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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.

Glucocorticoids potently inhibit expression of many inflammatory mediators, and have been very widely used to treat both acute and chronic inflammatory diseases for more than seventy years. However, they can have several unwanted effects, amongst which immunosuppression is one of the most common. Here we investigated effects of the synthetic glucocorticoid dexamethasone on the responses of primary mouse bone marrow-derived macrophages to the pro-inflammatory agonist lipopolysaccharide (LPS). At the mRNA level, dexamethasone inhibited the LPS-induced expression of more than 100 genes that are involved in cell-intrinsic defence against viral pathogens. Expression of most of the corresponding proteins was also reduced by dexamethasone. This antiviral disarmament occurred at two distinct levels. First, dexamethasone strongly and dose-dependently inhibited the expression of the type I interferon IFNβ by LPS-activated macrophages. IFNβ mediates an autocrine positive feedback loop in LPS-treated macrophages, promoting the expression of antiviral genes and other interferon-stimulated genes. Hence reduction of IFNβ expression contributes to impaired expression of antiviral genes. Dexamethasone also acted downstream of IFNβ to inhibit expression of a subset of interferon-regulated genes. We tested a number of hypotheses based on previous publications, but found that no single mechanism could account for more than a small fraction of the broad suppressive impact of dexamethasone on macrophage type I interferon signaling, underlining the complexity of this pathway. Preliminary experiments indicated that dexamethasone exerted similar inhibitory effects on primary human monocyte-derived or alveolar macrophages.

Recent research on extracellular vesicles (EVs) has provided new insights into pathogenesis and potential therapeutic options for acute respiratory distress syndrome (ARDS). EVs are membrane-bound anuclear structures that carry important intercellular communication mechanisms, allowing targeted transfer of diverse biologic cargo, including protein, mRNA, and microRNA, among several different cell types. In this review, we discuss the important role EVs play in both inducing and attenuating inflammatory lung injury in ARDS as well as in sepsis, the most important clinical cause of ARDS. We discuss the translational challenges that need to be overcome before EVs can also be used as prognostic biomarkers in patients with ARDS and sepsis. We also consider how EVs may provide a platform for novel therapeutics in ARDS.

Recent studies have indicated that extracellular vesicles (EV) may play a role in the pathogenesis of Acute Respiratory Distress Syndrome (ARDS). EV have been identified as potential biomarkers of disease severity and prognosis in other pulmonary diseases. We sought to characterize the EV phenotype within ARDS patient broncho-alveolar lavage fluid (BAL), and to determine whether BAL EV could be utilized as a potential biomarker in ARDS. BAL was collected from sepsis patients with and without ARDS, and from esophagectomy patients post-operatively (of whom a subset later developed ARDS during hospital admission). BAL EV were characterized with regards to size, number and cell of origin. Sepsis patients with ARDS had significantly higher numbers of CD14+/CD81+ monocyte-derived BAL EV than sepsis patients 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 to 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 ICU admission (p=0.027). Thus, CD14+/CD81+ BAL EV 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 EV to ARDS pathogenesis.

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

Acute respiratory distress syndrome (ARDS) is an inflammatory disorder of the lungs, with sepsis as the predominant aetiology. Despite advances in ventilation strategies, mortality for moderate to severe ARDS remains at 40–46% [1]. ARDS is associated with neutrophil influx into alveoli. Persistently high neutrophil and low alveolar macrophage (AM) numbers in bronchoalveolar lavage (BAL) fluid are associated with greater mortality [2]. While the inflammatory alveolar environment of early ARDS initially delays apoptosis, these neutrophils ultimately undergo apoptosis within alveoli [3]. Efficient efferocytosis of apoptotic neutrophils by AMs is critical for resolution of inflammation [3]. Apoptotic neutrophils may accumulate in ARDS due to defective AM efferocytosis and/or overwhelmed efferocytosis capacity, then undergo secondary necrosis, releasing inflammatory mediators into the alveolar space [4]. This may contribute to the prolonged inflammation observed in ARDS. No study has previously assessed AM efferocytosis in ARDS; however, monocyte-derived macrophages (MDMs) from ARDS patients do have impaired efferocytosis [5]. We investigated whether ARDS patients have impaired AM efferocytosis and increased alveolar neutrophil apoptosis., ARDS patients have decreased alveolar macrophage efferocytosis, which is associated with increased alveolar inflammation, and may contribute to worse clinical outcomes, including mortality. Upregulation of efferocytosis may offer a therapeutic strategy. https://bit.ly/2Q7REdM

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).

