The role of NFATc2 in chronic autoimmune neuroinflammation

Abstract

Dimethyl fumarate (DMF), a fumaric acid ester with potential immunomodulatory and neuroprotective effect, was recently approved as treatment for relapsing–remitting multiple sclerosis (MS). DMF ameliorates the clinical course of experimental autoimmune encephalomyelitis (EAE), the murine model of MS, where it exerts a neuroprotective action, reducing demyelination and axonal loss. We hypothesized that these effects are mediated, at least in part, through its action on microglia. We used a microglial cell line (N9) activated with lipopolysaccharide (LPS) to analyze the effect of monomethyl fumarate (MMF), a bioactive metabolite of DMF, in vitro. We show that MMF reverts the molecular phenotype of LPS-activatedmicroglia from pro(M1-like) to anti(M2-like) inflammatory. Thus, MMF significantly reduced production and expression of neurotoxic molecules, TNF-alpha, IL1-beta and iNOS, by activated microglia, while significantly increasing the production of receptors typically expressed by alternatively activated microglia, NURR1, CD200R and CX3CR1. We validated MMF effect at functional level, with the observation that MMF increases microglial internal calcium concentration and phagocytosis, itself associated with an increased expression of TREM2, which facilitates debris clearance in the absence of inflammation. Previous studies suggest thatMMF effects are related to activation of the Nrf2-anti-oxidant pathway; however such a possibility may not account for inhibitory effects on pro-inflammatory cytokines in microglia. In line with the observation that MMF upregulates NURR1, a molecule involved in inhibition of NFkB, we postulated that MMF exerts its anti-inflammatory effect through inhibiting NFkB. MMF is an agonist of HCA2 and activation of this receptor leads to an increase in intracellular Ca2+, which we observed upon exposure of activated microglia to MMF. We therefore addressed the possibility that MMF could signal through binding to HCA2.We show that HCA2 is expressed onN9 and blocking HCA2 reverts the effect ofMMF on the expression of pro-inflammatory cytokines and of NURR1. We have postulated and validated a downstream pathway activated by internal Ca2+ increase induced through MMF binding to HCA2, which leads to inhibition of NFkB activation by de-acetylation via the AMPK/Sirt axis. We show in vivo and ex vivo that treatment of EAE-affected mice with DMF upregulates M2-phenotype markers in the brain, and confirm the neuroprotective effect of MMF.