Fig. 2

Effect of splenocytes depleted of Tregs on bleomycin (BLM)-induced murine pulmonary fibrosis. a–f Splenocyte suspensions (1 × 10⁵ cells) obtained from C57BL/6 mice were incubated with 1 μL of an anti-CD25 monoclonal antibody (mAb) for 20 min on ice, after which they were injected via the caudal vein on day 14 post-BLM treatment. On day 28, the mice were sacrificed and the lungs were removed and subjected to hematoxylin and eosin (HE) and Masson’s trichrome staining. g The extent of lung fibrosis was measured by quantitative histology according to Ashcroft’s method to determine the effect of CD25 neutralization with the anti-CD25 mAb. Treatment of splenocytes with the anti-CD25 mAb abrogated the anti-fibrotic effect of splenocytes in BLM-induced pulmonary fibrosis. *P < 0.01. n = 7 mice/group. h The hydroxyproline content in the lungs was measured on day 28. A similar trend in the numerical score was observed in the lung hydroxyproline content, although statistical significance was not achieved. n = 4 mice for each group. Splenocytes were subjected to FACS analysis to identify Tregs (i) and macrophages (j). Tregs accounted for approximately 11% of the splenocytes. Macrophages consisted of approximately 10% splenocytes, most of which had the M1 phenotype