Background Internationally, researchers have called for evidence to support tackling health inequalities during the COVID19 pandemic. UK Office for National Statistics data suggests that patients in regions of most deprived overall Index of Multiple Deprivation Score (IMDS) are twice as likely to die of COVID19 than other causes. The Intensive Care National Audit and Research Centre (ICNARC) report that Black, Asian and Minority Ethnic (BAME) patients account for 34% of critically ill COVID19 patients nationally despite constituting 14% of the population. This paper is the first to explore the roles of social determinants of health, including specific IMDS sub-indices with indicators for household overcrowding deprivation (Barriers to Housing and Services subindex (BHS)), indoor housing quality deprivation and outdoor air pollution deprivation (Living Environment subindex (LE)) as modulators of presentation, Intensive Care Unit(ITU) admission and outcomes among COVID19 patients of all ethnicities. Methods An in-depth retrospective cohort study of 408 hospitalised COVID19 patients admitted to Queen Elizabeth Hospital, Birmingham was conducted. Quantitative data analyses including two-step cluster analyses were applied. Results Patients admitted from highest LE deprivation sub-indices were at increased risk of presenting with multi-lobar pneumonia and, in turn, ITU admission. Patients admitted from highest BHS deprivation sub-indices were at increased risk of ITU admission. BAME patients were more likely, than white patients, to present with multi-lobar pneumonia, be admitted to ITU and be admitted from highest BHS and LE deprivation indices. Comorbidities and frailty significantly increased the risk of death among COVID19 patients irrespective of deprivation. Conclusions Air pollution and housing quality deprivation are potential modulators of presentation with multi-lobar pneumonia. Household overcrowding deprivation and presentation with multi-lobar pneumonia are potential modulators of ITU admission. Patents of BAME ethnicity are more likely to be admitted from regions of highest air pollution, housing quality and household overcrowding deprivation; this is likely to contribute an explanation towards the higher ITU admissions reported among COVID19 BAME patients. Consideration of Charlson Comorbidity and Clinical Frailty Scores on admission supports clinicians in stratifying high risk patients. These findings have urgent implications for supporting front line clinical decisions, disseminating practical advice around applying social distancing messages at the household level and informing wider pandemic strategy. This study has been cited by several national and international public bodies including Public Health England and UK Parliament as evidence to support the COVID19 strategic response.

International audience; Few patients with bacteremia from a nonpulmonary source develop acute respiratory distress syndrome (ARDS). However, the mechanisms that protect the lung from injury in bacteremia have not been identified. We simulated bacteremia by adding Streptococcus pneumoniae to the perfusate of the ex vivo perfused human lung model. In contrast to a pneumonia model in which bacteria were instilled into the distal air spaces of one lobe, injection of high doses of S. pneumoniae into the perfusate was not associated with alveolar epithelial injury as demonstrated by low protein permeability of the alveolar epithelium, intact alveolar fluid clearance, and the absence of alveolar edema. Unexpectedly, the ex vivo human lung rapidly cleared large quantities of S. pneumoniae even though the perfusate had very few intravascular phagocytes and lacked immunoglobulins or complement. The bacteria were cleared in part by the small number of neutrophils in the perfusate, alveolar macrophages in the airspaces, and probably by interstitial pathways. Together, these findings identify one mechanism by which the lung and the alveolar epithelium are protected from injury in bacteremia.

