Specific Inhibition of Orai1-mediated Calcium Signalling Resolves Inflammation and Clears Bacteria in an Acute Respiratory Distress Syndrome Model.

Rationale: Acute respiratory distress syndrome (ARDS) has an unacceptably high mortality rate (35%) and is without effective therapy. Orai1 is a Ca2+ channel involved in store-operated Ca2+ entry (SOCE), a process that exquisitely regulates inflammation. Orai1 is considered a druggable target, but no Orai1-specific inhibitors exist to date. Objectives: To evaluate whether ELD607, a first-in-class Orai1 antagonist, can treat ARDS caused by bacterial pneumonia in preclinical models. Methods: ELD607 pharmacology was evaluated in HEK293T cells and freshly isolated immune cells from patients with ARDS. A murine acute lung injury model caused by bacterial pneumonia was then used: mice were infected with Pseudomonas aeruginosa, Staphylococcus aureus, methicillin-resistant S. aureus, or multidrug-resistant P. aeruginosa and then treated with ELD607 intranasally. Measurements and Main Results: ELD607 specifically inhibited SOCE in HEK293T cells with a half-maximal inhibitory concentration of 9 nM. ELD607 was stable in ARDS airway secretions and inhibited SOCE in ARDS immune cells. In vivo, inhaled ELD607 significantly reduced neutrophilia and improved survival. Surprisingly, Orai1 inhibition by ELD607 caused a significant reduction in lung bacteria, including methicillin-resistant S. aureus. ELD607 worked as an immunomodulator that reduced cytokine levels, reduced neutrophilia, and promoted macrophage-mediated resolution of inflammation and clearance of bacteria. Indeed, when alveolar macrophages were depleted with inhaled clodronate, ELD607 was no longer able to resolve inflammation or clear bacteria. Conclusions: These data indicate that specific Orai1 inhibition by ELD607 may be a novel approach to reduce multiorgan inflammation and treat antibiotic-resistant bacteria.

E-cigarette vapor renders neutrophils dysfunctional due to filamentous actin accumulation.

BACKGROUND: Electronic cigarette (e-cigarette) use continues to rise despite concerns of long-term effects, especially the risk of developing lung diseases such as chronic obstructive pulmonary disease. Neutrophils are central to the pathogenesis of chronic obstructive pulmonary disease, with changes in phenotype and function implicated in tissue damage. OBJECTIVE: We sought to measure the impact of direct exposure to nicotine-containing and nicotine-free e-cigarette vapor on human neutrophil function and phenotype. METHODS: Neutrophils were isolated from the whole blood of self-reported nonsmoking, nonvaping healthy volunteers. Neutrophils were exposed to 40 puffs of e-cigarette vapor generated from e-cigarette devices using flavorless e-cigarette liquids with and without nicotine before functions, deformability, and phenotype were assessed. RESULTS: Neutrophil surface marker expression was altered, with CD62L and CXCR2 expression significantly reduced in neutrophils treated with e-cigarette vapor containing nicotine. Neutrophil migration to IL-8, phagocytosis of Escherichia coli and Staphylococcus aureus pHrodo bioparticles, oxidative burst response, and phorbol 12-myristate 13-acetate-stimulated neutrophil extracellular trap formation were all significantly reduced by e-cigarette vapor treatments, independent of nicotine content. E-cigarette vapor induced increased levels of baseline polymerized filamentous actin levels in the cytoplasm, compared with untreated controls. CONCLUSIONS: The significant reduction in effector neutrophil functions after exposure to high-power e-cigarette devices, even in the absence of nicotine, is associated with excessive filamentous actin polymerization. This highlights the potentially damaging impact of vaping on respiratory health and reinforces the urgency of research to uncover the long-term health implications of e-cigarettes.

CD14-positive extracellular vesicles in bronchoalveolar lavage fluid as a new biomarker of acute respiratory distress syndrome.

