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Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis.

Heazlewood CK, Cook MC, Eri R, Price GR, Tauro SB, Taupin D, Thornton DJ, Png CW, Crockford TL, Cornall RJ, Adams R, Kato M, Nelms KA, Hong NA, Florin TH, Goodnow CC, McGuckin MA - PLoS Med. (2008)

Bottom Line: The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure.This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair.Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress.

View Article: PubMed Central - PubMed

Affiliation: Mucin and IBD Research Teams, Mucosal Diseases Program, Mater Medical Research Institute, and the University of Queensland, Aubigny Place, Mater Health Services, South Brisbane, Queensland, Australia.

ABSTRACT

Background: MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis.

Methods and findings: By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p < 0.01) and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1beta, TNF-alpha, and IFN-gamma was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-gamma, TNF-alpha, and IL-13). This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar accumulation of nonglycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in noninflamed intestinal tissue. Although our study demonstrates that mucin misfolding and ER stress initiate colitis in mice, it does not ascertain the genetic or environmental drivers of ER stress in human colitis.

Conclusions: Characterisation of the mouse models we created and comparison with human disease suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of human colitis and that clinical studies combining genetics, ER stress-related pathology and relevant environmental epidemiology are warranted.

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Related in: MedlinePlus

Spontaneous Colitis in Mice with Muc2 Mutations(A) Incidence of premature death or colitis-associated pathology requiring humane killing (chiefly rectal prolapse) in Winnie (n = 309) and Eeyore (n = 355) mice. Mice entering experiments or killed for other reasons were treated as censored observations (designated by upward ticks), and the number of uncensored mice remaining at 50 d intervals is included underneath the graph. Incidence rates in the two strains were compared using the Mantel Log-rank test.(B) Weight of the colon after removal of luminal faecal material in C57BL/6 (WT), Winnie (Win), and Eeyore (Eey) mice at 6 (Eey 6–9 wk), 12, and 18 (WT and Win only) wk of age, n = 4–9; box plots show median, quartiles, and range.(C) Histological colitis scores (see Materials and Methods) in WT and Win mice at 6, 12, and 18 wk of age, n = 4–6; scores from individual mice are shown.(D–K) Histology of normal distal colon from a C57BL/6 mouse (D and E) and examples of inflammation in the rectum (F–I) and distal large intestine (J and K) of untreated Winnie mice showing leukocytic infiltration (G and I), occasional branching crypts (F and H), crypt abscesses (J and K) and focal ulcerations (I); scale bars = 20 μm. Note the layer covering the mucosal surface in (F) is a granulocytic serous exudate.Statistics (B and C): p-values for Kruskal-Wallis nonparametric analysis are shown, Dunn's multiple comparison test versus wild type, ** p < 0.01, *** p < 0.001; versus Win at 6 wk, + p < 0.05; versus Eey at 6 wk, # p < 0.05.
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pmed-0050054-g003: Spontaneous Colitis in Mice with Muc2 Mutations(A) Incidence of premature death or colitis-associated pathology requiring humane killing (chiefly rectal prolapse) in Winnie (n = 309) and Eeyore (n = 355) mice. Mice entering experiments or killed for other reasons were treated as censored observations (designated by upward ticks), and the number of uncensored mice remaining at 50 d intervals is included underneath the graph. Incidence rates in the two strains were compared using the Mantel Log-rank test.(B) Weight of the colon after removal of luminal faecal material in C57BL/6 (WT), Winnie (Win), and Eeyore (Eey) mice at 6 (Eey 6–9 wk), 12, and 18 (WT and Win only) wk of age, n = 4–9; box plots show median, quartiles, and range.(C) Histological colitis scores (see Materials and Methods) in WT and Win mice at 6, 12, and 18 wk of age, n = 4–6; scores from individual mice are shown.(D–K) Histology of normal distal colon from a C57BL/6 mouse (D and E) and examples of inflammation in the rectum (F–I) and distal large intestine (J and K) of untreated Winnie mice showing leukocytic infiltration (G and I), occasional branching crypts (F and H), crypt abscesses (J and K) and focal ulcerations (I); scale bars = 20 μm. Note the layer covering the mucosal surface in (F) is a granulocytic serous exudate.Statistics (B and C): p-values for Kruskal-Wallis nonparametric analysis are shown, Dunn's multiple comparison test versus wild type, ** p < 0.01, *** p < 0.001; versus Win at 6 wk, + p < 0.05; versus Eey at 6 wk, # p < 0.05.

Mentions: The progressive incidence of rectal prolapse and colitis-associated mortality in Winnie and Eeyore mice is shown in Figure 3A. At 1 y approximately 40% of Eeyore and 25% of Winnie mice had died or were humanely killed due to the development of rectal prolapse or debility (based on a multifactorial scoring system, see Materials and Methods), whereas no wild-type littermates developed rectal prolapses. Proximal and distal colon were thickened and colon weight was greater in Winnie and Eeyore than in wild-type mice, and the thickening and weight increased progressively from 6 to 18 wk of age (Figure 3B). Despite its thickening, the colon was not shortened in mutant mice (Figure S2). Colitis was assessed histologically in Winnie mice at 6, 12, and 18 wk of age, revealing mild inflammation in the large intestine (Figure 3C). The inflammatory infiltrate was usually mild and did not become more severe with age. Classical signs of murine colitis, including crypt elongation, neutrophilic infiltrates, goblet cell loss, crypt abscesses, and focal epithelial erosions were present, particularly in the distal large intestine (examples shown in Figure 3F–3K).


Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis.

