Systematic perturbation screens decode regulators of inflammatory macrophage states and identify a role forTNFmRNA m6A modification

ABSTRACT Macrophages adopt dynamic cell states with distinct effector functions to maintain tissue homeostasis and respond to environmental challenges. During chronic inflammation, macrophage polarization is subverted towards sustained inflammatory states which contribute to disease, but there is limited understanding of the regulatory mechanisms underlying these disease-associated states. Here, we describe a systematic functional genomics approach that combines genome-wide phenotypic screening in primary murine macrophages with transcriptional and cytokine profiling of genetic perturbations in primary human monocyte-derived macrophages (hMDMs) to uncover regulatory circuits of inflammatory macrophage states. This process identifies regulators of five distinct inflammatory states associated with key features of macrophage function. Among these, the mRNA m6A writer components emerge as novel inhibitors of a TNFα-driven cell state associated with multiple inflammatory pathologies. Loss of m6A writer components in hMDMs enhances TNF transcript stability, thereby elevating macrophage TNFα production. A PheWAS on SNPs predicted to impact m6A installation on TNF revealed an association with cystic kidney disease, implicating an m6A-mediated regulatory mechanism in human disease. Thus, systematic phenotypic characterization of primary human macrophages describes the regulatory circuits underlying distinct inflammatory states, revealing post-transcriptional control of TNF mRNA stability as an immunosuppressive mechanism in innate immunity. Competing Interest Statement S.M.H., S.M.C., S.X., C.E., J.K., M.C., C.C., A.L., J.P.F., M.C., M.K., L.H., E.F., A.N., S.Z.W., B.Y., H. H., R.N.P, K.G.S., and S.J.T. are employees of Genentech. A.M. is an employee of Gilead Sciences. J.K. is an employee of Alector Therapeutics. J.L. is an employee of Sana Biotechnology.