Pirfendone reduces F4/80⁺macrophage accumulation and TGF-β deposition in lung. (A-C) Serial sections of lungs harvested on day 56 post-transplantation were stained with trichrome staining (A, top panels), anti-F4/80 antibody (A, middle panels), and anti–TGF-β antibody (A, bottom panels). F4/80-positive cell number and TGF-β staining area were quantified in (B) and (C). Pirfenidone reduced macrophages infiltration and TGF-β production in lung. (D-E) Mice were transplanted as described in Figure 1. cGVHD mice were treated by pirfenidone for a week from days 28 to 35. On day 35, LAP expression in alveolar macrophages (D) and tissue macrophages (E) were analyzed by flow cytometry. Pirfenidone significantly reduced LAP expression in both alveolar and tissue macrophages. (F) Donor B6 mice are CD45.1 and B10.BR CD45.2 recipient mice were used. Lung sections were fixed and stained with anti-CD45.1 (donor marker, red), CD68 (gray), and CD206 (green). Arrows pointed at cells that are CD45.1⁺CD68⁺CD206⁺. This result suggests that infiltrating macrophages in the lung are donor-derived M2 macrophages. (G) Macrophage migration was assessed in a transwell assay. IL-17A or MCP-1was used as a chemoattractant to induce migration. Pirfenidone (0.25 or 0.5 mg/mL) or vehicle was added to cell culture medium and migration medium. Pirfenidone inhibited IL-17A and MCP-1 induced J774 migration. Five to 8 mice were analyzed for each group in each assay. Results are representative of at least 2 experiments with similar results. Migration assay result is representative of 3 experiments with similar results. *P ≤ .05, **P < .01, ***P < .001; data are shown as the mean ± SEM.