Recent studies have indicated that extracellular vesicles (EVs) may play a role in the pathogenesis of acute respiratory distress syndrome (ARDS). EVs have been identified as potential biomarkers of disease severity and prognosis in other pulmonary diseases. We sought to characterize the EV phenotype within bronchoalveolar lavage (BAL) fluid of patients with ARDS, and to determine whether BAL EV could be used as a potential biomarker in ARDS. BAL was collected from patients with sepsis with and without ARDS, and from esophagectomy patients postoperatively (of whom a subset later developed ARDS during hospital admission). BAL EVs were characterized with regard to size, number, and cell of origin. Patients with sepsis-related ARDS had significantly higher numbers of CD14+/CD81+ monocyte-derived BAL EV than patients with sepsis without ARDS (P = 0.015). However, the converse was observed in esophagectomy patients who later developed ARDS (P = 0.003). Esophagectomy patients who developed ARDS also had elevated CD31+/CD63+ and CD31+/CD81+ endothelial-derived BAL EV (P ≤ 0.02) compared with esophagectomy patients who did not develop ARDS. Further studies are required to determine whether CD31+ BAL EV may be a predictive biomarker for ARDS in esophagectomy patients. CD14+/CD81+ BAL EV numbers were significantly higher in those patients with sepsis-related ARDS who died during the 30 days following intensive care unit admission (P = 0.027). Thus, CD14+/CD81+ BAL EVs are a potential biomarker for disease severity and mortality in sepsis-related ARDS. These findings provide the impetus to further elucidate the contribution of these EVs to ARDS pathogenesis.

Cigarette smoke exposure and alveolar macrophages: mechanisms for lung disease.

Cigarette smoking is the leading cause of preventable death worldwide. It causes chronic lung disease and predisposes individuals to acute lung injury and pulmonary infection. Alveolar macrophages are sentinel cells strategically positioned in the interface between the airway lumen and the alveolar spaces. These are the most abundant immune cells and are the first line of defence against inhaled particulates and pathogens. Recently, there has been a better understanding about the ontogeny, phenotype and function of alveolar macrophages and their role, not only in phagocytosis, but also in initiating and resolving immune response. Many of the functions of the alveolar macrophage have been shown to be dysregulated following exposure to cigarette smoke. While the mechanisms for these changes remain poorly understood, they are important in the understanding of cigarette smoking-induced lung disease. We review the mechanisms by which smoking influences alveolar macrophage: (1) recruitment, (2) phenotype, (3) immune function (bacterial killing, phagocytosis, proteinase/anti-proteinase release and reactive oxygen species production) and (4) homeostasis (surfactant/lipid processing, iron homeostasis and efferocytosis). Further understanding of the mechanisms of cigarette smoking on alveolar macrophages and other lung monocyte/macrophage populations may allow novel ways of restoring cellular function in those patients who have stopped smoking in order to reduce the risk of subsequent infection or further lung injury.

Lactate cross-talk in host-pathogen interactions.

Lactate is the main product generated at the end of anaerobic glycolysis or during the Warburg effect and its role as an active signalling molecule is increasingly recognised. Lactate can be released and used by host cells, by pathogens and commensal organisms, thus being essential for the homeostasis of host-microbe interactions. Infection can alter this intricate balance, and the presence of lactate transporters in most human cells including immune cells, as well as in a variety of pathogens (including bacteria, fungi and complex parasites) demonstrates the importance of this metabolite in regulating host-pathogen interactions. This review will cover lactate secretion and sensing in humans and microbes, and will discuss the existing evidence supporting a role for lactate in pathogen growth and persistence, together with lactate's ability to impact the orchestration of effective immune responses. The ubiquitous presence of lactate in the context of infection and the ability of both host cells and pathogens to sense and respond to it, makes manipulation of lactate a potential novel therapeutic strategy. Here, we will discuss the preliminary research that has been carried out in the context of cancer, autoimmunity and inflammation.

Understanding potential mechanisms of harm: the drivers of electronic cigarette-induced changes in alveolar macrophages, neutrophils, and lung epithelial cells.

