Cecilia M O’Kane

Aspirin therapy in patients with acute respiratory distress syndrome (ARDS) is associated with reduced intensive care unit mortality: a prospective analysis

IntroductionAcute respiratory distress syndrome (ARDS) is a common clinical syndrome with high mortality and long-term morbidity. To date there is no effective pharmacological therapy. Aspirin therapy has recently been shown to reduce the risk of developing ARDS, but the effect of aspirin on established ARDS is unknown.MethodsIn a single large regional medical and surgical ICU between December 2010 and July 2012, all patients with ARDS were prospectively identified and demographic, clinical, and laboratory variables were recorded retrospectively. Aspirin usage, both pre-hospital and during intensive care unit (ICU) stay, was included. The primary outcome was ICU mortality. We used univariate and multivariate logistic regression analyses to assess the impact of these variables on ICU mortality.ResultsIn total, 202 patients with ARDS were included; 56 (28%) of these received aspirin either pre-hospital, in the ICU, or both. Using multivariate logistic regression analysis, aspirin therapy, given either before or during hospital stay, was associated with a reduction in ICU mortality (odds ratio (OR) 0.38 (0.15 to 0.96) P = 0.04). Additional factors that predicted ICU mortality for patients with ARDS were vasopressor use (OR 2.09 (1.05 to 4.18) P = 0.04) and APACHE II score (OR 1.07 (1.02 to 1.13) P = 0.01). There was no effect upon ICU length of stay or hospital mortality.ConclusionAspirin therapy was associated with a reduced risk of ICU mortality. These data are the first to demonstrate a potential protective role for aspirin in patients with ARDS. Clinical trials to evaluate the role of aspirin as a pharmacological intervention for ARDS are needed.

Keratinocyte growth factor in acute lung injury to reduce pulmonary dysfunction – a randomised placebo-controlled trial (KARE): study protocol

BackgroundAcute lung injury is a common, devastating clinical syndrome associated with substantial mortality and morbidity with currently no proven therapeutic interventional strategy to improve patient outcomes. The objectives of this study are to test the potential therapeutic effects of keratinocyte growth factor for patients with acute lung injury on oxygenation and biological indicators of acute inflammation, lung epithelial and endothelial function, protease:antiprotease balance, and lung extracellular matrix degradation and turnover.Methods/designThis will be a prospective, randomised, double-blind, allocation-concealed, placebo-controlled, phase 2, multicentre trial. Randomisation will be stratified by presence of severe sepsis requiring vasopressors. Patients in an ICU fulfilling the American–European Consensus Conference Definition of acute lung injury will be randomised in a 1:1 ratio to receive an intravenous bolus of either keratinocyte growth factor (palifermin, 60 μg/kg) or placebo (0.9% sodium chloride solution) daily for a maximum of 6 days. The primary endpoint of this clinical study is to evaluate the efficacy of palifermin to improve the oxygenation index at day 7 or the last available oxygenation index prior to patient discontinuation from the study.A formal statistical analysis plan has been constructed. Analyses will be carried out on an intention-to-treat basis. A single analysis is planned at the end of the trial. P = 0.05 will be considered statistically significant and all tests will be two-sided. For continuously distributed outcomes, differences between groups will be tested using independent-sample t tests, analysis of variance and analysis of covariance with transformation of variables to normality or nonparametric equivalents. The trial will be reported in line with the Consolidated Standards of Reporting Trials (Consort 2010 guidelines).Trial registrationhttp://ISRCTN95690673

Unexpected Role for Adaptive αβTh17 Cells in Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) is a devastating disorder characterized by increased alveolar permeability with no effective treatment beyond supportive care. Current mechanisms underlying ARDS focus on alveolar endothelial and epithelial injury caused by products of innate immune cells and platelets. However, the role of adaptive immune cells in ARDS remains largely unknown. In this study, we report that expansion of Ag-specific αβTh17 cells contributes to ARDS by local secretion of IL-17A, which in turn directly increases alveolar epithelial permeability. Mice with a highly restrictive defect in Ag-specific αβTh17 cells were protected from experimental ARDS induced by a single dose of endotracheal LPS. Loss of IL-17 receptor C or Ab blockade of IL-17A was similarly protective, further suggesting that IL-17A released by these cells was responsible for this effect. LPS induced a rapid and specific clonal expansion of αβTh17 cells in the lung, as determined by deep sequencing of the hypervariable CD3RβVJ region of the TCR. Our findings could be relevant to ARDS in humans, because we found significant elevation of IL-17A in bronchoalveolar lavage fluid from patients with ARDS, and rIL-17A directly increased permeability across cultured human alveolar epithelial monolayers. These results reveal a previously unexpected role for adaptive immune responses that increase alveolar permeability in ARDS and suggest that αβTh17 cells and IL-17A could be novel therapeutic targets for this currently untreatable disease.

