Vitamin B12 Deficiency Alters the Gut Microbiota in a Murine Model of Colitis

Abstract

Purpose: Inflammatory bowel disease (IBD) refers to a spectrum of autoimmune diseases, which result in chronic intestinal inflammation. Previous findings suggest a role for diet, nutrition and dysbiosis of the gut microbiota in both the development and progression of the condition. Vitamin B12 is a key cofactor of methionine synthase and is produced solely by microbes. Previous work links increased levels of homocysteine, a substrate of methionine synthase, MetH, to IBD indicating a potential role for vitamin B12 deficiency in intestinal injury and inflammation. This study assessed the role of vitamin B12 in shaping the gut microbiota and determining responses to intestinal injury using a reproducible murine model of colitis. Methods: The effects of vitamin B12 supplementation and deficiency were assessed in vivo; 3-week-old post-weanling C57Bl/6 mice were divided into three dietary treatment groups: (1) sufficient vitamin B12 (50 mg/Kg), (2) deficient vitamin B12 (0 mg/Kg) and (3) supplemented vitamin B12 (200 mg/Kg) for a period of 4 weeks. Intestinal injury was induced with 2% dextran sodium sulphate (DSS) via drinking water for 5 days. The impact of varying levels of dietary vitamin B12 on gut microbiota composition was assessed using 16S rRNA gene sequencing from fecal samples collected at day 0 and day 28 of the dietary intervention, and 7 days following induction of colitis on day 38, when blood and colonic tissues were also collected. Results: No significant alterations were found in the gut microbiota composition of disease-free animals in response to dietary interventions. By contrast, after DSS-induced colitis, >30 genera were significantly altered in vitamin B12 deficient mice. Altered B12 levels produced no significant effect on composite disease-activity scores; however, administration of a B12 deficient diet resulted in reduced DSS-induced epithelial tissue damage. Conclusions: Vitamin B12 supplementation does not alter the gut microbiota composition under healthy conditions, but does contribute to differential microbial responses and intestinal dysbiosis following the induction of experimental colitis.

Keywords: colitis; inflammation; inflammatory bowel disease; microbiome; vitamin B12.

Figure 1

Graphical representation of the experimental design for vitamin B12 administration in a dextran sodium sulphate (DSS)-induced mouse model of colitis. Three-week-old female C57BL/6 mice were provided specific diets containing varying levels of vitamin B12 for 4 weeks. DSS was administered for 5 days followed by 2 days of water alone. Fecal pellets were obtained on days 0, 28, and 38. Blood and colon samples were collected at the time of sacrifice on day 38.

Figure 2

Vitamin B12 levels in serum and the effect of dietary vitamin B12 on weight. (A) Serum B12 levels measured at day 29 pre-DSS treatment and post-DSS treatment on day 38 show significant increased levels with supplementation and decreased levels with deficient diets in both control and DSS mice (P < 0.0001). (B-C) Body weight changes in the three dietary groups from Control (B) and DSS (C) mice (one-way ANOVA, *p < 0.05, n = 5).

Figure 3

Markers of colonic epithelial injury. (A) Representative hematoxylin and eosin staining of colonic sections showing normal morphology in control groups and marked tissue damage. (B) Representative images depicting the presence of Muc2 punctate staining in colonic tissue from control mice and loss of Muc2 staining in animals administered DSS and fed either vitamin B12 sufficient or vitamin B12 supplemented diets. (C) Colon length measurements post-mortem were similar between all diet groups for both control and DSS-induced colitis mice. Significant differences between colon lengths in vitamin B12 deficient and vitamin B12 supplemented mice exposed to DSS were observed (two-way ANOVA, *P < 0.05, **P < 0.01, n = 5), values are expressed as means + SEM.

Figure 4

Vitamin B12 deficiency affects DSS-induced colitis tissue damage. (A) Composite histopathology scores of colonic tissues, graded by tissue damage (TD), inflammatory cell infiltrates (ICI) and loss of goblet cells (GC). (B) Tissue damage score between B12 dietary groups post DSS-treatment, B12 deficient pups show decreased tissue damage (One-way ANOVA, P = 0.021, n = 5). Normalized mRNA fold change expression ΔΔCt, between control mice and DSS treated mice. (C) Anti-inflammatory cytokine IL-10. Significant increase in IL-10 between B12 deficient mice post DSS compared to control (unpaired t-test, n = 3, P = 0.005). (D) Pro-inflammatory TNF-α, increased post DSS in all treatment groups but no significant difference between treatment group was found. Statisticall signigicance represented as *p < 0.05. Values are expressed as means + SEM.

Figure 5

Effects of varying levels of dietary vitamin B12 on fecal microbial diversity and composition. Alpha diversity: Shannon index (A), OTU table rarefied 12,000. No significant differences between dietary groups post 4 weeks of intervention, or post DSS treatment. Chao1 index (B). Significant effects were found for both B12 diet (F_{2, 24} = 5.79, P = 0.009) and diet and time interaction (F_{4, 24} = 5.5, P = 0.002), Two-way ANOVA followed by Tukey's post-hoc test. Beta diversity: Binary Jaccard (C) and Bray-Curtis (D) were analyzed on OTU table rarefied to 12,000 reads. Significant differences were found within dietary intervention groups when comparing four weeks of dietary invention to baseline, however no differences between vitamin B12 diet intervention groups were identified at any time point. PERMANOVA P < 0.05, statistical singnicance represented as *p < 0.05.

Figure 6

(A) Dominant shifts in the top 20 taxa relative abundance at the genus level in response to dietary vitamin B12 treatments and DSS-induced colitis. (B) Hierarchical clustering of top 25 taxa at the genus level at days 0, 28, and for DSS-induced colitis, day 38. Clustering was observed for vitamin B12 supplemented animals at day 28, which was eliminated following DSS-induced colitis. Clustering of the vitamin B12 deficient group was noted following induction of colitis at day 38. Relative abundance of taxa was significantly different between treatment groups at day 38 following DSS-induced colitis (Kruskal Wallis, FDR <0.1) (C) Bacteroides, (D) unclassified member of the Erysipelotrichaceae family, and (E) Mycoplasma.