Mice with global deletion of NOX2 develop lung inflammation and a subset of CD11b^high AMs. (A) BAL leukocyte count, flow cytometric analysis of the percentage of polymorphonuclear leukocytes (PMNs) and total PMNs in 3 mL BAL from 4- and 12-week-old WT and Cybb^KO male and female mice (n ≥ 7 in each group). (B) Flow cytometric analysis of total AMs (CD45⁺Siglec F⁺CD11c⁺ cells) and total eosinophils (EOS) (CD45⁺Siglec F⁺CD11c⁻ cells) from 3 mL of BAL (n ≥ 7). (C) ELISA of IL-1β and CXCL2 in BAL (n ≥ 3 in each group). (D) Gating strategy to identify CD11b⁺ AMs in the BAL (CD45⁺SIglec F⁺ CD11c⁺CD11b^high). (E) Percentage of AMs that are CD11b^high in BAL samples from different groups (n ≥ 4). (F) CD11b expression by qRT-PCR in flow-sorted AMs of different groups. WTL, WT CD11b^low AMs; Cybb^KO L, Cybb^KO CD11b^low AMs; and Cybb^KO H, Cybb^KO CD11b^high AMs (n = 3). (G) Lung sections from 12-week-old WT and Cybb^KO mice, as indicated, stained with hematoxylin-eosin showing intracellular (arrowhead) and extracellular eosinophilic crystals (arrow) in Cybb^KO mice. The rightmost panel shows lung tissue from a Cybb^KO mouse with small infiltrates adjacent to some of the crystals. Scale bars, 100 μm. Representative images from ≥5 mice of each genotype. Bar graph data are expressed as mean ± standard error of the mean. The Student t test was performed for samples distributed in 2 groups. *P < .05; **P < .01; ***P < .001. One-way analysis of variance was performed followed by Tukey’s post hoc analysis comparing multiple groups. Data represent at least 2 independent experiments. *P < .05; **P < .01; ***P < .001; ****P < .0001. ELISA, enzyme linked immunosorbent assay.