Novel Probiotic Bacterium Rouxiella badensis subsp. acadiensis (Canan SV-53) Modulates Gut Immunity through Epigenetic Mechanisms

Abstract

Gut immune system homeostasis is crucial to overall host health. Immune disturbance at the gut level may lead to systemic and distant sites' immune dysfunction. Probiotics and prebiotics consumption have been shown to improve gut microbiota composition and function and enhance gut immunity. In the current study, the immunomodulatory and anti-inflammatory effects of viable and heat-inactivated forms of the novel probiotic bacterium Rouxiella badensis subsp. acadiensis (Canan SV-53), as well as the prebiotic protocatechuic acid (PCA) derived from the fermentation of blueberry juice by SV-53, were examined. To this end, female Balb/c mice received probiotic (viable or heat-inactivated), prebiotic, or a mixture of viable probiotic and prebiotic in drinking water for three weeks. To better decipher the immunomodulatory effects of biotics intake, gut microbiota, gut mucosal immunity, T helper-17 (Th17) cell-related cytokines, and epigenetic modulation of Th17 cells were studied. In mice receiving viable SV-53 and PCA, a significant increase was noted in serum IgA levels and the number of IgA-producing B cells in the ileum. A significant reduction was observed in the concentrations of proinflammatory cytokines, including interleukin (IL)-17A, IL-6, and IL-23, and expression of two proinflammatory miRNAs, miR-223 and miR425, in treated groups. In addition, heat-inactivated SV-53 exerted immunomodulatory properties by elevating the IgA concentration in the serum and reducing IL-6 and IL-23 levels in the ileum. DNA methylation analysis revealed the role of heat-inactivated SV-53 in the epigenetic regulation of genes related to Th17 and IL-17 production and function, including Il6, Il17rc, Il9, Il11, Akt1, Ikbkg, Sgk1, Cblb, and Smad4. Taken together, these findings may reflect the potential role of the novel probiotic bacterium SV-53 and prebiotic PCA in improving gut immunity and homeostasis. Further studies are required to ascertain the beneficial effects of this novel bacterium in the inflammatory state.

Keywords: DNA methylation; Th17 cell; gut immunity; gut microbiome; miRNA; prebiotic; probiotic SV-53.

Figure 1

The effect of the probiotic and prebiotic intake on gut mucosal immunity. Female Balb/c mice were treated with the SV-53, PCA, or SV-53 + PCA mixture administered in their drinking water for three weeks. Serum levels of IgA and IgG and the number of IgA⁺ and IgG⁺ B cells in the ileum tissues of mice were measured via ELISA and direct immunofluorescence, respectively; (A) the serum concentration of IgA, (B) the number of IgA⁺ B cells, (C) the serum concentration of IgG, and (D) the number of IgG⁺ B cells. One-way ANOVA followed by Dunnett’s multiple comparisons were used to compare groups. All values are mean ± SEM. N = 6, * p < 0.05, ** p < 0.01 and **** p < 0.0001 vs. control. (E) Immunofluorescence images of histological sections of the ileum stained with the appropriate dilution of anti-IgA antibody (1:100) and imaged using a fluorescent light microscope at 40× magnification.

Figure 2

The effect of the probiotic and prebiotic intake on selected cytokines and miRNAs in the ileum tissues of mice. Female Balb/c mice were treated with the SV-53, PCA, or SV-53 + PCA mixture in drinking water for three weeks. The concentrations of (A) IL-17A, (B) IL-6, (C) IL-23, and (D) IL-10 in the ileum tissues of mice were measured via ELISA. The frequencies of (E) IL-17A⁺, (F) IL-6⁺, and (G) IL-10⁺ cells were determined via immunofluorescence. The expression of (H) miR-223 and (I) miR-425 was measured via RT-qPCR. One-way ANOVA followed by Dunnett’s multiple comparisons was used to compare groups. All values are mean ± SEM. N = 6, * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control.

Figure 3

The effect of the probiotic and prebiotic intake on the gut microbiome. Female Balb/c mice were treated with the SV-53, PCA, or SV-53 + PCA mixture in drinking water for three weeks. Then, cecum contents of mice were used to analyze the gut microbiome via shallow shotgun sequencing; (A) alpha diversity, (B) beta diversity, (C) bacterial composition at the phylum level (ten most abundant phyla), and (D,E) differentially abundant species. n = 5 in the control group and n = 6 in the treatment groups.

Figure 4

The effect of the heat-inactivated probiotic intake on gut mucosal immunity. Female Balb/c mice were treated with live SV-53 or heat-inactivated SV-53 (HI-SV-53) in drinking water for three weeks. Then, the serum levels of IgA and IgG, and the number of IgA⁺ and IgG⁺ B cells in the ileum tissues of mice were measured by ELISA and immunofluorescence, respectively; (A) the concentrations of IgA, (B) the number of IgA⁺ B cells, (C) the concentrations of IgG, and (D) the number of IgG⁺ B cells. One-way ANOVA followed by Dunnett’s multiple comparisons were used to compare groups. All values are mean ± SEM. N = 6, * p < 0.05 and *** p < 0.001 vs. control.

Figure 5

The effect of the heat-inactivated probiotic on selected cytokines and miRNAs expression in the ileum tissues of mice. Female Balb/c mice were treated with live SV-53 or heat-inactivated SV-53 (HI-SV-53) in drinking water for three weeks. The concentrations of (A) IL-17A, (B) IL-6, (C) IL-23, and (D) IL-10 in the ileum tissues of mice were measured via ELISA. The frequencies of (E) IL-17A⁺, (F) IL-6⁺, and (G) IL-10⁺ cells were determined via immunofluorescence. The expression of (H) miR-223 and (I) miR-425 were measured via RT-qPCR. One-way ANOVA followed by Dunnett’s multiple comparisons was used to compare groups. All values are mean ± SEM. N = 6, * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control.

Figure 6

The effect of the heat-inactivated probiotic on DNA methylation. Female Balb/c mice were treated with live SV-53 or heat-inactivated SV-53 (HI-SV-53) administered via drinking water for three weeks. (A) Pathways enrichment analysis visualized by Enrichr, (B) boxplots of differentially methylated genes related to Th17 cells differentiation and function. * p < 0.05 and ** p < 0.01 vs. control.