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.

Proteomic, biomechanical and functional analyses define neutrophil heterogeneity in systemic lupus erythematosus.

OBJECTIVES: Low-density granulocytes (LDGs) are a distinct subset of proinflammatory and vasculopathic neutrophils expanded in systemic lupus erythematosus (SLE). Neutrophil trafficking and immune function are intimately linked to cellular biophysical properties. This study used proteomic, biomechanical and functional analyses to further define neutrophil heterogeneity in the context of SLE. METHODS: Proteomic/phosphoproteomic analyses were performed in healthy control (HC) normal density neutrophils (NDNs), SLE NDNs and autologous SLE LDGs. The biophysical properties of these neutrophil subsets were analysed by real-time deformability cytometry and lattice light-sheet microscopy. A two-dimensional endothelial flow system and a three-dimensional microfluidic microvasculature mimetic (MMM) were used to decouple the contributions of cell surface mediators and biophysical properties to neutrophil trafficking, respectively. RESULTS: Proteomic and phosphoproteomic differences were detected between HC and SLE neutrophils and between SLE NDNs and LDGs. Increased abundance of type 1 interferon-regulated proteins and differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs. The cell surface of SLE LDGs was rougher than in SLE and HC NDNs, suggesting membrane perturbances. While SLE LDGs did not display increased binding to endothelial cells in the two-dimensional assay, they were increasingly retained/trapped in the narrow channels of the lung MMM. CONCLUSIONS: Modulation of the neutrophil proteome and distinct changes in biophysical properties are observed alongside differences in neutrophil trafficking. SLE LDGs may be increasingly retained in microvasculature networks, which has important pathogenic implications in the context of lupus organ damage and small vessel vasculopathy.

Targeting HIF2α-ARNT hetero-dimerisation as a novel therapeutic strategy for pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a destructive disease of the pulmonary vasculature often leading to right heart failure and death. Current therapeutic intervention strategies only slow disease progression. The role of aberrant hypoxia-inducible factor (HIF)2α stability and function in the initiation and development of pulmonary hypertension (PH) has been an area of intense interest for nearly two decades.Here we determine the effect of a novel HIF2α inhibitor (PT2567) on PH disease initiation and progression, using two pre-clinical models of PH. Haemodynamic measurements were performed, followed by collection of heart, lung and blood for pathological, gene expression and biochemical analysis. Blood outgrowth endothelial cells from idiopathic PAH patients were used to determine the impact of HIF2α-inhibition on endothelial function.Global inhibition of HIF2a reduced pulmonary vascular haemodynamics and pulmonary vascular remodelling in both su5416/hypoxia prevention and intervention models. PT2567 intervention reduced the expression of PH-associated target genes in both lung and cardiac tissues and restored plasma nitrite concentration. Treatment of monocrotaline-exposed rodents with PT2567 reduced the impact on cardiovascular haemodynamics and promoted a survival advantage. In vitro, loss of HIF2α signalling in human pulmonary arterial endothelial cells suppresses target genes associated with inflammation, and PT2567 reduced the hyperproliferative phenotype and overactive arginase activity in blood outgrowth endothelial cells from idiopathic PAH patients. These data suggest that targeting HIF2α hetero-dimerisation with an orally bioavailable compound could offer a new therapeutic approach for PAH. Future studies are required to determine the role of HIF in the heterogeneous PAH population.

C5a impairs phagosomal maturation in the neutrophil through phosphoproteomic remodeling.

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.

A nebulised antitumour necrosis factor receptor-1 domain antibody in patients at risk of postoperative lung injury: A randomised, placebo-controlled pilot study.

BACKGROUND: Tumour necrosis factor receptor 1 (TNFR1) signalling mediates the cell death and inflammatory effects of TNF-α. OBJECTIVE: The current clinical trial investigated the effects of a nebulised TNFR1 antagonist (GSK2862277) on signs of lung injury in patients undergoing oesophagectomy. DESIGN: Randomised double-blind (sponsor unblind), placebo-controlled, parallel group study. SETTING: Eight secondary care centres, the United Kingdom between April 2015 and June 2017. PATIENTS: Thirty-three patients undergoing elective transthoracic oesophagectomy. INTERVENTIONS: Patients randomly received a single nebulised dose (26 mg) of GSK2862277 (n = 17) or placebo (n = 16), given 1 to 5 h before surgery; 14 and 16, respectively competed the study. MAIN OUTCOME MEASUREMENTS: Physiological and biochemical markers of lung injury, pharmacokinetic and safety endpoints were measured. The primary endpoint was the change from baseline in pulmonary vascular permeability index (PVPI) at completion of surgery, measured using single-indicator transpulmonary thermodilution. Adjusted point estimates and 95% credible intervals (analogous to conventional confidence intervals) were constructed for each treatment using Bayesian statistical models. RESULTS: The mean change (with 95% credible intervals) from baseline in PVPI on completion of surgery was 0.00 (-0.23, 0.39) in the placebo and 0.00 (-0.24, 0.37) in the GSK2862277 treatment groups. There were no significant treatment-related differences in PaO2/FiO2 or Sequential Organ Failure Assessment score. Levels of free soluble TNFR1, Macrophage Inflammatory Protein-1 alpha and total protein were significantly reduced in the bronchoalveolar lavage fluid of patients treated with GSK2862277 (posterior probability of decrease with GSK2862277 vs. placebo:≥0.977; equivalent to P < 0.05). The frequency of adverse events and serious adverse events were distributed evenly across the two treatment arms. CONCLUSION: Pre-operative treatment with a single 26 mg inhaled dose of GSK2862277 did not result in significantly lower postoperative alveolar capillary leak or extra vascular lung water. Unexpectedly small increases in transpulmonary thermodilution-measured PVPI and extra vascular lung water index at completion of surgery suggest less postoperative lung injury than historically reported, which may have also compromised a clear assessment of efficacy in this trial. GSK2862277 was well tolerated, resulted in expected lung exposure and reduced biomarkers of lung permeability and inflammation. TRIAL REGISTRATION: clinicaltrials.gov: NCT02221037.