IntroductionAcute respiratory distress syndrome (ARDS) is the major cause of mortality in patients with SARS-CoV-2 pneumonia. It appears that development of ‘cytokine storm’ in patients with SARS-CoV-2 pneumonia precipitates progression to ARDS. However, severity scores on admission do not predict severity or mortality in patients with SARS-CoV-2 pneumonia. Our objective was to determine whether patients with SARS-CoV-2 ARDS are clinically distinct, therefore requiring alternative management strategies, compared with other patients with ARDS. We report a single-centre retrospective study comparing the characteristics and outcomes of patients with ARDS with and without SARS-CoV-2.MethodsTwo intensive care unit (ICU) cohorts of patients at the Queen Elizabeth Hospital Birmingham were analysed: SARS-CoV-2 patients admitted between 11 March and 21 April 2020 and all patients with community-acquired pneumonia (CAP) from bacterial or viral infection who developed ARDS between 1 January 2017 and 1 November 2019. All data were routinely collected on the hospital’s electronic patient records.ResultsA greater proportion of SARS-CoV-2 patients were from an Asian ethnic group (p=0.002). SARS-CoV-2 patients had lower circulating leucocytes, neutrophils and monocytes (pDiscussionThe clinical syndromes and respiratory mechanics of SARS-CoV-2 and CAP-ARDS are broadly similar. However, SARS-CoV-2 patients initially have a lower requirement for vasopressor support, fewer circulating leukocytes and require prolonged ventilation support. Further studies are required to determine whether the dysregulated inflammation observed in SARS-CoV-2 ARDS may contribute to the increased duration of respiratory failure.

ARDS is the major cause of mortality in patients with SARS-CoV-2 pneumonia. We report a single-centre study comparing the characteristics of ARDS patients with and without SARS-CoV-2. A greater proportion of SARS-CoV-2 patients were from an Asian ethnic group (p=0.002). SARS-CoV-2 patients had lower circulating leukocytes, neutrophils and monocytes (p

Few patients with bacteremia from a nonpulmonary source develop acute respiratory distress syndrome (ARDS). However, the mechanisms that protect the lung from injury in bacteremia have not been identified. We simulated bacteremia by adding Streptococcus pneumoniae to the perfusate of the ex vivo perfused human lung model. In contrast to a pneumonia model in which bacteria were instilled into the distal air spaces of one lobe, injection of high doses of S. pneumoniae into the perfusate was not associated with alveolar epithelial injury as demonstrated by low protein permeability of the alveolar epithelium, intact alveolar fluid clearance, and the absence of alveolar edema. Unexpectedly, the ex vivo human lung rapidly cleared large quantities of S. pneumoniae even though the perfusate had very few intravascular phagocytes and lacked immunoglobulins or complement. The bacteria were cleared in part by the small number of neutrophils in the perfusate, alveolar macrophages in the airspaces, and probably by interstitial pathways. Together, these findings identify one mechanism by which the lung and the alveolar epithelium are protected from injury in bacteremia.

Supplemental Digital Content is available in the text., 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.

Acute respiratory distress syndrome (ARDS) is a major cause of acute respiratory failure that develops following several clinical disorders, including pneumonia, sepsis, aspiration and major trauma. Despite numerous clinical trials, there is still no effective pharmacotherapy available for ARDS patients. However, recent research on extracellular vesicles provides new insights into pathogenesis, prognosis, and potential therapeutic options for ARDS. Extracellular vesicles are membrane-bound anuclear structures which constitute a recently recognized and important intercellular communication mechanism, allowing targeted transfer of diverse biologic cargo between different cell types. There is new evidence that extracellular vesicles play an important role in the pathogenesis of ARDS and also potentially a protective role. In this chapter, we highlight recent translational and clinical studies that have advanced our understanding of the critical role extracellular vesicles play in both inducing and attenuating inflammatory lung injury in ARDS. This review also considers the wide range of potential clinical applications for extracellular vesicles, ranging from use as biomarkers of lung injury to therapeutic agents for ARDS. Extracellular vesicles represent a new frontier for research in ARDS.

