Dietary Supplementation throughout Life with Non-Digestible Oligosaccharides and/or n-3 Poly-Unsaturated Fatty Acids in Healthy Mice Modulates the Gut-Immune System-Brain Axis

Abstract

The composition and activity of the intestinal microbial community structures can be beneficially modulated by nutritional components such as non-digestible oligosaccharides and omega-3 poly-unsaturated fatty acids (n-3 PUFAs). These components affect immune function, brain development and behaviour. We investigated the additive effect of a dietary combination of scGOS:lcFOS and n-3 PUFAs on caecal content microbial community structures and development of the immune system, brain and behaviour from day of birth to early adulthood in healthy mice. Male BALB/cByJ mice received a control or enriched diet with a combination of scGOS:lcFOS (9:1) and 6% tuna oil (n-3 PUFAs) or individually scGOS:lcFOS (9:1) or 6% tuna oil (n-3 PUFAs). Behaviour, caecal content microbiota composition, short-chain fatty acid levels, brain monoamine levels, enterochromaffin cells and immune parameters in the mesenteric lymph nodes (MLN) and spleen were assessed. Caecal content microbial community structures displayed differences between the control and dietary groups, and between the dietary groups. Compared to control diet, the scGOS:lcFOS and combination diets increased caecal saccharolytic fermentation activity. The diets enhanced the number of enterochromaffin cells. The combination diet had no effects on the immune cells. Although the dietary effect on behaviour was limited, serotonin and serotonin metabolite levels in the amygdala were increased in the combination diet group. The combination and individual interventions affected caecal content microbial profiles, but had limited effects on behaviour and the immune system. No apparent additive effect was observed when scGOS:lcFOS and n-3 PUFAs were combined. The results suggest that scGOS:lcFOS and n-3 PUFAs together create a balance-the best of both in a healthy host.

Keywords: PUFA; SCFA; behaviour; early life; fructo-oligosaccharide; galacto-oligosaccharide; healthy mice; intestinal microbiota; omega-3 fatty acids; prebiotics.

Figure 1

Schematic overview of the experimental protocol and the executed behavioural tests. From day of birth, the dams received a control diet, a 3% scGOS:lcFOS (9:1)-enriched diet, a n-3 PUFA diet or a combination diet containing 3% scGOS:lcFOS and n-3 PUFAs. The pups were weaned when 3 weeks old and continued on the allocated diet to the end of the experiment. During adolescence and early adulthood, a battery of behavioural tests was conducted. Organs were collected after the last behavioural test. GB: grooming behaviour, SI: social interaction test, OF: open field test, and MB: marble burying test.

Figure 2

Caecum content alpha-diversity and Firmicutes-to-Bacteroidetes ratio for the control and dietary mice groups. Alpha-diversity indices were examined at the taxonomic level of genus. Alpha-diversity indices rarefied to 45,000 sequences per sample. Analysis of the (A) Shannon index and (B) evenness both indicated a significant dietary effect across all groups, with the combination diet diversity significantly higher than scGOS1cFOS. (C) Richness diversity was not significantly different across groups. At the taxonomic level of phylum, the (D) Firmicutes-to-Bacteroidetes ratio significantly decreased in the combination diet mice compared to the control mice. Data were square root transformed for statistics. (A–D): Data shown as the mean +/− SEM. Analysed by one-way ANOVA and Sidak’s multiple comparisons post hoc test. * p < 0.05, ** p < 0.01. n = 8–10 mice per group.

Figure 3

Visual of the non-multi-dimensional scaling (nMDS) plot depicting caecum content microbial community structures between control and dietary mice samples. A significant microbial community structure was observed between the control mice and the three dietary treatments. For statistical details, reference the analysis of similarity (ANOSIM) calculations in Table 1. Identified taxa with Pearson’s correlation (>0.6) were strongly associated with either the control or dietary interventions.

