Differential Cytokine-Induced Responses of Polarized Microglia

The role of select pro- and anti-inflammatory mediators in driving microglial cell polarization into classically (M1), or alternatively, (M2) activated states, as well as the subsequent differential responses of these induced phenotypes, was examined. Expression of PD-L1, MHC-II, MHC-I, arginase 1 (Arg-1), and inducible nitric oxide synthase (iNOS) was assessed using multi-color flow cytometry. We observed that both pro- and anti-inflammatory mediators induced PD-L1 expression on non-polarized microglia. Moreover, IFN-γ stimulated significant MHC class I and II expression on these cells. Interestingly, we observed that only IL-4 treatment induced Arg-1 expression, indicating M2 polarization. These M2 cells were refractory to subsequent depolarization and maintained their alternatively activated state. Furthermore, PD-L1 expression was significantly induced on these M2-polarized microglia after treatment with pro-inflammatory mediators, but not anti-inflammatory cytokines. In addition, we observed that only LPS induced iNOS expression in microglial cells, indicating M1 polarization. Furthermore, IFN-γ significantly increased the percentage of M1-polarized microglia expressing iNOS. Surprisingly, when these M1-polarized microglia were treated with either IL-6 or other anti-inflammatory cytokines, they returned to their non-polarized state, as demonstrated by significantly reduced expression of iNOS. Taken together, these results demonstrate differential responses of microglial cells to mediators present in dissimilar microenvironments.