Annexin A1 mimetic peptide controls the inflammatory and fibrotic effects of silica particles in mice

Abstract

Background and purpose: Endogenous glucocorticoids are pro-resolving mediators, an example of which is the endogenous glucocorticoid-regulated protein annexin A1 (ANXA1). Because silicosis is an occupational lung disease characterized by unabated inflammation and fibrosis, in this study we tested the therapeutic properties of the N-terminal ANXA1-derived peptide annexin 1-(2-26) (Ac2-26) on experimental silicosis.

Experimental approach: Swiss-Webster mice were administered silica particles intranasally and were subsequently treated with intranasal peptide Ac2-26 (200 μg per mouse) or dexamethasone (25 μg per mouse) for 7 days, starting 6 h post-challenge. Ac2-26 abolished the leukocyte infiltration, collagen deposition, granuloma formation and generation of pro-inflammatory cytokines evoked by silica; these variables were only partially inhibited by dexamethasone.

Key results: A clear exacerbation of the silica-induced pathological changes was observed in ANXA1 knockout mice as compared with their wild-type (WT) littermate controls. Incubation of lung fibroblasts from WT mice with Ac2-26 in vitro reduced IL-13 or TGF-β-induced production of CCL2 (MCP-1) and collagen, but this peptide did not affect the production of CCL2 (MCP-1) by stimulated fibroblasts from formyl peptide receptor type 1 (FPR1) knockout mice. Ac2-26 also inhibited the production of CCL2 (MCP-1) from fibroblasts of FPR2 knockout mice.

Conclusions and implications: Collectively, our findings reveal novel protective properties of the ANXA1 derived peptide Ac2-26 on the inflammatory and fibrotic responses induced by silica, and suggest that ANXA1 mimetic agents might be a promising strategy as innovative anti-fibrotic approaches for the treatment of silicosis.

Figure 1

Analysis of endogenous ANXA1 during silica-induced lung fibrosis in WT (ANXA1^+/+) and ANXA1 knockout (ANXA1^−/−) mice. (A–D) Picrosirius Red-stained sections of lung tissue after silica and saline challenge. (E) Quantification of total content of tissue collagen by the Sircol technique. (F) Basal levels of lung resistance and (G) elastance. All analyses were performed on day 7 after silica challenge (10 mg per animal). Scale bar = 200 μm. Values represent the mean ± SEM from seven animals. *P < 0.05 compared with the respective saline-challenged control group; ⁺P < 0.05 compared with ANXA1^+/+ silica-challenged group.

Figure 2

Airway hyper-reactivity measured as resistance (A) and elastance (B) induced by provocation with increasing concentrations of methacholine in mice challenged with silica (10 mg) and treated with dexamethasone (DEXA; 25 μg per mouse) or peptide Ac2-26 (200 μg per mouse). Animals instilled with saline were used as controls. The analyses were made on day 8 after silica challenge. Values represent mean ± SEM from seven animals. ⁺P < 0.05 as compared with saline-challenged group. *P < 0.05 as compared with silica-challenged group.

Figure 3

Histological sections of mouse lung on day 8 after saline (A) or silica challenge (B) and subsequently given intranasal treatment with dexamethasone (DEXA; 25 μg per mouse) (C) and peptide Ac2-26 (200 μg per mouse) (D). Morphometric analysis and total tissue collagen content are seen in panels E and F respectively. Slides were stained with haematoxylin-eosin. Scale bar = 200 μM. Values represent mean ± SEM from seven animals. *P < 0.05 as compared with saline-challenged group. ⁺P < 0.05 as compared with silica-challenged group.

Figure 4

Production of (A) CXCL1 (KC), (B) CCL2 (MCP-1), (C) TGF-β, (D) TNF-α and (E) IFN-γ in lung samples of mice treated with dexamethasone (DEXA; 25 μg per mouse) and peptide Ac2-26 (200 μg per mouse) from 6 h to 7 days after silica challenge (10 mg). Animals instilled with saline were used as controls. The analyses were made on day 8 after silica challenge. Values represent mean ± SEM from seven animals. *P < 0.05 as compared with saline-challenged group. ⁺P < 0.05 as compared with silica-challenged group.

Figure 5

Immunohistochemical localization of α-SMA-positive cells in the lung sections of mice challenged with saline (A), silica (B), silica treated with dexamethasone (25 μg per mouse) (C) and silica treated with Ac2-26 peptide (200 μg per mouse) (D). Treatment was performed for seven consecutive days, starting 6 h post-silica challenge (10 mg). The analyses were made 1 day after the last administration, on day 8 after silica challenge. The inset shows negative control in which primary antibody was omitted. Scale bar = 200 μM.

Figure 6

Activation of cultured lung fibroblasts recovered from ANXA1 WT (ANXA1^+/+) mice. Production of collagen (upper panels) and CCL2 (MCP-1) (lower panels) after stimulation with IL-13 (40 ng·mL⁻¹) (A, C) and TGF-β (10 ng·mL⁻¹) (B, D). Cells were exposed to peptide Ac2-26 (1–30 μM) 1 h before stimulation, and the analyses performed in the supernatant 24 h later. Values represent data from four to five independent experiments. Means were significantly different as analysed by anova. (A) F-value = 4.59 (P < 0.0031); (B) F-value = 2.65 (P = 0.0468); (C) F-value = 3.4 (P < 0.00025) and (D) F-value = 4.45 (P < 0.0037).

Figure 7

Activation of cultured lung fibroblasts recovered from FPR1 WT (FPR1^+/+) (A, C) and knockout (FPR1^−/−) mice (B, D). Production of collagen (upper panels) and CCL2 (MCP-1) (lower panels) after stimulation with IL-13 (40 ng·mL⁻¹) and TGF-β (10 ng·mL⁻¹). Cells were exposed to peptide Ac2-26 (15 and 30 μM) 1 h before stimulation and the analyses performed in the supernatant 24 h later. Values represent data from three to four independent experiments. Means were significantly different as analysed by anova. (A) F-value = 8.27 (P < 0.0003); (B) F-value = 4.27 (P = 0.011); (C) F-value = 18.00 (P < 0.0001) and (D) F-value = 13.36 (P < 0.0001).

Figure 8

Activation of cultured lung fibroblasts recovered from FPR2 WT (FPR2^+/+) (A, C) and knockout (FPR2^−/−) mice (B, D). Production of collagen (upper panels) and CCL2 (MCP-1) (lower panels) after stimulation with IL-13 (40 ng·mL⁻¹) and TGF-β (10 ng·mL⁻¹). Cells were exposed to peptide Ac2-26 (15 and 30 μM) 1 h before stimulation, and the analyses performed in the supernatant 24 h later. Values represent data from three to four independent experiments. Means were significantly different as analysed by anova. (A) F-value = 14.57 (P < 0.0001); (B) F-value = 12.95 (P = 0.0001); (C) F-value = 5.14 (P < 0.0027) and (D) F-value = 9.22 (P < 0.0002).