Gut microbiota-mediated polyphenol metabolism is restrained by parasitic whipworm infection and associated with altered immune function in mice

Figures & data

Figure 1. Proanthocyanidins downregulate immune-related genes and affect immune cell hyperplasia in duodenum tissues of Heligmosomoides polygyrus-infected mice with no effect on worm burdens.

a) Schematic of experimental design. Mice in proanthocyanidin (PAC)-treated groups received 200 mg/kg PAC derived from grape pomace every 2^nd day, whilst control mice received water. Mice infected with H. polygyrus (Hp) were inoculated with 200 larvae on day 0. b) Top ten up- and down-regulated genes (Z-scores) in the duodenum of H. polygyrus-infected mice relative to uninfected control mice, identified by RNA-sequencing (for all genes adjusted p value < 0.05 by DeSeq2). c) Top 5 up- and down-regulated gene pathways (q value < 0.05) in duodenal tissue identified by gene set enrichment analysis in mice infected with H. polygyrus, relative to uninfected mice. d) Specific expression of mast cell-related genes in the duodenum of H. polygyrus-infected mice and PAC-dosed H. polygyrus-infected mice, relative to uninfected control mice. Shown are means ± S.E.M. of FPKM values from RNA-Seq analsyis. e) Expression of Mcpt1 as measured by qPCR. Fold changes are relative to uninfected, control mice. Shown are median values. f) Enumeration of Mcpt1-positive mast cells in the duodenum. Shown are median values. g) H. polygyrus-specific serum IgG1 levels in serum. Shown are median values. h) Worm burdens in H. polygyrus- infected mice with and without PAC treatment. Shown are median values. A)-G), n = 5 per treatment group from a single experiment, H) n = 10 per group, pooled from two independent experiments. *p < 0.05, **p < 0.01, by Mann-Whitney tests or Kruskal-Wallis tests with Dunn’s post-hoc testing.

Figure 2. Proanthocyanidins modulate immune responses in Trichuris muris-infected mice.

a) Schematic of experimental design. Mice in proanthocyanidin (PAC)-treated groups received 300 mg/kg PAC derived from grape seeds every 2^nd day, whilst control mice received water. Mice infected with Trichuris muris (Tm) were inoculated with 20 eggs on day 0, 14 and 28. b) Top ten up- and down-regulated genes (Z-scores) in the cecum of T. muris-infected mice relative to uninfected control mice, identified by RNA-sequencing (for all genes adjusted p value < 0.05 by DeSeq2). c) Top 5 up- and down-regulated gene pathways (q value < 0.05) in cecal tissue identified by gene set enrichment analysis in mice infected with T. muris, relative to uninfected mice. d) Top ten up- and down-regulated genes (Z-scores) in the cecum of PAC-dosed mice relative to water-dosed control mice, identified by RNA-sequencing (for all genes adjusted p value < 0.05 by DeSeq2). e) Top 5 up- and down-regulated gene pathways (q value < 0.05) in cecal tissue identified by gene set enrichment analysis in PAC-dosed mice relative to water-dosed control mice. f) Top 5 up- and down-regulated gene pathways (q value < 0.05) in cecal tissue identified by gene set enrichment analysis in PAC-dosed mice infected with T. muris, relative to mice infected with T. muris alone. g) Expression of Ifit3b and Irgm1 measured by qPCR. Fold changes are relative to uninfected, control mice. h) IL-6 induced by T. muris antigens in lymphocytes isolated from mesenteric lymph nodes in T. muris-infected mice, with or without PAC dosing. i) T. muris-specific serum IgG2a levels in serum. J) Adult and larval worm burdens in T. muris-infected mice For all panels, n = 5-8 per group, pooled from two independent experiments. g) – h) – Shown are means ± S.E.M. *p < 0.05 by un-paired t test. I) – j) – Shown are median values. *p < 0.05, **p < 0.01 by Mann-Whitney test.

Figure 3. Proanthocyanidins alter T cell populations in the mesenteric lymph nodes in helminth-infected mice.

Impact of proanthocyanidins (PAC) on the total number of cells (a), and proportions of TCRβ⁺ CD4⁺T-bet⁺, TCRβ⁺ CD4⁺GATA3⁺ and TCRβ⁺ CD4⁺Foxp3⁺ T-cells in the mesenteric lymph nodes (MLN) of Heligmosomoides polygyrus (Hp; B) and and Trichuris muris (Tm; C) infected mice. b) n = 5 per treatment group from a single experiment. c) n = 5-8 per group, pooled from two independent experiments. Shown are means ± S.E.M. * p<0.05 by unpaired t-test. Gating strategy is shown in Supplementary Figure 8.

