To further evaluate bacterial penetration of the inner mucus and the closeness of bacteria to the epithelial STA-9090 cells, a scoring system from 0�C5 was used. Here 0 means no bacteria penetrating the inner mucus layer and 5 means a large number of bacteria in direct contact with the epithelium. This scoring system is explained and exemplified in Fig. S1. Using this system, blinded tissue sections were evaluated by two independent examiners and the average score is presented in Fig. 5A. A high score of 4 representing a large number of bacteria penetrating down to the epithelium, was reached already after 12 h. During the following 12 h there was a transient decline in the score value. This variation could be due to the murine diurnal rhythm of activity and drinking.

To evaluate this possibility, the DSS concentration was measured in mucosal scrapings from mouse colon exposed to DSS. The scrapings were extracted in guanidinium chloride and analyzed by AgPAGE for large molecules (Fig. 5B). The guanidinium chloride insoluble Alcian blue stained Muc2 band showed no alterations (data not shown). The guanidinium chlorides soluble fraction contained the DSS that had accumulated in the distal colon in addition to some Muc2 (marked M). Fig. 5B show high amounts of DSS at 12 h, 36 h and 120 h, but low levels at 18 h and 24 h. Thus there is a direct co-variation between a high bacterial penetration score and high relative amounts of DSS in the mucus. This suggests a direct relation between the DSS amounts in the colon mucus and bacterial penetration of the inner mucus layer.

These results further confirm that the DSS effect is fast and direct just as observed for the DSS treated mucus on the tissue explants. The in vivo experiments also suggest that the effect is reversible, at least at the standard early time points for evaluating DSS treatment. Figure 5 High number of bacteria penetrating the inner mucus layer co-varies with high amounts of DSS in the colon mucus. DSS and bacteria are in contact with the epithelium after 12 h of DSS exposure In the explant system the DSS was able Brefeldin_A to diffuse into the firm mucus and when tested in vivo FITC labeled DSS was observed at the epithelial surface. The FITC conjugated DSS was given to the mice in the drinking water and already after 12 h a substantial amount had reached down to the epithelial cells (Fig. 6A). The penetration of bacteria into the mucus is thus simultaneous with DSS penetration into the mucus, suggesting that DSS alter the mucus properties in such a way that it allows bacteria to penetrate the otherwise impermable mucus. As shown in Fig. 6B an enormous bacterial load is observed on the epithelial surface already after 12 h DSS treatment.