis development and effector function.Conditional deletion of YTHDF2 in double-positive thymocytes resulted in a substantial reduction of mature iNKTcells in the thymus liver and spleen. The transition from early to late stage of iNKT cell development wassubstantially blocked in Ythdf2-deficient mice. Single-cell RNA sequencing (scRNA-seq) of thymic iNKT cellsidentified the transcriptional factor Bach2 which is critical for T and NK cell development and maturation as apotential target of YTHDF2 in regulating iNKT cell development. Furthermore we found that loss of YTHDF2exacerbates acute liver injury with a significant increase in TNF- a key inflammatory cytokine involved in liverinjury. Surprisingly the elevated TNF- did not originate from iNKT cells other T cell subsets or NK cells butfrom monocyte-derived macrophages (MDMs) in the liver. RNA-seq analysis of liver iNKT cells revealed thatCsf1 encoding colony-stimulating factor 1 (CSF1) which is secreted by iNKT cells and critical for macrophagedifferentiation and function regulates TNF- production by MDMs in the liver. Based on these findings wehypothesize that YTHDF2 acts as a key regulator of iNKT cell development and iNKT cell-mediated liver injury.This hypothesis will be addressed in two Specific Aims. Aim 1: Elucidate how YTHDF2 regulates iNKT celldevelopment. We hypothesize that YTHDF2 deficiency inhibits iNKT cell development by blocking them atimmature stages through a cell-intrinsic mechanism involving Bach2. Aim 2: Elucidate how YTHDF2 regulatesiNKT cell-driven liver injury. We hypothesize that Ythdf2-deficient iNKT cells exacerbate liver injury in a cell-extrinsic mechanism through the CSF1-MDMs-TNF- axis. Collectively this project will improve ourunderstanding of how YTHDF2 and RNA m6A modifications impact innate immune cells particularly iNKT cellsand their role in liver diseases. It will also advance knowledge of the cellular and molecular mechanismsunderlying the immunopathogenesis of autoimmune hepatitis.