Edwin R. Chilvers

Summary: Neutrophils are the most abundant circulating leukocyte and are crucial to the initial innate immune response to infection. One of their key pathogen‐eliminating mechanisms is phagocytosis, the process of particle engulfment into a vacuole‐like structure called the phagosome. The antimicrobial activity of the phagocytic process results from a collaboration of multiple systems and mechanisms within this organelle, where a complex interplay of ion fluxes, pH, reactive oxygen species, and antimicrobial proteins creates a dynamic antimicrobial environment. This complexity, combined with the difficulties of studying neutrophils ex vivo, has led to gaps in our knowledge of how the neutrophil phagosome optimizes pathogen killing. In particular, controversy has arisen regarding the relative contribution and integration of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase‐derived antimicrobial agents and granule‐delivered antimicrobial proteins. Clinical syndromes arising from dysfunction in these systems in humans allow useful insight into these mechanisms, but their redundancy and synergy add to the complexity. In this article, we review the current knowledge regarding the formation and function of the neutrophil phagosome, examine new insights into the phagosomal environment that have been permitted by technological advances in recent years, and discuss aspects of the phagocytic process that are still under debate.

Many individual genetic risk loci have been associated with multiple common human diseases. However, the molecular basis of this pleiotropy often remains unclear. We present an integrative approach to reveal the molecular mechanism underlying the PROCR locus, associated with lower coronary artery disease (CAD) risk but higher venous thromboembolism (VTE) risk. We identify PROCR-p.Ser219Gly as the likely causal variant at the locus and protein C as a causal factor. Using genetic analyses, human recall-by-genotype and in vitro experimentation, we demonstrate that PROCR-219Gly increases plasma levels of (activated) protein C through endothelial protein C receptor (EPCR) ectodomain shedding in endothelial cells, attenuating leukocyte–endothelial cell adhesion and vascular inflammation. We also associate PROCR-219Gly with an increased pro-thrombotic state via coagulation factor VII, a ligand of EPCR. Our study, which links PROCR-219Gly to CAD through anti-inflammatory mechanisms and to VTE through pro-thrombotic mechanisms, provides a framework to reveal the mechanisms underlying similar cross-phenotype associations.

