Date Published: March 4, 2008
Publisher: Public Library of Science
Author(s): Chad K Heazlewood, Matthew C Cook, Rajaraman Eri, Gareth R Price, Sharyn B Tauro, Douglas Taupin, David J Thornton, Chin Wen Png, Tanya L Crockford, Richard J Cornall, Rachel Adams, Masato Kato, Keats A Nelms, Nancy A Hong, Timothy H. J Florin, Christopher C Goodnow, Michael A McGuckin, Stefan Schreiber
Abstract: BackgroundMUC2 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 FindingsBy 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-1β, TNF-α, and IFN-γ 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-γ, TNF-α, 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.ConclusionsCharacterisation 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.
Partial Text: Intestinal goblet cells produce the viscous mucus layer covering the intestinal epithelium. In the large intestine mucus completely fills the crypts and forms a thick coating over the mucosal surface. MUC2 mucin is the major macromolecular constituent of intestinal mucus, and is responsible for its high viscosity and forming a protease-resistant matrix that retains molecules vital to host defence. MUC2 contains a large central O-glycosylated domain and N- and C-terminal cysteine-rich domains that homo-oligomerise , forming a complex molecular lattice [2,3]. MUC2 is N-glycosylated in the ER, where initial homo-oligomerisation occurs , and the complex then moves into the Golgi apparatus where O-glycosylation, the final stages of oligomerisation, and packaging into granules occur . Mucins stored intracytoplasmically in granules form the characteristic goblet cell theca from where they are secreted constitutively and in response to stimuli.
This study demonstrates that the intestinal secreted mucin is prone to misfolding/aberrant assembly and that misfolding causes substantial ER stress, morphological goblet cell pathology, premature goblet cell apoptosis, and the development of chronic intestinal inflammation. We have described two strains of mice with ENU-induced allelic variants of Muc2 and phenotypes that closely model human colitis. To our knowledge Winnie and Eeyore are the first animal models of spontaneous intestinal inflammation arising due to single missense mutations in any gene. Their phenotype is characterized by diarrhoea, decreased goblet cell number, smaller goblet cell thecae, accumulation of Muc2 precursor in ER vacuoles within the goblet cell cytoplasm, increased epithelial cell proliferation and apoptosis, depletion of the secreted mucus layer, and spontaneous inflammation. The resulting pattern of inflammation with intestinal production of IL-1β and TNF-α, and the production of both Th1 (TNF-α and IFN-γ) and Th2 (IL-13) cytokines by MLN leukocytes, resembles the complex and somewhat inconsistent patterns of cytokine production described in UC [44–52]. The IL-13 could be sourced from natural killer T cells, which have been shown to produce IL-13 in UC , and in oxazolone-induced colitis in mice, which is considered a UC-like model , or from natural killer cells, which are known to produce IL-13 in the intestine during some infections . The increased intestinal permeability and increased coating of luminal bacteria with immunoglobulin also mirror the IBD phenotype [24,55].