Profibrogenic macrophage-targeted delivery of mitochondrial protector via exosome formula for alleviating pulmonary fibrosis

Abstract

Pulmonary fibrosis (PF) is a devastating lung disease with limited treatment options. During this pathological process, the profibrogenic macrophage subpopulation plays a crucial role, making the characterization of this subpopulation fundamentally important. The present study revealed a positive correlation between pulmonary macrophages with higher mitochondrial mass (Mø^mitohigh) and fibrosis. Among the Mø^mitohigh subpopulation of CD206⁺ M2, characterized by higher expression of dynamin 1-like (Drp1), as determined by flow cytometry and RNA-seq analysis, a therapeutic intervention was developed using an exosome-based formula composed of pathfinder and therapeutics. A pathfinder exosome called "exosome^MMP19 (Exo^MMP19)", was constructed to display matrix metalloproteinase-19 (MMP19) on the surface to locally break down the excessive extracellular matrix (ECM) in the fibrotic lung. A therapeutic exosome called "exosome ^therapeutics (Exo^Tx)", was engineered to display D-mannose on the surface while encapsulating siDrp1 inside. Prior delivery of Exo^MMP19 degraded excessive ECM and thus paved the way for Exo^Tx to be delivered into Mø^mitohigh, where Exo^Tx inhibited mitochondrial fission and alleviated PF. This study has not only identified Mø^mitohigh as profibrotic macrophages but it has also provided a potent strategy to reverse PF via a combination of formulated exosomes.

Keywords: Drp1; Exosomes; Macrophages; Mitochondrial fission; Pulmonary fibrosis.

Graphical abstract

Scheme 1

Schematic illustration of the study. Exo^MMP19, the pathfinder exosome, and Exo^Tx, the therapeutic exosome, were engineered. As prior delivery of Exo^MMP19 degraded excessive ECM, the way was paved for Exo^Tx delivery into Mø^mitohigh, the novel profibrogenic macrophage subtype we identified. The formulated exosome therapy inhibited mitochondrial fission and alleviated pulmonary fibrosis efficiently.

Fig. 1

Drp1 was highly expressed in Mø^mitohigh of mice with PF. (A) Schematic illustration of the process for RNA-Seq analysis. (B) Flow cytometric analysis of lung Mito^high macrophages after intratracheal administration of PBS/BLM. Representative data from three independent experiments. (C) Volcano plot showing DEG between Mø^mitolow and Mø^mitohigh in fibrotic lung tissues. (D) GSEA analysis of Mø^mitolow and Mø^mitohigh. NES, normalized enrichment score. P, Nominal P value. (E) Heat map diagram of mitochondrial fission related genes expression in Mø^mitolow and Mø^mitohigh. n = 3. (F) qRT-PCR analysis of mitochondrial fission related genes in Mø^mitolow and Mø^mitohigh in fibrotic lung tissues. Data are expressed as mean ± SEM of three independent experiments. *p < 0.05 by two-tailed unpaired Student's t-test.

Fig. 2

Interference with Drp1 reversed mitochondrial dysfunction and downregulates profibrotic genes in vitro. (A) Schematic illustration of the experiment. BMDMs were transfected with PBS/siNC/siDrp1. (B) qRT-PCR analysis of relative expression of Drp1 in BMDMs transfected with PBS/siNC/siDrp1. Data are expressed as mean ± SEM of three independent experiments. *p < 0.05 by One-way ANOVA. (C) Western blot analysis of Drp1 expression in BMDMs transfected with PBS/siNC/siDrp1. GAPDH served as the loading control. Representative data from three independent experiments. (D) Flow cytometric analysis of mitochondrial mass in BMDMs transfected with PBS/siNC/siDrp1. Representative data from three independent experiments. (E) Flow cytometric analysis of mtROS in BMDMs transfected with PBS/siNC/siDrp1. Representative data from three independent experiments. (F) Flow cytometric analysis of Δψm in BMDMs transfected with PBS/siNC/siDrp1. Representative data from three independent experiments. (G) Representative TEM images of mitochondrial morphology in BMDMs transfected with PBS/siNC/siDrp1. Scale bars: 1 μm. (H) Calculated mitochondria per cell and mitochondrial length. Data are expressed as mean ± SEM of three independent experiments in which 30 cells were analyzed. *p < 0.05 by One-way ANOVA. (I) qRT-PCR analysis of relative expression of Tgfβ1, Pdgfα, Chi3l1, Tnfα, IL1β and Ccn2 in BMDMs transfected with PBS/siNC/siDrp1. Data are expressed as mean ± SEM of three independent experiments. *p < 0.05 by One-way ANOVA.

