To collect bronchoalveolar lavage fluid (BALF), the trachea was cannulated, the lungs were lavaged three times with PBS (0.7 ml each time), and ~ 1.5 ml of the instilled fluid was consistently recovered. The samples with insufficient recovery (< 70%) were omitted. Total cell numbers were counted with a standard hemocytometer.

BALF was centrifuged at 3000 rpm for 5 min at 4 °C. After centrifugation, supernatants were collected and stored in − 80 °C until the subsequent analysis, while cell pellets were used to prepare cytospins. Smears of BALF cells were prepared with cytocentrifugation using Cytofuge2 (StatSpin, Norwood, MA, USA) at 1000 rpm for 5 min and then stained with May-Gruenwald Giemsa stain. Cell differentiation was examined by counting at least 100 cells using standard hemocytologic criteria to classify the cells as monocytes/macrophages, neutrophils, or lymphocytes.

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