Heazlewood CK, Cook MC, Eri R, Price GR, Tauro SB, Taupin D, Thornton DJ, Png CW, Crockford TL, Cornall RJ, Adams R, Kato M, Nelms KA, Hong NA, Florin TH, Goodnow CC, McGuckin MA - PLoS Med. (2008)

Spontaneous Colitis in Mice with Muc2 Mutations(A) Incidence of premature death or colitis-associated pathology requiring humane killing (chiefly rectal prolapse) in Winnie (n = 309) and Eeyore (n = 355) mice. Mice entering experiments or killed for other reasons were treated as censored observations (designated by upward ticks), and the number of uncensored mice remaining at 50 d intervals is included underneath the graph. Incidence rates in the two strains were compared using the Mantel Log-rank test.(B) Weight of the colon after removal of luminal faecal material in C57BL/6 (WT), Winnie (Win), and Eeyore (Eey) mice at 6 (Eey 6–9 wk), 12, and 18 (WT and Win only) wk of age, n = 4–9; box plots show median, quartiles, and range.(C) Histological colitis scores (see Materials and Methods) in WT and Win mice at 6, 12, and 18 wk of age, n = 4–6; scores from individual mice are shown.(D–K) Histology of normal distal colon from a C57BL/6 mouse (D and E) and examples of inflammation in the rectum (F–I) and distal large intestine (J and K) of untreated Winnie mice showing leukocytic infiltration (G and I), occasional branching crypts (F and H), crypt abscesses (J and K) and focal ulcerations (I); scale bars = 20 μm. Note the layer covering the mucosal surface in (F) is a granulocytic serous exudate.Statistics (B and C): p-values for Kruskal-Wallis nonparametric analysis are shown, Dunn's multiple comparison test versus wild type, ** p < 0.01, *** p < 0.001; versus Win at 6 wk, + p < 0.05; versus Eey at 6 wk, # p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2270292&req=5

pmed-0050054-g003: Spontaneous Colitis in Mice with Muc2 Mutations(A) Incidence of premature death or colitis-associated pathology requiring humane killing (chiefly rectal prolapse) in Winnie (n = 309) and Eeyore (n = 355) mice. Mice entering experiments or killed for other reasons were treated as censored observations (designated by upward ticks), and the number of uncensored mice remaining at 50 d intervals is included underneath the graph. Incidence rates in the two strains were compared using the Mantel Log-rank test.(B) Weight of the colon after removal of luminal faecal material in C57BL/6 (WT), Winnie (Win), and Eeyore (Eey) mice at 6 (Eey 6–9 wk), 12, and 18 (WT and Win only) wk of age, n = 4–9; box plots show median, quartiles, and range.(C) Histological colitis scores (see Materials and Methods) in WT and Win mice at 6, 12, and 18 wk of age, n = 4–6; scores from individual mice are shown.(D–K) Histology of normal distal colon from a C57BL/6 mouse (D and E) and examples of inflammation in the rectum (F–I) and distal large intestine (J and K) of untreated Winnie mice showing leukocytic infiltration (G and I), occasional branching crypts (F and H), crypt abscesses (J and K) and focal ulcerations (I); scale bars = 20 μm. Note the layer covering the mucosal surface in (F) is a granulocytic serous exudate.Statistics (B and C): p-values for Kruskal-Wallis nonparametric analysis are shown, Dunn's multiple comparison test versus wild type, ** p < 0.01, *** p < 0.001; versus Win at 6 wk, + p < 0.05; versus Eey at 6 wk, # p < 0.05.
Mentions: The progressive incidence of rectal prolapse and colitis-associated mortality in Winnie and Eeyore mice is shown in Figure 3A. At 1 y approximately 40% of Eeyore and 25% of Winnie mice had died or were humanely killed due to the development of rectal prolapse or debility (based on a multifactorial scoring system, see Materials and Methods), whereas no wild-type littermates developed rectal prolapses. Proximal and distal colon were thickened and colon weight was greater in Winnie and Eeyore than in wild-type mice, and the thickening and weight increased progressively from 6 to 18 wk of age (Figure 3B). Despite its thickening, the colon was not shortened in mutant mice (Figure S2). Colitis was assessed histologically in Winnie mice at 6, 12, and 18 wk of age, revealing mild inflammation in the large intestine (Figure 3C). The inflammatory infiltrate was usually mild and did not become more severe with age. Classical signs of murine colitis, including crypt elongation, neutrophilic infiltrates, goblet cell loss, crypt abscesses, and focal epithelial erosions were present, particularly in the distal large intestine (examples shown in Figure 3F–3K).

Bottom Line: The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure.This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair.Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress.

View Article: PubMed Central - PubMed

Affiliation: Mucin and IBD Research Teams, Mucosal Diseases Program, Mater Medical Research Institute, and the University of Queensland, Aubigny Place, Mater Health Services, South Brisbane, Queensland, Australia.

ABSTRACT

Background: MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis.

Methods and findings: By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p < 0.01) and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1beta, TNF-alpha, and IFN-gamma was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-gamma, TNF-alpha, and IL-13). This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar accumulation of nonglycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in noninflamed intestinal tissue. Although our study demonstrates that mucin misfolding and ER stress initiate colitis in mice, it does not ascertain the genetic or environmental drivers of ER stress in human colitis.

Conclusions: Characterisation of the mouse models we created and comparison with human disease suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of human colitis and that clinical studies combining genetics, ER stress-related pathology and relevant environmental epidemiology are warranted.

Show MeSH
Related in: MedlinePlus