Bosutinib Therapy Ameliorates Lung Inflammation and Fibrosis in Experimental Silicosis

Abstract

Silicosis is an occupational lung disease for which no effective therapy exists. We hypothesized that bosutinib, a tyrosine kinase inhibitor, might ameliorate inflammatory responses, attenuate pulmonary fibrosis, and thus improve lung function in experimental silicosis. For this purpose, we investigated the potential efficacy of bosutinib in the treatment of experimental silicosis induced in C57BL/6 mice by intratracheal administration of silica particles. After 15 days, once disease was established, animals were randomly assigned to receive DMSO or bosutinib (1 mg/kg/dose in 0.1 mL 1% DMSO) by oral gavage, twice daily for 14 days. On day 30, lung mechanics and morphometry, total and differential cell count in alveolar septa and granuloma, levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-4, transforming growth factor (TGF)-β, and vascular endothelial growth factor in lung homogenate, M1 and M2 macrophages, total leukocytes, and T cells in BALF, lymph nodes, and thymus, and collagen fiber content in alveolar septa and granuloma were analyzed. In a separate in vitro experiment, RAW264.7 macrophages were exposed to silica particles in the presence or absence of bosutinib. After 24 h, gene expressions of arginase-1, IL-10, IL-12, inducible nitric oxide synthase (iNOS), metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1, and caspase-3 were evaluated. In vivo, in silicotic animals, bosutinib, compared to DMSO, decreased: (1) fraction area of collapsed alveoli, (2) size and number of granulomas, and mononuclear cell granuloma infiltration; (3) IL-1β, TNF-α, IFN-γ, and TGF-β levels in lung homogenates, (4) collagen fiber content in lung parenchyma, and (5) viscoelastic pressure and static lung elastance. Bosutinib also reduced M1 cell counts while increasing M2 macrophage population in both lung parenchyma and granulomas. Total leukocyte, regulatory T, CD4⁺, and CD8⁺ cell counts in the lung-draining lymph nodes also decreased with bosutinib therapy without affecting thymus cellularity. In vitro, bosutinib led to a decrease in IL-12 and iNOS and increase in IL-10, arginase-1, MMP-9, and TIMP-1. In conclusion, in the current model of silicosis, bosutinib therapy yielded beneficial effects on lung inflammation and remodeling, therefore resulting in lung mechanics improvement. Bosutinib may hold promise for silicosis; however, further studies are required.

Keywords: lung fibrosis; lung mechanics; lymphocytes; macrophage polarization; regulatory T cells; tyrosine kinase inhibitor.

Figure 1

Study design. Sixty-four C57BL/6 female mice were divided into two groups: control (CTRL, n = 32) received sterile saline (50 μL) intratracheally (i.t.), while silicosis group (SIL, n = 32) received silica particles (20 mg in 50 μL saline, i.t.). Fifteen days after disease induction, the animals were randomized to receive dimethyl sulfoxide (DMSO 1% in saline solution, 100 μL, oral gavage) or bosutinib (BOS 1 mg/kg body weight in DMSO 1%, 100 μL, oral gavage).

Figure 2

Lung mechanics. Static lung elastance (Est,L) (A), resistive (ΔP1) (B), and viscoelastic pressure (ΔP2) (C). White bars, DMSO gray bars, BOS. Values are means + SD of eight animals per group. ^*Significantly different from CTRL-DMSO group (p < 0.05). ^**Significantly different from SIL-DMSO (p < 0.05).

Figure 3

Lung histology. Representative photomicrographs (light microscopy) of lung parenchyma stained with hematoxylin and eosin from CTRL-DMSO, CTRL-BOS, SIL-DMSO, and SIL-BOS animals. G, granuloma. Arrows denote areas of alveolar collapse. Original magnification 200 ×. Scale bars = 200 μm.

