Fig. 2

NOS2 deficiency increases the infiltration of macrophages and neutrophils in RSA59 infected mice brains. Flow cytometry analysis was performed on brains from WT and NOS2-/- mock and RSA59 infected (10,000 PFUs) brains at day 9/10 p.i. Infiltrating peripheral cells were distinguished by CD45hi gating from live cell populations gated from singlets. Similarly, CD45lo gate was used to extract brain resident cell populations. Representative flow cytometry contour plots showing percentages of CD45lo CD11b + microglia populations and their graphical representation in mock and RSA59 infected WT and NOS2-/- groups are given in (A). Percentages of CD45hi CD11b + Ly6G- macrophages and CD45hi CD11b + Ly6G + neutrophils are presented in representative contour plots and quantification of their numbers in graphical form in (B). (C) shows the representative contour plots of CD45hi CD3 + CD4 + T cells and its graphical representation of the absolute numbers. Similarly (D) shows the representative contour plots of CD45hi CD3 + CD8 + T cells and its graphical representation of the absolute numbers. Transcript levels of TNFα, IFNγ and Ifit2 assessed in WT and NOS2-/- mice brains are shown in (E). (Real time quantitative PCR experiment was performed in triplicates and average expression per sample as calculated by ΔCT method was plotted. Each dot corresponds to one biological sample.) Statistical analysis was performed using Two- Way ANOVA with Sidak’s multiple comparison test between mock and infected WT and NOS2-/- groups. Results were expressed as Mean ± SEM. n = 3 to 4 for MI and 5 to 7 for Infected for A, B, C and D. n = 3 for MI and 3 to 4 for Infected for E..*Asterisk represents statistical significance. p < 0.05 was considered as significant. *p < 0.05 and**p < 0.01