Towards personalised anti-microbial and immune approaches to infections in acute care. Can real-time genomic-informed diagnosis of pathogens, and immune-focused therapies improve outcomes for patients? An observational, experimental study protocol.

INTRODUCTION: Infection causes a vast burden of disease, with significant mortality, morbidity and costs to health-care systems. However, identifying the pathogen causative infection can be challenging, resulting in high use of broad-spectrum antibiotics, much of which may be inappropriate. Novel metagenomic methods have potential to rapidly identify pathogens, however their clinical utility for many infections is currently unclear. Outcome from infection is also impacted by the effectiveness of immune responses, which can be impaired by age, co-morbidity and the infection itself. The aims of this study are twofold: To compare diversity of organisms identified and time-to-result using metagenomic methods versus traditional culture -based techniques, to explore the potential clinical role of metagenomic approaches to pathogen identification in a range of infections.To characterise the ex vivo function of immune cells from patients with acute infection, exploring host and pathogen-specific factors which may affect immune function and overall outcomes. METHODS: This is a prospective observational study of patients with acute infection. Patients with symptoms suggestive of an acute infection will be recruited, and blood and bodily fluid relevant to the site of infection collected (for example, sputum and naso-oropharyngeal swabs for respiratory tract infections, or urine for a suspected urinary tract infection). Metagenomic analysis of samples will be compared to traditional microbiology, alongside the antimicrobials received. Blood and respiratory samples such as bronchoalveolar lavage will be used to isolate immune cells and interrogate immune cell function. Where possible, similar samples will be collected from matched participants without a suspected infection to determine the impact of infection on both microbiome and immune cell function.

Dysregulated Neutrophil Phenotype and Function in Hospitalised Non-ICU COVID-19 Pneumonia.

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.

ERS International Congress 2021: highlights from the Respiratory Infections Assembly.

The European Respiratory Society International Congress 2021 took place virtually for the second year running due to the coronavirus pandemic. The Congress programme featured more than 400 sessions and 3000 abstract presentations, covering the entire field of respiratory science and medicine. In this article, early career members of the Respiratory Infections Assembly summarise a selection of sessions across a broad range of topics, including presentations on bronchiectasis, non-tuberculosis mycobacteria, tuberculosis, cystic fibrosis and COVID-19.

Hypoxia Increases the Potential for Neutrophil-mediated Endothelial Damage in Chronic Obstructive Pulmonary Disease.

Rationale: Patients with chronic obstructive pulmonary disease (COPD) experience excess cardiovascular morbidity and mortality, and exacerbations further increase the risk of such events. COPD is associated with persistent blood and airway neutrophilia and systemic and tissue hypoxia. Hypoxia augments neutrophil elastase release, enhancing capacity for tissue injury. Objective: To determine whether hypoxia-driven neutrophil protein secretion contributes to endothelial damage in COPD. Methods: The healthy human neutrophil secretome generated under normoxic or hypoxic conditions was characterized by quantitative mass spectrometry, and the capacity for neutrophil-mediated endothelial damage was assessed. Histotoxic protein concentrations were measured in normoxic versus hypoxic neutrophil supernatants and plasma from patients experiencing COPD exacerbation and healthy control subjects. Measurements and Main Results: Hypoxia promoted PI3Kγ-dependent neutrophil elastase secretion, with greater release seen in neutrophils from patients with COPD. Supernatants from neutrophils incubated under hypoxia caused pulmonary endothelial cell damage, and identical supernatants from COPD neutrophils increased neutrophil adherence to endothelial cells. Proteomics revealed differential neutrophil protein secretion under hypoxia and normoxia, and hypoxia augmented secretion of a subset of histotoxic granule and cytosolic proteins, with significantly greater release seen in COPD neutrophils. The plasma of patients with COPD had higher content of hypoxia-upregulated neutrophil-derived proteins and protease activity, and vascular injury markers. Conclusions: Hypoxia drives a destructive "hypersecretory" neutrophil phenotype conferring enhanced capacity for endothelial injury, with a corresponding signature of neutrophil degranulation and vascular injury identified in plasma of patients with COPD. Thus, hypoxic enhancement of neutrophil degranulation may contribute to increased cardiovascular risk in COPD. These insights may identify new therapeutic opportunities for endothelial damage in COPD.

