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Bacterial pathogens are confronted with a range of challenges at the site of infection, including exposure to antibiotic treatment and harsh physiological conditions, that can alter the fitness benefits and costs of acquiring antibiotic resistance. Here, we develop an experimental system to recapitulate resistance gene acquisition by Staphylococcus aureus and test how the subsequent evolution of the resistant bacterium is modulated by antibiotic treatment and oxygen levels, both of which are known to vary extensively at sites of infection. We show that acquiring tetracycline resistance was costly, reducing competitive growth against the isogenic strain without the resistance gene in the absence of the antibiotic, for S. aureus under hypoxic but not normoxic conditions. Treatment with tetracycline or doxycycline drove the emergence of enhanced resistance through mutations in an RluD-like protein-encoding gene and duplications of

Rationale: Sonographic septations are assumed to be important clinical predictors of outcome in pleural infection but the evidence for this is sparse. The inflammatory and fibrinolysis-associated intrapleural pathway(s) leading to septation formation have not been studied in a large cohort of pleural fluid (PF) samples with confirmed pleural infection, matched with ultrasound and clinical outcome data. Objectives: To assess the presence and severity of septations against baseline PF Plasminogen-Activator Inhibitor-1 (PAI-1) and other inflammatory and fibrinolysis-associated proteins as well as to correlate these with clinically important outcomes. Methods: We analysed 214 pleural fluid samples from the PILOT study, a prospective observational pleural infection study, for inflammatory and fibrinolysis-associated proteins using the Luminex platform. Multivariate regression analyses were utilised to assess association of pleural biological markers with septation presence and severity (on ultrasound), and clinical outcomes. Results: PF PAI-1 level was the only protein independently associated with septation presence (p=

This study was funded by an investigator-initiated research grant from Insmed (Bridgewater, NJ, USA). The authors acknowledge the funding and logistical support from the UK National Institute for Health and Care Research. Background: Neutrophil serine proteases are involved in the pathogenesis of COVID-19 and increased serine protease activity has been reported in severe and fatal infection. We investigated whether brensocatib, an inhibitor of dipeptidyl peptidase-1 (DPP-1; an enzyme responsible for the activation of neutrophil serine proteases), would improve outcomes in patients hospitalised with COVID-19. Methods: In a multicentre, double-blind, randomised, parallel-group, placebo-controlled trial, across 14 hospitals in the UK, patients aged 16 years and older who were hospitalised with COVID-19 and had at least one risk factor for severe disease were randomly assigned 1:1, within 96 h of hospital admission, to once-daily brensocatib 25 mg or placebo orally for 28 days. Patients were randomly assigned via a central web-based randomisation system (TruST). Randomisation was stratified by site and age (65 years or ≥65 years), and within each stratum, blocks were of random sizes of two, four, or six patients. Participants in both groups continued to receive other therapies required to manage their condition. Participants, study staff, and investigators were masked to the study assignment. The primary outcome was the 7-point WHO ordinal scale for clinical status at day 29 after random assignment. The intention-to-treat population included all patients who were randomly assigned and met the enrolment criteria. The safety population included all participants who received at least one dose of study medication. This study was registered with the ISRCTN registry, ISRCTN30564012. Findings: Between June 5, 2020, and Jan 25, 2021, 406 patients were randomly assigned to brensocatib or placebo; 192 (47·3%) to the brensocatib group and 214 (52·7%) to the placebo group. Two participants were excluded after being randomly assigned in the brensocatib group (214 patients included in the placebo group and 190 included in the brensocatib group in the intention-to-treat population). Primary outcome data was unavailable for six patients (three in the brensocatib group and three in the placebo group). Patients in the brensocatib group had worse clinical status at day 29 after being randomly assigned than those in the placebo group (adjusted odds ratio 0·72 [95% CI 0·57-0·92]). Prespecified subgroup analyses of the primary outcome supported the primary results. 185 participants reported at least one adverse event; 99 (46%) in the placebo group and 86 (45%) in the brensocatib group. The most common adverse events were gastrointestinal disorders and infections. One death in the placebo group was judged as possibly related to study drug. Interpretation: Brensocatib treatment did not improve clinical status at day 29 in patients hospitalised with COVID-19. Publisher PDF

Background and objectivesLong-term prophylactic antibiotics are often used to prevent bacterial infections. However, supporting evidence for this is not always robust. Including parents in decisions relating to medication is key to medicines optimisation. Parental concern regarding medication is a major determinant of poor adherence. This study explores parental experiences of having a child prescribed prophylactic antibiotics and how that affects their antibiotic use behaviour.MethodsWe conducted a prospective, single-centre, exploratory, qualitative study at Sheffield Children’s Hospital. Through 15 interviews, involving 18 participants, we explored parental ‘lived experiences’ and attitudes towards azithromycin prophylaxis prescribed for various respiratory conditions. Thematic analysis was conducted.ResultsThe overriding factor influencing parental decisions about the uptake of antibiotic prophylaxis is wanting their child to be well now. The main concern voiced by parents is that of antibiotic resistance given their children are high users of antibiotics. This is however seen as a problem for the future, not the present. Preparing families adequately helps prevent practical difficulties relating to medication. Facilitating ‘normalisation’ of prophylaxis through daily routines and minimising disruption to the family environment may reduce parental anxiety, promote adherence and result in easing of potential restrictions to the child’s daily activities.ConclusionGrounded in our deeper understanding, we propose a behavioural model that describes phases parents go through while having a child on prophylactic antibiotics. Time invested in holistically addressing the parental experience and having an awareness of potential issues parents face, may facilitate medication adherence, reduce anxieties and improve doctor-parent relationships.

