Chronic low-grade inflammation is a pathological characteristic of obesity-related conditions such as metabolic syndrome, type 2 diabetes mellitus, and cardiovascular disease and is noted by increased and sustained systemic levels of proinflammatory cytokines with no resolution (1–3). The accumulation and pro-inflammatory activation of macrophages in expanding adipose tissue, particularly around the abdomen, is considered a major contributor to chronic low-grade inflammation and promotes a detrimental shift in glucose and lipid metabolism (4–6). In addition to central adiposity, altered glucose metabolism, lipid metabolism, and hypertension are key components of metabolic syndrome, which proceeds and underlies many major chronic diseases (7). As an early driver of metabolic syndrome and chronic disease progression, intervention strategies are needed to counter inflammatory stimuli and chronic low-grade inflammation in metabolically at-risk populations.

The composition of, and host interactions, with the gut microbiota are of growing research and clinical interest. The gut microbiota comprises a diverse group of bacteria (and other microbes) in the human gastrointestinal tract that is altered in metabolic states associated with obesity and related conditions (8–11). A strong body of evidence in human and animal models supports the contribution of the gut microbiota to obesity development through increased energy harvest (8–10, 12–14). The composition and functionality of the gut microbiota, and ability to influence the host, is largely dependent on genetic and environmental factors (15, 16). Host diet, as a modifiable environmental factor, has been shown to influence gut microbial composition (16–19). For example, a high-fat diet (HFD) is often utilized in animal models to promote obesity and has been shown to concomitantly promote inflammation in the colon, adipose tissue, skeletal muscle, and the liver (20–22). Increased gut barrier permeability has been proposed as the connection between the gut microbiota and host inflammation. Alterations in gut bacteria, mucus bilayer composition, intestinal epithelial cells tight junctions, and local immune cells can all impact permeability (23–25). Chronic exposure to dietary fat is another factor that may negatively impact tight junction proteins and mucosal layer integrity, promoting increased translocation of bacterial constituents into the lamina propria and a subsequent induction of inflammatory responses establishing a positive feedback loop (23, 24, 26). Thus, frequent perturbations to barrier function can have profound clinical implications in diseases with localized inflammation and may partially explain a rise in systemic inflammation.

To combat inflammation, incorporation of functional foods may serve as a beneficial dietary strategy for metabolically at-risk populations to reduce the onset and progression of chronic disease (27, 28). Polyphenols can act as antioxidants which reduce cellular damage by free radicals and alter cellular signaling affecting inflammatory gene expression (29). A majority of polyphenols reach the large intestine and are subject to bacterial degradation into simpler phenolic metabolites (30). Polyphenols and phenolic derivatives can positively impact gut barrier integrity which may impact downstream proinflammatory processes (31) but may exert anti-inflammatory properties systemically (32). Importantly, polyphenols and their derivatives can modulate microbial diversity (33) and in turn, microbial metabolism can influence their bioavailability (34–36).

The black chokeberry, or Aronia melanocarpa, is of particular interest as an antioxidant-rich food. This hardy shrub produces fruit with the highest known antioxidant capacity for fresh fruit as measured by oxygen radical absorbance capacity (37). Commercial varieties are derived from a cross between Aronia melanocarpa, which is native to the United States, and European Mountain Ash (Sorbus aucuparia) sometimes classified as Aronia mitschurinii (38). Aronia melanocarpa fruit has a high polyphenolic content (39) with almost four times the anthocyanin content of blueberries, a lauded natural antioxidant-rich food (40). The antioxidant capacity of Aronia fruit (technically a pome fruit, like apples) stems from anthocyanins, procyanidins, and hydroxycinnamic acids. Aronia polyphenols have been previously shown to reduce inflammatory stimuli (41–45), the expression and concentration of pro-inflammatory cytokines (43, 44, 46, 47), and influence the colonic environment (45).

Dietary strategies, such as increased antioxidant availability through consuming polyphenolic-rich foods, present possible targeted therapeutic avenues for early chronic disease prevention. Dietary patterns can modulate gut microbiota composition and function, which are important in mediating host responses through production of bioactive compounds. Therefore, we hypothesized that compounds in host systemic circulation may provide key insights into the dynamic interplay between the gut microbiome and the host. In this study, we recruited healthy but metabolically at-risk human subjects and assessed their metabolic and systemic inflammatory profile. We created a humanized mouse model of chronic low-grade inflammation by transplanting stool from a human donor with high or low systemic inflammation into germ-free mice to account for variability in host genetics and host gut microbiota. We then introduced Aronia or control juice to their offspring and added a HFD as an inflammatory stimulus. Using advanced analytical measurement techniques including 16S rRNA gene sequencing and ultra-high performance liquid chromatography mass spectrometry, we investigated whether ingestion of polyphenolic rich Aronia juice could offer protective effects against HFD, and if that protection is dependent on gut microbial alterations including the inflammation phenotype of the human donors.

