Title: Impact of aging on pro-resolving pathways in neutrophils: role of extracellular vesicles.
Funder: Dunhill Medical Trust

Abstract: Background/scope: This project aims to address the problem of inflammageing, an increased inflammatory state that affects people as they age. We hypothesise that this may be due, at least in part, to a dysregulation of the pro-resolving pathways that function to limit and switch off inflammation in healthy young people. We have chosen to focus on a well characterised pathway (AnnexinA1) that is operative during the resolution of inflammation in healthy young individuals. AnxA1 has a multitude of protective effects, including limiting leukocyte recruitment, promoting clearance of bacteria and dead cells (to contain and dampen the immune response) and restoring tissue function. This protein is released from neutrophils via extracellular vesicles (EVs) that can be delivered to neighbouring cell types to exert beneficial effects. Our preliminary data indicates that levels of AnxA1 are decreased in neutrophils as we age, yet it is not currently known how this pathway is impacted by the ageing process. Unmet need: Inflammageing reduces the ability of our immune system to mount a sufficient inflammatory response rendering the aged individual more susceptible to injury and infection. This has been highlighted by the current COVID-19 pandemic with an increased burden of infection related mortality with increased age. Understanding how we can restore normal immune function in aged individuals will pave the way for new therapeutic advances in the elderly population. New Knowledge: There is currently limited data as to how neutrophil-derived resolution pathways are impacted by the ageing process. This project will provide new information on the phenotype of neutrophils and their EVs in aged individuals, whether the pro-resolving AnxA1 pathway is impeded during ageing and importantly whether neutrophil function can be enhanced by FPR2 agonists. Research Questions: The objectives of this proposal are to: 1: Determine whether levels of the pro-resolving protein AnnexinA1 and its receptor are altered in neutrophils as we age. 2: Determine whether FPR2 agonists restore function in neutrophils of aged individuals 3: Compare neutrophil-derived extracellular vesicle (EV) generation/content from aged individuals and young healthy individuals. 4: Assess the effect of neutrophil-derived EVs on endothelial phenotype and leukocyte trafficking. These objectives will challenge our overarching hypothesis that AnxA1 levels decline in neutrophils (and their EVs) as we age, contributing to the decline in ability to recover from or resolve inflammation. Methodologies: The proposed investigations will be performed using human blood samples from healthy young controls and aged individuals. Levels of AnxA1 and its receptor in both neutrophils and neutrophil-derived EVs will be assessed by multi-colour flow cytometry and highly sensitive specialist equipment for EV analysis. Functional effects of neutrophil-derived EVs will be assessed on endothelial cell phenotype (vascular permeability transwell assays and live imaging of leukocyte recruitment under flow) as well as leukocyte functionality in whole blood (phagocytosis of bacteria). Proposed Impact: Whilst this project will not have an immediate impact on the lives of older people, understanding how neutrophil function is altered during ageing and the impact this has on the ability to fight infection will undoubtably influence future clinical direction. Indeed, there are already clinical trials investigating the efficacy of new FPR2 agonists with positive outcomes in phase 1 trials indicating they are safe and well-tolerated in humans. Thus, increasing our knowledge on this pathway during ageing opens opportunities for therapeutic interventions for inflammatory conditions in the elderly.

Lay abstract: Advanced age is a major risk factor for most chronic diseases and infections in humans, yet the reasons why this occurs are not fully understood. We now know that the body makes protective molecules as part of a normal healthy response to infection or inflammation, however this process becomes defective with ageing. One of these protective molecules is called Annexin A1, which instructs white blood cells to destroy bacteria during infections. We hypothesise that the amount of Annexin A1 produced by our body declines with age and that if we supplement these levels, we will be able to restore normal white blood cell function and subsequently reduce the risk of infections and chronic illness and improve the quality of life in elderly individuals. For this study, we will use a small amount of blood from healthy young and old people to compare the levels of AnnexinA1 on white blood cells; we will also explore how these cells communicate and influence other cells via tiny packages termed microvesicles. This is important as multiple cell types are involved in the immune response to infection or inflammation therefore monitoring their cross-talk is vital for obtaining a full overview of the health status of these cells.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM