Gut microbial metabolites as key mediators in inflammatory bowel disease

Figure 1 Viability of Caco-2 cells (% of control). A: Caco-2 cells treated with various concentrations (0-10 mmol/L) of metabolites; B: Caco-2 cells treated with different concentrations (0-7%) of dextran sulfate sodium (DSS); C: Caco-2 cells treated with metabolites together with 3% DSS, 3% DSS considered as 100% cell viability. Values are shown as medians and interquartile range (from min to max). All assays were performed in four independent experiments, each consisting of 5-10 biological replicates per group. ^aP < 0.05, ^bP < 0.01 vs the control, and ^cP < 0.05 vs cells treated with butyric, propionic, and valeric acids combination, Kruskal-Wallis test with post hoc Dunn’s test, corrected with Benjamini-Hochberg procedure. DSS: Dextran sulfate sodium; But: Butyric acid; Prop: Propionic acid; Val: Valeric acid; BPV: Combination of butyric, propionic, and valeric acids.

Figure 2 Effect of 10 μg/mL lipopolysaccharides on indicators of the intestinal barrier integrity and inflammation in Caco-2 cells. A: Transepithelial electrical resistance; B: Apparent permeability coefficient (P_app) for monolayer permeability to fluorescein isothiocyanate-dextran; C: Gene expression of tight junction proteins and pro-inflammatory cytokines; D: Interleukin-8 content in culture medium. Values are shown as medians and interquartile range (from min to max). All assays were performed in four independent experiments, each comprising 5-6 biological replicates per group. ^aP < 0.05, ^bP < 0.01 vs the control, Mann-Whitney U test. TEER: Transepithelial electrical resistance; P_app: Apparent permeability coefficient; LPS: Lipopolysaccharides; IL-8: Interleukin-8; TNF-α: Tumor necrosis factor-α.

Figure 3 Effect of metabolites with 10 μg/mL lipopolysaccharides on the barrier integrity and inflammation in the Caco-2 cells. A: Apparent permeability coefficient (P_app) for monolayer permeability to fluorescein isothiocyanate-dextran; B: Interleukin-8 content in culture medium; C: Gene expression of tight junction proteins and pro-inflammatory cytokines. Values are shown as medians and interquartile range (from min to max). All assays were performed in three independent experiments, each with 4-9 biological replicates per group. ^aP < 0.05 vs the control (10 μg/mL lipopolysaccharides), ^bP < 0.01 vs the control (10 μg/mL lipopolysaccharides), ^cP < 0.05 vs lipopolysaccharides + combination of butyric, propionic, and valeric acids group, ^dP < 0.01 vs lipopolysaccharides + combination of butyric, propionic, and valeric acids group, Kruskal-Wallis test with post hoc Dunn’s test, corrected with Benjamini-Hochberg procedure. P_app: Apparent permeability coefficient; LPS: Lipopolysaccharides; But: Butyric acid; Prop: Propionic acid; Val: Valeric acid; BPV: Combination of butyric, propionic, and valeric acids; IL-8: Interleukin-8; TNF-α: Tumor necrosis factor-α.

Figure 4 Muc2 knockout mice as an in vivo model of inflammatory bowel disease - increased intestinal permeability and colon inflammation. A and B: Differences in colon length between wild-type mice (C57BL/6) and mice with mucin 2 gene knockout (Muc2^–/–) mice; C: Hematoxylin-eosin (top panel) and alcian blue (bottom-panel)-stained colonic sections (10 × magnification) of C57BL/6 and Muc2^-/- mice; D: Intestinal permeability of C57BL/6 and Muc2^-/- mice, expressed in μg/mL of fluorescein isothiocyanate-dextran in blood; E: Expression of pro- and anti-inflammatory cytokines and Foxp3 transcriptional factor in colon of C57BL/6 and Muc2^-/- mice; F: Intestinal content of inflammatory interleukin-1β and anti-inflammatory interleukin-10 in C57BL/6 and Muc2^-/- mice; G: Regulatory T cells content in mesenteric lymph nodes in C57BL/6 and Muc2^-/- mice; H: Ratio of M1 (CD80, iNOS) and M2 (CD209, Arginase-1) peritoneal macrophages in C57BL/6 and Muc2^-/- mice. All assays were performed in two independent experiments, each consisting of 4-11 biological replicates (mice) per group. ^aP < 0.05, ^bP < 0.01 vs the control (C57BL/6 mice), Mann-Whitney U test. FITC: Fluorescein isothiocyanate; Treg: Regulatory T cells; IL: Interleukin; TNF-α: Tumor necrosis factor-α; TGF-β: Transforming growth factor β.

