The influence of nutrition on human immunity is not fully understood, despite the known impact of diet on host physiology through fuel provision and microbiota modulation. The microbiota, shaped significantly by nutrition, can influence host resilience. Existing studies often examine only single diets, ignoring the variability in individual responses. This research addresses this gap by employing a highly controlled clinical setting where participants consumed distinct (ketogenic and vegan) diets sequentially for two weeks in random order. This is, to the authors' knowledge, the first multi-omics study exploring the effects of these diets on humans, aiming to enhance understanding of diet-based therapeutic options for disease prevention and treatment.

Previous research has established the link between nutrition and the gut microbiome, with diet significantly influencing microbiome composition and function in experimental models. However, the effects of nutritional interventions on the human immune system remain under-explored, particularly using a multi-omics approach. Studies have often focused on single diets, neglecting the high inter-individual variability in responses to dietary changes. Prior work highlighted the impact of ketogenic diets on TMAO and other cardio-metabolic risk markers, as well as the varied responses of individuals to different diets, underlining the need for more comprehensive studies exploring multiple diets in a controlled setting.

Twenty participants were admitted to the NIH Clinical Center for a crossover study. Each participant consumed ad libitum either a ketogenic (75.8% fat, 10% carbohydrate) or vegan (10.3% fat, 75.2% carbohydrate) diet for two weeks, with the order randomized. Both diets included non-starchy vegetables and minimal processed food. Baseline food intake was assessed via a questionnaire. Blood samples were collected at various time points for flow cytometry (n=7), bulk RNA sequencing (RNA-seq) (n=6), and SomaLogic proteomic analysis (n=20). Fecal samples (n=10) were collected for microbiome metagenomic sequencing and metabolomic analyses were conducted on blood and urine samples (n=20). Note that due to sample availability, not all assays were performed on every participant. Flow cytometry focused on major immune cell types, excluding neutrophils. RNA-seq of whole blood examined gene expression changes. Blood transcription module (BTM) and Hallmark analyses were employed to identify functional pathways enriched. The SomaLogic assay quantified the abundance of ~1300 proteins in plasma. Microbiome metagenomic sequencing analyzed changes in microbiota composition and function, and functional pathways were evaluated by mapping reads to Enzyme Commission (EC) numbers. Targeted metabolomics analysis examined plasma and urine samples using UPLC-MS/MS. Statistical analyses included linear mixed effects models, paired t-tests, PERMANOVA and others, with multiple-testing corrections employed.

Dietary intervention induced significant changes in lymphoid composition, irrespective of diet order. A significant decrease in naive CD8 T cells and an increase in activated CD4 T cells, effector CD4 T cells, and effector CD8 T cells were observed following both diets. Each diet also produced distinct changes: ketogenic diets increased activated regulatory T cells and CD16+ natural killer (NK) cells, while vegan diets increased activated T helper cells and activated NK cells. Bulk RNA sequencing revealed a polarization in pathway enrichment: ketogenic diets upregulated pathways related to adaptive immunity (T cell activation, B cells, plasma cells, NK cells), including oxidative phosphorylation, while vegan diets upregulated pathways associated with innate immunity and antiviral responses (type I interferon signatures). Proteomic analysis via SomaLogic showed that the ketogenic diet had the most significant effect on protein abundance. The ketogenic diet impacted proteins from blood, brain, and bone marrow, whereas both diets affected proteins from liver and secondary lymphoid organs. Microbiome analysis showed no significant differences in diversity but revealed shifts in composition and function following ketogenic diets. Ketogenic diets downregulated microbial amino acid and vitamin metabolism pathways, likely due to increased amino acid intake. Metabolomic analysis revealed that the ketogenic diet had a stronger impact on plasma metabolites than the vegan diet, with lipids being the most affected. Both diets upregulated amino acid biosynthesis pathways, but the ketogenic diet upregulated BCAA pathways, and vegan diets upregulated alanine, aspartate, and glutamate metabolism. A highly interconnected network analysis, integrating proteomic, metabolomic, and microbiome data, revealed relationships between lipids, amino acids, and immune-related factors, with adaptive immunity-related factors more abundant after ketogenic diets.

This study demonstrates that short-term dietary intervention dramatically alters host immunity, overriding genetic, age, sex, and BMI effects. The observed lack of influence from diet order suggests that two weeks is sufficient to significantly change immunity, microbiome composition, and metabolic profiles. However, the long-term effects and the impact on tissue immunity outside the blood remain to be investigated. The stronger impact of the ketogenic diet might be attributed to the shift in primary energy source from carbohydrates to fats and ketones. The increased innate immunity after vegan diets could stem from both qualitative and quantitative differences in nutrition. Further investigation into the impact of caloric reduction is needed. The significant impact of the ketogenic diet on the gut microbiome, with downregulation of amino acid metabolism pathways, highlights functional trade-offs between the microbiota and the host. The study's preliminary findings suggest different effects of these diets on cancer-related pathways, though further research is needed to confirm these associations. The convergence of altered pathways in multiple datasets underscores diet's crucial role in shaping human health.

This study provides the first multi-omics analysis of the impact of ketogenic and vegan diets on human health, demonstrating striking and divergent effects on immunity and the microbiome. Ketogenic diets strongly impacted the proteome and microbiome, while both diets significantly changed host immunity and metabolism. Future research should focus on the long-term effects of these diets, the impact on tissue immunity, and further exploration of the mechanistic relationship between diet, the microbiome, and disease.

This study's relatively small sample size limits the generalizability of findings. The lack of a washout period between diets is another limitation, though the consistent results irrespective of diet order suggest that the impacts observed were primarily due to the dietary intervention. The analysis of immune signatures was limited to peripheral blood, leaving the potential effects on tissue immunity open for further study.