The lungs are exposed to a range of environmental toxins (including cigarette smoke, air pollution, asbestos) and pathogens (bacterial, viral and fungal), and most respiratory diseases are associated with local or systemic hypoxia. All of these adverse factors can trigger endoplasmic reticulum (ER) stress. The ER is a key intracellular site for synthesis of secretory and membrane proteins, regulating their folding, assembly into complexes, transport and degradation. Accumulation of misfolded proteins within the lumen results in ER stress, which activates the unfolded protein response (UPR). Effectors of the UPR temporarily reduce protein synthesis, while enhancing degradation of misfolded proteins and increasing the folding capacity of the ER. If successful, homeostasis is restored and protein synthesis resumes, but if ER stress persists, cell death pathways are activated. ER stress and the resulting UPR occur in a range of pulmonary insults and the outcome plays an important role in many respiratory diseases. The UPR is triggered in the airway of patients with several respiratory diseases and in corresponding experimental models. ER stress has been implicated in the initiation and progression of pulmonary fibrosis, and evidence is accumulating suggesting that ER stress occurs in obstructive lung diseases (particularly in asthma), in pulmonary infections (some viral infections and in the setting of the cystic fibrosis airway) and in lung cancer. While a number of small molecule inhibitors have been used to interrogate the role of the UPR in disease models, many of these tools have complex and off-target effects, hence additional evidence (eg, from genetic manipulation) may be required to support conclusions based on the impact of such pharmacological agents. Aberrant activation of the UPR may be linked to disease pathogenesis and progression, but at present, our understanding of the context-specific and disease-specific mechanisms linking these processes is incomplete. Despite this, the ability of the UPR to defend against ER stress and influence a range of respiratory diseases is becoming increasingly evident, and the UPR is therefore attracting attention as a prospective target for therapeutic intervention strategies.

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.

Background Hyperpolarised gas magnetic resonance imaging (MRI) can be used to assess ventilation patterns. Previous studies have shown the image-derived metric of ventilation defect per cent (VDP) to correlate with forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) and FEV1 in asthma. Objectives The aim of this study was to explore the utility of hyperpolarised xenon-129 (129Xe) ventilation MRI in clinical care and examine its relationship with spirometry and other clinical metrics in people seen in a severe asthma service. Methods 26 people referred from a severe asthma clinic for MRI scanning were assessed by contemporaneous 129Xe MRI and spirometry. A subgroup of 18 patients also underwent reversibility testing with spirometry and MRI. Quantitative MRI measures of ventilation were calculated, VDP and the ventilation heterogeneity index (VHI), and compared to spirometry, Asthma Control Questionnaire 7 (ACQ7) and blood eosinophil count. Images were reviewed by a multidisciplinary team. Results VDP and VHI correlated with FEV1, FEV1/FVC and forced expiratory flow between 25% and 75% of FVC but not with ACQ7 or blood eosinophil count. Discordance of MRI imaging and symptoms and/or pulmonary function tests also occurred, prompting diagnostic re-evaluation in some cases. Conclusion Hyperpolarised gas MRI provides a complementary method of assessment in people with difficult to manage asthma in a clinical setting. When used as a tool supporting clinical care in a severe asthma service, occurrences of discordance between symptoms, spirometry and MRI scanning indicate how MRI scanning may add to a management pathway., This article demonstrates the feasibility of using 129Xe MRI in clinical practice. Discordance between symptoms, spirometry and MRI can support the use of further treatment or suggest coexisting breathing control issues or laryngeal disorders. https://bit.ly/3ky4oXP

Klebsiella pneumoniae is a Gram-negative bacterium that is increasingly difficult to treat due to the emergence of multidrug resistant strains. In a recent article, Ding et al demonstrate that prekallikrein depletion in mice followed by intranasal instillation of K. pneumoniae leads to a reduced bacterial burden and prolonged host survival, together with evidence of reduced distant organ damage. These effects are apparently independent of the role of prekallikrein in the contact system, and are associated with transcriptional changes relevant to innate immunity in the lung, established prior to infection. This study highlights the importance of further investigating the role of prekallikrein and other contact cascade components in host defence to counter K. pneumoniae (and perhaps other pathogens), with an overall aim of identifying potential therapeutic targets relevant to pulmonary infection with such resistant pathogens. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Activated phosphoinositide 3-kinase delta syndrome (APDS) is a novel primary immunodeficiency (PID) caused by heterozygous gain of function mutations in PI3Kδ catalytic p110δ (PIK3CD) or regulatory p85α (PIK3R1) subunits leading to APDS1 and APDS2, respectively. Patients with APDS present a spectrum of clinical manifestations, particularly recurrent respiratory infections and lymphoproliferation. We searched PubMed, Web of Science, and Scopus databases for APDS patients and screened for eligibility criteria. A total of 243 APDS patients were identified from 55 articles. For all patients, demographic, clinical, immunologic, and molecular data were collected. Overall, 179 APDS1 and 64 APDS2 patients were identified. The most common clinical manifestations were respiratory tract infections (pneumonia (43.6%), otitis media (28.8%), and sinusitis (25.9%)), lymphoproliferation (70.4%), autoimmunity (28%), enteropathy (26.7%), failure to thrive (20.6%), and malignancy (12.8%). The predominant immunologic phenotype was hyper-IgM syndrome (48.1%). Immunologic profiling showed decreased B cells in 74.8% and CD4+ T cells in 64.8% of APDS patients. The c.3061 G>A (p. E1021K) mutation in APDS1 with 85% frequency and c.1425+1 G> (A, C, T) (p.434–475del) mutation in APDS2 with 79% frequency were hotspot mutations. The majority of APDS patients were placed on long-term immunoglobulin replacement therapy. Immunosuppressive agents such as rituximab, tacrolimus, rapamycin, and leniolisib were also administered for autoimmunity and inflammatory complications. In addition, hematopoietic stem cell transplantation (HSCT) was used in 12.8% of patients. APDS has heterogynous clinical manifestations. It should be suspected in patients with history of recurrent respiratory infections, lymphoproliferation, and raised IgM levels. Moreover, HSCT should be considered in patients with severe and complicated clinical manifestations with no or insufficient response to the conventional therapies., This work was supported by vice chancellor for research, Alborz University of Medical Sciences, under Grant No. IR.ABZUMS.REC.1397.051.

