OBJECTIVE:Community-acquired pneumonia (CAP) is a leading cause of hospitalisation in older adults and is associated with a high likelihood of adverse outcomes. Given the ageing population and lack of therapeutic advances in CAP, new strategies to manage the burden of this disease are needed. Neutrophil dysfunction has been widely demonstrated in CAP and is associated with poor outcomes. We hypothesised that impaired glycolytic metabolism was driving neutrophil dysfunction in older adults with CAP. METHODS:To investigate the mechanism underlying neutrophil dysfunction in CAP, we recruited older adults with CAP and sepsis, age-matched controls and healthy young adults to assess neutrophil function and glycolytic metabolism in peripheral blood neutrophils. RESULTS:We demonstrate that neutrophils from older donors with CAP display a broad range of functional defects, including inaccurate migration to interleukin 8, impaired respiratory burst in response to phorbol 12-myristate 13-acetate and increased spontaneous degranulation compared with age-matched controls. Glycolysis (assessed by extracellular flux and RNA-sequencing) was not significantly altered between age-matched groups; however, basal rates of neutrophil glycolysis were significantly higher in patients with CAP and older adult controls compared with healthy young adults, and stimulated glycolysis was significantly higher in young adults compared with older adults with and without CAP. CONCLUSIONS:Our findings suggest that neutrophil dysfunction in older adults with CAP may be implicated in poor outcomes, irrespective of glycolytic metabolism.

Background: Smoking cessation remains the only effective strategy for slowing the progression of Chronic Obstructive Pulmonary Disease (COPD). E-cigarettes (EC) have been shown effective in smoking cessation trials, however real-world data suggests long-term usage after cessation is common. Acute exposure studies suggest signals of harm in immune cells from young healthy volunteers, yet data is lacking on the impact of vaping on COPD patient neutrophils. Hypothesis: EC exposure, despite being less harmful than tobacco smoking, impairs neutrophil functions, indicating that EC exposure may enhance the pathophysiology of COPD patients. Methods: Neutrophils were isolated from the blood of healthy older adults and COPD patients by Percoll density gradient separation, then exposed to a total of 40 EC puffs (4 seconds on, 30 seconds off), in a modified hypoxia chamber. Viability was analysed by flow cytometry; annexin V and Propidium iodide. NETosis was measured using SYTOX® green, following a 3hour phorbol myristate acetate (PMA) incubation. Results: EC exposure did not affect viability of neutrophils from healthy older adult age matched controls (AMC) or COPD patient neutrophils. Baseline and PMA stimulated NETosis was lower in in COPD patient neutrophils compared to AMC, indicating an overall lower capacity to produce ex vivo. Our preliminary data suggests nicotine containing EC exposure reduced PMA stimulated NETosis in both groups. Conclusion: Exposure to EC vapour impairs the capacity to produce NETs in response in both healthy older adult and COPD patient neutrophils. Further reduction in COPD neutrophil NETosis following exposure to EC, suggests additional harm in this group.

Neutrophil responses are critical during inflammatory and infective events, and neutrophil dysregulation has been associated with poor patient outcomes. Immunometabolism is a rapidly growing field that has provided insights into cellular functions in health and disease. Neutrophils are highly glycolytic when activated, with inhibition of glycolysis associated with functional deficits. There is currently very limited data available assessing metabolism in neutrophils. Extracellular flux (XF) analysis assesses real time oxygen consumption and the rate of proton efflux in cells. This technology allows for the automated addition of inhibitors and stimulants to visualise the effect on metabolism. We describe optimised protocols for an XFe96 XF Analyser to (i) probe glycolysis in neutrophils under basal and stimulated conditions, (ii) probe phorbol 12-myristate 13-acetate induced oxidative burst, and (iii) highlight challenges of using XF technology to examine mitochondrial function in neutrophils. We provide an overview of how to analyze XF data and identify pitfalls of probing neutrophil metabolism with XF analysis. In summary we describe robust methods for assessing glycolysis and oxidative burst in human neutrophils and discuss the challenges around using this technique to assess mitochondrial respiration. XF technology is a powerful platform with a user-friendly interface and data analysis templates, however we suggest caution when assessing neutrophil mitochondrial respiration.

