MOESM1 of Campylobacter jejuni infection of conventionally colonized mice lacking nucleotide-oligomerization-domain-2

Additional file 1: Figure S1. Kinetic of intestinal C. jejuni loads in perorally infected conventionally colonized NOD2-/- mice. (A) Wildtype (WT; white circles) and (B) NOD2-/- mice (black circles) were perorally infected with C. jejuni strain 81-176 on three consecutive days (d0, 1 and 2). Pathogenic colonization densities were assessed in fecal samples (CFU, colony forming units per gram) over time post infection as indicated by culture. Medians (black bars) and levels of significance (p values) determined by Mann-Whitney U test are indicated. Numbers of mice harboring C. jejuni out of the total number of analyzed animals are given in parentheses. Data were pooled from four independent experiments

MOESM2 of Campylobacter jejuni infection of conventionally colonized mice lacking nucleotide-oligomerization-domain-2

Additional file 2: Figure S2. Colonic mucin-2 mRNA expression levels in C. jejuni infected conventionally colonized NOD2-/- mice. Wildtype (WT; white circles) and NOD2-/- mice (black circles) were perorally infected with C. jejuni strain 81-176 on three consecutive days (d0, 1 and 2). Mucin-2 (MUC-2) mRNA expression levels were determined in colonic ex vivo biopsies at day 14 post infection by Real Time PCR and expressed in Arbitrary Units (fold expression). Naive (N) mice served as uninfected controls. Medians (black bars) and numbers of analyzed animals (in parentheses) are indicated. Data were pooled from four independent experiments

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

Pro-inflammatory cytokine responses in spleens of NOD2 deficient mice following ileitis induction.

<p>Systemic pro-inflammatory cytokine responses were assessed by measuring (<b>A</b>) TNF-α and (<b>B</b>) IFN-γ levels in <i>ex vivo</i> biopsies of spleens derived from C57BL/6 wildtype (WT; black circles) and NOD2 deficient (NOD2-/-; white circles) mice seven days following ileitis induction (ILE). Naive mice served as negative controls (N). Numbers of analyzed mice (in parentheses), means (black bars) and levels of significance (<i>P</i>-values) as compared to the respective groups (determined by Mann-Whitney-U test) are indicated. Data shown are representative for three independent experiments.</p

Acute ileitis in <i>T. gondii</i> infected NOD2 deficient mice.

<p>In order to induce acute ileitis, C57BL/6 wildtype (WT; black circles) and NOD2 deficient (NOD2-/-; white circles) were perorally infected with <i>T. gondii</i> at day 0. (<b>A</b>) Relative body weight loss between day 7 post infection (p.i.) and day 0 were determined (in %). (<b>B</b>) Absolute small intestinal lengths were measured in naïve (N) and <i>T. gondii</i> infected mice with ileitis (ILE). (<b>C</b>) Histopathological mucosal changes were assessed in ileal paraffin sections, and (<b>D</b>) <i>T. gondii</i> DNA determined in <i>ex vivo</i> ileal biopsies. Numbers of analyzed mice are given in parentheses. Means (black bars) and significance levels (<i>p</i>-values) determined by Mann-Whitney-U test are indicated. Data shown are representative for three independent experiments.</p

Intestinal immune cell responses following ileitis induction in NOD2 deficient mice.

<p>Immune cell responses were assessed microscopically in ileal paraffin sections derived from C57BL/6 wildtype (WT; black circles) and NOD2 deficient (NOD2-/-; white circles) seven days following ileitis induction (ILE) applying <i>in situ</i> immunohistochemistry. The average numbers of ileal (<b>A</b>) apoptotic cells (positive for caspase-3), (<b>B</b>) neutrophilic granulocytes and monocytes (positive for MPO-7), (<b>C</b>) macrophages (positive for F4/80), (<b>D</b>) T-lymphocytes (positive for CD3), and (<b>E</b>) regulatory T-cells (Treg, positive for FOXP3) were determined microscopically in six high power fields (HPF, 400× magnification) per animal. Naive mice served as negative controls (N). Numbers of analyzed mice (in parentheses), means (black bars) and levels of significance (<i>P</i>-values) as compared to the respective groups (determined by Mann-Whitney-U test) are indicated. Data shown are representative for three independent experiments.</p

Intestinal microbiota composition of NOD2 deficient mice following ileitis induction.

