PROJECT SUMMARY Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by focal T cell and myeloid cell infiltrates leading to demyelination and loss of neurologic function. Activated microglia and macrophages are the predominant inflammatory cells in active or chronic MS plaques, and they persist in secondary-progressive MS. Despite advances in our understanding of MS pathophysiology, there are minimal disease-modifying treatments or preventions for innate-mediated, secondary-progressive forms of MS. We recently found that mutually antagonistic pathways driven by TGFβ and APOE signaling in microglia, dictate a phenotypic switch between homeostatic (M0) and neurodegenerative (MGnD) phenotypes. Using a systems biology approach, we identified major transcriptional and epigenetic regulators of M0- and MGnD- microglia. Finally, we reveal a new role for TGFβ-IFNγ signaling in the reprogramming of peripheral monocytes into microglia-like cells with the M0-molecular signature. Based on our published and preliminary data, we hypothesize that modulating the APOE-TGFβ/IFNγ pathway will restore the homeostatic-tolerogenic microglia and ameliorate EAE. We will address our hypothesis in the following aims: Aim 1: Replenish M0-homeostatic microglia via APOE-TGFβ/IFNγ signaling in EAE In this Aim, we will determine 1) the impact of TGFβ/IFNγ signaling on the replenishment of M0-microglia in EAE; 2) the regulatory networks controlled by APOE-TGFβ signaling underlying microglia phenotype regulation in EAE; and 3) fate-map analysis of microglia phenotype switch during peak and recovery stage of EAE. Aim 2: Reprogram monocytes into MG-like cells via APOE-TGFβ/IFNγ pathway in EAE and from MS patients In this Aim, we will determine 1) whether targeting Apoe in Ly6CHi monocytes facilitates the acquisition of the M0-microglial transcriptional program; 2) the molecular mechanisms underlying TGFβ/IFN-mediated reprogramming of Ly6CHi monocytes; and 3) whether modulation of the APOE-TGFβ/IFN pathway in CD14+/CD16– monocytes isolated from MS subjects induces the M0-microglial transcriptional program to serve as a novel therapeutic approach for progressive MS. Successful completion will result in: 1) identification of molecular mechanisms mediated by APOE-TGFβ/IFNγ signaling in microglia and monocyte phenotype regulation that contribute to the development, progression and resolution of EAE, and 2) understanding the role of APOE-TGFβ/IFNγ signaling in reprogramming of peripheral monocytes into homeostatic microglia-like cells in EAE and from MS patients.