Background: Electronic cigarette (e-cigarette) use continues to rise despite concerns of long-term effects, especially the risk of developing lung diseases such as chronic obstructive pulmonary disease. Neutrophils are central to the pathogenesis of chronic obstructive pulmonary disease, with changes in phenotype and function implicated in tissue damage. Objective: We sought to measure the impact of direct exposure to nicotine-containing and nicotine-free e-cigarette vapor on human neutrophil function and phenotype. Methods: Neutrophils were isolated from the whole blood of self-reported nonsmoking, nonvaping healthy volunteers. Neutrophils were exposed to 40 puffs of e-cigarette vapor generated from e-cigarette devices using flavorless e-cigarette liquids with and without nicotine before functions, deformability, and phenotype were assessed. Results: Neutrophil surface marker expression was altered, with CD62L and CXCR2 expression significantly reduced in neutrophils treated with e-cigarette vapor containing nicotine. Neutrophil migration to IL-8, phagocytosis of Escherichia coli and Staphylococcus aureus pHrodo bioparticles, oxidative burst response, and phorbol 12-myristate 13-acetate-stimulated neutrophil extracellular trap formation were all significantly reduced by e-cigarette vapor treatments, independent of nicotine content. E-cigarette vapor induced increased levels of baseline polymerized filamentous actin levels in the cytoplasm, compared with untreated controls. Conclusions: The significant reduction in effector neutrophil functions after exposure to high-power e-cigarette devices, even in the absence of nicotine, is associated with excessive filamentous actin polymerization. This highlights the potentially damaging impact of vaping on respiratory health and reinforces the urgency of research to uncover the long-term health implications of e-cigarettes. (J Allergy Clin Immunol 2024;153:320-9.)

Rational: Evidence of neutrophil dysfunction in COVID-19 is based on transcriptomics. Cell functions are interwoven pathways, so understanding the effect of COVID-19 across neutrophil function may identify therapeutic targets. We examined neutrophil phenotype and function in 41 hospitalised, non-ICU COVID-19 patients versus 23 age-matched controls (AMC) and 26 community acquired pneumonia (CAP) patients. Methods: Isolated neutrophils underwent ex vivo analyses for migration, phagocytosis and NETosis, and the effect of PI3K inhibition. Circulating DNAse 1 activity and levels of cfDNA were measured. Results: Compared to AMC and CAP, COVID-19 neutrophils demonstrated elevated transmigration (p=0.0397, A) and NETosis (p=0.0366, B), but impaired phagocytosis (p=0.0236, C) associated with impaired ROS generation (p<0.0001). COVID-19 and CAP patients showed increased systemic markers of NETosis including increased cfDNA (p=0.0153) and impaired DNAse activity (p<0.0.001, D). Ex vivo inhibition of PI3K γ and δ reduced NET release by COVID-19 neutrophils (p=0.0156). Conclusion: COVID-19 is associated with neutrophil dysfunction across all main effector functions, with elevated migration, impaired antimicrobial responses and elevated NETosis. These changes represent a clear mechanism for tissue damage and highlight that targeting neutrophil function via PI3k may help modulate COVID-19 severity.

Background: In the UK, around 2.7 million people use electronic (e-)cigarettes despite uncertainties in their long-term safety. Neutrophils play a central role in the pathophysiology of chronic obstructive pulmonary disease (COPD), a smoking-associated lung disease. Objective: To measure the impact of direct e-cigarette vapour exposure on the function and phenotype of human neutrophils. Methods: Peripheral blood neutrophils from non-smoker/vaper participants were exposed to 40 puffs (4s on/30s off) of e-cigarette vapour generated from e-cigarettes loaded with flavourless e-liquids with and without nicotine using second and third generation devices. Neutrophil functions were measured by microscopy and flow cytometry. Results: Neutrophil migration to CXCL8, phagocytosis of E. coli and S. aureus pHrodo bioparticles and generation of reactive oxygen species (ROS) were significantly reduced by e-cigarette vapour. Neutrophil extracellular trap (NET) formation was suppressed. Oxidative burst and glycolytic reserve were significantly impaired. Neutrophil phenotypic markers including CD62L were altered, suggesting an activated and immature phenotype. Conclusions: Neutrophils are functionally supressed after exposure to vapour from high power e-cigarette devices, even in the absence of nicotine. This is likely driven by the reported presence of reactive carbonyl species in the vapour of e-cigarettes, the production of which is directly tied to device power output. This work highlights the potentially damaging impact of vaping on respiratory health and increases the urgency of further research to uncover the long-term health implications of e-cigarettes.