Inflammation, ageing and diseases of the lung: Potential therapeutic strategies from shared biological pathways.

Lung diseases disproportionately affect elderly individuals. The lungs form a unique environment: a highly elastic organ with gaseous exchange requiring the closest proximity of inhaled air containing harmful agents and the circulating blood. The lungs are highly susceptible to senescence, with age and 'inflammageing' creating a pro-inflammatory environment with a reduced capacity to deal with challenges. While lung diseases may have disparate causes, the burden of ageing and inflammation provides a common process that can exacerbate seemingly unrelated pathologies. However, these shared pathways may also provide a common route to treatment, with increased interest in drugs that target ageing processes across respiratory diseases. In this review, we will examine the evidence for the increased burden of lung disease in older adults, the structural and functional changes seen with advancing age and assess what our expanding knowledge of inflammation and ageing pathways could mean for the treatment of lung disease. LINKED ARTICLES: This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.

Assessment of Alveolar Macrophage Dysfunction Using an in vitro Model of Acute Respiratory Distress Syndrome.

Background: Impaired alveolar macrophage (AM) efferocytosis may contribute to acute respiratory distress syndrome (ARDS) pathogenesis; however, studies are limited by the difficulty in obtaining primary AMs from patients with ARDS. Our objective was to determine whether an in vitro model of ARDS can recapitulate the same AM functional defect observed in vivo and be used to further investigate pathophysiological mechanisms. Methods: AMs were isolated from the lung tissue of patients undergoing lobectomy and then treated with pooled bronchoalveolar lavage (BAL) fluid previously collected from patients with ARDS. AM phenotype and effector functions (efferocytosis and phagocytosis) were assessed by flow cytometry. Rac1 gene expression was assessed using quantitative real-time PCR. Results: ARDS BAL treatment of AMs decreased efferocytosis (p = 0.0006) and Rac1 gene expression (p = 0.016); however, bacterial phagocytosis was preserved. Expression of AM efferocytosis receptors MerTK (p = 0.015) and CD206 (p = 0.006) increased, whereas expression of the antiefferocytosis receptor SIRPα decreased following ARDS BAL treatment (p = 0.036). Rho-associated kinase (ROCK) inhibition partially restored AM efferocytosis in an in vitro model of ARDS (p = 0.009). Conclusions: Treatment of lung resection tissue AMs with ARDS BAL fluid induces impairment in efferocytosis similar to that observed in patients with ARDS. However, AM phagocytosis is preserved following ARDS BAL treatment. This specific impairment in AM efferocytosis can be partially restored by inhibition of ROCK. This in vitro model of ARDS is a useful tool to investigate the mechanisms by which the inflammatory alveolar microenvironment of ARDS induces AM dysfunction.

New Pharmacological Tools to Target Leukocyte Trafficking in Lung Disease.

Infection and inflammation of the lung results in the recruitment of non-resident immune cells, including neutrophils, eosinophils and monocytes. This swift response should ensure clearance of the threat and resolution of stimuli which drive inflammation. However, once the threat is subdued this influx of immune cells should be followed by clearance of recruited cells through apoptosis and subsequent efferocytosis, expectoration or retrograde migration back into the circulation. This cycle of cell recruitment, containment of threat and then clearance of immune cells and repair is held in exquisite balance to limit host damage. Advanced age is often associated with detrimental changes to the balance described above. Cellular functions are altered including a reduced ability to traffic accurately towards inflammation, a reduced ability to clear pathogens and sustained inflammation. These changes, seen with age, are heightened in lung disease, and most chronic and acute lung diseases are associated with an exaggerated influx of immune cells, such as neutrophils, to the airways as well as considerable inflammation. Indeed, across many lung diseases, pathogenesis and progression has been associated with the sustained presence of trafficking cells, with examples including chronic diseases such as Chronic Obstructive Pulmonary Disease and Idiopathic Pulmonary Fibrosis and acute infections such as Pneumonia and Pneumonitis. In these instances, there is evidence that dysfunctional and sustained recruitment of cells to the airways not only increases host damage but impairs the hosts ability to effectively respond to microbial invasion. Targeting leukocyte migration in these instances, to normalise cellular responses, has therapeutic promise. In this review we discuss the current evidence to support the trafficking cell as an immunotherapeutic target in lung disease, and which potential mechanisms or pathways have shown promise in early drug trials, with a focus on the neutrophil, as the quintessential trafficking immune cell.

