Ketogenic Diets Alter the Gut Microbiome Resulting in Decreased Intestinal Th17 Cells

Abstract

Very low-carbohydrate, high-fat ketogenic diets (KDs) induce a pronounced shift in metabolic fuel utilization that elevates circulating ketone bodies; however, the consequences of these compounds for host-microbiome interactions remain unknown. Here, we show that KDs alter the human and mouse gut microbiota in a manner distinct from high-fat diets (HFDs). Metagenomic and metabolomic analyses of stool samples from an 8-week inpatient study revealed marked shifts in gut microbial community structure and function during the KD. Gradient diet experiments in mice confirmed the unique impact of KDs relative to HFDs with a reproducible depletion of bifidobacteria. In vitro and in vivo experiments showed that ketone bodies selectively inhibited bifidobacterial growth. Finally, mono-colonizations and human microbiome transplantations into germ-free mice revealed that the KD-associated gut microbiota reduces the levels of intestinal pro-inflammatory Th17 cells. Together, these results highlight the importance of trans-kingdom chemical dialogs for mediating the host response to dietary interventions.

Keywords: Th17 cells; adipose tissue; bifidobacteria; intestinal immunity; ketogenic diet; ketone bodies; ketone ester; microbiome; β-hydroxybutyrate.

Conflict of interest statement

Declaration of Interests J.C.N. and E.V. are co-founders with equity interest of BHB Therapeutics, which is developing products related to ketone bodies. P.J.T. is on the scientific advisory boards for Kaleido, Pendulum, Seres, and SNIPRbiome; there is no direct overlap between the current study and these consulting duties.

Copyright © 2020 Elsevier Inc. All rights reserved.

Figures

Figure 1.. A ketogenic diet alters the human gut microbial community structure relative to an isocaloric control diet.

(A) Daily diet composition during the baseline diet (BD) and ketogenic diet (KD) stages respectively, represented in percent calories. (B) Plasma levels of the ketone bodies acetoacetate (AcAc) and βHB (βHB) significantly increased during KD across all 17 study participants (***P < 0.001; paired t-test). (C) Reproducible shifts in gut microbial community structure are observed across study participants on the first principle coordinate (PCo1) of PhILR Euclidean distances between the two diet stages (P = 0.001, r2 = 0.024, ADONIS with Subject ID as stratum), despite significant inter-individual variation in community structure between subjects (P = 0.001, r2 = 0.845, ADONIS testing for Subject ID). (D) KD alters the relative abundance of the major phyla in the human gut microbiota (*P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant; paired Wilcoxon test). (E) Fold change of genera whose abundances were significantly altered by KD (FDR 1H NMR spectra of fecal metabolite profiles between BD and KD diet stages across 17 study participants. The OPLS-DA model was validated with a 7-fold cross validation method; R2 value represents predictive power (R2>0.5 suggests the model is robust) and Q2>0.4 value suggests the model is valid. (G) GC-MS analysis of human fecal short-chain fatty acids (SCFAs) did not reveal differences in SCFA concentration between BD (n = 15 samples) and KD (n = 15 samples) diet stages. Statistical analyses performed using paired t-tests for each SCFA. (H) Fecal DNA content is not significantly different between the two diet stages (ns = not significant; paired t-test). Data are presented as mean±SEM. Data points represent single samples (C, F-G) or the average of samples per study participant on each diet stage (D-E, H). See also Figure S1, Tables S1 and S3.

Figure 2.. Metagenomic sequencing reveals decreased abundance of bifidobacterial species on the ketogenic diet in our human cohort.

(A) Heatmap showing fold change of the 50 most abundant species, comparing the ketogenic diet (KD) to baseline diet (BD) stages across all 17 subjects in our human study. Bacterial species are ordered by the magnitude and direction of the average fold change, from the most negative (top) to positive (bottom). (B) The ketogenic diet results in consistent reductions in the abundance of bifidobacterial species in our human cohort. Each line connects paired samples between the two diet arms from each study subject, for bifidobacterial species detected in a study subject’s gut microbiota. See also Table S2.

Figure 3.. The impact of a ketogenic diet on the gut microbiota is distinct from a high-fat diet.