Using gnotobiotic mice, we were able to elucidate the impact of Aronia juice supplementation on the gut microbiome and the serum metabolome before and during a high-fat diet. Success in this study relied heavily on our ability to humanize mice by transferring microbiomes from human stool donors who were metabolically similar but had a different inflammatory profile as well as the ability to transfer the donor microbiome to second-generation pups. Additionally, we were able to generate unique metabolomic profiles from each donor specific mouse population. By exploring the beta diversity of the microbiomes along with the grouping of the global metabolomes and specific microbial populations and metabolites, our analysis indicated that two distinct humanized mouse populations were created. This is a significant observation that has broad impacts on the use of animal models to study inflammation.

Each mouse population had a unique microbial profile that closely resembled that of the original human stool donor (Figure 2). Human stool donors were metabolically similar (Table 1), with the specific donors selected based on their distinct low and high systemic inflammatory profile. A higher microbial diversity was observed in the selected human LO microbiome profile vs. HI microbiome profile, a trait that was recapitulated in the mice and helped separate the microbial communities into two distinct groups at baseline (Figure 4). The transfer of microbiomes from inoculated germ-free mice to the second-generation without declines in diversity has been previously observed (19). Additionally, our findings echo previous research which found that low gut bacterial diversity in humans correlated with a pronounced inflammatory phenotype (65). High bacterial diversity is a generally accepted indicator of a healthy gut and is thought to contribute to the resistance of microbial communities to ecological perturbations and the ability to return to equilibrium (10). Along with distinct microbial communities, metabolomic analysis indicated discrete global profiles in each donor population. Metabolomic profiles of the mice grouped based on donor and revealed the presence of discriminating metabolites (Figure 3; Supplementary Table S2). One of discriminating metabolites, indolepyruvate, was upregulated in the LO mice and is only known to be produced by specific Clostridium species associated with the LO microbiome. Conversely, 3-hydroxyproline was upregulated in HI mice. 3-hydroxyproline is an imino acid shown to stimulate inflammation and modify macrophage signaling (66). 3-hydroxyproline has also been shown to be diet dependent in mammals, yet mice in this study received the same food, indicating a microbial influence promoting 3-hydroxyproline production (67).

After 2 weeks of juice supplementation and normal diet, a shift was detected in the gut microbiota of second-generation mice (Figure 5). The control and Aronia juice both contained glucose, fructose, and sorbitol, and the introduction of these carbohydrates was also marked by a pronounced shift in β-diversity after 2 weeks across both LO and HI mice. The impact of Aronia on β-diversity was minimal, though we detected a statistical main effect with Aronia juice for an increase in α-diversity (Figure 4). We observed that bacterial changes during the Aronia supplementation period were not donor-specific and that Aronia promoted an increase in the abundance of Eisenbergiella and Faecalibacillus. Eisenbergiella is negatively correlated with inflammatory gene expression (68) and one study has found Faecalibacillus to positively associate with hypertension (69). The metabolic capacity of either genus is still largely unknown; thus, additional research is required to better understand the metabolic capacity of these two bacterial taxa and particularly, their capacity to metabolize (poly)phenolic compounds and relationship to health outcomes.

