Inorganic nitrate attenuates the systemic inflammatory response in typhoid vaccine-induced endothelial dysfunction in healthy volunteers

Abstract

Background: Inflammatory responses underlie the development of endothelial dysfunction in CVD, however, therapeutics that might target this pathway have not been forthcoming. A key pathogenic mechanism mediating endothelial dysfunction is a reduction in bioavailable (eNOS‐derived) nitric oxide (NO). Activation of the non‐canonical pathway for in‐vivo NO generation might offer an approach to improve NO levels and recover vascular function in pre‐clinical models of CVD. Whether this might occur in humans is unknown. Purpose: We hypothesize that consumption of inorganic nitrate will lead to increases in bioavailable NO and thus attenuate the inflammatory pathways leading to typhoid vaccine‐induced endothelial dysfunction in healthy volunteers. Methods: Healthy male volunteers were recruited (n=78) and randomized to receive either beetroot juice containing 8‐10mmol nitrate or placebo (nitrate‐deplete) juice once daily for 6 days. Participants underwent serial measurements of BP, FMD and GTN‐induced brachial artery dilatation, and haematology and biochemistry, before and after typhoid vaccination. Blood, urine and saliva nitrite and nitrate were quantified using ozone chemiluminescence, and leukocyte flow cytometry analysis was conducted. Results: 8‐hours post‐vaccine endothelial function was depressed in placebo‐treated volunteers, however this was prevented in nitrate‐treated volunteers. This dysfunction was due to impaired endothelial function since responses to GTN were unaffected either by vaccination or dietary intervention (p=0.981). Dietary nitrate resulted in an increase in plasma (p<0.0001), urine (p=0.0006) and saliva (p<0.0001) nitrate, and urine (p=0.0354) and saliva (p<0.0001) nitrite levels. There was a reduction in the proportions of CD14++/CD16+intermediate monocytes in nitratetreated participants after vaccine (p=0.016, change from baseline between groups). In the nitrate‐treated group, less CD14++/CD16+ intermediate monocyte CD62L expression was identified post‐vaccine (p=0.0122), compared to placebo, with no difference in soluble plasma CD62L between groups (p=0.875). CD11b median fluorescence intensity was increased in CD3+/CD4+ T‐lymphocytes in nitrate‐treated volunteers (p=0.0095). Conclusions: Dietary nitrate reduced BP, as previously shown, indicating efficacy of the intervention. Importantly, we also now show for the first time that inorganic nitrate suppresses the systemic inflammatory response, specifically by reducing the numbers and activation state of CD14++/CD16+ intermediate monocytes. Furthermore, an increased expression of CD3+/CD4+ T‐cell CD11b and preserved FMD in healthy volunteers treated with nitrate, suggests an anti‐inflammatory phenotype, induced by the intervention, leading to improved endothelial function. Inorganic dietary nitrate modulates endothelial function through the attenuation of inflammatory responses and may be of potential therapeutic benefit in patients with established CAD.