Abstract

Inorganic dietary nitrate, found abundantly in green leafy and some root vegetables, elicits several beneficial physiological effects, including a reduction in blood pressure and improvements in blood flow through nitrate–nitrite–nitric oxide signaling. Recent animal and human studies have shown that dietary nitrate and nitrite also modulate inflammatory processes and immune cell function and phenotypes. Chronic low-grade inflammation and immune dysfunction play a critical role in cardiovascular disease. This review outlines the current evidence on the efficacy of nitrate-rich plant foods and other sources of dietary nitrate and nitrite to counteract inflammation and promote homeostasis of the immune and vascular systems. The data from these studies suggest that immune cells and immune–vasculature interactions are important targets for dietary interventions aimed at improving, preserving, or restoring cardiovascular health.

INTRODUCTION

Inflammation is an integral component of the innate immune response to both infectious pathogens and tissue damage.1–3 Under normal physiological conditions, inflammation is a tightly coordinated and dynamic process that initiates pathogen killing and plays a key role in the repair, regeneration, and adaptive remodeling of injured tissue.3–5 Central to restoring homeostasis, self-regulatory mechanisms resolve the inflammatory process once the infection or inflammatory stimulus is eliminated or controlled.3 In contrast to properly controlled acute inflammatory responses, chronic inflammation is characterized by failure to resolve the inflammatory process, owing to dysfunctional resolving mechanisms and/or continuous exposure to inflammatory stimuli.3 Chronic inflammation is also closely associated with oxidative stress,6 which is defined as an imbalance between oxidants and antioxidants in favor of the former, leading to molecular damage and/or disruption of redox signaling and control.7 Unresolved inflammation results in tissue destruction, fibrosis, and necrosis.3 Importantly, chronic low-grade inflammation contributes to the pathology of several age-related conditions,6,8 as well as a wide range of chronic diseases.9–11 Unresolved inflammatory responses contribute especially to the early stages of the development of cardiovascular diseases, which remain a leading cause of morbidity, disability, and mortality in modern societies.6,12,13 Solid evidence exists that diet composition, nutrients, and non-nutrient food components (such as plant-derived bioactive compounds) modulate inflammatory processes.11 Counteracting chronic inflammation through dietary interventions, therefore, represents a key lifestyle strategy for maintaining cardiovascular health and preventing cardiovascular disorders such as atherosclerosis and hypertension.

Another biological process that is critical for cardiovascular health is the ability of endothelial nitric oxide synthase (eNOS) in the vascular endothelium to produce nitric oxide (NO) through the L-arginine-NOS pathway.6,14,15 Nitric oxide is an important signaling molecule with various physiological functions in the human body.15–17 By regulating blood flow and maintaining vascular integrity, NO plays a key role for cardiovascular homeostasis.16 A decline in the production and/or bioavailability of NO is associated with endothelial dysfunction, vascular aging, cardiovascular risk factors, and established cardiovascular disease.6,15 In addition to endogenous NO production through the L-arginine-NOS pathway, NO is generated from inorganic dietary nitrate through the enterosalivary nitrate–nitrite–NO pathway.14,15,18 In this pathway, nitrate, contained abundantly in green leafy vegetables and some root vegetables, is absorbed in the small intestine and enters the blood circulation.14,16,19 Parts of the circulating nitrate are excreted by the kidney, but about 25% of the nitrate is taken up and secreted by the salivary glands and is subsequently reduced to nitrite by commensal bacteria in the mouth.14,16,19,20 The nitrite is then swallowed, absorbed through the intestinal tract, and further reduced to NO and other bioactive nitrite intermediates by enzymatic and nonenzymatic mechanisms in the blood and tissues.14,16,19 In contrast to NOS-dependent NO generation, nitrite reduction to NO in the nitrate–nitrite–NO pathway is oxygen independent.14,16,19 Nitric oxide generation through the nitrate–nitrite–NO pathway is also accelerated in low-oxygen or acidic conditions, which is beneficial during tissue ischemia, which occurs, for example, in patients with chronic cardiovascular disease.14,16 Epidemiological studies suggest that the protective cardiovascular effects of a diet rich in green leafy vegetables are related to the high nitrate content of this type of vegetable.20,21

Following the recognition of the importance of NO bioavailability for cardiovascular health, there has been increasing interest in the preventative and therapeutic potential of inorganic dietary nitrate as an additional source of NO.14,15 A number of studies have shown beneficial effects of dietary nitrate in physiological and clinical settings, and several reviews of these studies are available.14,16,18–20,22,23 Especially with regard to cardiovascular health, strong evidence exists that dietary nitrate reduces blood pressure and improves other markers of vascular function in animal models and healthy human populations.16,18 Evidence in clinical populations at risk of cardiovascular disease is still limited,18,24 but longer-term clinical trials are currently ongoing to address this issue.16

Cell culture, animal, and human studies have emerged in the past few years demonstrating that modulation of inflammation and the immune system might be an important mechanism through which dietary nitrate exerts its cardiovascular benefits.25–30 This review combines and integrates current evidence for increased dietary nitrate intake as a preventative and therapeutic intervention to improve immune function and maintain or restore cardiovascular homeostasis. It summarizes and discusses studies that have investigated effects of dietary nitrate or nitrite on various biomarkers of inflammation, with the major focus on animal and human studies. To present a contemporary view, the specific focus is on available data on the modulation of immune–vasculature interactions through inorganic nitrate or nitrite.

IMMUNE AND VASCULAR SYSTEMS IN HEALTH AND DISEASE

The following section briefly outlines how the vascular system influences immune cells in homeostasis and during inflammation. The sequence and timing of distinct inflammatory stages from initiation to resolution of inflammation have been covered elsewhere.3 This review focuses instead on processes during inflammation that play a role in the pathophysiology of cardiovascular disorders, particularly atherosclerosis and hypertension. It highlights the mechanisms modulated by NO as well as others that might provide potential targets for interventions with dietary nitrate.

Homeostatic functions of the vasculature and immune cells

The major homeostatic functions of the vascular system include the control of blood flow and pressure, regulation of the exchange of macromolecules between blood and tissues, and prevention of inappropriate activation of leukocytes.1 The entire vascular system is lined by a monolayer of endothelial cells.1 The vascular endothelium synthesizes and secretes various biologically active molecules (including NO produced by eNOS) that act in an autocrine and paracrine fashion to modulate the function and health of arteries and surrounding tissues.6 In healthy arteries, the secretory profile of the endothelium is characterized by molecules that favorably modulate vasodilation and coagulant and inflammatory processes.6 In their basal state, endothelial cells prevent interactions with leukocytes, mainly by controlling the expression and secretion of molecules that activate or assist in recruiting leukocytes, including various chemotactic cytokines (chemokines) and adhesion molecules.1 In addition, the basal production of NO by the endothelium through eNOS exerts anti-inflammatory and antiadhesive effects.31

During inflammation, endothelial cells, in conjunction with vascular smooth muscle cells and pericytes, play a central role in recruiting and directing circulating leukocytes to inflamed tissue sites.1,32 The recruitment of leukocytes to the endothelium is mediated by mechanosignaling between adhesion molecules (eg, intracellular adhesion molecule [ICAM] 1 and 2; vascular cell adhesion molecule [VCAM] 1; E-selectin; and P-selectin), chemokines expressed by endothelial cells (eg, chemokine, CC motif, ligand 2 [CCL2]), and integrins expressed by leukocytes (eg, macrophage-1 antigen [Mac-1]).32–34 The recruitment cascade ultimately leads to leukocyte transmigration across the endothelial barrier.32,33 This interplay between cells of the microvasculature and leukocytes in physiological and pathophysiological conditions is an emerging target for preventing or treating chronic low-grade inflammation.32 Data from animal studies have suggested that dietary nitrate might be a promising candidate for favorably modulating immune–vasculature interactions.26,27,30

Vascular inflammation and oxidative stress

Inflammation, particularly vascular inflammation, is closely linked with oxidative stress.6,35 Oxidative stress activates proinflammatory and damage-associated signaling pathways.7,35,36 Conversely, inflammation induces and amplifies oxidative stress through the increased generation of reactive oxygen and nitrogen species by immune cells.16,35 Reactive species are important physiological signaling molecules, and they contribute to normal immune function (eg, destruction of cell debris, defense against pathogens).7,37 However, excess production of reactive species (eg, superoxide anion and hydrogen peroxide) may lead to oxidative stress if the function of antioxidant defense systems is insufficient, leading to oxidative damage to molecules, cells, and tissues.7 Chronic vascular inflammation and oxidative stress are important pathophysiological mechanisms underlying reductions in vascular NO bioavailability, vascular aging, vascular dysfunction, and the development or progression of cardiovascular disorders.6,16,32 Several studies have shown that supplementation with inorganic nitrate and nitrite attenuates oxidative stress in various disorders, including models of vascular aging and cardiovascular disease.16,25,30,38,39 Reducing oxidative stress, in turn, might be an important mechanism by which dietary nitrate or nitrite favorably modulates immune cell function,40 reduces proinflammatory processes25,38 and organ injuries,30 and improves vascular function.38

Role of the immune system and inflammation in atherosclerosis

Atherosclerosis is the main pathophysiological process underlying cardiovascular disease and is a leading cause of mortality and morbidity worldwide.6,12 The contribution of inflammation to the pathogenesis of atherosclerosis is widely recognized.9,41 Various immune cell subsets involved in innate and adaptive immune responses play key roles in initiating, promoting, and advancing atherosclerosis.9,41 In the early stages of atherosclerotic lesion formation, endothelial activation (eg, through hyperlipidemia) initiates an innate immune response and the recruitment of early inflammatory cells such as monocytes and, potentially, neutrophils to the endothelium.33,36 After transendothelial migration and entry into the intima, monocytes transform into macrophages and are further regulated by damage- or pathogen-associated molecular patterns (DAMPs or PAMPs) and various cytokines, including tumor necrosis factor α (TNF-α), interleukin (IL) 1, and IL-6.36 Critical processes in the progression of atherosclerosis include the following: (1) accumulation of macrophages in developing plaques, (2) uptake of lipids by macrophages, (3) formation of macrophage foam cells, (4) stimulation of smooth muscle cell growth, (5) fibrosis, and (6) macrophage cell death.33

There is increasing evidence that immune cells, particularly macrophages, display marked and dynamic phenotypic plasticity.4,9,42 This plasticity in the phenotypic and functional characteristics of macrophages is primarily dependent on the microenvironment of the macrophages in tissues and atherosclerotic plaques.42 Macrophage populations that differ phenotypically include macrophages that display a more proinflammatory phenotype (traditionally referred to as classically activated or M1 macrophages) and macrophages that display a more anti-inflammatory and inflammation-resolving phenotype (traditionally referred to as M2 macrophages).42 The shifts between these phenotypes are critical for either promoting or resolving inflammation.4,33,42 The plasticity of macrophages and, possibly, other leukocytes such as neutrophils43 and the signaling mechanisms that regulate leukocyte phenotype and function in atherosclerotic plaques may be potential target processes for interventions to prevent the progression of atherosclerosis.32,33

M1 macrophages produce large quantities of NO and superoxide anions through inducible NOS (iNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, respectively.16,42 The combined and excessive production of both NO and superoxide anions generates the highly reactive radical peroxynitrite,15,16 further exacerbates oxidative stress and inflammation, and reduces the vascular bioavailability of NO.6,16,32 Notably, when activated macrophages are treated in vitro with nitrite, reductions in the formation of superoxide anions through NADPH oxidase, iNOS expression, and peroxynitrite production have been observed.40 More research is required to investigate the underlying mechanisms and clinical implications of these effects and to determine whether dietary nitrate influences leukocyte function and phenotypic characteristics.

In animal models, treatment with dietary nitrate or nitrite has been shown to prevent vascular inflammation caused by hypercholesterolemia,25 stabilize atherosclerotic plaques through anti-inflammatory mechanisms,26 and decrease leukocyte recruitment in vascular inflammation.27 These findings suggest a therapeutic potential of dampening inflammation through modulation of vascular–immune interactions in atherosclerosis by enhancing the nitrate–nitrite–NO pathway.

