干扰素调节因子5（IRF5）调控小鼠骨髓来源巨噬细胞的分化

Sun Kang, Qu Jianguo, Chen Jixiang, Dang Shengchun, He Songbing, Zhang Jin, Xie Rong, Wang Yun, Zhang Jianxin

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2017 Feb;33(2):168-73.

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.

目的

探讨小鼠骨髓来源的M1和M2巨噬细胞中干扰素调节因子5（IRF5）的表达差异以及靶向IRF5基因的小干扰RNA（IRF5siRNA）对巨噬细胞分化的影响。

方法

用干扰素-γ（IFN-γ）和脂多糖（LPS）处理，将小鼠骨髓来源的巨噬细胞分化为M1巨噬细胞；用白细胞介素-4（IL-4）处理，将小鼠骨髓来源的巨噬细胞分化为M2巨噬细胞。通过流式细胞术检测分化效率。采用定量实时聚合酶链反应（RT-PCR）分析M1和M2巨噬细胞中IRF5、白细胞介素-12（IL-12）、肿瘤坏死因子-α（TNF-α）、诱导型一氧化氮合酶（iNOS）、精氨酸酶1（Arg1）、巨噬细胞甘露糖受体（MMR）mRNA的表达。此外，用IRF5 siRNA感染巨噬细胞，然后通过RT-PCR检测M1和M2中上述蛋白的mRNA表达，并通过蛋白质印迹法检测蛋白表达。分析结果以评估巨噬细胞的极化状态。

结果

流式细胞术检测巨噬细胞的分化比例为81.7%。RT-PCR显示，M1巨噬细胞中IRF5、IL-12、iNOS mRNA的表达明显高于M2巨噬细胞，TNF-α mRNA在M1巨噬细胞中也较高。M2巨噬细胞中Arg1、MMR mRNA的表达明显高于M1巨噬细胞。用IRF5 siRNA沉默IRF5后，巨噬细胞中IRF5、IL-12、TNF-α和iNOS mRNA的表达显著降低，而Arg-1、MMR mRNA的表达增加。蛋白质印迹法显示，IRF5、IL-12、TNF-α和iNOS蛋白的表达显著降低，而Arg1和MMR蛋白的表达升高。巨噬细胞的极化转变为M2状态。