Role of RNA modification in innate immune cell development and function

PROJECT SUMMARY 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 key component of the liver-resident immune cells and contribute to the pathogenesis and progression of various liver diseases, such as autoimmune hepatitis. Unraveling the mechanisms that regulate iNKT cell development and function could provide therapeutic options for treating liver diseases. N6-methyladenosine (m6A) is the most prevalent 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 and others have demonstrated that YTHDF2 plays a critical role in regulating innate immune cell functions. However, its role in iNKT cells and iNKT cell-mediated liver disease remains poorly understood. Our strong preliminary data 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 iNKT cells in the thymus, liver, and spleen. The transition from early to late stage of iNKT cell development was substantially blocked in Ythdf2-deficient mice. Single-cell RNA sequencing (scRNA-seq) of thymic iNKT cells identified the transcriptional factor Bach2, which is critical for T and NK cell development and maturation, as a potential target of YTHDF2 in regulating iNKT cell development. Furthermore, we found that loss of YTHDF2 exacerbates acute liver injury, with a significant increase in TNF-α, a key inflammatory cytokine involved in liver injury. Surprisingly, the elevated TNF-α did not originate from iNKT cells, other T cell subsets, or NK cells, but from monocyte-derived macrophages (MDMs) in the liver. RNA-seq analysis of liver iNKT cells revealed that Csf1, encoding colony-stimulating factor 1 (CSF1), which is secreted by iNKT cells and critical for macrophage differentiation and function, regulates TNF-α production by MDMs in the liver. Based on these findings, we hypothesize 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 cell development. We hypothesize that YTHDF2 deficiency inhibits iNKT cell development by blocking them at immature stages through a cell-intrinsic mechanism involving Bach2. Aim 2: Elucidate how YTHDF2 regulates iNKT cell-driven liver injury. We hypothesize that Ythdf2-deficient iNKT cells exacerbate liver injury in a cell- extrinsic mechanism through the CSF1-MDMs-TNF-α axis. Collectively, this project will improve our understanding of how YTHDF2 and RNA m6A modifications impact innate immune cells, particularly iNKT cells, and their role in liver diseases. It will also advance knowledge of the cellular and molecular mechanisms underlying the immunopathogenesis of autoimmune hepatitis.