Purpose of review Neutrophils priming has been long studied in vitro. Recent studies describe it in vivo. In pathophysiological conditions, complex, heterogeneous characteristics of priming are described in the last few years. Recent findings Priming can occur systemically when insults such as sepsis or trauma result in an array of circulating mediators and circulating primed neutrophils seem to exert detrimental effects either directly, or indirectly by interacting with other cells, thereby contributing to the development of organ dysfunction. Local priming of neutrophils augments their ability to clear infection, but may also lead to local bystander tissue injury, for example, in the inflamed joint. The complexity, heterogeneity and dynamic nature of inflammatory responses and the accessibility of cells from local sites make neutrophil priming challenging to study in human disease; however, recent advances have made significant progress to this field. Summary Herein, we summarize the literature regarding neutrophil priming in selected conditions. In some diseases and in the setting of specific genetic influences, the priming repertoire seems to be restricted, with only some neutrophil functions upregulated. A greater understanding of the nature of neutrophil priming and its role in human disease is required before this process becomes tractable to therapeutic intervention.

Studies of chest computed tomography (CT) in patients with primary antibody deficiency syndromes (ADS) suggest a broad range of bronchial pathology. However, there are as yet no multicentre studies to assess the variety of bronchial pathology in this patient group. One of the underlying reasons is the lack of a consensus methodology, a prerequisite to jointly document chest CT findings. We aimed to establish an international platform for the evaluation of bronchial pathology as assessed by chest CT and to describe the range of bronchial pathologies in patients with antibody deficiency. Ffteen immunodeficiency centres from 9 countries evaluated chest CT scans of patients with ADS using a predefined list of potential findings including an extent score for bronchiectasis. Data of 282 patients with ADS were collected. Patients with common variable immunodeficiency disorders (CVID) comprised the largest subgroup (232 patients, 82.3%). Eighty percent of CVID patients had radiological evidence of bronchial pathology including bronchiectasis in 61%, bronchial wall thickening in 44% and mucus plugging in 29%. Bronchiectasis was detected in 44% of CVID patients aged less than 20 years. Cough was a better predictor for bronchiectasis than spirometry values. Delay of diagnosis as well as duration of disease correlated positively with presence of bronchiectasis. The use of consensus diagnostic criteria and a pre-defined list of bronchial pathologies allows for comparison of chest CT data in multicentre studies. Our data suggest a high prevalence of bronchial pathology in CVID due to late diagnosis or duration of disease.

Rationale: Pulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome. Circulating BMP9 (bone morphogenetic protein 9) is emerging as an important regulator of pulmonary vascular homeostasis. Objectives:To determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity and whether loss of endogenous BMP9 occurs during LPS challenge. Methods: A BMP9-neutralizing antibody was administrated to healthy adult mice, and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human lung endothelial cells. The impact of BMP9 administration was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and from endotoxemic mice. Measurements and Main Results: Subacute neutralization of endogenous BMP9 in mice (N = 12) resulted in increased lung vascular permeability (P = 0.022), interstitial edema (P = 0.0047), and neutrophil extravasation (P = 0.029) compared with IgG control treatment (N = 6). In pulmonary endothelial cells, BMP9 regulated transcriptome pathways implicated in vascular permeability and cell-membrane integrity. Augmentation of BMP9 signaling in mice (N = 8) prevented inhaled LPS-induced lung injury (P = 0.0027) and edema (P < 0.0001). In endotoxemic mice (N = 12), endogenous circulating BMP9 concentrations were markedly reduced, the causes of which include a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human patients with sepsis (N = 10), circulating concentratons of BMP9 were also markedly reduced (P < 0.0001). Conclusions: Endogenous circulating BMP9 is a pulmonary endothelial-protective factor, downregulated during inflammation. Exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in lung injury.

RationalePulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome (ARDS), which carries a high mortality. Circulating bone morphogeneic protein 9 (BMP9) is emerging as an important regulator of pulmonary vascular homeostasis.ObjectiveTo determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity, and whether loss of endogenous BMP9 occurs during lipopolysacharride (LPS)-induced lung inflammation and permeability.MethodsA BMP9-neutralizing antibody was administrated to healthy adult mice and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human primary lung endothelial cells. Impact of BMP9 was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and endotoxemic mice.Main ResultsSubacute neutralization of endogenous BMP9 in mice resulted in increased lung vascular permeability, interstitial edema and neutrophil extravasation. In lung endothelial cells, BMP9 regulated a programme of gene expression and pathways controlling vascular permeability and cell membrane integrity. Augmentation of BMP9 signalling in mice with exogenous BMP9 prevented inhaled LPS-caused lung injury and edema. In endotoxemic mice, endogenous BMP9 levels were markedly reduced, due to a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human sepsis patients, circulating levels of BMP9 were also markedly reduced.ConclusionsEndogenous circulating BMP9 is a pulmonary endothelial protective factor, down-regulated during inflammation. Supplementation with exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in the setting of lung injury.Short summaryScientific Knowledge on the SubjectIncreased pulmonary endothelial permeability is a major factor in the development of acute respiratory distress syndrome (ARDS). Evidence is emerging that circulating BMP9, secreted from the liver, might protect the pulmonary endothelium from injury. For example, loss of BMP9 levels or signalling receptor contributes to the development of pulmonary arterial hypertension. The role of endogenous BMP9 in endothelial permeability remains unclear.What This Study Adds to the FieldHere we show that subacute neutralization of endogenous BMP9 leads to lung vascular injury, including enhanced permeability and neutrophil extravasation. BMP9 levels were markedly reduced in the setting of inflammation in mice and humans. Conversely, exogenous supplementation of BMP9 protected the lung from LPS-induced injury. This study suggests that exogenous BMP9 could offer a novel approach to prevent increased pulmonary endothelial permeability in the setting of lung injury and ARDS.