Not only were changes in the gut microbiome makeup observed after 2 weeks of supplementation, but mouse metabolism was also altered as well. Prior to juice introduction mouse metabolic profiles had grouped by donor (Figure 3; Supplementary Table S2). However, by 2 weeks profiles indicated a significant shift and began to cluster by juice group (Figure 7; Supplementary Table S2). Indoleacrylic acid was upregulated with Aronia only in mice with the low inflammation gut microbiome donor. Indoleacrylic acid is a tryptophan catabolism metabolite with potent antioxidant, anti-inflammatory, and enhanced gut barrier function effects. This metabolite is only produced by three bacterial species of the Peptostreptococcus genus and one species of the Clostridium genus, which pinpoints the sources of this microbiome dependent health benefit of Aronia (70). Further, the metabolic switch was characterized by an increase in phosphatidylcholines (PC) in Aronia treated mice. Increased PC composition has been indicated in a variety of positive health benefits. In addition to their role as structural components of cellular membranes, PCs can counter LPS-induced inflammation by inhibiting tumor necrosis factor expression as well as limiting inflammatory responses by suppressing pro-inflammatory pathway signaling (71–73). These positive benefits are augmented by the ability of PCs to impact membrane composition and fluidity. Barriers in the intestinal mucus are ameliorated with higher PC composition and a decrease in PC composition is seen in inflammatory conditions such as inflammatory bowel disease and ulcerative colitis (74–76). This observation is likely due to PC-specific modulation of membrane composition and a PC-induced increase in membrane fluidity which contributes to normal cell function. Reduction in PCs has been indicated in rigid membranes and promoted aging processes (77–79). The increased serum PCs seen in Aronia treated mice may be due to complexation of phosphatidylcholines with polyphenolic compounds and polyphenolic derivatives. Anthocyanins are larger polyphenols which do not pass easily through the intestinal barrier by diffusion, thereby limiting bioaccessibility to the host. Formation of phyto-phospholipid complexes, which improves membrane permeability, miscibility, and absorption of polyphenols from the gut for host bioavailability (80–82). Given we did not observe phosphatidylcholine increases in control mice receiving the same diet, we propose that the increase in phosphatidylcholine with Aronia supplementation was facilitated through the complexation process of polyphenolic compounds present in Aronia juice.

To elicit an inflammatory response reflective of a Western diet, the normal diet (13.3% fat) was switched to a high-fat diet (41.7% fat). This allowed for a comparison of Aronia supplementation with and without an inflammatory stimulus and provided a model to determine the possible Aronia driven effects during an HFD. High fat diets induce reproducible shifts in the gut microbiome independently of fluctuations in body weight (83, 84). As expected, we observed a substantial global shift in the microbial community with the introduction of HFD across both donor groups (Figure 5B). This effect was donor-dependent as HFD facilitated a prominent shift in the β-diversity of the microbiota to which the LO INF mice were more resistant. Further, a key finding in this study was the donor-dependent protective effect of Aronia supplementation against a loss of α-diversity during the HFD, with LO INF mice less susceptible to losses in α-diversity (Figure 4B). A greater α-diversity and increased presence of beneficial taxa such as Parasutterella, Faecalibacterium, and Clostridium Cluster XIVa (Supplementary Figure S3) may have partially contributed to this protective effect in LO INF mice.

The mice continued the experimental juice for 6 weeks concomitant with HFD. After 6 weeks, additional juice-dependent changes in the gut microbiota were observed. The impact of Aronia supplementation on the global microbial composition during HFD was modest as indicated by our constrained ordination (Figure 5B). However, specific taxonomic groups were unique to Aronia supplementation, with a greater abundance of Faecalibacillus, Howardella, and an unclassified member of Eggerthellaceae during the last 6 weeks (juice and HFD). The unclassified Eggerthellaceae genus increased 7-fold which aligns with the known ability of the Eggerthellaceae family to metabolize (poly)phenols. The genus has also been positively related to lipid metabolism (85). The functions of Howardella and Faecalibacillus are poorly known and present new genera to explore in the realm of (poly)phenol metabolism. The only genus to decrease with Aronia was Bifidobacterium, with losses approximately 4.7-fold compared to control juice. Bifidobacterium species have been previously found to metabolize anthocyanins, which suggests that a HFD may compromise the metabolism of anthocyanins by reducing Bifidobacterium (86).

The influence of HFD was also seen in an analysis of the metabolomic profiles. Profiles from week two and week four show major shifts in metabolic activity regardless of the donor microbiome or juice (Supplementary Figure S4). A comparison of the top discriminating features using hierarchical clustering between weeks two and four showed strong grouping pre- and post-HFD introduction. Significant changes were observed indicating a large-scale shift in metabolic activity due to the HFD and the associated inflammatory stimulus. A closer look at specific discriminating metabolites revealed that sphingomyelin was upregulated after the HFD introduction. Sphingolipids are important components of cellular membranes and are signaling molecules important in the regulation of inflammatory pathways (87, 88). Although definitive clarity on the effects of sphingolipids on inflammation requires additional study, initial results have demonstrated tumor necrosis factor-dependent increases in sphingomyelin (89). Upregulation of sphingomyelin induces production of sphingomyelin-1-phosphate through the action of the kinases Sphk1 and Sphk2 in the sphingolipid-to-glycerolipid pathway (90). Increases in sphingomyelin-1-phosphate have also been associated with immune recruitment, lymphocyte activity, inflammation and progression of inflammatory diseases and cancers (91). Reduced sphingolipids were previously shown to increase intestinal inflammation in irritable bowel disease subjects though interestingly, host-derived sphingolipids were increased systemically (88). Therefore, it is possible that the upregulation of sphingomyelin we detected was an early stress response to increased dietary fat load, mediated by elevated LPS-stimulation of TLR4 and proinflammatory activation.

