Figure 4

Figure 4

Neratinib treatment in a mouse LPS-induced acute lung injury model increases the rates of macrophage efferocytosis and reduces the number of neutrophil corpses in bronchoalveolar lavage fluid. Schematic of the treatment protocol (A). Cytospins of BAL were stained with Kwik-Diff and examined by light microscopy, and neutrophils, macrophages, and lymphocytes were identified by morphology (B). Total cells in BAL were counted using the hemocytometer counting chamber (C), and the percentage of neutrophils (D), macrophages (E), and lymphocytes (F) in BAL were unchanged between treatment groups. The engulfment of cell debris by macrophages is visible as inclusions within intracellular vesicles, which can be identified with Kwik-Diff staining and light microscopy (G, red arrows). Many macrophages contained vesicles, but these were empty of debris (G, left panel). Cell debris may be broken into small pieces within a vesicle (G, middle panel, left) or fill the vesicles (G, middle panel, right). Macrophages may contain one or more vesicles with inclusions; in some cases, too many to count accurately (G, right panel); if above 6, the number of inclusions was recorded as 6. Both the percentage of macrophages containing any inclusions (H) and the total number of inclusions per 100 macrophages (I) were increased in the neratinib treatment group. Cells in BAL were also analyzed by flow cytometry, and the percentage of TO-PRO-3+ cells (J) and TO-PRO-3+ neutrophils (Ly6G+ cells) (K) was reduced in neratinib-treated mice. Concentrations of the cytokines IL-6 (L) and KC (M) were analyzed in BAL supernatant by ELISA and were unchanged between treatment groups. Each data point in the graphs represents data from one mouse (n = 6 per treatment group); bars show mean ± standard deviation. Unpaired t-tests were used for statistical analysis; p-values were indicated, where p <0.05.