The tongue microbiome and nitric oxide bioavailability across the human lifespan

Context: The human digestive system contains trillions of microbes collectively called the gut microbiota. Important discoveries have been made through better understanding the role the gut microbiota has in maintaining physical and mental health. Imbalances in the oral microbial community, and poor dental health, have been associated with impaired cardiovascular health, but the role of the oral microbiota as a potential modulator of human health is not well understood. One mechanism that may link the oral microbiota to good health is the nitrate-nitrite-nitric oxide (NO) reduction pathway. This, for its first step, crucially relies on the reduction of nitrate, which has been ingested through the diet, to nitrite by bacteria residing in the mouth. Among many other physiological roles, NO regulates vascular endothelial function, and therefore blood pressure (BP). In the UK, at least 25% of adults and more than 50% of people over 60 years old have elevated BP and this is considered a significant cause of premature morbidity and mortality. Epidemiological studies indicate that a greater consumption of high nitrate foodstuffs (specifically, green leafy vegetables and some fruits, such as in the 'Mediterranean' or '5 a day' diets) may protect against adverse cardiovascular events, and it is known that dietary nitrate supplementation significantly reduces BP in both young and older adults. Differences between individuals in the ability to reduce nitrate to nitrite and in the effects on BP indicate that the benefits derived from a high nitrate diet may be contingent on individuals having the 'right' oral microbiota.

The ability to produce NO in the body through the classical pathway involving nitric oxide synthase (NOS) enzymes is impaired in older age, leading to a gradual increase in BP. We propose that the nitrate-nitrite-NO pathway has significant potential to compensate for dysfunctional NOS in older age. Little is known, however, about how the symbiotic relationship between the human host and the oral microbiota might change with ageing and how the microbiota could be harnessed to maintain cardiovascular health during ageing.

Aims and Objectives: We aim to investigate whether the prevalence of specific nitrate-reducing bacteria residing on the surface of the tongue influences the cardiovascular benefits that may be derived from the consumption of a high nitrate diet and whether there are differences between young and older adults. This builds on our preliminary data which indicate differences in the oral microbiome between young and older adults as well as correlations between the prevalence of specific oral bacteria in an individual and their ability to reduce a standardised dietary nitrate bolus to nitrite and the observed reduction in BP. This suggests that augmentation of this nitrate-nitrite-NO pathway through manipulation of diet and/or the oral microbiome might compensate for a reduced ability to generate NO endogenously with older age.

Objectives:
1) Identify the bacterial species responsible for the reduction of inorganic nitrate to nitrite in the human oral cavity.
2) Compare the oral microbiomes and nitrate reduction ability of young and older adults.
3) Establish the in vivo effects of dietary nitrate supplementation and antibacterial mouthwash on the oral microbiome, NO bioavailability/bioactivity and indices of cardiovascular health (blood pressure, arterial stiffness, flow-mediated dilation).
4) Assess the effects of nitrate, antibacterial mouthwash and the sweetening agent, xylitol, on the quantities and relative abundances of oral bacteria in an in vitro biofilm model.

Potential Applications and Benefits: The results of this work, which will be undertaken in collaboration with industrial sponsor DuPont Nutrition & Health, may ultimately lead to the development of prebiotic and/or probiotic interventions that help to maintain NO homeostasis and cardiovascular health across the human lifespan.

