A pro-resolving phenotype underpins the anti-inflammatory effects of inorganic nitrate

Abstract

Introduction Increasing evidence highlights the critical role of chronic inflammation in cardiovascular disease (CVD). Targeting inflammatory pathways in patients with CVD has been associated with improved CV function in pre-clinical (Gee, 2017), early clinical (Yndestad, 2006; Velmurugan 2013; Jones, 2016) and large phase III studies (Ridker, 2017). The resolution of inflammation is an active process and its failure has also been proposed to contribute to CVD progression. At least one mechanism thought to underlie this failure is dysfunction of the canonical pathway for anti-inflammatory nitric oxide (NO) production. Restoring NO through provision of inorganic nitrate (NO3-) and subsequent bioactivation via the non-canonical pathway may offer therapeutic benefit. Aim To test whether dietary NO3–derived NO accelerates resolution of inflammation. Methods Randomised, double-blind, placebo-controlled, parallel limb study of 8–10mmol dietary NO3- supplementation versus NO3–deplete placebo beetroot juice in 36 healthy male volunteers (NCT03183830). Using a cantharadin-induced skin blister model (Day, 2001), acute (24h) and chronic (72h)-phase blisters were harvested pre- and post-treatment. Blister exudate was analysed for leucocyte activation state (CD11b, CD62L, CD162) by flow cytometry and cytokine/chemokine composition by ELISA. Ozone chemiluminescence established NO3-/NO2- levels in key biological matrices: plasma, urine and saliva. Results 9.3mmol inorganic NO3- led to a significant rise (versus placebo, p<0.001) of NO3-/NO2- in plasma, saliva and urine NO2- (p<0.02). No differences were seen in blister volumes, cell counts or markers of systemic inflammation. Whilst no differences were seen in the proportions of cellular infiltrate in 24h blisters, there were significant reductions of neutrophil (p=0.017) and intermediate monocyte proportions (p=0.001) and cellular adhesion molecules across inflammatory, intermediate and resolving monocytes at 72h (Figure 1). Generally, no differences in blister cytokine/chemokine profile was evident except for borderline significant suppression of TNFα at 24hrs with dietary NO3- treatment (P=0.057). Conclusion Whilst dietary inorganic NO3- does not impair the essential host defence response it does accelerate resolution: enhanced pro- to anti-inflammatory monocyte subtype switching and curtailed neutrophil recruitment, likely via attenuated TNFα production. These actions offer a novel, easy to administer, approach to influence inflammatory responses without impairing host defence. FUNDunding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Derek Willoughby Trust and British Heart Foundation