Role of the immune system and inflammation in hypertension

Hypertension, or high blood pressure, is the leading risk factor for mortality and morbidity globally.13 According to the 2017 American College of Cardiology/American Heart Association Clinical Practice Guidelines for High Blood Pressure in Adults, stage 1 hypertension is defined as an average systolic blood pressure of 130 to 139 mm Hg or a diastolic blood pressure of 80 to 89 mm Hg, and stage 2 hypertension as an average systolic blood pressure of ≥ 140 mm Hg or a diastolic blood pressure of ≥ 90 mm Hg.44

The pathophysiology of hypertension is complex and multifactorial; it involves genetic and environmental/lifestyle-related factors (the latter of which includes sedentary behavior and unhealthy dietary habits).16,35,45 Abnormal function of the kidneys, vasculature, and central (sympathetic) nervous system have been implicated in the pathogenesis of hypertension.35,45,46 Furthermore, as early as about half a century ago, it was suggested that immunity and inflammation contribute to hypertension.47 In the past few years, several lines of evidence to support this concept, including experimental hypertension models and cross-sectional human studies, have emerged.35,46 Taken together, these investigations suggest that the development and progression of high blood pressure involve the transmigration and accumulation of innate and adaptive immune cells in the affected tissues, including kidneys and arteries.35,46 According to a model developed from these findings, initial stimuli that activate innate immunity in prehypertension (high-normal blood pressure) and transient hypertension may include DAMPs, Toll-like receptor expression, and episodic tissue accumulation and infiltration of immune cells.35 Cytokines released from these immune cells promote vascular and renal dysfunction (including impairments in the renin–angiotensin–aldosterone system and sodium reabsorption) and oxidative stress.46 Chronic oxidative stress, in turn, results in the formation of modified proteins (eg, γ-ketoaldehydes) that activate an adaptive immune response.35,46 Established hypertension is associated with the interaction between the innate and the adaptive immune systems, resulting in chronic vascular and renal inflammation and end-organ damage.35,46

The most consistent outcome of increased dietary nitrate consumption in human studies is the reduction in blood pressure in both normotensive and hypertensive individuals.14,16,48 Acute blood pressure–lowering effects of dietary nitrate, such as described by Larsen et al,49 have mainly been attributed to peripheral vasodilation mediated by NO.16,50 With regard to the longer-term effects of dietary nitrate on blood pressure, however, other mechanisms—such as modulation of immune cell function and oxidative stress by dietary nitrate—could play important roles.16,29,30 In light of the involvement of the immune system in the pathogenesis of hypertension, future research may focus on how dietary nitrate can be used to develop more effective and cost-efficient strategies to prevent or treat the disease.

DIETARY NITRATE AND THE IMMUNE SYSTEM IN CARDIOVASCULAR HEALTH AND DISEASE

Nitric oxide signaling in immune modulation

Central to the mechanisms underlying physiological NO signaling is the free radical nature of NO.15,19 The heme-centered enzyme soluble guanylyl cyclase is the major intracellular receptor of NO.16,17 When NO is generated in the vasculature (through eNOS, for example), it diffuses to the endothelium, where it activates soluble guanylyl cyclase in various cell types such as smooth muscle cells, circulating platelets, and leukocytes.17 Activated soluble guanylyl cyclase increases the production of the second messenger, cyclic guanosine monophosphate, which in turn relaxes smooth muscle cells and inhibits adhesion of platelets and leukocytes.16,17 In addition to its paracrine effects on adjacent cells, NO controls multiple intracellular functions (eg, mitochondrial function, reactive oxygen species production, and signaling) within the cells from which it was generated (eg, endothelial cells).17 Through the oxidation of NO to the more stable anions nitrite and nitrate (and reduction back to NO under hypoxic conditions), NO can also act in an endocrine-like fashion in cells more distant from the site of NO production.14–16,22 Additional NO signaling mechanisms include the generation of other bioactive nitrogen intermediates through nitrosation and nitration of proteins and lipids, leading to modifications in protein function and lipid signaling.15,16 Although excessive production of NO (from iNOS) and superoxide anions has toxic effects through the formation of peroxynitrite (as described above), NO itself can also act as an antioxidant by scavenging other more reactive radicals.15,16

Nitric oxide generated by eNOS exerts anti-inflammatory effects in experimental inflammatory models by preventing leukocyte activation and adhesion, reducing vascular permeability and leukocyte transmigration, and inhibiting expression of adhesion molecules.16,31 The exact mechanisms by which NO modulates leukocyte–endothelial interactions are not entirely known.15,27 However, on basis of the concept that NO is a potent anti-inflammatory mediator,31,51 a number of animal and human studies in the past few years have investigated whether vascular inflammation and immune homeostasis can be modulated through the dietary nitrate–nitrite–NO pathway (Figure 1).25,27 These studies are discussed below.

Figure 1

Proposed mechanisms by which inorganic dietary nitrate or nitrite exert anti-inflammatory effects, modulate immune cell phenotypes and function, and immune–endothelial interactions. Mechanisms shown are based on data from cell culture, animal, and human studies. Abbreviations: eNOS, endothelial nitric oxide synthase; NO2, nitrite; NO3, nitrate.

Epidemiological evidence linking inflammation, cardiovascular disease, and dietary nitrate

Vegetables, fruits, nuts, and whole grains form the basis of a plant-based diet and are components found abundantly in Dietary Approaches to Stop Hypertension (DASH), the Mediterranean diet, and vegetarian diets—all of which have been linked with cardiovascular health benefits.52–55 The individual food components of these diets that have been identified as most protective are vegetables, olive oil, fruits, and legumes.56 These foods contain a number of vitamins, minerals, phytochemicals, and fibers that could benefit cardiovascular health.57,58 A cardiovascular protective component that has recently been identified is inorganic nitrate. Inorganic nitrate is predominantly found in vegetables, particularly leafy green vegetables (eg, spinach, lettuce, rucola/rocket), beetroot, celery, and radish,59 and contributes to over 80% of the total nitrate consumed in the diet.19,21 It has been estimated that a diet rich in vegetables, such as the DASH diet, can contribute up to 1000 mg of dietary nitrate per day.21 Interestingly, habitual intakes of dietary nitrate above 53 mg/d may be enough to lower the long-term risk of cardiovascular disease.60–62

The role of dietary inorganic nitrate on immune function and inflammatory biomarkers in large cross-sectional and prospective cohort studies has yet to be examined. There is, however, evidence linking diets rich in vegetables to inflammatory biomarkers. Diets rich in vegetables have been inversely associated with inflammatory biomarkers, predominantly C-reactive protein (CRP).63 Conversely, in a cross-sectional study of 285 adolescent boys and girls (aged 13–17 y), there was no evidence for an association between vegetable intake and either plasma concentrations of CRP or markers of lipid peroxidation (prostaglandin F2a and F2-isoprostane).64 However, one vegetable serving more (½ cup) was associated with a reduction in the plasma concentration of IL-6 by 0.15 pg/mL and TNF-α by 0.13 pg/ mL.64 It is important to note that this population may not have had existing elevated levels of plasma CRP, prostaglandin F2a, and F2-isoprostane; therefore, an association may not have been detected. The association between vegetable intake and inflammatory biomarkers may be stronger in individuals with elevated levels of inflammation. When investigating diets rich in leafy green vegetables, ie, the largest source of nitrate from vegetables, there was evidence for an inverse association with plasma concentrations of CRP, IL-6, homocysteine, and soluble ICAM-1, but not soluble E-selectin, in an ethnically diverse population (n = 5089).65 In a cohort of middle-aged women (n = 657), a 1-serving increment in the intake of green leafy vegetables was associated with a 0.29 mg/dL lower plasma CRP concentration and a 0.11 pg/mL lower plasma IL-6 concentration.66 No relationship was observed between the intake of green leafy vegetables and the plasma concentrations of soluble TNF receptor 2, E-selectin, soluble ICAM-1 or soluble VCAM-1.66

It is important to note that these studies are observational in nature and cannot prove causation. Vegetable-rich diets are high in nitrate but also contain many vitamins, minerals, and phytochemicals, which are all likely to have an important role in inflammation, immune function, and cardiovascular health.11 Of interest are potential interactions between components. Nitrate and flavonoids may have additive or synergistic physiological effects,67 and the effect of nitrate could be abolished with coingestion of sulfur-containing compounds.68,69 These relationships are yet to be investigated in larger observational and prospective studies in individuals with chronic inflammation.

In conclusion, evidence from observational studies suggests that consumption of vegetable-rich diets plays a potential role in lowering inflammation. The lack of observational studies investigating nitrate and biomarkers of immune function and inflammation represents a significant gap in the literature.

Intervention studies on the effects of dietary nitrate and nitrite on vascular inflammation and immune–vasculature interactions

This section summarizes and discusses available evidence from animal (n = 13) and human studies (n = 8) on the effects of nitrate-rich plant foods and other sources of nitrate or nitrite on inflammation and immune function in the context of cardiovascular health. Most of these studies have measured biomarkers of inflammation, including soluble inflammatory mediators (eg, cytokines, chemokines, adhesion molecules, and acute-phase proteins such as CRP), within the blood circulation as well as blood cellular markers (eg, surface markers of leukocytes and platelets). However, this section also refers to key animal studies that have investigated tissue inflammation by evaluating histology or by measuring gene or protein tissue expression of inflammatory mediators, because these investigations offer insights into the effects of dietary nitrate and nitrite on inflammation within body compartments such as arterial tissue.

Animal studies

Animal studies have investigated the effects of nitrate or nitrite on soluble inflammatory markers, tissue inflammation by histological evaluation, tissue expression of inflammatory and chemotactic factors, phenotypic and functional characteristics of leukocytes, and leukocyte–vasculature interactions. The interventions used included sodium nitrite (n = 5), sodium nitrate (n = 7), potassium nitrate (n = 1), and a spinach supplement (n = 1). Nitrite content ranged from 33.5 mg/L to 100.5 mg/L. Nitrate content ranged from 15 mg/L to 924 mg/L. All animal studies were chronic (1 wk to 68 wk).

Soluble inflammatory mediators.

Soluble inflammatory markers are commonly used as markers of inflammation within various cardiovascular diseases.3 A number of animal models have been used to determine if dietary nitrate influences plasma cytokines in various models of disease (Table 125,,26,,38,,70–75).

Table 1

Effects of dietary nitrate or nitrite on soluble inflammatory mediators, tissue expression of inflammatory mediators, and histologically evaluated tissue inflammation in animal studiesa

Reference Animal model Intervention Nitrite dose Nitrate dose Duration Positive effects Negative effects or no effects
Stokes et al (2009)25 Hypercholesterolemic mice NaNO2 33.5 mg/L; 100.5 mg/L 3 wk ↓ CRP
Sindler et al (2011)38 Ageing mice NaNO2 33.5 mg/L 3 wk Aorta: ↓ IL-1β; ↓ IL-6; ↓ IFN-γ; ↓ TNF-α ↑ IL-6 in young nitrite-treated mice compared with young nontreated controls
Sindler et al (2015)70 Diabetic mice NaNO2 33.5 mg/L 5 wk Aorta: ↓ IL-6 No effect on circulating or aortic IL-1α; IL-1β; IL-6; IL-12; IL-13; MCP-1; TNF-α
Ohtake et al (2017)71 Postmenopausal mice NaNO2 33.5 mg/L; 100.5 mg/L 18 wk Ovarian adipose tissue: ↓ IL-6; ↓ TNF-α; ↓ MCP-1
Hezel et al (2015)72 Healthy mice NaNO3 62 mg/L 68 wk ↓ IL-10 No effect on IFN-γ; IL-1β; IL-6; CXCL1; IL-12-p70
Li et al (2016)73 Healthy mice Spinach-derived NO3 15, 30, and 60 mg/kg of BW/d 4 wk ↓ CRP; ↓ TNF-α; ↓ IL-6; ↓ ET-1
Yang et al (2017)74 Mice with IR-induced renal injury NaNO3 62 mg/kg of BW/d 2 wk ↓ IL-12p70; ↓ IL-1β; ↓ IL-6 No effect on TNF-α, IFN-γ, CXCL1, or IL-10
Histology: ↓ inflammation (kidney)
Khambata et al (2017)26 Atheroprone mice KNO3 924 mg/L 12 wk ↓ CXCL1; ↓ CXCL2; ↓ CCL2 No effect on circulating CCL5
Peritoneal lavage: ↑ IL-10 No effect on aortic CXCL1, CXCL5, CX3CL1, CCL2, or CXCL2
Histology: ↓ inflammation (atherosclerotic plaques)
Gheibi et al (2018)75 Obese, diabetic rats NaNO3 73 mg/L 8 wk ↓ IL-1β ↓ iNOS expression (soleus and epididymal adipose tissue)
Reference Animal model Intervention Nitrite dose Nitrate dose Duration Positive effects Negative effects or no effects
Stokes et al (2009)25 Hypercholesterolemic mice NaNO2 33.5 mg/L; 100.5 mg/L 3 wk ↓ CRP
Sindler et al (2011)38 Ageing mice NaNO2 33.5 mg/L 3 wk Aorta: ↓ IL-1β; ↓ IL-6; ↓ IFN-γ; ↓ TNF-α ↑ IL-6 in young nitrite-treated mice compared with young nontreated controls
Sindler et al (2015)70 Diabetic mice NaNO2 33.5 mg/L 5 wk Aorta: ↓ IL-6 No effect on circulating or aortic IL-1α; IL-1β; IL-6; IL-12; IL-13; MCP-1; TNF-α
Ohtake et al (2017)71 Postmenopausal mice NaNO2 33.5 mg/L; 100.5 mg/L 18 wk Ovarian adipose tissue: ↓ IL-6; ↓ TNF-α; ↓ MCP-1
Hezel et al (2015)72 Healthy mice NaNO3 62 mg/L 68 wk ↓ IL-10 No effect on IFN-γ; IL-1β; IL-6; CXCL1; IL-12-p70
Li et al (2016)73 Healthy mice Spinach-derived NO3 15, 30, and 60 mg/kg of BW/d 4 wk ↓ CRP; ↓ TNF-α; ↓ IL-6; ↓ ET-1
Yang et al (2017)74 Mice with IR-induced renal injury NaNO3 62 mg/kg of BW/d 2 wk ↓ IL-12p70; ↓ IL-1β; ↓ IL-6 No effect on TNF-α, IFN-γ, CXCL1, or IL-10
Histology: ↓ inflammation (kidney)
Khambata et al (2017)26 Atheroprone mice KNO3 924 mg/L 12 wk ↓ CXCL1; ↓ CXCL2; ↓ CCL2 No effect on circulating CCL5
Peritoneal lavage: ↑ IL-10 No effect on aortic CXCL1, CXCL5, CX3CL1, CCL2, or CXCL2
Histology: ↓ inflammation (atherosclerotic plaques)
Gheibi et al (2018)75 Obese, diabetic rats NaNO3 73 mg/L 8 wk ↓ IL-1β ↓ iNOS expression (soleus and epididymal adipose tissue)

Abbreviations and symbols: BW, body weight; CCL, C-C motif chemokine ligand; CRP, C-reactive protein; CXCL, chemokine (C-X-C motif) ligand; ET, endothelin; IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; IR, ischemia-reperfusion; KNO3, potassium nitrate; MCP, monocyte chemoattractant protein; MPO, myeloperoxidase; NaNO2, sodium nitrite; NaNO3, sodium nitrate; TNF, tumor necrosis factor; ↑, increased; ↓, decreased.

a

Experiments performed on circulating markers, unless otherwise stated.

