IL-18 enhances the recruitment of NK cells into the infected cecum LP thereby stimulating early cecal inflammation.

<p>(a) <i>Il18</i>^<i>-/-</i> mice and littermate controls were Sm-pretreated, infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 3–4 per group). RNA-Seq was performed on RNA extracted from complete cecum tissue. RNA-Seq analysis: The Volcano plot shows the induction (log₂ fold change) versus the -log₁₀ p-value for all chemokines. Chemokines able to induce NK cell recruitment are highlighted in red. (b) C57BL/6 WT mice were Sm-pretreated and either uninfected or infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 4 per group). <i>Cxcl9</i>, <i>Cxcl10</i>, <i>Cxcl11</i>, <i>Ccl3</i> and <i>Ccl4</i> mRNA levels in whole cecum tissue were measured by RT-qPCR. Results are presented relative to the expression of <i>Actb</i>. (c) Same as (b) but comparing cecum tissues from infected <i>Il18</i>^<i>-/-</i> mice vs infected littermate controls. (d and e) Flow cytometric analysis of isolated cecal LP cells from Sm-pretreated C57BL/6 WT mice, either uninfected or infected with 5x10⁷ CFU <i>S</i>.Tm (n = 5 per group). Single live cells were gated on CD45⁺ lymphocytes. (d) Representative dot plots and (e) quantification of NK1.1⁺ CD3^- cells. (f and g) Flow cytometric analysis of isolated cecal LP cells from <i>Il18</i>^-/- mice and littermates, Sm-pretreated and orally infected with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 5–6 per group). Single live cells were gated on CD45⁺ lymphocytes. (f) Representative dot plots and (g) quantification of NK1.1⁺ CD3^- cells. (h) C57BL/6 mice were infected for 12h with 5x10⁷ CFU <i>S</i>.Tm and isolated cecal LP cells of two mice were pooled for staining and isotype control staining; data are shown for one out of three independent experiments. CD45⁺ NK1.1⁺ CD3^- cells were characterized according to their surface expression of KLRG1, NKp46, CD122, TCRγδ and Thy1. (i) C57BL/6 mice were infected for 18h with 5x10⁷ CFU <i>S</i>.Tm. Isolated cecal LP cells of two mice each were pooled for fluorescence activated cell sorting. Live CD45⁺ CD3^- cells were further sorted according to their expression of NK1.1. <i>Tbx21</i>, <i>Eomes</i>, <i>Rorc</i>, <i>Gata3</i>, <i>Prf1 and Sell</i> transcripts were analyzed by RT-qPCR. Results are presented relative to the expression of <i>Actb</i>. (j and k) Flow cytometric analysis of isolated cecal LP cells from Sm-pretreated (j) Casp1/11^-/- or (k) Casp11^-/- mice. Single live cells were gated on CD45⁺ lymphocytes and NK1.1⁺ CD3^- cells were quantified. (l and m) C57BL/6 WT mice were injected intraperitoneally with anti-asialo GM1 antiserum (50μL antiserum/mouse; three consecutive days), anti-NK1.1 (10mg/kg; 2 consecutive days) or PBS and mice were infected orally with 5x10⁷ CFU <i>S</i>.Tm for 12h (n = 8–10 per group). (l) HE-stained cryosections of anti-asialo GM1 -treated or control mice, (m) pathological score; arrows indicate mice of representative HE-stained cryosections; SE = submucosal edema, L = lumen; scale bar = 100μm. Statistical analysis was performed using the Mann-Whitney-U test (ns = not significant, * = p<0.05; ** = p<0.01).