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.
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