Background: Idiopathic pulmonary fibrosis is an irreversible fibrosing lung disorder with a poor prognosis. Current treatments slow the rate of decline in lung function and may influence survival, but they have a significant side-effect profile and so additional therapeutic options are required. People with idiopathic pulmonary fibrosis have altered innate immunity and altered lung microbiota, with the bacterial burden relating to mortality. Two randomised controlled trials have demonstrated beneficial effects with co-trimoxazole (SEPTRIN^®; Essential Generics Ltd, Egham, UK; Chemidex Generics Ltd, Egham, UK), with the suggestion of an improvement in rates of survival.Objectives: To determine the clinical efficacy of co-trimoxazole in people with moderate to severe idiopathic pulmonary fibrosis.Design: A Phase II, double-blind, placebo-controlled, parallel-group, randomised multicentre study.Setting: UK specialist interstitial lung disease centres.Participants: Patients who were randomised had idiopathic pulmonary fibrosis diagnosed by a multidisciplinary team. In addition, patients had significant breathlessness (i.e. a Medical Research Council Dyspnoea Scale score of > 1) and impaired lung function (i.e. a forced vital capacity of < 75% predicted). Patients could be taking licensed medication for idiopathic pulmonary fibrosis, but were excluded if they had significant comorbidities, including airflow obstruction.Intervention: Oral co-trimoxazole, 960 mg twice per day (two 480-mg tablets twice per day), compared with placebo tablets (two tablets twice per day) for a median of 27 months (range 12–42 months). Otherwise, both trial groups had standard care.Main outcome measures: The primary outcome was the time to death (all causes), transplant or first non-elective hospital admission. Secondary outcomes were the individual components of the primary end point and the number of respiratory-related events. Questionnaires (the King’s Brief Interstitial Lung Disease questionnaire; the Medical Research Council Dyspnoea Scale; EuroQol-5 Dimensions, five-level version; the Leicester Cough Questionnaire; and the Cough Symptom Score) and lung function tests (forced vital capacity and diffusing capacity for carbon monoxide) were undertaken at baseline and at 12 months.Results: The trial randomised a total of 342 (295 male) patients (active treatment group,n = 170; placebo group,n = 172), using minimisation for hospital and receipt of licensed antifibrotic medication, from 39 UK hospitals. The patients had a mean (standard deviation) age of 71.3 years (7.47 years) and a mean forced vital capacity of 2.25 l (0.56 l). A total of 137 (40%) patients were taking pirfenidone (Esbriet, Roche Holding AG, Basel, Switzerland) and 116 (34%) were taking nintedanib (Ofev^®, Boehringer Ingelheim, Brackness, UK). There was one post-randomisation exclusion from the co-trimoxazole group, but no withdrawals. There was no difference in the time to event for the composite primary end point (co-trimoxazole: hazard ratio 1.2, 95% confidence interval 0.9 to 1.6;p = 0.319). Likewise, there was no difference in other event outcomes, lung function measurements or patient-reported outcomes, other than a beneficial effect on the total Leicester Cough Questionnaire score, the social domain of the Leicester Cough Questionnaire score and the chest domain of the King’s Brief Interstitial Lung Disease questionnaire in the adjusted analysis. The repeated-measures analysis showed a significant overall difference in Cough Symptom Score. There were significantly more reports of nausea, but fewer reports of diarrhoea, with co-trimoxazole; however, differences in frequency of hyperkalaemia, rash and headache were not significant. The limitations of the trial were that it was not possible to evaluate the lung microbiota, there were missing data for secondary end points and there was no health economic analysis.Conclusion: These results suggest that co-trimoxazole does not reduce the likelihood of death or number of hospitalisations among people with idiopathic pulmonary fibrosis with moderate to severe idiopathic pulmonary fibrosis. Further work is required to evaluate the effect in subgroups of individuals with idiopathic pulmonary fibrosis or the effect of antibiotics with different antibacterial properties.Trial registration: Current Controlled Trials ISRCTN17464641.Funding: This project was funded by the Efficacy and Mechanism Evaluation programme, a Medical Research Council and National Institute for Health Research (NIHR) partnership. This will be published in full inEfficacy and Mechanism Evaluation; Vol. 8, No. 9. See the NIHR Journals Library for further project information.

Neutrophils play an important role in the lung tumour microenvironment. We hypothesised that radiolabelled neutrophils coupled to single-photon emission CT (SPECT) may non-invasively quantify neutrophil uptake in tumours from patients with non-small cell lung cancer. We demonstrated increased uptake of radiolabelled neutrophils from the blood into tumours compared with non-specific uptake using radiolabelled transferrin. Moreover, indium-111-neutrophil activity in the tumour biopsies also correlated with myeloperoxidase (MPO)-positive neutrophils. Our data support the utility of imaging with In-111-labelled neutrophils and SPECT-CT to quantify neutrophil uptake in lung cancer.

Critical illness is accompanied by the release of large amounts of the anaphylotoxin, C5a. C5a suppresses antimicrobial functions of neutrophils which is associated with adverse outcomes. The signaling pathways that mediate C5a-induced neutrophil dysfunction are incompletely understood. Healthy donor neutrophils exposed to purified C5a demonstrated a prolonged defect (7 hours) in phagocytosis of Staphylococcus aureus . Phosphoproteomic profiling of 2712 phosphoproteins identified persistent C5a signaling and selective impairment of phagosomal protein phosphorylation on exposure to S . aureus . Notable proteins included early endosomal marker ZFYVE16 and V-ATPase proton channel component ATPV1G1. An assay of phagosomal acidification demonstrated C5a-induced impairment of phagosomal acidification, which was recapitulated in neutrophils from critically ill patients. Examination of the C5a-impaired protein phosphorylation indicated a role for the PI3K VPS34 in phagosomal maturation. Inhibition of VPS34 impaired neutrophil phagosomal acidification and killing of S . aureus . This study provides a phosphoproteomic assessment of human neutrophil signaling in response to S . aureus and its disruption by C5a, identifying a defect in phagosomal maturation and mechanisms of immune failure in critical illness.