Table 1

Effects of dietary nitrate or nitrite on soluble inflammatory mediators, tissue expression of inflammatory mediators, and histologically evaluated tissue inflammation in animal studiesa

Reference Animal model Intervention Nitrite dose Nitrate dose Duration Positive effects Negative effects or no effects
Stokes et al (2009)25 Hypercholesterolemic mice NaNO2 33.5 mg/L; 100.5 mg/L 3 wk ↓ CRP
Sindler et al (2011)38 Ageing mice NaNO2 33.5 mg/L 3 wk Aorta: ↓ IL-1β; ↓ IL-6; ↓ IFN-γ; ↓ TNF-α ↑ IL-6 in young nitrite-treated mice compared with young nontreated controls
Sindler et al (2015)70 Diabetic mice NaNO2 33.5 mg/L 5 wk Aorta: ↓ IL-6 No effect on circulating or aortic IL-1α; IL-1β; IL-6; IL-12; IL-13; MCP-1; TNF-α
Ohtake et al (2017)71 Postmenopausal mice NaNO2 33.5 mg/L; 100.5 mg/L 18 wk Ovarian adipose tissue: ↓ IL-6; ↓ TNF-α; ↓ MCP-1
Hezel et al (2015)72 Healthy mice NaNO3 62 mg/L 68 wk ↓ IL-10 No effect on IFN-γ; IL-1β; IL-6; CXCL1; IL-12-p70
Li et al (2016)73 Healthy mice Spinach-derived NO3 15, 30, and 60 mg/kg of BW/d 4 wk ↓ CRP; ↓ TNF-α; ↓ IL-6; ↓ ET-1
Yang et al (2017)74 Mice with IR-induced renal injury NaNO3 62 mg/kg of BW/d 2 wk ↓ IL-12p70; ↓ IL-1β; ↓ IL-6 No effect on TNF-α, IFN-γ, CXCL1, or IL-10
Histology: ↓ inflammation (kidney)
Khambata et al (2017)26 Atheroprone mice KNO3 924 mg/L 12 wk ↓ CXCL1; ↓ CXCL2; ↓ CCL2 No effect on circulating CCL5
Peritoneal lavage: ↑ IL-10 No effect on aortic CXCL1, CXCL5, CX3CL1, CCL2, or CXCL2
Histology: ↓ inflammation (atherosclerotic plaques)
Gheibi et al (2018)75 Obese, diabetic rats NaNO3 73 mg/L 8 wk ↓ IL-1β ↓ iNOS expression (soleus and epididymal adipose tissue)
Reference Animal model Intervention Nitrite dose Nitrate dose Duration Positive effects Negative effects or no effects
Stokes et al (2009)25 Hypercholesterolemic mice NaNO2 33.5 mg/L; 100.5 mg/L 3 wk ↓ CRP
Sindler et al (2011)38 Ageing mice NaNO2 33.5 mg/L 3 wk Aorta: ↓ IL-1β; ↓ IL-6; ↓ IFN-γ; ↓ TNF-α ↑ IL-6 in young nitrite-treated mice compared with young nontreated controls
Sindler et al (2015)70 Diabetic mice NaNO2 33.5 mg/L 5 wk Aorta: ↓ IL-6 No effect on circulating or aortic IL-1α; IL-1β; IL-6; IL-12; IL-13; MCP-1; TNF-α
Ohtake et al (2017)71 Postmenopausal mice NaNO2 33.5 mg/L; 100.5 mg/L 18 wk Ovarian adipose tissue: ↓ IL-6; ↓ TNF-α; ↓ MCP-1
Hezel et al (2015)72 Healthy mice NaNO3 62 mg/L 68 wk ↓ IL-10 No effect on IFN-γ; IL-1β; IL-6; CXCL1; IL-12-p70
Li et al (2016)73 Healthy mice Spinach-derived NO3 15, 30, and 60 mg/kg of BW/d 4 wk ↓ CRP; ↓ TNF-α; ↓ IL-6; ↓ ET-1
Yang et al (2017)74 Mice with IR-induced renal injury NaNO3 62 mg/kg of BW/d 2 wk ↓ IL-12p70; ↓ IL-1β; ↓ IL-6 No effect on TNF-α, IFN-γ, CXCL1, or IL-10
Histology: ↓ inflammation (kidney)
Khambata et al (2017)26 Atheroprone mice KNO3 924 mg/L 12 wk ↓ CXCL1; ↓ CXCL2; ↓ CCL2 No effect on circulating CCL5
Peritoneal lavage: ↑ IL-10 No effect on aortic CXCL1, CXCL5, CX3CL1, CCL2, or CXCL2
Histology: ↓ inflammation (atherosclerotic plaques)
Gheibi et al (2018)75 Obese, diabetic rats NaNO3 73 mg/L 8 wk ↓ IL-1β ↓ iNOS expression (soleus and epididymal adipose tissue)

Abbreviations and symbols: BW, body weight; CCL, C-C motif chemokine ligand; CRP, C-reactive protein; CXCL, chemokine (C-X-C motif) ligand; ET, endothelin; IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; IR, ischemia-reperfusion; KNO3, potassium nitrate; MCP, monocyte chemoattractant protein; MPO, myeloperoxidase; NaNO2, sodium nitrite; NaNO3, sodium nitrate; TNF, tumor necrosis factor; ↑, increased; ↓, decreased.

a

Experiments performed on circulating markers, unless otherwise stated.

The first major study that investigated the effect of nitrite on inflammatory markers was conducted by Stokes et al.25 Using a hypercholesterolemic mouse model, the investigators administered sodium nitrite in drinking water at a concentration of either 50 mg/L or 150 mg/L for 3 weeks. Both doses of nitrite reduced circulating levels of CRP.25 Similarly, a dose-dependent reduction in plasma CRP was observed in healthy mice given a freeze-dried, spinach-derived supplement containing varying levels of nitrate, as measured by high-performance liquid chromatography.73 This study also showed lowered plasma concentrations of the proinflammatory cytokines TNF-α and IL-6 and of the vasoconstrictive molecule endothelin-1, with the greatest effect seen in the group that received the highest concentration of nitrate (60 mg/kg).73 Sodium nitrate administration of 100 mg/L for 8 weeks to obese, diabetic rats reduced the serum concentration of the proinflammatory cytokine IL-1β75. In a robust, placebo-controlled study by Khambata et al,26 the effects of 12 weeks of nitrate at a concentration of 925 mg/L were tested on atherosclerosis-prone, apolipoprotein E knockout mice. A reduction in plasma concentrations of leukocyte chemoattractants and an enhanced anti-inflammatory response (increased IL-10 concentration) in peritoneal lavage fluid following acute inflammation challenges were observed in the nitrate-fed mice26. The authors of this study proposed that dietary nitrate upregulated the expression and activity of IL-10, which provided an accelerated resolution of acute inflammatory reactions.26 In contrast to these findings, healthy mice given sodium nitrate for 68 weeks showed a reduction in the anti-inflammatory cytokine IL-10, coupled with an absence of changes to proinflammatory cytokines (IL-1β, IL-6, and IL-12p70).72 Similarly, the same dose of sodium nitrite as used in the study by Stokes et al (50 mg/L) for 5 weeks did not reduce circulating concentrations of proinflammatory cytokines (including IL-1β, IL-6, TNF-α) or the proatherogenic molecule monoctye chemoattractant protein 1 (MCP-1) in diabetic mice, despite an improvement in endothelial functioning as measured by endothelium-dependent dilation.70

In disease models not specifically related to cardiovascular disease, some evidence exists to provide proof of concept that dietary nitrate modulates inflammation. In a mouse model investigating the effects of nitrate on renal ischemia-reperfusion injury, sodium nitrate given for 2 weeks prior to ischemic insult decreased levels of proinflammatory markers 24 hours post injury (cytokine IL-12p70) as well as 2 weeks post injury (IL-1β and IL-6) in nitrate-treated mice.74 No changes to other proinflammatory cytokines (TNF-α, IFN-γ, KC-GRO) or the anti-inflammatory cytokine IL-10 were observed.74 Although this study was conducted in a renal model as opposed to cardiovascular models of disease, this research may provide evidence of an anti-inflammatory effect of chronic nitrate ingestion, particularly within tissues.

Tissue inflammation.

Recent studies have investigated the effects of inorganic nitrate or nitrite on tissue inflammation by evaluating histology (ie, leukocyte tissue infiltration) or by assessing gene and protein expression levels of inflammatory and chemotactic factors in animal tissues (Table 1 and Table 225–27,30,74,76,77). Most studies have reported favorable effects on inflammation in tissue derived from animals fed with nitrate or nitrite. In one of the studies that investigated the effects of nitrate histologically, Carlström et al30 used a high-salt diet to induce hypertension in Sprague-Dawley rats with compromised kidney function.30 Daily doses of sodium nitrate at 6.2 and 62 mg/kg of body weight administered for 11 weeks attenuated histological signs of kidney inflammation (ie, leukocyte infiltration of renal tissue).30 Reductions in macrophage infiltration in the kidney were also seen in a mouse model of renal ischemia-reperfusion injury after 2 weeks of sodium nitrate administration (62 mg/kg of body weight/d).74 In a mouse model of chronic, colonic inflammation, reductions in histological signs of inflammation were observed following supplementation with both sodium nitrate (620 mg/L) and sodium nitrite (46 mg/L).76 Studies on the effects of nitrate on atherosclerotic plaques have yielded conflicting results. Khambata et al26 observed a reduction in macrophage accumulation within atherosclerotic plaques of artheroprone apolipoprotein E knockout mice fed nitrate from potassium nitrate (924 mg/L) for 12 weeks. In contrast, supplementation with nitrate from sodium nitrate (720 mg/L) for 14 weeks did not affect macrophage infiltration of artherosclerotic plaques in low-density lipoprotein receptor knockout mice.77 Future investigations that compare different nitrate and nitrite supplementation regimens (eg, with regard to the duration of supplementation) in the same genetic models might help explain the reason for this discrepancy.

A number of studies have examined the effects of nitrate or nitrite on gene and protein expression of inflammatory mediators within tissues. Ohtake et al71 observed reductions in the transcriptions of proinflammatory cytokines in visceral fat in postmenopausal mice fed sodium nitrite at a concentration of either 33 mg/L or 100.5 mg/L for 18 weeks. Sindler et al38 investigated the effects of sodium nitrite, administered at 50 mg/L, for 3 weeks on age-associated vascular dysfunction by comparing tissue expression of inflammatory markers in old mice vs in young mice. Sodium nitrite reduced aortic arch protein expression levels of proinflammatory cytokines (IL-1β, IL-6, TNF-α, and IFN-γ) in old mice to levels comparable with those in young, healthy mice.38 Reductions in aortic IL-6 were also seen in diabetic mice given the same dose of sodium nitrite (50 mg/L) for 5 weeks (although interestingly, no changes to circulating IL-6 were observed).70 In atheroprone mice, 12 weeks of potassium nitrate ingestion led to an anti-inflammatory change (increased IL-10 messenger RNA [mRNA]) in aortic arch tissue, although no changes in mRNA expression of other inflammatory and chemotactic cytokines were observed.26

Considering the effect of inflammation in cardiovascular disease, reductions of inflammatory markers seen in other disease models may provide insight into processes that influence the cardiovascular system. Jädert et al27 have shown a number of anti-inflammatory effects of dietary nitrate and nitrite in tissues. In an experimental model of tissue injury, they observed a reduction in the tissue expression of the neutrophil-associated enzyme myeloperoxidase, used as a quantitative measure of neutrophil tissue infiltration, following administration of sodium nitrate at a concentration of 124 mg/L for a week. In a more recent study, potassium nitrate ingestion was associated with reduced myeloperoxidase activity in homogenized mesentery tissue and in cells from pellets of peritoneal lavage fluid.26 In a separate mouse study by Jädert et al76 that investigated the effects of nitrite and nitrate on colonic inflammation, 7-day administration of either sodium nitrite at 46 mg/L or sodium nitrate at 620 mg/L was associated with lower colonic expression of proinflammatory pathways (iNOS and NF-κB p65). Similarly, sodium nitrate supplementation for 8 weeks in diabetic rats decreased mRNA expression of iNOS in both the soleus muscle and epididymal adipose tissue.75 Inducible NOS is highly expressed in several inflammatory states, including atherosclerosis, and has been implicated in the cardiovascular inflammatory process.16 Reductions in iNOS expression may indicate a less inflammatory profile. In a study conducted on mice to simulate postmenopausal metabolic syndrome, 18 weeks of nitrite supplementation reduced mRNA levels of inflammatory and atherogenic cytokines (IL-6, TNF-α, MCP-1) in ovarian adipose tissue.71

Leukocyte–vasculature interactions and phenotypic and functional characteristics of leukocytes.

