MUC2 positive cells on domes of mouse, rat and human Peyer's patches.

<p>Fluorescent staining of Muc2 reveals mucin containing cells in the FAE of a mouse (A), rat (B) and human (C) ileal PP. Bars = 50 µm. Inset in panel C shows the MUC2 positive cells at higher magnification (bar = 10 µm). Muc2 staining is green, nuclei are blue and FAE is indicated by dashed lines. (D) MUC2 positive cells and nuclei in FAE were counted in sections from 5 mice, 5 rats and 5 humans. Values are presented as median (25^th and 75^th percentile). The percentage of goblet cells was larger in human FAE compared to mouse FAE (<i>P</i><0.001, ***) and rat domes (<i>P</i><0.05, *).</p

Transmission electron micrographs of mouse, rat and human Peyer's patches show secreting goblet cells.

<p>(A) Secreting goblet cell in mouse FAE. (B) M cell in mouse FAE. (C) Secreting goblet cell in a rat FAE. (D) Two M cells next to each other in a rat FAE. (E) Secreting goblet cell in human FAE. (F) Mucus on top of a human FAE, mucus border indicated by black arrow and mucus marked by black star. Bars = 2 µm.</p

Mouse ileal Peyer's patches are covered by a mucus layer.

<p>(A) Stereo microscope image of an ileal explant containing a PP. Domes are indicated by black arrows. Charcoal particles were added to visualize the otherwise transparent mucus layer (bar = 0.5 mm). (B) Two mucus filled goblet cells (black arrows) in a dome stained by PAS (bar = 10 µm). (C) Mucus on top of the FAE was removed and remaining mucus thickness measured every 20 minutes for an hour (open circles; n = 6) or mucus thickness was measured at time 0 and 20 min and a combination of carbachol and PGE₂, 10 µM of each, was perfused after the second measurement (arrow, closed circles; n = 6). (D) Initial mucus thickness was measured on the villi of the PP, mucus was removed and remaining mucus thickness measured at time 20 min. Half the number of explants were left unstimulated (open circles; n = 10) and half of the explants were stimulated with carbachol and PGE₂ (10 µM of each; arrow), and mucus thickness was measured at time 40 and 60 min (closed circles; n = 10). (E) Mucus penetrability to beads the size of bacteria was assessed by confocal imaging of mouse ileal explants containing a PP. Tissue is visualized in blue and beads are red (0.5 µm), purple (1 µm) and green (2 µm). (F) To clarify how beads penetrate to the FAE surface, a flat section of the epithelium (blue) is shown. Note how some beads (red, purple and green) are suspended in the mucus. Bars in E and F = 50 µm.</p

CHimi and TMC/siRNA nanoparticle complexation capacity.

<p>(<b>A</b>) Nanoparticle complexation capacity determined by detecting free siRNA migration in an agarose gel electrophoresis. Free siRNA was used as positive control. Nanoparticles with different N/P ratios were tested. (<b>B</b>) SYBRGold exclusion assay. The complexation capacity of the prepared nanoparticles was analysed at different N/P rations and at two different pHs (n = 3; average ± SD). * p<0.01.</p

Mucus penetrability of nanoparticles in mouse distal colon explants.

<p>(<b>A</b>) Representative Z – stack projections of TMC/siRNA nanoparticles in distal colon explants and the corresponding normalised intensity plots; tissue is blue and nanoparticles are red. Scale bars 100 µm. (<b>B</b>) Percentage of the total fluorescence intensity of TMC and CHimi2/siRNA nanoparticles in each plan (tissue, lower half, and upper half) at each time point. Data are presented as means ± SD (n = 3).</p

The basal electrochemical properties of the distal colonic explants measured in Ussing chambers during <i>C. rodentium</i> infection.

<p>Baseline PD (A), Im (B) and Rp (C) of distal colonic tissue during <i>C. rodentium</i> infection. Statistics: ANOVA, Dunnet’s post hoc test, * = p<0.05, ** = p<0.01.</p

Change in membrane current (ΔIm) after stimulation with forskolin and carbachol with and without pre-treatment with the CFTR inhibitor GlyH-101.

<p>(mean±SEM, n = 6). As the Caco-2 cells did not respond to carbachol, the ability of GlyH-101 to inhibit the response was not analyzed in this cell line.</p

mRNA levels of mucins and some related proteins in distal colon during <i>C. rodentium</i> infection.

<p>Muc1 (A), Muc2 (B), Muc4 (C), Muc13 (D), Clca-3 (E), Cftr (F), and Best-2 (G). Expression data were normalized against the Hprt-1 housekeeping gene. Fold changes were calculated using ΔΔCT with mean of CT from three uninfected mice as control.</p

<i>C. rodentium</i> density, colitis score and mucus thickness and growth during infection.

<p>A: Colony forming units (CFU) of <i>C. rodentium</i> were analyzed in fecal pellets collected from individual mice. Compared to non-infected, all time points had an increased amount of <i>C. rodentium</i> (p<0.05). The total amount of luminal bacteria in the distal colon was scored in DAPI stained sections: Score 3 = same density as non-infected animals, 2 = medium density, 1 = low density and 0 = no bacteria detected (*p<0.05). Statistics: ANOVA, Dunnet’s post hoc test. B: The thickness of mucus layer changed during infection with lowest thickness between day 4 to 10 and the highest at day 14 post infection, whereas goblet cells depletion and colitis increased to the highest score at day 10 and 14 post infection, respectively. C: The thickness of the inner mucus layer changed during the course of infection, and reached its highest thickness at the start of the decrease in fecal <i>C. rodentium</i> density (CFU/gram feces). The distal colon explant was mounted and the thickness measured with a scaled micropipette, p<0.05 # vs day 14 and 19, * vs day 0. C: The <i>ex vivo</i> growth of the adherent mucus layer measured for the first 15 minutes after mounting the distal colon tissue. No significant differences were observed between the different time points (n = 5–12). Statistics: ANOVA, Tukey’s post hoc test.</p

Summary of changes during the course the infection.

<p>The presented data are normalized against uninfected controls, which are set to 1. The actual data points and error bars have been presented previously in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084430#pone-0084430-g001" target="_blank">Figures 1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084430#pone-0084430-g007" target="_blank">7</a>. Panel A: Mucus thickness as measured with a scaled micropipette (grey filled area under a black line), mucin material present in lumen as measured with Alcian blue/PAS stain in fixed sections (––) and secretory responses to forskolin (Rp▴, Im Δ) and carbachol (Rp ▪, Im □). Panel B: Muc2 mRNA (grey filled area under a black line), mucin material present in tissue storage as measured with Alcian blue/PAS stain in fixed sections (––), ratio of Best-2 mRNA (•) and ratio of Rp in response to carbachol vs forskolin (○).</p