Pleiotropic antifibrotic actions of aspirin-triggered resolvin D1 in the lungs

Abstract

Introduction: Pulmonary fibrosis is a destructive, progressive disease that dramatically reduces life quality of patients, ultimately leading to death. Therapeutic regimens for pulmonary fibrosis have shown limited benefits, hence justifying the efforts to evaluate the outcome of alternative treatments.

Methods: Using a mouse model of bleomycin (BLM)-induced lung fibrosis, in the current work we asked whether treatment with pro-resolution molecules, such as pro-resolving lipid mediators (SPMs) could ameliorate pulmonary fibrosis. To this end, we injected aspirin-triggered resolvin D1 (7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E19Z-docosahexaenoic acid; ATRvD1; i.v.) 7 and 10 days after BLM (intratracheal) challenge and samples were two weeks later.

Results and discussion: Assessment of outcome in the lung tissues revealed that ATRvD1 partially restored lung architecture, reduced leukocyte infiltration, and inhibited formation of interstitial edema. In addition, lung tissues from BLM-induced mice treated with ATRvD1 displayed reduced levels of TNF-α, MCP-1, IL-1-β, and TGF-β. Of further interest, ATRvD1 decreased lung tissue expression of MMP-9, without affecting TIMP-1. Highlighting the beneficial effects of ATRvD1, we found reduced deposition of collagen and fibronectin in the lung tissues. Congruent with the anti-fibrotic effects that ATRvD1 exerted in lung tissues, α-SMA expression was decreased, suggesting that myofibroblast differentiation was inhibited by ATRvD1. Turning to culture systems, we next showed that ATRvD1 impaired TGF-β-induced fibroblast differentiation into myofibroblast. After showing that ATRvD1 hampered extracellular vesicles (EVs) release in the supernatants from TGF-β-stimulated cultures of mouse macrophages, we verified that ATRvD1 also inhibited the release of EVs in the bronco-alveolar lavage (BAL) fluid of BLM-induced mice. Motivated by studies showing that BLM-induced lung fibrosis is linked to angiogenesis, we asked whether ATRvD1 could blunt BLM-induced angiogenesis in the hamster cheek pouch model (HCP). Indeed, our intravital microscopy studies confirmed that ATRvD1 abrogates BLM-induced angiogenesis. Collectively, our findings suggest that treatment of pulmonary fibrosis patients with ATRvD1 deserves to be explored as a therapeutic option in the clinical setting.

Keywords: ATRvD1; angiogenesis; fibrosis; inflammation; lung; macrophages; microparticles; tissue repair.

Figure 1

ATRvD1 treatment restores bleomycin (BLM)-induced lung morphology and inflammation. (A) Representative lung sections from saline (SAL) (left), BLM (center; 0.06 U/mouse in a final volume of 30 μl of SAL), and BLM plus ATRvD1 treatment (right; on day 7, 2.5 μg/kg and a booster on day 10 with 0.5 μg/kg) obtained 14 days after BLM injection and stained with hematoxylin and eosin. Scale bar = 50 μm. (B, C) Inflammatory cells and cytokines, MMP-9, and TIMP-1 were quantified in the BAL fluid and lung tissue, respectively, 14 days after SAL, BLM, or BLM plus ATRvD1 treatment as described in (A). Results represent mean + SEM from two independent experiments. *P ≤ 0.05 vs. the SAL group; ^# P ≤ 0.05 vs. the BLM group.

Figure 2

ATRvD1 rescues the cell numbers and phenotype in the lung and BAL of BLM-injected mice. (A) Total cell count quantified in the supernatant after enzymatic digestion of the lungs (left) and after BAL harvest (right). (B) Dot-plot images show the gate strategy for monocyte and neutrophil analysis: (i) forward and side scatter plots show a gated region representing leukocytes; (ii) forward scatter gated region of singlet cells; (iii) forward scatter and CD11b cell expression gated on live cells; (iv) contour plot of Ly6C⁺ and Ly6G⁺ cells. The bottom graphs show the absolute number of the neutrophil (Ly6G^highLy6C^int/CD11b⁺) and monocyte (Ly6C^highLy6G⁻/CD11b⁺) populations in the lungs and BAL. (C) Dot-plot images show the gate strategy for alveolar macrophages and eosinophils: (i) forward and side scatter plots show a gated region representing leukocytes; (ii) forward scatter gated region of singlet cells; (iii) contour plot of CD11c and SiglecF gated on live cells. The bottom graphs show the absolute number of the alveolar macrophage (SiglecF^highCD11c^high) and eosinophil (SiglecF^highCD11c⁻) populations in the lungs and BAL. (D) Absolute number and MFI of CD86⁺ or CD206⁺ cells in the SiglecF^highCD11c^high population. Results represent mean + SEM from two independent experiments. *P ≤ 0.05 vs. the SAL group; ^# P ≤ 0.05 vs. the BLM group.