Neutrophils are key effector cells of innate immunity, rapidly recruited to defend the host against invading pathogens. Neutrophils may kill pathogens intracellularly, following phagocytosis, or extracellularly, by degranulation and the release of neutrophil extracellular traps; all of these microbicidal strategies require the deployment of cytotoxic proteins and proteases, packaged during neutrophil development within cytoplasmic granules. Neutrophils operate in infected and inflamed tissues, which can be profoundly hypoxic. Neutrophilic infiltration of hypoxic tissues characterises a myriad of acute and chronic infectious and inflammatory diseases, and as well as potentially protecting the host from pathogens, neutrophil granule products have been implicated in causing collateral tissue damage in these scenarios. This review discusses the evidence for the enhanced secretion of destructive neutrophil granule contents observed in hypoxic environments and the potential mechanisms for this heightened granule exocytosis, highlighting implications for the host. Understanding the dichotomy of the beneficial and detrimental consequences of neutrophil degranulation in hypoxic environments is crucial to inform potential neutrophil-directed therapeutics in order to limit persistent, excessive, or inappropriate inflammation.

Background Inflamed environments are typically hypercellular, rich in pro-inflammatory cytokines, and profoundly hypoxic. While the effects of hypoxia on neutrophil longevity and function have been widely studied, little is known about the consequences of this stimulus on eosinophils. Objective We sought to investigate the effects of hypoxia on several key aspects of eosinophil biology; namely secretion, survival, and their sensitivity to glucocorticosteroids (GCS), agents which normally induce eosinophil apoptosis. Methods Eosinophils derived from patients with asthma/atopy or healthy controls were incubated under normoxia and hypoxia, with or without glucocorticoids. Activation was measured by flow cytometry, ELISA of cultured supernatants and F-actin staining; apoptosis and efferocytosis by morphology and flow cytometry, and GCS efficacy by apoptosis assays and qPCR. Results Hypoxic incubation (3 kPa) caused: (i) stabilisation of HIF-2α and up-regulation of hypoxia regulated genes including BNIP3 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3) and GLUT1 (glucose transporter 1), (ii) secretion of pre-formed IL-8, and Charcot Leyden crystal (CLC) formation, that was most evident in eosinophils derived from atopic and asthmatic donors, (iii) enhanced F-actin formation, (iv) marked prolongation of eosinophil lifespan (via a NF-κB and Class I PI3-kinase-dependent mechanism), and (v) complete abrogation of the normal pro-apoptotic effect of dexamethasone and fluticasone furoate. This latter effect was evident despite preservation of GCS-mediated gene transactivation under hypoxia. Conclusion and Clinical Relevance These data indicate that hypoxia promotes an eosinophil pro-inflammatory phenotype by enhancing eosinophil secretory function, delaying constitutive apoptosis and importantly, antagonising the normal pro-apoptotic effect of GCS. Since eosinophils typically accumulate at sites that are relatively hypoxic, particularly during periods of inflammation, these findings may have important implications to understanding the behaviour these cells in vivo. This article is protected by copyright. All rights reserved.

Neutrophils are key to host defence, and impaired neutrophil function predisposes to infection with an array of pathogens, with Staphylococcus aureus a common and sometimes life-threatening problem in this setting. Both infiltrating immune cells and replicating bacteria consume oxygen, contributing to the profound tissue hypoxia that characterises sites of infection. Hypoxia in turn has a dramatic effect on both neutrophil bactericidal function and the properties of S. aureus, including the production of virulence factors. Hypoxia thereby shapes the host–pathogen interaction and the progression of infection, for example promoting intracellular bacterial persistence, enabling local tissue destruction with the formation of an encaging abscess capsule, and facilitating the establishment and propagation of bacterial biofilms which block the access of host immune cells. Elucidating the molecular mechanisms underlying host–pathogen interactions in the setting of hypoxia will enable better understanding of persistent and recalcitrant infections due to S. aureus and may uncover novel therapeutic targets and strategies.

Background: Neutrophilic airway infiltration is central to chronic obstructive pulmonary disease (COPD) pathogenesis. COPD patient sputum contains high levels of neutrophil-derived microvesicles (NMVs). NMVs can be internalised by and activate target cells, however, little is known about their interaction with the lung epithelium. Methodology: NMVs were isolated from human neutrophils treated with PBS, 10µM fMLP, or 50% cigarette smoke extract (CSE) for 1h. Matrix metalloproteinase-9 (MMP-9) levels were determined by western blotting (n=4). Internalisation of fluorescently-labelled NMVs by BEAS-2B bronchial epithelial cells after 2h was quantified using confocal microscopy and flow cytometry (n=5), and IL-8 and MCP-1 secretion after 24h was quantified by ELISA (n=3). Plasma MVs from exacerbating COPD patients (n=6) and age-matched controls (n=5) were analysed by multi-colour flow cytometry to determine cellular origin and surface MMP-9 expression. Results: CSE generated more NMVs than fMLP. NMVs from stimulated cells had high levels of active MMP-9, were internalised by BEAS-2B cells (34.1±15.0%) and significantly increased epithelial IL-8 (>12-fold; p6-fold; p In patient plasma, NMVs represented 22.5% of the total MVs, and surface expression of MMP-9 was detected (117.4±75.8 MMP-9+NMVs/µl), however, levels did not differ between patients and controls. Conclusion: NMVs contain proteases associated with COPD progression and induce pro-inflammatory activation of lung epithelial cells, indicating a potential pathogenic role in the disease. Systemic NMV levels may not correlate with levels in the lungs and local production of NMVs may be key.

Spencer et al. report the first description of human IL-6R deficiency in two patients presenting with recurrent infections, atopy, elevated IgE, and abnormal acute-phase responses., IL-6 excess is central to the pathogenesis of multiple inflammatory conditions and is targeted in clinical practice by immunotherapy that blocks the IL-6 receptor encoded by IL6R. We describe two patients with homozygous mutations in IL6R who presented with recurrent infections, abnormal acute-phase responses, elevated IgE, eczema, and eosinophilia. This study identifies a novel primary immunodeficiency, clarifying the contribution of IL-6 to the phenotype of patients with mutations in IL6ST, STAT3, and ZNF341, genes encoding different components of the IL-6 signaling pathway, and alerts us to the potential toxicity of drugs targeting the IL-6R.

Immunometabolism and regulation of mitochondrial reactive oxygen species (mROS) are critical determinants of the immune effector phenotype of differentiated macrophages. Mitochondrial function requires dynamic fission and fusion, but whether effector function is associated with altered dynamics during bacterial responses is unknown. We show that macrophage mitochondria undergo fission after 12 h of progressive ingestion of live Streptococcus pneumoniae (pneumococci). Fission is associated with progressive reduction in oxidative phosphorylation but increased mROS generation. Fission is enhanced by mROS production, PI3Kγ signaling and by cathepsin B, but not by inflammasome activation or IL-1β generation. Reduced fission following PI3Kγ or cathepsin B inhibition is associated with reduced mROS generation and bacterial killing. Fission is associated with Parkin recruitment to mitochondria, but not mitophagy. Fission occurs upstream of apoptosis induction and independently of caspase activation. During macrophage innate responses to live bacteria mitochondria shift from oxidative phosphorylation and ATP generation to mROS production and microbicidal responses with a progressive shift towards mitochondrial fission.

Non-typeable Haemophilus influenzae (NTHi) is a frequent cause of lower respiratory tract infection in people with chronic obstructive pulmonary disease (COPD). Pellino proteins are a family of E3 ubiquitin ligases that are critical regulators of TLR signaling and inflammation. The aim of this study was to identify a role for Pellino-1 in airway defense against NTHi in the context of COPD. Pellino-1 is rapidly upregulated by LPS and NTHi in monocyte-derived macrophages (MDMs) isolated from individuals with COPD and healthy control subjects, in a TLR4 dependent manner. C57BL/6 Peli1-/- and wild-type (WT) mice were subjected to acute (single LPS challenge) or chronic (repeated LPS and elastase challenge) airway inflammation followed by NTHi infection. Both WT and Peli1-/- mice develop airway inflammation in acute and chronic airway inflammation models. Peli1-/- animals recruit significantly more neutrophils to the airway following NTHi infection which is associated with an increase in the neutrophil chemokine, KC, in bronchoalveolar lavage fluid as well as enhanced clearance of NTHi from the lung. These data suggest that therapeutic inhibition of Pellino-1 may augment immune responses in the airway and enhance bacterial clearance in individuals with COPD.

GM‐CSF is important in regulating acute, persistent neutrophilic inflammation in certain settings, including lung injury. Ligand binding induces rapid internalization of the GM‐CSF receptor (GM‐CSFRα) complex, a process essential for signaling. Whereas GM‐CSF controls many aspects of neutrophil biology, regulation of GM‐CSFRα expression is poorly understood, particularly the role of GM‐CSFRα in ligand clearance and whether signaling is sustained despite major down‐regulation of GM‐CSFRα surface expression. We established a quantitative assay of GM‐CSFRα surface expression and used this, together with selective anti‐GM‐CSFR antibodies, to define GM‐CSFRα kinetics in human neutrophils, and in murine blood and alveolar neutrophils in a lung injury model. Despite rapid sustained ligand‐induced GM‐CSFRα loss from the neutrophil surface, which persisted even following ligand removal, pro‐survival effects of GM‐CSF required ongoing ligand‐receptor interaction. Neutrophils recruited to the lungs following LPS challenge showed initially high mGM‐CSFRα expression, which along with mGM‐CSFRβ declined over 24 hr; this was associated with a transient increase in bronchoalveolar lavage fluid (BALF) mGM‐CSF concentration. Treating mice in an LPS challenge model with CAM‐3003, an anti‐mGM‐CSFRα mAb, inhibited inflammatory cell influx into the lung and maintained the level of BALF mGM‐CSF. Consistent with neutrophil consumption of GM‐CSF, human neutrophils depleted exogenous GM‐CSF, independent of protease activity. These data show that loss of membrane GM‐CSFRα following GM‐CSF exposure does not preclude sustained GM‐CSF/GM‐CSFRα signaling and that this receptor plays a key role in ligand clearance. Hence neutrophilic activation via GM‐CSFR may play an important role in neutrophilic lung inflammation even in the absence of high GM‐CSF levels or GM‐CSFRα expression., GM‐CSF released during ALI stimulates neutrophil recruitment, induces down‐regulation of GM‐CSFR, and GM‐CSF consumption by neutrophils, yet sustained anti‐apoptotic signals require continued GM‐CSF stimulation.

In the original version of this article unfortunately two authors were missing: Dr. Jurgen Weidemann and Dr. Daniel Berthold. The correct list of authors is presented above.

