Macrophage Exosomes Transfer Angiotensin II Type 1 Receptor to Lung Fibroblasts Mediating Bleomycin-Induced Pulmonary Fibrosis
Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disease characterized by inflammation and excessive extracellular matrix (ECM) deposition. Lung fibroblasts play a pivotal role in the initiation and progression of pulmonary fibrosis through their proliferation and synthesis of ECM components. Inhibiting fibroblast activation has emerged as a promising therapeutic strategy for pulmonary fibrosis. Macrophages, essential inflammatory regulators, are known to promote fibrogenesis in various organs, including the lungs. However, the mechanisms by which macrophages communicate with lung fibroblasts remain largely unexplored. Exosomes, nanometer-sized vesicles that mediate intercellular communication, have been implicated in chronic inflammatory lung diseases, including pulmonary fibrosis. This study investigates the role of exosomes in mediating the crosstalk between macrophages and lung fibroblasts, focusing on the angiotensin II (Ang II)/angiotensin II type 1 receptor (AT1R) axis and its contribution to bleomycin (BLM)-induced pulmonary fibrosis.
Role of Exosomes in Pulmonary Fibrosis
Exosomes are secreted by nearly all cell types and carry bioactive molecules, including proteins, lipids, RNA, and DNA. They play a crucial role in intercellular communication and have been linked to the progression of chronic inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. In IPF patients, exosomes exhibit altered levels of fibrogenic and anti-fibrotic microRNAs (miRNAs), suggesting their involvement in fibrotic processes. However, the specific mechanisms by which exosomes contribute to lung fibrosis remain unclear.
Macrophages and Pulmonary Fibrosis
Macrophages are key players in the pathogenesis of fibrotic lung diseases. They promote fibrosis by activating lung fibroblasts through paracrine signaling. Previous studies have demonstrated that macrophages exert pro-fibrotic effects in various organs, including the heart, kidneys, liver, and lungs. In the lungs, macrophages are thought to activate fibroblasts through the secretion of cytokines and other mediators. However, whether exosomes mediate this interaction between macrophages and fibroblasts has not been explored.
The Ang II/AT1R Axis in Pulmonary Fibrosis
The renin-angiotensin system (RAS) is a critical regulator of inflammation and fibrosis. The Ang II/AT1R axis, a component of RAS, is upregulated in pulmonary fibrosis and contributes to disease progression. Ang II, a potent pro-fibrotic factor, binds to AT1R to activate downstream signaling pathways, including the transforming growth factor-beta (TGF-β)/Smad pathway, which promotes collagen synthesis and fibrosis. Recent studies have shown that exosomes derived from cardiac fibroblasts increase Ang II production and AT1R expression in cardiomyocytes, leading to pathological cardiac hypertrophy. This study hypothesizes that macrophage-derived exosomes may promote collagen synthesis in lung fibroblasts by upregulating the Ang II/AT1R axis.
Experimental Design and Methods
To investigate the role of exosomes in pulmonary fibrosis, the study employed both in vivo and in vitro models. In vivo, a BLM-induced lung fibrosis model was established in male C57 mice. The effects of GW4869, an exosome inhibitor, on lung fibrosis were assessed. Additionally, macrophage-derived exosomes were injected into mice to evaluate their pro-fibrotic effects. In vitro, exosomes were isolated from Ang II-stimulated macrophages and used to treat lung fibroblasts. The expression of collagen I, AT1R, TGF-β, and phosphorylated Smad2/3 (p-Smad2/3) in fibroblasts was examined to determine the impact of exosomes on fibroblast activation.
Results: Exosomes Inhibitor Attenuates Pulmonary Fibrosis
In the BLM-induced pulmonary fibrosis model, GW4869 significantly attenuated fibrosis, as evidenced by reduced Ashcroft scores, hydroxyproline content, and collagen I expression. These findings suggest that exosomes play a pro-fibrotic role in BLM-induced lung fibrosis. Furthermore, the infiltration of macrophages and the levels of Ang II and AT1R were increased in BLM-treated mice, and these effects were reversed by GW4869. These results indicate that exosomes mediate the pro-fibrotic effects of macrophages in pulmonary fibrosis.
