解放军医学杂志2025，Vol.50Issue(2)：176-187,12.DOI:10.11855/j.issn.0577-7402.0108.2024.0805
嗜肺军团菌抑制小鼠巨噬细胞内体溶酶体融合的机制探讨
Investigating the mechanism of Legionella pneumophila inhibiting endosome-lysosome fusion in mouse macrophages based on transcriptome sequencing
摘要
Abstract
Objective To explore the pathogenic mechanisms of Legionella pneumophila(L.pneumophila)infection inhibiting the fusion of endosome-lysosome fusion in mouse macrophages.Methods Twelve C57 mice were randomly divided into control group and L.pneumophila infection group(n=6 each).After anesthesia,an equal volume of physiological saline or L.pneumophila solution was administered nasally.Body weight changes were monitored for 3 consecutive days,and the lungs were extracted to assess injury.Hematoxylin and eosin(HE)staining and immunohistochemical staining were performed to observe the pathological characteristics of lung tissue in both groups.Transcriptome sequencing was utilized to analyze differentially expressed genes(DEGs)and associated signaling pathways in lung tissues.Mouse bone marrow macrophages(BMDMs)were isolated and co-cultured with L.pneumophila,with infection status confirmed by immunofluorescence staining.Transcriptome sequencing was employed to analyze DEGs and enriched related signaling pathways before and after infection.Core genes involved in the post-infection signaling pathway were identified,and the consistency of their mRNA expression levels in vivo and in vitro was verified using RT-qPCR.The expression of relevant proteins was detected by Western Blotting,and bacterial proliferation assays were conducted to evaluate the intracellular replication of L.pneumophila.Results Compared with control group,the body weight of mice in L.pneumophila infection group significantly decreased(P<0.001)on the second and third day post-infection.Edema and red hepatoid degeneration were observed in both left and right lung tissues,with lesion areas spreading from the hilum to the lung periphery.HE staining revealed increased inflammatory cell infiltration in the alveolar spaces,thickening of alveolar septa and increased fibrin exudation in L.pneumophila infection group.Immunohistochemistry results showed a significant increase in myeloperoxidase(MPO)activity in the lung tissue infected mice(P<0.001).Transcriptome sequencing identified 2550 DEGs,with 1444 up-regulated genes and 1106 down-regulated genes.KEGG enrichment analysis indicated that these DEGs were mainly involved in pathways related to tumor necrosis factor,rheumatoid arthritis,Rap1,PI3K-Ak,and phagosome pathways.Immunofluorescence results showed in vitro proliferation of L.pneumophila within mouse BMDMs.Transcriptome sequencing identified 2550 DEGs,including 1677 up-regulated genes and 873 down-regulated genes.KEGG enrichment analysis showed that enrichment in pathway related to transcription dysregulation in cancer,PI3K-Akt and phagosome pathways.Thirteen core genes,including tubulin β1(Tubb1),were identified from the overlap between mouse lung tissue and BMDMs.RT-qPCR results demonstrated a significant decrease in Tubb1 expression in both lung tissue and BMDMs infected with L.pneumophila(P<0.001).Western Blotting results revealed significant decreases in Rab7,Tubb1,and LAMP2 protein expression(P<0.05),and increases in iNOS and MPO expression(P<0.05).Intracellular proliferation experiments indicated that L.pneumophila gradually increased within BMDMs over time.Conclusion The potential mechanism of L.pneumophila infection in mouse macrophages involves the down-regulation of Rab7/Tubb1/LAMP2 which inhibits the endosome-lysosome fusion.关键词
嗜肺军团菌/微管蛋白β1/内体溶酶体融合/转录组测序/巨噬细胞Key words
Legionella pneumophila/tubulin beta 1/endosome-lysosome fusion/transcriptomics/macrophages分类
基础医学引用本文复制引用
陈民佳,曹秀琴,贺瑞霞,陈海霞,杨志伟..嗜肺军团菌抑制小鼠巨噬细胞内体溶酶体融合的机制探讨[J].解放军医学杂志,2025,50(2):176-187,12.基金项目
This work was supported by the National Natural Science Foundation of China(82060362),and the Ningxia Natural Science Foundation(2023AAC03182) 国家自然科学基金(82060362) （82060362）
宁夏自然科学基金(2023AAC03182) （2023AAC03182）
