M2型巨噬细胞通过CCL22-CCR4信号促进肺泡上皮细胞EMT  

M2 macrophages promote epithelial-mesenchymal transition via CCL22-CCR4 signaling in alveolar epithelial cells

作　　者：王璐 徐婷贞[1] 周林水[1] 朱渊红[1] WANG Lu;XU Ting-zhen;ZHOU Lin-shui;ZHU Yuan-hong(The First Affiliated Hospital of Zhejiang Chinese Medical University(Zhejiang Provincial Hospital of Traditional Chinese Medicine),Hangzhou 310006,China)

机构地区：[1]浙江中医药大学附属第一医院(浙江省中医院),浙江杭州310006

出　　处：《中国病理生理杂志》2022年第10期1848-1855,共8页Chinese Journal of Pathophysiology

基　　金：浙江省自然科学基金资助项目(No.LQ20H270014,No.LY19H270002);国家自然科学基金资助项目(No.82104587,No.81904172);浙江中医药大学校级中青年创新科研基金(No.KC201931)。

摘　　要：目的:探讨M2型巨噬细胞通过CCL22-CCR4轴对肺泡上皮细胞上皮-间充质转化(epithelial-mesenchymal transition,EMT)的影响,并探讨其机制。方法:取THP-1单核细胞,经特定细胞因子分化成为M0、M1和M2型巨噬细胞,通过Transwell小室对不同表型巨噬细胞和肺泡上皮细胞(A549细胞)进行共培养(0、24、36、48或72 h),分别采用CCK-8法、划痕实验、Annexin V-FITC/PI双染法检测各组上皮细胞活力、迁移、凋亡水平;ELISA检测各组细胞上清C-C基序趋化因子22(C-C motif chemokine 22,CCL22)的表达;RT-qPCR和/或Western blot检测各组细胞上皮、间充质标志物和趋化因子受体4(C-C motif chemokine receptor 4,CCR4)水平,以及Smad2/3信号激活水平;采用CCR4抑制剂,进一步验证CCL22-CCR4-Smad2/3信号通路在M2型巨噬细胞介导的肺泡上皮细胞EMT中的作用。结果:与正常对照组A549细胞相比,M1型巨噬细胞可显著抑制A549细胞活力、迁移能力并诱导其凋亡(P<0.01);M2型巨噬细胞则可促进A549细胞迁移并抑制其细胞凋亡(P<0.05)。与此同时,M2型巨噬细胞共培养可导致A549向间充质细胞形态改变,上皮标志物E-cadherin表达减少,间充质标志物fibronectin、α-SMA表达增多(P<0.05)。ELISA结果显示,与M2型巨噬细胞共培养后,细胞上清中CCL22表达增多;Western blot结果显示M2型巨噬细胞可促进A549细胞CCR4表达上调及Smad2/3信号通路的激活(P<0.05);当CCR4被抑制后,M2型巨噬细胞诱导上皮细胞EMT的作用显著减弱,并且Smad2/3磷酸化蛋白表达也显著下调(P<0.01)。结论:M2型巨噬细胞促进上皮细胞EMT,其机制可能与激活CCL22-CCR4-Smad2/3信号通路有关。AIM:This study aimed to investigate the effect and potential mechanism of M2 macrophages on promoting the epithelial-mesenchymal transition(EMT)in alveolar epithelial cells(A549).METHODS:Human myeloid leukemia mononuclear THP-1 cells were used to induce mature M0,M1,and M2 macrophages.Macrophages and A549 cells were co-cultured in Transwell plates for 0,24,36,48 or 72 h.CCK-8,Scratch wound healing,and Annexin V-FITC/PI assays were used to assess the cell proliferation,migration ability,and apoptosis rate,respectively.The expression levels of C-C motif chemokine 22(CCL22)in the supernatant of each group were determined by ELISA and the protein levels of EMT cell markers and C-C motif chemokine receptor 4(CCR4),Smad2/3,and phosphorylated Smad2/3were measured by RT-qPCR and/or Western blot.Furthermore,a CCR4 inhibitor was used to validate the role of the CCL22-CCR4 axis.RESULTS:Compared with the control cells,M1 macrophages inhibited cell viability and migration capacity but increased the apoptosis rate of A549 cells(P<0.01).M2 macrophages promoted A549 cell migration and suppressed the apoptosis rate(P<0.05).Compared with A549 cells,cells co-cultured with M2 macrophages demonstrated morphological alteration such as fibroblastic appearance,upregulated expression of fibronectin andα-SMA and downregulated expression of E-cadherin were also observed(P<0.05).ELISA showed that the levels of CCL22 in cell supernatants from A549 cells cultured with M2 macrophages increased.Furthermore,M2 macrophage increased CCR4 levels and activated Smad2/3 signaling pathway in A549 cells(P<0.01).Stimulation of EMT and Smad2/3 signaling pathway induced by M2 macrophage were blocked by CCR4 inhibitor in A549 cells(P<0.01).CONCLUSION:M2 macrophages contributes to EMT through CCL22-CCR4-Smad2/3 signaling pathway in alveolar epithelial cells.

关 键 词：肺纤维化 上皮间充质转化 巨噬细胞极化 M2型巨噬细胞 CCL22-CCR4信号通路 

分 类 号：R563[医药卫生—呼吸系统] R363.2[医药卫生—内科学]