Figure 5 Effect of metabolites on gut permeability and inflammation in the colon of Muc2^-/- mice. A: Evaluation of metabolites toxicity by alanine aminotransferase and aspartate aminotransferase activity in blood; B: Effect of metabolites on intestinal permeability of Muc2^-/- mice, expressed in μg/mL of fluorescein isothiocyanate-dextran in blood; C: Effect of metabolites on n (%) of regulatory T cells in the mesenteric lymph nodes of Muc2^-/- mice; D: Effect of metabolites on expression of pro- and anti-inflammatory cytokines and Foxp3 transcriptional factor in colon of Muc2^-/- mice; E: Effect of metabolites on cytokine content in colon of Muc2^-/- mice; F: Effect of metabolites on expression of markers of pro-inflammatory M1 (CD80, iNOS) and anti-inflammatory M2 (CD209, Arginase-1) peritoneal macrophages. Values are shown as medians and interquartile range (from min to max). All assays were performed in one experiment, consisted of 4-11 biological replicates (mice) per group. ^aP < 0.05 vs the control (Muc2^-/- mice), ^bP < 0.01 vs the control (Muc2^-/- mice), ^cP < 0.05 vs Muc2^-/- mice treated with butyric, propionic, and valeric acids combination, Kruskal-Wallis test with post hoc Dunn’s test, corrected with Benjamini-Hochberg procedure. ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; FITC: Fluorescein isothiocyanate; Treg: Regulatory T cells; But: Butyric acid; Prop: Propionic acid; Val: Valeric acid; BPV: Combination of butyric, propionic, and valeric acids; IL: Interleukin; TNF-α: Tumor necrosis factor-α.

Figure 6 The abundance of the main bacterial phyla in all studied groups of mice. A: Sum of the median level of reads for each phylum presented as cumulative bar chart; B: Abundance of the main bacterial families, genus and species in Bacillota phylum presented as heat map; C: Abundance of the main bacterial families, genus and species in Bacteroidota phylum presented as heat map; D: Abundance of the main bacterial genus and species in Actinomycetota phylum (pink underline), Verrucomicrobiota phylum (black underline), Thermodesulfobacteriota phylum (orange underline), Pseudomonadota phylum (yellow underline) presented as heat map. Values out of range are marked in dark blue color: 20% abundance for Lachnospiraceae family, 12% abundance for Muribaculaceae family, 10% abundance for Akkermansia genus. The metagenomic assay was performed in a single experiment with 4 mice per group. ^aP < 0.05 between Muc2^-/- mice vs the control (C57BL/6 mice), Mann-Whitney U test; ^bP < 0.05 between Muc2^-/- mice treated with metabolites vs the control (Muc2^-/- mice without treatment), ^cP < 0.01 between Muc2^-/- mice treated with metabolites vs the control (Muc2^-/- mice without treatment), Kruskal-Wallis test with post hoc Dunn’s test, corrected with Benjamini-Hochberg procedure. But: Butyric acid; Prop: Propionic acid; Val: Valeric acid.

Figure 7 Heatmap of the changes in metabolite concentration in the blood serum of all studied groups of mice. A: Hydroxycarboxylic acids; B: Long-chain fatty acids; C: Amino acids and their derivatives; D: Other metabolites. Red indicates an increase in metabolite concentration in the blood; green indicates a decrease. The metabolomic assay was performed in one experiment, consisted of 4 mice per group. ^aP < 0.05 between Muc2^-/- mice vs the control (C57BL/6 mice), Mann-Whitney U test; ^bP < 0.05 between Muc2^-/- mice treated with metabolites vs the control (Muc2^-/- mice without treatment), ^cP < 0.01 between Muc2^-/- mice treated with metabolites vs the control (Muc2^-/- mice without treatment), ^dP < 0.05 between Muc2^-/- mice treated with butyric, propionic and valeric acids vs Muc2^-/- mice treated with butyric, propionic and valeric acids combination, Kruskal-Wallis test with post hoc Dunn’s test, corrected with Benjamini-Hochberg procedure. But: Butyric acid; Prop: Propionic acid; Val: Valeric acid; BPV: Combination of butyric, propionic and valeric acids.

Figure 8 Biological activity of 2-hydroxybutyric acid in the pathogenesis of inflammatory bowel disease on Caco-2 cell model. A: Viability of Caco-2 cells treated with 2-hydroxybutyric acid together with 3% dextran sulfate sodium; B: Apparent permeability coefficient (P_app) for monolayer permeability to fluorescein isothiocyanate-dextran; C: Gene expression of pro-inflammatory cytokine interleukin-8 and tight junction protein ZO-1; D: Interleukin-8 content in culture medium. Values are shown as medians and interquartile range (from min to max). All assays were performed in three independent experiments, each of which consisted of 5-11 biological replicates per group. ^aP < 0.05 vs the control, Mann-Whitney U test. DSS: Dextran sulfate sodium; LPS: Lipopolysaccharides; P_app: apparent permeability coefficient; 2-HB: 2-hydroxybutyric acid; IL-8: Interleukin-8.

Figure 9 Biological activity of 2-hydroxybutyric acid in the pathogenesis of inflammatory bowel disease in Muc2^-/- mice. A: Gene expression of pro-inflammatory cytokines in colon; B: Intestinal content of inflammatory interleukin-1β; C: n (%) of regulatory T cells in the mesenteric lymph nodes; D: Effect of 2-hydroxybutyric acid on CD80 expression in peritoneal macrophages. Values are shown as medians and interquartile range (from min to max). All assays were performed in one experiment, consisted of 4-11 biological replicates (mice) per group. ^aP < 0.05 vs the control, Mann-Whitney U test. Treg: Regulatory T cells; 2-HB: 2-hydroxybutyric acid; IL: Interleukin; TNF-α: Tumor necrosis factor-α.