The Mitochondrial Basis Of Profibrotic Macrophage Heterogeneity In Pulmonary Fibrosis And The Intervention Strategy Based On Exosomes

BackgroundIdiopathic pulmonary fibrosis（IPF）is a chronic and progressive interstitial lung disease.However,the etiology and pathogenesis of IPF remain incompletely elucidated,and the therapies are limited.In fibrotic lung tissues,macrophages secrete profibrotic factors,promoting the proliferation and activation of myofibroblasts and leading to the secretion of excess extracellular matrix（ECM）,ultimately resulting in dysregulated wound healing and remodeling of lung tissues.Macrophages exhibit a high degree of heterogeneity and plasticity.The M1 and M2 paradigm have become insufficient to fully explain the heterogeneity and complexity of macrophage phenotypes in fibrotic lung tissues.Mitochondria are closely associated with macrophage function.Therefore,this study proposed to reclassify lung macrophages into M?^mitolow and M?^mitohigh based on mitochondrial mass,and explored the function differs,molecular mechanisms,and targeted intervention strategies of these two subpopulations.ObjectivesTo elucidate the mitochondrial heterogeneity of macrophages in lung and its relationship with pulmonary fibrosis（PF）.To explore the molecular regulatory mechanisms underlying the heterogeneity of mitochondrial mass in macrophages.To develop a strategy for targeted regulation of mitochondrial mass in macrophages and alleviating PF.Methods1.A mouse model of Bleomycin（BLM）-induced PF was established,and the changes in M?^mitolow and M?^mitohigh in the lung tissues were explored via flow cytometry and q RT-PCR to clarify their function differs in PF.Transcriptomics techniques were applied to identify the key target for the increase in mitochondrial mass of M?^mitohigh.2.The mouse lung epithelial cells were treated with BLM/PBS,and the supernatants were then introduced into macrophages to establish a co-culture model,thereby investigating the effects of dynamin-related protein 1（Drp1）on the mitochondrial mass and profibrotic function of macrophages.3.Based on the surface markers of M?^mitohigh,the engineered exosomes targeting M?^mitohigh and delivering si Drp1 were constructed via molecular cloning techniques,systematically evaluating the inhibitory effect on PF.Results1.In PF mice,lung macrophages exhibited mitochondrial heterogeneity,and can be classified into M?^mitolow and M?^mitohigh based on mitochondrial mass.M?^mitohigh exhibited a more potent profibrotic function,with a significant increase in the lung tissue of PF mice.The expression of Drp1 m RNA was elevated in M?^mitohigh,which was a key factor in the increase of mitochondrial mass.2.Suppressing the expression of Drp1 m RNA in macrophages can reduce mitochondrial mass,restore mitochondrial function and inhibit the expression of genes related to profibrotic function.3.In the lung tissues of PF mice,the expression of M2 marker CD206 was higher in M?^mitohigh compared to M?^mitolow,suggesting that there was a certain degree of overlap between M?^mitohighand M2 macrophages,yet they were not entirely consistent.Targeting CD206⁺macrophages represented a relatively specific approach for targeting M?^mitohigh.4.Through the sequential administration of Exo^MMP19（the pathfinder exosome）and Exo^Tx（the therapeutic exosome）,the expression of Drp1 in M?^mitohigh was effectively suppressed,thereby inhibiting mitochondrial fission and ultimately alleviating PF.ConclusionIn fibrotic lung tissues,mitochondrial mass in macrophages possessed heterogeneity,and M?^mitohigh exhibited a more potent profibrotic function.Drp1 represented a pivotal target in regulating mitochondrial mass and profibrotic functions of macrophages.A combined exosome-based drug delivery strategy to inhibit the profibrotic effect of M?^mitohigh represented an effective approach for alleviating PF.