Regulation of microglial immune function by store-operated calcium channels

Abstract

Microglia is the immune effector cell in central nervous system (CNS) that participates in tissue repair, inflammatory responses and neuronal degeneration. Microglia differentiates into active state which shows not only morphology but increased chemotaxis response, phagocytosis capacity, and cytokine release1. Most important signaling in differentiation of immune cell is sustained calcium increase in cytosol which is maintained by SOCE (Store Operated Calcium Entry). Recently, molecular identity of SOC (Store Operated Channel) has revealed that Orais (Orai 1, 2, 3) and STIMs (STIM1, 2) constitute SOC. Orais are the calcium channel which show high selectivity to Ca2+, and STIMs are the sensor of stored calcium in the ER (Endoplasmic Reticulum) membrane3. In this study, we demonstrate that SOC mediated by Orai1 and STIM1, not by TRPCs influences the immune function of microglia, especially IL-6 cytokine secretion. In this study, primary microglia cells from neonatal mouse brain were used, which show 99.8% purity with glial fibrillary acidic protein (GFAP) immune-staining. Primary microglia has Orai 1, 2, 3, and STIM1, but not any TRPCs and STIM2. To identify the role of SOC in microglia immune functions, microglia was treated with siRNA Orai1 and STIM1, and then was measured for phagocytosis activity and cytokine secretion by ELISA (Enzyme-linked Immunosorbent Assay). Microglial phagocytosis activity was not affected by knock down of Orai1 and STIM1 in both UDP treated and untreated microglial cell. In ELISA assay, TNF-α secretion was not siginificantly affected by knock-down of Orai1 and STIM1, however IL-6 secretion was dramatically decreased by siRNA Orai1 and STIM1 treatment. To evaluate the SOC effect on the upstream signaling in immune function, phosphorylation of IκB signaling was measured in STIM1 knock down condition. These results indicate that SOC constituted by Orai1 and STIM1 influences the immune fuction governed by chronic calcium increase, but not transient increase, in microglia.