Electronic (e-) cigarettes are growing in popularity despite uncertainties regarding their long-term health implications. The link between cigarette smoking and initiation of chronic lung disease took decades to unpick so in vitro studies mimicking e-cigarette exposure aim to detect early indicators of harm. In response to e-cigarette exposure, alveolar macrophages adopt a proinflammatory phenotype of increased secretion of proinflammatory cytokines, reduction in phagocytosis, and efferocytosis and reactive oxygen species generation. These effects are largely driven by free radical exposure, changes in PI3K/Akt signaling pathways, nicotine-induced reduction in phagocytosis receptors, and impaired lipid homeostasis leading to a foam-like lipid-laden phenotype. Neutrophils exhibit disrupted chemotaxis and transmigration to chemokines, reduced phagocytosis and bacterial killing, and an increase in protease secretion without corresponding antiproteases in response to e-cigarette exposure. This is driven by an altered ability to respond and to polarize toward chemoattractants, an activation of the p38 MAPK signaling pathway and inability to assemble NADPH oxidase. E-cigarettes induce lung epithelial cells to display decreased ciliary beat frequency and ion channel conductance as well as changes in chemokine secretion and surface protein expression. Changes in gene expression, mitochondrial function, and signaling pathways have been demonstrated in lung epithelial cells to explain these changes. Many functional outputs of alveolar macrophages, neutrophils, and lung epithelial cells have not been fully explored in the context of e-cigarette exposure and the underlying driving mechanisms are poorly understood. This review discusses current evidence surrounding the effects of e-cigarettes on alveolar macrophages, neutrophils, and lung epithelial cells with particular focus on the cellular mechanisms of change.

Incidence and risk factors of acute kidney injury in COVID-19 patients with and without acute respiratory distress syndrome (ARDS) during the first wave of COVID-19: a systematic review and Meta-Analysis.

BACKGROUND: Acute kidney injury (AKI) is common among patients with COVID-19. However, AKI incidence may increase when COVID-19 patients develop acute respiratory distress syndrome (ARDS). Thus, this systematic review and meta-analysis aimed to assess the incidence and risk factors of AKI, need for kidney replacement therapy (KRT), and mortality rate among COVID-19 patients with and without ARDS from the first wave of COVID-19. METHODS: The databases MEDLINE and EMBASE were searched using relevant keywords. Only articles available in English published between December 1, 2019, and November 1, 2020, were included. Studies that included AKI in COVID-19 patients with or without ARDS were included. Meta-analyses were conducted using random-effects models. RESULTS: Out of 618 studies identified and screened, 31 studies met the inclusion criteria. A total of 27,500 patients with confirmed COVID-19 were included. The overall incidence of AKI in patients with COVID-19 was 26% (95% CI 19% to 33%). The incidence of AKI was significantly higher among COVID-19 patients with ARDS than COVID-19 patients without ARDS (59% vs. 6%, p < 0.001). Comparing ARDS with non-ARDS COVID-19 cohorts, the need for KRT was also higher in ARDS cohorts (20% vs. 1%). The mortality among COVID-19 patients with AKI was significantly higher (Risk ratio = 4.46; 95% CI 3.31-6; p < 0.00001) than patients without AKI. CONCLUSION: This study shows that ARDS development in COVID-19-patients leads to a higher incidence of AKI and increased mortality rate. Therefore, healthcare providers should be aware of kidney dysfunction, especially among elderly patients with multiple comorbidities. Early kidney function assessment and treatments are vital in COVID-19 patients with ARDS.

Neutrophils in community-acquired pneumonia: parallels in dysfunction at the extremes of age.

