Trajectory of monocyte-to-macrophage differentiation and GPR183 expression after experimental emptying of the alveolar niche. (A) Overview of SPAM deleter mice. In Siglecf^Lox-DTR-Lox mice, SiglecF⁺ alveolar macrophages and eosinophils express DTR. In Epx^CreSiglecf^Lox-DTR-Lox mice (SPAM deleter mice), Epx^Cre activity excises the floxed DTR cassette in eosinophils, resulting in alveolar macrophage–specific DTR expression. This allows specific depletion of alveolar macrophages in an inducible manner after DT administration. (B and C) Frequency of alveolar macrophages and other immune cells in the BAL fluid (BALF) of SPAM deleter mice after intratracheal administration of 100 pg DT as determined by flow cytometry. BAL cells in panel B were gated as live CD45⁺ single cells. Myeloid cells in panel C were gated by excluding CD11b⁻CD11c⁻ cells and were further separated into alveolar macrophages (CD11c⁺SiglecF⁺), CD64⁺ non-alveolar macrophages (CD11c⁻SiglecF⁻CD64⁺), and neutrophils (CD11c⁻SiglecF⁻CD11b⁺Ly6G⁺). Nonmyeloid cells (CD11b⁻CD11c⁻) were separated into T (CD3⁺B220⁻) and B cells (CD3^-B220⁺). Alveolar macs, alveolar macrophages. Data are from a single experiment with n = 5–6 mice per time point. (D) Experimental setup for single-cell RNA sequencing of lung cells from SPAM deleter mice. Lungs were harvested at the indicated time points before and after alveolar macrophage depletion with 40 ng intratracheal DT. Purified CD45⁻ non-hematopoietic cells as well as resident CD45⁺CD64⁺ and CD45⁺CD64⁻ hematopoietic cells were used for single-cell RNA sequencing as described in the Materials and methods. Resident hematopoietic cells were isolated based on being protected from intravascular cell labeling after intravenous injection of anti-mouse CD45 antibody. AM, alveolar macrophages. (E) Combined UMAP of 117,715 lung cells from SPAM deleter mice at 0, 12, 24, and 48 h and on day 5, 8, and 14 after alveolar macrophage depletion with DT as determined by single-cell RNA sequencing. See Fig. S2 C for markers used to annotate the cell clusters. (F) UMAP showing monocyte and macrophage clusters (26,817 cells) in the lung of SPAM deleter mice before and after alveolar macrophage depletion with DT. See Fig. S2 D for markers used to annotate the monocyte-macrophage clusters. (G)Gpr183 expression in the monocyte and macrophage clusters from panel F. IMs, interstitial macrophages; MoMac, monocyte-macrophages; MoAM or mono-AMs, monocyte-derived alveolar macrophages. Data in panels E–G are from one single-cell RNA-sequencing experiment with n = 4 mice per time point. Panels A and D were adapted from Servier Medical Art.