Timing of Tributyrin Supplementation Differentially Modulates Gastrointestinal Inflammation and Gut Microbial Recolonization Following Murine Ileocecal Resection

Abstract

Background: Gastrointestinal surgery imparts dramatic and lasting imbalances, or dysbiosis, to the composition of finely tuned microbial ecosystems. The aim of the present study was to use a mouse ileocecal resection (ICR) model to determine if tributyrin (TBT) supplementation could prevent the onset of microbial dysbiosis or alternatively enhance the recovery of the gut microbiota and reduce gastrointestinal inflammation.

Methods: Male wild-type (129 s1/SvlmJ) mice aged 8-15 weeks were separated into single cages and randomized 1:1:1:1 to each of the four experimental groups: control (CTR), preoperative TBT supplementation (PRE), postoperative TBT supplementation (POS), and combined pre- and postoperative supplementation (TOT). ICR was performed one week from baseline assessment with mice assessed at 1, 2, 3, and 4 weeks postoperatively. Primary outcomes included evaluating changes to gut microbial communities occurring from ICR to 4 weeks.

Results: A total of 34 mice that underwent ICR (CTR n = 9; PRE n = 10; POS n = 9; TOT n = 6) and reached the primary endpoint were included in the analysis. Postoperative TBT supplementation was associated with an increased recolonization and abundance of anaerobic taxa including Bacteroides thetaiotomicorn, Bacteroides caecimuris, Parabacteroides distasonis, and Clostridia. The microbial recolonization of PRE mice was characterized by a bloom of aerotolerant organisms including Staphylococcus, Lactobacillus, Enteroccaceae, and Peptostreptococcacea. PRE mice had a trend towards decreased ileal inflammation as evidenced by decreased levels of IL-1β (p = 0.09), IL-6 (p = 0.03), and TNF-α (p < 0.05) compared with mice receiving TBT postoperatively. In contrast, POS mice had trends towards reduced colonic inflammation demonstrated by decreased levels of IL-6 (p = 0.07) and TNF-α (p = 0.07). These changes occurred in the absence of changes to fecal short-chain fatty acid concentrations or histologic injury scoring.

Conclusions: Taken together, the results of our work demonstrate that the timing of tributyrin supplementation differentially modulates gastrointestinal inflammation and gut microbial recolonization following murine ICR.

Keywords: Crohn’s disease; ileocecal resection; inflammatory bowel disease; microbiome; tributyrin.

Figure 1

Overview of study design and changes in weight, food, and water intake. (a) Study design overview. Column graphs represent median +/− SEM. (b) Mouse weights from baseline to week 4 across intervention groups. (c) Percent change in weight relative to baseline across intervention groups. (d) Differences in weekly food intake across intervention groups. (e) Differences in weekly water intake across intervention groups. All p-values were two-sided with statistical significance defined as p < 0.05. * represents significance in paired analysis for percent change relative to baseline for all groups; $\varnothing$ represents significance in paired analysis for percent change relative to baseline for control group alone. ns represents values that are not significant.

Figure 2

Difference in microbial abundance at the phylum level along changes in alpha and beta diversity from BL to W4. (a) Phylum-level differences in relative microbial abundance between groups over time. (b) Within- and between- group changes in α-diversity using Chao1 and Shannon indices. (c) Between-group differences from baseline to week 4 in β-diversity using weighted UniFrac analysis.

Figure 3

Volcano plots demonstrating significant differences (p adj. < 0.05) in relative abundance of microbial taxa from W1 to W4. (a) CTR group volcano plot. (b) PRE group. (c) POS group volcano plot. (d) TOT group volcano plot. NS represents values that are not significant.

Figure 4

Concentrations of fecal short-chain fatty acids (SCFAs) from BL to W4 by intervention group. Column graphs represent median +/− SEM. (a) Total SCFAs. (b) Acetate. (c) Propionate. (d) Butyrate. (e) Proportion of SCFAs. * represents p < 0.05.

Figure 5

Tissue-weight-adjusted cytokine concentrations in perianastomotic ileal and colonic tissue homogenate. Box-and-whisker plots represent the distribution of each group at W4. The median is represented by the middle line while the upper and lower borders of the box plot identify the 75th and 25th percentile, respectively. The whiskers correspond to the maximal and minimal values. (a) Heatmap of ileal tissue cytokine concentrations after logarithmic transformation of data. (b–e) Concentration of IL-1 β, IL-6, IL-10, and TNF-α, respectively, per gram of dry ileal tissue. (f) Heatmap of colonic tissue cytokine concentrations after logarithmic transformation of data. (g–j) Concentration of IL-1 β, IL-6, IL-10, and TNF-α, respectively, per gram of dry colonic tissue.