Macrophage Infiltration Correlates with Lung Fibrosis
The study also examined the temporal changes in macrophage infiltration during BLM-induced lung fibrosis. BLM-treated mice showed increased infiltration of inflammatory cells, loss of alveolar structure, and thickening of alveolar septa over time. The number of macrophages in lung tissue and bronchoalveolar lavage fluid (BALF) was significantly increased, peaking at day 28 post-BLM treatment. These findings confirm that macrophage infiltration is closely associated with the progression of lung fibrosis.
Macrophage Exosomes Promote Collagen Synthesis in Fibroblasts
Exosomes derived from Ang II-stimulated macrophages were characterized using transmission electron microscopy and nanoparticle tracking analysis. These exosomes were round, vesicle-like, and ranged in size from 40 to 150 nm. They expressed exosomal markers such as Alix, CD9, and CD63 but were negative for calnexin, confirming their identity as exosomes. When lung fibroblasts were treated with these exosomes, they exhibited increased uptake of exosomes and elevated levels of collagen I, indicating that macrophage exosomes promote collagen synthesis in fibroblasts.
Activation of the Ang II/AT1R Axis and TGF-β/Smad Pathway
The study further explored the molecular mechanisms underlying the pro-fibrotic effects of macrophage exosomes. Treatment with exosomes increased the expression of AT1R, TGF-β, and p-Smad2/3 in lung fibroblasts, suggesting that exosomes activate the Ang II/AT1R axis and the TGF-β/Smad pathway. The AT1R blocker irbesartan (IR) reversed these effects, confirming that the pro-fibrotic effects of exosomes are mediated through the Ang II/AT1R axis.
Direct Transfer of AT1R by Exosomes
To determine how exosomes increase AT1R expression in fibroblasts, the study examined AT1R mRNA levels in fibroblasts treated with exosomes. No significant change in AT1R mRNA was observed, indicating that the increase in AT1R protein is not due to transcriptional upregulation. Instead, exosomes were found to contain AT1R protein, which was directly transferred to fibroblasts. This was confirmed by silencing AT1R in macrophages, which abolished the ability of exosomes to induce AT1R expression in fibroblasts. Additionally, GFP-tagged AT1R in macrophages was detected in fibroblasts after exosome treatment, providing further evidence that exosomes directly deliver AT1R to fibroblasts.
Ang II Enhances Exosome Production
The study also investigated the role of Ang II in exosome production. Ang II stimulation increased the secretion of exosomes from macrophages, as evidenced by elevated levels of exosomal markers Alix and AT1R. This suggests a positive feedback loop in which Ang II promotes exosome production, and exosomes, in turn, enhance Ang II signaling in fibroblasts. This loop may contribute to the sustained activation of the Ang II/AT1R axis and the progression of pulmonary fibrosis.
In Vivo Effects of Macrophage Exosomes
To further validate the pro-fibrotic effects of macrophage exosomes, exosomes were injected into the tail vein of mice. Exosome-treated mice exhibited increased hydroxyproline content, macrophage infiltration, and activation of the Ang II/AT1R axis and TGF-β pathway. These effects were reversed by IR, confirming that exosomes promote lung inflammation and fibrosis through the Ang II/AT1R axis.
Conclusion
This study demonstrates that AT1R-containing exosomes derived from macrophages play a critical role in BLM-induced pulmonary fibrosis. These exosomes activate the Ang II/AT1R axis and the TGF-β/Smad pathway in lung fibroblasts, promoting collagen synthesis and fibrosis. The findings reveal a novel mechanism by which macrophages communicate with fibroblasts through exosomes, highlighting the potential of targeting exosome production or the Ang II/AT1R axis as a therapeutic strategy for pulmonary fibrosis. The study also identifies a positive feedback loop between Ang II and exosome production, which may sustain fibrotic signaling in the lungs. These insights provide a foundation for further research into the role of exosomes in fibrotic diseases and the development of targeted therapies.
doi.org/10.1097/CM9.0000000000001605
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