NOX2-deleted CD11b^high AMs acquire an inflammatory transcriptional signature. (A) Multidimensional scaling plot of RNA-seq transcriptomes in 2 different dimensions showing the differences in gene signatures among the indicated groups of samples of AMs sorted from 4- and 12-week-old mice (n = 3 independent samples in each group). (B) Heat maps showing relative expression of the top 500 altered genes in AMs in the different groups, as indicated (n = 3 independent samples in each group). (C) Hallmark gene sets with significant changes in GSEA between Cybb^KO CD11b^high and WT CD11b^low AMs from 12-week-old mice. False-discovery rate (FDR)q <0.05 was considered significant (n = 3 independent samples for each group). (D) GSEA plot showing differences in the hallmark inflammatory response gene sets between AMs from 12-week-old Cybb^KO CD11b^high and Cybb^KO CD11b^low, as well as Cybb^KO CD11b^low and WT CD11b^low mice, as indicated. FDRq <0.05 considered significant (n = 3 independent samples for each group). (E) Heat map from RNA-seq data showing relative expression of selected genes in Cybb^KO and WT AMs from 12-week-old mice (n = 3 independent samples for each group). (F) qRT-PCR of gene expression in flow-sorted AMs from WT and Cybb^KO mice of different ages for selected genes. WTL, WT CD11b^low AM; Cybb^KO L, Cybb^KO CD11b^low AM; and Cybb^KO H, Cybb^KO CD11b^high AM (n = 3 samples for each group). (G) Heat map from AM RNA-seq data for selected M1 and M2 genes, as indicated. (H) qRT-PCR for expression of selected genes in flow sorted AMs, as indicated, from Ncf2^fl/fl and Ncf2^LysMcre mice (n ≥ 3 samples for each group). Adjusted P values: *P < .05; **P < .01; ***P < .001. Bar graph data are expressed as the mean ± standard error of the mean. One-way analysis of variance was performed, followed by Tukey’s post hoc analysis. *P < .05; **P < .01; ***P < .001; ****P < .0001. FDR, false-discovery rate.