"Science means constantly walking a tight rope" Heinrich Rohrer, physicist, 1933. Community-acquired pneumonia (CAP) is the leading cause of death from infectious disease worldwide and disproportionately affects older adults and children. In high-income countries, pneumonia is one of the most common reasons for hospitalisation and (when recurrent) is associated with a risk of developing chronic pulmonary conditions in adulthood. Pneumococcal pneumonia is particularly prevalent in older adults, and here, pneumonia is still associated with significant mortality despite the widespread use of pneumococcal vaccination in middleand high-income countries and a low prevalence of resistant organisms. In older adults, 11% of pneumonia survivors are readmitted within months of discharge, often with a further pneumonia episode and with worse outcomes. In children, recurrent pneumonia occurs in approximately 10% of survivors and therefore is a significant cause of healthcare use. Current antibiotic trials focus on short-term outcomes and increasingly shorter courses of antibiotic therapy. However, the high requirement for further treatment for recurrent pneumonia questions the effectiveness of current strategies, and there is increasing global concern about our reliance on antibiotics to treat infections. Novel therapeutic targets and approaches are needed to improve outcomes. Neutrophils are the most abundant immune cell and among the first responders to infection. Appropriate neutrophil responses are crucial to host defence, as evidenced by the poor outcomes seen in neutropenia. Neutrophils from older adults appear to be dysfunctional, displaying a reduced ability to target infected or inflamed tissue, poor phagocytic responses and a reduced capacity to release neutrophil extracellular traps (NETs); this occurs in health, but responses are further diminished during infection and particularly during sepsis, where a reduced response to granulocyte colony-stimulating factor (G-CSF) inhibits the release of immature neutrophils from the bone marrow. Of note, neutrophil responses are similar in preterm infants. Here, the storage pool is decreased, neutrophils are less able to degranulate, have a reduced migratory capacity and are less able to release NETs. Less is known about neutrophil function from older children, but theoretically, impaired functions might increase susceptibility to infections. Targeting these blunted responses may offer a new paradigm for treating CAP, but modifying neutrophil behaviour is challenging; reducing their numbers or inhibiting their function is associated with poor clinical outcomes from infection. Uncontrolled activation and degranulation can cause significant host tissue damage. Any neutrophil-based intervention must walk the tightrope described by Heinrich Rohrer, facilitating necessary phagocytic functions while preventing bystander host damage, and this is a significant challenge which this review will explore.

Simvastatin Improves Neutrophil Function and Clinical Outcomes in Pneumonia. A Pilot Randomized Controlled Clinical Trial.

Rationale: Population studies suggest improved sepsis outcomes with statins, but the results of randomized controlled trials in patients with sepsis and organ dysfunction in critical care settings have broadly been negative. In vitro data suggest that statins modulate age-related neutrophil functions, improving neutrophil responses to infection, but only in older patients and at high doses.Objectives: To determine if high-dose simvastatin improves neutrophil functions and is safe and tolerated in hospitalized older adults with community-acquired pneumonia with sepsis (CAP + S) not admitted to critical care.Methods: We conducted a randomized, double-blind, placebo-controlled pilot study of simvastatin 80 mg or placebo for 7 days for patients with CAP + S aged 55 years or older admitted to a secondary care hospital. The Day 4 primary endpoint was change in neutrophil extracellular trap formation (NETosis). Day 4 secondary endpoints included neutrophil chemotaxis, safety and tolerability, Sequential Organ Failure Assessment score, mortality, readmission, and markers of tissue degradation/inflammation.Measurements and Main Results: Four days of simvastatin adjuvant therapy in patients with CAP + S was associated with improvements in systemic neutrophil function (NETosis and chemotaxis), a reduction in systemic neutrophil elastase burden, and improved Sequential Organ Failure Assessment scores compared with placebo. A post hoc analysis demonstrated that simvastatin therapy was associated with improved hospitalization-free survival compared with placebo. Simvastatin was well tolerated in this elderly and multimorbid patient group with common coprescription of macrolide antibiotics.Conclusions: This pilot study supports high-dose simvastatin as an adjuvant therapy for CAP + S in an older and milder disease cohort than assessed previously. A definitive multicenter study is now warranted in this population to assess the likelihood of benefit and harm.Clinical trial registered with EudraCT (2012-00343-29).

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.

Effect of Co-trimoxazole (Trimethoprim-Sulfamethoxazole) vs Placebo on Death, Lung Transplant, or Hospital Admission in Patients With Moderate and Severe Idiopathic Pulmonary Fibrosis: The EME-TIPAC Randomized Clinical Trial.

