A Multiomics Evaluation of the Countermeasure Influence of 4-Week Cranberry Beverage Supplementation on Exercise-Induced Changes in Innate Immunity

Abstract

Objectives: This study examined the effect of a 4-week unsweetened cranberry beverage (CRAN) (317 mg polyphenols) versus placebo beverage (PLAC) ingestion (240 mL/day) on moderating exercise-induced changes in innate immunity.

Methods: Participants included 25 male and female non-elite cyclists. A randomized, placebo-controlled, double-blind crossover design was used with two 4-week supplementation periods and a 2-week washout period. Supplementation periods were followed by an intensive 2.25 h cycling bout. Six blood samples were collected before and after supplementation (in an overnight fasted state) and at 0 h, 1.5 h, 3 h, and 24 h post-exercise. Stool and urine samples were collected pre- and post-supplementation. Outcome measures included serum creatine kinase, myoglobin, and cortisol, complete blood counts, plasma untargeted proteomics, plasma-targeted oxylipins, untargeted urine metabolomics, and stool microbiome composition via whole genome shotgun (WGS) sequencing.

Results: Urine CRAN-linked metabolites increased significantly after supplementation, but no trial differences in alpha or beta microbiota diversity were found in the stool samples. The 2.25 h cycling bout caused significant increases in plasma arachidonic acid (ARA) and 53 oxylipins (FDR q-value < 0.05). The patterns of increase for ARA, four oxylipins generated from ARA-cytochrome P-450 (CYP) (5,6-, 8,9-, 11,12-, and 14,15-diHETrEs), two oxylipins from linoleic acid (LA) and CYP (9,10-DiHOME, 12,13-DiHOME), and two oxylipins generated from LA and lipoxygenase (LOX) (9-HODE, 13-HODE) were slightly but significantly higher for the CRAN versus PLAC trial (all interaction effects, p < 0.05). The untargeted proteomics analysis showed that two protein clusters differed significantly between the CRAN and PLAC trials, with CRAN-related elevations in proteins related to innate immune activation and reduced levels of proteins related to the regulation of the complement cascade, platelet activation, and binding and uptake of ligands by scavenger receptors. No trial differences were found for cortisol and muscle damage biomarkers.

Conclusions: CRAN versus PLAC juice resulted in a significant increase in CRAN-related metabolites but no differences in the gut microbiome. CRAN supplementation was associated with a transient and modest but significant post-exercise elevation in selected oxylipins and proteins associated with the innate immune system.

Keywords: cranberry; exercise; gut microbiome; immunity; metabolomics; oxylipins; proteomics.

Figure 1

Study participant flow diagram.

Figure 2

(a) OPLSDA analysis of 24 h urine samples collected before and after the 4-week supplementation period. R2X = 0.142, R2Y = 0.98, Q2 = 0.416. Data represent metabolite changes when comparing post- and pre-supplementation urine samples. (b) Urine metabolites that were higher in the cranberry trial supported the trial separation data from (a). Metabolites were selected that met these criteria: VIP > 2.0, FDR p < 0.05, high ontology annotation, and literature confirmation as a cranberry-related metabolite. Box plots for the six metabolites show the distribution of the 4-week change in peak intensity for the cranberry and placebo trials.

Figure 2

(a) OPLSDA analysis of 24 h urine samples collected before and after the 4-week supplementation period. R2X = 0.142, R2Y = 0.98, Q2 = 0.416. Data represent metabolite changes when comparing post- and pre-supplementation urine samples. (b) Urine metabolites that were higher in the cranberry trial supported the trial separation data from (a). Metabolites were selected that met these criteria: VIP > 2.0, FDR p < 0.05, high ontology annotation, and literature confirmation as a cranberry-related metabolite. Box plots for the six metabolites show the distribution of the 4-week change in peak intensity for the cranberry and placebo trials.

Figure 3

Violin plots for the change in the sum of 4 plasma DiHETrEs between the cranberry and placebo trials (time effect p < 0.001, interaction effect p = 0.003). The horizontal lines represent the median and interquartile range. Wider sections of the violin plot represent a higher probability that study participants will take on the given value.

Figure 4

Untargeted proteomics heat map for the cranberry and placebo trials across six timepoints (T1 = pre-supplementation, T2 = post-4 weeks supplementation, T3 = 0 h post-exercise, T4 = 1.5 h post-exercise, T5 = 3 h post-exercise, and T6 = 24 h post-exercise).

Figure 5

(a) Cluster A (elevated in the cranberry trial) visualizes the protein–protein interaction (PPI) network for proteins enriched in reactome pathway analysis. The size and color of the nodes indicate the p-value from the two-way ANOVA analysis. Proteins with four or more interactions and statistical significance were chosen as key proteins in the pathway. (b) Cluster B (lower in the cranberry trial) visualizes the protein–protein interaction (PPI) network for proteins enriched in reactome pathway analysis. The size and color of the nodes indicate the p-value from the two-way ANOVA analysis. Proteins with four or more interactions and statistical significance were chosen as key proteins in the pathway. See Table 4 and Table 5 for an explanation of the abbreviations.

Figure 6

Violin plots for the pre- and post-4 week supplementation of stool sample microbiome alpha diversity (observed richness or the number of taxa) with cranberry and placebo beverages. Time effect p = 0.650; interaction effect p = 0.302. The horizontal lines represent the median and interquartile range. Wider sections of the violin plot represent a higher probability that study participants would take on the given value.