肺纤维化微环境中肺泡巨噬细胞的蛋白质组学分析

Proteomic Analysis of Alveolar Macrophages in Pulmonary Fibrosis Microenvironment

目的肺泡巨噬细胞(alveolar macrophages,AMs)能够处理表面活性物质来维持肺泡膨胀开放,并充当防御病原体入侵的第一道免疫防线,对肺部微环境稳态的维持至关重要。已有研究表明,肺纤维化过程中,单核来源的AMs持续释放促炎因子和趋化因子,招募更多免疫细胞到受损区域,维持并加剧炎症,从而发挥负面作用。目前,大多数研究聚焦于肺纤维化微环境AMs的基因表达水平,而在蛋白质功能和调控方面的报道较少。本研究旨在探讨正常生理条件下与肺纤维化后AMs的差异表达蛋白(differentially expressed proteins,DEPs),以便更全面地理解AMs在肺纤维化发展过程中的作用。方法本研究建立博来霉素诱导肺纤维化小鼠模型,利用流式细胞分选技术收集来自生理盐水对照组和肺纤维化模型的AMs(每个样本2.5×105个细胞),通过无标记蛋白质组学方法获得蛋白质表达谱。结果通过将生理盐水组与已公开的生理AMs蛋白质组数据比对发现,本研究产出的蛋白质组数据质量较高,能够满足研究需求。综合分析结果显示,与对照组相比,博来霉素组AMs有778种蛋白质表达上调。此外,上调的DEPs中富集通路包括I-κB/NF-κB通路、炎症反应调节通路、吞噬调节通路、TGF-β通路和HIF-1通路,表明肺纤维化微环境中的AMs具有促炎和促纤维化功能。对DEPs的蛋白质-蛋白质相互作用网络分析表明,Tlr2和Pycard之间的相互作用是AMs促炎表型的控制节点,从而导致肺纤维化进展。结论本研究探讨了AMs在肺纤维化微环境中蛋白质表达谱的变化。结果发现,AMs显著上调多条与炎症和纤维化关联的通路蛋白,并提示Tlr2和Pycard的相互作用是AMs表现出高度促炎活性的控制节点。

Objective Alveolar macrophages(AMs)are critical for maintaining the homeostasis of pulmonary microenvironment.They process surfactants to ensure alveoli patency,and also serve as the first line of immune defense against pathogen invasion.Available studies have shown that monocyte-derived AMs continuously release pro-inflammatory cytokines and chemokines,recruiting other immune cells to the damaged area during pulmonary fibrosis.These monocyte-derived AMs maintains and amplifies inflammation,playing a negative role in pulmonary fibrosis progression.Current researches have predominantly focused on the gene expression levels of AMs in pulmonary fibrosis microenvironment,with less emphasis on the function and regulation of proteins.This study aims to investigate the differentially expressed proteins(DEPs)of AMs under normal physiological conditions and after pulmonary fibrosis,in order to gain a more comprehensive understanding of the role of AMs in the progression of pulmonary fibrosis.Methods Firstly,the construction of bleomycin-induced pulmonary fibrosis mouse models was evaluated through using measurements such as body mass,lung coefficient,lung wet-to-dry mass ratio,H&E staining and Masson staining.Subsequently,AMs from both the saline controls and the pulmonary fibrosis models(2.5×105 cells per sample)were collected using FACS sorting,and protein expression profiles of these cells were obtained through label-free proteomics approach.The quality of the proteomic data was assessed by comparing our saline control proteomic data with public proteomic data of physiological AMs.Thirdly,DEPs analysis between the saline controls and the bleomycin groups was carried out using R package Prostar.Functional enrichment analyses of significantly upregulated DEPs were performed using R package Clusterprofiler for GO and KEGG pathways.Finally,the STRING database was used to explore the protein-protein interaction networks related to phagocytosis regulation,inflammatory response regulation,and I-κB/NF-κB signaling pathway.The expression levels of Tlr2 and Pycard were detected respectively by FACS and western blotting.Results Compared to the saline controls,mice in the bleomycin groups exhibited a lower average body mass,extensive infiltration of inflammatory cells,and deposition of collagen in the lungs.This indicates that bleomycin successfully induced pulmonary fibrosis in mouse models.The proteomic data of AMs obtained from these models was of high quality and fulfilled the research requirements.A comprehensive analysis showed that 778 proteins were upregulated in pulmonary fibrosis groups compared with control groups.Moreover,the signal pathways enriched in up-regulated DEPs were related to the I-κB/NF-κB pathway,inflammatory response regulation,phagocytosis regulation,TGF-βsignaling,and HIF-1 pathway,indicating that AMs in pulmonary fibrosis microenvironment exerted pro-inflammatory and pro-fibrotic functions.Protein protein interaction network analysis of the DEPs suggested that the interactions between Tlr2 and Pycard were control nodes for the pro-inflammatory phenotype of AMs,thereby contributing to pulmonary fibrosis progression.Further validation by FACS and Western blotting respectively confirmed that the expression levels of Tlr2 and Pycard in AMs were significantly increased after pulmonary fibrosis.Conclusion This study investigates the changes in the protein expression profile of AMs in the pulmonary fibrosis microenvironment.The results show that AMs notably enhanced the activity of various pathways associated with inflammation and fibrosis,suggesting that the interaction between Tlr2 and Pycard serves as a key control node for the highly pro inflammatory behavior of AMs.