Introduction and objective Heightened endothelial cell permeability is a feature of life-threatening conditions such as sepsis and acute respiratory distress syndrome. However, there is a lack of pharmacological therapies targeting this pathological hyper-permeability. Bone morphogenetic protein 9 (BMP9) is a circulating vascular quiescence factor, signalling on vascular endothelial cells (ECs) through activin receptor-like kinase 1 (ALK1) and BMP receptor type II (BMPRII). Since recombinant BMP9 affords protection from endothelial permeability induced by inflammatory mediators, our objective was to investigate whether endogenous BMP9 plays a constitutive role in maintaining endothelial barrier function. Methods Wild-type mice were treated with BMP9 neutralising antibody at 5 mg/kg to assess: 1) rapid vascular leak using live imaging in mouse cremaster muscle and 2) more sustained vascular leak using Evans Blue extravasation, neutrophil accumulation and lung histology. Changes in endogenous BMP9 during LPS-induced endotoxemia were assessed by mRNA expression in the liver and protein levels in circulation. Anti-BMP9 effects on endothelial VE-cadherin were assessed by immunostaining. BMP9 signalling on EC receptors was profiled using microarray. Effects of BMP9 administration were investigated using an intranasal LPS-induced lung injury model. Results Selective inhibition of circulating BMP9 alone resulted in a rapid ( Conclusions Endogenous circulating BMP9 plays a constitutive role in maintaining lung endothelial barrier function. Remarkably, inhibition of BMP9 alone was sufficient to increase lung vascular permeability and promote neutrophil extravasation. The reduction in circulating BMP9 associated with LPS-induced inflammation, and the rescue of lung permeability with exogenous BMP9 suggests that supplementation of this factor may be a useful therapeutic approach in conditions associated with lung endothelial injury.

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease., Author summary The prevalence of methicillin resistant Staphylococcus aureus (MRSA) in both hospitals and the wider community places a huge weight on healthcare providers. To discover new control regimes, it is therefore important to understand how the pathogen behaves within the relevant environment of the host. This is often hampered by the ability to obtain sufficient ex vivo pathogen samples for study. We have developed a method to isolate S. aureus from the infected host to be able to analyse cellular morphology and structure. S. aureus, isolated from an infected kidney abscess are smaller in size, with thicker cell walls than exponentially growing cells in vitro. Their cell wall peptidoglycan also is less crosslinked. These features suggested the role of components controlling cell wall homeostasis as being important for infections. We tested the role of PBP4, known to increase cell wall crosslinking and found a pbp4 mutant to have increased survival in macrophages and fitness within the murine host. Conversely the peptidoglycan hydrolase SagB, whose loss results in thinner cell walls was attenuated in the murine systemic model of infection, with concomitant loss of fitness within macrophages. Our study reveals an important adaptation to the host environment and the role of those components involved in cell wall homeostasis in vivo.

Streptococcus pneumoniaeis a major cause of pneumonia and a leading cause of death world-wide. Antibody-mediated immune responses can offer protection against repeated exposure toS. pneumoniae, yet vaccines only offer partial protection. Patients with Activated PI3Kδ Syndrome (APDS) are highly susceptible toS. pneumoniae. We generated a conditional knockin mouse model of this disease and identified a CD19+B220−B cell subset that is induced by PI3Kδ signaling, is resident in the lungs, and which promotes increased susceptibility toS. pneumoniaeduring the early phase of infection via an antibody-independent mechanism. We show that an inhaled PI3Kδ inhibitor improves survival rates followingS. pneumoniaeinfection in wild-type mice and in mice with activated PI3Kδ. These results suggest that a subset of B cells in the lung can promote the severity ofS. pneumoniaeinfection, representing a novel therapeutic target.

The activation status of neutrophils can cycle from basal through primed to fully activated (“green‐amber‐red”), and at least in vitro, primed cells can spontaneously revert to a near basal phenotype. This broad range of neutrophil responsiveness confers extensive functional flexibility, allowing neutrophils to respond rapidly and appropriately to varied and evolving threats throughout the body. Primed and activated cells display dramatically enhanced bactericidal capacity (including augmented respiratory burst activity, degranulation and longevity), but this enhancement also confers the capacity for significant unintended tissue injury. Neutrophil priming and its consequences have been associated with adverse outcomes in a range of disease states, hence understanding the signalling processes that regulate the transition between basal and primed states (and back again) may offer new opportunities for therapeutic intervention in pathological settings. A wide array of host‐ and pathogen‐derived molecules is able to modulate the functional status of these versatile cells. Reflecting this extensive repertoire of potential mediators, priming can be established by a range of signalling pathways (including mitogen‐activated protein kinases, phosphoinositide 3‐kinases, phospholipase D and calcium transients) and intracellular processes (including endocytosis, vesicle trafficking and the engagement of adhesion molecules). The signalling pathways engaged, and the exact cellular phenotype that results, vary according to the priming agent(s) to which the neutrophil is exposed and the precise environmental context. Herein we describe the signals that establish priming (in particular for enhanced respiratory burst, degranulation and prolonged lifespan) and describe the recently recognised process of de‐priming, correlating in vitro observations with in vivo significance.

Defining features of chronic airway diseases include abnormal and persistent inflammatory processes, impaired airway epithelial integrity and function, and increased susceptibility to recurrent respiratory infections. Phosphoinositide 3-kinases (PI3K) are lipid kinases, which contribute to multiple physiological and pathological processes within the airway, with abnormal PI3K signalling contributing to the pathogenesis of several respiratory diseases. Consequently, the potential benefit of targeting PI3K isoforms has received considerable attention, being viewed as a viable therapeutic option in inflammatory and infectious lung disorders. The class I PI3K isoform, PI3Kδ (Phosphoinositide 3-kinases δ) is of particular interest given its multiple roles in modulating innate and adaptive immune cell functions, airway inflammation and corticosteroid sensitivity. In this mini-review, we explore the role of PI3Kδ in airway inflammation and infection, focusing on oxidative stress, ER stress, histone deacetylase 2 and neutrophil function. We also describe the importance of PI3Kδ in adaptive immune cell function, as highlighted by the recently described Activated PI3K Delta Syndrome, and draw attention to some of the potential clinical applications and benefits of targeting this molecule.

