Fig. 5

NOX2 is negative regulator of de novo differentiation of M1 macrophages. A The expression of M1 and M2 surface markers in NOX2-deficient BMDM. BMDM derived from BM cells of WT and NOX2 KO mice were stimulated with LPS and IFN-γ for 12 and 24 h to drive M1 polarization, and used for evaluating the expression of surface markers by flow cytometric analysis. Values in the histograms show the average MFI ± SEM of each surface molecule expression in macrophage population after gating on CD11b ⁺ F4/80 ⁺ cells. B and C Enhanced Ag-presentation of NOX2-deficient BMDM. BMDM derived from the BM cells of WT and NOX2 KO mice was either stimulated with (M1) or without (M0) LPS and IFN-γ for 12 h, and co-cultured with purified OT-II CD4 ⁺ T cells at the varying ratios in the presence of OVA_323-339 peptide for 24 h and 72 h to assess the generation of IL-2 and IFN-γ-producing cells, respectively. The Ag-presentation of BMDM to OT-II CD4 ⁺ T cells was evaluated by intracellular IL-2 and IFN-γ staining combined with surface CD4 staining. Values in dot-plots show the mean ± SEM percentage of IL-2 and IFN-γ-producing CD4 ⁺ T cells after gating on CD4 ⁺ cells. D Enhanced activation of OT-II CD4 ⁺ T cells stimulated with NOX2-deficient BMDM. The activation markers of OT-II CD4 ⁺ T cells stimulated with BMDM derived from WT and NOX2 KO mice were determined by flow cytometric analysis after surface staining with CD4 and activation marker antibodies. Data was expressed by the average MFI ± SEM levels of each activation marker. E Suppression of M1 macrophage polarization by NOX2-generated H₂O₂. BMDM derived from WT and NOX2 KO mice was stimulated with LPS and IFN-γ in the absence or presence of H₂O₂ (300 µM) for 18 h. The production of M1 effector cytokines (IL-12p40, TNF-α, IL-6) was determined by cytokine ELISA using culture media. F Facilitated de novo differentiation of M1 macrophage by JEV infection. BMDM derived from BM cells of WT and NOX2 KO mice were infected with JEV (5 MOI) in the presence of IFN-γ (10 ng/ml) for 48 h. The M1 polarization of infected BMDM was evaluated by intracellular IL-12p40 and iNOS staining combined with surface CD11b and F4/80 staining. G Higher production of M1 effector cytokines from NOX2-deificent macrophage by JEV infection. BMDM derived from WT and NOX2 KO mice was infected with JEV in the presence of IFN-γ, and the production of M1 effector cytokines (TNF-α, IL-6, IL-12p70) was determined by cytokine ELISA using culture media 48 h later. H Suppressed replication of JEV in NOX2-deficient macrophages. BMDM derived from WT and NOX2 KO mice were infected with JEV (1.0 MOI). Total RNA was extracted from infected BMDM and subjected to determine JEV replication with real-time qRT-PCR at the indicated time points post-infection. JEV RNA replication was expressed as viral RNA copy number targeted on JEV NS1 gene per microgram of total RNA. Data show the mean ± SEM of the levels derived from at least 2 individual experiments (n = 5–6). *p < 0.05, **p < 0.01, ***p < 0.001 between the levels derived from WT and NOX2 KO mice