Rationale: Infection with the SARS-CoV2 virus is associated with elevated neutrophil counts. Evidence of neutrophil dysfunction in COVID-19 is based on transcriptomics or single functional assays. Cell functions are interwoven pathways, and understanding the effect across the spectrum of neutrophil function may identify therapeutic targets. Objectives: Examine neutrophil phenotype and function in 41 hospitalised, non-ICU COVID-19 patients versus 23 age-matched controls (AMC) and 26 community acquired pneumonia patients (CAP). Methods: Isolated neutrophils underwent ex vivo analyses for migration, bacterial phagocytosis, ROS generation, NETosis and receptor expression. Circulating DNAse 1 activity, levels of cfDNA, MPO, VEGF, IL-6 and sTNFRI were measured and correlated to clinical outcome. Serial sampling on day three to five post hospitalization were also measured. The effect of ex vivo PI3K inhibition was measured in a further cohort of 18 COVID-19 patients. Results: Compared to AMC and CAP, COVID-19 neutrophils demonstrated elevated transmigration (p = 0.0397) and NETosis (p = 0.0332), and impaired phagocytosis (p = 0.0036) associated with impaired ROS generation (p < 0.0001). The percentage of CD54+ neutrophils (p < 0.001) was significantly increased, while surface expression of CD11b (p = 0.0014) and PD-L1 (p = 0.006) were significantly decreased in COVID-19. COVID-19 and CAP patients showed increased systemic markers of NETosis including increased cfDNA (p = 0.0396) and impaired DNAse activity (p < 0.0001). The ex vivo inhibition of PI3K γ and δ reduced NET release by COVID-19 neutrophils (p = 0.0129). Conclusions: COVID-19 is associated with neutrophil dysfunction across all main effector functions, with altered phenotype, elevated migration and NETosis, and impaired antimicrobial responses. These changes highlight that targeting neutrophil function may help modulate COVID-19 severity.

Electronic (e-) cigarettes are growing in popularity despite uncertainties regarding their long-term health implications. The link between cigarette smoking and initiation of chronic lung disease took decades to unpick so in vitro studies mimicking e-cigarette exposure aim to detect early indicators of harm. In response to e-cigarette exposure, alveolar macrophages adopt a proinflammatory phenotype of increased secretion of proinflammatory cytokines, reduction in phagocytosis, and efferocytosis and reactive oxygen species generation. These effects are largely driven by free radical exposure, changes in PI3K/Akt signaling pathways, nicotine-induced reduction in phagocytosis receptors, and impaired lipid homeostasis leading to a foam-like lipidladen phenotype. Neutrophils exhibit disrupted chemotaxis and transmigration to chemokines, reduced phagocytosis and bacterial killing, and an increase in protease secretion without corresponding antiproteases in response to e-cigarette exposure. This is driven by an altered ability to respond and to polarize toward chemoattractants, an activation of the p38 MAPK signaling pathway and inability to assemble NADPH oxidase. E-cigarettes induce lung epithelial cells to display decreased ciliary beat frequency and ion channel conductance as well as changes in chemokine secretion and surface protein expression. Changes in gene expression, mitochondrial function, and signaling pathways have been demonstrated in lung epithelial cells to explain these changes. Many functional outputs of alveolar macrophages, neutrophils, and lung epithelial cells have not been fully explored in the context of e-cigarette exposure and the underlying driving mechanisms are poorly understood. This review discusses current evidence surrounding the effects of e-cigarettes on alveolar macrophages, neutrophils, and lung epithelial cells with particular focus on the cellular mechanisms of change.

RationalInfection with the SARS-CoV2 virus is associated with elevated neutrophil counts. Evidence of neutrophil dysfunction in COVID-19 is based predominantly on transcriptomics or single functional assays. Cell functions are interwoven pathways, and so understanding the effect of COVID-19 across the spectrum of neutrophil function may identify therapeutic targets to treat disease.ObjectivesExamine neutrophil phenotype and functional capacity in COVID-19 patients versus age-matched controls (AMC).MethodsIsolated neutrophils from 41 non-ICU COVID-19 patients and 23 AMC underwent ex vivo analyses for migration, phagocytosis of Streptococcus pneumoniae, reactive oxygen species (ROS) generation, neutrophil extracellular trap formation (NETosis) and cell surface receptor expression. Serum DNAse 1 activity was measured, alongside circulating levels of cell-free (cf)DNA, myeloperoxidase (MPO), VEGF, IL-6 and sTNFRI. All measurements were correlated to clinical outcome. Serial sampling on day 3–5 post hospitalisation were also measured.ResultsCompared to AMC, COVID-19 neutrophils demonstrated elevated transmigration (p=0.0397) and NETosis (p=0.0366), but impaired phagocytosis (p=0.0236) associated with impaired ROS generation (p<0.0001). Surface expression of CD54 (p<0.0001) and CD11c (p=0.0008) was significantly increased and CD11b significantly decreased (p=0.0229) on COVID-19 patient neutrophils. On day 3–5 follow-up, levels of senescent neutrophils increased compared to day 1 (indicated by decreased CXCR2 and elevated CXCR4 expression (p=0.0332)). COVID-19 patients showed increased systemic markers of NETosis including increased cfDNA (p=0.0153) and impaired DNAse activity (p<0.0.001). MPO, VEGF, sTNFRI, and IL-6 (p<0001) were elevated in COVID-19, which positively correlated with disease severity by 4C score.ConclusionCOVID-19 is associated with neutrophil dysfunction across all main effector functions, with altered phenotype, elevated migration, impaired antimicrobial responses and elevated NETosis. These changes represent a clear mechanism for tissue damage and highlight that targeting neutrophil function may help modulate COVID-19 severity. Please refer to page A189 for declarations of interest related to this abstract.