Rationale and Hypothesis HIV-1-seropositive individuals receiving highly active antiretroviral therapy (HAART) have an increased incidence of chronic obstructive pulmonary disease (COPD), independent of smoking history. Although HIV-1 infection is associated with impaired redox homeostasis and increased pro-inflammatory cytokine expression in the lungs despite HAART, the mechanisms driving HIV-1-associated COPD are poorly understood. Free HIV-1 envelope glycoprotein gp120 is detectable in bronchoalveolar lavage fluid from HAART-treated individuals. gp120 displays affinity (tropism) for either CCR5 or CXCR4 chemokine receptors, and has been implicated as a mediator of inflammation and oxidative stress in various HIV-1-associated disease processes. We hypothesised that gp120 directly induces bronchial epithelial cell oxidative stress, and drives airway inflammation indirectly via alveolar macrophages, a response which is augmented following secondary exposure to pro-inflammatory stimuli such as bacterial pathogens. Objectives To explore the mechanisms and consequences of gp120 interactions with bronchial epithelial cells and macrophages. Methods An immortalised bronchial epithelial cell line (BEAS-2B), primary bronchial epithelial cells (PBECs) or monocyte-derived macrophages (MDMs) from healthy volunteers were treated with recombinant gp120 (CCR5- or CXCR4-tropic, 100 ng/mL) for 24–48 hour. BEAS-2B were primed (or not) with IL-1β. MDMs were co-cultured with confluent BEAS-2B cells at a ratio of 1:5 in the presence or absence of LPS (100 ng/ml). Cytokine outputs were quantified by ELISA, and cellular reactive oxygen species (ROS) production assessed by confocal microscopy using CellROX or MitoSOX reagents. Findings Picomolar concentrations of CXCR4- but not CCR5-tropic gp120 induced CXCL8 release from IL-1β-primed BEAS-2B monocultures and upregulated cellular ROS production in both BEAS-2Bs and PBECs, consistent with expression of CXCR4 but not CCR5 on these cells. gp120 stimulation of BEAS-2B/MDM co-cultures caused no detectable changes in cytokine release. However, co-cultures primed with gp120 (of either tropism) and stimulated with LPS demonstrated significant CXCL8 release at 48 hours, reflecting MDM expression of both chemokine receptors. Impaired redox homeostasis and upregulated inflammatory responses may contribute to gp120-mediated airway epithelial dysfunction, and may drive neutrophil recruitment in this setting. Conclusion HIV-1 gp120 influences key airway cell interactions to disturb redox homeostasis and inflammatory responses at concentrations equivalent to those found in the lungs of individuals receiving long-term HAART.

Introduction COPD is a progressive neutrophilic lung disease associated with increased risk of cardiovascular complications. Neutrophil elastase (NE) is implicated in COPD pathogenesis but the precise mechanisms of neutrophil-mediated tissue damage are unknown, particularly with respect to systemic manifestations. Inflamed COPD airways are profoundly hypoxic. We therefore hypothesised that hypoxia synergises with inflammatory cytokines to promote a destructive neutrophil phenotype with enhanced capacity for tissue damage, both locally and systemically. Methods Neutrophils isolated from exacerbating COPD patients and age/sex-matched healthy volunteers were incubated under normoxia (21% O2) or hypoxia (0.8% O2) for 4 hours, before treatment with priming (PAF) and stimulating (fMLP) agents, with/without PI3Kinase inhibitors. NE activity was measured by Enzchek assay. Neutrophil supernatants were incubated with primary human pulmonary artery endothelial cells (HPAEC); cell damage was assessed by confocal microscopy. Normoxic/hypoxic neutrophil supernatants underwent tandem mass tag-labelled mass spectrometry (TMT-MS), and identified protein abundance was quantified. Neutrophil-derived microparticles (NDMPs) were isolated by ultra-centrifugation and quantified by NanoSight nanoparticle tracking technology. Results Hypoxia increased NE release in a PI3K-dependent manner, with significantly more NE secreted by hypoxic neutrophils from exacerbating COPD patients versus healthy controls (p Conclusions Hypoxia augments NE release in a PI3K-dependent manner, further increased during COPD exacerbations, and hypoxic neutrophil supernatants injure endothelial cells in vitro. Unbiased characterisation of hypoxic neutrophil secretomes identified several upregulated proteins which may contribute to cellular/tissue damage. In addition to degranulation, NDMP release may underpin differential protein secretion under hypoxia. Hypoxia engenders a neutrophil phenotype with potential to cause local and distant tissue damage in COPD; novel targets in the hypoxic neutrophil secretome may identify new therapeutic opportunities.

Rational and Hypothesis Human Rhinovirus (HRV) infections are major contributors to the increased morbidity burden associated with asthma and COPD acute exacerbations. There are currently no effective treatments or vaccines targeting exacerbations, therefore understanding the host-virus interactions that drive cellular damage will help identify potential therapeutic targets. Viral infections alter the airway environment through increased production of inflammatory mediators, defensive factors and viral proteins. This Results in the upregulation of cellular processes such as the unfolded protein response (UPR), an ER (endoplasmic reticulum) stress pathway that acts to alleviate ER stress caused by increased demands on protein synthesis. In the event that UPR fails to restore cellular homeostasis, pro-apoptotic pathways are activated. Many viruses induce ER stress and have evolved mechanisms to modify UPR to promote their own replication. Interestingly, the mechanisms and consequences of HRV-induced ER stress in bronchial epithelial cells have yet to be explored. We therefore hypothesised that HRV infection induces and manipulates ER stress processes within bronchial epithelial cells. Objectives To explore the mechanisms and consequences of HRV-induced ER stress within bronchial epithelial cells. Methods The immortalised bronchial epithelial cell line, BEAS-2B was infected with HRV for 1 hour at MOI 1.5. Induction and subcellular localisation of ER stress markers (GRP78 and ATF4) were measured at various time points by western blotting and confocal microscopy. Tunicamycin (a known ER stress inducer) and filtered HRV were included as positive and negative controls respectively. Findings Virally infected BEAS-2B cells induced ER stress as evidenced by the significant induction of the UPR chaperone protein, GRP78 at 24 hour. ATF4, a transcriptional activator of UPR target genes, redistributed from a cytoplasmic location to perinuclear regions, as assessed by immunofluorescence and confocal microscopy. Translocation was seen from as early as 1 hour following treatment with Tunicamycin, but this response was relatively delayed in HRV-infected BEAS-2B cells, with ATF4 redistributing to perinuclear regions from 8 hour post infection. Conclusion Our data demonstrate for the first time HRV-induced ER stress within bronchial epithelial cells, and suggest that HRV may manipulate ER stress pathways to facilitate its own replication.

