Finding Needles in the Gut Microbiota’s Haystack

Despite the seeming recency of our recognition of the connection between the gut microbiota and atherosclerotic cardiovascular disease (ASCVD), the concept of ingesting beneficial microbes to change the gut microbiota and prevent or treat human disease is not a new one. Roman author and philosopher Pliny the Elder’s Natural History, first published around the year 77, described the use of fermented milk to treat intestinal problems. Over 1800 years later, Nobel Prize winner Élie Mechnikov proposed that Bulgarian peasants were outliving Bulgarian nobility, despite harsher living conditions, due to lactic acid-producing bacteria (Lactobacillus bulgaris) in yogurt commonly consumed by the peasants that were observed to be present in the colon.¹ Around the same, Henri Tissier discovered a Bifidobacterium in the gut bacteria of infants who were breastfed that he associated with reduced gastrointestinal illness.²

Research connecting the gut microbiota and ASCVD, including precursor steps to the development of clinical ASCVD, endothelial dysfunction, and vascular stiffening, began appearing more frequently than a handful of publications a year less than two decades ago. Mechanistic animal and cell-based data, along with human cross-sectional studies implicate differences in the gut microbiota composition and altered microbiota metabolite production in response to dietary changes³ as critical drivers of a significant component of the excessive oxidative stress,^4,5 reduced nitric oxide,⁴ increased inflammation,^3,6,7 and cellular senescence⁸ that drive the development of ASCVD.

These data drive continued enthusiasm for oral probiotics, live micro-organisms, to improve cardiovascular health. However, a critical barrier to progress is figuring out which probiotic to give to whom? The human gut microbiota contains an estimated 10¹³ microbes with the latest estimates suggesting at least approximately 3600 different species.⁹ In humans, the gut microbiota composition has significant differences based on sex, age, and prevalent disease states.¹⁰ With all of these variables, how do we rationally determine which microbes could be beneficial in preventing and mitigating ASCVD?

To address this question, Luo et al performed an elegant set of experiments, leveraging careful human cross-sectional studies to inform mechanistic studies in mice to identify Flavonifractor plautii’s potential favorable effects on vascular stiffness as well as its potential mechanism of action through suppression of vascular inflammation and matrix metalloproteinase-2 (MMP-2) expression.¹¹ F. plautii is a gram-positive rod and member of the Clostridiales family that is commonly found in the human gut flora. The research team began by using shotgun metagenomic sequencing of stool DNA to compare the gut microbiome species richness and genus level composition of 44 individuals with elevated arterial stiffness [brachial-ankle pulse wave velocity (baPWV) ≥ 14 m/sec] and 44 age and sex-matched controls recruited from a community center in southern China. Reassuringly, similar findings related to the diversity of the microbiome were found in a validation cohort of 179 subjects from a separate local population. They identified increased expression of F. plautii as the microbial species with the strongest ability to discern between those categorized as having normal vascular compliance versus increased vascular stiffness. This finding was validated in a multivariable-adjusted model with baPWV as a continuous variable and F. plautii as the only microbial species found to correlate (inversely) with baPWV, systolic blood pressure, and diastolic blood pressure.

Co-occurrence analysis further identified F. plautii as a backbone species of positive interactions that were part of a more diverse, more complex network of gut microbiota in healthy controls, which was notably absent in arterial stiffness subjects. Mediation analyses showed that F. plautii association with vascular stiffness was, in part, independent of blood pressure. Using MetaCyc to map genes to metabolites and metabolic pathways, 41 metabolic pathways were identified as associated with baPWV, nearly all of which correlated with F. plautii expression. Using liquid chromatography–mass spectrometry, 54 metabolites were determined to be associated with baPWV, and two of these metabolites, cis-aconitic acid (CAA) and 2,5-furandicarboxylic acid, were positively associated with F. plautii expression in fecal samples.

Taken together, the experimental procedures to this point suggest a potential mechanistic connection between F. plautii and vascular stiffness that could be mediated by CAA and 2,5-furandicarboxylic acid. Many times, in the literature of this field, this is where the story ends. However, Luo et al take their work several steps further, leveraging animal models to further test the strength of the evidence for causal connections between F. plautii, CAA and/or 2,5-furandicarboxylic acid, and vascular stiffening.