Rational: Infection with the SARS-CoV2 virus is associated with elevated neutrophil counts. Evidence of neutrophil dysfunction in COVID-19 is based on transcriptomics. Further insight into SARS-CoV-2 effects on neutrophil biology at the functional level could identify therapeutic targets to treat severe disease. Objectives: Examine neutrophil phenotype and functional capacity in COVID-19 patients versus age-matched controls (AMC) Methods: Isolated neutrophils from 41 non-ICU COVID-19 patients and 23 AMC underwent ex vivo analyses for migration, bacterial phagocytosis, ROS generation, NET formation (NETosis) and cell surface receptor expression. DNAse 1 activity was measured, alongside circulating levels of cfDNA, MPO, VEGF, IL-6 and sTNFRI. All measurements were correlated to clinical outcome. Serial sampling on day 3-5 post hospitalisation were also measured. Results: Compared to AMC, COVID-19 neutrophils demonstrated elevated migration (p=0.0397) and NETosis (p=0.036), but impaired phagocytosis (p=0.023) associated with impaired ROS generation (p<0.0001). Surface expression of CD54 (p<0.0001) and CD11c (p=0.0008) was significantly increased and CD11b significantly decreased (p=0.022) on COVID-19 neutrophils. COVID-19 patients showed increased systemic markers of NETosis including cfDNA (p=0.015) and impaired DNAse activity (p<0.0.001). MPO, VEGF, sTNFRI, and IL-6 (p<0001) were elevated in COVID-19, which positively correlated with disease severity by 4C score. Conclusion: Circulating neutrophils from COVID-19 patients display altered phenotype, elevated migration, impaired anti-microbial responses and elevated NETosis. Targeting neutrophil function may help modulate COVID-19 severity.

Neutrophil dysfunction has been described with age, appears exaggerated in infection, with altered phosphoinositol signaling a potential mechanism. However, functional aging is heterogeneous. Frailty is a negative health status and is more common in older adults. We hypothesized that neutrophil migration may be compromised in frailty, associated with the degree of frailty experienced by the older person. We compared measures of frailty, neutrophil function, and systemic inflammation in 40 young and 77 older community-dwelling adults in the United Kingdom. Systemic neutrophils exhibited an age-associated reduction in the accuracy of migration (chemotaxis) which was further blunted with frailty. The degree of migratory inaccuracy correlated with physical (adjusted hand grip strength) and cognitive (Stroop test) markers of frailty. Regression analysis demonstrated that age, Charlson comorbidity index, and frailty index were able to predict neutrophil chemotaxis. Reduced chemotaxis of neutrophils from frail adults could be reversed using selective PI3K inhibitors. Exposure of neutrophils from young adults to plasma from chronically inflamed frail older adults could not recapitulate the migratory deficit in vitro, and there were no relationships with systemic inflammation and neutrophil dysfunction. Frailty exaggerated the neutrophil deficits seen with advanced age but aspects of the frailty-associated deficit in neutrophil function are rescuable and thus potentially form a therapeutic target to improve outcomes from infection in older adults.