Background: Human bone marrow mesenchymal stem cell (MSC) administration reduces inflammation in pre-clinical models of lung injury, however clinical efficacy in patients with acute respiratory distress syndrome (ARDS) has not been shown. Upregulation of the glucocorticoid activating enzyme, 11β hydroxysteroid dehydrogenase type 1 (HSD-1) within the alveolar space elevates local anti-inflammatory cortisol levels, and promotes alveolar macrophage efferocytosis. Administration of HSD-1 transgenic MSCs (tMSCs) that overexpress HSD-1 may enhance local cortisol activation. This combined cellular and gene therapy may be more effective at reducing inflammation in ARDS than cellular therapy alone. Methods: Molecular cloning was used to create a recombinant lentiviral vector containing the HSD-1 and green fluorescent protein (GFP) transgenes. MSCs were transfected using this lentivirus, then transfection efficiency was assessed using flow-cytometry. HSD-1 transgene expression was assessed using immunofluorescence and western blotting. Thin layer chromatography was used to assess HSD-1 function in tMSCs. Bi-lineage differentiation and flow-cytometry were used to determine whether tMSCs maintained a stem cell phenotype. Results: A recombinant lentiviral vector was created containing the HSD-1 and GFP transgenes under the control of a tetracycline promoter. MSCs were successfully transfected, with a transfection efficiency of 91.1%. HSD-1 transgene expression was confirmed by immunofluorescence and western blot. Functional HSD-1 activity was evident within tMSCs, with predominant reductase cortisol activation, following treatment with the transcriptional activator doxycycline. HSD-1 reductase activity was maintained for 72 hours after doxycycline was removed from tMSCs. HSD-1 tMSCs maintained the capacity for osteogenic and adipogenic differentiation, and maintained expression of MSC surface markers. Conclusions: We successfully transduced tMSCs to express the HSD-1 transgene that functions predominantly as a reductase. Administration of HSD-1 tMSCs to in vivo models of ARDS may attenuate inflammation through activation of cortisol in the alveolar space.

Introduction The role of alveolar macrophages (AMs) in ARDS pathogenesis is unclear. Our previous work with ARDS patients has shown that cortisol:cortisone ratios are significantly lower in bronchoalveolar lavage fluid (BALF) compared to plasma, indicating dysregulated alveolar steroid metabolism. AMs normally express 11β hydroxysteroid dehydrogenase type 1 (HSD-1) which converts cortisone to cortisol. We investigated whether AM HSD-1 activity is decreased in ARDS and the relationship with mortality and AM function. Methods AMs were isolated from the BALF of ventilated ICU patients with ARDS (n=24) or Sepsis (n=12, control group at risk of ARDS). AMs were used in efferocytosis and phagocytosis assays; using labelled apoptotic neutrophils or pHrodo particles (E. Coli and S. Aureus) respectively. Thin layer chromatography assays measured AM HSD-1 reductase activity. BAL neutrophils were assessed for apoptosis and viability using flow cytometry. Results AMs had significantly lower HSD-1 reductase activity in ARDS compared to sepsis (0.102 vs 0.966 pM/hr, p=0.038). ARDS patients who died during admission had significantly lower AM HSD-1 reductase activity compared to survivors (0.0585 vs 0.566 pM/hr, p=0.0189, figure 1). AM efferocytic index was significantly impaired in ARDS compared to sepsis (4.95% vs 24.3%, p=0.0001). Direct correlation is seen between AM HSD-1 activity and efferocytic index, across both patient groups (R2=0.6249, p=0.0195). A greater proportion of BAL neutrophils were apoptotic in ARDS compared to Sepsis (39.2% vs 19%, p=0.04). No significant difference in AM phagocytic index was seen between ARDS and Sepsis. Conclusions ARDS onset is associated with decreased AM HSD-1 reductase activity. Cortisol stimulates efferocytosis,1 therefore reduced AM HSD-1 reductase activity may cause the impaired efferocytosis we observed. This impaired efferocytosis likely contributes to the alveolar accumulation of apoptotic neutrophils we observed. These persistent apoptotic neutrophils likely undergo secondary necrosis, causing the prolonged inflammation seen in ARDS. This may explain why lower AM HSD-1 reductase activity is associated with greater mortality in ARDS. Therapeutic strategies to upregulate alveolar HSD-1 activity may restore AM efferocytosis function, decrease inflammation, and reduce mortality in ARDS. Work to modify HSD-1 activity in vivo is ongoing using transgenic mesenchymal stem cells which overexpress HSD-1. Reference 1. PMID: 22615206.