<p>Main bacterial groups of the commensal intestinal microbiota were quantified by molecular analysis of fecal samples derived from C57BL/6 wildtype (WT; black circles) and NOD2 deficient (NOD2-/-; white circles) mice before (N, naïve) and seven days after ileitis induction by peroral <i>T. gondii</i> infection (ILE). Quantitative Real-Time-PCR analyses amplified bacterial 16S rRNA variable regions and 16S rRNA gene numbers/ng DNA from the following bacterial groups: (<b>A</b>) Total eubacterial load, (<b>B</b>) <i>Enterobacteria</i>, (<b>C</b>) <i>Enterococci</i>, (<b>D</b>) <i>Lactobacilli</i>, (<b>E</b>) <i>Bifidobacteria</i>, (<b>F</b>) <i>Bacteroides/Prevotella</i> spp., (<b>G</b>) <i>Clostridium leptum</i> group, (<b>H</b>) <i>Clostridium coccoides</i> group, and (<b>I</b>) <i>Mouse intestinal Bacteroidetes</i>. Numbers of mice harboring the respective bacterial 16S rRNA out of the total number of analyzed animals are given in parentheses. Medians and significance levels (<i>p</i>-values) determined by Mann-Whitney-U test are indicated. Data shown are representative for three independent experiments.</p

Small intestinal cytokine responses in NOD2 deficient mice following ileitis induction.

<p>(<b>A</b>) Nitric oxide, (<b>B</b>) IFN-γ, (<b>C</b>) TNF-α, and (<b>D</b>) IL-10 levels were determined in <i>ex vivo</i> ileal biopsies derived from C57BL/6 wildtype (WT; black circles) and NOD2 deficient (NOD2-/-; white circles) seven days following ileitis induction (ILE) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105120#s4" target="_blank">methods</a>. Naive mice served as negative controls (N). Numbers of analyzed mice (in parentheses), means (black bars) and levels of significance (<i>P</i>-values) as compared to the respective groups (determined by Mann-Whitney-U test) are indicated. Data shown are representative for three independent experiments.</p

Impact of the <i>cj0268c</i> gene on colonic inflammatory and immune cell responses following infection of gnotobiotic IL-10^−/− mice.

<p>Gnotobiotic IL-10^−/− mice were generated by antibiotic gut decontamination and perorally infected with <i>C. jejuni</i> NCTC11168 (11168-WT, closed circles), mutant strain NCTC11168::<i>cj0268c</i> (Δ0268c, open circles), or the complemented strain NCTC11168::<i>cj0268c</i>-comp-<i>cj0268c</i> (Δ0268cCompl, crossed circles) as described (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090148#s4" target="_blank">methods</a>). The average numbers of apoptotic cells (positive for caspase-3, panel A), T lymphocytes (positive for CD3, panel B), regulatory T cells (Treg, positive for Foxp3, panel C), B lymphocytes (positive for B220, panel D), neutrophils (positive for MPO7, panel E), and macrophages/monocytes (positive for F4/80, panel F) from at least six high power fields (HPF, 400x magnification) per animal were determined microscopically in immunohistochemically stained colon sections at day 6 p.i. uninfected animals (Naïve; open diamonds) served as negative controls. Numbers of analyzed animals are given in parentheses. Means (black bars) and levels of significance (<i>P</i>-values) determined by the Mann-Whitney-U test are indicated. Data shown were pooled from three independent experiments.</p

<i>C. jejuni</i> knockout mutant NCTC11168::<i>cj0268c</i> colonization along the gastrointestinal tract of gnotobiotic IL-10^−/− mice.

<p>Gnotobiotic IL-10^−/− mice were generated by antibiotic gut decontamination and perorally infected with <i>C. jejuni</i> NCTC11168 (11168-WT, closed circles), mutant strain NCTC11168::<i>cj0268c</i> (Δ0268c, open circles), or the complemented strain NCTC11168::<i>cj0268c</i>-comp-<i>cj0268c</i> (Δ0268cCompl, crossed circles) as described (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090148#s4" target="_blank">methods</a>). The pathogen densities in distinct compartments of the gastrointestinal tract were determined by quantification of live <i>C. jejuni</i> in luminal samples taken from stomach, duodenum, ileum, and colon at day 6 p.i. by cultural analysis (CFU, colony forming units). Medians (black bars) are indicated and numbers of animals harbouring the respective <i>C. jejuni</i> strain out of the total number of analyzed animals given in parentheses. Data shown were pooled from three independent experiments.</p