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, 14 (11), e0225079
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Comparison of the Fecal, Cecal, and Mucus Microbiome in Male and Female Mice After TNBS-induced Colitis

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Comparison of the Fecal, Cecal, and Mucus Microbiome in Male and Female Mice After TNBS-induced Colitis

Ariangela J Kozik et al. PLoS One.

Abstract

Crohn's Disease and Ulcerative Colitis are chronic, inflammatory conditions of the digestive tract, collectively known as Inflammatory Bowel Disease (IBD). The combined influence of lifestyle factors, genetics, and the gut microbiome contribute to IBD pathogenesis. Studies of the gut microbiome have shown significant differences in its composition between healthy individuals and those with IBD. Due to the high inter-individual microbiome variation seen in humans, mouse models of IBD are often used to investigate potential IBD mechanisms and their interplay between host, microbial, and environmental factors. While fecal samples are the predominant material used for microbial community analysis, they may not be the ideal sample to use for analysis of the microbiome of mice with experimental colitis, such as that induced by 2, 4, 6 trinitrobenzesulfonic acid (TNBS). As TNBS is administered intrarectally to induce colitis and inflammation is confined to the colon in this model, we hypothesized that the microbiome of the colonic mucus would most closely correlate with TNBS colitis severity. Based on our previous research, we also hypothesized that sex would be associated with both disease severity and microbial differences in mice with chronic TNBS colitis. We examined and compared the fecal, cecal content, and colonic mucus microbiota of 8-week old male and female C57BL/6J wild-type mice prior to and after the induction of TNBS colitis via 16S rRNA gene sequencing. We found that the colonic mucus microbiome was more closely correlated with disease severity than were alterations in the fecal and cecal microbiomes. We also found that the microbiomes of the feces, cecum, and mucus were distinct, but found no significant differences that were associated with sex in either compartment. Our findings highlight the importance of sampling colonic mucus in TNBS-induced colitis. Moreover, consideration of the differential impact of sex on the microbiome across mouse strains may be critical for the appropriate application of TNBS colitis models and robust comparisons across studies in the future.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1
A) Bar Graph colitis scores in TNBS (n = 18) and SHAM (n = 18) treated mice. ****T-test p<0.0001. Values plotted as Mean ± SEM B) Bar graph of differences in colitis score by sex (female n = 8, male n = 10) in TNBS treated mice. T-test *p<0.05. Values plotted as Mean ± SEM C) Hemotoxilin and eosin stained representative photomicrographs of colon showing a mixed inflammatory cell infiltrate in the mucosa of male TNBS treated animals (#) and necrosis and loss of glands, in addition to mild, mixed inflammation (*) in the mucosa of female TNBS treated mice. 20x magnification. Scale bar 100 microns.
Fig 2
Fig 2
A) Significant differences in beta diversity in the feces of TNBS and SHAM treated mice post-colitis calculated using the Bray Curtis metric. Treatment separates along PC1, which explains approximately 30% of the total variation in the data. Significance found with PERMANOVA p<0.05. PERMDISP indicates dispersion does not contribute to significance. B) Significant differences in beta diversity in the colon mucus of TNBS and SHAM treated mice post-colitis calculated using Weighted UniFrac. Treatment separates along PC1, which explains approximately 76% of the total variation in the data. Significance found with PERMANOVA p<0.05. PERMDISP indicates dispersion does not contribute to significance.
Fig 3
Fig 3. PCoA reveals significant differences in beta diversity by body site, regardless of treatment.
Site separates along PC1 and PC2, explaining 20% and 12.6% of the total variation, respectively. Significance found with PERMANOVA (p<0.05). Permdisp indicates that dispersions does not contribute to significance.
Fig 4
Fig 4. Plots of significantly different taxa across body sites and treatment groups.
Overall significance found using ANCOM, and values were corrected for multiple comparisons using False Discovery Rate. Follow-up pairwise tests were performed using Welch’s t-test. *p<0.05, **p < .01, ***p<0.001, ****p<0.0001.
Fig 5
Fig 5. Canonical Correspondence Analysis on the relative taxon abundances reveals that colitis score is more closely associated with the mucus microbiome than the fecal microbiome.
Feces is closely associated with CCA1, which explains 52.6% of the constrained variation. TNBS treatment and colitis score was associated with CCA2 and CCA3, which explains 23.8% and 15.2% of the constrained variation, respectively. Significance found using ANOVA p = 0.001.

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The author(s) received no specific funding for this work.
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