Respiratory viral pandemics result in large numbers of cases of acute respiratory failure arising from a single etiology, thus reducing the heterogeneity of precepting insult and allowing improved insight into the variation of host responses. In 2009-2011, an influenza pandemic occurred, with pH1N1 infecting millions of people worldwide. Here, we have used novel bioinformatic methods to combine clinical, protein biomarker, and genomic data from patients with influenza-associated acute respiratory failure to identify three mechanistically discrete sub-types with significantly different clinical outcomes. The three endotypes identified can be described as “neutrophil-driven” (16.3%), “adaptive” (51.9%), and “endothelial leak” (31.7%). The neutrophil driven patients display evidence of innate immune activation with associated multi-organ dysfunction and reduced 30-day survival. These patients could be differentiated from the adaptive endotype by an alteration in the GAIT-mechanism, a late transcriptional regulatory response to IFN-γ that acts to suppress innate immunity by reducing caeruloplasmin mRNA translation. Patients with the neutrophil-driven endotype had significantly increased IFN-γ levels but appeared unable to suppress their innate immune response. The endothelial leak endotype could be distinguished from both the neutrophil driven and adaptive endotypes by alterations in Slit-Robo signalling, a pathway important in the maintenance of endothelial barrier integrity; Although patients with this endotype required mechanical ventilation, they did not develop multi-organ failure in the manner of the neutrophil-driven endotype patients, and had significantly better clinical outcomes. Importantly, the endotypes identified were stable over 48 hours opening up the possibility of stratified interventional clinical trials in the future. One Sentence Summary: We have identified three new mechanistically distinct subtypes of influenza associated acute respiratory failure, with differential clinical outcomes.

Rationale: Pulmonary 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. Objective: To 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. 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 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 Results: Subacute 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. Conclusions: Endogenous 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 summary: Scientific Knowledge on the Subject: Increased 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 Field: Here 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.

Treatment with the CXCR4 antagonist, plerixafor (AMD3100), has been proposed for clinical use in patients with WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome and in pulmonary fibrosis. However, there is controversy with respect to the impact of plerixafor on neutrophil dynamics in the lung, which may affect its safety profile. In this study, we investigated the kinetics of endogenous neutrophils by direct imaging, using confocal intravital microscopy in mouse bone marrow, spleen, and lungs. Neutrophils are observed increasing their velocity and exiting the bone marrow following plerixafor administration, with a concomitant increase in neutrophil numbers in the blood and spleen, while the marginated pool of neutrophils in the lung microvasculature remained unchanged in terms of numbers and cell velocity. Use of autologous radiolabeled neutrophils and SPECT/CT imaging in healthy volunteers showed that plerixafor did not affect GM-CSF-primed neutrophil entrapment or release in the lungs. Taken together, these data suggest that plerixafor causes neutrophil mobilization from the bone marrow but does not impact on lung marginated neutrophil dynamics and thus is unlikely to compromise respiratory host defense both in humans and mice.

Genome-wide association studies have identified many individual genetic loci associated with multiple complex traits and common diseases. There are, however, few examples where the molecular basis of such pleiotropy has been elucidated. To address this challenge, we describe an integrative approach, focusing on the p.Ser219Gly (rs867186 A>G) variant in the PROCR gene (encoding the endothelial protein C receptor, EPCR), which has been associated with lower coronary artery disease (CAD) risk but higher venous thromboembolism (VTE) risk. In a phenome scan of 12 cardiometabolic diseases and 24 molecular factors, we found that PROCR -219Gly associated with higher plasma levels of zymogenic and activated protein C as well as coagulation factor VII. Using statistical colocalization and Mendelian randomization analyses, we uncovered shared genetic etiology across activated protein C, factor VII, CAD and VTE, identifying p.S219G as the likely causal variant at the locus. In a recall-by-genotype study of 52 healthy volunteers stratified by p.S219G, we detected 2.5-fold higher soluble EPCR levels and 1.2-fold higher protein C levels in plasma per effect allele, suggesting the allele induces EPCR shedding from the membrane of endothelial cells. Finally, in cell adhesion assays, we found that increasing concentrations of activated protein C, but not soluble EPCR, reduced leukocyte–endothelial cell adhesion, a marker for vascular inflammation. These results support a role for protein C as a causal factor in arterial and venous diseases, suggesting that PROCR -219Gly protects against CAD through anti-inflammatory mechanisms while it promotes VTE risk through pro-thrombotic mechanisms. Overall, our study illustrates a multi-modal approach that can help reveal molecular underpinnings of cross-disease associations.