In addition to its effects on cytokines, the vasculature, and other tissues, NOS-dependent NO has been implicated in modulating function and phenotypes of various immune cell subpopulations.37 This has stimulated research interest in the potential to modulate immune cell function and phenotypes through the dietary nitrate–nitrite–NO pathway.78 Many leukocyte subsets are associated with specific and often varied physiological functions, many of which are linked to cardiovascular pathologies.79 Leukocyte subsets are determined by up- or downregulation of surface markers, typically measured using flow cytometry.80 The Mac-1 complex is one particular cell adhesion molecule expressed on leukocytes that has been implicated in adherence to the endothelium through factors that may be downregulated by dietary nitrate. The Mac-1 complex (also known as αMβ2 and identified by measuring expression of CD11b/CD18 proteins) undergoes conformational changes to bind to endothelial cells through surface molecules such as ICAM-1 and ICAM-2.81 The Mac-1 integrin is highly expressed on neutrophils.82 Reduced expression of CD11b may therefore indicate a more anti-inflammatory (or, alternatively, a less proinflammatory) neutrophil phenotype. Neutrophils expressing less CD11b are less likely to roll along the endothelium83 or produce myeloperoxidase.84 These CD11b-expressing neutrophils may also play a role in the phenotypic transition of proinflammatory M1 macrophages to anti-inflammatory M2 macrophages.85

Recently, Khambata et al26 reported preliminary evidence of an effect of dietary nitrate on leukocyte expression on some of these surface markers. They investigated the effects of 12 weeks of nitrate fed at a concentration of 924 mg/L in mice fed a normal chow diet vs in mice fed a high-fat diet. In the mice fed a normal chow diet, a reduction in CD11b expression on neutrophils was noted following the 12-week feeding period. Additionally, circulating numbers of inflammatory monocytes were decreased 24 hours after TNF-α–induced inflammation, with a concurrent decrease in the number of monocytes within artherosclerotic plaques.26 Because neutrophils with increased levels of CD11b surface expression are considered to be proinflammatory neutrophils (particularly in cardiovascular risk states such as hyperlipidemia86), reductions in CD11b expression may indicate a less inflammatory phenotype of neutrophils.

In support of a potential role of the dietary nitrate–nitrite–NO pathway in counteracting unwanted leukocyte transendothelial migration, various effects of dietary nitrate and nitrite on interactions between endothelial cells and immune cells have been observed. These effects include a decrease in leukocyte adhesion,25,27 leukocyte emigration,26,27,76 and numbers of rolling leukocytes26 (Table 2). In the study conducted by Jädert et al,27 the effects on leukocyte adhesion and emigration were associated with a decrease in P-selectin expression in ileal tissues.27 In a separate cell culture arm of this study, nitrite was shown to attenuate ICAM-1 upregulation in human dermal microvascular endothelial cells that were exposed to TNF-α.27 This suggests that nitrite reduces leukocyte interactions with the endothelium through a mechanism partly mediated by downregulation of endothelial cell adhesion molecules.27 Notably, despite attenuating vascular inflammation, nitrite did not alter the animals’ ability to clear a Staphylococcus aureus skin infection.27 The results from this study indicate that dietary modulation of the nitrate–nitrite–NO pathway may suppress low-grade chronic inflammation while not impeding the body’s immune defense against harmful pathogens.27 In a subsequent study using a dextran sodium sulfate–induced colitis model of disease in mice, Jädert et al76 showed that dietary nitrite and nitrate were both associated with a reduction in the infiltration of inflammatory cells into colonic tissue.

Table 2

Effects of dietary nitrate or nitrite on blood cellular markers of inflammation, leukocyte–vasculature interactions, and histologically evaluated tissue inflammation in animal studies

Reference Animal model Intervention Nitrite dose Nitrate dose Duration Positive effects Negative/no effects
Stokes et al (2009)25 Hypercholesterolemic mice NaNO2 33.5 mg/L; 100.5 mg/L 3 wk ↓ Leukocyte emigration and adhesion No effect on total circulating leukocyte count
Jädert et al (2014)76 Mice with DSS-induced colitis NaNO2; NaNO3 46 mg/L 620 mg/L 1 wk Colon: ↓ iNOS and NF-κB p65 expression;
Histology: ↓ inflammation (colon)
Carlström et al (2011)30 Rats with salt-induced hypertension NaNO3 6.2 mg/kg of BW/d; 11 wk Histology: ↓ inflammation (kidney)
62 mg/kg of BW/d
Jädert et al (2012)27 Healthy rats and mice NaNO3 124 mg/L 1 wk ↓ Leukocyte rolling and emigration No effect on adherent cells, velocities of rolling cells, or clearance of bacterial skin infection
Ileum: ↓ MPO; ↓ P-selectin expression
Histology: ↓ inflammation (HDMECs)
Marsch et al (2016)77 LDL-receptor KO mice NaNO3 730 mg/L 14 wk ↓ Plasma NO2 to baseline; no effect on granulocytes, monocytes, or lymphocytes; no histological changes in atherosclerotic plaques
Khambata et al (2017)26 Atheroprone mice KNO3 924 mg/L 12 wk ↓ Leukocyte rolling and adherence; ↓ MPO activity; ↓ neutrophil CD11b No effect on CD162 or CD62L
No effect on circulating neutrophils, monocytes, or lymphocytes
Histology: ↓ macrophages in atherosclerotic plaques
Yang et al (2017)74 Mice with IR–induced renal injury NaNO3 62 mg/kg of BW/d 2 wk ↓ BMDM superoxide generation
Histology: ↓ macrophage infiltrate (kidney)
Reference Animal model Intervention Nitrite dose Nitrate dose Duration Positive effects Negative/no effects
Stokes et al (2009)25 Hypercholesterolemic mice NaNO2 33.5 mg/L; 100.5 mg/L 3 wk ↓ Leukocyte emigration and adhesion No effect on total circulating leukocyte count
Jädert et al (2014)76 Mice with DSS-induced colitis NaNO2; NaNO3 46 mg/L 620 mg/L 1 wk Colon: ↓ iNOS and NF-κB p65 expression;
Histology: ↓ inflammation (colon)
Carlström et al (2011)30 Rats with salt-induced hypertension NaNO3 6.2 mg/kg of BW/d; 11 wk Histology: ↓ inflammation (kidney)
62 mg/kg of BW/d
Jädert et al (2012)27 Healthy rats and mice NaNO3 124 mg/L 1 wk ↓ Leukocyte rolling and emigration No effect on adherent cells, velocities of rolling cells, or clearance of bacterial skin infection
Ileum: ↓ MPO; ↓ P-selectin expression
Histology: ↓ inflammation (HDMECs)
Marsch et al (2016)77 LDL-receptor KO mice NaNO3 730 mg/L 14 wk ↓ Plasma NO2 to baseline; no effect on granulocytes, monocytes, or lymphocytes; no histological changes in atherosclerotic plaques
Khambata et al (2017)26 Atheroprone mice KNO3 924 mg/L 12 wk ↓ Leukocyte rolling and adherence; ↓ MPO activity; ↓ neutrophil CD11b No effect on CD162 or CD62L
No effect on circulating neutrophils, monocytes, or lymphocytes
Histology: ↓ macrophages in atherosclerotic plaques
Yang et al (2017)74 Mice with IR–induced renal injury NaNO3 62 mg/kg of BW/d 2 wk ↓ BMDM superoxide generation
Histology: ↓ macrophage infiltrate (kidney)

Abbreviations and symbols: BMDM, bone marrow-derived macrophage; BW, body weight; CD, cluster of differentiation; DSS, dextran sodium sulfate; HDMEC, human dermal microvascular endothelial cell; iNOS, inducible nitric oxide synthase; IR, ischemia-reperfusion; KNO3, potassium nitrate; KO, knockout; LDL, low-density lipoprotein; MPO, myeloperoxidase; NaNO2, sodium nitrite; NaNO3, sodium nitrate; NF-κB; nuclear factor κB; NO2, nitrite; ↑, increased; ↓, decreased.

Table 2

Effects of dietary nitrate or nitrite on blood cellular markers of inflammation, leukocyte–vasculature interactions, and histologically evaluated tissue inflammation in animal studies

Reference Animal model Intervention Nitrite dose Nitrate dose Duration Positive effects Negative/no effects
Stokes et al (2009)25 Hypercholesterolemic mice NaNO2 33.5 mg/L; 100.5 mg/L 3 wk ↓ Leukocyte emigration and adhesion No effect on total circulating leukocyte count
Jädert et al (2014)76 Mice with DSS-induced colitis NaNO2; NaNO3 46 mg/L 620 mg/L 1 wk Colon: ↓ iNOS and NF-κB p65 expression;
Histology: ↓ inflammation (colon)
Carlström et al (2011)30 Rats with salt-induced hypertension NaNO3 6.2 mg/kg of BW/d; 11 wk Histology: ↓ inflammation (kidney)
62 mg/kg of BW/d
Jädert et al (2012)27 Healthy rats and mice NaNO3 124 mg/L 1 wk ↓ Leukocyte rolling and emigration No effect on adherent cells, velocities of rolling cells, or clearance of bacterial skin infection
Ileum: ↓ MPO; ↓ P-selectin expression
Histology: ↓ inflammation (HDMECs)
Marsch et al (2016)77 LDL-receptor KO mice NaNO3 730 mg/L 14 wk ↓ Plasma NO2 to baseline; no effect on granulocytes, monocytes, or lymphocytes; no histological changes in atherosclerotic plaques
Khambata et al (2017)26 Atheroprone mice KNO3 924 mg/L 12 wk ↓ Leukocyte rolling and adherence; ↓ MPO activity; ↓ neutrophil CD11b No effect on CD162 or CD62L
No effect on circulating neutrophils, monocytes, or lymphocytes
Histology: ↓ macrophages in atherosclerotic plaques
Yang et al (2017)74 Mice with IR–induced renal injury NaNO3 62 mg/kg of BW/d 2 wk ↓ BMDM superoxide generation
Histology: ↓ macrophage infiltrate (kidney)
Reference Animal model Intervention Nitrite dose Nitrate dose Duration Positive effects Negative/no effects
Stokes et al (2009)25 Hypercholesterolemic mice NaNO2 33.5 mg/L; 100.5 mg/L 3 wk ↓ Leukocyte emigration and adhesion No effect on total circulating leukocyte count
Jädert et al (2014)76 Mice with DSS-induced colitis NaNO2; NaNO3 46 mg/L 620 mg/L 1 wk Colon: ↓ iNOS and NF-κB p65 expression;
Histology: ↓ inflammation (colon)
Carlström et al (2011)30 Rats with salt-induced hypertension NaNO3 6.2 mg/kg of BW/d; 11 wk Histology: ↓ inflammation (kidney)
62 mg/kg of BW/d
Jädert et al (2012)27 Healthy rats and mice NaNO3 124 mg/L 1 wk ↓ Leukocyte rolling and emigration No effect on adherent cells, velocities of rolling cells, or clearance of bacterial skin infection
Ileum: ↓ MPO; ↓ P-selectin expression
Histology: ↓ inflammation (HDMECs)
Marsch et al (2016)77 LDL-receptor KO mice NaNO3 730 mg/L 14 wk ↓ Plasma NO2 to baseline; no effect on granulocytes, monocytes, or lymphocytes; no histological changes in atherosclerotic plaques
Khambata et al (2017)26 Atheroprone mice KNO3 924 mg/L 12 wk ↓ Leukocyte rolling and adherence; ↓ MPO activity; ↓ neutrophil CD11b No effect on CD162 or CD62L
No effect on circulating neutrophils, monocytes, or lymphocytes
Histology: ↓ macrophages in atherosclerotic plaques
Yang et al (2017)74 Mice with IR–induced renal injury NaNO3 62 mg/kg of BW/d 2 wk ↓ BMDM superoxide generation
Histology: ↓ macrophage infiltrate (kidney)

Abbreviations and symbols: BMDM, bone marrow-derived macrophage; BW, body weight; CD, cluster of differentiation; DSS, dextran sodium sulfate; HDMEC, human dermal microvascular endothelial cell; iNOS, inducible nitric oxide synthase; IR, ischemia-reperfusion; KNO3, potassium nitrate; KO, knockout; LDL, low-density lipoprotein; MPO, myeloperoxidase; NaNO2, sodium nitrite; NaNO3, sodium nitrate; NF-κB; nuclear factor κB; NO2, nitrite; ↑, increased; ↓, decreased.

