Experimental depletion of alveolar macrophages in SPAM deleter mice. (A) Concentrations of GM-CSF, M-CSF, CCL2, and IL-1β protein in the BAL fluid (BALF) of SPAM deleter mice at the indicated time points after intratracheal administration of 100 ng DT. Data are from a single experiment with n = 4–6 mice per time point. (B) Concentrations of the indicated cytokines and chemokines in the BALF of WT (Epx^CreSiglecf^+/+) and SPAM deleter (Epx^CreSiglecf^Lox-DTR-Lox) mice 24 h after DT administration. Data are from a single experiment with n = 6 mice per time point. Data are represented as mean ± SEM. **P < 0.01 by two-tailed Mann–Whitney test. (C) Dot plot showing expression of curated marker genes to annotate the lung cell clusters in Fig. 3 E and Fig. 8 A from SPAM deleter mice as determined by single-cell RNA sequencing. (D) Dot plot showing expression of the top five differentially expressed genes for the lung monocyte-macrophage clusters in Fig. 3 F from SPAM deleter mice as determined by single-cell RNA sequencing. (E) Repopulation of alveolar macrophages after experimental depletion in SPAM deleter mice as shown by single-cell RNA sequencing. Red dots overlaid on the UMAP from Fig. 3 F show cells present in the lung of SPAM deleter mice at the indicated time points before and after alveolar macrophage depletion with DT. Data in panels C–E are from one single-cell RNA-sequencing experiment with n = 4 mice per time point.