Figure 4

Significant genera taxa-specific relative abundances across mice groups (caecum content). The abundances of eight significant genera across mice groups are identified as: (A) Allobaculum, (B) S24-7 Unclassified, (C) Oscillospira, (D) Ruminococcaceae Unclassified, (E) Turicibacter, (F) Akkermanisa, (G) Lachnospiraceae Unclassified, and (H) Rikenellaceae Unclassified. (A–H): Data shown as individual data points and median. Assessed for significance using Kruskal–Wallis test controlling for false-discovery rate (FDR): * FDR-P < 0.05, ** FDR-P < 0.01. n = 8–10 mice per group.

Figure 5

Caecum content predicted and targeted short-chain fatty acids metabolite concentrations between mice groups. Genera taxa metabolite predictive models depicting the relative abundances of (A) acetate- (B) propionate- and (C) butyrate-producing taxa were examined in the control and dietary mice groups. Targeted SCFA and BCFA graphs depict (D) total SCFA (mM/kg); (E) acetate (mM/kg); (F) propionate (mM/kg); (G) butyrate (mM/kg); (H) valerate (mM/kg); (I) iso-butyrate (mM/kg) and (J) iso-valeric acid (mM/kg) in the mice groups. (A–C): Data shown as individual data points and median. (D,E,G): Data shown as the mean +/− SEM. (F,H–J): Data shown as box-and-whiskers Tukey plots. (A–C,F,H–J): Analysed by Kruskal–Wallis and Dunn’s multiple comparisons post hoc tests. (D,E,F): Analysed by one-way ANOVA and Sidak’s multiple comparisons post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001. (A–J): n = 8–10 mice per group. SCFA: short-chain fatty acids.

Figure 6

Serotonin-producing cells in the jejunum, ileum and colon. (A) In the jejunum, the number of serotonin-producing cells increased in the scGOS:lcFOS, n-3 PUFA and the combination diet groups compared to control. (B) In the ileum, the diets did not affect the serotonin-producing cells. (C) In the colon, the number of serotonin-producing cells was significantly decreased in the combination diet group compared to the n-3 PUFA group. (D–F) Representative pictures (control group) of the jejunum (D), ileum (E) and colon (F). (A–C): Data shown as the mean +/− SEM. Analysed by one-way ANOVA and Sidak’s multiple comparisons post hoc test. * p < 0.05, ** p < 0.01, **** p < 0.0001. n = 6 mice per group.

Figure 7

The dietary effect of scGOS:lcFOS, n-3 PUFAs and the combination of scGOS:lcFOS and n-3 PUFAs on activated CD4 cells, Th1 cells and Th2 cells in the MLN. (A) The percentage of activated CD4 (CD69⁺ CD4⁺) cells is significantly increased in the n-3 PUFA group compared to control. (B) The percentage of Th1 cells (CXCR3⁺ CD4⁺) tended to a decrease in the combination diet group compared to the scGOS:lcFOS group. (C) The percentage of Th2 cells (T1ST2⁺ CD4⁺) tended to a decrease in the combination diet group compared to the n-3 PUFA group. An outlier in the control group was excluded by use of ROUT analysis. (A–C): Data shown as the mean +/− SEM. Analysed by one-way ANOVA and Sidak’s multiple comparisons post hoc test. ** p < 0.01. n = 5–10 mice per group, 2 samples in the control group, 3 samples in the n-3 PUFA group and 2 samples in the combination diet group were excluded due to low number of viable cells. Th1: T helper 1 cells. Th2: T helper 2 cells.

Figure 8

The amygdala levels of tryptophan, 5-HT, 5-HIAA and the serotonin turnover. (A) The tryptophan level did not differ between the dietary groups. (B,C) The 5-HT and 5-HIAA levels were significantly increased in the combination diet group compared to the scGOS:lcFOS group. (D) The serotonin turnover was unchanged in the dietary groups. (A–D): Data shown as the mean +/− SEM. Analysed by one-way ANOVA and Sidak’s multiple comparisons post hoc test. * p < 0.05, ** p < 0.01. n = 4–5 samples per group, samples were pooled in pairs, in order to reach detection minimum, each sample contained two left brains. 5-HT: serotonin. 5-HIAA: 5-hydroxyindoleacetic acid (serotonin metabolite).