Dysregulated alveolar inflammation & neutrophil accumulation are characteristic of ARDS, however the role of alveolar macrophages (AMs) in pathogenesis is unclear. We investigated if AM efferocytic & phagocytic functions are impaired in ARDS. Methods: AMs were isolated from the bronchoalveolar lavage fluid of ventilated ICU patients with ARDS (n=5) or Sepsis (n=8, control group at risk of ARDS). AMs were used in efferocytosis & phagocytosis assays using labelled apoptotic neutrophils or pHrodo particles (E. Coli & S. Aureus) respectively. Thin layer chromatography assays determined AM conversion of cortisone to cortisol, thus measuring 11s hydroxysteroid dehydrogenase type 1 (HSD1) reductase activity in AMs. Results: ARDS AM efferocytic index was significantly impaired compared to sepsis AMs (Figure 1, medians 6.1% vs 21.0%, p=0.006). No significant difference in AM phagocytic index between ARDS & Sepsis patients was seen. ARDS AMs had significantly lower HSD1 reductase activity than sepsis AMs (medians 102 vs 853 fM/hr, p=0.02). Conclusions: Impaired AM efferocytosis in ARDS likely contributes to apoptotic neutrophil accumulation & prolonged inflammation. Cortisol stimulates efferocytosis1, therefore reduced HSD1 reductase activity in ARDS AMs may cause impaired efferocytosis. Upregulation of alveolar HSD1 activity may restore AM efferocytic function & aid ARDS resolution. References: 1) PMID: 22615206

Dysregulated alveolar inflammation is characteristic of ARDS, however the role of alveolar macrophages (AMs) in pathogenesis is unclear. We investigated whether AM efferocytic function is impaired in ARDS, and if this correlates with alveolar accumulation of apoptotic neutrophils. Methods: Bronchoalveolar lavage (BAL) was performed on ventilated ICU patients with ARDS (n=8) or Sepsis (n=10, control group at risk of ARDS). BAL neutrophils were assessed for apoptosis & viability using flow cytometry. BAL AMs were isolated for use in efferocytosis and phagocytosis assays, using labelled apoptotic neutrophils or pHrodo bioparticles (E. Coli & S. Aureus) respectively. Results: A greater proportion of BAL neutrophils were apoptotic in ARDS compared to Sepsis patients (39.2% vs 19%, p=0.04). AM efferocytic index was significantly impaired in ARDS compared to Sepsis patients (Fig 1, median 4.9% vs 23.5%, p=0.001). No significant difference in AM phagocytic index was seen between ARDS & Sepsis patients. Conclusions: The onset of ARDS is associated with impaired AM efferocytosis, which contributes to the accumulation of apoptotic neutrophils within alveoli. These persistent apoptotic neutrophils likely undergo secondary necrosis, causing the prolonged inflammation seen in ARDS. Therapeutic strategies to enhance AM efferocytosis may thereby aid resolution of inflammation in ARDS.

The process of vaping may increase the cytotoxic effects of e-cigarette liquid (ECL) and increase the negative effects on alveolar macrophages yet many studies have used only unvaped fluid. We sought to examine the potential cytotoxic and functional effects of vaped and unvaped e-cigarette fluid on alveolar macrophages (AM). AMs were treated with e-cigarette vapour condensate (ECVC)/ECL +/- nicotine. Viability, apoptosis/necrosis, cytokine release, phagocytosis and ROS generation were then assessed. AM culture with ECL/ECVC resulted in dose dependent reduction in cell viability. ECVC was cytotoxic at lower concentrations than ECL (0.8% vs 5%, n=6). 24hr culture with 0.8% ECVC resulted in a 25% increase in apoptosis and necrosis (p ECVC is significantly more toxic to AMs than non-vaped ECL. Elevated inflammatory cytokine production and ROS suggests ECIG users may be inducing a chronic inflammatory state in vapour exposed AMs. Impaired phagocytic ability following ECVC exposure suggests AMs in the lung of ECIG users may show impaired bacterial clearance. Taken together this suggests ECIG users may experience more frequent and more severe bacterial infections due to impaired innate immune response by AMs.

Epithelial–mesenchymal transition (EMT) is a process when epithelial cells gradually transform into mesenchymal-like cells losing their epithelial functionality and characteristics. EMT is thought to be involved in the pathogenesis of numerous lung diseases ranging from developmental disorders, fibrotic tissue remodelling to lung cancer. The most important question—namely what is the importance and contribution of EMT in the pathogenesis of several chronic lung conditions (asthma, COPD, bronchiolitis obliterans syndrome and lung fibrosis)—is currently intensely debated. This review gives a brief insight into the mechanism and assessment methods of EMT in various pulmonary diseases and summarises the recent literature highlighting the controversial experimental data and conclusions.

