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BACKGROUND

Members of the interferon regulatory factor (IRF) family are transcriptional regulators that play vital roles in the inflammatory response of macrophages. IRF1, IRF3, and IRF9 regulate the expression of immune-responsive gene 1 (IRG1) in macrophages. However, the role of IRF2 in the inflammatory response of macrophages remains somewhat contradictory. The regulatory relationship between IRF2 and IRG1 and their role in macrophages remain unclear. This study aimed to explore the role of IRF2 and IRG1 in macrophages.

METHODS

Overexpression plasmids of IRF2 (IRF2-pcDNA3.1) and silencing siRNA targeting IRF2 and IRG1 (si-IRF2, si-IRG1) were constructed and transfected into RAW264.7 cells respectively. Subsequently, cells were treated with LPS and IFN-γ for 24 h. The expression of IRF2, IRG1, iNOS, IL-6, and BCL-xl was detected using Western blotting and qRT-PCR. Cell viability, migration and apoptosis were determined by CCK-8, transwell and flow cytometry. The IRG1 promoter region was cloned into the pGL3-basic plasmid. A dual-luciferase reporter assay was performed to verify the regulatory relationship between IRF2 and IRG1.

RESULTS

IRF2 overexpression inhibited the expression of IL-6 and iNOS, cell migration, and apoptosis and elevated the expression of BCL-xl, IRG1, and cell viability of LPS- and IFN-γ-induced macrophages. IRF2 silencing had an opposite effect. IRF2 activated the promoter activity of IRG1. The inhibitory effects on LPS- and IFN-γ-induced proinflammatory responses, cell migration, apoptosis, and enhancing effects on the cell viability of over-expressed IRF2 were reversed by IRG1 silencing.

CONCLUSION

IRF2 promoted the promoter activity of IRG1 and regulated directly the expression of IRG1. IRF2 inhibited LPS and IFN-γ-induced pro-inflammatory responses, cell migration and apoptosis, enhanced cell viability in macrophages through regulating IRG1. IRF2 affected LPS- and IFN-γ-induced pro-inflammatory responses, cell viability, migration and apoptosis of macrophages by regulating IRG1.

BACKGROUND

METHODS

RESULTS

CONCLUSION

背景

干扰素调节因子（IRF）家族成员是转录调节因子，在巨噬细胞的炎症反应中发挥重要作用。IRF1、IRF3和IRF9调节巨噬细胞中免疫反应基因1（IRG1）的表达。然而，IRF2在巨噬细胞炎症反应中的作用仍存在一定矛盾。IRF2与IRG1之间的调控关系及其在巨噬细胞中的作用尚不清楚。本研究旨在探讨IRF2和IRG1在巨噬细胞中的作用。

方法

构建IRF2的过表达质粒（IRF2-pcDNA3.1）以及靶向IRF2和IRG1的沉默siRNA（si-IRF2、si-IRG1），并分别转染至RAW264.7细胞中。随后，用脂多糖（LPS）和干扰素-γ（IFN-γ）处理细胞24小时。采用蛋白质免疫印迹法和实时定量聚合酶链反应（qRT-PCR）检测IRF2、IRG1、诱导型一氧化氮合酶（iNOS）、白细胞介素-6（IL-6）和凋亡抑制蛋白（BCL-xl）的表达。通过细胞计数试剂盒-8（CCK-8）、Transwell小室和流式细胞术检测细胞活力、迁移和凋亡情况。将IRG1启动子区域克隆至pGL3-basic质粒中。进行双荧光素酶报告基因检测以验证IRF2与IRG1之间的调控关系。

结果

IRF2过表达抑制了LPS和IFN-γ诱导的巨噬细胞中IL-6和iNOS的表达、细胞迁移和凋亡，并提高了BCL-xl、IRG1的表达以及细胞活力。IRF2沉默则产生相反的效果。IRF2激活了IRG1的启动子活性。IRG1沉默可逆转过表达IRF2对LPS和IFN-γ诱导的促炎反应、细胞迁移、凋亡的抑制作用以及对细胞活力的增强作用。