A Specific Mutation in Muc2 Determines Early Dysbiosis in Colitis-Prone Winnie Mice

Abstract

Background: Inflammatory bowel disease (IBD), including Crohn disease (CD) and ulcerative colitis (UC), is a multifactorial disorder characterized by chronic inflammation and altered gut barrier function. Dysbiosis, a condition defined by dysregulation of the gut microbiome, has been reported in patients with IBD and in experimental models of colitis. Although several factors have been implicated in directly affecting gut microbial composition, the genetic determinants impacting intestinal dysbiosis in IBD remain relatively unknown.

Methods: We compared the microbiome of normal, uninflamed wild-type (WT) mice with that of a murine model of UC (ie, Winnie strain). Winnie mice possess a missense mutation in Muc2 that manifests in altered mucus production as early as 4 weeks of age, with ensuing colonic inflammation. To better address the potential role of mutant Muc2 in promoting dysbiosis in Winnie mice, we evaluated homozygous mutant mice (Winnie-/-) with their WT littermates that, after weaning from common mothers, were caged separately according to genotype. Histologic and inflammatory status were assessed over time, along with changes in their respective microbiome compositions.

Results: Dysbiosis in Winnie mice was already established at 4 weeks of age, before histologic evidence of gut inflammatory changes, in which microbial communities diverged from that derived from their mothers. Furthermore, dysbiosis persisted until 12 weeks of age, with peak differences in microbiome composition observed between Winnie and WT mice at 8 weeks of age. The relative abundance of Bacteroidetes was greater in Winnie compared with WT mice. Verrucomicrobia was detected at the highest relative levels in 4-week-old Winnie mice; in particular, Akkermansia muciniphila was among the most abundant species found at 4 weeks of age.

Conclusions: Our results demonstrate that mutant genetic determinants involved in the complex regulation of intestinal homeostasis, such as that observed in Winnie mice, are able to promote early gut dysbiosis that is independent from maternal microbial transfer, including breastfeeding. Our data provide evidence for intestinal dysbiosis attributed to a Muc2-driven mucus defect that leads to colonic inflammation and may represent an important target for the design of future interventional studies.

Keywords: dysbiosis; inflammatory bowel disease (IBD); microbiome; murine model of ulcerative colitis (UC).

FIGURE 1.

Total body weight of Winnie mice is consistently decreased compared with age- and sex-matched WT mice. Weight of Winnie and age-matched WT mice from 4 to 16 weeks in male (A) and female (B) mice. Inset graphs (right panels) depict differences in individual mice, comparing 16-week-old WT (●) and Winnie (▪) mice. ^***P < 0.001; ^**P < 0.01 using unpaired 2-tailed Student t tests.

FIGURE 2.

Aberrant morphologic and histologic features in colons from Winnie vs WT mice. Representative images of whole colons (A) and measurements of colon length (B) and colon weight (C) of 16-week-old Winnie and WT male mice. D and E, Colon length/mouse weight and colon weight/mice weight indices, respectively. F, Hematoxylin and eosin staining of distal colon sections shows abundant mucus-secreting goblet cells with moderate inflammation only in 16-week-old Winnie mice and evident signs of erosion within the intestinal epithelium compared with WT controls. Scale bar = 100 µm. No signs of inflammation are present in 4- and 8-week-old mice. ^***P < 0.001; *P < 0.05 (Student t test).

FIGURE 3.

Increased immune cell infiltration within the lamina propria of Winnie compared with WT mice. A, Representative density plot for LP cells, with analyses performed on viable cells by excluding the 7-AAD⁺ population. Graphs (right panels) represent percentages of CD45.2⁺ MHC II⁺ (i) and CD45.2⁺ MHC II^- (ii) cells in WT and Winnie mice. B, Relative expression of Cd45 in colons of Winnie and WT mice. Bars represent mean relative expression ± SEM (n = 3) for each genotype. *P < 0.05 (Student t test).

FIGURE 4.

Changes in bacterial composition occur as early as 4 weeks in Winnie compared with WT mice. A, Average relative abundance (%) at 4, 8, and 16 weeks of age. The group “other” encompasses all phyla with relative abundance <0.01%. B, Values of relative abundance (%). Significance was calculated comparing bacterial abundance in Winnie vs WT at the respective time points. ^***P < 0.001; ^**P < 0.01; *P < 0.05 (Student t test).

FIGURE 5.

Winnie mice differ in the profile of genera compared with WT mice. Relative abundance (%) of total (16S rRNA) bacteria, differentially found (P < 0.05) at the genus level in fecal samples of WT and Winnie (W) mice at 4, 8, and 16 weeks of age.

FIGURE 6.

Winnie mice differ in the profile of species compared with WT mice. Relative abundance (%) of total (16S rRNA) bacteria, differentially found (P < 0.05) at the species level in fecal samples of Winnie (W) and WT mice at 4, 8, and 16 weeks of age.