Georgia Walton

Aberrant neutrophil functions in stable chronic obstructive pulmonary disease: the neutrophil as an immunotherapeutic target.

Chronic obstructive pulmonary disease (COPD) is a common, progressive and debilitating chronic inflammatory condition affecting the lungs, with significant systemic manifestations and co-morbidities. Smoking cigarettes is the main risk factor, but only a fifth of smokers have clinically significant airflow obstruction and the inflammation persists after smoking cessation. This suggests that smoking (and exposure to other inhaled toxins) may be necessary but not sufficient to cause COPD. Neutrophils are believed central to COPD and their accumulation and degranulation are associated with tissue damage, increased inflammation and disordered tissue repair. It was assumed that neutrophil activity and function were appropriate in COPD, responding to the presence of high levels of inflammation in the lung. However more recent studies of neutrophil function (including migration, reactive oxygen species generation, degranulation, phagocytosis and extracellular trap (NET) production) suggest that there is a general impairment in COPD neutrophil responses that predispose towards increased inflammation and reduced bacterial clearance. This may be amenable to correction and manipulating neutrophil intracellular pathways (such as phosphoinositide-3-kinase signalling) appears to restore some key COPD neutrophil responses. Targeting neutrophil intra-cellular signalling may provide a means to normalise neutrophil behaviour in COPD. This could lead to improvements in disease outcomes by reducing extraneous inflammatory burden. However further studies are needed to determine if these findings are relevant in vivo and whether this would impact positively upon health and disease.

Repurposing Treatments to Enhance Innate Immunity. Can Statins Improve Neutrophil Functions and Clinical Outcomes in COPD

Drug classes used in the treatment of Chronic Obstructive Pulmonary Disease (COPD) have not changed for many years, and none to date have shown disease-modifying activity. Statins are used to help reduce cardiovascular risk, which is high in many patients with COPD. Their use has been associated with improvements in some respiratory manifestations of disease and reduction in all-cause mortality, with greatest reductions seen in patients with the highest inflammatory burden. The mechanism for these effects is poorly understood. Neutrophils are key effector cells in COPD, and correlate with disease severity and inflammation. Recent in vitro studies have shown neutrophil functions are dysregulated in COPD and this is thought to contribute both to the destruction of lung parenchyma and to the poor responses seen in infective exacerbations. In this article, we will discuss the potential utility of statins in COPD, with a particular emphasis on their immune-modulatory effects as well as presenting new data regarding the effects of statins on neutrophil function in vitro.

Pulmonary Infections in the Elderly Lead to Impaired Neutrophil Targeting, Which Is Improved by Simvastatin

Rationale: Dysregulated neutrophil functions with age and sepsis are described. Statins are associated with improved infection survival in some observational studies, but trials in critically ill patients have not shown benefit. Statins also alter neutrophil responses in vitro. Objectives: To assess neutrophil migratory accuracy with age during respiratory infections and determine if and how a statin intervention could alter these blunted responses. Methods: The migratory accuracy of blood neutrophils from young (aged <35 yr) and old (aged >60 yr) patients in health and during a lower respiratory tract infection, community‐acquired pneumonia, and pneumonia associated with sepsis was assessed with and without simvastatin. In vitro results were confirmed in a double‐blind randomized clinical trial in healthy elders. Cell adhesion markers were assessed. Measurements and Main Results: In vitro neutrophil migratory accuracy in the elderly deteriorated as the severity of the infectious pulmonary insult increased, without recovery at 6 weeks. Simvastatin rescued neutrophil migration with age and during mild to moderate infection, at high dose in older adults, but not during more severe sepsis. Confirming in vitro results, high‐dose (80‐mg) simvastatin improved neutrophil migratory accuracy without impeding other neutrophil functions in a double‐blind randomized clinical trial in healthy elders. Simvastatin modified surface adhesion molecule expression and activity, facilitating accurate migration in the elderly. Conclusions: Infections in older adults are associated with prolonged, impaired neutrophil migration, potentially contributing to poor outcomes. Statins improve neutrophil migration in vivo in health and in vitro in milder infective events, but not in severe sepsis, supporting their potential utility as an early intervention during pulmonary infections. Clinical trial registered with www.clinicaltrialsregister.eu (2011‐002082‐38).

