In this systematic investigation of neutrophils from healthy individuals of different age we could clearly demonstrate that neutrophil characteristics, potential of activation and ability to undergo NET formation change with human age. We observed a shift in neutrophil subpopulations towards decreased mature LDNs, but also lower ability to upregulate the activation marker CD11b upon stimulation with IO and PMA with increasing age. This may relate to altered neutrophil functions with increasing human age and contribute to the well-known higher susceptibility to pathogens in persons of advanced age. NET formation also correlated with age and was lowest in younger persons. The risk of thrombosis also increases with age and neutrophils of individuals at younger age might be less prone to prothrombotic NET formation.
Data on LDN frequency in healthy controls are rarely available and LDN counts vary between reports (24, 25). HDN and LDN counts showed no age related differences, however, percentages of mature CD62Lhigh/CD16high LDNs correlated negatively and percentage of newly released CD16low LDNs positively with age. Sagiv and colleagues observed that neutrophils, especially newly released neutrophils, are increased in patients with cancer and show a pro-tumorigenic phenotype (26). However, none of our healthy volunteers had active or a history of cancer. Additionally, newly released neutrophils are less potent in their pathogen defence (27) and might thus contribute to higher infection susceptibility of the elderly. The decreased potency of the immune system is furthermore associated with increased incidence of patients with cancer and metastasis (28).
An association with age and decreased ability to upregulate CD66b upon PMA stimulation of HDNs was observed. Expression levels of the activation marker CD11b of HDNs were also negatively associated with age. Upregulation of CD11b expression on HDNs was found to be distinctively more prominent in younger individuals upon stimulation with different stimulants at various concentrations. Data on age-related CD66b expression levels in neutrophils have to our knowledge not been published to date. Data on age-related changes of CD11b, which is part of the integrin αMβ2 (CD11b/CD18) complex and crucial for neutrophil adhesion, are available, yet scarce. Two publications showed that baseline levels of CD11b are not significantly altered in humans with higher age (16, 29). Furthermore, age-related expression data on CD11b and CD66b levels upon in vitro stimulation have not been published to date. Our results support the notion that neutrophil functions of pathogen defence, which are attributed to HDNs rather than LDNs, are less effectively activated in older individuals and may hence in part explain the higher infection rate within the older population.
Data on DNA release after 3 h stimulation at 37 °C is likely to correspond to the release of NETs, although we did not measure any NET specific marker in this experiment. Upon low dose PMA stimulation, levels of released DNA were elevated in isolated PMNs with increasing individuals’ age and also in isolated PBMCs harbouring the LDN fraction. HDNs within the PMN fraction were isolated with a median purity of 97.05% (IQR: 96.05–97.95), while LDNs within the PBMC fraction showed a median abundance of 1.95% (IQR: 1.30–2.95). Further purification of PBMCs via magnetic-bead sampling or other experimental purification steps would result in pure LDNs but would also pre-activate cells. Therefore, it has to be considered that LDNs could not be separately analysed and other cells within the PBMC layer may release their DNA as well. Since this method was not specific for measuring only NETs but all extracellular DNA, it can be assumed that distinct amounts of DNA originate from neutrophils but also from other cells such as eosinophils, mast cells, and monocyte/macrophages, which have all previously been described to also form extracellular traps in a process termed ETosis (27, 30–32). Higher levels of DNA release upon increased individuals’ age could be explained by the elevated number of immature, newly released neutrophils that were previously reported to release their DNA also spontaneously (33, 34), regardless of the stimulus or the respective concentrations.
While DNA release was high in AG2 as well as AG3 (frequently exhibiting comparable median levels, see Fig. 2), the NET marker H3Cit was highest in the middle aged group of 45–54 years (in baseline, unstimulated and IO stimulated samples). Considering the lower level of H3Cit positive LDNs in AG3, it could be argued that older people have higher numbers of immature neutrophils that most likely have released their NETs spontaneously in circulation and therefore could not be activated again ex vivo (33, 34). Another potential explanation for this discrepancy is that neutrophils may change to another pathway of NET induction, without the generation of H3Cit. In vitro studies have shown that distinct activation pathways via NADPH oxidase (35), myeloperoxidase and neutrophil elastase and/or via mitochondrial ROS production (36) may lead to NET formation in the absence of H3Cit generation. Thus, measurements of DNA release (in particular of the highly pure PMN fraction) may more reliably and comprehensively reflect the NET forming capacity of isolated neutrophils than H3Cit levels. Regarding the link of NET formation and venous thrombosis, which has been investigated before (37, 38), it is known that age is an important risk factor for VTE (39). One hypothesis is the increased blood coagulability (39). But also, the increased release of DNA by HDNs (or possibly also other cell types) may contribute to this increased risk.
Some limitations of this study have to be addressed: first, the rather small number of participants and second, the chosen classification into age groups. This healthy control cohort was designed to age and sex-match patients of other studies. The exclusion criteria were a history of cancer, bleeding disorders, a history of thrombotic events and infectious diseases 6 weeks prior to the blood draw. The resulting healthy control cohort was then divided in three age groups of approximately even participant numbers. Third, we did not cover exercise routine with the structured questionnaire, as it is known that exercise has many different effects on neutrophil numbers and behaviour. Regular exercise leads to an increase in endogenous pyrogen, a known neutrophil priming agent (40) and increases the numbers of circulating neutrophils (41). Lastly, LDN results have to be interpreted with caution, as there were fewer analysed events compared to HDNs. This study bears a number of strengths, one most important is that it is the first study that systematically analysed age related changes in neutrophils. The persons entering the study were well defined with strict in- and exclusion criteria, as diseases, that might impact neutrophil function, were excluded, e.g. known malignancy or recent history of an infectious disease.