摘要
目的探究结核分枝杆菌(Mycobacterium tuberculosis,Mtb)毒力因子Rv1983刺激巨噬细胞释放的外泌体对Mtb感染的影响及潜在作用机制。方法超速离心法提取Mtb Rv1983蛋白刺激巨噬细胞分泌的外泌体,并与未感染巨噬细胞共培养,碘化丙啶染色法检测对巨噬细胞活力影响,脂质过氧化荧光探针法检测脂质活性氧(reactive oxygen species,ROS)水平,比色法测定细胞内脂质过氧化产物丙二醛(malondialdehyde,MDA)含量和亚铁离子(Fe2+)浓度,Western blot检测外泌体中酰基辅酶A合成酶长链家族成员4(acyl-CoA synthetase long chain family member 4,ACSL4)蛋白表达情况。提取Mtb Rv1983刺激ACSL4干扰后的巨噬细胞分泌的外泌体,并与未感染巨噬细胞共培养,流式细胞术和Western blot检测对巨噬细胞极化的影响。平板集落试验、吞噬溶酶体共定位试验分析对巨噬细胞吞噬杀伤能力影响。结果与对照组相比,Rv1983诱导巨噬细胞释放的外泌体可促进未感染巨噬细胞发生铁死亡,表现为细胞内脂质ROS、MDA和Fe2+水平升高,而它们被铁死亡抑制剂Fer-1显著抑制。Rv1983诱导巨噬细胞释放ACSL4高表达的外泌体。干扰巨噬细胞ACSL4后,经Rv1983刺激所产生的外泌体中ACSL4降低,诱导未感染巨噬细胞铁死亡明显减少。Rv1983诱导巨噬细胞释放的外泌体可诱导巨噬细胞向M2型极化,表现为CD206和Arg1表达升高,巨噬细胞吞噬杀伤能力减弱,而干扰ACSL4后分离的外泌体或Fer-1与Rv1983诱导巨噬细胞释放的外泌体联合处理则减少了巨噬细胞向M2型极化,巨噬细胞吞噬杀伤能力增强。结论Mtb Rv1983蛋白刺激巨噬细胞释放的外泌体中ACSL4表达上调诱导未感染巨噬细胞铁死亡,引起巨噬细胞M2极化,吞噬杀伤能力减弱,促进Mtb的感染。
ObjectiveTo explore the effect of exosomes released by Mycobacterium tuberculosis(Mtb)Rv1983-stimulated macrophages on tuberculosis infection and its potential mechanism.MethodsExosomes(Rv1983-M-Exo)released by macrophages following Mtb Rv1983 stimulation were extracted by hypervelocity centrifugation and then co-cultured with uninfected macrophages.Macrophage activity was detected by prodium iodide(PI)staining.The level of lipid reactive oxygen species(ROS)was detected by lipid peroxidation fluorescence probe.The expression of ferrous ions(Fe 2+)and malondialdehyde(MDA)were detected by colorimetry assay.The protein expression of acyl-CoA synthetase long chain family member 4(ACSL4)in Rv1983-M-Exo was detected by Western blot.Exosomes(Rv1983-M+siACSL4-Exo)were isolated from Rv1983-stimulated macrophages with interfered ACSL4 expression Rv1983 and co-cultured with uninfected macrophages.The effect of exosomes on the polarization of macrophages was detected by flow cytometry and Western blot.The effects of exosomes on phagocytosis and killing ability of macrophages were analyzed by plate colony assay and BCG-phagocytosis lysosome co-localization assay.ResultsCompared with macrophage-derived exosomes(M-Exo),Rv1983-M-Exo promoted ferroptosis in uninfected macrophages,manifested by increased levels of intracellular lipid ROS,MDA and Fe 2+,which were significantly inhibited by ferroptosis inhibitor Fer-1.Rv1983 induced macrophages to release exosomes with high expression of ACSL4.Interfering the expression of ACSL4 in macrophages,the concentration of ACSL4 in the Rv1983-M+siACSL4-Exo was significantly reduced.When the Rv1983-M+siACSL4-Exo was co-cultured with uninfected macrophages,the ferroptosis of macrophages was significantly reduced.Rv1983-M-Exo promoted M2 macrophage polarization which showed up-regulation of CD206 and Arg1 expression,and decreased the phagocytosis and killing ability of macrophages,while Rv1983-M+siACSL4-Exo or Fer-1 in combination with Rv1983-M-Exo reduced the expression of CD206 and Arg1 in ma
作者
蔡秦真
袁纯辉
王军
庹文彬
谢思
商宇
郭夏
向贇
Cai Qinzhen;Yuan Chunhui;Wang Jun;Tuo Wenbin;Xie Si;Shang Yu;Guo Xia;Xiang Yun(Department of Clinical Laboratory,Wuhan Children′s Hospital(Wuhan Maternal and Child Healthcare Hospital),Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430016,China)
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2024年第12期1018-1027,共10页
Chinese Journal of Microbiology and Immunology
基金
武汉市卫健委医学科研项目(WX21Q50,WX21M03,WX22Q08)
国家自然科学基金(82202536)
武汉市科技局自然科学基金(2022020801020573)。