The counter-intuitive role of the neutrophil in the acute respiratory distress syndrome.

INTRODUCTION: Neutrophils are the primary effectors of the innate immune system but are profoundly histotoxic cells. The acute respiratory distress syndrome (ARDS) is considered to be a prime example of neutrophil-mediated tissue injury. SOURCES OF DATA: The information presented in this review is acquired from the published neutrophil cell biology literature and the longstanding interest of the senior authors in ARDS pathogenesis and clinical management. AREAS OF AGREEMENT: Investigators in the field would agree that neutrophils accumulate in high abundance in the pulmonary microcirculation, lung interstitium and alveolar airspace of patients with ARDS. ARDS is also associated with systemic neutrophil priming and delayed neutrophil apoptosis and clearance of neutrophils from the lungs. In animal models, reducing circulating neutrophil numbers ameliorates lung injury. AREAS OF CONTROVERSY: Areas of uncertainty include how neutrophils get stuck in the narrow pulmonary capillary network-whether this reflects changes in the mechanical properties of primed neutrophils alone or additional cell adhesion molecules, the role of neutrophil sub-sets or polarization states including pro-angiogenic and low-density neutrophils, whether neutrophil extracellular trap (NET) formation is beneficial (through bacterial capture) or harmful and the potential for neutrophils to participate in inflammatory resolution. The latter may involve the generation of specialized pro-resolving molecules (SPMs) and MMP-9, which is required for adequate matrix processing. GROWING POINTS: Different and possibly stable endotypes of ARDS are increasingly being recognized, yet the relative contribution of the neutrophil to these endotypes is uncertain. There is renewed and intense interest in understanding the complex 'new biology' of the neutrophil, specifically whether this cell might be a valid therapeutic target in ARDS and other neutrophil-driven diseases and developing understanding of ways to enhance the beneficial role of the neutrophil in the resolution phase of ARDS. AREAS TIMELY FOR DEVELOPING RESEARCH: Aside from treatment of the precipitating causes of ARDS, and scrupulous fluid, infection and ventilation management, there are no pharmacological interventions for ARDS; this represents an urgent and unmet need. Therapies aimed at reducing overall neutrophil numbers risk secondary infection; hence better ways are needed to reverse the processes of neutrophil priming activation, hyper-secretion and delayed apoptosis while enhancing the pro-resolution functions of the neutrophil.

Real-time deformability cytometry reveals sequential contraction and expansion during neutrophil priming.

It has become increasingly apparent that the biomechanical properties of neutrophils impact on their trafficking through the circulation and in particularly through the pulmonary capillary bed. The retention of polarized or shape-changed neutrophils in the lungs was recently proposed to contribute to acute respiratory distress syndrome pathogenesis. Accordingly, this study tested the hypothesis that neutrophil priming is coupled to morpho-rheological (MORE) changes capable of altering cell function. We employ real-time deformability cytometry (RT-DC), a recently developed, rapid, and sensitive way to assess the distribution of size, shape, and deformability of thousands of cells within seconds. During RT-DC analysis, neutrophils can be easily identified within anticoagulated "whole blood" due to their unique granularity and size, thus avoiding the need for further isolation techniques, which affect biomechanical cell properties. Hence, RT-DC is uniquely suited to describe the kinetics of MORE cell changes. We reveal that, following activation or priming, neutrophils undergo a short period of cell shrinking and stiffening, followed by a phase of cell expansion and softening. In some contexts, neutrophils ultimately recover their un-primed mechanical phenotype. The mechanism(s) underlying changes in human neutrophil size are shown to be Na+ /H+ antiport-dependent and are predicted to have profound implications for neutrophil movement through the vascular system in health and disease.

C5a anaphylatoxin and its role in critical illness-induced organ dysfunction.

Critical illness is an aetiologically and clinically heterogeneous syndrome that is characterised by organ failure and immune dysfunction. Mortality in critically ill patients is driven by inflammation-associated organ damage and a profound vulnerability to nosocomial infection. Both factors are influenced by the activated complement protein C5a, released by unbridled activation of the complement system during critical illness. C5a exerts deleterious effects on organ systems directly and suppresses antimicrobial functions of key immune cells. Whilst several recent reports have added key knowledge of the cellular signalling pathways triggered by C5a, there remain a number of areas that are incompletely understood and therapeutic opportunities are still being evaluated. In this review, we summarise the cellular basis for C5a-induced vulnerability to nosocomial infection and organ dysfunction. We focus on cells of the innate immune system, highlighting the major areas in need of further research and potential avenues for targeted therapies.