极化过程影响巨噬细胞的铁代谢

Polarized activation affects iron metabolism in macrophages

本文旨在研究巨噬细胞极化过程对自身铁代谢调节的影响。用20 ng/mLγ干扰素(interferon gamma,IFN-γ)刺激猪肺泡巨噬细胞(3D4/2细胞)24 h,诱导其为M1型巨噬细胞,另外用10 ng/mL白细胞介素4(interleukin-4,IL-4)联合10 ng/mL巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)共同刺激3D4/2细胞24 h,诱导其为M2型巨噬细胞,用实时定量PCR、免疫荧光染色法和Western bolt检测巨噬细胞炎症因子和铁代谢相关蛋白的表达。收集M1/M2型巨噬细胞培养液上清液与猪肠上皮IPEC-J2细胞共培养,用CCK-8法检测IPEC-J2的增殖能力。给M1/M2型巨噬细胞外源添加柠檬酸铁铵(ammonium ferric citrate,FAC),用异硫氰酸荧光素标记的葡聚糖(fluorescein isothiocyanate-dextran,FITC-dextran)及流式细胞术检测3D4/2细胞吞噬能力。结果显示,和对照组相比,M1型巨噬细胞铁调素、铁蛋白重链、轻链和脂质运载蛋白-2 mRNA表达水平上调,胞内铁含量增加,显示出铁保留的表型,并且铁可增强M1型巨噬细胞的吞噬能力;M2型巨噬细胞上述基因mRNA表达水平无显著变化,而铁转运蛋白和转铁蛋白受体mRNA表达水平上调,胞内铁含量减少,显示出铁释放的表型,并且其培养液上清液能促进IPEC-J2细胞增殖。上述结果提示,M1型巨噬细胞倾向于将铁保留在细胞内,减少胞外铁含量,从而抑制胞外菌的增殖;而M2型巨噬细胞倾向于释放铁,有助于周围细胞的增殖,从而促进组织修复。

The aim of this study was to investigate the effects of polarization program on the ability of macrophages to regulate iron metabolism.M1 and M2 macrophages were propagated in vitro from porcine alveolar macrophages 3D4/2 and polarized by cytokines.The 3D4/2 macrophages were treated with 20 ng/mL interferon gamma(IFN-γ)and 10 ng/mL interleukin-4(IL-4)combined with 10 ng/mL macrophage colony-stimulating factor(M-CSF)to induce polarization to M1 and M2,respectively.After incubation for 24 h,the expression levels of inflammatory factors and iron-metabolism genes were determined using real-time qPCR,Western bot and immunofluorescence.The M1/M2 macrophages culture media supernatant was collected and used to treat porcine intestinal epithelial cells IPEC-J2.The proliferation ability of IPEC-J2 was detected using CCK-8 assay kit.Following exogenous addition of ammonium ferric citrate(FAC)to M1/M2 macrophages,the phagocytic function of macrophages was detected using fluorescein isothiocyanate-dextran(FITC-dextran)and flow cytometry.The results showed that,compared with control,M1 macrophages had higher mRNA levels of iron storage proteins(ferritin heavy and light polypeptide,i.e.FtH and FtL),hepcidin and lipocalin-2,as well as iron content.Moreover,iron enhanced the ability of M1 macrophages to phagocytize FITC-dextran.There was no significant change in these m RNA expression levels in M2 macrophages,but the mRNA expression levels of ferroportin and transferrin receptor were up-regulated.In addition,the conditioned media supernatant from M2 macrophages promoted cell proliferation of IPEC-J2.These findings indicate that M1 macrophages tend to lock iron in the cell and reduce extracellular iron content,thereby inhibiting the proliferation of extracellular bacteria.While M2 macrophages tend to excrete iron,which contributes to the proliferation of surrounding cells and thus promotes tissue repair.