Fig. 6

Targeting MARCO⁺ AMs significantly controlled lung fibrosis development by reducing CCL6 in the aged mice. A Experimental protocol for Clodronate liposomes, anti-MARCO or anti-CCL6 treatment. Bleomyclin (BLM, 2.5 mg/kg, i.n.) was used to induce lung fibrosis in mice. Clodronate liposomes (50 μL/mouse, one time per 3 days), anti-MARCO (100 μg/mouse, one time per 7 days) or anti-CCL6 mAb (100 μg/mouse, one time per 7 days) were used to treat the aged mice 7 days before BLM treatment. Lung and serum samples were harvested 21 days post-BLM treatment. B Histopathology of lung tissue by Masson Trichrome staining for Clodronate liposomes-treated and anti-MARCO-treated aged mice. Ashcroft scores were used to indicate the degree of fibrosis. Scale bar, 100 μm. The hydroxyproline in lung tissue was detected by using hydroxyproline microplate assay kit. The mRNA expression levels of Col1a1, Timp1 and α-SMA in lung tissue were detected by using real-time PCR. C Co-staining of MARCO and CCL6 in the aged lung tissues detected by immunofluorescence for clodronate liposomes-treated and anti-MARCO-treated aged mice, compared with the control. The arrows indicate the positive cells in the lung tissues. Scale bar, 25 μm. MARCO⁺ CCL6⁺ cell numbers were counted and analyzed. Each symbol represents the average of 10 fields of vision (63 ×) from an individual sample. There were 6 mice in each group. D The levels of CCL6 in the serum (n = 6 for each group) and BALF (1.0 mL/mouse, n = 5 for each group) were detected by ELISA. E Histopathology of lung tissue by Masson Trichrome staining for CCL6-neutralized aged mice. Ashcroft scores were used to indicate the degree of fibrosis. Scale bar, 100 μm. The hydroxyproline in lung tissue was detected by using hydroxyproline microplate assay kit. The mRNA expression levels of Col1a1, Timp1 and α-SMA in lung tissue were detected by using real-time PCR. There were 6 mice in each group. Data are shown as the mean ± SEM. Comparisons by unpaired two-tailed Student’s t-test. ** p < 0.01, *** p < 0.001, **** p < 0.0001