Figure 3.

Pad2 deficiency promoted Alveolar macrophages (AMs) towards M2 polarization and increased phagocytosis. The Pad2^-/- and WT mice were infected with 2.5 X 10⁶ CFU Pseudomonas aeruginosa (PA) 19660/mouse for 24 hours. (A) AMs from Pad2^-/- and WT mice were lysed, and the expression of classical cytokine genes (Tnfα, Il6, and Il10) and macrophage polarization signature genes (Nos2, Ccl2, Mrc1, and Arg1) was quantified by qRT-PCR. (B) BALF was collected from both groups of mice, and the levels of inflammation cytokines (TNF-α, IL-6, and IL-10) were determined using ELISA. (C) AMs were isolated from Pad2^-/- and WT mice, cell lysates from AMs were analyzed by western blotting to assess the protein levels of macrophage polarization markers, iNOS, and Arg-1. (D) AMs were subjected to immunofluorescence staining to evaluate the expression of macrophage polarization markers, iNOS, and Arg-1. (E) Isolated AMs from Pad2^-/- and WT mice were analyzed by flow cytometry to quantify the distribution of M1 and M2 macrophage populations. (F) Quantification of the proportion of M1 and M2 macrophages. (G) The phagocytic capacity of AMs was measured using pHrodo Red E. coli BioParticles. Data are representative of three independent experiments expressed as means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.01.