These findings on the influence of nitrate on immune cell infiltration were corroborated by Yang et al,74 who showed that ingestion of sodium nitrate at a concentration of 62 mg/kg for 2 weeks prior to renal ischemic injury in mice reduced macrophage infiltration into renal tissues. In a separate arm of the same study by Yang et al,74 bone marrow–derived macrophages cultured from blood obtained from mice fed a high-nitrate diet for 2 weeks showed a less proinflammatory phenotype, as evidenced by reduced intracellular IL-6 and reduced superoxide generation. The total circulating leukocyte number seems to be unaffected by nitrate ingestion,25,26,77 although a study that used a comparatively large dose of potassium nitrate in mice reported a decrease in the total leukocyte count, with a decrease in neutrophils and an increase in monocytes within artherosclerotic plaques.26

Taken together, the results from the animal studies suggest that dietary nitrate and nitrite modulate inflammation and shift the inflammatory status toward a more anti-inflammatory profile. Further research is warranted to examine the mechanism of action underlying the effects of dietary nitrate and nitrite on inflammatory mediators and on leukocyte phenotypes, functions, and interactions with the vasculature.

Human studies

To date, 8 human intervention studies have examined whether the positive effects of dietary nitrate on inflammation and immune function observed in animal studies translate into preventative and therapeutic benefits in human populations. These studies have investigated the effects of nitrate or nitrite on soluble inflammatory markers and on blood cellular markers of inflammation and leukocyte–platelet interactions. The interventions performed used sodium nitrate (n = 1), sodium nitrite (n = 1), potassium nitrate (n = 2), beetroot (n = 3), and a proprietary blend consisting of beetroot powder, Hawthorn berry extract, L-citrulline, and sodium nitrite (n = 1). The amount of nitrate in these dietary sources ranged from 375 mg to 744 mg, with the proprietary blend study not specifying the nitrate dose. The sodium nitrite study used 2 doses, 80 mg/d and 160 mg/d. Three studies investigated the acute effects of nitrate (single dose and effects within 24 hours), and 6 studies investigated chronic effects (3 days to 10 weeks; multiple doses).

Soluble inflammatory mediators.

Experimental studies in humans have produced mixed results with regard to soluble inflammatory markers within the blood circulation (Table 328,87–91). Macrophage migratory inhibition factor is one inflammatory cytokine that has been suggested to play a major role in atherogenesis through effects on immune cells.92 It contributes to atherosclerosis through early activation of endothelial cells, upregulation of endothelial cell surface adhesion molecules, and elevation of the levels of proinflammatory chemokines.92 Four weeks of daily administration of nitrate (as part of sodium nitrate), administered at 9.3 mg/kg of body weight in individuals with moderate cardiovascular risk, resulted in lower plasma macrophage migratory inhibition factor.89 Contrary to this study, other studies showed cardiovascular benefits of dietary nitrate, but without significant improvements in inflammatory markers.88,90,91 Healthy, older adults given either 80 mg or 160 mg of sodium nitrite per day for 10 weeks demonstrated no changes in circulating inflammatory markers (including high-sensitivity CRP [hsCRP]), despite a significant improvement of vascular function.88 Similarly, in a placebo-controlled study in individuals with hypercholesterolemia given 250 mL nitrate-rich beetroot juice for 6 weeks, no overall effect on hsCRP was found.91 However, stratification of the study participants into low, medium, and high baseline hsCRP groups showed a trend for reductions of hsCRP in the medium and high groups following the nitrate treatment.91 In another study, a population of older adults (median age, 56 years) with a moderate risk of cardiovascular disease was given a proprietary blend of beetroot and Hawthorn berry twice daily for 30 days.90 The intake of this supplement led to a significant increase in both plasma nitrite and nitrate but had no overall effect on plasma CRP concentrations.90 Interestingly, however, only 4 individuals in this cohort had elevated CRP at baseline, and all 4 participants showed marked reductions in CRP by the study conclusion.

Table 3

Human studies investigating the effects of dietary nitrate on soluble inflammatory mediators

Reference Participants Sex and mean age Intervention Nitrate dose Duration Positive effects Negative effects or no effects
Ashor et al (2016)87 Obese Young group: 8 M, 2 F; 31.4 ± 1.8 y KNO3 4.3 mg/kg of BW (≈ 426 mg) Acute: 3 h ↓ E-selectin, ↓ P-selectin No change to ICAM-3 or thrombomodulin; trend for ↓ IL-6
Old group 5 M, 5 F; 64 ± 1.3 y
DeVan et al (2016)88 Healthy 80 mg nitrite group: 5 M, 5 F; 60 ± 2 y NaNO2 80 mg/d Chronic: 10 wk Improved vascular function associated with metabolomic changes No change to circulating markers (including CRP and ET-1)
160 mg/d
160 mg nitrite group: 6 M, 5 F; 63 ± 2 y
Placebo group: 6 M, 4 F; 62 ± 3 y
Rammos et al (2014)89 Moderate CV risk Nitrate group: 7 M, 4 F; 63.7 ± 2 y NaNO3 9.3 mg/kg of BW/d Chronic: 4 wk ↓ MIF
Placebo group: 6 M, 4 F; 62.6 ± 1.3 y
Asgary et al (2016)28 Prehypertensiveb Raw beetroot group: sex NR; 55 ± 11.39 yCooked beetroot group: sex NR; 53.33 ± 10.28 y Raw beetroot; cooked beetroot; 250 g Not measured Chronic: 2 wk ↓ ICAM-1, ↓ VCAM-1, ↓ E-selectin, ↓ IL-6, ↓ hsCRP, ↓ TNF-α
Zand et al (2011)90 ≥ 3 CV risk factors Neo40 group: 23 Proprietary formulation (Neo40 Daily) NR Chronic: 4 wk ↓ TG No effect on CRP, except in 4 patients with baseline elevated CRP who had a ↓ in CRP
Placebo group: 7
Total group: M (63%), F (37%); median age 56 y; range, 42–79 y
Velmurugan et al (2016)91 Hypercholesterolemicb Nitrate group: 12 M, 21 F; 53 ± 10 y Beetroot juice 375 mg/d Chronic: 6 wk No effect on hsCRP or CXCL1
Placebo group: 12 M, 22 F; 53 ± 12 y
Reference Participants Sex and mean age Intervention Nitrate dose Duration Positive effects Negative effects or no effects
Ashor et al (2016)87 Obese Young group: 8 M, 2 F; 31.4 ± 1.8 y KNO3 4.3 mg/kg of BW (≈ 426 mg) Acute: 3 h ↓ E-selectin, ↓ P-selectin No change to ICAM-3 or thrombomodulin; trend for ↓ IL-6
Old group 5 M, 5 F; 64 ± 1.3 y
DeVan et al (2016)88 Healthy 80 mg nitrite group: 5 M, 5 F; 60 ± 2 y NaNO2 80 mg/d Chronic: 10 wk Improved vascular function associated with metabolomic changes No change to circulating markers (including CRP and ET-1)
160 mg/d
160 mg nitrite group: 6 M, 5 F; 63 ± 2 y
Placebo group: 6 M, 4 F; 62 ± 3 y
Rammos et al (2014)89 Moderate CV risk Nitrate group: 7 M, 4 F; 63.7 ± 2 y NaNO3 9.3 mg/kg of BW/d Chronic: 4 wk ↓ MIF
Placebo group: 6 M, 4 F; 62.6 ± 1.3 y
Asgary et al (2016)28 Prehypertensiveb Raw beetroot group: sex NR; 55 ± 11.39 yCooked beetroot group: sex NR; 53.33 ± 10.28 y Raw beetroot; cooked beetroot; 250 g Not measured Chronic: 2 wk ↓ ICAM-1, ↓ VCAM-1, ↓ E-selectin, ↓ IL-6, ↓ hsCRP, ↓ TNF-α
Zand et al (2011)90 ≥ 3 CV risk factors Neo40 group: 23 Proprietary formulation (Neo40 Daily) NR Chronic: 4 wk ↓ TG No effect on CRP, except in 4 patients with baseline elevated CRP who had a ↓ in CRP
Placebo group: 7
Total group: M (63%), F (37%); median age 56 y; range, 42–79 y
Velmurugan et al (2016)91 Hypercholesterolemicb Nitrate group: 12 M, 21 F; 53 ± 10 y Beetroot juice 375 mg/d Chronic: 6 wk No effect on hsCRP or CXCL1
Placebo group: 12 M, 22 F; 53 ± 12 y

Abbreviations and symbols: BW, body weight; CV, cardiovascular; ET-1, endothelin-1; F, female; CXCL, chemokine (C-X-C motif) ligand V; hsCRP, high-sensitivity C-reactive protein; ICAM, intracellular adhesion molecule; IL, interleukin; KNO3, potassium nitrate; M, male; MIF, macrophage migratory inhibitory factor; NaNO2, sodium nitrite; NaNO3, sodium nitrate; NR, not reported; TG, triglycerides; TNF, tumor necrosis factor; VCAM, vascular cell adhesion molecule.

a

Age of study participants is indicated as mean  ±  standard error of the mean, unless otherwise stated.

b

Participant ages described as mean  ±  standard deviation.

Table 3

Human studies investigating the effects of dietary nitrate on soluble inflammatory mediators

Reference Participants Sex and mean age Intervention Nitrate dose Duration Positive effects Negative effects or no effects
Ashor et al (2016)87 Obese Young group: 8 M, 2 F; 31.4 ± 1.8 y KNO3 4.3 mg/kg of BW (≈ 426 mg) Acute: 3 h ↓ E-selectin, ↓ P-selectin No change to ICAM-3 or thrombomodulin; trend for ↓ IL-6
Old group 5 M, 5 F; 64 ± 1.3 y
DeVan et al (2016)88 Healthy 80 mg nitrite group: 5 M, 5 F; 60 ± 2 y NaNO2 80 mg/d Chronic: 10 wk Improved vascular function associated with metabolomic changes No change to circulating markers (including CRP and ET-1)
160 mg/d
160 mg nitrite group: 6 M, 5 F; 63 ± 2 y
Placebo group: 6 M, 4 F; 62 ± 3 y
Rammos et al (2014)89 Moderate CV risk Nitrate group: 7 M, 4 F; 63.7 ± 2 y NaNO3 9.3 mg/kg of BW/d Chronic: 4 wk ↓ MIF
Placebo group: 6 M, 4 F; 62.6 ± 1.3 y
Asgary et al (2016)28 Prehypertensiveb Raw beetroot group: sex NR; 55 ± 11.39 yCooked beetroot group: sex NR; 53.33 ± 10.28 y Raw beetroot; cooked beetroot; 250 g Not measured Chronic: 2 wk ↓ ICAM-1, ↓ VCAM-1, ↓ E-selectin, ↓ IL-6, ↓ hsCRP, ↓ TNF-α
Zand et al (2011)90 ≥ 3 CV risk factors Neo40 group: 23 Proprietary formulation (Neo40 Daily) NR Chronic: 4 wk ↓ TG No effect on CRP, except in 4 patients with baseline elevated CRP who had a ↓ in CRP
Placebo group: 7
Total group: M (63%), F (37%); median age 56 y; range, 42–79 y
Velmurugan et al (2016)91 Hypercholesterolemicb Nitrate group: 12 M, 21 F; 53 ± 10 y Beetroot juice 375 mg/d Chronic: 6 wk No effect on hsCRP or CXCL1
Placebo group: 12 M, 22 F; 53 ± 12 y
Reference Participants Sex and mean age Intervention Nitrate dose Duration Positive effects Negative effects or no effects
Ashor et al (2016)87 Obese Young group: 8 M, 2 F; 31.4 ± 1.8 y KNO3 4.3 mg/kg of BW (≈ 426 mg) Acute: 3 h ↓ E-selectin, ↓ P-selectin No change to ICAM-3 or thrombomodulin; trend for ↓ IL-6
Old group 5 M, 5 F; 64 ± 1.3 y
DeVan et al (2016)88 Healthy 80 mg nitrite group: 5 M, 5 F; 60 ± 2 y NaNO2 80 mg/d Chronic: 10 wk Improved vascular function associated with metabolomic changes No change to circulating markers (including CRP and ET-1)
160 mg/d
160 mg nitrite group: 6 M, 5 F; 63 ± 2 y
Placebo group: 6 M, 4 F; 62 ± 3 y
Rammos et al (2014)89 Moderate CV risk Nitrate group: 7 M, 4 F; 63.7 ± 2 y NaNO3 9.3 mg/kg of BW/d Chronic: 4 wk ↓ MIF
Placebo group: 6 M, 4 F; 62.6 ± 1.3 y
Asgary et al (2016)28 Prehypertensiveb Raw beetroot group: sex NR; 55 ± 11.39 yCooked beetroot group: sex NR; 53.33 ± 10.28 y Raw beetroot; cooked beetroot; 250 g Not measured Chronic: 2 wk ↓ ICAM-1, ↓ VCAM-1, ↓ E-selectin, ↓ IL-6, ↓ hsCRP, ↓ TNF-α
Zand et al (2011)90 ≥ 3 CV risk factors Neo40 group: 23 Proprietary formulation (Neo40 Daily) NR Chronic: 4 wk ↓ TG No effect on CRP, except in 4 patients with baseline elevated CRP who had a ↓ in CRP
Placebo group: 7
Total group: M (63%), F (37%); median age 56 y; range, 42–79 y
Velmurugan et al (2016)91 Hypercholesterolemicb Nitrate group: 12 M, 21 F; 53 ± 10 y Beetroot juice 375 mg/d Chronic: 6 wk No effect on hsCRP or CXCL1
Placebo group: 12 M, 22 F; 53 ± 12 y

Abbreviations and symbols: BW, body weight; CV, cardiovascular; ET-1, endothelin-1; F, female; CXCL, chemokine (C-X-C motif) ligand V; hsCRP, high-sensitivity C-reactive protein; ICAM, intracellular adhesion molecule; IL, interleukin; KNO3, potassium nitrate; M, male; MIF, macrophage migratory inhibitory factor; NaNO2, sodium nitrite; NaNO3, sodium nitrate; NR, not reported; TG, triglycerides; TNF, tumor necrosis factor; VCAM, vascular cell adhesion molecule.

a

Age of study participants is indicated as mean  ±  standard error of the mean, unless otherwise stated.

b

Participant ages described as mean  ±  standard deviation.

