Exosomes derived from M2 macrophages regulate airway inflammation by modulating epithelial cell proliferation and apoptosis

Abstract

Background: Asthma is a chronic inflammatory disease characterized by airway remodeling and immune dysregulation. This study aimed to explore the mechanisms by which M2 macrophage-derived exosomes (M2Φ-Exos) regulate airway inflammation in asthma by modulating epithelial cell proliferation and apoptosis.

Methods: M2Φ-Exos were extracted and characterized by morphology, size, and marker protein expression. In vitro, the effects of M2Φ-Exos on House Dust Mites (HDM)-stimulated mouse lung epithelial cells (MLE-12s) were evaluated using western blotting to analyze Proliferating Cell Nuclear Antigen (PCNA), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and cleaved caspase-3 expression. In vivo, M2Φ-Exos were administered to HDM-induced asthmatic mice to assess their impact on airway inflammation, epithelial remodeling, and proliferation-apoptosis balance using immunohistochemistry, immunofluorescence, and western blotting. Cytokine levels in lung tissue and bronchoalveolar lavage fluid (BALF) were measured by qRT-PCR and ELISA.

Results: M2Φ-Exos displayed typical cup-shaped morphology, an average diameter of 115.5 nm, and expressed marker proteins CD9, TSG101, and CD63. MLE-12 cells internalized M2Φ-Exos, leading to reduced abnormal proliferation and apoptosis in HDM-stimulated cells. In asthmatic mice, M2Φ-Exos alleviated airway inflammation and epithelial thickening while reducing PCNA, cleaved caspase-3, and Bax levels and increasing Bcl-2 expression. M2Φ-Exos suppressed pro-inflammatory cytokines (IL-4, IL-5, IL-13) and Transforming growth factor (TGF)-β, while enhancing anti-inflammatory cytokine IFN-γ and IL-10.

Conclusion: These findings demonstrate that M2Φ-Exos regulate the imbalance in epithelial proliferation and apoptosis in asthma, reducing inflammation and mitigating tissue remodeling, and provide new insights into potential therapeutic strategies for asthma management.

Keywords: Apoptosis; Asthma; Exosome; M2 macrophage; Proliferation.

Fig. 1

Identification of M2Φ and M2Φ-Exosomes. A Morphological analysis of M2Φ under a microscope. Scale bar = 100 nm. B-G Gene expression of M2Φ-associated markers (Arg1, Mrc1, and YM1) and M1Φ-associated markers (TNF-α, IL-1β and CD86) quantified by RT-qPCR. H Expression of surface biomarkers F4/80 and CD206 on M2Φ, determined by flow cytometry. I Extraction process of M2Φ-derived exosomes (M2Φ-Exos). J Transmission electron microscopy (TEM) of exosomes isolated from the supernatant of M2Φ. Scale bar = 100 nm. K Size distribution of M2Φ-Exos measured by nanoparticle tracking analysis (NTA). L Western blot analysis of exosome marker proteins CD9, TSG101, and CD63, along with the endoplasmic reticulum marker protein Calnexin. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

M2-Exosomes inhibit the proliferation of MLE-12 cells induced by HDM. A Immunofluorescence staining showing the uptake of PKH-26-labeled M2Φ-Exosomes (red) by MLE-12 cells (DAPI, blue; PKH-26, red; BF, bright field). Control: untreated cells (without PKH26). Scale bar = 10 μm. B Cell viability of MLE-12 cells analyzed by the CCK-8 assay following treatment with varying concentrations of M2Φ-Exosomes. C Migration ability of MLE-12 cells assessed by wound healing assays at 24 h and 48 h post-treatment. D Quantitative analysis of wound healing in MLE-12 cells. E Proliferation of MLE-12 cells analyzed by EdU incorporation assay. Scale bar = 50 μm. F Quantification of EdU-positive cells. G Immunofluorescence staining for PCNA in MLE-12 cells. Scale bar = 10 μm. H Quantification of the region of interest (ROI) mean intensity of PCNA. I Western blot analysis of PCNA expression in the three groups. J Quantification of grayscale values from Western blot analysis. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

M2Φ-Exosomes reduce apoptosis in MLE-12 cells induced by HDM. A Apoptosis of MLE-12 cells was analyzed using Annexin V-FITC/PI staining by flow cytometry. B Flow cytometric analysis of apoptosis in MLE-12 cells. C Representative immunofluorescence images of TUNEL staining in the three groups. Scale bar = 10 μm. D Quantification of the region of interest (ROI) mean intensity of TUNEL staining. E Immunofluorescence staining for Cleaved-caspase3 in MLE-12 cells. Scale bar = 20 μm. F Quantification of ROI mean intensity of Cleaved-caspase3. G Representative western blots of caspase 3 and Cleaved-caspase3 proteins, and H, I quantification of grayscale values. J Representative western blots of Bax and Bcl2 proteins, and K, L quantification of grayscale values. N = 3/group. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant

Fig. 4

M2Φ-Exosomes inhibit lung injury in HDM-induced asthmatic mice. A Schematic representation of the process used to establish the HDM-induced allergic asthma mouse model. M2Φ-Exosomes were administered intranasally as a reference treatment. B Representative H&E staining of lung tissue sections from the three experimental groups. Scale bars: 50 μm and 20 μm. C Lung inflammation scores in the mice across the different groups. D Collagen fibers in mouse lung tissues visualized by Masson’s trichrome staining. Scale bars: 50 μm and 20 μm. E Quantification of collagen volume fraction. F Total cell count in the bronchoalveolar lavage fluid (BALF). G Eosinophil count in the BALF. H Total serum IgE levels in the mice, as analyzed by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

M2Φ-Exosomes Inhibit Aberrant Airway Epithelial Cell Proliferation in Asthmatic Mice. A Intranasal administration of DiR-labeled M2Φ-Exosomes was detected via fluorescence imaging. B Localization of DiR-labeled M2Φ-Exosomes within lung tissues was observed using fluorescence imaging. C Representative Western blots showing PCNA protein levels in lung tissues. D Quantification of grayscale values of PCNA expression. E Immunohistochemical (IHC) staining of PCNA-positive proliferating cells in lung tissues. Scale bar: 50 μm and 20 μm. F Quantification of immunohistochemical staining. *P < 0.05, **P < 0.01

Fig. 6

M2Φ-Exosomes inhibit apoptosis of airway epithelial cells in asthmatic mice. A Representative images of TUNEL staining in lung tissue from asthmatic mice. Scale bar = 100 μm. B Quantification of the region of interest (ROI) mean intensity of TUNEL staining. C Immunofluorescence staining for Cleaved-caspase3 in lung tissue of mice. Scale bar = 50 μm. D Quantification of ROI mean intensity of Cleaved-caspase3. E Representative western blots showing caspase 3 and Cleaved-caspase3 protein expression. F, G Quantification of grayscale values for caspase 3 and Cleaved-caspase3. H Representative western blots of Bax and Bcl2 protein expression. I, J Quantification of grayscale values for Bax and Bcl2. K IHC staining for Cleaved-caspase3-positive cells in lung tissues. Scale bars = 50 μm and 20 μm. L Quantification of immunohistochemical staining. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant

Fig. 7

M2Φ-Exosomes regulate inflammatory cytokines in asthmatic mice. Quantification of mRNA levels for (A) IL-4, (B) IL-5, (C) IL-13, (D) TGF-β, (E) IL-10 and (F) IFN-γ in lung tissue using qRT-PCR. Expression of (G) IL-4, (H) IL-5, (I) IL-13, (J) TGF-β, (K) IL-10 and (L) IFN-γ in BALF of mice detected by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001