Using antibiotic-treated mice and fecal microbial transplant (FMT) with microbiota from human control donors and elevated arterial stiffness donors, Luo et al found that mice receiving FMT from control donors had significantly lower arterial stiffness. Mice receiving FMT from increased arterial stiffness donors showed increased arterial stiffness, increased blood pressure, enhanced MMP-2 expression associated with large artery elastic fiber disruption, and increased pro-inflammatory monocyte chemoattractant protein-1 (MCP-1) and NF-κB expression compared to mice who received FMT from humans with normal stiffness. Dietary supplementation with F. plautii reversed these effects in mouse models both with and without hypertension induced by angiotensin II infusion. Further, F. plautii supplementation only increased circulating CAA and not 2,5-furandicarboxylic acid, suggesting only the change in CAA in the human plasma samples was likely to reflect F. plautii metabolic activity. Finally, CAA supplementation alone had nearly identical effects as F. plautii supplementation, leading the authors to conclude that CAA was likely a critical driver of F. plautii’s favorable effects on vascular compliance through reduced vascular inflammation and MMP-2 expression.

Luo et al’s efforts provide a translational “roadmap” for the discovery of microbial species in the human microbiota (summarized in the Figure), with the potential to include in oral probiotics for the improvement of cardiovascular health. The approach involves connecting observational human phenotypical data to (1) sequencing and characterization of the gut microbiome; (2) informatics tools to analyze differences in metabolic pathways affected by microbiome differences between human groups; and (3) untargeted metabolomics. These data may reveal connections between the targeted phenotype and relevant microbial species and metabolites that can inform mechanistic studies in preclinical models to uncover mechanisms of effect. Following a systematic approach to oral probiotic discovery that integrates human data early in the process holds promise for speeding the development and testing of probiotics as therapeutics for a wide range of diseases, including ASCVD. Additionally, metabolites identified as mediators of beneficial microbial effects could be leveraged for development into novel pharmacological agents to benefit cardiovascular health. Existing data repositories with curated human microbiomes and matching metabolomic data along with advances in tools to analyze connections between the microbiome and metabolites could be leveraged to efficiently uncover additional promising microbes for the treatment of human disease.

Figure 1: Proposed model of discovery for new beneficial microbial species.

Inclusion of human phenotypic observational data early in the discovery process to guide discovery of new microbial species that can be developed as potential therapeutics.

So, in F. plautii, has Luo et al found a proverbial needle in the human gut microbiota haystack, or is it just another straw? Possibly. The study has some limitations that require consideration. The study population is small (89 subjects). Therefore, the discovery work in human samples required loosening of acceptable false discovery rates in some of the human microbiome analyses. This is reasonable for discovery work in this area given the size of the datasets and expense of these analyses but suggests additional validation studies should be considered. The study population also has unique attributes (located in southern China, healthy other than some who were current smokers, and some with untreated mild hypertension). Diet, environmental exposures, disease states, sex hormones, and medications all considerably impact the composition of the gut microbiota which in turn influences the metabolic activity of the microbiota. Therefore, the connection between F. plautii, CAA, and human vascular stiffening observed by Luo et al cannot be generalized outside the small human population studied, as noted by the authors. Measures of vascular stiffness like baPWV are a combination of fixed structural effects and dynamic activity of vascular endothelium, including nitric oxide production.¹² The impact of F. plautii and CAA on dynamic endothelial function were not well-delineated in the study and merit additional investigation to determine a more complete spectrum of action. CAA’s mechanism of action for the suppression of MMP-2 remains to be determined by additional studies. Finally, several case reports describe systemic infection caused by F. plautii, most commonly associated with immunosuppressed states.¹³ These case reports suggest human testing of F. plautii as a therapeutic should be limited to immunocompetent individuals.

In summary, Lou et al need to be commended for their ability to introduce a novel species that may have beneficial capabilities on arterial stiffness. They successfully showed an inverse association of F. plautii to baPWV measurements in humans and established causality in a murine model. We anticipate that their results will be met with eagerness for further translational studies involving F. plautii and vascular function.