Together, these findings also support the notion that nitrate or nitrite supplementation does not interfere with normal immune function.27 Importantly, all of these studies reported beneficial physiological effects on their respective primary outcomes, including reduced circulating triglycerides90 and increased flow-mediated dilatation.91,93 Overall, there is evidence to suggest potentially beneficial effects of nitrate on inflammatory markers, particularly in individuals at risk or with elevated levels of inflammatory markers such as CRP. Importantly, there may be a (currently unknown) threshold for inflammation above or below which dietary nitrate may provide benefit.

Furthermore, in a study without a nitrate-depleted placebo, Asgary et al found that 250 g of either cooked or raw beetroot, given for 2 weeks to healthy individuals, reduced biomarkers of systemic inflammation and endothelial dysfunction, including ICAM-1, VCAM-1, E-selectin, IL-6, hsCRP, and TNF-α.28 These effects were seen in response to consumption of both juiced and cooked beetroot, with more-pronounced effects observed with raw beetroot, suggesting that preparation methods may influence the bioavailability of phytochemicals, including dietary nitrate, within beetroot. It is important to note that the anti-inflammatory effects in this study cannot be attributed solely to nitrate but may be due to other nutrients and phytochemicals within beetroot (eg, betalains, flavonoids) or perhaps even additive or synergistic effects between all of these bioactive compounds. Interactive effects of dietary nitrate with other food components that affect inflammation remain to be tested.

Blood cellular markers of inflammation, and leukocyte–platelet interactions.

Animal and cell culture studies have shown that dietary nitrate (in vivo) and nitrite (in vitro) positively modulates leukocyte–vasculature interactions27 and the functional and phenotypic status of circulating leukocytes.26 On the basis of evidence from these studies, Raubenheimer et al29 have recently investigated whether this translates into benefits in a human population. The most important findings of this study in healthy older adults were that, 3 hours following the consumption of beetroot juice, expression of CD11b on granulocytes, monocyte–platelet aggregation, and blood pressure were all decreased.29 Interactions between leukocytes and platelets, such as monocyte–platelet aggregation, play an important role in the development of inflammation in cardiovascular disease.94 The benefits of dietary nitrate as an anticoagulant agent through its effects on platelets have been established,50,91,95 although the precise mechanisms remain unclear.

A physiologically noteworthy and adverse clinical consequence of increased platelet activation is the effect on immune cells toward a shift to more adhesive and inflammatory phenotypes.96 In this context, recent studies have investigated the effect of dietary nitrate on the inflammatory and thrombotic profile and on biomarkers of leukocyte–platelet interactions (Table 429,87,91,97) Ashor et al87 compared the effects of potassium nitrate (7 mg/kg) against potassium chloride over a 3-hour timeframe in 2 obese cohorts who were older (64.0±1.3 years) or younger (31.4±1.8 years). The authors observed that older participants receiving nitrate had reductions in circulating E-selectin and P-selectin to levels similar to those found in the younger cohort.87 A similar trend for a reduction in IL-6 in the older cohort was also noted.87 Circulating blood cells, such as leukocytes and platelets, bind to the endothelium through adhesive cell surface markers such as P-selectin.98 In a study investigating the acute effects of 493 mg of nitrate from potassium nitrate over a 3-hour period, Velmurugan et al97 found a significant decrease in platelet expression of P-selectin. Reductions in the expression of endothelial cell adhesion molecules and/or blood cell markers may indicate a less adhesive vascular environment, reducing the number of adherent immune cells and leading to better outcomes for cardiovascular patients. Dietary nitrate from vegetables has been shown to be promising as a dietary compound to reduce platelet activation through reduced platelet aggregation activity, lowered P-selectin levels, and reduced platelet–leukocyte aggregate formation.50,91,97 All 3 of these platelet-related effects are beneficial to cardiovascular health, and platelet–leukocyte aggregation is a reliable marker of platelet activation.93 Velmurugan et al91 found that 6 weeks of daily nitrate-rich beetroot juice or nitrate-depleted placebo given to hypercholesterolemic patients resulted in improved vascular function, reductions in monocyte–platelet aggregates, and a trend toward a decrease in stimulated P-selectin in platelets.

Table 4

Human studies investigating the effects of dietary nitrate on blood cellular markers of inflammation and leukocyte–platelet interactions

Reference Participants Sex and mean age Intervention Nitrate dose Duration Positive effects Negative effects or no effects
Velmurugan et al (2013)97 Healthy 12 M; 26 ± 0.8 y; KNO3 493 mg Acute: 3 h ↓ P-selectin (platelets) No effect in women
12 F; 24.1 ± 1.9 y
Raubenheimer (2017)29 Healthy 5 M, 7 F; median age, 64 y; range, 57–71 y Beetroot juice 744 mg Acute: 6 h ↓ PMA; ↓ CD11b (granulocytes); ↑ intermediate monocytes No change in P-selectin (platelets), PGA, classical monocytes, or nonclassical monocytes
Ashor et al (2016)87 Obese Young group: 8 M, 2 F; 31.4 ± 1.8 y KNO3 4.3 mg/kg of BW (≈ 426 mg) Acute: 3 h ↓ PBMC superoxide generation
Old group: 5 M, 5 F; 64 ± 1.3 y
Velmurugan et al (2016)91 Hypercholesterolemicb Nitrate group: 12 M, 21 F; 53 ± 10 y Beetroot juice 375 mg/d Chronic:6 wk ↓ PMA No effect on hsCRP, CXCL1, or platelet P-selectin
Placebo group: 12 M, 22 F; 53 ± 12 y
Reference Participants Sex and mean age Intervention Nitrate dose Duration Positive effects Negative effects or no effects
Velmurugan et al (2013)97 Healthy 12 M; 26 ± 0.8 y; KNO3 493 mg Acute: 3 h ↓ P-selectin (platelets) No effect in women
12 F; 24.1 ± 1.9 y
Raubenheimer (2017)29 Healthy 5 M, 7 F; median age, 64 y; range, 57–71 y Beetroot juice 744 mg Acute: 6 h ↓ PMA; ↓ CD11b (granulocytes); ↑ intermediate monocytes No change in P-selectin (platelets), PGA, classical monocytes, or nonclassical monocytes
Ashor et al (2016)87 Obese Young group: 8 M, 2 F; 31.4 ± 1.8 y KNO3 4.3 mg/kg of BW (≈ 426 mg) Acute: 3 h ↓ PBMC superoxide generation
Old group: 5 M, 5 F; 64 ± 1.3 y
Velmurugan et al (2016)91 Hypercholesterolemicb Nitrate group: 12 M, 21 F; 53 ± 10 y Beetroot juice 375 mg/d Chronic:6 wk ↓ PMA No effect on hsCRP, CXCL1, or platelet P-selectin
Placebo group: 12 M, 22 F; 53 ± 12 y

Abbreviations: BW, body weight; CD, cluster of differentiation; CXCL, chemokine (C-X-C motif) ligand; F, female; KNO3, potassium nitrate; M, male; PBMC, peripheral blood mononuclear cells; PGA, platelet-granulocyte aggregates; PMA, platelet-monocyte aggregates; ↑, increased; ↓, decreased.

a

Age of study participants is indicated as mean  ±  standard error of the mean, unless otherwise stated.

b

Participant ages described as mean  ±  standard deviation.

Table 4

Human studies investigating the effects of dietary nitrate on blood cellular markers of inflammation and leukocyte–platelet interactions

Reference Participants Sex and mean age Intervention Nitrate dose Duration Positive effects Negative effects or no effects
Velmurugan et al (2013)97 Healthy 12 M; 26 ± 0.8 y; KNO3 493 mg Acute: 3 h ↓ P-selectin (platelets) No effect in women
12 F; 24.1 ± 1.9 y
Raubenheimer (2017)29 Healthy 5 M, 7 F; median age, 64 y; range, 57–71 y Beetroot juice 744 mg Acute: 6 h ↓ PMA; ↓ CD11b (granulocytes); ↑ intermediate monocytes No change in P-selectin (platelets), PGA, classical monocytes, or nonclassical monocytes
Ashor et al (2016)87 Obese Young group: 8 M, 2 F; 31.4 ± 1.8 y KNO3 4.3 mg/kg of BW (≈ 426 mg) Acute: 3 h ↓ PBMC superoxide generation
Old group: 5 M, 5 F; 64 ± 1.3 y
Velmurugan et al (2016)91 Hypercholesterolemicb Nitrate group: 12 M, 21 F; 53 ± 10 y Beetroot juice 375 mg/d Chronic:6 wk ↓ PMA No effect on hsCRP, CXCL1, or platelet P-selectin
Placebo group: 12 M, 22 F; 53 ± 12 y
Reference Participants Sex and mean age Intervention Nitrate dose Duration Positive effects Negative effects or no effects
Velmurugan et al (2013)97 Healthy 12 M; 26 ± 0.8 y; KNO3 493 mg Acute: 3 h ↓ P-selectin (platelets) No effect in women
12 F; 24.1 ± 1.9 y
Raubenheimer (2017)29 Healthy 5 M, 7 F; median age, 64 y; range, 57–71 y Beetroot juice 744 mg Acute: 6 h ↓ PMA; ↓ CD11b (granulocytes); ↑ intermediate monocytes No change in P-selectin (platelets), PGA, classical monocytes, or nonclassical monocytes
Ashor et al (2016)87 Obese Young group: 8 M, 2 F; 31.4 ± 1.8 y KNO3 4.3 mg/kg of BW (≈ 426 mg) Acute: 3 h ↓ PBMC superoxide generation
Old group: 5 M, 5 F; 64 ± 1.3 y
Velmurugan et al (2016)91 Hypercholesterolemicb Nitrate group: 12 M, 21 F; 53 ± 10 y Beetroot juice 375 mg/d Chronic:6 wk ↓ PMA No effect on hsCRP, CXCL1, or platelet P-selectin
Placebo group: 12 M, 22 F; 53 ± 12 y

Abbreviations: BW, body weight; CD, cluster of differentiation; CXCL, chemokine (C-X-C motif) ligand; F, female; KNO3, potassium nitrate; M, male; PBMC, peripheral blood mononuclear cells; PGA, platelet-granulocyte aggregates; PMA, platelet-monocyte aggregates; ↑, increased; ↓, decreased.

a

Age of study participants is indicated as mean  ±  standard error of the mean, unless otherwise stated.

b

Participant ages described as mean  ±  standard deviation.

Taken together, the data from human studies provide evidence of a potential preventative and therapeutic role of dietary nitrate and nitrite in counteracting excessive inflammation. More research is needed to understand the underlying mechanisms and to examine if an inflammatory threshold exists whereby dietary nitrate may be beneficial and under which physiological and pathophysiological conditions dietary nitrate has the most pronounced effect.

Clinical relevance.

Dietary nitrate supplementation has been suggested as a novel, cost-efficient, and natural strategy to therapeutically modulate NO bioavailability in states of NO insufficiency, including settings of age-associated disorders and cardiovascular disease.6 The potential therapeutic value of dietary nitrate is highlighted by the findings of Jones et al,99 who showed that intracoronary nitrite administration following primary percutaneous intervention for acute myocardial infarction reduced neutrophil activation, as evidenced by reduced CD11b expression on circulating neutrophils.99 This particular study also found lower hsCRP levels, reduced plasma levels of neutrophil and monocyte chemoattractants (CXCL1, CXCL5, CCL2), and sustained long-term reductions in major adverse cardiac events 3 years after the intervention.99 Evidence from animal studies demonstrates that nitrite is stored in cardiac tissues and reduces infarct severity after myocardial infarction.100 A diet high in nitrates may therefore act as a potential reservoir of NO that can lead to cardioprotective effects. Further studies investigating the translational implications of these effects are required.

