IFNγ expression by NK cells is IL-18-dependent but not required for mounting tissue inflammation during the first 12h of the infection.

(a) Volcano plot of all cytokines differentially expressed in the cecal mucosa of the Il18^-/- mice and littermate shown in Fig 2A. We plotted log₂ (fold change) against -log₁₀ (p-value). NK cell effector cytokines are highlighted in red. (b-d) Il18^-/- mice and littermate controls were Sm-pretreated and infected orally with 5x10⁷ CFU S.Tm for 12h (b) or 18h (c-d). (b) Ifng, Iigp1 and Cxcl10 transcripts in whole cecum tissue were analyzed by RT-qPCR. Results are presented relative to the expression of Actb (n = 8–9 per group). (c) IFNγ protein concentration in whole cecum tissue lysates (n = 5 per group) as measured by CBA; dashed line = detection limit. (d) Quantification of IFNγ-producing cells by flow cytometric analysis of isolated cecal LP cells (n = 6 per group). (e and f) C57BL/6 mice were infected for 18h with 5x10⁷ CFU S.Tm and cecal LP cells were isolated for staining. Data are shown from one out of three independent experiments. (e) Representative dot plot of IFNγ-expressing cells, pre-gated on single live lymphocytes. (f) FACS-analysis of CD3 and NK1.1 surface marker expression by IFNγ⁺ cell populations. (g) Ifng^-/-, Ifngr1^-/- and littermate controls were Sm-pretreated, infected orally with 5x10⁷ CFU S.Tm for 12h and pathological scores were assessed (n = 6–8 per group). (h) Mesenteric lymph node loads as determined by plating of organs from (left) Ifng^-/- and (right) Il18^-/- mice and their littermate controls. Mice were Sm-pretreated and infected orally with 5x10⁷ CFU S.Tm for 12h or 72h. Statistical analysis was performed using the Mann-Whitney-U test (ns = not significant, * = p<0.05; ** = p<0.01; *** = p<0.001).