Bacterial infections after lung transplantation cause airway epithelial injury and are associated with an increased risk of developing bronchiolitis obliterans syndrome. The damaged epithelium is a source of alarmins that activate the innate immune system, yet their ability to activate fibroblasts in the development of bronchiolitis obliterans syndrome has not been evaluated. Two epithelial alarmins were measured longitudinally in bronchoalveolar lavages from lung transplant recipients who developed bronchiolitis obliterans syndrome and were compared to stable controls. In addition, conditioned media from human airway epithelial cells infected with Pseudomonas aeruginosa was applied to lung fibroblasts and inflammatory responses were determined. Interleukin‐1 alpha (IL‐1α) was increased in bronchoalveolar lavage of lung transplant recipients growing P. aeruginosa (11.5 [5.4–21.8] vs. 2.8 [0.9–9.4] pg/mL, p, This study demonstrates that a Pseudomonas aeruginosa challenge of lung epithelial cells causes the release of IL‐1α and the induction of an inflammatory phenotype in lung fibroblasts, and provides evidence of an association between infection with P. aeruginosa, IL‐1α levels in bronchoalveolar lavage fluid, and the development of bronchiolitis obliterans syndrome following lung transplantation.

A novel actinomycete, strain N1286T, isolated from a lung transplant patient with a pulmonary infection, was provisionally assigned to the genus Nocardia . The strain had chemotaxonomic and morphological properties typical of members of the genus Nocardia and formed a distinct phyletic line in the Nocardia 16S rRNA gene tree. Isolate N1286T was most closely related to Nocardia farcinica DSM 43665T (99.8 % gene sequence similarity) but could be distinguished from the latter by the low level of DNA–DNA relatedness. These strains were also distinguishable on the basis of a broad range of phenotypic properties. It is concluded that strain N1286T represents a novel species of the genus Nocardia for which the name Nocardia kroppenstedtii sp. nov. is proposed. The type strain is N1286T ( = DSM 45810T = NCTC 13617T).

Idiopathic pulmonary fibrosis (IPF), sarcoidosis and systemic sclerosis can each cause diffuse parenchymal lung disease involving alveolar epithelial abnormalities, and manifest as interstitial lung fibrosis. Systemic sclerosis is an autoimmune connective tissue disease and sarcoidosis is a multisystem granulomatous disease; the development of lung fibrosis in both conditions generally indicates a poor prognosis. IPF is a progressive disease which is thought to occur after recurrent injury to the alveolar epithelium followed by dysregulation of cellular homoeostasis, abnormal repair and excessive deposition of extracellular matrix, ultimately leading to loss of lung function.1 Mortality 3–5 years after diagnosis of IPF is 50%, and treatment options are currently limited. Seibold et al 2 have previously shown that a variant single-nucleotide polymorphism (SNP) in the promoter region of an airway mucin gene ( MUC5B ) is associated with greatly increased production of MUC5B, and with pulmonary fibrosis. This suggested that dysregulated MUC5B expression in the lung may be key to the pathogenesis of pulmonary fibrosis. One hypothesis is that an excess of MUC5B causes bronchiolar plugging and impairs mucosal host defence, reducing clearance …

Introduction and objectives Mesenchymal stem cells (MSC) have well-established anti-inflammatory properties and could potentially be used therapeutically in Acute Respiratory Distress Syndrome (ARDS).1 MSC conditioned medium (MSC-CM) has been found to reproduce the beneficial effects of MSCs.2 However, the impact of expansion on MSC secretome remains elusive. In the present study we assessed the expression of four potent MSC paracrine factors after prolonged in vitro culture, while investigating the effects of passaging on the in vitro properties of MSCs. Methods Human Bone marrow-derived MSCs were expanded in vitro in αMEM with 16% FBS. Conditioned medium was collected from each passage and stored at −40°C. Scratch assays were undertaken using A549 cells treated with MSC-CM and control media. Proliferation of A549 cells was assessed via BrdU assay. Gene expression of expanded MSCs was assessed by RT-qPCR. VEGF and Angiopoietin in the MSC-CM were quantified by ELISA. Results RT-qPCR revealed that MSCs express the anti-inflammatory genes PTGES2, FGF7 and ANGPT1. The expression of these genes doubled after 8 days in culture and subsequently decreased. (P Conclusion We conclude that MSCs of early passages are more potent angiogenic inducers, while promoting wound healing in vitro. Thus, we provide important insights into the changes occurring after prolonged in vitro expansion, underlining the importance of using low passage MSCs in clinical trials for ARDS. In agreement with published data, we also found that MSCs do not induce cellular proliferation in the absence of stimulation. References Antunes MA, et al. Mesenchymal stem cell trials for pulmonary diseases. J Cell Biochem 2014;115:1023–1032. Kordelas L, et al. MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease. LeukaemiaLeukemia 2014;28:970–973.