CONCLUSION

Historically, inorganic nitrate has been considered a detrimental dietary component, owing to the formation of nitrite and the possible generation of carcinogenic nitrosamines.101 However, the potential relationship between nitrate intake and cancer in humans has not been substantiated.19 Rather, the initial negative view on dietary nitrate has been challenged by accumulating evidence suggesting that an increased consumption of nitrate-containing vegetables could be a key component of lifestyle interventions to promote, preserve, or even restore cardiovascular health. Recent clinical findings support the concept that an increased consumption of nitrate-rich vegetables translates to a reduction in the long-term risk of cardiovascular disease.

Emerging data suggest that the benefits of dietary nitrate on the cardiovascular system are partially exerted through modulation of immune and inflammatory aspects. Further animal studies are needed to advance the understanding of the underlying molecular and cellular mechanisms. Additional translational research is also warranted for verifying these effects in experimental and clinical settings in humans.

In particular, more long-term, randomized, placebo-controlled clinical studies measuring a combination of multiple markers of inflammation—in addition to functional and clinical outcomes—are required to determine if nitrate-rich plant foods reduce the risk of developing chronic low-grade inflammation. Furthermore, investigating whether dietary nitrate modulates responses to inflammatory challenges, such as acute metabolic inflammation following a high-fat meal, will provide important information on the effect on immune homeostasis. Future research could also take advantage of “omics” technologies (eg, blood immune cell and plasma proteomics) to investigate immune- and inflammation-related molecular biomarker panels and biological networks in response to dietary nitrate ingestion. Another emerging aspect that warrants further investigation is the relationship between the gut microbiome and the immune system in the context of cardiovascular health and disease.102 In light of data suggesting that dietary nitrate affects the intestinal and oral microbiota,16,102 this could be an indirect mechanism through which nitrate contained in plant-based foods modulates immune function.

The evidence in this review suggests that inorganic nitrate may be a key vegetable component for reducing chronic inflammation and promoting immune and cardiovascular homeostasis. Together, this is a promising and important area of research. It is likely that advancing the understanding of the role of dietary nitrate in modulating immune function will expand a variety of potential targets for treating and preventing cardiovascular disease, age-related disorders, and other diseases associated with chronic inflammation.

Acknowledgments

Author contributions. K.R. conducted the literature review and contributed text and table content. C.B. contributed to the manuscript conception and table content and designed the figure. L.B. contributed text content. K.-H.W. and J.M.P. contributed to the manuscript conception. O.N. contributed to the manuscript conception and text content and supported K.R. in conducting the literature review. All authors were involved in drafting and revising the manuscript and approved the final version of the submitted manuscript.

Funding/support. No external funds supported this work.

Declaration of interest. The authors have no relevant interests to declare.

References

1

Pober
JS
,
Sessa
WC.
Inflammation and the blood microvascular system
.
Cold Spring Harb Perspect Biol.
2014
;
7
:
a016345.

2

Walsh
NP
,
Gleeson
M
,
Shephard
RJ
, et al. .
Position statement. Part one: immune function and exercise
.
Exerc Immunol Rev.
2011
;
17
:
6
63
.

3

Calder
PC
,
Ahluwalia
N
,
Albers
R
, et al. .
A consideration of biomarkers to be used for evaluation of inflammation in human nutritional studies
.
Br J Nutr.
2013
;
109(suppl 1)
:
S1
34
.

4

Peake
JM
,
Neubauer
O
,
Della Gatta
PA
, et al. .
Muscle damage and inflammation during recovery from exercise
.
J Appl Physiol (1985)
.
2017
;
122
:
559
570
.

5

Tidball
JG.
Regulation of muscle growth and regeneration by the immune system
.
Nat Rev Immunol.
2017
;
17
:
165
178
.

6

Seals
DR
,
Kaplon
RE
,
Gioscia-Ryan
RA
, et al. .
You're only as old as your arteries: translational strategies for preserving vascular endothelial function with aging
.
Physiology (Bethesda)
.
2014
;
29
:
250
264
.

7

Sies
H
,
Berndt
C
,
Jones
DP.
Oxidative stress
.
Annu Rev Biochem.
2017
;
86
:
715
748
.

8

Seals
DR
,
Justice
JN
,
LaRocca
TJ.
Physiological geroscience: targeting function to increase healthspan and achieve optimal longevity
.
J Physiol.
2016
;
594
:
2001
2024
.

9

Libby
P
,
Hansson
GK.
Inflammation and immunity in diseases of the arterial tree: players and layers
.
Circ Res.
2015
;
116
:
307
311
.

10

Pedersen
BK.
The diseasome of physical inactivity—and the role of myokines in muscle–fat cross talk
.
J Physiol.
2009
;
587
:
5559
5568
.

11

Minihane
AM
,
Vinoy
S
,
Russell
WR
, et al. .
Low-grade inflammation, diet composition and health: current research evidence and its translation
.
Br J Nutr.
2015
;
114
:
999
1012
.

12

Libby
P
,
Bornfeldt
KE
,
Tall
AR.
Atherosclerosis: successes, surprises, and future challenges
.
Circ Res.
2016
;
118
:
531
534
.

13

Rahimi
K
,
Emdin
CA
,
MacMahon
S.
The epidemiology of blood pressure and its worldwide management
.
Circ Res.
2015
;
116
:
925
936
.

14

Woessner
MN
,
McIlvenna
LC
,
Ortiz de Zevallos
J
, et al. .
Dietary nitrate supplementation in cardiovascular health: an ergogenic aid or exercise therapeutic?
Am J Physiol Heart Circ Physiol.
2018
;
314
:
H195
H212
.

15

Lundberg
JO
,
Gladwin
MT
,
Weitzberg
E.
Strategies to increase nitric oxide signalling in cardiovascular disease
.
Nat Rev Drug Discov.
2015
;
14
:
623
641
.

16

Carlström
M
,
Lundberg
JO
,
Weitzberg
E.
Mechanisms underlying blood pressure reduction by dietary inorganic nitrate
.
Acta Physiol (Oxf).
2018
;
224
:e13080.

17

Ghimire
K
,
Altmann
HM
,
Straub
AC
, et al. .
Nitric oxide: what's new to NO?
Am J Physiol Cell Physiol.
2017
;
312
:
C254
C262
.

18

Blekkenhorst
LC
,
Bondonno
NP
,
Liu
AH
, et al. .
Nitrate, the oral microbiome, and cardiovascular health: a systematic literature review of human and animal studies
.
Am J Clin Nutr
.
2018
;
107
:
504
522
.

19

Weitzberg
E
,
Lundberg
JO.
Novel aspects of dietary nitrate and human health
.
Annu Rev Nutr.
2013
;
33
:
129
159
.

20

Bondonno
CP
,
Croft
KD
,
Ward
N
, et al. .
Dietary flavonoids and nitrate: effects on nitric oxide and vascular function
.
Nutr Rev.
2015
;
73
:
216
235
.

21

Hord
NG
,
Tang
Y
,
Bryan
NS.
Food sources of nitrates and nitrites: the physiologic context for potential health benefits
.
Am J Clin Nutr.
2009
;
90
:
1
10
.

22

McNally
B
,
Griffin
JL
,
Roberts
LD.
Dietary inorganic nitrate: from villain to hero in metabolic disease?
Mol Nutr Food Res.
2016
;
60
:
67
78
.

23

Jones
AM
,
Thompson
C
,
Wylie
LJ
, et al. .
Dietary nitrate and physical performance
.
Annu Rev Nutr.
2018
;
38
:
303
328
.

24

Kapil
V
,
Khambata
RS
,
Robertson
A
, et al. .
Dietary nitrate provides sustained blood pressure lowering in hypertensive patients: a randomized, phase 2, double-blind, placebo-controlled study
.
Hypertension
.
2015
;
65
:
320
327
.

25

Stokes
KY
,
Dugas
TR
,
Tang
Y
, et al. .
Dietary nitrite prevents hypercholesterolemic microvascular inflammation and reverses endothelial dysfunction
.
Am J Physiol Heart Circ Physiol.
2009
;
296
:
H1281
H1288
.

26

Khambata
RS
,
Ghosh
SM
,
Rathod
KS
, et al. .
Antiinflammatory actions of inorganic nitrate stabilize the atherosclerotic plaque
.
Proc Natl Acad Sci USA.
2017
;
114
:
E550
E559
.

27

Jädert
C
,
Petersson
J
,
Massena
S
, et al. .
Decreased leukocyte recruitment by inorganic nitrate and nitrite in microvascular inflammation and NSAID-induced intestinal injury
.
Free Radic Biol Med
.
2012
;
52
:
683
692
.

28

Asgary
S
,
Afshani
MR
,
Sahebkar
A
, et al. .
Improvement of hypertension, endothelial function and systemic inflammation following short-term supplementation with red beet (Beta vulgaris L.) juice: a randomized crossover pilot study
.
J Hum Hypertens.
2016
;
30
:
627
632
.

29

Raubenheimer
K
,
Hickey
D
,
Leveritt
M
, et al. .
Acute effects of nitrate-rich beetroot juice on blood pressure, hemostasis and vascular inflammation markers in healthy older adults: a randomized, placebo-controlled crossover study
.
Nutrients
.
2017
;
9:1270
. doi:10.3390/nu9111270

30

Carlström
M
,
Persson
AE
,
Larsson
E
, et al. .
Dietary nitrate attenuates oxidative stress, prevents cardiac and renal injuries, and reduces blood pressure in salt-induced hypertension
.
Cardiovasc Res.
2011
;
89
:
574
585
.

31

Hickey
MJ
,
Kubes
P.
Role of nitric oxide in regulation of leucocyte-endothelial cell interactions
.
Exp Physiol.
1997
;
82
:
339
348
.

32

Kreuger
J
,
Phillipson
M.
Targeting vascular and leukocyte communication in angiogenesis, inflammation and fibrosis
.
Nat Rev Drug Discov.
2016
;
15
:
125
142
.

33

Randolph
GJ.
Mechanisms that regulate macrophage burden in atherosclerosis
.
Circ Res.
2014
;
114
:
1757
1771
.

34

Petri
B
,
Phillipson
M
,
Kubes
P.
The physiology of leukocyte recruitment: an in vivo perspective
.
J Immunol.
2008
;
180
:
6439
6446
.

35

Rodriguez-Iturbe
B
,
Pons
H
,
Johnson
RJ.
Role of the immune system in hypertension
.
Physiol Rev.
2017
;
97
:
1127
1164
.

36

Hopkins
PN.
Molecular biology of atherosclerosis
.
Physiol Rev.
2013
;
93
:
1317
1542
.

37

Bogdan
C.
Nitric oxide and the immune response
.
Nat Immunol.
2001
;
2
:
907
916
.

38

Sindler
AL
,
Fleenor
BS
,
Calvert
JW
, et al. .
Nitrite supplementation reverses vascular endothelial dysfunction and large elastic artery stiffness with aging
.
Aging Cell
.
2011
;
10
:
429
437
.

39

Gao
X
,
Yang
T
,
Liu
M
, et al. .
NADPH oxidase in the renal microvasculature is a primary target for blood pressure-lowering effects by inorganic nitrate and nitrite
.
Hypertension
.
2015
;
65
:
161
170
.

40

Yang
T
,
Peleli
M
,
Zollbrecht
C
, et al. .
Inorganic nitrite attenuates NADPH oxidase-derived superoxide generation in activated macrophages via a nitric oxide-dependent mechanism
.
Free Radic Biol Med
.
2015
;
83
:
159
166
.

41

Weber
C
,
Zernecke
A
,
Libby
P.
The multifaceted contributions of leukocyte subsets to atherosclerosis: lessons from mouse models
.
Nat Rev Immunol.
2008
;
8
:
802
815
.

42

Tabas
I
,
Bornfeldt
KE.
Macrophage phenotype and function in different stages of atherosclerosis
.
Circ Res.
2016
;
118
:
653
667
.

43

Scapini
P
,
Cassatella
MA.
Social networking of human neutrophils within the immune system
.
Blood
.
2014
;
124
:
710
719
.

44

Whelton
PK
,
Carey
RM.
The 2017 American College of Cardiology/American Heart Association clinical practice guideline for high blood pressure in adults
.
JAMA Cardiol.
2018
;
3
:
352
353
.

45

Rossier
BC
,
Bochud
M
,
Devuyst
O.
The hypertension pandemic: an evolutionary perspective
.
Physiology (Bethesda)
.
2017
;
32
:
112
125
.

46

McMaster
WG
,
Kirabo
A
,
Madhur
MS
, et al. .
Inflammation, immunity, and hypertensive end-organ damage
.
Circ Res.
2015
;
116
:
1022
1033
.

47

Olsen
F.
Inflammatory cellular reaction in hypertensive vascular disease in man
.
Acta Pathol Microbiol Scand A
.
1972
;
80
:
253
256
.

48

Siervo
M
,
Lara
J
,
Ogbonmwan
I
, et al. .
Inorganic nitrate and beetroot juice supplementation reduces blood pressure in adults: a systematic review and meta-analysis
.
J Nutr.
2013
;
143
:
818
826
.

49

Larsen
FJ
,
Ekblom
B
,
Sahlin
K
, et al. .
Effects of dietary nitrate on blood pressure in healthy volunteers
.
N Engl J Med.
2006
;
355
:
2792
2793
.