Pre-emptive or early adjuvant simvastatin therapy in elderly patients with infection and sepsis

Abstract Background The incidence, severity, morbidity, and mortality associated with sepsis increases with age, and statin treatment improves outcomes during infection. We characterised the effect of age and acute infection on key neutrophil functions, assessed whether physiologically relevant doses of simvastatin altered neutrophil functions, and, if benefits were seen, when statins could be used during septic infection. Methods Neutrophils extracted from the whole blood of healthy volunteers and patients with a lower respiratory tract infection (LRTI), pneumonia, or sepsis were assessed for migratory accuracy, phagocytosis, and production of neutrophil extracellular traps (NET) before and after in-vitro treatment with simvastatin. In addition, neutrophil function was assessed in healthy elderly volunteers, who were receiving simvastatin (80 mg/day for 2 weeks) or placebo as part of a crossover, double-blind, randomised controlled trial. Here we present data for neutrophil migration. Findings Neutrophils from healthy volunteers (n=70, aged 21–94 years) showed preserved chemokinesis (random movement) but reduced chemotaxis (directed migration) ( r 2 =−0·48, p Interpretation Neutrophil function in elderly people is compromised in health, and deteriorates further during infective episodes in accordance with the severity of the disease. Migratory accuracy can be improved with short-term in-vitro simvastatin therapy in health and mild infection but not in sepsis. Our data suggest that statin therapy might be a preventive or an early adjuvant intervention rather than a treatment in established sepsis. We are testing in a clinical trial whether simvastatin 80 mg for 7 days modifies neutrophil responses in elderly patients with pneumonia and sepsis. Funding British Journal of Anaesthesia, Royal College of Anaesthetists.

S96 Simvastatin as an adjuvant therapy for infection and sepsis–in-vitro and in-vivo studies suggest pre-emptive / early therapy in the elderly

Ageing is associated with increased episodes of sepsis and poorer outcomes. Statins are associated with improved outcomes during infection. We aimed to characterise the impact of age and acute severe infection on key neutrophil functions, assess whether physiologically relevant doses of simvastatin altered neutrophil functions and if benefits were seen, when during a septic episode statins could be utilised. Methods Neutrophils from healthy volunteers and patients with lower respiratory tract infections (LRTI), pneumonia and sepsis were assessed for migratory accuracy, phagocytosis and neutrophil extracellular trap production before and after in-vitro treatment with simvastatin. Healthy elderly volunteers received 80mg simvastatin or placebo in a cross over double-blind randomised controlled trial and neutrophil functions were assessed. Data presented is for migration. Results Neutrophils from healthy subjects (n = 70, aged 21–94) demonstrated preserved neutrophil movement) (R2 = -0.48, p < 0.0001) towards chemoattractants (data shown for IL-8). Neutrophil chemotaxis decreased after 60yrs (comparing <35 to >65yrs: mean difference (MD)1.25μm/min, p = 0.02). There was a progressive decrease in neutrophil chemotaxis in old patients with a LRTI, pneumonia and severe sepsis (MD compared to healthy control; LRTI (n = 10), 0.7μm/min, p = 0.04; pneumonia (n = 5), MD1.1μm/min, p = 0.02; sepsis (n = 22) MD1.6μm/min, p = 0.01) with “septic neutrophils” unable to mount targeted chemotaxis. Improvements to baseline were seen following recovery. In-vitro treatment of neutrophils from healthy older people with simvastatin (1µM) restored “old” neutrophil chemotaxis to that of “young” cells. Simvastatin also restored neutrophil migration from old patients with LRTI and pneumonia to baseline but not in patients with sepsis. Two weeks of oral simvastatin 80mg once daily therapy in healthy old volunteers (Age>65,n = 20) increased the accuracy of neutrophil migration (MD1.68μm/min, p = 0.02) replicating benchwork. Conclusions “Elderly” neutrophil function is compromised in health, and deteriorates during infective episodes, in accordance with the severity of the insult. Migratory accuracy can be improved with simvastatin therapy however neutrophil function in sepsis patients cannot be modulated during short term in-vitro therapy. Our data suggest statin therapy might be a preventative or an early adjuvant intervention rather than a treatment in established sepsis. We are testing whether simvastatin 80mg for seven days modifies neutrophil responses in elderly patients with pneumonia and sepsis (SNOOPI Trial). Abstract S96 Figure 1. Simavastatin 80mg once daily for 14 days improves directional migration (chemostaxis) of neutrophils from healthy elderly volunteers towards IL-8. *Student’s t-Test

Statin therapy in patients with community-acquired pneumonia.

ABSTRACT Community-acquired pneumonia (CAP) is the leading cause of death from infection in developed countries. There is evidence of an association between improved survival from infection and statin use. The possible beneficial effects of statins are complicated by the common use of macrolide antibiotics for pneumonia, with current guidance suggesting that concurrent macrolide and statin use is contraindicated. We conducted an observational study of statin use in patients with CAP. Of 2,067 patients with CAP, 30.4% were on statin therapy at admission. Statin users were more likely to survive the admission (p<0.001). In addition, we conducted a survey of doctors and found that knowledge regarding concurrent macrolide and statin use was lacking. These data suggest a potential role of statins in the management of CAP. Further research using high-dose statins is required to assess their safe use in subjects with mild to moderate infections.

