AdMSC-derived exosomes alleviate acute lung injury via transferring mitochondrial component to improve homeostasis of alveolar macrophages

Abstract

Rationale: Aberrant activation of macrophages with mitochondria dismiss was proved to be associated with pathogenesis of ALI (acute lung injury). Exosomes from adipose-derived mesenchymal stem cells (AdMSC-Exos) have been distinguished by their low immunogenicity, lack of tumorigenicity, and high clinical safety, but their role in treating ALI and the mechanism involved need to be defined. In this study, we sought to investigate whether the mitochondrial donation from AdMSC-Exos provides profound protection against LPS-induced ALI in mice, accompanied by improvement of macrophage mitochondrial function. Methods: C57BL/6 mice were orotracheally instilled with LPS (1 mg/kg). AdMSC-Exos were administered via the tail vein 4 h after LPS inhalation. Flow cytometry, H&E, Quantitative Real-Time PCR, immunofluorescence (IF), confocal microscopy imaging was conducted to investigate lung tissue inflammation and macrophage mitochondrial function. And further observe the transfer of exosomes and the effect on mitochondrial function of MH-S cells through in vitro experiments. Results: AdMSC-Exos can transfer the stem cell-derived mitochondria components to alveolar macrophages in a dose-dependent manner. Likely through complementing the damaged mitochondria, AdMSC-Exos exhibited the ability to elevate the level of mtDNA, mitochondrial membrane potential (MMP), OXPHOS activity and ATP generation, while reliving mROS stress in LPS-challenged macrophages. Restoring mitochondrial integrity via AdMSC-Exos treatment enabled macrophages shifting to anti-inflammatory phenotype, as featured with the down-regulation of IL-1β, TNF-α and iNOS secretion and increase in production of anti-inflammatory cytokines IL-10 and Arg-1. As we depleted alveolar macrophages using clodronate liposomes, the protective role for AdMSC-Exos was largely abrogated. Conclusions: AdMSC-Exos can effectively donate mitochondria component improved macrophages mitochondrial integrity and oxidative phosphorylation level, leading to the resumption of metabolic and immune homeostasis of airway macrophages and mitigating lung inflammatory pathology.

Keywords: acute lung injury; alveolar macrophages; exosomes; mitochondrial function; stem cells.

Figure 1

AdMSC-Exos are capable of transferring mitochondria to macrophages. (A) To isolate exosomes, conditioned media (CM) were subjected to successive differential centrifugation. (B) Nanoparticle tracking analysis of vesicles derived from AdMSCs. The mean protein concentration and mean particle concentration of the AdMSC-Exos were 2.44 mg/mL and 2.33 × 10⁹ particles/mL, respectively. (C) Transmission electron microscopy analysis of vesicles derived from AdMSCs (Scale bar, 100 nm). (D) Exosome representative markers CD9, CD63 and TSG101 were detected by western blot. Both lanes were loaded with 40 μg of proteins. (E) The experimental schematic of AdMSC-Exos transfers mitochondria to macrophages. Mito Tracker Red was used to pre-label the mitochondria in AdMSCs, and then the exosomes were separated from the cell culture supernatant, and the exosomes were incubated with MH-S cells for 12 h, and the phagocytosis of exosomes by macrophages was analyzed by flow cytometry. (F) The confocal laser scanning microscope micrographs showing Mito-Red staining of mitochondria in adipose mesenchymal stem cells. Scale bar, 50 μm. Mito-red: red, DAPI: blue. (G) The confocal laser scanning microscope micrographs showing that AdMSC-Exos containing stem cell mitochondrial fragments are internalized by macrophages. Left scale bar, 25 μm; Right scale bar, 5 μm. Mito-red: red, HSP60: green, DAPI: blue. (H) The co-localization of mitochondrial DNA and mitochondria in MH-S cells was examined by confocal microscope. Among them, nDNA and mtDNA are dyed green by Draq5, and functional mitochondria are dyed red by Mito Tracker Red. We deleted mtDNA in MH-S cells using low-dose EtBr (Materials and Methods), and then transfer the exosomes to mtDNA-deleted MH-S cells. The EtBr + PBS group is a negative control for exosomes (Scale bar, 10 μm). (I) Representative FACS plots of Mito Red on MH-S cells at increasing amounts of AdMSC-Exos. All the data are expressed as the mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Human mitochondria DNA transfers through AdMSC-exos and improves macrophage mitochondrial fitness. (A) Mitochondrial DNA expression on MH-S cells at increasing amounts of AdMSC-Exos. (B) Left panel, representative agarose gel electrophoresis image of the 16.5-kbp whole mtDNA genome from purified AdMSC-Exos. Right panel, the average number of copies of human mitochondrial DNA in exosomes derived from human adipose-derived mesenchymal stem cells. After treatment with Deoxyribonuclease (DNase) and ethidium bromide (EtBr), mtDNA in AdMSC-Exos is deleted. (C) Left panel, the expression of human mtDNA PCR amplified product 109-bp ND1 and 154-bp ND5 detected in human MSCs-derived exosomes. Right panel, after transferring AdMSC-Exos ± DNase to MH-S cells, qPCR detects the copy number of human mitochondrial DNA in MH-S cells. (D) AdMSC and purified AdMSC-Exos were blotted for proteins associated with mtDNA (TFAM), for proteins located in the outer mitochondrial membrane (TOM20 and VDAC), the inner mitochondrial membrane (NDUFV2), the endoplasmic reticulum (CALR) and the lysosomal (LAMP1). (E) The histogram shows the relative ratio of the expression level of the target protein to that of an internal reference. All the data are expressed as the mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Transferring of mitochondrial component through AdMSC-Exos improves macrophage mitochondrial function. (A) Representative transmission electron micrographs (TEM) showing mitochondrial amount, morphology and cristae. The histogram represents the quantification of the size and number of mitochondria. Scale bar, 1 μm. (B) Flow cytometry analysis of the effect of exosomes on MH-S cell apoptosis. (C) Flow cytometry analysis of the effect of exosomes on the proliferation of MH-S cells. (D) Mitochondrial DNA expression in MH-S cells. (E) Expression of mitochondrial ATP production in MH-S cells. (F) Effects of AdMSC-Exos on oxidative phosphorylation of MH-S cells detected by Extracellular flux analysis. (G) Flow cytometry and quantification of mitochondrial reactive oxygen species (ROS) levels by staining with Mito Sox. (H) Flow cytometry of mitochondria staining with Mito Tracker Red and Mito Tracker green (Green⁺/Red^-). (I) Heatmap showing the expression of mitochondrial respiratory chain complex-related genes in MH-S cells detected by qPCR. Primer sequences are reported in Table s1. All the data are expressed as the mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

