Interferon-gamma ameliorates experimental autoimmune encephalomyelitis by inducing homeostatic adaptation of microglia

Compelling evidence has shown that interferon (IFN)-? has dual effects in multiple sclerosis and in its animal model of experimental autoimmune encephalomyelitis (EAE), with results supporting both a pathogenic and beneficial function. However, the mechanisms whereby IFN-? may promote neuroprotection in EAE and its effects on central nervous system (CNS)-resident cells have remained an enigma for more than 30 years. In this study, the impact of IFN-? at the peak of EAE, its effects on CNS infiltrating myeloid cells (MC) and microglia (MG), and the underlying cellular and molecular mechanisms were investigated. IFN-? administration resulted in disease amelioration and attenuation of neuroinflammation associated with significantly lower frequencies of CNS CD11b+ myeloid cells and less infiltration of inflammatory cells and demyelination. A significant reduction in activated MG and enhanced resting MG was determined by flow cytometry and immunohistrochemistry. Primary MC/MG cultures obtained from spinal cord of IFN-?-treated EAE mice that were ex vivo re-stimulated with a low dose (1 ng/ml) of IFN-? and neuroantigen promoted a significantly higher induction of CD4+ regulatory T (Treg) cells associated with increased transforming growth factor (TGF)-ß secretion. Additionally, IFN-?-treated primary MC/MG cultures produced significantly lower nitrite in response to LPS challenge than control MC/MG. IFN-?-treated EAE mice had a significantly higher frequency of CX3CR1high MC/MG and expressed lower levels of program death ligand 1 (PD-L1) than PBS-treated mice. Most CX3CR1highPD-L1lowCD11b+Ly6G- cells expressed MG markers (Tmem119, Sall2, and P2ry12), indicating that they represented an enriched MG subset (CX3CR1highPD-L1low MG). Amelioration of clinical symptoms and induction of CX3CR1highPD-L1low MG by IFN-? were dependent on STAT-1. RNA-seq analyses revealed that in vivo treatment with IFN-? promoted the induction of homeostatic CX3CR1highPD-L1low MG, upregulating the expression of genes associated with tolerogenic and anti-inflammatory roles and down-regulating pro-inflammatory genes. These analyses highlight the master role that IFN-? plays in regulating microglial activity and provide new insights into the cellular and molecular mechanisms involved in the therapeutic activity of IFN-? in EAE. Overall design: CD11b+Ly6G-CX3CR1highPD-L1lowcells obtained from SC of EAE mice treated with either IFN-? or PBS