Fig 1.
Nintedanib and A-770041 inhibit the phosphorylation of lymphocyte-specific protein tyrosine kinase (Lck) of murine CD4+ T-cells.
CD4+ T-cells were collected from murine spleens using magnetic activated cell sorting. CD4+ T-cells were stimulated by anti-CD3/CD28 antibodies and incubated with different concentrations of nintedanib (0–300 nM) (A)(B) or A-770041 (0–1000 nM) (C)(D) for 5 minutes. The phosphorylation of Lck was analyzed by the Simple WesTM system (n = 4). *p<0.05 versus groups treated without nintedanib or A-770041; **p<0.01 versus groups treated without nintedanib or A-770041.
Fig 2.
A-770041 attenuates BLM-induced lung fibrosis.
Mice received a single transbronchial instillation of 3.0 mg/kg BLM on Day 0, and A-770041 (5 mg/kg/d) or vehicle was administered daily. Mice were separately treated with A-770041 from days 0 to 10 (early phase), days 11 to 21 (late phase) or days 0 to 21 (full treatment). On Day 21, mice were killed, and their lungs were collected. Lung sections were stained with (A) hematoxylin and eosin. (B) The fibrotic changes in the lungs were quantified with a numerical fibrotic score (Ashcroft score) histopathologically (n = 6). (C) The hydroxyproline contents in lung tissue were measured by a hydroxyproline assay (n = 6). *p<0.05 versus groups treated with BLM without A-770041; **p<0.01 versus groups treated with BLM without A-770041.
Fig 3.
A-770041 reduces the percentage of TGF-β-producing CD4+ T-cells in BLM treated lungs.
Mice received a single transbronchial instillation of 3.0 mg/kg BLM on Day 0, and BALF was collected on days 0, 7, 14, and 21. Lymphocyte counts (A) and the CD4/8 ratio (C) in the BALF were examined by flow cytometry (n = 5). The percentages of Il-17+ CD4+ T-cells and TGF-β1+ CD4+ T-cells infiltrating lung tissues were examined by flow cytometry (B, D, E). After BLM treatment, A-770041 (5 mg/kg/day) or vehicle was administered daily, and lungs were collected on day 7 (n = 5). The percentages of Il-17+ CD4+ T-cells and TGF-β1+ CD4+ T-cells infiltrating lung tissues with or without A-770041 were examined by flow cytometry (F, G). The concentration of TGF-β in BALF was examined by an ELISA. *: p<0.05 versus samples of day 7; **: p<0.01 versus samples of day 0; #: p<0.01 versus group treated with BLM without A-770041; ##: p<0.05 versus group treated with BLM without A-770041.
Fig 4.
A-770041 and nintedanib inhibit the production of TGF-β in regulatory T-cells.
Tregs and TGF-β1-producing Tregs infiltrating BLM-treated lung tissues were examined by flow cytometry. Tregs were identified as CD3e+ CD4+ Foxp3+ TGF-β1+ cells, and the gating strategy for the identification of TGF-β1-producing Tregs is shown (A). Mice received a single transbronchial instillation of 3.0 mg/kg BLM on Day 0, and lungs were collected on days 0, 7, 14, and 21. Time courses of the percentages of whole Treg cells to CD4+ T-cells (B) and that of TGF-β1+ Tregs to Tregs (C) were evaluated (Day 0, n = 4; Day 7, n = 8; Day 14, n = 8; Day 21, n = 6). After BLM treatment, A-770041 (5 mg/kg/day), nintedanib (60 mg/kg/day), or vehicle was administered daily, and lungs were collected on day 7 (n = 5). The percentages of whole Tregs to CD4+ T-cells (D) and that of TGF-β1+ Tregs to Tregs (E) were evaluated by flow cytometry. *p<0.05 versus Day 0 samples; **p<0.05 versus group treated with BLM without A-770041 or nintedanib.
Fig 5.
A-770041 inhibit the production of TGF-β in Tregs in vitro.
Tregs obtained from murine spleen were stimulated by anti-CD3/CD28 antibodies and incubated with different concentrations of A-770041 (0, 100, 500 nM) for 24 h. Concentrations of TGF-β in the supernatant were determined by an ELISA (n = 4). The expression of Tgfb mRNA in incubated cells was examined by qPCR (n = 4).