Monocytes and Macrophages in Alpha-1 Antitrypsin Deficiency.

Alpha-1 antitrypsin deficiency (AATD) is a genetic condition characterised by low circulating levels of alpha-1 antitrypsin (AAT), a serine proteinase inhibitor. The most common deficiency variants are the S and Z mutations, which cause the accumulation of misfolded AAT in hepatocytes resulting in endoplasmic reticular stress and insufficient release of AAT into the circulation (<11µmol/L). This leads to liver disease, as well as an increased risk of emphysema due to unopposed proteolytic activity of neutrophil-derived serine proteinases in the lungs. AATD has been traditionally viewed as an inflammatory disorder caused directly by a proteinase-antiproteinase imbalance in the lung, but increasing evidence suggests that low AAT levels may affect other cellular functions. Recently, AAT polymers have been identified in both monocytes and macrophages from AATD patients and evidence is building that these cells may also play a role in the development of AATD lung disease. Alveolar macrophages are phagocytic cells that are important in the lung immune response but are also implicated in driving inflammation. This review explores the potential implications of monocyte and macrophage involvement in non-liver AAT synthesis and the pathophysiology of AATD lung disease.

Mitigating Health Risks to Reopen a Clinical Research Laboratory During the COVID-19 Pandemic: A Framework.

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.

Targeting defective pulmonary innate immunity - A new therapeutic option?

Chronic pulmonary conditions now account for 1 in 15 deaths in the US and mortality is increasing. Chronic obstructive pulmonary disease (COPD) is due to become the 3rd largest cause of mortality by 2030 and mortality from other respiratory conditions such as asthma, idiopathic pulmonary fibrosis and cystic fibrosis are not reducing. There is an urgent need for novel therapies to address this problem as many of the current strategies targeting inflammation are not sufficient. The innate immune system of the lung is an important defence against invading pathogens, but in many chronic pulmonary diseases, this system mounts an inappropriate response. In COPD, macrophages are increased in number, but fail to clear pathogens correctly and become highly activated. This leads to increased damage and remodelling of the airways. In idiopathic fibrosis, there is a switch of macrophage phenotype to a cell that promotes abnormal repair. Neutrophils also display dysfunction in COPD where aberrant migratory profiles may lead to increased damage to lung tissue and emphysema; while in cystic fibrosis the proteolytic lung environment damages neutrophil receptors leading to ineffective phagocytosis and migration. Targeting the innate immune system to restore 'normal function' could have enormous benefits. Improving phagocytosis of pathogens could reduce exacerbations and hence the associated decline in lung function, and novel therapeutics such as sulforaphane appear to do this in vitro. Other natural products such as resveratrol and derivatives also have anti-inflammatory properties. Statins have traditionally been used to manage cholesterol levels in hypercholesterolaemia, however these molecules also have beneficial effects on the innate immune cells. Statins have been shown to be anti-inflammatory and restore aberrant neutrophil chemotaxis in aged cells. Other possible agents that may be efficacious are senolytics. These compounds include natural products such as quercetin which have anti-inflammatory properties but can also suppress viral replication. As viruses have been shown to suppress phagocytosis of macrophages, it is possible that these compounds could have benefit during viral exacerbations to protect this innate response. These compounds demonstrate that it is possible to address defective innate responses in the lung but a better understanding of the mechanisms driving defective innate immunity in pulmonary disease may lead to improved therapeutics.

Defective bacterial phagocytosis is associated with dysfunctional mitochondria in COPD macrophages.

