Mucins in Infectious Colitis Figure 5. Tissue localization of mucins in the mid large intestine. The apical surface of the mid large intestinal epithelium and crypts are lined by the cell surface mucins Muc13 and Muc17 as well as a small amount of Muc4 (brown). The goblet cells contain Alcian blue positive material (blue = highly charged carbohydrate structures, purple = medium charged carbohydrate structures), which mainly is Muc2 (brown) and Muc4. The photographs were taken using a 206magnification. The quantification scores for Muc1, and Muc13 were significantly different between non-infected mice (#) and mice infected with C. rodentium for 12 days (N) (* p,0.05, 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.g005 interspersed with the more inflamed elongated crypts. In infected animals, the distal colonic crypts were depleted of AB, Muc2 and Muc4 positive material (p,0.05 for AB/PAS, Figure 6). In some areas the goblet cell thecae were depleted from the base of crypts but some goblet cell thecae remained at the top of crypts, and there was a massive amount of secreted mucus and shed goblet cells (see arrow in Figure 5) in the lumen. The mid large intestine also tended to be depleted of stored Alcian blue positive material at the bottom of the crypt and partially of the purple goblets from mid length to top (Figure 6), although the variability between animals was larger in this location. The more elongated crypts tended to be more depleted of mucins linking goblet cell depletion with localized infection and/or inflammation. In the C. rodentium infected animals the apical Muc1 expression in the lower half of the crypt was increased in the mid and distal colon, and de novo expression occurred apically on the surface epithelium in 3/4 animals (p,0.05, Figures 5–6 and Supplemen- tary Figure 1). Although the apical Muc13 expression was similar to that of non-infected animals, the cytoplasmic expression was significantly decreased (Figures 5–6), consistent with increased apical turn-over of this cell surface mucin. Similarly, Muc17 levels in the cytoplasm of the crypt base also decreased in the mid and distal large intestine with infection and the apical expression in the surface epithelium also decreased (Figures 5–6). C. rodentium binding to Muc2 In the healthy intestine, Muc2 is the main secreted mucin composing the mucus layer, which is continuously washing the intestinal surface to clear trapped material. Using a microtiter based assay, we demonstrated that murine Muc2 can bind to C. rodentium (Figure 7A). Although it cannot be excluded that the insoluble Muc2 complex used in this study could contain small amounts of contamination with other reduction sensitive poly- meric complexes (for example other mucins), large amounts of bacteria were found in the secreted Muc2 in C. rodentium infected animals (Figure 7B–D), consistent with Muc2 binding to C. rodentium. Salmonella St Paul, Campylobacter jejuni and Clostridium difficile induce MUC1 in human distal colon during infection Distal colonic biopsies from four patients infected with S. St Paul or C. jejuni were positive for MUC1, whereas the age, sex and site matched control samples had no or very little detectable MUC1 (p,0.05, Figure 8A and B). Similarly, the MUC1 expression in distal colonic biopsies from two IBD patients with acute C. difficile had higher levels of MUC1 compared to biopsies from the same site in the absence of C. difficile infection, although low levels of MUC1 were still present in the IBD control biopsies (Figure 8C). No MUC1 was demonstrated in one IBD patient infected with Salmonella enteritidis. Thus, MUC1 is induced in response to infection by a range of different intestinal pathogens in humans, in a similar manner to the induction we demonstrated experi- mentally in C. rodentium infection in mice. Discussion This work demonstrates that six mucins have distinctly different expression patterns throughout the murine intestinal tract, indicating that they are differentially regulated and are likely to make different functional contributions to the mucosal barrier. The cellular content of most mucins changes in response to infection, with Muc1 being the only mucin to be induced whereas all other mucins were depleted, albeit to differing degrees. As mucins are strategically positioned between the vulnerable mucosa and the bacterial contents of the intestinal lumen [46], the changes in mucin concentrations and the consequent changes in mucin binding to bacteria are likely to affect both host-commensal and host-pathogen interactions. Muc1/MUC1 appears to play a significant role in host defense in acute infection. In the murine infection model of C. jejuni, Muc1 is upregulated acutely in the intestine in response to infection [6]. We now show that C. rodentium infection also induces Muc1 in mouse colon, and similarly that S. St Paul, C. jejuni and C. difficile induced MUC1 in human colon during infection. Thus, Muc1/ MUC1 is induced in response to infection by a range of different pathogens in both mice and humans. As Muc12/2 mice have a higher rate of systemic C. jejuni infection [6], Muc1/MUC1 induction appears to be an effective host defense used by both human and mouse innate immunity. MUC1 expression in biopsies from inflammatory bowel disease patients with chronic inflamma- tion was also higher during acute infection compared to the inflammatory bowel disease control biopsies. This indicates that MUC1 is upregulated in response to acute infection and that chronic inflammation by itself is not sufficient to substantially upregulate MUC1. Previous studies have shown that mucins bind to several pathogens [6,22,23] and we show here that mucins bind to C. rodentium. Increased secretion of mucin in response to infection has been demonstrated using in vitro airway epithelial cells co-cultured with Burkholderia cenocepacia and Listeria monocytogenes inoculated rat ileal loops [47,48]. The depletion in the amount of Alcian blue positive mucus material as well as Muc2, Muc4, Muc13 and Muc17 we detected here is also likely to occur as a consequence of increased secretion and therefore be part of the host defence. In a recently published study, Muc2 depletion in C. rodentium infected mice was shown to be dependent on T- and B-cells [49]. However, for such a host defence to be effective the mucus must be replenished. In our infected specimens we saw large amounts of shed whole cells, including goblet cells (Figure 5). The factors causing premature death of the goblet cells with infection are unknown, although it appears that C. rodentium directly interact with and infect murine goblet cells during infection [49] and may therefore result in goblet cell death. Shedding of these cells would delay replenishment of the mucus layer providing an advantage to the mucosal pathogen. In addition, several pathogens have been shown to specifically inhibit mucin synthesis [50,51]. A further possibility is that the goblet cells experience endoplasmic reticulum stress due to the enormous amount of protein synthesis that they effect during infection and that the consequent unfolded protein PLoS ONE | www.plosone.org 9 December 2008 | Volume 3 | Issue 12 | e3952