Figure 3.

Hem-1 is required for the development of AMs. (A) Diagram depicting the development of tissue-resident AMs (Mat-AMs) from FMs and pre-AMs. GM-CSF, Pparg (pparg), Bach2, and TGFβ stimulate AM development (Kopf et al., 2015). (B and C) Flow cytometric analyses of AMs in BALF from adult Hem1^fl/flLysMCre⁺ and LysMCre⁺ mice showing forward scatter–A (FSC-A) histogram (left) and mean FSC (right; B) and mean fluorescence intensity (MFI) of surface marker expression (C; gated as in D). (D) Gating strategy (top) and bar graphs showing the number of CD45.2⁺ cells (bottom left) and percentages of cells in lungs from PND3 WT and Hem1^−/− mice (bottom right). (E and F) Gene expression as measured by real-time PCR in BALF-derived AMs from adult Hem1^fl/flLysMCre^+/+ and LysMCre^+/+ mice after 24 h of LPS (10 μg/ml) stimulation. (G) Pparg expression in FACS-purified lung monocytes from PND3 Hem1^fl/flLysMCre⁺ and LysMCre⁺ mice after 24 h in vitro GM-CSF (40 ng/ml) stimulation. (H) GM-CSF levels in lungs from PND3 Hem1^fl/flLysMCre⁺ and LysMCre⁺ mice, as measured by ELISA. (I) Percentages of BrdU⁺CD45.2⁺CD11c⁺CD64⁺ cells in BALF from adult Hem1^fl/flLysMCre⁺ and LysMCre⁺ mice after 4 d of daily BrdU injections. Shown data are mean ± SEM from n = 4–9 mice/genotype/≥2 independent exps. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; Student’s two-tailed t tests. Monos, monocytes; Rel., relative.