Modeling IBD in the mouse through random germline mutagenesis

Abstract

Inflammatory bowel diseases (IBD), including both ulcerative colitis and Crohn's disease, have strong genetic components. In mice, several mutations are known to favor IBD or to inhibit intestinal inflammation. But a comprehensive picture of the pathogenesis of IBD cannot be assembled based on the limited information so far available from genetic analyses. To unveil new genes important for the pathogenesis of IBD, we took an unbiased, hypothesis-free view of IBD, using germline mutagenesis to analyze the phenomenon in mice. We screen for mutations that cause susceptibility to IBD on a defined genetic background using the Dextran sodium sulfate (DSS) colitis model, positionally clone these mutations, and form hypotheses concerning the participation of each validated gene in the disease pathogenesis. A total of 5846 mice have been screened by placing them on 1% DSS in drinking water for a period of 7 days. During this time period all animals are weighed daily, and extreme outliers are retrieved for further study. A total of 16 transmissible mutations causing hypersensitivity to DSS have been detected. Of these, 6 have been identified, and fall into 5 genes. Failure of epithelial integrity is sensed by Toll-like receptors (TLR) on epithelial cells. Wound closure also additionally requires water transport via aquaporin 3, signaling via the EGF receptor pathway, and an intact unfolded protein response, which depends on Mbtps1, also known as the site-1 protease. Relatively common mutations affecting the structure of Mucin-2 also enhance susceptibility to colitis, evidently by increasing ER stress. In addition, genes with unknown functions have been identified causing increased susceptibility to DSS colitis. One of the mutant mice identified in this screen was homozygous for a mutation in an uncharacterized gene. The gene is predicted to encode a protein with an unstructured cytoplasmic N-terminal domain and a C-terminal domain containing five transmembrane α helices connected by short loops. The mutation lies in the splice acceptor site of intron 3 of the seven exon gene, and affects a thymine base five nucleotides from the next exon. cDNA sequencing revealed that the mutation results in skipping of exon 4. Splicing of exon 3 to exon 5 creates a frameshift and a premature stop codon. The Klein-Zschocher mouse showed severe weight loss after administration of 1% DSS in the drinking water. Periodic acid-Schiff stain staining of small intestines and colons of Klein-Zschocher mice revealed reduced numbers of Paneth and goblet cells in naïve mutant mice. Furthermore, electron microscopy of the small intestine and colon of naive Klein-Zschocher mice showed abnormal vesicles in Paneth and goblet cells of the mutant mice. Defective Paneth cells of Klein-Zschocher mutant mice show enhanced autophagy as demonstrated by LC3 Western blotting. In addition, the intestinal permeability was increased 85% in Klein-Zschocher mice demonstrating epithelial barrier dysfunction in these mice. Disturbed epithelial integrity together with abnormal Paneth and goblet cells explain the increased susceptibility in the DSS colitis model. Taken together, using random mutagenesis and the DSS mouse model we found new genes and new functions of known genes that might play a role in IBD. A coherent picture of the essential events required for homeostasis has begun to emerge.