Fig. 2. BCG-disA-OE elicits greater macrophage reprogramming, phagocytic activity, and autophagy than BCG-WT in human and murine macrophages.

a Percentages of inflammatory M1- and immunosuppressive M2-macrophages and M-MDSCs arising from primary murine macrophages. b Percentages of inflammatory, TNF-α⁺ M1, and IL-6⁺ inflammatory macrophages (M1-like), and c CD206⁺CD124⁺ and IL-10⁺ immunosuppressive macrophages (M2-like) arising from primary human macrophages. Data were collected after 24 h exposures at MOI of 20:1 as determined by flow-cytometry using gating schemes shown in Fig. S3–S9. Data are presented as mean values ± S.E.M. (n = 4 independent biological replicate experiments). d Phagocytic activity in human primary macrophages in representative confocal photomicrographs showing intracellular uptake of FITC-labeled IgG-opsonized latex beads (green) with nuclei stained blue. Data are represented as mean values ± S.E.M (n = 3 independent biological replicate experiments). e Autophagy induction and f quantification by BCG-LC3B colocalization in primary murine macrophages shown by representative confocal photomicrographs. Autophagy was measured by LC3B puncta or g. p62 colocalization with BCG appearing in yellow. FITC-labeled BCG strains are stained green, LC3B or p62 autophagic puncta (red), and nuclei blue. h Quantification of BCG-p62 colocalization. Cells were fixed using 4% paraformaldehyde 6 h after infection (MOI 10:1), and images obtained with an LSM700 confocal microscope and Fiji software processing. Quantification was measured by mean fluorescence intensity. Data represented for all confocal microscopy studies (phagocytosis, LC3B-BCG colocalization and p62-BCG colocalization) as mean values ± S.E.M. (n = 3 independent biological replicate experiments). All statistical analyses done using two-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Data shown are for BCG-Tice; similar findings were observed for BCG-Pasteur as shown in Figs. S2 and S7.