MOESM2 of The IL-23/IL-22/IL-18 axis in murine Campylobacter jejuni infection

Additional file 2: Figure S2. Extraintestinal translocation of viable intestinal commensal E. coli in perorally C. jejuni strain 81-176 infected mice lacking IL-23p19, IL-22 or IL-18. Conventional wildtype (WT), IL-23p19−/−, IL-22−/− and IL-18−/− mice were perorally infected with C. jejuni strain 81-176 by gavage at day 0 and day 1. Intestinal translocation of viable E. coli derived from the commensal intestinal microbiota to extraintestinal compartments was assessed by determining bacterial loads (CFU, colony forming units per gram) in (A) spleen (B) liver (C) kidney, and (D) cardiac blood at day 14 postinfection by culture. Numbers of mice harboring E. coli out of the total number of analyzed animals are given in parentheses, and medians (black bars) are indicated. Data were pooled from three independent experiments

Colonic IL-17A and IL-1β mRNA expression in <i>C</i>. <i>jejuni</i> strain 81–176 infected gnotobiotic mice lacking IL-23p19, IL-22 or IL-18.

<p><b>(A)</b> IL-17A and <b>(B)</b> IL-1β mRNA levels were determined in colonic <i>ex vivo</i> biopsies at day (d) 8 (black circles) postinfection by Real Time PCR and expressed in Arbitrary Units (fold expression). Naive (N) mice served as uninfected controls (white circles). Medians (black bars), level of significance (p-value) determined by Mann-Whitney U test, and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from four independent experiments.</p

IL-23p19, IL-22, and IL-18 mRNA expression in colonic <i>ex vivo</i> biopsies derived from <i>C</i>. <i>jejuni</i> strain 81–176 infected gnotobiotic mice lacking IL-23p19, IL-22 or IL-18.

<p><b>(A)</b> IL-23p19, <b>(B)</b> IL-22, and <b>(C)</b> IL-18 mRNA levels were determined in colonic <i>ex vivo</i> biopsies at day (d) 8 postinfection (black circles) by Real Time PCR and expressed in Arbitrary Units (fold expression). Naive (N) mice served as uninfected controls (white circles). Medians (black bars), level of significance (p-value) determined by Mann-Whitney U test, and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from four independent experiments.</p

Colonic immune cell responses in <i>C</i>. <i>jejuni</i> strain 81–176 infected gnotobiotic mice lacking IL-23p19, IL-22 or IL-18.

<p>The average number of colonic epithelial cells positive for <b>(A)</b> CD3 (T Lymphocytes), <b>(B)</b> FOXP3 (Regulatory T Cells, Tregs), <b>(C)</b> B220 (B Lymphocytes), and <b>(D)</b> F4/80 (Macrophages / Monocytes) from at least six high power fields (HPF, 400x magnification) per animal was determined microscopically in immunohistochemically stained colonic paraffin sections at day (d) 8 (black circles) postinfection. Naive (N) mice served as uninfected controls (white circles). Medians (black bars), level of significance (p-value) determined by Mann-Whitney U test, and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from four independent experiments.</p

Systemic pro-inflammatory cytokine secretion in <i>C</i>. <i>jejuni</i> strain 81–176 infected gnotobiotic mice lacking IL-23p19, IL-22 or IL-18.

<p><b>(A)</b> TNF, <b>(B)</b> IFN-γ, and <b>(C)</b> IL-6 concentrations were determined in serum samples taken from gnotobiotic wildtype (WT), IL-23p19^-/-, IL-22^-/- and IL-18^-/- mice at day (d) 8 (black circles) postinfection. Naive (N) mice served as uninfected controls (white circles). Medians (black bars), level of significance (p-value) determined by Mann-Whitney U test, and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from four independent experiments.</p

Apoptotic and proliferating cells in the colonic epithelium of <i>C</i>. <i>jejuni</i> strain 81–176 infected gnotobiotic mice lacking IL-23p19, IL-22 or IL-18.

<p>The average number of colonic <b>(A)</b> apoptotic cells (positive for caspase-3, Casp3) and <b>(B)</b> proliferating cells (positive for Ki67) from at least six high power fields (HPF, 400x magnification) per mouse was determined microscopically in immunohistochemically stained colonic paraffin sections at day (d) 8 (black circles) postinfection. Naive (N) mice served as uninfected controls (white circles). Medians (black bars), level of significance (p-value) determined by Mann-Whitney U test, and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from four independent experiments.</p

Pro-inflammatory cytokine secretion in colonic <i>ex vivo</i> biopsies derived from <i>C</i>. <i>jejuni</i> strain 81–176 infected gnotobiotic mice lacking IL-23p19, IL-22 or IL-18.

<p><b>(A)</b> TNF, <b>(B)</b> IFN-γ, and <b>(C)</b> IL-6 concentrations were determined in supernatants of colonic <i>ex vivo</i> biopsies derived from gnotobiotic wildtype (WT), IL-23p19^-/-, IL-22^-/- and IL-18^-/- mice at day (d) 8 (black circles) postinfection. Naive (N) mice served as uninfected controls (white circles). Medians (black bars), level of significance (p-value) determined by Mann-Whitney U test, and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from four independent experiments.</p

Colonic lengths of perorally <i>C</i>. <i>jejuni</i> strain 81–176 infected gnotobiotic mice lacking IL-23p19, IL-22 or IL-18.

<p>Absolute colonic lengths of mice were determined at day of necropsy (day (d) 8 postinfection). Naive (N) mice served as uninfected controls. Numbers of analyzed animals (in parentheses), means (black bars) and level of significance (p-value) determined by Mann-Whitney U test are indicated. Data were pooled from four independent experiments.</p

Intestinal <i>C</i>. <i>jejuni</i> loads in perorally infected gnotobiotic mice lacking IL-23p19, IL-22 or IL-18.

<p>Gnotobiotic wildtype (WT), IL-23p19^-/-, IL-22^-/- and IL-18^-/- mice were generated by broad-spectrum antibiotic treatment and perorally infected with <i>C</i>. <i>jejuni</i> strain 81–176 by gavage at day 0 and day 1. Pathogenic loads (colony forming units (CFU) per gram) were determined in <b>(A)</b> colonic luminal samples and <b>(B)</b> homogenates of mesenteric lymphnodes (MLN) at day 8 postinfection by culture. Numbers of analyzed mice (in parentheses), medians (black bars) and level of significance (p-value) determined by Mann-Whitney U test are indicated. Data were pooled from four independent experiments.</p

<i>Cytokineproduction</i> by mLN cells during acute <i>H</i>. <i>p</i><i>. bakeri</i> infection.

<p>IL-4 (A), IL-13 (B), IFN-γ (C), IL-10 (D), TGF-β (E) and IL-17A (F) were measured in mLN culture supernatants with (ConA, H.p.b-Ag) or without (-) stimulation. Cells were derived from naïve controls and mice at day 14 post infection. Mean + SEM is shown for 5 mice per group. H.p.b. Ag: <i>H</i>. <i>p</i>. <i>bakeri</i> antigen; Con A: concanavalin A; * p < 0.05; ** p < 0.005.</p