Increased reactive oxygen species (ROS) have been implicated in the pathophysiology of chronic obstructive pulmonary disease (COPD). This study examined the effect of exogenous and endogenous oxidative stress on macrophage phagocytosis in patients with COPD.Monocyte-derived macrophages (MDMs) were generated from non-smoker, smoker and COPD subjects, differentiated in either granulocyte macrophage-colony stimulating factor (G-Mφ) or macrophage-colony stimulating factor (M-Mφ). Alveolar macrophages were isolated from lung tissue or bronchoalveolar lavage fluid. Macrophages were incubated in ±200 µM H2O2 for 24 h, then exposed to fluorescently labelled Haemophilus influenzae or Streptococcus pneumoniae for 4 h, after which phagocytosis, mitochondrial ROS (mROS) and mitochondrial membrane potential (ΔΨm) were measured.Phagocytosis of bacteria was significantly decreased in both G-Mφ and M-Mφ from COPD patients compared with from non-smoker controls. In non-smokers and smokers, bacterial phagocytosis did not alter mROS or ΔΨm; however, in COPD, phagocytosis increased early mROS and decreased ΔΨm in both G-Mφ and M-Mφ. Exogenous oxidative stress reduced phagocytosis in non-smoker and COPD alveolar macrophages and non-smoker MDMs, associated with reduced mROS production.COPD macrophages show defective phagocytosis, which is associated with altered mitochondrial function and an inability to regulate mROS production. Targeting mitochondrial dysfunction may restore the phagocytic defect in COPD.

Human Rhinovirus Impairs the Innate Immune Response to Bacteria in Alveolar Macrophages in Chronic Obstructive Pulmonary Disease.

Rationale: Human rhinovirus (HRV) is a common cause of chronic obstructive pulmonary disease (COPD) exacerbations. Secondary bacterial infection is associated with more severe symptoms and delayed recovery. Alveolar macrophages clear bacteria from the lung and maintain lung homeostasis through cytokine secretion. These processes are defective in COPD. The effect of HRV on macrophage function is unknown. Objectives: To investigate the effect of HRV on phagocytosis and cytokine response to bacteria by alveolar macrophages and monocyte-derived macrophages (MDM) in COPD and healthy control subjects. Methods: Alveolar macrophages were obtained by bronchoscopy and MDM by adherence. Macrophages were exposed to HRV16 (multiplicity of infection 5), polyinosinic:polycytidylic acid (poly I:C) 30 µg/ml, IFN-ß 10 µg/ml, IFN-γ 10 µg/ml, or medium control for 24 hours. Phagocytosis of fluorescently labeled Haemophilus influenzae or Streptococcus pneumoniae was assessed by fluorimetry. CXCL8 (IL-8), IL-6, TNF-α (tumor necrosis factor-α), and IL-10 release was measured by ELISA. Measurements and Main Results: HRV significantly impaired phagocytosis of H. influenzae by 23% in MDM (n = 37; P = 0.004) and 18% in alveolar macrophages (n = 20; P < 0.0001) in COPD. HRV also significantly reduced phagocytosis of S. pneumoniae by 33% in COPD MDM (n = 20; P = 0.0192). There was no effect in healthy control subjects. Phagocytosis of H. influenzae was also impaired by poly I:C but not IFN-ß or IFN-γ in COPD MDM. HRV significantly reduced cytokine responses to H. influenzae. The IL-10 response to H. influenzae was significantly impaired by poly I:C, IFN-ß, and IFN-γ in COPD cells. Conclusions: HRV impairs phagocytosis of bacteria in COPD, which may lead to an outgrowth of bacteria. HRV also impairs cytokine responses to bacteria via the TLR3/IFN pathway, which may prevent resolution of inflammation leading to prolonged exacerbations in COPD.

Comparison of fluticasone propionate and budesonide on COPD macrophage and neutrophil function.

