Fig. 4

Attenuated JE progression is associated with increased accumulation of M1 macrophages in NOX2-ablated mice. A Increased accumulation of M1 macrophages in peritoneal cavity and spleen of NOX2 KO mice. B Accumulated number of M1 macrophages in peritoneal cavity and spleen. Leukocytes were prepared from the peritoneal cavity and spleen of Wild-type (WT) and NOX2 KO mice 1 and 3 dpi, and briefly stimulated with LPS (200 ng/ml) for 6 h. M1 macrophages were then detected by intracellular IL-12p40 and iNOS staining combined with surface CD11b and F4/80 staining. C Reduced ROS levels in sera of JEV-infected NOX2 KO mice. ROS levels were determined by ROS ELISA kit using sera derived from JEV-infected WT and NOX2 KO mice at 2nd dpi. D Intracellular ROS levels of CD11b ⁺ F4/80 ⁺ macrophages derived from WT and NOX2 KO mice. Intracellular ROS levels in macrophages derived from peritoneal cavity and spleen were assessed using total ROS assay kit 2 days after JEV infection. Values in histograms show the average MFI ± SEM of total ROS in macrophages population after gating on CD11b ⁺ F4/80 ⁺ cells. E Enhanced M1 polarization of NOX2-deficient macrophage. BMDM derived from BM cells of WT and NOX2 KO mice were stimulated with LPS and IFN-γ for 6 and 12 h to drive M1 polarization. The M1 polarization of BMDM was evaluated by intracellular IL-12p40 and iNOS staining combined with surface CD11b and F4/80 staining. F Increased expression of M1 effector molecules in NOX2-deficient macrophages. Total RNA extracted from M1-polarized BMDM driven from WT and NOX2 KO mice was used for determining the expression of M1 and M2 effector molecules with real-time qRT-PCR. The expression of M1 and M2 effector molecules was normalized to β-actin expression and displayed as the average of at least four independent samples, according to the indicated color on a log₂ scale. G Higher production of M1 effector cytokines from NOX2-deficient macrophages. BMDM derived from WT and NOX2 KO mice was stimulated with LPS and IFN-γ form the indicated times and the production of M1 effector cytokines (TNF-α, IL-6, IL-12p70) was determined by cytokine ELISA using culture media. 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