50

Webb
AJ
,
Patel
N
,
Loukogeorgakis
S
, et al. .
Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite
.
Hypertension
.
2008
;
51
:
784
790
.

51

Ahluwalia
A
,
Foster
P
,
Scotland
RS
, et al. .
Antiinflammatory activity of soluble guanylate cyclase: cGMP-dependent down-regulation of P-selectin expression and leukocyte recruitment
.
Proc Natl Acad Sci USA.
2004
;
101
:
1386
1391
.

52

Liyanage
T
,
Ninomiya
T
,
Wang
A
, et al. .
Effects of the Mediterranean diet on cardiovascular outcomes—a systematic review and meta-analysis
.
PLoS One.
2016
;
11
:
e0159252.

53

Salehi-Abargouei
A
,
Maghsoudi
Z
,
Shirani
F
, et al. .
Effects of Dietary Approaches to Stop Hypertension (DASH)-style diet on fatal or nonfatal cardiovascular diseases—incidence: a systematic review and meta-analysis on observational prospective studies
.
Nutrition.
2013
;
29
:
611
618
.

54

Huang
T
,
Yang
B
,
Zheng
J
, et al. .
Cardiovascular disease mortality and cancer incidence in vegetarians: a meta-analysis and systematic review
.
Ann Nutr Metab.
2012
;
60
:
233
240
.

55

Hu
FB.
Plant-based foods and prevention of cardiovascular disease: an overview
.
Am J Clin Nutr.
2003
;
78
:
544S
551S
.

56

Grosso
G
,
Marventano
S
,
Yang
J
, et al. .
A comprehensive meta-analysis on evidence of Mediterranean diet and cardiovascular disease: are individual components equal?
Crit Rev Food Sci Nutr.
2017
;
57
:
3218
3232
.

57

Threapleton
DE
,
Greenwood
DC
,
Evans
CE
, et al. .
Dietary fibre intake and risk of cardiovascular disease: systematic review and meta-analysis
.
BMJ.
2013
;
347
:
f6879.

58

Kim
Y
,
Je
Y.
Flavonoid intake and mortality from cardiovascular disease and all causes: a meta-analysis of prospective cohort studies
.
Clin Nutr ESPEN
.
2017
;
20
:
68
77
.

59

Blekkenhorst
LC
,
Prince
RL
,
Ward
NC
, et al. .
Development of a reference database for assessing dietary nitrate in vegetables
.
Mol Nutr Food Res
.
2017
;
61
:1600982. doi:10.1002/mnfr.201600982

60

Blekkenhorst
LC
,
Bondonno
CP
,
Lewis
JR
, et al. .
Association of dietary nitrate with atherosclerotic vascular disease mortality: a prospective cohort study of older adult women
.
Am J Clin Nutr.
2017
;
106
:
207
216
.

61

Bondonno
CP
,
Blekkenhorst
LC
,
Prince
RL
, et al. .
Association of vegetable nitrate intake with carotid atherosclerosis and ischemic cerebrovascular disease in older women
.
Stroke
.
2017
;
48
:
1724
1729
.

62

Liu
AH
,
Bondonno
CP
,
Russell
J
, et al. .
Relationship of dietary nitrate intake from vegetables with cardiovascular disease mortality: a prospective study in a cohort of older Australians
.
Eur J Nutr
.
2018
. doi:10.1007/s00394-018-1823-x

63

Barbaresko
J
,
Koch
M
,
Schulze
MB
, et al. .
Dietary pattern analysis and biomarkers of low-grade inflammation: a systematic literature review
.
Nutr Rev.
2013
;
71
:
511
527
.

64

Holt
EM
,
Steffen
LM
,
Moran
A
, et al. .
Fruit and vegetable consumption and its relation to markers of inflammation and oxidative stress in adolescents
.
J Am Diet Assoc.
2009
;
109
:
414
421
.

65

Nettleton
JA
,
Steffen
LM
,
Mayer-Davis
EJ
, et al. .
Dietary patterns are associated with biochemical markers of inflammation and endothelial activation in the Multi-Ethnic Study of Atherosclerosis (MESA)
.
Am J Clin Nutr.
2006
;
83
:
1369
1379
.

66

Song
Y
,
Li
TY
,
van Dam
RM
, et al. .
Magnesium intake and plasma concentrations of markers of systemic inflammation and endothelial dysfunction in women
.
Am J Clin Nutr
.
2007
;
85
:
1068
1074
.

67

Gago
B
,
Lundberg
JO
,
Barbosa
RM
, et al. .
Red wine-dependent reduction of nitrite to nitric oxide in the stomach
.
Free Radic Biol Med.
2007
;
43
:
1233
1242
.

68

Dewhurst-Trigg
R
,
Yeates
T
,
Blackwell
JR
, et al. .
Lowering of blood pressure after nitrate-rich vegetable consumption is abolished with the co-ingestion of thiocyanate-rich vegetables in healthy normotensive males
.
Nitric Oxide.
2018
;
74
:
39
46
.

69

Kuhnle
GG
,
Luben
R
,
Khaw
KT
, et al. .
Sulfate, nitrate and blood pressure— an EPIC interaction between sulfur and nitrogen
.
Pharmacol Res
.
2017
;
122
:
127
129
.

70

Sindler
AL
,
Cox-York
K
,
Reese
L
, et al. .
Oral nitrite therapy improves vascular function in diabetic mice
.
Diab Vasc Dis Res.
2015
;
12
:
221
224
.

71

Ohtake
K
,
Ehara
N
,
Chiba
H
, et al. .
Dietary nitrite reverses features of postmenopausal metabolic syndrome induced by high-fat diet and ovariectomy in mice
.
Am J Physiol Endocrinol Metab.
2017
;
312
:
E300
E308
.

72

Hezel
MP
,
Liu
M
,
Schiffer
TA
, et al. .
Effects of long-term dietary nitrate supplementation in mice
.
Redox Biol.
2015
;
5
:
234
242
.

73

Li
T
,
Lu
X
,
Sun
Y
, et al. .
Effects of spinach nitrate on insulin resistance, endothelial dysfunction markers and inflammation in mice with high-fat and high-fructose consumption
.
Food Nutr Res
.
2016
;
60
:
32010.

74

Yang
T
,
Zhang
XM
,
Tarnawski
L
, et al. .
Dietary nitrate attenuates renal ischemia-reperfusion injuries by modulation of immune responses and reduction of oxidative stress
.
Redox Biol
.
2017
;
13
:
320
330
.

75

Gheibi
S
,
Jeddi
S
,
Carlström
M
, et al. .
Effects of long-term nitrate supplementation on carbohydrate metabolism, lipid profiles, oxidative stress, and inflammation in male obese type 2 diabetic rats
.
Nitric Oxide
.
2018
;
75
:
27
41
.

76

Jädert
C
,
Phillipson
M
,
Holm
L
, et al. .
Preventive and therapeutic effects of nitrite supplementation in experimental inflammatory bowel disease
.
Redox Biol
.
2014
;
2
:
73
81
.

77

Marsch
E
,
Theelen
TL
,
Janssen
BJ
, et al. .
The effect of prolonged dietary nitrate supplementation on atherosclerosis development
.
Atherosclerosis
2016
;
245
:
212
221
.

78

Lundberg
JO
,
Carlström
M
,
Larsen
FJ
, et al. .
Roles of dietary inorganic nitrate in cardiovascular health and disease
.
Cardiovasc Res.
2011
;
89
:
525
532
.

79

Swirski
FK
,
Nahrendorf
M.
Leukocyte behavior in atherosclerosis, myocardial infarction, and heart failure
.
Science
.
2013
;
339
:
161
166
.

80

Horan
PK
,
Slezak
SE
,
Poste
G.
Improved flow cytometric analysis of leukocyte subsets: simultaneous identification of five cell subsets using two-color immunofluorescence
.
Proc Natl Acad Sci USA.
1986
;
83
:
8361
8365
.

81

Phillipson
M
,
Heit
B
,
Colarusso
P
, et al. .
Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade
.
J Exp Med.
2006
;
203
:
2569
2575
.

82

Kolaczkowska
E
,
Kubes
P.
Neutrophil recruitment and function in health and inflammation
.
Nat Rev Immunol.
2013
;
13
:
159
175
.

83

Sumagin
R
,
Prizant
H
,
Lomakina
E
, et al. .
LFA-1 and Mac-1 define characteristically different intralumenal crawling and emigration patterns for monocytes and neutrophils in situ
.
J Immunol
.
2010
;
185
:
7057
7066
.

84

Lau
D
,
Mollnau
H
,
Eiserich
JP
, et al. .
Myeloperoxidase mediates neutrophil activation by association with CD11b/CD18 integrins
.
Proc Natl Acad Sci USA
.
2005
;
102
:
431
436
.

85

Sica
A
,
Mantovani
A.
Macrophage plasticity and polarization: in vivo veritas
.
J Clin Invest.
2012
;
122
:
787
795
.

86

Soehnlein
O
,
Steffens
S
,
Hidalgo
A
, et al. .
Neutrophils as protagonists and targets in chronic inflammation
.
Nat Rev Immunol.
2017
;
17
:
248
261
.

87

Ashor
AW
,
Chowdhury
S
,
Oggioni
C
, et al. .
Inorganic nitrate supplementation in young and old obese adults does not affect acute glucose and insulin responses but lowers oxidative stress
.
J Nutr
.
2016
;
146
:
2224
2232
.

88

DeVan
AE
,
Johnson
LC
,
Brooks
FA
, et al. .
Effects of sodium nitrite supplementation on vascular function and related small metabolite signatures in middle-aged and older adults
.
J Appl Physiol (1985)
.
2016
;
120
:
416
425
.

89

Rammos
C
,
Hendgen-Cotta
UB
,
Sobierajski
J
, et al. .
Dietary nitrate reverses vascular dysfunction in older adults with moderately increased cardiovascular risk
.
J Am Coll Cardiol
.
2014
;
63
:
1584
1585
.

90

Zand
J
,
Lanza
F
,
Garg
HK
, et al. .
All-natural nitrite and nitrate containing dietary supplement promotes nitric oxide production and reduces triglycerides in humans
.
Nutr Res
.
2011
;
31
:
262
269
.

91

Velmurugan
S
,
Gan
JM
,
Rathod
KS
, et al. .
Dietary nitrate improves vascular function in patients with hypercholesterolemia: a randomized, double-blind, placebo-controlled study
.
Am J Clin Nutr
.
2016
;
103
:
25
38
.

92

Zernecke
A
,
Bernhagen
J
,
Weber
C.
Macrophage migration inhibitory factor in cardiovascular disease
.
Circulation
.
2008
;
117
:
1594
1602
.

93

van Gils
JM
,
Zwaginga
JJ
,
Hordijk
PL.
Molecular and functional interactions among monocytes, platelets, and endothelial cells and their relevance for cardiovascular diseases
.
J Leukoc Biol
.
2009
;
85
:
195
204
.

94

Rondina
MT
,
Weyrich
AS
,
Zimmerman
GA.
Platelets as cellular effectors of inflammation in vascular diseases
.
Circ Res.
2013
;
112
:
1506
1519
.

95

Burnley-Hall
N
,
Abdul
F
,
Androshchuk
V
, et al. .
Dietary nitrate supplementation reduces circulating platelet-derived extracellular vesicles in coronary artery disease patients on clopidogrel therapy: a randomised, double-blind, placebo-controlled study
.
Thromb Haemost.
2018
;
118
:
112
122
.

96

Cerletti
C
,
de Gaetano
G
,
Lorenzet
R.
Platelet–leukocyte interactions: multiple links between inflammation, blood coagulation and vascular risk
.
Mediterr J Hematol Infect Dis.
2010
;
2
:
e2010023.

97

Velmurugan
S
,
Kapil
V
,
Ghosh
SM
, et al. .
Antiplatelet effects of dietary nitrate in healthy volunteers: involvement of cGMP and influence of sex
.
Free Radic Biol Med
.
2013
;
65
:
1521
1532
.

98

Collins
RG
,
Velji
R
,
Guevara
NV
, et al. .
P-selectin or intercellular adhesion molecule (ICAM)-1 deficiency substantially protects against atherosclerosis in apolipoprotein E–deficient mice
.
J Exp Med.
2000
;
191
:
189
194
.

99

Jones
DA
,
Khambata
RS
,
Andiapen
M
, et al. .
Intracoronary nitrite suppresses the inflammatory response following primary percutaneous coronary intervention
.
Heart
.
2017
;
103
:
508
516
. doi:10.1136/heartjnl-2016-309748

100

Bryan
NS
,
Calvert
JW
,
Elrod
JW
, et al. .
Dietary nitrite supplementation protects against myocardial ischemia-reperfusion injury
.
Proc Natl Acad Sci USA
.
2007
;
104
:
19144
19149
.

101

Tannenbaum
SR
,
Sinskey
AJ
,
Weisman
M
, et al. .
Nitrite in human saliva. Its possible relationship to nitrosamine formation
.
J Natl Cancer Inst.
1974
;
53
:
79
84
.

102

Galla
S
,
Chakraborty
S
,
Mell
B
, et al. .
Microbiotal-host interactions and hypertension
.
Physiology (Bethesda).
2017
;
32
:
224
233
.

This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)