Figure 4

Fraction area of granuloma in lung parenchyma (A) and percent total cell count (B), mononuclear cells (C), and neutrophils (D). All values were computed in 10 random, non-coincident fields of views per mouse. Boxes show the interquartile (25–75%) range, whiskers encompass the range (minimum–maximum), and horizontal lines represent median values of 8 animals per group. ^**Significantly different from SIL-DMSO (p < 0.05).

Figure 5

Macrophage polarization: M1 and M2. Representative immunohistochemistry photomicrographs of M1 (iNOS-positive) and M2 (arginase-positive) macrophages (right panels). Quantification of M1 (A) and M2 (B) (global score: severity × extension) in lung tissue of CTRL-DMSO, CTRL-BOS, SIL-DMSO, SIL-BOS. Arrows denote M1 or M2 cells. White bars, DMSO; gray bars, BOS. Boxes show the interquartile (25–75%) range, whiskers encompass the range (minimum–maximum), and horizontal lines represent median values of 8 animals per group. ^*Significantly different from CTRL-DMSO group (p < 0.05). ^**Significantly different from SIL-DMSO (p < 0.05).

Figure 6

Levels of cytokines and growth factors. Quantification of protein levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-4, transforming growth factor (TGF)-β, and vascular endothelial growth factor (VEGF) quantified by ELISA in the lung homogenate. Black circle, DMSO; black square, BOS. Symbols represent individual animals. Lines represent median (interquartile range) of 8 animals per group. ^*Significantly different from C-DMSO (p < 0.05). ^**Significantly different from SIL-DMSO (p < 0.05).

Figure 7

Expression of interleukin (IL)-10 (A), arginase (Arg)-1 (B), IL-12 (C), inducible nitric oxide synthase (iNOS) (D), metalloproteinase (MMP)-9 (E), tissue inhibitor of metalloproteinase (TIMP) (F), and caspase-3 (G) in macrophages exposed to saline or silica and treated with DMSO or bosutinib (BOS). White bars, DMSO; gray bars, BOS. Values are means + SD of 8 animals per group. ^*Significantly different from C-DMSO (p < 0.05). ^**Significantly different from SIL-DMSO (p < 0.05).

Figure 8

Total inflammatory cell counts in bronchoalveolar lavage fluid (BALF) (A), lymph node (B), and thymus tissue (C). Black circle, DMSO; black square, BOS. Symbols represent individual animals. Lines represent median (interquartile range) of 6 or 8 animals per group. ^*Significantly different from C-DMSO (p < 0.05). ^**Significantly different from SIL-DMSO (p < 0.05).

Figure 9

Regulatory T cells, helper T cells, and killer T cells in bronchoalveolar lavage fluid (BALF), lymph nodes, and thymus tissue. Quantification of total CD4⁺CD25⁺Foxp3⁺ cells (first vertical line), CD4⁺ cells (second vertical line), and CD8⁺ cells (third vertical line) in BALF (A,D,G), lymph node (B,E,H), and thymus tissue (C,F,I), respectively, of CTRL-DMSO, CTRL-BOS, SIL-DMSO, and SIL-BOS animals. White bars, DMSO; gray bars, BOS. Values are means + SD of 8 animals per group. ^*Significantly different from C-DMSO (p < 0.05). ^**Significantly different from SIL-DMSO (p < 0.05).

Figure 10

Collagen fibers. Representative photomicrographs (polarized light microscopy) of lung parenchyma and granuloma tissue stained with Picrosirius Red (right panels). Collagen fibers are shown in orange. Original magnification 200 ×. Scale bars = 200 μm. Left panels depict collagen fiber content quantification in lung parenchyma (upper panel, A) and granuloma (lower panel, B). White bar, DMSO; gray bar, BOS. Boxes show the interquartile (25–75%) range, whiskers encompass the range (minimum–maximum), and horizontal lines represent median values of 8 animals per group. ^*Significantly different from C-DMSO (p < 0.05). ^**Significantly different from SIL-DMSO (p < 0.05).