Fig 1.
Intestinal microbiota composition in human microbiota associated mice suffering from acute ileitis.
Human microbiota associated (hma) were perorally infected with T. gondii strain ME49 to induce acute ileitis as described in methods. Main intestinal bacterial groups abundant in the ileum (A, B) and the colon lumen (C, D) of hma mice were quantitatively assessed applying both culture (A, C) and culture-independent (i.e. molecular 16S rRNA based; B, D) methods 7 days following ileitis induction (ILE, filled circles). Noninfected hma mice served as controls (N, open circles). Numbers of enterobacteria (EB), enterococci (EC), Gram-positive rods (GPR), Bacteroides / Prevotella spp. (B/P), Clostridium / Eubacterium spp. (C/E) and the total bacterial loads (TL) are expressed as colony forming units per gram feces (CFU / g). 16S rRNA of the main intestinal bacterial groups including enterobacteria (EB), enterococci (EC), lactobacilli (LB), bifidobacteria (Bif), Bacteroides / Prevotella species (B/P), Clostridium coccoides group (Clocc), Clostridium leptum group (Clept), Mouse Intestinal Bacteroides (MIB) and the total eubacterial loads (TL) are expressed as gene numbers per ng DNA. Numbers of animals harboring the respective bacterial group out of the total number of analyzed mice are given in parentheses. Medians (black bars) and significance levels (p-values) determined by Student’s t test and Mann-Whitney U test are indicated. Data shown are pooled from three independent experiments.
Fig 2.
Clinical, macroscopic and microscopic sequelae in human microbiota associated mice suffering from acute ileitis.
Human microbiota associated (hma) mice were perorally infected with T. gondii strain ME49 to induce acute ileitis (ILE; filled symbols). Noninfected hma mice served as controls (N, open symbols). Clinical, macroscopic and microscopic intestinal changes were assessed at day 7 following ileitis induction: (A) Abundance of blood was determined in fecal samples by the Guajac (Haemoccult) method. Means, standard deviations and numbers of fecal blood positive mice out of the total numbers of analyzed animals are given in parentheses. (B) Absolute small intestinal lengths were measured (in cm) and (C) histopathological changes were determined in H&E stained ileal paraffin sections applying a standardized scoring system (see methods). Scores ≥4 (dotted line) indicate severe inflammation with necrosis. (D) The average numbers of apoptotic cells (positive for caspase 3; Casp3+) from at least six high-power fields (HPF, 400 x magnification) per animal were determined microscopically in immunohistochemically stained ileal paraffin sections. Numbers of animals (in parentheses), medians and significance levels (p-values) determined by Students-t test and Mann-Whitney U test are indicated. Data shown were pooled from three independent experiments.
Fig 3.
Small intestinal immune cell responses in human microbiota associated mice suffering from acute ileitis.
Human microbiota associated (hma) mice were perorally infected with T. gondii strain ME49 to induce acute ileitis (ILE; grey boxes). Noninfected hma mice served as controls (N, white boxes). The average numbers of ileal (A) T lymphocytes (positive for CD3), (B) regulatory T cells (positive for FOXP3), (C) B lymphocytes (positive for B220), and (D) macrophages (positive for F4/80) from six high power fields (HPF, 400 x magnification) per animal were determined microscopically in immunohistochemically stained ileal paraffin sections at day 7 post ileitis induction. Box plots represent the 75th % and 25th % percentiles of the medians (black bar inside the boxes). Total range and significance levels (p-values) determined by the Student’s t test and Mann-Whitney U test and numbers of mice (in parentheses) are indicated. Data shown were pooled from three independent experiments.
Fig 4.
Intestinal cytokine responses in human microbiota associated mice suffering from acute ileitis.
Human microbiota associated (hma) mice were perorally infected with T. gondii strain ME49 to induce acute ileitis (ILE; grey boxes). Noninfected hma mice served as controls (N, white boxes). At day 7 post ileitis induction secretion of distinct pro- and anti-inflammatory cytokines (as indicated) were determined in ex vivo biopsies derived from distinct intestinal compartments including (A) ileum (B) colon and (C) mesenteric lymph nodes (MLN). Box plots represent the 75th % and 25th % percentiles of the medians (black bar inside the boxes). Total range and significance levels (p-values) determined by the Student’s t test and Mann-Whitney U test and numbers of mice (in parentheses) are indicated. Data shown were pooled from three independent experiments.
Fig 5.
Translocating intestinal bacteria in human microbiota associated mice suffering from acute ileitis.
Human microbiota associated (Hma) mice were perorally infected with T. gondii strain ME49 to induce acute ileitis (ILE, filled bars). Noninfected hma mice served as controls (N, open bars). At day 7 post ileitis induction rates of viable intestinal bacterial species translocating to extra-intestinal and systemic compartments were determined by cultivation of ex vivo biopsies derived from mesenteric lymph nodes (MLN), liver, lung and spleen and of cardiac blood. Mean translocation rates (in %) ± standard deviations and numbers of culture-positive samples out of the total number of analyzed animals are indicated in parentheses. Data shown were pooled from three independent experiments.
Fig 6.
Intestinal bacteria translocating to extra-intestinal compartments in human microbiota associated mice suffering from acute ileitis.
Human microbiota associated (hma) mice were perorally infected with T. gondii strain ME49 to induce acute ileitis (ILE, filled circles). Noninfected hma mice served as controls (N, open circles). At day 7 post ileitis induction viable intestinal bacteria translocating to (A) MLN, (B) extra-intestinal (i.e. liver and lung) and (C) systemic compartments (i.e. spleen and blood) were quantitatively determined by culture of respective ex vivo biopsies and cardiac blood. Numbers of enterobacteria (EB), enterococci (EC), Gram-positive rods (GPR), Bacteroides / Prevotella species (B/P), Clostridium / Eubacterium species (C/E) and the total bacterial loads (TL) were expressed as colony forming units per gram organ homogenate or ml blood (CFU/g; CFU/ml). Numbers of culture-positive samples out of the total number of analyzed mice are given in parentheses. Medians (black bars) and significance levels (p-values) determined by Mann-Whitney U test are indicated. Data shown were pooled from three independent experiments.
Fig 7.
Extra-intestinal cytokine responses in human microbiota associated mice suffering from acute ileitis.
Human microbiota associated (hma) mice were perorally infected with T. gondii strain ME49 to induce acute ileitis (ILE; grey boxes). Noninfected hma mice served as controls (N, white boxes). At day 7 post ileitis induction secretion of distinct pro-inflammatory cytokines (as indicated) were determined in ex vivo biopsies derived from distinct extra-intestinal compartments including (A) liver and (B) kidney. Box plots represent the 75th % and 25th % percentiles of the medians (black bar inside the boxes). Total range and significance levels (p-values) determined by the Student’s t test and Mann-Whitney U test and numbers of mice (in parentheses) are indicated. Data shown were pooled from three independent experiments.
Fig 8.
Systemic cytokine responses in human microbiota associated mice suffering from acute ileitis.
Human microbiota associated mice were perorally infected with T. gondii strain ME49 to induce acute ileitis (ILE; grey boxes). Noninfected hma mice served as controls (N, white boxes). At day 7 following ileitis induction secretion of distinct pro- and anti-inflammatory cytokines were determined in systemic compartments including (A) serum and (B) spleen (as indicated). Box plots represent the 75th % and 25th % percentiles of the medians (black bar inside the boxes). Total range and significance levels (p-values) determined by the Student’s t test and Mann-Whitney U test and numbers of mice (in parentheses) are indicated. Data shown were pooled from three independent experiments.