Importance: Idiopathic pulmonary fibrosis (IPF) has a poor prognosis and limited treatment options. Patients with IPF have altered lung microbiota, with bacterial burden within the lungs associated with mortality; previous studies have suggested benefit with co-trimoxazole (trimethoprim-sulfamethoxazole). Objective: To determine the efficacy of co-trimoxazole in patients with moderate and severe IPF. Design, Setting, and Participants: Double-blind, placebo-controlled, parallel randomized trial of 342 patients with IPF, breathlessness (Medical Research Council dyspnea scale score >1), and impaired lung function (forced vital capacity ≤75% predicted) conducted in 39 UK specialist interstitial lung disease centers between April 2015 (first patient visit) and April 2019 (last patient follow-up). Interventions: Study participants were randomized to receive 960 mg of oral co-trimoxazole twice daily (n = 170) or matched placebo (n = 172) for between 12 and 42 months. All patients received 5 mg of folic acid orally once daily. Main Outcomes and Measures: The primary outcome was time to death (all causes), lung transplant, or first nonelective hospital admission. There were 15 secondary outcomes, including the individual components of the primary end point respiratory-related events, lung function (forced vital capacity and gas transfer), and patient-reported outcomes (Medical Research Council dyspnea scale, 5-level EuroQol 5-dimension questionnaire, cough severity, Leicester Cough Questionnaire, and King's Brief Interstitial Lung Disease questionnaire scores). Results: Among 342 individuals who were randomized (mean age, 71.3 years; 46 [13%] women), 283 (83%) completed the trial. The median (interquartile range) duration of follow-up was 1.02 (0.35-1.73) years. Events per person-year of follow-up among participants randomized to the co-trimoxazole and placebo groups were 0.45 (84/186) and 0.38 (80/209), respectively, with a hazard ratio of 1.2 ([95% CI, 0.9-1.6]; P = .32). There were no statistically significant differences in other event outcomes, lung function, or patient-reported outcomes. Patients in the co-trimoxazole group had 696 adverse events (nausea [n = 89], diarrhea [n = 52], vomiting [n = 28], and rash [n = 31]) and patients in the placebo group had 640 adverse events (nausea [n = 67], diarrhea [n = 84], vomiting [n = 20], and rash [n = 20]). Conclusions and Relevance: Among patients with moderate or severe IPF, treatment with oral co-trimoxazole did not reduce a composite outcome of time to death, transplant, or nonelective hospitalization compared with placebo. Trial Registration: ISRCTN Identifier: ISRCTN17464641.

Wnt signaling regulates trans-differentiation of stem cell like type 2 alveolar epithelial cells to type 1 epithelial cells.

BACKGROUND: Type 2 alveolar epithelial cells (AT2s) behave as stem cells and show clonal proliferation upon alveolar injury followed by trans-differentiation (TD) into Type 1 alveolar epithelial cells (AT1s). In the present study we identified signaling pathways involved in the physiological AT2-to-AT1 TD process. METHODS: AT2 cells can be isolated from human lungs and cultured in vitro where they undergo TD into AT1s. In the present study we identified signaling pathways involved in the physiological AT2-to-AT1 TD process using Affymetrix microarray, qRT-PCR, fluorescence microscopy, and an in vitro lung aggregate culture. RESULTS: Affymetrix microarray revealed Wnt signaling to play a crucial role in the TD process. Wnt7a was identified as a ligand regulating the AT1 marker, Aquaporin 5 (AQP5). Artificial Neural Network (ANN) analysis of the Affymetrix data exposed ITGAV: Integrin alpha V (ITGAV), thrombospondin 1 (THBS1) and epithelial membrane protein 2 (EMP2) as Wnt signaling targets. CONCLUSIONS: Wnt signaling targets that can serve as potential alveolar epithelial repair targets in future therapies of the gas exchange surface after injury. As ITGAV is significantly increases during TD and is regulated by Wnt signaling, ITGAV might be a potential target to speed up the alveolar healing process.