Bronchiolitis obliterans syndrome is characterized by fibrotic obliteration of small airways which severely impairs graft function and survival after lung transplantation. Bronchial epithelial cells from the transplanted lung can undergo epithelial to mesenchymal transition and this can be accentuated by activated macrophages. Macrophages demonstrate significant plasticity and change phenotype in response to their microenvironment. In this study we aimed to identify secretory products from macrophages that might be therapeutic targets for limiting the inflammatory accentuation of epithelial to mesenchymal transition in bronchiolitis obliterans syndrome. TNFα, IL-1β and IL-8 are elevated in bronchoalveolar lavage from lung transplant patients prior to diagnosis of bronchiolitis obliterans syndrome. Classically activated macrophages secrete more TNFα and IL-1β than alternatively activated macrophages and dramatically accentuate TGF-β1-driven epithelial to mesenchymal transition in bronchial epithelial cells isolated from lung transplant patients. Blocking TNFα, but not IL-1β, inhibits the accentuation of epithelial to mesenchymal transition. In a pilot unblinded therapeutic intervention in five patients with progressive bronchiolitis obliterans syndrome, anti-TNFα treatment improved forced expiratory volume in 1 second and 6-min walk distances in four patients. Our data identify TNFα as a potential new therapeutic target in bronchiolitis obliterans syndrome deserving of a randomized placebo controlled clinical trial.

Lung transplantation is a well-established treatment option for selected patients with end-stage lung disease, leading to improved survival and improved quality of life. The last 20 years have seen a steady growth in number of lung transplantation procedures performed worldwide. The increase in clinical activity has been associated with tremendous progress in the understanding of cellular and molecular processes that limit both short- and long-term outcomes. This review gives a comprehensive overview of the current status of lung transplantation for the referring physician. It demonstrates that careful selection of potential recipients, optimisation of their condition prior to transplant, use of carefully assessed donor organs, excellent surgery and meticulous long-term follow-up are all essential ingredients in determining a successful outcome.

Introduction Survival after lung transplantation is limited to a median of 5-year due to development of bronchiolitis obliterans syndrome (BOS). BOS is the clinical manifestation of chronic allograft dysfunction, characterised by inflammation and fibrosis of small/medium-sized airways leading to airflow obstruction. Numerous insults to the transplanted lungs have been associated with BOS development. Alarmins are cell derived danger signals released from damaged tissue which activate innate and adaptive immune responses. We hypothesised that the release of alarmins into the airway compartment after lung transplantation may contribute to BOS pathogenesis. Methods A retrospective longitudinal study of 52 lung transplant recipients from 2005 to present was performed (26 recipients developed BOS; 26 remained free from BOS). All recipients had lung function and bronchoalveolar lavage (BAL) performed at 1, 3, 6 and 12 months post transplant. Further samples were taken if the diagnostic criteria for BOS were fulfilled. A total of 214 BAL samples were analysed by ELISA for the alarmins Interleukin-1a (IL-1a) and High Motility Group-Box1 (HMG-B1). Data were analysed using Mann–Whitney tests. Results Both BOS and non-BOS recipients with culture positive BAL samples had significantly higher concentrations of IL-1a and HMG-B1 than culture negative samples. Due to the confounding effect of infection, our analysis excluded 90 BAL samples with positive cultures. Concentrations of IL-1a were significantly higher in culture negative BAL from BOS patients (median 2.411, range [AJF1] 0.073–19.078 pg/ml) than from Non-BOS patients (median 1.424, range [AJF2] 1.159–17.41 pg/ml; p=0.001). No significant difference in HMG-B1 concentrations between the two groups was observed (BOS median 58.906, range 0–197.5; Non-BOS median 76.25, range 0–211.563 ng/ml; p=0.2378). Longitudinal measurements of IL-1a in BOS patients showed significantly higher levels 3 months before or after BOS diagnosis (median 3.935, range 1.122–13.544 pg/ml), compared to >3 months before BOS diagnosis (median 2.015, range 0.073–14.669 pg/ml; p=0.0153). There was no such difference in HMG-B1 concentrations (p=0.9164). Conclusions An increase in the alarmin IL-1a, but not HMG-B1, is associated with BOS development. The cellular source of IL-1a requires further evaluation but may be a marker of airway epithelial injury and/or play a mechanistic role in BOS development via its secretion by other cell types.

