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

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

CHimi and TMC/siRNA nanoparticle characterization.

<p>(<b>A</b>) Size (hydrodynamic diameter), polydispersity index (PDI) and charge (ZP, zeta potential) of CHimi and TMC/siRNA nanoparticles determined using a Zetasizer Nano ZS at pH 5.5 and 7.4, respectively (n = 3; average ± SD). (<b>B</b>) Transmission electron microscopy images of CHimi2 and TMC nanoparticles. Scale bar 500 nm.</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

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

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

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

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