ResolvinD1 stimulates epithelial wound repair and inhibits TGF-ß-induced EMT whilst reducing fibroproliferation and collagen production.

Acute and chronic inflammatory lung diseases are often associated with epithelial cell injury/loss and fibroproliferative responses. ResolvinD1 (RvD1) is biosynthesized during the resolution phase of inflammatory response and exerts potent anti-inflammatory and promotes resolution of inflammatory lung diseases. The aim of this study was to investigate whether RvD1 exerts protective effects on alveolar epithelial cell function/differentiation and protects against fibroproliferative stimuli. Primary human alveolar type II cells were used to model the effects of RvD1 in vitro upon wound repair, proliferation, apoptosis, transdifferentiation, and epithelial-mesenchymal transition (EMT). Effects of RvD1 upon primary human lung fibroblast proliferation, collagen production, and myofibroblast differentiation were also examined. RvD1 promoted alveolar type II (ATII) cell wound repair and proliferation. RvD1 protected ATII cells against sFas-ligand/TNF-α-induced apoptosis and inhibition on cell proliferation and viability. RvD1 promoted ATII cells transdifferentiation. Moreover, we demonstrate that RvD1 inhibited EMT in response to TGF-ß. Furthermore RvD1 inhibited human lung fibroblast proliferation, collagen production, and myofibroblast differentiation induced by both TGF-ß and bronchoalveolar lavage fluid from acute respiratory distress syndrome (ARDS) patients. The effects of RvD1 were PI3-kinase dependent and mediated via the resolvin receptor. RvD1 seems to promote alveolar epithelial repair by stimulating ATII cells wound repair, proliferation, reducing apoptosis, and inhibiting TGF-ß-induced EMT. While RvD1 reduced fibroproliferation, collagen production, and myofibroblast differentiation. Together, these results suggest a potential new therapeutic strategy for preventing and treating chronic diseases (such as idiopathic pulmonary fibrosis) as well as the fibroproliferative phase of ARDS by targeting RvD1 actions that emphasizes natural resolution signaling pathways.

Pro-inflammatory effects of e-cigarette vapour condensate on human alveolar macrophages.

OBJECTIVE: Vaping may increase the cytotoxic effects of e-cigarette liquid (ECL). We compared the effect of unvaped ECL to e-cigarette vapour condensate (ECVC) on alveolar macrophage (AM) function. METHODS: AMs were treated with ECVC and nicotine-free ECVC (nfECVC). AM viability, apoptosis, necrosis, cytokine, chemokine and protease release, reactive oxygen species (ROS) release and bacterial phagocytosis were assessed. RESULTS: Macrophage culture with ECL or ECVC resulted in a dose-dependent reduction in cell viability. ECVC was cytotoxic at lower concentrations than ECL and resulted in increased apoptosis and necrosis. nfECVC resulted in less cytotoxicity and apoptosis. Exposure of AMs to a sub-lethal 0.5% ECVC/nfECVC increased ROS production approximately 50-fold and significantly inhibited phagocytosis. Pan and class one isoform phosphoinositide 3 kinase inhibitors partially inhibited the effects of ECVC/nfECVC on macrophage viability and apoptosis. Secretion of interleukin 6, tumour necrosis factor α, CXCL-8, monocyte chemoattractant protein 1 and matrix metalloproteinase 9 was significantly increased following ECVC challenge. Treatment with the anti-oxidant N-acetyl-cysteine (NAC) ameliorated the cytotoxic effects of ECVC/nfECVC to levels not significantly different from baseline and restored phagocytic function. CONCLUSIONS: ECVC is significantly more toxic to AMs than non-vaped ECL. Excessive production of ROS, inflammatory cytokines and chemokines induced by e-cigarette vapour may induce an inflammatory state in AMs within the lung that is partly dependent on nicotine. Inhibition of phagocytosis also suggests users may suffer from impaired bacterial clearance. While further research is needed to fully understand the effects of e-cigarette exposure in humans in vivo, we caution against the widely held opinion that e-cigarettes are safe.