BACKGROUND: Inhaled corticosteroid use is associated with increased rates of pneumonia in COPD patients. The underlying mechanism is unknown, although recent data suggest that pneumonia is more frequent in patients treated with fluticasone propionate (FP) than budesonide. Macrophages and neutrophils from COPD patients are deficient in clearing bacteria, and this might explain increased bacterial colonization in COPD. Inhaled corticosteroid may further suppress this response; therefore, we examined the effect of FP and budesonide on phagocytosis of common respiratory pathogens by monocyte-derived macrophages (MDMs) and neutrophils. METHODS: MDMs from COPD patients (n=20-24) were preincubated with FP or budesonide for 1 or 18 hours, after which phagocytosis of fluorescently labeled inert beads or heat-killed Haemophilus influenzae/Streptococcus pneumoniae were measured fluorimetrically after 1 or 4 hours. Additionally, CXCL8, IL6, and TNFα concentrations in supernatants by ELISA, MDM-scavenger-receptor expression by flow cytometry, and MDM ability to kill bacteria were measured. Neutrophils from COPD patients (n=8) were preincubated with corticosteroids for 1 hour and bacteria phagocytosis measured by flow cytometry. RESULTS: After 1 hour's preincubation, neither corticosteroid altered MDM phagocytosis of beads or H. influenzae; however, budesonide (10-7 M) increased S. pneumoniae phagocytosis by 23% (P<0.05). After 18 hours' preincubation, neither corticosteroid altered MDM phagocytosis of any prey, although H. influenzae phagocytosis by budesonide was significantly greater compared to FP at 10-6 and 10-5 M (P<0.05). The 1-hour preincubation with either corticosteroid inhibited bacteria-induced CXCL8 release (at 10-7 and 10-5 M, P<0.05); however, this effect was lost at 18-hour preincubation. There was no change in receptor expression, bacterial killing, or neutrophil phagocytosis by either corticosteroid. CONCLUSION: These data suggest that dissolved FP and budesonide do not have an overall effect on MDM or neutrophil phagocytosis of bacteria.

Sputum microbiome temporal variability and dysbiosis in chronic obstructive pulmonary disease exacerbations: an analysis of the COPDMAP study.

BACKGROUND: Recent studies suggest that lung microbiome dysbiosis, the disease associated disruption of the lung microbial community, might play a key role in chronic obstructive pulmonary disease (COPD) exacerbations. However, characterising temporal variability of the microbiome from large longitudinal COPD cohorts is needed to better understand this phenomenon. METHODS: We performed a 16S ribosomal RNA survey of microbiome on 716 sputum samples collected longitudinally at baseline and exacerbations from 281 subjects with COPD at three UK clinical centres as part of the COPDMAP consortium. RESULTS: The microbiome composition was similar among centres and between stable and exacerbations except for a small significant decrease of Veillonella at exacerbations. The abundance of Moraxella was negatively associated with bacterial alpha diversity. Microbiomes were distinct between exacerbations associated with bacteria versus eosinophilic airway inflammation. Dysbiosis at exacerbations, measured as significant within subject deviation of microbial composition relative to baseline, was present in 41% of exacerbations. Dysbiosis was associated with increased exacerbation severity indicated by a greater fall in forced expiratory volume in one second, forced vital capacity and a greater increase in CAT score, particularly in exacerbations with concurrent eosinophilic inflammation. There was a significant difference of temporal variability of microbial alpha and beta diversity among centres. The variation of beta diversity significantly decreased in those subjects with frequent historical exacerbations. CONCLUSIONS: Microbial dysbiosis is a feature of some exacerbations and its presence, especially in concert with eosinophilic inflammation, is associated with more severe exacerbations indicated by a greater fall in lung function. TRIAL REGISTRATION NUMBER: Results, NCT01620645.

Macrophage Dysfunction in Respiratory Disease.

