Importance and regulation of the colonic mucus barrier in a mouse model of colitis

Abstract

The colonic mucus layer serves as an important barrier and prevents colonic bacteria from invading the mucosa and cause inflammation. The regulation of colonic mucus secretion is poorly understood. The aim of this study was to investigate the role of the mucus barrier in induction of colitis. Furthermore, regulation of mucus secretion by luminal bacterial products was studied. The colon of anesthetized Muc2(-/-), Muc1(-/-), wild-type (wt), and germ-free mice was exteriorized, the mucosal surface was visualized, and mucus thickness was measured with micropipettes. Colitis was induced by DSS (dextran sodium sulfate, 3%, in drinking water), and disease activity index (DAI) was assessed daily. The colonic mucosa of germ-free and conventionally housed mice was exposed to the bacterial products LPS (lipopolysaccharide) and PGN (peptidoglycan). After DSS induction of colitis, the thickness of the firmly adherent mucus layer was significantly thinner after 5 days and onward, which paralleled the increment of DAI. Muc2(-/-) mice, which lacked firmly adherent mucus, were predisposed to colitis, whereas Muc1(-/-) mice were protected with significantly lower DAI by DSS compared with wt mice. The mucus barrier increased in Muc1(-/-) mice in response to DSS, whereas significantly fewer T cells were recruited to the inflamed colon. Mice housed under germ-free conditions had an extremely thin adherent colonic mucus layer, but when exposed to bacterial products (PGN or LPS) the thickness of the adherent mucus layer was quickly restored to levels observed in conventionally housed mice. This study demonstrates a correlation between decreasing mucus barrier and increasing clinical symptoms during onset of colitis. Mice lacking colonic mucus (Muc2(-/-)) were hypersensitive to DSS-induced colitis, whereas Muc1(-/-) were protected, probably through the ability to increase the mucus barrier but also by decreased T cell recruitment to the afflicted site. Furthermore, the ability of bacteria to regulate the thickness of the colonic mucus was demonstrated.

Fig. 1.

The adherent mucus layer in wild-type mice decreased whereas disease activity index increased with days on dextran sulfate sodium (DSS). A: adherent mucus thickness (μm) in wild-type (C57Bl/6) mice without (untreated, n = 11) or with 3% DSS addition to the drinking water for 6–8 days (n = 9). B: disease activity index of the mice treated with DSS (solid lines) and the decreasing mucus thickness at different time points during DSS treatment (open bars) (n = 27). C and D: histological tissue sections stained with hematoxylin-eosin from untreated and DSS-treated C57Bl/6 mice, respectively. *P < 0.05; bars correspond to 100 μm.

Fig. 2.

Muc2-deficient mice lack adherent colonic mucus and develop a more severe colitis. Disease activity index in wild-type (wt; n = 8) and Muc2-deficient mice (n = 5) before (A) and 2–4 days following DSS treatment (B). C: adherent mucus thickness (μm) of untreated (n = 9) and DSS-treated Muc2-deficient mice (n = 5). Please note that the DSS treatment had to be interrupted already after 2–4 days because of severe rectal bleeding and weight loss in Muc2-deficient mice. D and E: histological samples stained with periodic acid-Schiff (PAS) from untreated and DSS-treated Muc2-deficient mice, respectively. F: bacterial penetrations estimated by bacterial culturing of spleens isolated from DSS-treated wt (n = 2) or Muc2^−/− mice (n = 2). *P < 0.05; bars correspond to 100 μm.

Fig. 3.

Muc1-deficient mice are partly protected against DSS colitis by increased adherent mucus thickness and decreased T cell infiltration. A: adherent colonic mucus thickness (μm) in Muc1-deficient mice with (n = 7) or without (n = 7) DSS treatment for 6–8 days. B: disease activity index in wild-type (n = 9) or Muc1-deficient mice (n = 7) after DSS treatment. *P < 0.05. C and D: representative histological samples stained with hematoxylin-eosin from Muc1-deficient mice with or with DSS treatment. E: immunohistological staining with an anti-CD3 mAb (as indicated by arrows) and quantification of infiltrated T cells in the DSS-treated wild-type (n = 3) or Muc1-deficient (n = 5) colonic mucosa (number of infiltrated T cells per mm² colonic mucosa). Significantly fewer T cells were observed in the mucosa of Muc1^−/− mice. Bars correspond to 100 μm.

Fig. 4.

Germ-free mice lack firmly adherent colonic mucus, but this mucus layer is restored by bacterial products. A and B: thickness of the adherent mucus in the colon in NMRI mice housed conventionally (n = 9) or under germ-free conditions (n = 11) either with luminal saline or the bacterial products LPS (conventional; n = 5, germ-free; n = 7) or peptidoglycan (PGN; conventional; n = 4, germ-free; n = 4). The mucus thickness is expressed in μm; *P < 0.05. C and D: representative histological samples from a conventional and a germ-free mouse stained with PAS solution. Arrows point at PAS-stained mucins. Bars correspond to 100 μm.