Vitamin D to Prevent Lung Injury Following Esophagectomy-A Randomized, Placebo-Controlled Trial.

OBJECTIVES: Observational studies suggest an association between vitamin D deficiency and adverse outcomes of critical illness and identify it as a potential risk factor for the development of lung injury. To determine whether preoperative administration of oral high-dose cholecalciferol ameliorates early acute lung injury postoperatively in adults undergoing elective esophagectomy. DESIGN: A double-blind, randomized, placebo-controlled trial. SETTING: Three large U.K. university hospitals. PATIENTS: Seventy-nine adult patients undergoing elective esophagectomy were randomized. INTERVENTIONS: A single oral preoperative (3-14 d) dose of 7.5 mg (300,000 IU; 15 mL) cholecalciferol or matched placebo. MEASUREMENTS AND MAIN RESULTS: Primary outcome was change in extravascular lung water index at the end of esophagectomy. Secondary outcomes included PaO2:FIO2 ratio, development of lung injury, ventilator and organ-failure free days, 28 and 90 day survival, safety of cholecalciferol supplementation, plasma vitamin D status (25(OH)D, 1,25(OH)2D, and vitamin D-binding protein), pulmonary vascular permeability index, and extravascular lung water index day 1 postoperatively. An exploratory study measured biomarkers of alveolar-capillary inflammation and injury. Forty patients were randomized to cholecalciferol and 39 to placebo. There was no significant change in extravascular lung water index at the end of the operation between treatment groups (placebo median 1.0 [interquartile range, 0.4-1.8] vs cholecalciferol median 0.4 mL/kg [interquartile range, 0.4-1.2 mL/kg]; p = 0.059). Median pulmonary vascular permeability index values were significantly lower in the cholecalciferol treatment group (placebo 0.4 [interquartile range, 0-0.7] vs cholecalciferol 0.1 [interquartile range, -0.15 to -0.35]; p = 0.027). Cholecalciferol treatment effectively increased 25(OH)D concentrations, but surgery resulted in a decrease in 25(OH)D concentrations at day 3 in both arms. There was no difference in clinical outcomes. CONCLUSIONS: High-dose preoperative treatment with oral cholecalciferol was effective at increasing 25(OH)D concentrations and reduced changes in postoperative pulmonary vascular permeability index, but not extravascular lung water index.

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

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

Sepsis Induces a Dysregulated Neutrophil Phenotype That Is Associated with Increased Mortality.

BACKGROUND: Neutrophil dysfunction in sepsis has been implicated in the pathogenesis of multiorgan failure; however, the role of neutrophil extracellular traps (NETs) remains uncertain. We aimed to determine the sequential changes in ex vivo NETosis and its relationship with mortality in patients with sepsis and severe sepsis. METHODS: This was a prospective observational cohort study enrolling 21 healthy age-matched controls and 39 sepsis and 60 severe sepsis patients from acute admissions to two UK hospitals. Patients had sequential bloods for the ex vivo assessment of NETosis in response to phorbol-myristate acetate (PMA) using a fluorometric technique and chemotaxis using time-lapse video microscopy. Continuous data was tested for normality, with appropriate parametric and nonparametric tests, whilst categorical data was analysed using a chi-squared test. Correlations were performed using Spearman's rho. RESULTS: Ex vivo NETosis was reduced in patients with severe sepsis, compared to patients with sepsis and controls (p = 0.002). PMA NETosis from patients with septic shock was reduced further (p < 0.001) compared to controls. The degree of metabolic acidosis correlated with reduced NETosis (p < 0.001), and this was replicated when neutrophils from healthy donors were incubated in acidotic media. Reduced NETosis at baseline was associated with an increased 30-day (p = 0.002) and 90-day mortality (p = 0.014) in sepsis patients. These findings were accompanied by defects in neutrophil migration and delayed apoptosis. Resolution of sepsis was not associated with the return to baseline levels of NETosis or migration. CONCLUSIONS: Sepsis induces significant changes in neutrophil function with the degree of dysfunction corresponding to the severity of the septic insult which persists beyond physiological recovery from sepsis. The changes induced lead to the failure to effectively contain and eliminate the invading pathogens and contribute to sepsis-induced immunosuppression. For the first time, we demonstrate that reduced ex vivo NETosis is associated with poorer outcomes from sepsis.

