Mucins in Infectious Colitis Figure 4. Tissue localization of mucins in the proximal large intestine. The apical surface of the epithelial cells of the crypts in the leaf-like folds of the proximal large intestine is lined by the cell surface mucins Muc4, Muc13 and Muc17 (brown). The goblet cells contain Alcian blue positive material (blue), which mainly is Muc2 (brown), although the goblet cells of the surface also contain Muc4. The photographs were taken using a 206 magnification. The quantification scores for any of these mucins were not significantly different between non-infected mice (#) and mice infected with C. rodentium for 12 days (N) in these areas of the intestine. (Mann Whitney U test, n = 7). SCL = subcellular localization, RC = region of crypt. The mucin stains are shown in serial sections on the same tissue area, whereas the mucin stain scores represent the average stain scores for the entire specimen for each individual mouse. doi:10.1371/journal.pone.0003952.g004 feces (Figure 1A). As fecal C. rodentium CFU counts were maximal at 12 days and the fecal C. rodentium load decreased at later time points (data not shown), we sacrificed the mice on day 12. C. rodentium was detected in intestinal tissues of 7/8 mice with approximately 100 fold higher CFU in the colon compared to small intestine (108 vs 106, Figure 1B). In 6/8 animals C. rodentium was detected in the liver and/or spleen, demonstrating that live bacteria cross the intestinal barrier. The mice did not lose any weight, and the infection only induced mild diarrhea with no rectal bleeding at any time (data not shown). In the small intestine, only 1/8 mice developed mild inflammation with irregular crypt architecture, 10–25% goblet cell loss, increased leukocytes in the lamina propria and slightly increased levels of polymorphonuclear cells in the lamina propria. In the caecum and proximal colon, 6/8 animals showed signs of mild colitis (Figure 1D). The distal colon was most affected by the infection with moderate crypt loss, crypt abscesses, discrete lesions, increased crypt length, .50% goblet cell loss, transmural extension of inflammatory infiltrates and high levels of polymor- phonuclear cells in the lamina propria (Figure 1D). Non-infected control mice did not show any signs of colitis (data not shown). Mice infected with C. rodentium normally mount an IgG response that eradicates the bacteria after 3–4 weeks [43]. By day 12 most mice in this study had an increased Ig response to C. rodentium (Figure 2). However, at this time point the Ig response had not cleared the C. rodentium in any of the animals as all the animals with an increase in C. rodentium reactive Ig had both high intestinal CFU counts and C. rodentium present in their spleen and/or liver. Mucin expression in the healthy murine intestinal tract and following C. rodentium infection Mucin expression was evaluated by Alcian blue/periodic acid Schiff (AB/PAS) staining and by immunohistochemistry for the Muc2 and Muc5AC secreted mucins, and the Muc1, Muc3/17 (hereto Muc17), Muc4 and Muc13 cell surface mucins in the small intestine, caecum, proximal-, mid- and distal large intestine of uninfected and infected mice. Muc5ac was not expressed in normal or infected intestine (stomach control sections were strongly positive). Small Intestine. AB stains acidic mucins blue and the PAS stains neutral mucins pink whilst mixtures of neutral and acidic mucins appear purple. In the small intestine, the goblet cells varied from purple to blue and tended to be bluer towards the bottom of the crypts with more purple/fushia towards the top (Figure 3), indicating that the carbohydrates are more highly charged towards the bottom of the crypts. All goblet cells expressed Muc2 and most of the villous goblet cells expressed Muc4 with weaker staining in the crypt goblet cells. Enterocytes expressed the Muc4, Muc13 and Muc17 mucins, but not Muc1, with the Muc13 antibody giving the strongest stain, which decorated the surface of the entire glandular and villous epithelium. The presence of Muc4 in goblet cells and enterocytes is similar to that described for human tissue, and is consistent with previously published work showing that MUC4 can be produced both in membrane bound and soluble forms [40,41]. No discernable changes were seen in expression of any of the mucins in the infected small intestine, despite the presence of a low density of C. rodentium. Caecum. Caecal goblet cells were strongly AB positive and contained Muc2 and Muc4 (Figure 3). Muc4 was weakly expressed on the epithelial surface, whereas Muc13 was strongly expressed on the apical surface and in the cytoplasm of caecal epithelial cells and Muc17 was apically expressed on the surface cells but not in the crypts. Like the small intestine, Muc1 was not expressed in the caecum and there were no changes in mucin expression in response to infection. Proximal large intestine. In the proximal large intestine the two distinct crypt and surface lineages of goblet cells were both strongly AB positive and contained Muc2, whereas Muc4 was present in the goblet cells on the surface, but to a lesser degree in the crypts (Figure 4). The enterocytes and their apical surfaces were both weakly positive for Muc4 and more strongly positive for Muc13, whereas Muc17 was restricted to the apical surface of the surface epithelium, and Muc1 was not expressed (Figure 4). Although no statistically significant differences in mucin expression were detected between non-infected and C. rodentium infected proximal colon, the Muc2/AB filled goblet cell thecae had a higher variability in size in the infected animals with groups of crypts being either hypertrophic and bulging, whilst other groups of crypts were partially depleted of mucin (Figure 4). This suggests that both Muc2 production and secretion is increased in the proximal large intestine of infected mice. Saponification prior to mild PAS staining to reveal O-acetylated mucin oligosaccharides revealed O-acetylation in a proportion of the goblet cell lineage found at the base of the crypts of the proximal and mid-colon, as previously reported [44]. Post-infection the loss of goblet cells was reflected in reduced overall mild PAS staining, however, in the remaining cells of the basal goblet cell lineage there was an increase in mild PAS staining without prior saponification, indicating a reduction of O-acetylation in those cells following infection (Figure S1). Mid and distal large intestine. In the mid and distal large intestine, the upper half of the crypts contain the majority of the goblet cells, whereas the lower half of the crypts contain cells of secretory type but with a more vacuolated morphology [45]. AB/ PAS staining of the goblet cells revealed medium charged carbohydrates from the middle of the crypt to the top, whereas the bottom of the crypts had a small amount of highly charged carbohydrate chains (Figures 5 and 6). As for the other regions of the intestine these goblet cells expressed Muc2 and to a lesser extent Muc4. In the mid- and distal colon, the apical surface of the lower half of the crypt was weakly positive for Muc1 (Figures 5–6 and Figure S2, intensity score 0–0.5). Muc13 and Muc17 were highly expressed in the cytoplasm of the cells with secretory vacuolated morphology in lower half of the crypt and also expressed on the apical surface of cells in the crypts and the surface epithelium (Figure 6). Muc17 was also present cytoplasmically in the bottom half of the crypt (Figures 5–6). Thus, the tissue localization of Muc17 is similar to that of Muc13 in the mid and distal large intestine, whereas differences occur in the more proximal parts of the intestine. C. rodentium caused focal damage and elongation of crypts in the mid and distal colon, with patches of almost normal epithelium PLoS ONE | www.plosone.org 7 December 2008 | Volume 3 | Issue 12 | e3952