AdMSC-Exos promotes functional shift of endotoxin-challenged macrophages to anti-inflammatory phenotype via mitochondria transfer. MH-S cells were pretreated with AdMSC-Exos or PBS for 30 min, and then subjected to LPS (100 ng/mL) stimulation for the time periods, as indicated. (A) Relative mRNA of pro-inflammatory and anti-inflammatory cytokines were assayed by qPCR. (B) Expression of MHC II and CD206 on MH-S cells 12 h post-LPS exposure was detected by flow cytometry. Representative histograms and average relative mean fluorescence intensity (MFI) are depicted. (C) Relative levels of pro-inflammatory and anti-inflammatory cytokines were assayed by ELISA. (D, E) Western blot analysis of phosphorylated and total IKKα, IκBα, p65, JNK, ERK and p38 levels in MH-S cells that were pretreated with AdMSC-Exos or PBS for 30 min and then stimulated with LPS for the indicated time periods. (F) The effect of nucleosome inhibition on the anti-inflammatory effect of AdMSC-exos. (G) The effect of proteasome inhibition on the anti-inflammatory effect of AdMSC-exos. All the data are expressed as the mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

The delivery of AdMSC-Exos alleviates lung inflammation and injury in mice. C57BL/6 mice (n = 5 mice/group) were challenged with LPS (1 mg/kg, intratracheally) for 4 h, and then tail vein injected with PBS or Exos (10 μg/mL). 24 h later, mice were sacrificed and subjected to the functional analysis. (A) Representative H&E staining of lung tissues (Scale bar, 50 μm). The histogram shows the lung tissue pathological damage score. (B) BALF cell counts; (C) MPO level in lungs; (D) total protein level; and (E) cytokine concentration in the BAL fluid. (F) Flow cytometry analysis of Alveolar macrophages (CD11c⁺ Siglec F⁺) in BAL fluid (Gate on CD11b^- CD64⁺). (G) Flow cytometry analysis of macrophages (CD11b⁺ F4/80⁺) in BAL fluid. (H) Flow cytometry analysis of neutrophils (CD11b⁺ Ly6G⁺) in BAL fluid. (I) Survival rate of the mice that were challenged with LPS at a high dose (10 mg/kg, i.t.) and then treated with Exos or PBS (Injection via tail vein). Kaplan-Meier survival plots were depicted (n = 15 mice/group). All the data are expressed as the mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

AdMSC-Exos treatment improves macrophage mitochondrial function in vivo. (A) Ex vivo images of vital organs at 2 and 12 h after MitoRed-exos post injection. (B) Representative confocal laser scanning microscopy micrographs showing the colocalization of F4/80 immunostaining (green) with internalized AdMSC-Exos (red). Scale bar, 10 μm. DAPI: blue. (C) Mitochondrial DNA expression in Mouse alveolar macrophages. (D) Assay of ATP generation in Mouse alveolar macrophages. (E) Effects of AdMSC-Exos on mitochondrial viability and quantity in Mouse alveolar macrophages by flow cytometry. (F) Flow cytometry and quantification of Mouse alveolar macrophages mitochondrial ROS levels by staining with Mito Sox. (G) Expression of mitochondrial-associated protein in mouse alveolar macrophages. Shown are representative data from two independent experiments. All the data are expressed as the mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

The therapeutic effect of AdMSC-Exos on ALI largely depends on alveolar macrophages. (A) The experimental schematic of Alveolar macrophage depletion experiment (n = 5 mice/group). (B) Flow cytometry analysis of Alveolar macrophages (CD11c⁺ Siglec F⁺) in the BAL fluid from different groups. (C) Representative H&E staining of lung tissues (Scale bar, 50 μm). The histogram shows the lung tissue pathological damage score. (D) BALF cell counts; (E) MPO level in lungs; (F) total protein level in the BAL fluid. (G) cytokine concentration in the BAL fluid. (H) Flow cytometry analysis of Alveolar macrophages (CD11c⁺ Siglec F⁺) in BAL fluid (Gate on CD11b^- CD64⁺). (I) Flow cytometry analysis of macrophages (CD11b⁺ F4/80⁺) in BAL fluid. (J) Flow cytometry analysis of neutrophils (CD11b⁺ Ly6G⁺) in BAL fluid. All the data are expressed as the mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001.