BACKGROUND:The COVID-19 pandemic has led to many countries implementing lockdown procedures, resulting in the suspension of laboratory research. With lockdown measures now easing in some areas, many laboratories are preparing to reopen. This is particularly challenging for clinical research laboratories due to the dual risk of patient samples carrying the virus that causes COVID-19, SARS-CoV-2, and the risk to patients being exposed to research staff during clinical sampling. To date, no confirmed transmission of the virus has been confirmed within a laboratory setting; however, operating processes and procedures should be adapted to ensure safe working of samples of positive, negative, or unknown COVID-19 status. OBJECTIVE:In this paper, we propose a framework for reopening a clinical research laboratory and resuming operations with the aim to maximize research capacity while minimizing the risk to research participants and staff. METHODS:This framework was developed by consensus among experienced laboratory staff who have prepared to reopen a clinical research laboratory. RESULTS:Multiple aspects need to be considered to reopen a clinical laboratory. We describe our process to stratify projects by risk, including assessment of donor risk and COVID-19 clinical status, the COVID-19 status of the specific sample type, and how to safely process each sample type. We describe methods to prepare the laboratory for safe working including maintaining social distancing through signage, one-way systems and access arrangements for staff and patients, limiting staff numbers on site and encouraging home working for all nonlaboratory tasks including data analysis and writing. Shared equipment usage was made safe by adapting booking systems to allow for the deployment of cleaning protocols. All risk assessments and standard operating procedures were rewritten and approved by local committees, and staff training was initiated to ensure compliance. CONCLUSIONS:Laboratories can adopt and adapt this framework to expedite reopening a clinical laboratory during the current COVID-19 pandemic while mitigating the risk to research participants and staff.

Abstract Introduction Sleep quantity and continuity vary across the lifespan. Actigraphy is reliable, ecologically valid, and is the most widely-used behavioral measure of sleep in research and personal health monitoring. The extent to which age is associated with actigraphy-assessed sleep has not been evaluated across the lifespan. The aim of this meta-analysis was to evaluate the associations between age and actigraphy-assessed sleep in relatively healthy individuals. Methods A systematic search of PubMed, Embase.com, Cochrane CENTRAL, and PsycINFO using “actigraphy” and “sleep” terms provided 7,079 titles/abstracts, which were screened to exclude studies of only individuals with mental health disorders, medical conditions, sleep disorders, or shift workers. We evaluated 1,379 full-text articles for reports on the association between age and actigraphy-assessed sleep duration, efficiency, timing, and/or regularity. Overall, 88 articles met these criteria (182 effect sizes; N=18,443). Four meta-analyses were conducted, examining sleep duration (k=86), sleep efficiency (k=58), bedtime (k=27), and wake-up time (k=11). There were insufficient numbers of studies (less than 5) to evaluate sleep midpoint or sleep regularity. We tested continent of the study, study design, actigraphy device type, and number of nights of data collection as moderators of meta-analytic associations. Results With increasing age, sleep duration was shorter (r = -0.13) and sleep efficiency was lower (r = -0.06). Bedtime was later with age for ages 6-21 (r = 0.31) and earlier for ages 22 and up (r = -0.65). Wake-up time was not associated with age for ages 6-21 (r = 0.20) but was earlier with increasing age for ages 22 and up (r = -0.71). The strength of these associations was modified by continent and study design, but not by type of actigraphy device or number of nights of data collection. Conclusion Weak associations between age and actigraphy-assessed duration and efficiency suggest that inadequate sleep quantity or poor sleep continuity should not be dismissed as typical consequences of aging. Large associations between age and sleep timing, despite a small literature, highlights a promising area for further study, particularly to determine the age at which sleep timing shifts from delaying to advancing. Support MAB was supported by T32 HL07560.

Airway neutrophilia is a common feature of many chronic inflammatory lung diseases and is associated with disease progression, often regardless of the initiating cause. Neutrophils and their products are thought to be key mediators of the inflammatory changes in the airways of patients with chronic obstructive pulmonary disease (COPD) and have been shown to cause many of the pathological features associated with disease, including emphysema and mucus hypersecretion. Patients with COPD also have high rates of bacterial colonisation and recurrent infective exacerbations, suggesting that neutrophil host defence mechanisms are impaired, a concept supported by studies showing alterations to neutrophil migration, degranulation and reactive oxygen species production in cells isolated from patients with COPD. Although the role of neutrophils is best described in COPD, many of the pathological features of this disease are not unique to COPD and also feature in other chronic inflammatory airway diseases, including asthma, cystic fibrosis, alpha-1 anti-trypsin deficiency, and bronchiectasis. There is increasing evidence for immune cell dysfunction contributing to inflammation in many of these diseases, focusing interest on the neutrophil as a key driver of pulmonary inflammation and a potential therapeutic target than spans diseases. This review discusses the evidence for neutrophilic involvement in COPD and also considers their roles in alpha-1 anti-trypsin deficiency, bronchiectasis, asthma, and cystic fibrosis. We provide an in-depth assessment of the role of the neutrophil in each of these conditions, exploring recent advances in understanding, and finally discussing the possibility of common mechanisms across diseases.