Platelet CLEC-2 protects against lung injury via effects of its ligand podoplanin on inflammatory alveolar macrophages in the mouse.

There is no therapeutic intervention proven to prevent acute respiratory distress syndrome (ARDS). Novel mechanistic insights into the pathophysiology of ARDS are therefore required. Platelets are implicated in regulating many of the pathogenic processes that occur during ARDS; however, the mechanisms remain elusive. The platelet receptor CLEC-2 has been shown to regulate vascular integrity at sites of acute inflammation. Therefore the purpose of this study was to establish the role of CLEC-2 and its ligand podoplanin in a mouse model of ARDS. Platelet-specific CLEC-2-deficient, as well as alveolar epithelial type I cell (AECI)-specific or hematopoietic-specific podoplanin deficient, mice were established using cre-loxP strategies. Combining these with intratracheal (IT) instillations of lipopolysaccharide (LPS), we demonstrate that arterial oxygen saturation decline in response to IT-LPS in platelet-specific CLEC-2-deficient mice is significantly augmented. An increase in bronchoalveolar lavage (BAL) neutrophils and protein was also observed 48 h post-IT-LPS, with significant increases in pro-inflammatory chemokines detected in BAL of platelet-specific CLEC-2-deficient animals. Deletion of podoplanin from hematopoietic cells but not AECIs also reduces lung function and increases pro-inflammatory chemokine expression following IT-LPS. Furthermore, we demonstrate that following IT-LPS, platelets are present in BAL in aggregates with neutrophils, which allows for CLEC-2 interaction with podoplanin expressed on BAL inflammatory alveolar macrophages. Taken together, these data suggest that the platelet CLEC-2-podoplanin signaling axis regulates the severity of lung inflammation in mice and is a possible novel target for therapeutic intervention in patients at risk of developing ARDS.

Evidence for chemokine synergy during neutrophil migration in ARDS.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterised by pulmonary oedema, respiratory failure and severe inflammation. ARDS is further characterised by the recruitment of neutrophils into the lung interstitium and alveolar space. OBJECTIVES: The factors that regulate neutrophil infiltration into the inflamed lung and our understanding of the pathomechanisms in ARDS remain incomplete. This study aimed at determining the role of the chemokine (C-C motif) ligand (CCL)2 and CCL7 in ARDS. METHODS: CCL2 and CCL7 protein levels were measured in bronchoalveolar lavage (BAL) fluid obtained from lipopolysaccharide(LPS)-challenged human volunteers and two separate cohorts of patients with ARDS. Neutrophil chemotaxis to ARDS BAL fluid was evaluated and the contribution of each was assessed and compared with chemokine (C-X-C motif) ligand 8 (CXCL8). Chemokine receptor expression on neutrophils from blood or BAL fluid of patients with ARDS was analysed by flow cytometry. RESULTS: CCL2 and CCL7 were significantly elevated in BAL fluid recovered from LPS-challenged volunteers and patients with ARDS. BAL fluid from patients with ARDS was highly chemotactic for human neutrophils and neutralising either CCL2 or CCL7 attenuated the neutrophil chemotactic response. Moreover, CCL2 and CCL7 synergised with CXCL8 to promote neutrophil migration. Furthermore, neutrophils isolated from the blood or BAL fluid differentially regulated the cell surface expression of chemokine (C-X-C motif) receptor 1 and C-C chemokine receptor type 2 during ARDS. CONCLUSION: This study highlights important inflammatory chemokines involved in regulating neutrophil migration, which may have potential value as therapeutic targets for the treatment of ARDS.