A gut-vascular barrier controls the systemic dissemination of bacteria

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Science  13 Nov 2015:
Vol. 350, Issue 6262, pp. 830-834
DOI: 10.1126/science.aad0135

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  1. Fig. 1 Characterization of the GVB.

    (A) Intestinal blood vessels permeability to 4 kD and 70 kD FITC-dextran was analyzed by intravital two-photon microscopy in untreated or Salmonella-infected mice. Images were acquired every 30 s for 10 min. Arrowheads indicate fluorophore extravasation. Scale bar, 20 μm. Images are representative of three mice per group. (B) Localization of indicated TJ proteins (green) on endothelial cells (CD31, red) in the mouse intestine. Scale bars: (i and iv) 10 μm; (ii and iii) 20 μm. (C) AJ proteins expression on intestinal vessels. Scale bars, 10 μm. (D) Confocal images of C57BL/6J mice intestine stained with CD31 (red) and GFAP (green) (scale bar, 30 μm) or CD31 (gray), α-SMA (green), and desmin (red). Scale bars: (right) 20 μm, (left) 10 μm. In each section, actin filaments or nuclei were stained with phalloidin or 4′,6′-diamidino-2-phenylindole (DAPI) (blue), respectively. For panels (B) to (D), images are representative of six different mice.

  2. Fig. 2 PV1 expression correlates with Salmonella systemic dissemination.

    (A) C57BL/6J mice were orally infected or not with S. typhimurium ΔaroA, and PV1 (red) expression was evaluated in small intestinal (SI) blood vessels stained with CD34 (green). Cell nuclei were stained with DAPI (blue). Scale bars, 50 μm. (B) Mean fluorescence intensity of PV1 was measured to quantify the up-regulation of the protein in the intestinal vessels. AU, arbitrary units. Statistical comparisons were based on one-way analysis of variance (ANOVA). *P < 0.001. Each part of the SI was compared with its nontreated counterpart; #,§P < 0.001 ileum (I) was compared with duodenum (D) (#) or with jejunum (J) (§) at the same time point. (C and D) S. typhimurium dissemination in PPs, MLNs, spleen, and liver (C) and ALT in the serum (D) were evaluated at the indicated time points after infection. Two independent experiments are shown, each data point representing an individual mouse (n = 4 to 8). Error bars represent SEM. Student’s unpaired t test was used to determine statistical significance. NT, not treated.

  3. Fig. 3 Salmonella interferes with activation of the Wnt/β-catenin signaling pathway via a Spi2-mediated mechanism.

    (A) Primary lung endothelial cells were infected with WT S. typhimurium, a Spi2 mutant strain (ΔssaV), or a Spi1 mutant strain (ΔinvA). Alternatively, cells were treated with recombinant Wnt3a as a positive control. The expression of Axin2 was assessed by quantitative reverse transcription polymerase chain reaction. Results are pooled from three independent experiments. Error bars represent SEM. Statistical significance between untreated and treated samples was evaluated using one-way ANOVA. Error bars represent SEM. (B) β-Catenin gain-of-function mice (black symbols) or β-cateninlox(ex3)/lox(ex3) control mice (gray symbols) were orally infected with S. typhimurium ΔaroA. Colony-forming units (CFUs) in PPs, MLNs, spleen, and liver were determined at the indicated time points. Two independent experiments are pooled; each data point represents an individual mouse (n = 3 to 9). (C) Bacterial counts in PPs, MLNs, liver, spleen, SI, and colon at the indicated time points after C57BL/6J mice infection with 1010 S. typhimurium ΔssaV. NT, not treated. Each dot represents one mouse (n = 5 to 8). Error bars represent SEM. Statistical significance was evaluated by Student’s unpaired t test. *P < 0.05 **P < 0.01.

  4. Fig. 4 GVB is present in the human intestine and is disrupted in patients with celiac disease.

    (A) Confocal images and three-dimensional reconstruction of human healthy ileum stained with CD31 (green) and GFAP (red). Tissue sections were also stained for pericyte markers α-SMA (antibody against smooth muscle actin) (left) and desmin (right). (B and C) Immunofluorescence of three representative duodenal biopsies from celiac disease patients with high (B) or normal (C) levels of ALT and aspartate transaminase (AST). PV1 (green) stainings are shown. All sections are counterstained with DAPI (blue) to visualize cell nuclei. Scale bars, 50 μm. (D) PV1 expression quantified in 6 samples from celiac disease patients with high ALT and AST, 9 from patients with low ALT and AST, and 10 from healthy individuals. One-way ANOVA and t test were used to determine statistical significance. *P < 0.05.