Figure 9

Anxiety-like behaviour assessed by marble burying and explorative behaviour in the open field. (A) Number of buried marbles by mice receiving the control, scGOS:lcFOS, n-3 PUFA or combination diet in adolescence and early adulthood (B) Explorative behaviour, the frequency the mice receiving the control, scGOS:lcFOS, n-3 PUFA or combination diet entered the centre of the open field in adolescence and early adulthood. (C) Explorative behaviour, the time the mice receiving the control, scGOS:lcFOS, n-3 PUFA or combination diet spent in the centre of the open field. (A–C): Data shown as the mean +/− SEM. Analysed with mixed models, controlled for repeated measures, litter effect and Sidak’s multiple comparisons post hoc test. a = * p < 0.05 compared with early adulthood within diet group, b = ** p < 0.01 compared with early adulthood within diet group, c = *** p < 0.001 compared with early adulthood within diet group. (A–C): n = 8–10 mice per group.

Figure 10

The datasheet of the entire study was analysed regarding marble burying (number of marbles buried) at 8 weeks of age (early adulthood). (A) After running the REFS algorithm 10 times, the best signature is at 9 features, with an average global accuracy of all classifiers of 0.73. (B) The optimal associated receiver-operating characteristic (ROC) curve for the best-performing classifier Ridge demonstrates an area under the curve (AUC) of 0.84 ± 0.17. (C) The 9 features separating the two labels: 0 = Adlercreutzia, 1 = DH: tryptophan (nmol/gr), 2 = Lachnospiraceae Other, 3 = Dehalobacterium, 4 = Th17 cells in MLN, 5 = Activated Th2 cells in spleen, 6 = α-diversity (Shannon index), 7 = PFC: noradrenaline (nmol/gr), 8 = PFC: noradrenaline (nmol/gr), and 9 = Th1 cells in MLN. Label 0: number of buried marbles <10. Label 1: number of buried marbles ≥10.

Figure 11

Indications of which feature combination significantly influence repetitive and anxiety-like behaviour and explorative behaviour evaluated by marble burying (A) and open field behaviour (B) tests, respectively. (A) The alpha-diversity and relative abundances of the genera Adlercreutzia and Dehalobacterium, changes in Th1 and Th17 cells in MLN, changes in activated Th2 cells in spleen, and tryptophan levels in dorsal hippocampus significantly predict changes in repetitive behaviour. (B) The relative abundances of the genera Oscillospira, Ruminococcus, Odoribacter, Turibacter, Lachnospiraceae other/Unclassified and Adlercreutzia and changes in serotonin and dopamine metabolism in PFC and amygdala significantly predict changes in explorative behaviour. DA: dopamine; 5HT: serotonin; NA: noradrenaline; Th: Thelper; TRP: tryptophan.

Figure 12

The datasheet of the entire study was analysed regarding the open field test (number of entries in the open field) at 8 weeks of age (early adulthood). (A): After running the REFS algorithm 10 times, the best signature is at 16 features with average accuracy of all classifiers of 0.77. (B): The optimal associated ROC curve for the best-performing classifier Ridge demonstrates an area under the curve (AUC) of 0.82 ± 0.20. (C): The 16 features separating the two labels: 0 = Cyanobacteria Unclassified, 1 = Oscillospira, 2 = Odoribacter, 3 = Turicibacter, 4 = AM: (DOPAC + HVA)/DA, 5 = PFC: 5HIAA/5HT, 6 = Lachnospiraceae Unspecified, 7 = Allobaculum, 8 = DH: noradrenaline (nmol/gr), 9 = Lactobacillus, 10 = PFC: 5-HIAA (nmol/gr), 11 = Lachnospiraceae Other, 12 = Ruminococcus, 13 = Adlercreutzia, 14 = PFC: HVA (nmol/gr), and 15 = AM: (DOPAC + HVA + 3MT)/DA. Label 0: number of entries to open field ≥ 10. Label 1: number of entries to open field < 10.