Fig. 3

Drp1 was highly expressed in CD206⁺ macrophages. (A) Venn diagram of highly-expressed cell surface genes of Mø^mitohigh compared to Mø^mitolow. (B) qRT-PCR analysis of macrophage markers in Mø^mitolow and Mø^mitohigh in fibrotic lung tissues. Data are expressed as mean ± SEM of three independent experiments. *p < 0.05 by two-tailed unpaired Student's t-test. (C) Schematic illustration of the process for lung flow cytometric analysis and cell sorting. (D) Flow cytometric analysis of mice lung CD206⁺ macrophages in Mø^mitohigh after BLM-induced PF. Representative data from three independent experiments. (E) Representative multiplexed immunofluorescence images of Drp1 in macrophages of fibrotic lung tissues. Scale bar: 50 μm. n = 3.

Fig. 4

Exo^Target can target CD206⁺ macrophages in vivo. (A) Schematic illustration of the experimental procedure. (B) Biodistribution of DiR-labeled Exo^None/Exo^Traptavidin/Exo^Target among organs after the intratracheal administration. n = 5. (C) Schematic illustration of the experimental procedure. (D) Representative fluorescence images of the DiI-labeled Exo^None/Exo^Traptavidin/Exo^Target in lung tissues. Scale bar:10 μm. n = 5. (E) The percentage of triple-positive cells in DiI-positive cells. n = 5. *p < 0.05 by One-way ANOVA. (F) Flow cytometric analysis of DiO-positive CD206⁺ macrophages in fibrotic lung tissues of mice treated with Exo^None/Exo^Traptavidin/Exo^Target. Representative data from five independent experiments.

Fig. 5

Exo^Tx decreased mitochondrial mass and downregulated the profibrotic genes in vivo. (A) Flow cytometric analysis of mitochondrial mass in CD206⁺ macrophages in fibrotic lung tissues of mice treated with PBS/Exo^siNC/Exo^Tx. Representative data from five independent experiments. (B) Representative TEM images of mitochondrial morphology in lung macrophages treated with PBS/Exo^siNC/Exo^Tx. Scale bars: 1 μm. (C) Calculated mitochondria per cell and mitochondrial length. Data are expressed as mean ± SEM of five independent experiments in which 50 cells were analyzed. *p < 0.05 by One-way ANOVA. (D) Schematic illustration of the process for lung cells sorting after exosomes treated. (E) qRT-PCR analysis of Drp1 in lung CD206⁺ macrophages of BLM-induced mice treated with PBS/Exo^siNC/Exo^Tx. Data are expressed as mean ± SEM of five independent experiments. *p < 0.05 by One-way ANOVA. (F) qRT-PCR analysis of Tgfβ1, Pdgfα, Chi3l1, Tnfα, IL1β and Ccn2 in lung CD206⁺ macrophages of BLM-induced mice treated with PBS/Exo^siNC/Exo^Tx. Data are expressed as mean ± SEM of five independent experiments. *p < 0.05 by One-way ANOVA.

Fig. 6

Exo^MMP19degraded excessive ECM and improved the targeting efficacy of Exo^Target. (A) Schematic illustration of the experimental procedure. (B) Western blot analysis of fibronectin, Col1a1 and α-SMA of L929 cells. GAPDH served as the loading control. Representative data from three independent experiments. (C) Schematic illustration of the experimental procedure. (D) Biodistribution of DiR-labeled Exo^None/Exo^{Empty vector}/Exo^MMP19 among organs after the intratracheal administration. n = 5. (E) Schematic illustration of the experimental procedure. (F) Representative fluorescence images of the DiI-labeled Exo^None/Exo^{Empty vector}/Exo^MMP19 in lung tissues. Scale bar:20 μm. n = 5. (G) Col1a1 relative MFI was analyzed. MFI, mean fluorescence intensity. n = 5. *p < 0.05 by One-way ANOVA. (H) Schematic illustration of the experimental procedure. (I) Representative fluorescence images of the DiI-labeled Exo^Target in lung tissues. Scale bar:10 μm. n = 5. (J) The percentage of triple-positive cells in DiI-positive cells. n = 5. *p < 0.05 by two-tailed unpaired Student's t-test. (K) Flow cytometric analysis of DiO-positive CD206⁺ macrophages in fibrotic lung tissues of mice treated with Exo^Target + Exo^None or Exo^Target + Exo^MMP19. Representative data from five independent experiments.

Fig. 7

Therapeutic efficacy of Exo^Tx combined with Exo^MMP19in vivo. (A) Schematic illustration of the experimental procedure and exosomes treatment time course. (B) Survival curve of the mice after treatments. n = 20. *p < 0.05. (C) Representative H&E, Masson's trichrome, Sirius red staining images and immunohistochemical staining of α-SMA of lung tissues in different groups. Scale bar: 200 μm. n = 5. (D) qRT-PCR analysis of Fn1, Col1a1 and Acta2 of lung tissues with different treatments. *p < 0.05. Data are expressed as mean ± SEM of five independent experiments. *p < 0.05 by One-way ANOVA. (E) Western blot analysis of fibronectin, Col1a1 and α-SMA of lung tissues with different treatments. GAPDH served as the loading control. Representative data from five independent experiments.