This proposal is based on the overarching hypothesis that the constitution of the human oral microbiome modulates nitric oxide (NO) bioavailability and, consequently, indices of cardiovascular health. We aim to investigate whether the prevalence of specific nitrate-reducing bacteria residing on the surface of the tongue influences the cardiovascular benefits that may be derived from the consumption of a high nitrate diet and whether there are differences between young and older adults. This builds on our preliminary findings which indicate differences in the oral microbiome for young and older adults as well as inter-relationships between the prevalence of specific oral bacteria in an individual and their ability to reduce a standardised dietary nitrate bolus to nitrite and the reduction in resting blood pressure. This suggests that augmentation of this nitrate-nitrite-NO pathway through manipulation of diet and/or the oral microbiome might compensate for a reduced ability to generate NO endogenously with older age. We shall use next generation sequencing of 16S rRNA genes to characterise the oral microbiome, including nitrate-reducing bacteria, in young and older adults and use chemiluminescence to measure plasma and saliva [nitrate] and [nitrite]. We will recruit healthy young (18-30 years) and older (65-80 years) adults to undergo dietary supplementation with nitrate-rich and nitrate-depleted (placebo) beetroot juice in a double-blind cross-over design to test the effects of the nitrate content of the diet on the abundance of nitrate-reducing bacterial species and indices of cardiovascular health (blood pressure, arterial stiffness, flow-mediated dilatation). We shall also assess the effects of daily antibacterial mouthwash use on the microbiome and NO bioavailability. The above in vivo work will be supported by parallel in vitro experiments designed to evaluate the effects of these same interventions as well as xylitol on bacterial biofilms.

This study will establish how lifestyle choices including diet may influence the oral microbiome, and subsequently increase or decrease the ability of young and older adults to activate dietary nitrate (obtained from a vegetable-rich diet) to derive cardiovascular health benefits. The academic team (3 Professors, 2 Senior Lecturers, 2 Postdoctoral Research Fellows and a Research Nurse shared between the College of Life and Environmental Sciences and the Medical School at the University of Exeter and the Dental School at Cardiff University) and their industrial collaborators, DuPont Nutrition & Health, will benefit through the potential discovery and development of new classes of prebiotics and alternative sweeteners. This multi-disciplinary research group will use the results to generate high-impact publications and for continued research funding applications to expand on the applications of the microbiome-human cross-talk and develop prebiotic and probiotic interventions, designed primarily to impact cardiovascular and metabolic health across the lifespan. The post-doctoral research fellows and the research nurse will benefit from a multi-disciplinary research environment and from the research training provided by the senior academics, as well as study management training from the Clinical Trials Unit. Furthermore, they will develop unique skill sets using state-of-the-art methodologies in our laboratories and in the industrial sponsor's research laboratory at DuPont Kantvik, thus significantly enhancing future career prospects beyond this project.
Our research will benefit the wider NO biology, nutrition, exercise science/physiology and microbiome and health research fields. We will share our findings and methodological advances at international conferences and encourage the project team to attend scientific meetings outside their normal specialism to add further interdisciplinary value to the work, and develop opportunities for expanded collaborations with both academic and industrial bodies. We intend to publish the work in open-access international peer-reviewed journals that we shall highlight on our university web sites and through social media channels. The proposed work will also be of benefit to researchers investigating the microbiota throughout the entire alimentary tract. We will deposit microbial gene sequence data in relevant databases that are accessible to scientists or the general public as appropriate.

More than 25% of all UK adults and more than 50% of people over 60 years of age have elevated blood pressure. Dietary interventions that help maintaining normal blood pressure across the lifespan could significantly reduce the risk of developing cardiovascular diseases. Our research may impact the general public in the future (within ~10 years) by enabling development of a cost-effective strategy to limit the adverse effects of vascular aging, with important implications for quality of life in older age and the burden on the UK and world economies.

The proposed work will extend Exeter's impact on the 'functional foods' and sports supplements industry. Scientific evidence generated by our research group on the benefits of nitrate-rich foods for cardiovascular health and exercise performance has resulted in the creation of new product lines and increased sales of nitrate supplements. The intellectual property generated from this research will impact DuPont R&D activities (particularly at Kantvik Active Nutrition Group) and product development.

The proposed research will have direct impact on the study participants, who will be recruited from the 'Exeter 10,000' database and the Exeter 'University of the 3rd Age', as well as among the University of Exeter student population. We will host regular coffee mornings to provide information on nutrition and health and feedback on the study outcomes to participants and we will use newsletters to broadcast our messages more widely amongst the local community.