Neutrophilic Inflammation in the Pathogenesis of Chronic Obstructive Pulmonary Disease

Abstract Current paradigms of chronic obstructive pulmonary disease (COPD) treatment suggest stratifying patients by their symptoms, utilising three main drug classes, but it is unclear if this approach will substantially alter the progression of the disease in the long term. More treatment options are needed which target the underlying pathology of the condition. Whilst many inflammatory cells are implicated in COPD, the neutrophil is by far the most abundant and has been extensively associated with disease pathogenesis. Neutrophil products are thought to be key mediators of inflammatory changes in the airways of COPD patients, causing pathological features such as emphysema and hypersecretion of mucus. High rates of bacterial colonisation and recurrent infective exacerbations of COPD, as well as evidence of neutrophil-associated host damage suggest that neutrophil functions may be impaired in COPD. This concept is supported by studies demonstrating impaired migratory accuracy and increased degranulation and reactive oxygen species release, with some evidence of altered cellular signalling pathways which might be exploitable as therapeutic targets. This review discusses our evolving understanding of neutrophil function in both health and COPD and highlights the role of this cell in disease pathogenesis, to determine whether this key inflammatory mediator represents a viable therapeutic target to prevent disease progression.

S114 Simvastatin improves neutrophil migratory targeting in COPD: in vitro studies supporting Statin use as a potential adjuvant therapy

Introduction Statin use in COPD is associated with a reduction in all cause mortality, with greatest reductions seen in patients with the highest inflammatory burden. However, the mechanism for these effects is poorly understood, as statin treatment has not been found to lower systemic inflammation and in vitro studies of cellular effects use concentrations that exceed the therapeutic range. Neutrophils are key effector cells in COPD, and correlate with disease severity and inflammation. Recent in vitro studies have shown neutrophil migratory accuracy to be reduced in COPD. This is thought to contribute to the destruction of lung parenchyma and the poor responses seen in infective exacerbations. We aimed to characterise neutrophil migration in COPD and assess whether physiologically relevant concentrations of simvastatin altered neutrophil migration. Abstract S114 Figure1 Simvastatin improves neutrophil migration in COPD. Legend. Isolated neutrophils from COPD patients (n = 13) migrated towards IL8 (100nM) or fMLP (100nM) following incubation with carrier control or Simvastatin (1nM or 1uM). Measurements were taken from 10 randomly selected cells from each individual. The average results for each subject were calculated, and an overall average was used for comparisons across groups using analysis of variance. Bars represent the mean migratory parameter with standard deviation shown as the error line. * = significant difference in migratory parameter from carrier control data across groups (p<0.05). Methods Neutrophils were isolated from COPD patients and healthy smoking age-matched controls (age > 60yrs, n = 13 per group) and incubated with 1nM - 1μM Simvastatin or with a carrier control before migratory dynamics were assessed towards IL8 and fMLP using time-lapse photography. Data is expressed as means with standard deviation in parentheses. Results COPD neutrophils displayed reduced chemotaxis (directional speed of migration) and reduced chemotactic accuracy (Chemotactic Index - a vector analysis of migratory tracks) compared to cells from healthy age-matched controls (HC) in the presence of IL-8 and f-MLP, replicating previous work. For example, Chemotactic Index: IL8; HC, 0.42CU (0.03), COPD 0.22CU (0.05), p = 0.002: fMLP; HC, 0.34CU (0.05), COPD, 0.18CU (0.03) p = 0.014). Treatment with Simvastatin significantly improved the chemotactic ability of COPD neutrophils in a dose response with greatest improvement seen at the highest concentration (e.g. Chemotaxis to IL8, Carrier control 0.8um/min (0.2), 1nM Simvastatin 1.3um/min (0.2), p = 0.04; 1uM Simvastatin 1.4um/min (0.2), p = 0.004). A similar improvement was seen in Chemotactic Accuracy (e.g. Chemotactic Index to fMLP, Carrier control 0.17CU (0.03), 1nM Simvastatin 0.26CU (0.02), p = 0.018; 1uM Simvastatin 0.31CU (0.03), p = 0.002). Conclusions Migratory accuracy of circulating neutrophils is reduced in COPD patients compared with healthy, smoking, age-matched controls but can be restored by treatment with therapeutic concentrations of Simvastatin in vitro . Our data suggest statin therapy might be an adjuvant intervention in COPD, modulating neutrophil responses.

