Estrogen receptor α (ERα) has been suggested to regulate anti-inflammatory signaling in brain microglia, the only resident immune cells in the brain. ERα conserves the phosphorylation motif at Ser216 within the DNA binding domain. Previously, Ser216 was found to be phosphorylated in neutrophils infiltrating into the mouse uterus and to enable ERα to regulate migration. Given the implication of this phosphorylation in immune regulation, ERα was examined in mouse microglia to determine if Ser216 is phosphorylated and regulates microglia’s inflammation. It was found that Ser216 was constitutively phosphorylated in microglia and demonstrated that in the absence of phosphorylated ERα in ERα KI brains microglia inflamed, confirming that phosphorylation confers ERα with anti-inflammatory capability. ERα KI mice were obese and weakened motor ability. Mixed glia cells were prepared from brains of 2-days-old neonates and cultured to mature and isolate microglia. An antibody against an anti-phospho-S216 peptide of ERα (αP-S216) was used to detect phosphorylated ERα in double immunofluorescence staining with ERα antibodies and a microglia maker Iba-1 antibody. A knock-in (KI) mouse line bearing the phosphorylation-blocked ERα S216A mutation (ERα KI) was generated to examine inflammation-regulating functions of phosphorylated ERα in microglia. RT-PCR, antibody array, ELISA and FACS assays were employed to measure expressions of pro- or anti-inflammatory cytokines at their mRNA and protein levels. Rotarod tests were performed to examine motor connection ability. Double immune staining of mixed glia cells showed that ERα is phosphorylated at Ser216 in microglia, but not astrocytes. Immunohistochemistry with an anti-Iba-1 antibody showed that microglia cells were swollen and shortened branches in the substantial nigra (SN) of ERα KI brains, indicating the spontaneous activation of microglia as observed with those of lipopolysaccharide (LPS)-treated ERα WT brains. Pro-inflammatory cytokines were up-regulated in the brain of ERα KI brains as well as cultured microglia, whereas anti-inflammatory cytokines were down-regulated. FACS analysis showed that the number of IL-6 producing and apoptotic microglia increased in those prepared from ERα KI brains. Times of ERα KI mice on rod were shortened in Rotarod tests. Blocking of Ser216 phosphorylation aggravated microglia activation and inflammation of mouse brain, thus confirming that phosphorylated ERα exerts anti-inflammatory functions. ERα KI mice enable us to further investigate the mechanism by which phosphorylated ERα regulates brain immunity and inflammation and brain diseases.

中文翻译：

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Ser216 磷酸化的雌激素受体 α 赋予小鼠小胶质细胞炎症功能。


雌激素受体α（ERα）被认为可以调节大脑小胶质细胞的抗炎信号传导，小胶质细胞是大脑中唯一的常驻免疫细胞。 ERα 保留 DNA 结合域内 Ser216 处的磷酸化基序。此前，人们发现 Ser216 在渗入小鼠子宫的中性粒细胞中被磷酸化，并使 ERα 能够调节迁移。鉴于这种磷酸化在免疫调节中的意义，我们在小鼠小胶质细胞中检查了 ERα，以确定 Ser216 是否被磷酸化并调节小胶质细胞的炎症。研究发现小胶质细胞中 Ser216 被组成性磷酸化，并证明在 ERα KI 中缺乏磷酸化 ERα 的情况下，小胶质细胞会发炎，证实磷酸化赋予 ERα 抗炎能力。 ERα KI 小鼠肥胖且运动能力减弱。从 2 天大的新生儿大脑中制备混合神经胶质细胞，并培养至成熟并分离小胶质细胞。使用抗 ERα 磷酸 S216 肽的抗体 (αP-S216) 在 ERα 抗体和小胶质细胞标记 Iba-1 抗体的双重免疫荧光染色中检测磷酸化 ERα。产生带有磷酸化阻断的 ERα S216A 突变 (ERα KI) 的敲入 (KI) 小鼠系，以检查小胶质细胞中磷酸化 ERα 的炎症调节功能。采用 RT-PCR、抗体阵列、ELISA 和 FACS 测定来测量促炎或抗炎细胞因子的 mRNA 和蛋白质水平的表达。进行旋转杆测试以检查电机连接能力。混合胶质细胞的双重免疫染色显示，小胶质细胞中的 ERα 在 Ser216 位点被磷酸化，但星形胶质细胞中则没有。 使用抗 Iba-1 抗体进行的免疫组织化学显示，ERα KI 大脑的实质黑质 (SN) 中的小胶质细胞肿胀且分支缩短，这表明与脂多糖 (LPS) 处理的 ERα WT 大脑中观察到的小胶质细胞自发激活一样。 ERα KI 大脑以及培养的小胶质细胞中促炎细胞因子上调，而抗炎细胞因子下调。 FACS 分析表明，在 ERα KI 脑中制备的细胞中，产生 IL-6 和凋亡的小胶质细胞数量增加。 Rotarod 试验中 ERα KI 小鼠在杆上的时间缩短了。阻断Ser216磷酸化会加剧小鼠大脑的小胶质细胞活化和炎症，从而证实磷酸化的ERα发挥抗炎功能。 ERα KI 小鼠使我们能够进一步研究磷酸化 ERα 调节脑免疫、炎症和脑疾病的机制。