In the healthy lung, macrophages maintain homeostasis by clearing inhaled particles, bacteria, and removing apoptotic cells from the local pulmonary environment. However, in respiratory diseases including chronic obstructive pulmonary disease (COPD), asthma, and cystic fibrosis, macrophages appear to be dysfunctional and may contribute to disease pathogenesis. In COPD, phagocytosis of bacterial species and apoptotic cells by both alveolar macrophages and monocyte-derived macrophages is significantly reduced, leading to colonization of the lung with pathogenic bacteria. COPD macrophages also release high levels of pro-inflammatory cytokines and chemokines, including CXCL8, TGFß, and CCL2, driving recruitment of other inflammatory cells including neutrophils and monocytes to the lungs and promoting disease progression.In asthma, defective phagocytosis and efferocytosis have also been reported, and macrophages appear to have altered cell surface receptor expression; however, it is as yet unclear how this contributes to disease progression but may be important in driving Th2-mediated inflammation. In cystic fibrosis, macrophages also display defective phagocytosis, and reduced bacterial killing, which may be driven by the pro-inflammatory environment present in the lungs of these patients.The mechanisms behind defective macrophage function in lung diseases are not currently understood, but potential mechanisms include alterations in phagocytic receptor expression levels, oxidative stress, but also the possibility that specific diseases are associated with a specific, altered, macrophage phenotype that displays reduced function. Identification of the mechanisms responsible may present novel therapeutic opportunities for treatment.

Differential Effects of p38, MAPK, PI3K or Rho Kinase Inhibitors on Bacterial Phagocytosis and Efferocytosis by Macrophages in COPD.

Pulmonary inflammation and bacterial colonization are central to the pathogenesis of chronic obstructive pulmonary disease (COPD). Defects in macrophage phagocytosis of both bacteria and apoptotic cells contribute to the COPD phenotype. Small molecule inhibitors with anti-inflammatory activity against p38 mitogen activated protein kinases (MAPKs), phosphatidyl-inositol-3 kinase (PI3K) and Rho kinase (ROCK) are being investigated as novel therapeutics in COPD. Concerns exist, however, about off-target effects. We investigated the effect of p38 MAPK inhibitors (VX745 and SCIO469), specific inhibitors of PI3K α (NVS-P13K-2), δ (NVS-P13K-3) or γ (NVS-P13K-5) and a ROCK inhibitor PF4950834 on macrophage phagocytosis, early intracellular killing of bacteria and efferocytosis of apoptotic neutrophils. Alveolar macrophages (AM) obtained from broncho-alveolar lavage (BAL) or monocyte-derived macrophages (MDM) from COPD patients (GOLD stage II/III) enrolled from a well characterized clinical cohort (MRC COPD-MAP consortium) or from healthy ex-smoker controls were studied. Both COPD AM and MDM exhibited lower levels of bacterial phagocytosis (using Streptococcus pneumoniae and non-typeable Haemophilus influenzae) and efferocytosis than healthy controls. None of the inhibitors altered bacterial internalization or early intracellular bacterial killing in AM or MDM. Conversely PF4950834, but not other inhibitors, enhanced efferocytosis in COPD AM and MDM. These results suggest none of these inhibitors are likely to exacerbate phagocytosis-related defects in COPD, while confirming ROCK inhibitors can enhance efferocytosis in COPD.

Enhanced monocyte migration to CXCR3 and CCR5 chemokines in COPD.

Chronic obstructive pulmonary disease (COPD) patients exhibit chronic inflammation, both in the lung parenchyma and the airways, which is characterised by an increased infiltration of macrophages and T-lymphocytes, particularly CD8+ cells. Both cell types can express chemokine (C-X-C motif) receptor (CXCR)3 and C-C chemokine receptor 5 and the relevant chemokines for these receptors are elevated in COPD. The aim of this study was to compare chemotactic responses of lymphocytes and monocytes of nonsmokers, smokers and COPD patients towards CXCR3 ligands and chemokine (C-C motif) ligand (CCL)5. Migration of peripheral blood mononuclear cells, monocytes and lymphocytes from nonsmokers, smokers and COPD patients toward CXCR3 chemokines and CCL5 was analysed using chemotaxis assays. There was increased migration of peripheral blood mononuclear cells from COPD patients towards all chemokines studied when compared with nonsmokers and smokers. Both lymphocytes and monocytes contributed to this enhanced response, which was not explained by increased receptor expression. However, isolated lymphocytes failed to migrate and isolated monocytes from COPD patients lost their enhanced migratory capacity. Both monocytes and lymphocytes cooperate to enhance migration towards CXCR3 chemokines and CCL5. This may contribute to increased numbers of macrophages and T-cells in the lungs of COPD patients, and inhibition of recruitment using selective antagonists might be a treatment to reduce the inflammatory response in COPD.