(A) Macronutrient composition (in percent calories) of the chow diet and three semi-purified diets: low-fat diet (LFD), high-fat diet (HFD) and ketogenic diet (KD). (B) Circulating βHB levels in mice on diets for 3 weeks (n = 6 mice/group, ***P < 0.001; one-way ANOVA with Tukey’s test). (C) Overall mean caloric intake represented as kcal per mouse per day over 3 weeks of dietary intervention (n = 6 mice/group, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant; one-way ANOVA with Tukey’s test). (D) Percent change in body weight compared to baseline after 3 weeks of dietary intervention (n = 6 mice/group, *P < 0.05; Kruskal-Wallis with Dunn’s test). (E) Principal coordinate analysis of PhILR Euclidean distances reveal significant effect of diet on community composition (P = 0.001, r2 = 0.60; ADONIS) and also significant differences in community composition comparing HFD to KD (P = 0.005, r2 = 0.45; ADONIS). (F) Relative abundance of the major phyla in the microbial communities of mice fed the respective diets (n = 4-6 mice/group, *P < 0.05, **P < 0.01; Kruskal-Wallis with Dunn’s test). (G) Fold change from baseline of genera whose abundances were significantly different between HFD and KD (FDR

Figure 4.. Modest changes in dietary carbohydrate associated with partial induction of ketogenesis is sufficient to alter gut microbial composition.

(A) Macronutrient composition (in percent calories) of four semi-purified diets in order of decreasing CHO content from 15% (HFD75) to 0% (KD). The CHO and fat content of each diet are shown below the barplots, with protein content matched at 10% kcal across all diets. (B) Circulating βHB levels in mice fed the respective diets for 3 weeks (n = 5 mice/group, *P < 0.05, **P < 0.01; one-way ANOVA with Tukey’s test). (C) Overall mean caloric intake, represented as kcal per mouse per day over 3 weeks of dietary intervention, was not significantly different between diet groups (n = 5 mice/group, ns = not significant; one-way ANOVA). (D) Body weights of mice after 3 weeks of dietary intervention were not significantly different between diet groups (n = 5 mice/group, ns = not significant; one-way ANOVA). (E) Changes in percent body fat, measured using EchoMRI at baseline and after 3 weeks of dietary intervention, were not significantly different between diet groups (n = 5 mice/group, ns = not significant; one-way ANOVA). (F) Principal coordinate analyses of PhILR Euclidean distances reveal significant differences in community composition comparing HFD75 to KD (P = 0.013, r2 = 0.15) and HFD85 to KD (P = 0.001, r2 = 0.13) but not HFD89 to KD (P = 0.066, r2 = 0.06). Each data point represents a single fecal sample, with the color and shape of the symbols representing diet group and day of fecal collection respectively (n = 5 mice/group; ADONIS testing for effect of diet and accounting for within-mouse replicates). (G) Relative abundance of the major phyla in the microbial communities of mice fed the respective diets for 3 weeks (n = 5 mice/group, *P < 0.05, **P < 0.01; Kruskal-Wallis with Dunn’s test). (H) Relative abundances of two genera (Bifidobacterium and Lactobacillus) that are significantly different comparing HFD89 to KD (FDR<0.05, DESeq2) are shown across diet groups (n = 5 mice/group, *P < 0.05, **P < 0.01; Kruskal-Wallis with Dunn’s test). Data are presented as mean±SEM. Each data point represents an individual, singly-housed animal (B-E, G-H).

Figure 5.. Ketone bodies directly inhibit gut bacterial growth.

