干扰素调节因子5(IRF5)调控小鼠骨髓源性巨噬细胞的极化

Interferon regulatory factor 5(IRF5) regulates the differentiation of bone marrow-derived macrophages in mice

目的诱导小鼠骨髓来源的巨噬细胞极化为M1型巨噬细胞和M2型巨噬细胞,检测干扰素调节因子5(IRF5)在M1型和M2型巨噬细胞中的表达差异,并用IRF5小干扰RNA(IRF5 siRNA)沉默巨噬细胞中IRF5基因表达,观察其极化状态的改变。方法用γ干扰素(IFN-γ)和脂多糖(LPS)诱导小鼠骨髓来源的巨噬细胞向M1型巨噬细胞分化,白细胞介素4(IL-4)诱导其向M2型巨噬细胞分化,实时定量PCR检测IRF5、IL-12、肿瘤坏死因子α(TNF-α)、诱导型一氧化氮合酶(i NOS)、精氨酸酶1(Arg1)和巨噬细胞甘露糖受体(MMR)mRNA在M1型和M2型巨噬细胞中的表达。用IRF5 siRNA转染巨噬细胞,实时定量PCR检测M1型巨噬细胞标志物IRF5、IL-12、TNF-α、i NOS和M2型巨噬细胞标志物Arg1、MMR mRNA的表达,评估其极化状态的改变。结果流式细胞术检测巨噬细胞分化率达81.7%,实时定量PCR检测显示M1型巨噬细胞中IRF5、IL-12、i NOS mRNA的表达量明显高于M2型巨噬细胞,TNF-α亦高于M2型巨噬细胞;M2型巨噬细胞中Arg1、MMR mRNA表达量同M1型巨噬细胞比较显著增高。IRF5 siRNA转染巨噬细胞后,IRF5、IL-12、TNF-α、i NOS mRNA的表达量显著降低,Arg1、MMR mRNA的表达量明显增加。Western blot法检测结果显示IRF5、IL-12、TNF-α、i NOS蛋白的表达量显著降低,Arg1、MMR蛋白的表达量明显增加,极化状态向M2型巨噬细胞偏移。结论 IRF5对巨噬细胞的极化调控有重要作用,可作为鉴别M1型巨噬细胞和M2型巨噬细胞的重要标志物。

Objective To investigate the expression differences of interferon regulatory factor 5（ IRF5） between M1 and M2 macrophages derived from mouse bone marrow and the effects of small interfering RNA targeting IRF5 gene（ IRF5siRNA） on macrophage differentiation. Methods With the treatment of IFN-γ and lipopolysaccharide（ LPS）,mouse bone marrow-derived macrophages were differentiated into M1 macrophages; while with the treatment of IL-4,mouse bone marrow-derived macrophages were differentiated into M2 macrophages. Differentiation efficiency was measured by flow cytometry. The expressions of IRF5,IL-12,tumor necrosis factor-α（ TNF-α）,inducible nitric oxide synthase（ i NOS）,arginase 1（ Arg1）,macrophage mannose receptor（ MMR） mRNAs in M1 and M2 macrophages were analyzed by quantitative real-time PCR（ RT-PCR）. Furthermore,macrophages were infected with IRF5 siRNA,and then the mRNA expressions of the above proteins in M1 and M2 were tested by RT-PCR,and the protein expressions were detected by Western blotting.The results were analyzed to evaluate the polarization state of macrophages. Results The differentiation proportion of macrophages measured by flow cytometry was 81. 7%. RT-PCR showed that the expressions of IRF5,IL-12,i NOS mRNAs were obviously higher in M1 macrophages than in M2,and TNF-α mRNA was also higher in M1 macrophages. The expressions of Arg1,MMR mRNAs were obviously higher in M2 macrophages than in M1 macrophages. After silencing the IRF5 by IRF5 siRNA,the expressions of IRF5, IL-12, TNF-α and i NOS mRNAs decreased remarkably in macrophages, while the expressions of Arg-1,MMR mRNAs increased. Western blotting revealed that the expressions of IRF5,IL-12,TNF-α and i NOS proteins were significantly reduced,while the expressions of Arg1 and MMR protein were raised. The polarization of macrophages shifted to M2 state. Conclusion IRF5 can be used as an important marker to identify M1 and M2 macrophages,and it has an important role in the regulation of macrophage differentiation.