Objective: To explore the mechanism by which estradiol modulates the immunophenotype of macrophages through the endoplasmic reticulum stress pathway.
Methods: Peritoneal macrophages isolated from C57 mice were cultured in the presence of 60 ng/mL interferon-γ (IFN-γ) followed by treatment with estradiol (1.0 nmol/L) alone, estradiol with estrogen receptor antagonist (Acolbifene, 4 nmol/L), estradiol with IRE1α inhibitor (4 μ 8 C), or estradiol with IRE1α agonist. After the treatments, the expression levels of MHC-Ⅱ, iNOS and endoplasmic reticulum stress marker proteins IRE1α, eIF2α and ATF6 in the macrophages were detected with Western blotting, and the mRNA levels of TGF-β, IL-6, IL-10 and TNF-α were detected with RT-PCR.
Results: Estrogen treatment of the macrophages significantly decreased the expressions of M1-related proteins MHC-Ⅱ (P=0.021) and iNOS (P < 0.001) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.004), increased the mRNA expression of TGF-β (P=0.002) and IL-10 (P=0.008), and up-regulated the protein expressions of IRE1α (P < 0.001) and its downstream transcription factor XBP-1 (P < 0.001). Addition of the estrogen inhibitor obviously blocked the effect of estrogen. Compared with estrogen treatment alone, combined treatment of the macrophages with estrogen and the IRE1α inhibitor 4 μ 8 C significantly up-regulated the protein expressions of MHC-Ⅱ (P=0.002) and iNOS (P=0.003) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.024), and obviously down-regulated the mRNA expression of TGF-β (P < 0.001) and IL-10 (P < 0.001); these changes were not observed in cells treated with estrogen and the IRE1α agonist.
Conclusion: Estrogen can inhibit the differentiation of murine macrophages into a pro-inflammatory phenotype by up-regulating the IRE1α-XBP-1 signaling axis, thereby producing an inhibitory effect on inflammatory response.
目的: 研究雌二醇其通过内质网应激通路影响巨噬细胞免疫特性的可能机制。
方法: 提取C57小鼠腹腔巨噬细胞，IFN-γ 60 ng/mL作用下体外培养巨噬细胞，分组检测雌激素对巨噬细胞影响：实验组为雌二醇（1.0 nmol/L）处理；抑制剂组为雌二醇（1.0 nmol/L）和雌激素受体拮抗剂（Acolbifene, 4 nmol/L）同时作用；对照组加等量PBS处理，培养48 h后通过Western blot法检测比较巨噬细胞极化蛋白MHC-Ⅱ、iNOS和内质网应激标志性蛋白IRE1α、eIF2α和ATF6的表达水平，通过RT-PCR法检测巨噬细胞TGF-β、IL-6、IL-10、TNFα mRNA水平变化。随后分组检测内质网应激对巨噬细胞的影响：雌激素处理组；雌激素和内质网IRE1α信号抑制剂（4 μ 8 C，50 μmol/L）处理组；IRE1α激动剂组（TG，0.2 nmol/L）处理组；对照组加等量PBS处理，随后检测巨噬细胞的分化情况。
结果: 和对照组比较，雌激素处理后巨噬细胞M1相关蛋白MHC-Ⅱ（P=0.021）和iNOS（P < 0.001）表达显著降低，促炎细胞因子TNF-α（P=0.003）和IL-6 mRNA（P=0.004）表达下调，抗炎的M2型细胞因子TGF-β（P=0.002）和IL-10（P=0.008）mRNA表达上升，同时发现内质网相关的IRE1α（P < 0.001）蛋白活化水平升高，其下游转录因子XBP-1（P < 0.001）表达上调，加入雌激素受体阻断剂能够改变这种变化。和单纯雌激素处理组比较，在培养基中加入雌激素的同时加入IRE1α抑制剂4 μ 8 C会导致巨噬细胞M1相关蛋白MHC-Ⅱ（P=0.002）和iNOS（P=0.003）表达显著上调，促炎细胞因子TNF-α（P=0.003）和IL-6 mRNA（P=0.024）表达上调，抗炎的M2型细胞因子TGF-β（P < 0.001）和IL-10（P < 0.001）mRNA表达下调，而激动IRE1α通路会矫正这种变化。
结论: 雌激素能够通过上调IRE1α-XBP-1信号轴抑制巨噬细胞促炎表型的分化，从而对炎症反应发挥抑制作用。
Keywords: endoplasmic reticulum stress pathway; estrogen; macrophages.