Introduction Evidence has implicated neutrophil elastase (NE), a proteolytic enzyme, as a key driver of the pulmonary vascular remodelling that underlies pulmonary arterial hypertension (PAH). Moreover, studies using animal models and explanted human lung tissue have demonstrated that inhibition of NE attenuates pulmonary hypertension. However, there has been little investigation into neutrophil function in PAH patients even though their azurophilic granules are the main physiological reservoir of NE. We investigated neutrophil phenotypes in patients with PAH versus healthy controls, with a focus on neutrophil degranulation. Methods Neutrophils were isolated from venous blood of PAH patients and healthy controls (HC) and treated with LPS; viability was assessed at 20 hours by morphology. Cell surface receptor expression was determined by flow cytometry. To evaluate degranulation, neutrophils were treated with priming agents, platelet activating factor (PAF, 1 µM) or tumour necrosis factor-α (TNF, 20 ng/ml), and subsequently stimulated with N-formylmethionyl-leucyl-phenylalanine (fMLP; 100 nM). NE release was measured by ELISA and released NE activity and myeloperoxidase (MPO) activity were determined by fluorogenic (Enzchek) and colorimetric (o-dianisidine oxidation) assays respectively. Results Neutrophil apoptosis 20 hours following stimulation with LPS was significantly lower in PAH (25.4%+/-2.2, n=12) versus HC samples (44.9%+/-4.7, n=9), p Conclusions Our Results indicate that neutrophil phenotype is altered in PAH, with a prolonged lifespan in response to a pro-inflammatory stimulus and increased release of NE. However, we did not detect a corresponding increase in NE activity, suggesting a concomitant increase in NE inhibitor release from PAH neutrophils. The potential role of this altered neutrophil phenotype in vascular remodelling requires further investigation.

Background Eosinophils are key mediators of allergic inflammation. The ability to localise and quantify eosinophilic inflammation in vivo would facilitate patient endotyping and evaluation of eosinophil-targeted therapeutics. We aimed to quantify eosinophil distribution and organ-specific uptake in healthy subjects, asthmatics, and patients with focal pulmonary eosinophilic inflammation. Methods We injected autologous radiolabelled eosinophils into 8 healthy volunteers, 15 asthmatics (7 obese and 7 non-obese), and 3 patients with focal eosinophilic inflammation and monitored eosinophil distribution (planar imaging, single photon emission computed tomography – SPECT)/CT). Lung accumulation of technetium-99 m-labelled eosinophils was quantified (Patlak-Rutland analysis). Whole body indium-111-labelled eosinophil distribution and loss were further assessed in 5 healthy volunteers and 7 asthmatics using a whole body counter. Findings Pulmonary eosinophil clearance was increased in patients with focal eosinophilia (0·0033 ml/min/ml; 95% CI −0·005–0·011; p=0.02) compared to asthmatics (0·0007 ml/min/ml; 95% CI 0·0003–0·0010; p=0.14) and controls (0·0003 ml/min/ml; 95% CI −7·5 × 10–5–0·0008). Absolute lung eosinophil migration was elevated in patients with focal inflammation (5932 eosinophils/min/ml; 95% CI −14351–26215, p=0.01) and asthma (364 eosinophils/min/ml; 95% CI 38–689; p=0.03) versus healthy volunteers (38 eosinophils/min/ml; 95% CI −11–87). Stratification of asthmatics based on BMI revealed increased pulmonary eosinophil clearance in obese (0·001 ml/min/ml; 95% CI 0·0007–0·001; p=0.02) versus non-obese asthmatics (0·0003 ml/min/ml; 95% CI −0·0002–0·0009). Interpretation Eosinophil radiolabelling can quantify pulmonary eosinophilic inflammation, with the potential for patient endotyping and testing eosinophil-targeted treatments. Funding Medical Research Council, Wellcome Trust, Asthma UK, Cambridge NIHR Biomedical Research Centre.

Staphylococcal infection and neutrophilic inflammation can act in concert to establish a profoundly hypoxic environment. In this review we summarise how neutrophils and Staphylococcus aureus are adapted to function under hypoxic conditions, with a particular focus on the impaired ability of hypoxic neutrophils to effect Staphylococcus aureus killing.

Eosinophils are mediators of allergic inflammation and are implicated in the pathogenesis of numerous conditions including asthma, parasitic infections, neoplasms, hyper-eosinophilic syndromes, vasculitic disorders, and organ-specific conditions. Assessing eosinophilic inflammation is therefore important in establishing a diagnosis, in monitoring and assessing response to treatment, and in testing novel therapeutics. Clinical markers of atopy and eosinophilic inflammation include indirect tests such as lung function, exhaled breath condensate analysis, fractional exhaled nitric oxide, serum immunoglobulin E levels and serum periostin. Direct measures, which quantify but do not anatomically localise inflammation include blood eosinophil counts, serum or plasma eosinophil cationic protein and sputum eosinophil levels. Cytology from bronchoalveolar lavage and histology from endobronchial and transbronchial biopsies are better at localising inflammation but are more invasive. Novel approaches using radiolabelled eosinophils with single-photon emission computed tomography, offer the prospect of non-invasive methods to localise eosinophilic inflammation.