miR-155通过调控巨噬细胞极化和ROS/NO分泌发挥抗菌作用

MiR-155 exerts bacteria killing effects by regulating the polarization and ROS/NO secretion in macrophages

目的:探讨miR-155通过调控巨噬细胞极化发挥清除病原菌的作用和机制。方法:使用脂多糖/干扰素-γ(lipopolysaccharide/interferon-γ,LPS/IFN-γ)或白细胞介素(interleukin,IL)-4分别处理RAW264.7细胞和骨髓来源巨噬细胞(bone marrow-derived macrophage,BMDM),24 h后检测miR-155表达水平;将RAW264.7细胞和BMDM分别过表达、抑制miR-155后,检测巨噬细胞极化表型转化,以及活性氧、活性氮和细胞因子释放;将miR-155过表达的BMDM进行M1型诱导,取上清培养大肠杆菌不同时间后,检测大肠杆菌菌落形成能力。结果:LPS/IFN-γ的添加可以明显促进miR-155在RAW264.7细胞和BMDM中的表达,而IL-4的诱导降低BMDM中miR-155水平;进一步在RAW264.7细胞和BMDM中过表达miR-155后,可促进M1型极化分子转录、抑制M2型极化分子标志物的表达;反过来,通过反义寡核苷酸抑制miR-155后,可以抑制M1型极化而促进M2型极化;同时,过表达miR-155的巨噬细胞分泌的一氧化氮(nitric oxide,NO)、活性氧(reactive oxygen species,ROS)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)显著性被促进;过表达miR-155的M1型极化BMDM培养上清明显可以减少大肠杆菌菌落形成数量。结论:miR-155通过调控巨噬细胞极化,影响其ROS/NO和细胞因子的分泌,参与巨噬细胞清除、杀伤病原菌的过程。

Objective To investigate the role and mechanisms of miR-155 in eliminating the pathogenic bacteria by regulating the polarization of macrophages.Methods RAW264.7 cells and bone marrow-derived macrophages(BMDM)were treated with lipopolysaccharide/interferon-γ(LPS/IFN-γ)or interleukin(IL)-4 respectively and the expression of miR-155 was detected after 24 h.Further,the polarization status of macrophages was determined when miR-155 was over-expressed or inhibited in RAW264.7 cells and BMDM.Simultaneously,the reactive oxygen species,reactive nitrogen and cytokine production were also accessed in macrophages with the same treatment.Escherichia coli was cultured in the supernatant of M1 polarized miR-155 over-expressing BMDM or control BMDM.The colony formation capacity of Escherichia coli was detected after incubation with different time.Results LPS/IFN-γcould significantly promote miR-155 expression in RAW264.7 cells and BMDM;meanwhile,IL-4 decreased the level of miR-155 in BMDM.The further results displayed that over-expression of miR-155 promoted the M1 polarization and inhibited M2 polarization markers in RAW264.7 cells and BMDM.Inhibition of miR-155 by using antisense oligonucleotides suppressed M1 polarization and promoted M2 polarization.Simultaneously,the production and secretion of nitric oxide(NO),reactive oxygen species(ROS)and tumor necrosis factor-α(TNF-α)were promoted significantly in macrophage over-expressed miR-155.The supernatant of M1-polarized macrophage with miR-155 over-expression could obviously promote bacteria killing and inhibit Escherichia coli colony forming ability.Conclusion MiR-155 regulates macrophage polarization,affects the production of NO,ROS and cytokines,and participates in the process of macrophage clearance and killing pathogens.