Title: The effect of age on monocyte phenotype and function
Funder: Dunhill Medical Trust

Abstract: Inflammageing, the process of chronic systemic low grade inflammation, characterised as elevated circulating IL-6, IL-8 and C Reactive protein (CRP) which occurs with increasing age. Inflammageing is known to be detrimental to the health of older adults, as elevated circulating inflammatory markers are associated with frailty, cognitive decline and all-cause morbidity and mortality. In addition, increased inflammation is detrimental to a functioning immune system with reduced vaccine efficacy and antigen-specific immunity. The origins of inflammageing in older adults is believed to be multifactorial. To date the majority of the research on inflammageing has focussed on the role of senescent cells, however what is clear is that mononuclear phagocytes and in particular monocytes may drive the inflammageing process and warrant further investigation. Monocytes historically were presumed to be precursor cells for macrophages and dendritic cells - although this can be true, monocytes are recognised as established immune effector cells in their own right. Human monocytes are defined by their expression of the cell surface markers CD14 and CD16. The classical monocytes are defined as CD14+CD16-, the intermediate monocytes are CD14+CD16+ and then the non-classical monocytes are defined as CD16+CD14-/lo. Studies, have shown that there is an increased frequency of CD16+ monocytes in the peripheral blood of older adults (≥65 years) , data from my lab has expanded this observation and shown that there is a significant increase in both intermediate and non-classical monocytes in older adults as compared to young (<​35 years). To date the majority of functional research on older monocytes has focussed on CD14+ monocyte populations – despite the fact that it is the CD16+ monocytes specifically increase with age. Although older monocytes produce similar inflammatory cytokines in response to one TLR/inflammatory stimuli. When the combination of two stimuli are used such as Sodium Chloride (NaCl; an inflammatory stimuli which is known to trigger inflammatory monocyte infiltration at the site of skin challenge) and Lipopolysaccharide (LPS; a Toll-like receptor (TLR) 4 agonist) were utilised older monocytes produce significantly more TNFα as compared to young. This increase in inflammatory cytokine production was due to the increased number of CD16+ non-classical monocytes found in older adults. As inflammageing is an age-associated phenomenon there is great potential that monocytes and in particular CD16+ monocytes contribute to the inflammatory environment. Therefore understanding how they are functionally different from younger individuals is vitally important. The aim of this research is to understand how ageing alters the phenotype and function of the monocytes. This project will utilise in vitro monocyte cultures and high dimensional proteomic assessment of monocyte populations ex vivo and post-stimulation to provide an in depth analysis of the effect of age on monocyte phenotype and function. It is expected that this project will identify if there is a general inflammatory defect in older monocyte populations or a more specific defect in certain signalling pathways. It is anticipated that the data generated from the proteome analysis will identify specific pathways/proteins which are differentially expressed in older monocytes which could be targeted therapeutically to reduce the inflammatory cytokine production and thus reduce the inflammageing process – and in turn improve health and immunity in older adults.

Lay abstract: Inflammageing is the process whereby as we get older we have an increased amount inflammatory proteins circulating in blood. This increase in inflammatory proteins is associated with worsening immunity, poor vaccine responses and quite shockingly increased risk of death. Thereby identifying therapeutic targets to block this inflammation is desperately needed to improve the health of older adults. Although there are many potential origins of inflammageing, there is one cell type which has been ignored, despite the fact it has the potential to drive inflammatory protein production and thus inflammageing – is a monocyte. Monocytes are circulating white blood cells which predominantly live in the blood, and respond to pathogen and inflammageing signals with a large increase in inflammatory protein production. Monocytes are known to exist in three different subtypes (flavours) and they are known as either classical, intermediate and non-classical. It has been observed by myself and others that there is a significant increase in the number of non-classical monocytes in the blood of older (≥65 years) adults as compared to younger (<​35 years). Yet the majority of research to date on the effect of age on monocyte function has focussed on the classical monocytes, actively excluding the non-classical monocytes. I have observed that when monocytes from older adults are cultured in a plate with a combination of two different stimuli, there is significantly more inflammatory protein production as compared to younger monocytes. This increase in inflammatory proteins is due to the increased number of inflammatory non-classical monocytes. However, further research is needed to understand if this is a general inflammatory defect in the older non-classical monocytes or whether its specific to the stimulation conditions I used. The overarching aim of this grant is to perform in depth analysis of older monocyte populations and compared them to younger monocytes. I will be using culturing techniques as well as an exciting new technique called proteomics which looks at protein levels within the cell, to assess if there are any differences between young and old. The ultimate aim of this research is to identify different proteins within older monocytes which drive inflammatory protein production, which in turn can be blocked with drugs and improve immunity and health of older adults.

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