(A) Chemical structure of the ketone ester (KE) comprising βHB linked to two 6-carbon medium chain fatty acids which are metabolized to βHB. (B) Circulating βHB levels in mice fed HFD, KE-supplemented HFD (HFD-KE), KD, or KE-supplemented KD (KD-KE) respectively for 3 weeks (n = 11-12 mice/group from two independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Kruskal-Wallis with Dunn’s test). (C) βHB concentrations in cecal contents of mice fed the respective diets for 3 weeks (n = 11-12 mice/group from two independent experiments, **P < 0.01, ***P < 0.001; Kruskal-Wallis with Dunn’s test). (D) βHB concentrations in colon tissues of mice fed the respective diets for 3 weeks (n = 7 mice/group, *P < 0.05, **P < 0.01, ****P < 0.0001; Kruskal-Wallis with Dunn’s test). (E) Overall mean caloric intake, represented as kcal per mouse per day over 3 weeks of dietary intervention, was not significantly different between groups (n = 4-5 mice/group, ns = not significant; Kruskal-Wallis test). (F) Percent change in body weight from baseline after 3 weeks of dietary intervention was significantly lower in mice fed the KE-supplemented diets (HFD-KE and KD-KE) compared to either HFD or KD (n = 4-5 mice/group, *P < 0.05, **P < 0.01, ***P < 0.001; Kruskal-Wallis with Dunn’s test). (G) Change in percent body fat, measured using EchoMRI at baseline and after 3 weeks, was significantly lower in mice fed HFD-KE compared to other diets (n = 4-5 mice/group, *P < 0.05, **P < 0.01; Kruskal-Wallis with Dunn’s test). (H) Principal coordinates analysis of PhILR Euclidean distances reveals distinct clustering of microbial communities between diet groups (P = 0.001, r2 = 0.41, ADONIS), and microbial communities on the HFD diet are significantly different from each of the other diet groups (P and r2 values are indicated on the PCoA plot, ADONIS). (I) Fold change of genera that are consistently altered in comparisons between HFD vs. KD and HFD vs. HFD-KE (FDR Bifidobacterium fold change in mice fed HFD or HFD-KE for 3 weeks (P = 0.037, rho = −0.762; Spearman’s rank correlation). (K) Growth of fecal suspensions from four healthy donors measured under anaerobic conditions with different concentrations of βHB (12.5, 25 and 50mM) or vehicle control (0mM) over 24 hours. The average growth curves among the four donors, each grown in quadruplicates per concentration, are shown with shaded areas representing standard error (using stat_smooth function in R) and optical density (600 nm) measured at 15 min intervals over 24 hours. (L) Centered log2-ratio (CLR) normalized abundances of the major phyla in four ex vivo communities incubated with 50 mM βHB or vehicle control (0 mM) for 16 hours. Each data point represents the average value from quadruplicates for a single donor community. Each line connects the microbial communities from the same donor, incubated with βHB or vehicle control (***P < 0.001, ns = not significant; paired t-test). (M) Fold change of genera whose abundances were significantly altered by 50mM βHB compared to vehicle control (FDR

Figure 6.. The KD-associated microbiota reduces gut Th17 cell accumulation in gnotobiotic mice.