S85 Neutrophil Cell Membrane Expression of Proteinase 3 and Its Relationship to Alpha-1-Antitrypsin Deficiency (A1ATD)

Introduction Serine proteinases such as proteinase 3 (PR3) and neutrophil elastase (NE) on the surface of activated neutrophils retain their activity, which may be central to the tissue damage in emphysema. However, little is known about this expression in A1ATD when it may be more critical. Aims To compare neutrophil surface expression of PR3 and NE in patients with A1ATD, usual COPD and healthy controls. To determine the influence of the local concentration of A1AT on neutrophil surface expression of PR3 and NE. Methods Clinically stable patients with A1ATD (n=9), COPD (n=6) and healthy controls (n=9) were recruited. Neutrophils were isolated from blood. Half were stimulated with fMLP and half were unstimulated. Membrane expression of NE and PR3 was measured by flow cytometry. Neutrophils isolated from six further healthy controls were stimulated in the presence of either normal (PiMM) or A1ATD plasma (PiZZ). Membrane expression of NE and PR3 was measured. Results PR3 expression on the surface of unstimulated neutrophils was greater in A1ATD patients (2365±305MFI) compared to healthy controls (1517±253MFI; p=0.048) and COPD patients (1360±315MFI; p=0.046). NE expression was similar between groups. PR3 expression on stimulated neutrophils was greater in A1ATD patients (5112±547MFI) compared to healthy controls (3411±541MFI; p=0.042), but not different to COPD patients (4723±1509MFI; p=0.78). NE expression was similar between groups. When neutrophils from healthy controls were stimulated in the presence of plasma, the surface expression of PR3 (but not NE) was greater (p=0.031) in the presence of PiZZ plasma (1921MFI) compared to PiMM plasma (1352MFI), but less than that observed without plasma. Conclusions Baseline neutrophil surface expression of PR3 is greater in A1ATD patients compared to healthy controls. Neutrophils express more PR3 when stimulated in an environment with low concentrations of A1AT, suggesting that membrane binding is dependent on the ability of A1AT to bind released PR3 but not NE. This may have clinical significance for A1ATD emphysema since active membrane-bound PR3 is resistant to inhibitors and can replicate the pathological features associated classically with NE. These findings may explain the association of Wegener’s granulomatosis (where PR3 is an autoantigen) with A1ATD.

S129 Neutrophil function and advancing age: the effects of Simvastatin in health and during pneumonia

Background Age is associated with a decline in immunity, including neutrophil function. This may partially explain the worsening clinical outcomes seen following pneumonia in the elderly. Statins may improve outcomes from pneumonia although it is unknown whether they influence neutrophil function at conventional therapeutic concentrations. This is crucial, as statins may be beneficial adjuvants during infections. Methods We studied the effect of 5 ng/ml Simvastatin (equivalent to 80 mg orally) on key neutrophil functions: speed and accuracy of migration using time-lapse imaging (μm/min), Neutrophil Extracellular Trap formation using cell-impermeable DNA-binding dye (in AFU) and generation of reactive oxygen species (ROS, in RLU). Results We studied 70 healthy subjects (aged 20–90 years, 10 in each decennial) and 6 young (<35 years) and 6 older (>65 years) patients during an admission with pneumonia. All studied neutrophil functions declined with age (eg, neutrophil chemotaxis, r =−0.7, p<0.001). Specifically, average neutrophil chemotaxis for subjects >65 yrs was 0.72 μm/min (SD ± 0.28, p<0.001) slower than subjects <35. These neutrophils produced less NETS (average difference =−1725AFU±283, p=0.007) and peak ROS (average difference −117RLU±31; p=0.04). Neutrophils from young patients with pneumonia displayed an up-regulation of function that was not seen in older pneumonia patients. There were no age-associated differences in the surface expression of chemo-attractant receptors, but there appears to be differential intracellular signalling with reduced expression of adhesion molecules. Incubating neutrophils from older subjects with Simvastatin improved all functions back to that seen in young subjects (chemotactic speed + 0.92 μm/min ±0.27, p=0.001: NET + 1386AFU±273, p=0.04: ROS +223RLU±39, p=0.005). Similar improvements were seen with neutrophils from older subjects with pneumonia. This was a dose-dependent phenomenon; not seen at higher concentrations of Simvastatin. Conclusion With age, there is a global deterioration in neutrophil function and no up-regulation when pneumonia is present, which may partially explain the age-associated mortality. Simvastatin up-regulates neutrophil function in the elderly, even during pneumonia. This up-regulation may explain the beneficial effects seen clinically. In vivo studies are warranted, to determine if simvastatin should be utilised during episodes of acute infection.