Figure 3

ATRvD1 impairs BLM-induced matrix protein deposition in the lung. Lungs were harvested on the 14th day after the SAL, BLM, or BLM + ATRvD1 challenges, as described in the Material and methods section. (A) Picrosirius red staining under bright field (superior images) and polarized light (lower images). (B) Hydroxyproline content in the lungs on the 14th day after mice were instilled with SAL, BLM, or BLM + ATRvD1 (top right). Results are expressed as micrograms per milligram of lung tissue. *P ≤ 0.05 compared with the SAL group; ^# P ≤ 0.05 compared with the BLM group. (C–E) Lung sections were immunostained for collagen type I and α-SMA by immunohistochemistry and counterstained with hematoxylin or for fibronectin and DAPI by immunofluorescence. (F) Primary lung fibroblasts were isolated, cultured, and incubated with the medium, TGF-β (10 ng/ml), or TGF-β + ATRvD1 (100 ng/ml) and then stained by immunofluorescence for α-SMA as described. Pictures are representative of each group, constituted of n = 4-5. Inserts in panels (C–E) represent the images obtained by immunostaining for the control isotype antibodies. (G, H) Total RNA was extracted from the lung tissue obtained from the SAL-, BLM-, or BLM + ATRvD1-treated mice or from the homogenate of 10⁶ primary mouse lung fibroblasts incubated with medium or TGF-β at 10, 50, and 100 ng/ml. Samples were prepared using the TRIzol reagent and quantitative real-time RT-qPCR for ALX expression determined as described in the Material and methods section. Scale bar = 50 μm.

Figure 4

ATRvD1 inhibits the release of extracellular vesicles (EVs) in vivo and in vitro. (A) EVs were isolated from the BAL obtained on day 14 after the SAL, BLM, and BLM + ATRvD1 treatments or from the conditioned medium harvested 24 h after the macrophages were incubated with TGF-β (10 ng/ml) or TGF-β + ATRvD1 (100 ng/ml). The EVs were stained for FITC-labeled annexin V and analyzed by flow cytometry. (B) Dot-plots and density-plot images show the gate strategy for EVs: (i) the gate region of large vesicles using 1-μm standard beads (left); (ii, iii) gates were defined from unlabeled vesicles (negative), being considered positive for the specific markers of the fluorescence captured following that point. (C) The EVs obtained from BAL were also stained for F4/80-PE (macrophages), SinglecF-alexa488 (eosinophils), Ly6G-APC (neutrophils), and CD3-alexa488 (lymphocytes) and analyzed by flow cytometry, and fluorescence was plotted as histogram and bar graphs of absolute number. Results represent mean + SEM from three independent experiments. *P ≤ 0.05 vs. the SAL/control; ^# P ≤ 0.05 vs. the treated groups (BLM or TGF-β).

Figure 5

ATRvD1 treatment inhibits BLM-induced angiogenesis in HCP. (A) Representative fluorescent light images of HCP preparation after SAL, BLM (1 U/100 µL/hamster – injected into the cheek pouch), BLM + ATRvD1 (25 µg/kg - 100 µL i.v.) treatments, and contralateral images of HCP in BLM-treated hamsters. The image area is 5 mm². (B–D) Microvascular alterations were quantified by measuring the fluorescence measurement (RFU), vascular area (VA), and total vascular length (TVL). Results represent mean ± SD from independent experiments. The p-value on the top of the BLM bar represents the comparison to the SAL group, while the others values were compared to the BLM group.