A 39-year-old man presented to the hospital in April 2011 with a 2-month history of tonsillitis, night sweats, fatigue, weight loss, shortness of breath on exertion and a dry cough. He was a non-smoker, previously fit and well with no regular medication. Examination of the respiratory, cardiovascular and gastrointestinal systems was normal; he appeared generally well. C reactive protein and erythrocyte sedimentation rate were raised. A CT of the thorax showed mediastinal thickening and mediastinal lymphadenopathy. Whole body (18)F-fluorodeoxyglucose positron emission tomography showed diffuse tracheobronchial activity. Tracheal and lymph node biopsies showed non-specific features. Lung function tests showed an obstructive picture. A diagnosis of relapsing polychondritis was made. Immunosuppressive treatment was started, initially with oral methotrexate and prednisolone, later progressing to intravenous methylprednisolone and intravenous cyclophosphamide. Repeat bronchoscopy showed improvement in inflammation; however, the patient's symptoms were not improved. The patient's symptoms and lung function currently remain stable on maintenance oral prednisolone.

Background Activation of the innate immune system plays a key role in exacerbations of chronic lung disease. Myeloid cells are classically considered to drive innate immune responses yet the potential of fibroblasts to act as immune cells has been postulated. We hypothesized that alarmins released from lung epithelium during environmental insults such as oxidant injury and viral infection might induce innate immune responses in lung fibroblasts. Methods Human bronchial epithelial cells (BECs) and human lung fibroblasts (HLFs) were cultured from brushings taken from lung transplant recipients and resected lung tissue respectively. Cytokine concentrations were measured by ELISA or multiplex platform (MSD). Gene expression was assessed by qRT-PCR. Wild-type and Il1a-/- mice were infected with Influenza (PR8). Data were analysed using t-Student or Mann-Whitney U Test. Correlations were assessed using Spearman rank correlation coefficient. Results Conditioned media from PBECs subjected to oxidant injury contained elevated levels of alarmins. Treatment of HLFs with conditioned media significantly upregulated proinflammatory cytokine expression. Anti-IL-1α or IL-1Ra significantly reduced induction of IL-8 (93% and 95%), IL-6 (90% and 91%), MCP-1 (92% and 93%) and GM-CSF (95% and 94%). Anti-IL-1β had no effect. Co-stimulation with Poly I:C significantly accentuated the IL-1 α induced inflammatory phenotype in HLFs. Bronchoalveolar lavage (BAL) form Influenza infected wild-type mice contains elevated levels of IL-1α and activates innate immune signalling in wild-type murine lung fibroblasts (MLFs) but not Il1r1-/- MLFs. BAL from Il1a-/- mice had no effect on MLFs and demonstrated a blunted neutrophilic response to Influenza. Clinically we show that IL-1α is increased in BAL of lung transplant recipients with infections and within 3 months of developing bronchiolitis obliterans syndrome (BOS) (p Conclusions We propose a new paradigm of innate immune signalling in exacerbations of lung disease, where epithelial damage triggers a potent inflammatory phenotype in resident fibroblasts. The pivotal role of IL-1α in this process is accentuated in the presence of viral infection. This novel pathway warrants further evaluation of its therapeutic potential to limit the repeated cycles of injury and exacerbation in chronic lung diseases.

Prospective Evaluation of Alveolar and Bronchiolar C4d Expression in Transbronchial Biopsies from Lung Transplanted Recipients J. Majo, R. Mahida, N. Thampy, F. Black, G. Meachery, J.L. Lordan, P.A. Corris, A.J. Fisher. Institute of Transplantation, Freeman Hospital, Newcastle upon Tyne, England, United Kingdom; Institute of Cellular Medecine, Newcastle University, Newcastle upon Tyne, England, United Kingdom.