Fig 5. Priming by IFN-γ promotes macrophage activation against K. pneumoniae.

(A) Alveolar macrophages from WT and Ifnar1^-/- mice were pretreated or not with 5 ng/ml mouse IFN-γ for 5 h, followed by infection (MOI = 70) for 1 h and RNA isolation. mRNA levels of Il12b, Cxcl10, Tnf and Il1b were quantitated by qPCR and normalized to Hprt. (B,C) Macrophages primed or unprimed with IFN-γ (10 ng/ml) for 2 h were infected (MOI = 70) for 90 min (including gentamicin treatment starting at 30 min after infection) (B) or 30 min without gentamicin treatment (C), and bacterial loads were determined as CFU per ml. (D-F) WT and Irf3^-/- mice were infected intranasally (5 x 10⁴ CFU of K. pneumoniae) for 48 h (n = 11 per genotype). Expression of indicated genes (D,E) and bacterial loads (F) in lungs were determined using qPCR and CFU assays, respectively. (G, H) WT mice received intranasally IFNβ (30 μl, 30,000 U) or PBS (n = 4 per treatment). Animals were euthanized 6 h following treatment. RNA was isolated from lungs and analyzed for expression of Mx1, Isg15 and Ifit1 (G) as well as Ifng, Il12b and Cxcl10 (H). Statistical evaluation in (A), (D), (E), (G) and (H): unpaired Student’s t test; error bars, mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. Statistical evaluation in (B), (C) and (F): Mann-Whitney test; horizontal bars represent median; ***, P < 0.001; ns, not significant.