Involvement of IL-4-induced intracellular zinc release in microglial M2 phenotype

[Speaker] Takaaki Aratake:1
[Co-author] Youichirou Higashi:1, Takahiro Shimizu:1, Shogo Shimizu:1, Yusuke Ueba:1, Tomoya Hamada:1, Zou Suo:1, Masaki Yamamoto:1, Yoshiki Nagao:1, Motoaki Saito:1
1:Pharmacology, Kochi Medical School, Kochi University, Japan

[Background] Microglia are the resident immune cells of the central nervous system. They are polarized into two opposite activation states known as neurodegenerative M1 and neuroprotective M2. Arginase (Arg)-1 expressed in M2 microglia reduces nitric oxide (NO) production by competing with inducible NO synthase for L-arginine (L-Arg), which contributes to attenuation of brain inflammation. On the other hand, recent researches demonstrated that excessive Arg-1 and depletion of L-Arg are observed in Alzheimer's disease (AD) in rodent models and patients. Brain zinc has been shown to promote M1 phenotype, but the role of zinc in the regulation of M2 phenotype is obscured. Here, we examined whether zinc regulates Arg-1 expression in M2 microglia.
[Methods] M2 polarization of microglia prepared from C57BL/6 mice was induced by interleukin (IL)-4 (10 ng/mL). Distribution of intracellular free zinc was visualized by FluoZin-3AM staining. Total mRNA was extracted from M2 microglia pretreated with a cell permeable zinc chelator, TPEN (0.5-1 microM) or extracellular zinc chelator, CaEDTA (25-100 microM), and then expression of Arg-1 was assessed by real-time polymerase chain reaction. Furthermore, phagocytic activity in M2 microglia pretreated with TPEN (1 microM) in presence of L-Arg (0.5-1 mM) was examined using yellow-green carboxylate latex beads.
[Results] FluoZin-3AM fluorescence was observed throughout the cytoplasm of microglia 6 h after IL-4 treatment. Arg-1 mRNA expression was increased significantly from 1 h after treatment and reached a peak at 6 h. Pretreatment with TPEN, but not CaEDTA, resulted in dramatic dose-dependent increase in level of Arg-1 in microglia 6 h after IL-4 stimulation. Microglial phagocytic activity of yellow-green carboxylate latex beads was induced 6 h after IL-4 stimulation, which decreased by TPEN pretreatment. However, the inhibitory effect of TPEN on phagocytic activity was inhibited by L-Arg supplementation.
These results suggest that intracellular zinc release acts as a negative regulator of Arg-1 expression in IL-4-induced M2 microglia and that these responses mediate microglial phagocytic activity via the prevention of L-Arg depletion.
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