Increased variability in mucus layer thickness during clearance.

<p>A: The variation in thickness (i.e. the range, as measured with a micropipette in tissue explants) within each sample was altered at the different time points of infection (p<0.05, ANOVA F-test). B: Muc2 (green) stained distal colon of a non-infected C57BL/6 mouse. The inner, highly organized Muc2 layer is indicated by the white bar. C: The section from panel B stained with DAPI. The inner mucus layer is visible as a non-stained black band. D: Muc2 stained distal colon of a mouse infected for 19 days with <i>C. rodentium</i>. The striated adherent inner Muc2 layer is indicated by white bars; one bar has been placed in an area where the inner mucus layer appears thin, and the other one in an area where it is thick. E: The section from panel D stained with DAPI. F: Muc2 stained distal colon of another mouse infected for 19 days with <i>C. rodentium</i>. The adherent Muc2 layer is disorganized. G: The section from panel F stained with DAPI. Sloughed off cells are visible within the inner mucus layer.</p

The response to forskolin and carbachol is altered during infection in distal colon explants studied in Ussing chambers.

<p>Changes in Im (A and C) and Rp (B and D) in response to forskolin (A–B) and carbachol (C–D) in distal colonic tissue during <i>C. rodentium</i> infection. Statistical analysis was performed on log transformed data: ANOVA Tukey’s post hoc test: p<0.05 #,*, p<0.01 ##,**p<0.001 ###,***, # vs day 19, * vs day 0).</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

Quantification of total mucus as determined by Alcian blue/PAS stain in the distal colon.

<p>The percentage of the lumen (A) surface epithelium (B), mid-crypt (C) and bottom of the crypt (D) that was Alcian blue/PAS positive was calculated using the Image J image analysis program, n = 7–12, statistics: ANOVA, p<0.05, # vs day 0, & vs day 14, * vs day 19.</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

List of primers for SyberGreen based RT-PCR.

<p>List of primers for SyberGreen based RT-PCR.</p

Ussing chamber responses are larger in a goblet cell-like <i>in vitro</i> model than in an enterocyte-like model after infection with <i>C. rodentium</i>.

<p>A: Membrane current (Im) response to forskolin of the goblet cell containing model (LS513) <i>vs</i> enterocyte like model (Caco-2) co-cultured with <i>C. rodentium</i> for 24 h (black bars) and cells without bacteria (white bars). B: Membrane current (Im) response of LS513 vs Caco-2 cells treated with <i>C. rodentium</i> for 30 min after insertion into the Ussing chamber and then stimulated with carbachol and forskolin. C: Transepithelial resistance (Rp) response of LS513 vs Caco-2 cells treated with <i>C. rodentium</i> for 30 min after insertion into the Ussing chamber and then stimulated with carbachol and forskolin. (2-tailed T-test, **p<0.01, ***p<0.001).</p

Mucus and bacteria during bacterial expulsion (day 14).

<p>A–C: The anti-Muc2C3 immunostained Muc2 mucin (green) formed an inner mucus layer (marked with a yellow bar in panel C) that is less well organized than in uninfected colon. Bacteria (FISH, eubacterial probe, hybridizing with both <i>C. rodentium</i> and other eubacteria) are mainly present in the outer mucus layer. Photos were taken with a Nikon Eclipse 90 i microscope, white bar = 50 µm. D–F: The mucus stained as in A–C showing the rod-shaped bacteria within the green mucus (white arrows), white bar = 50 µm. Photos taken with a LSM 510 META (Zeiss) confocal microscope with a 40x objective.</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