(A) B. adolescentis BD1 (BA) induces a robust Th17 population in the small intestine (SI) but not large intestine (colon) of mice fed HFD. Th17 populations are measured as IL-17a+ CD4+ live cell percentages (see gating strategy in Figure S7A). Black and grey bars represent germ-free (GF) and BA-monocolonized mice respectively (n = 4 mice/group, *P < 0.05, ns = not significant; Welch’s t-test). Representative flow cytometry plots of the IL-17a+ CD4+ live cell populations are shown to the left. (B) BA-monocolonized mice fed KD do not show intestinal Th17 cell induction compared to germ-free controls fed KD, in the SI and colon (n = 4 mice/group, ns = not significant; Welch’s t-test). Representative flow cytometry plots of the IL-17a+ CD4+ live cell populations are shown to the left. (C) Bacterial DNA content, expressed as 16s rRNA gene copies/ gram wet weight in the ileal contents from BA-monocolonized mice, is significantly lower in mice fed KD compared to HFD (n = 3-4 mice/group, *P < 0.05; Welch’s t-test). (D, E) No significant difference in Th1 populations in the SI and colon between germ-free (GF, black bars) and BA-monocolonized (BA, grey bars) mice fed (D) HFD and (E) KD respectively. Th1 populations are measured as IFNγ+ CD4+ live cell percentages (n = 3-4 mice/group, ns = not significant; Welch’s t-test). (F) Circulating βHB levels are significantly elevated in both GF and BA-monocolonized mice fed KD compared to HFD. Black and grey bars represent HFD and KD respectively (n = 4 mice/group, ***P < 0.001; Welch’s t-test). (G, H) Community composition at (G) phylum and (H) genus level of donor fecal samples used for gnotobiotic transplant, pooled from the same four individuals during the baseline diet (BD) and ketogenic diet (KD) stages respectively, and a third sample comprising the KD donor sample supplemented with B. adolescentis BD1 (KD+BA). (I) Fecal microbiota of mice colonized with the donor samples shown in panels G and H, sampled every two days with sacrifice occurring 12 days post-transplantation (n = 5-6 mice/group). (J) Abundance of B. adolescentis in the fecal microbiota of mice in the three transplant groups, sampled over the course of the experiment (n = 5-6 mice/group). BA abundance is significantly different between transplant groups (Pgroup = 1.8e-5; PKD+BAv.KD < 0.001; PKD+BAv.BD < 0.01; PBDv.KD = 0.053). Statistical analysis carried out using a linear mixed effects model with MouseID as random effect and Tukey’s test. (K) Mice that received the KD microbiota showed significantly lower levels of intestinal Th17 cells than either BD or KD+BA transplant groups. Tissues were harvested on day 12 post-transplantation. Representative flow cytometry plots of the IL-17a+ CD4+ live cell populations are shown to the left (n = 4-6 mice/group, **P < 0.01, ns = not significant; one-way ANOVA with Tukey’s test). (L) Th1 cell populations in the small intestine are not significantly different between transplant groups. Representative flow cytometry plots of the IFNγ+ CD4+ live cell populations are shown to the left (n = 4-6 mice/group, ns = not significant; one-way ANOVA with Tukey’s test). Data are presented as mean±SEM. Each data point represents an individual, singly-housed animal (A-F, K-L).

Figure 7.. The ketogenic diet and ketone ester feeding reduces intestinal Th17 cell accumulation in conventional mice.

(A) SPF mice fed either KD or ketone ester (KE)-supplemented diets show significantly lower frequencies of small intestinal Th17 cells compared to mice fed HFD for 3 weeks (n = 4-5 mice/group, *P < 0.05; one-way ANOVA with Tukey’s test). Representative flow cytometry plots of the IL-17a+ CD4+ live cell populations are shown to the left. (B, C) No significant differences in Th17 cell levels in the (B) colon and (C) spleen of mice fed the respective diets for 3 weeks (n = 4-5 mice/group, ns = not significant; one-way ANOVA with Tukey’s test). (D) SPF mice fed KD show significantly lower frequencies of small intestinal Th17 cells compared to mice fed similar high-fat, low-CHO diets shown in Figure 4A for 3 weeks (n = 4-5 mice/group, *P < 0.05, **P < 0.01, ****P < 0.0001; one-way ANOVA with Tukey’s test). Representative flow cytometry plots of the IL-17a+ CD4+ live cell populations are shown to the left. (E, F) No significant differences in (E) IFNγ+ CD4+ Th1 and (F) Foxp3+ CD4+ Treg cell populations in the small intestine between groups after 3 weeks of dietary intervention (n = 4-5 mice/group, ns = not significant; one-way ANOVA with Tukey’s test). (G) Th17 cell populations in the epididymal white adipose tissue of mice fed the respective diets for 3 weeks. Representative flow cytometry plots of the IL-17a+ CD4+ live cell populations in epididymal fat are shown to the left (n = 5 mice/group, ***P < 0.001, ****P < 0.0001; one-way ANOVA with Tukey’s test). (H) IFNγ+ CD4+ (Th1) cell populations in the epididymal white adipose tissue of mice fed the respective diets for 3 weeks (n = 5 mice/group, **P < 0.01; one-way ANOVA with Tukey’s test). (I) Foxp3+ CD4+ (Treg) cell populations in the epididymal white adipose tissue are not significantly different between groups after 3 weeks of dietary intervention (n = 5 mice/group, ns = not significant; one-way ANOVA with Tukey’s test). Data are presented as mean±SEM. Each data point represents an individual, singly-housed animal. See also Figure S7.