Despite uncertainties in their long term impact on lung health, use of electronic-cigarettes (EC) has increased rapidly in recent years. Previous studies have uncovered cytotoxic and pro-inflammatory effect of EC on alveolar macrophages, but the effect on neutrophil (PMN) function and viability is largely unknown. Methods: PMNs were isolated from the venous blood of healthy young volunteers. Using a novel vapour exposure system, PMN functions were investigated at a sub-cytotoxic exposure level(40 puffs EC vapour), generated from Kanger 2nd and 4th generation (gen) devices, using flavourless e-cigarette liquid. Apoptosis and necrosis were assessed by flow cytometry (Annexin V assay with propidium iodide (PI) staining). Phagocytosis was assessed by pHrodo assay (E.coli and S.Aureus). Chemotaxis to CXCL8 was assessed using an Insall chamber and video microscope. Results: Using a sub-cytotoxic vapour exposure, PMNs showed delayed neutrophil apoptosis and a trend towards reduced chemokinesis/chemotaxis to CXCL8 compared to untreated controls (81.2% reduction (chemotaxis), 52.4% reduction (chemokinesis) n=5, p=0.0625). These effects were more pronounced when using the 4th gen device (132.1% reduction (chemotaxis), 65.2% reduction (chemokinesis), n=4, p=0.0625). Phagocytosis of E.Coli and S.Aureus bioparticles was significantly increased (4.6% and 29.4% respectively) post-vaping when using 2nd gen devices. Conclusion: Sub-cytotoxic exposure of PMNs to EC vapour suppresses chemotaxis and delays apoptosis but significantly increases phagocytosis. This is a phenotype we have previously seen in patients with sepsis. Further, functional defects are more pronounced in 4th vs 2nd gen devices.

Background Despite uncertainties in their long term impact on lung health, the use of electronic-cigarettes (EC) has increased rapidly in recent years. In spite of the essential role of the neutrophil in the innate immune system, little is known about the effects of EC exposure on these cells. Our previous studies have uncovered cytotoxic and pro-inflammatory effects of EC on alveolar macrophages; this work aims to determine the effects on neutrophil (PMN) viability and function. Methods PMNs were isolated from the venous blood of healthy young volunteers. Using a novel EC vapour (ECV) exposure system, PMN were exposed to 40puffs EC vapour, generated from Kanger 2nd generation (2G) and 4th generation (4G) devices, using flavourless e-cigarette liquid. Apoptosis and necrosis was assessed by flow cytometry (Annexin V assay with propidium iodide (PI) staining). Phagocytosis was assessed by pHrodo assay (E.coli and S.Aureus). Chemotaxis to CXCL8 was assessed using an Insall chamber and video microscope. Nicotine content was assessed by GCFID. Results There were no significant differences in the viability of neutrophils immediately after exposure to 40 puffs ECV or after 4 hours. 24 hours post ECV exposure PMN cell death was elevated following 4GECV exposure (UTC; 20.4% apoptotic, 2GECV; 20.1% apoptotic, 4GECV; 57.3% apoptotic, n=3, not significant (ns)). Necrosis was also elevated in these samples (UTC; 6.49% necrotic, 4GECV; 17.8% necrotic, n=3, ns). Chemokinesis/chemotaxis to CXCL8 were significantly impaired after ECV exposure compared to UTC (2GECV; 70.7% reduction (chemotaxis), 51.6% reduction (chemokinesis) n=10, p<0.01). This effect was further exaggerated after 4GECV exposure (108.0% reduction (chemotaxis), 70.0% reduction (chemokinesis), n=9, p<0.0001). Phagocytosis was significantly decreased by 4GECV exposure compared to UTC (E.Coli 33.3% reduction, S.Aureus 71.5% reduction, n=6, p<0.05). Nicotine content analysis showed 4G devices delivered 2–4 times nicotine compared to 2G devices. Conclusion ECV exposure did not impact neutrophil viability at time 0 nor at 4hours, however PMNs showed delayed, exaggerated neutrophil apoptosis at 24hours. ECV exposure also impaired function, inhibiting both chemotaxis and phagocytosis immediately following exposure, more powerful 4G devices having greater functional implications. This neutrophil phenotype has previously been seen in patients with sepsis.