After the HFD introduction, metabolomic profiling indicated the continued influence of Aronia supplementation. However, the donor impact was stronger with high-fat diet than with normal diet and more donor-specific effects were discovered. Due to multiple strong effectors, examination of the metabolomic profiles show little separation by donor or by juice. Yet, an analysis of metabolomic profiles for each donor indicated different characteristics based on juice treatment. This provides evidence that microbiota from different donors interact uniquely with the polyphenol-rich Aronia fruit juice leading to disparate metabolomic profiles. A focused analysis at 8-weeks was completed and indicated donor and juice specific metabolites upregulated in the mouse models with Aronia supplementation again resulting in an increase in PCs (Figure 8B). To determine observed PC increases were influenced by alterations in microbial makeup or metabolic interactions with the polyphenolic compounds found in Aronia, TMAO was isolated and quantified. TMAO has been shown to be a biomarker for increased risk of cardiovascular disease and other chronic inflammatory diseases and is formed in the liver through the oxidation of TMA by FMO3 (92). TMA can only be generated by a subset of gut microbiota that possess cutC, a glycyl radical enzyme. The cleavage of choline by cutC forms TMA which can then be transported across the lumen. Our data indicates that at week 8, Aronia treated mice had a significantly lower TMAO concentration than control mice (Figure 9). Further analysis of the Aronia treated mice revealed that TMAO concentrations decreased more dramatically in the HI mice than in the LO mice showing a shift in microbial makeup presumably resulting in a decrease in microbes possessing cutC. This analysis indicated that both microbial makeup and phenolic compound availability had an impact on TMAO, and likely PC, concentrations.

While demonstrating several important gut microbiome and metabolic impacts of Aronia juice in humanized mice, we also acknowledge that our study has limitations. Our experiment was set up as a pilot study to demonstrate proof of concept demonstrating gut microbiota dependent impacts linked to inflammation phenotype. As a result, the limited number of mice in our study was dependent on litter sizes. While we demonstrated several key findings, we acknowledge that a greater sample size will be needed to further explore additional impacts. Similarly, both human gut microbiome donors were female, and our study may reflect sex-specific metabolic differences. Sexual dimorphism is present in diet, nutrient metabolism, and gut microbiome composition (93). Thus, our findings need to be confirmed in males and future research incorporating male and female gut microbiota donors is needed to evaluate whether there are differential responses to Aronia fruit juice supplementation.

In this study, we were able to successfully humanize a second-generation of germ-free mice inoculated with stool from human donors with different systemic inflammatory profiles, the results of which were reflected in disparate microbiota and metabolomic profiles. This allowed for a comparison of polyphenol-rich juice treatment, consisting of Aronia melanocarpa fruit juice between two distinct mouse populations. Using 16S rRNA sequencing and mass spectrometry analysis, donor specific microbial communities and metabolites demonstrated distinct responses to juice treatment and the subsequent introduction of an inflammatory stimulus through a HFD introduction. Importantly, Aronia juice offered protective effects against HFD that were microbiome dependent. Metabolomic responses were centered around an increase in the phosphatidylcholine with juice supplementation and sphingomyelin with the introduction of HFD. Further research in should consider replication with additional stool donors for better generalizability.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/, PRJNA596000, https://www.ncbi.nlm.nih.gov/, PRJNA906115, https://www.metabolomicsworkbench.org, ST004409.

The studies were conducted in accordance with the local legislation and institutional requirements. The studies involving humans were approved by the Montana State University Institutional Review Board. The participants provided their written informed consent to participate in this study. The animal study was approve by the Montana State University IACUC.

CY, SWa, ZM, BB, and MM: conceptualization. MM, CY, SWa, BB, and JP: methodology. SWi and JP: software, formal analysis, data curation, writing—original draft preparation, and visualization. SWi, JP, and HF: investigation. SWa, ZM, BB, and MM: resources. SWi, JP, CY, SWa, BB, and MM: writing—review and editing. SWi: project administration. SWa and MM: supervision. CY, SWa, ZM, BB, and MM: funding acquisition. All authors contributed to the article and approved the submitted version.

This research was supported by Montana State University Research Initiative 51040-MSUR12015-03 and USDA-NIFA 2017-67018-26367. Funding for the Montana State Mass Spectrometry Facility used in this publication was made possible in part by the MJ Murdock Charitable Trust, the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers P20GM103474 and S10OD28650, and the MSU Office of Research and Economic Development. Funding sources were not involved in the design, data collection and analysis, and writing of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.