Figure 4. Effects of proanthocyanidins during Trichuris muris infection in mice fed either chow or semi-synthetic diets.

a) Weight gain over the course of the 49 day experiment in mice fed either chow or semi-synthetic diets (SSD), and administered either proanthocyanidins (PAC) or water (control). Adult and larval T. muris counts (b-c), total cellularity of the mesenteric lymph nodes (d), proportions of T-bet⁺ (Th1) and GATA3⁺ (Th2) cells within the MLN TCRβ⁺ CD4⁺ population, and the Th1/Th2 ratio (e-g), serum IgG2a specific for T. muris antigen (h), and IL-6 secretion from MLN cells stimulated with T. muris antigen (i) at day 35 following the start of T. muris infection. n = 6 per treatment group from a single experiment. *p < 0.05, **p < 0.01, ***p<0.005 by two-way ANOVA with Tukey post-hoc testing. Shown are means ± S.E.M.

Figure 5. Impact of Heligmosomoides polygyrus (Hp) infection and proanthocyanidins (PAC) on the host cecal microbiota.

a) Violin plots illustrating the number of observed zOTUs and Shannon Diversity Index for each treatment group. * p< 0.05 by two-way ANOVA followed by Tukey post-hoc testing. Shown is median and interquartile range. B) Principal coordinates analysis based on Bray-Curtis dissimilarity metrics for control and Hp-infected mice fed with semi-synthetic diets and dosed with either PAC or water (control). Each data point on the PCoA plots indicate a sample, with ellipses showing 95% confidence interval. The percentage in brackets is the percentage of variation explained by each PCoA axis. Pairwise comparisons between treatment groups were performed using permutation MANOVAs on a distance matrix with p-value correction using the Holm method. p<0.05 was considered as significant. C) Relative abundance violin plots for zOTUs corresponding to Lactobacillus johnsonii, Turicibacter sanguinis, Ligilactobacillus animalis, and Bifidobacterium animalis subsp. lactis in uninfected and Hp-infected mice dosed with either PAC or water (control) (n = 4-5 per treatment group). The significance of difference was assessed by two-way ANOVA and Tukey post-hoc testing (* p<0.05; **p < 0.01). Shown is median and interquartile range.

Figure 6. Impact of Trichuris muris (Tm) infection and proanthocyanidins (PAC) on the host fecal microbiota.

a) Violin plots illustrating the number of observed zOTUs and Shannon Diversity Index for each treatment group. *** p < 0.001, ** p < 0.01 by two-way ANOVA followed by Tukey post-hoc testing. Shown is median and interquartile range. b) Principal coordinates analysis based on Bray-Curtis dissimilarity metrics for control and Tm-infected mice fed with semi-synthetic diets and dosed with either PAC or water (control). Each data point on the PCoA plots indicate a sample, with ellipses showing 95% confidence interval. The percentage in brackets is the percentage of variation explained by each PCoA axis. No ellipse was drawn for the Tm-infected mice dosed with PAC group as n = 3. Pairwise comparisons between treatment groups were performed using permutation MANOVAs on a distance matrix with p-value correction using the Holm method. p<0.05 was considered as significant. c) Relative abundance violin plots for zOTUs corresponding to Ligilactobacillus animalis, Turicibacter sanguinis, and Escherichia fergusonii in uninfected and T. muris-infected mice dosed with either PAC or water (control) (n = 3-7 per treatment group). **p < 0.01, *p < 0.05 by Tukey post-hoc testing following two-way ANOVA. Shown is median and interquartile range.

Figure 7. Short chain fatty acids and identification of proanthocyanidin metabolites in Trichuris muris infected mice.

a) Effect of proanthocyanidins (PAC) and Trichuris muris (Tm) infection on the concentrations of total short chain fatty acids (SCFA), acetic acid, propionic acid, and butyric acid in fecal samples. A significant (p < 0.05) interaction between diet and infection was noted for acetic acid, propionic acid and total SCFA by two-way ANOVA. *p < 0.05, # p = 0.06 by Tukey’s post-hoc test. b) Identification of PAC metabolites in the cecum of PAC-dosed uninfected and T. muris-infected mice relative to uninfected mice dosed with water. c) Caecal and d) serum concentrations of PAC-derived metabolites in PAC-dosed uninfected and T. muris infected mice. *p < 0.05 by unpaired t-test. n = 5-8 per group, pooled from two independent experiments. Shown are means ± S.E.M.

Data availability statement

RNA sequence data from cecum and duodenum are deposited at the NCBI Gene Expression Omnibus (GEO: Accession numbers GSE174756 and GSE176182). The raw 16S rRNA sequencing data can be accessed at Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) using the accession number PRJNA1044063.