Hydroxymethylglutaryl-CoA reductase inhibition with simvastatin in Acute lung injury to Reduce Pulmonary dysfunction (HARP-2) trial: study protocol for a randomized controlled trial

BackgroundAcute lung injury (ALI) is a common devastating clinical syndrome characterized by life-threatening respiratory failure requiring mechanical ventilation and multiple organ failure. There are in vitro, animal studies and pre-clinical data suggesting that statins may be beneficial in ALI. The Hydroxymethylglutaryl-CoA reductase inhibition with simvastatin in Acute lung injury to Reduce Pulmonary dysfunction (HARP-2) trial is a multicenter, prospective, randomized, allocation concealed, double-blind, placebo-controlled clinical trial which aims to test the hypothesis that treatment with simvastatin will improve clinical outcomes in patients with ALI.Methods/DesignPatients fulfilling the American-European Consensus Conference Definition of ALI will be randomized in a 1:1 ratio to receive enteral simvastatin 80 mg or placebo once daily for a maximum of 28 days. Allocation to randomized groups will be stratified with respect to hospital of recruitment and vasopressor requirement. Data will be recorded by participating ICUs until hospital discharge, and surviving patients will be followed up by post at 3, 6 and 12 months post randomization. The primary outcome is number of ventilator-free days to day 28. Secondary outcomes are: change in oxygenation index and sequential organ failure assessment score up to day 28, number of non pulmonary organ failure free days to day 28, critical care unit mortality; hospital mortality; 28 day post randomization mortality and 12 month post randomization mortality; health related quality of life at discharge, 3, 6 and 12 months post randomization; length of critical care unit and hospital stay; health service use up to 12 months post-randomization; and safety. A total of 540 patients will be recruited from approximately 35 ICUs in the UK and Ireland. An economic evaluation will be conducted alongside the trial. Plasma and urine samples will be taken up to day 28 to investigate potential mechanisms by which simvastatin might act to improve clinical outcomes.Trial registrationCurrent Controlled Trials ISRCTN88244364.

Simvastatin decreases the level of heparin-binding protein in patients with acute lung injury

BackgroundHeparin-binding protein is released by neutrophils during inflammation and disrupts the integrity of the alveolar and capillary endothelial barrier implicated in the development of acute lung injury and systemic organ failure. We sought to investigate whether oral administration of simvastatin to patients with acute lung injury reduces plasma heparin-binding protein levels and improves intensive care unit outcome.MethodsBlood samples were collected from patients with acute lung injury with 48 h of onset of acute lung injury (day 0), day 3, and day 7. Patients were given placebo or 80 mg simvastatin for up to 14 days. Plasma heparin-binding protein levels from patients with acute lung injury and healthy volunteers were measured by ELISA.ResultsLevels of plasma heparin-binding protein were significantly higher in patients with acute lung injury than healthy volunteers on day 0 (p = 0.011). Simvastatin 80 mg administered enterally for 14 days reduced plasma level of heparin-binding protein in patients. Reduced heparin-binding protein was associated with improved intensive care unit survival.ConclusionsA reduction in heparin-binding protein with simvastatin is a potential mechanism by which the statin may modify outcome from acute lung injury.Trial registrationCurrent controlled trials: ISRCTN70127774

Novel anti-tumour necrosis factor receptor-1 (TNFR1) domain antibody prevents pulmonary inflammation in experimental acute lung injury

Background Tumour necrosis factor alpha (TNF-α) is a pleiotropic cytokine with both injurious and protective functions, which are thought to diverge at the level of its two cell surface receptors, TNFR1 and TNFR2. In the setting of acute injury, selective inhibition of TNFR1 is predicted to attenuate the cell death and inflammation associated with TNF-α, while sparing or potentiating the protective effects of TNFR2 signalling. We developed a potent and selective antagonist of TNFR1 (GSK1995057) using a novel domain antibody (dAb) therapeutic and assessed its efficacy in vitro, in vivo and in a clinical trial involving healthy human subjects. Methods We investigated the in vitro effects of GSK1995057 on human pulmonary microvascular endothelial cells (HMVEC-L) and then assessed the effects of pretreatment with nebulised GSK1995057 in a non-human primate model of acute lung injury. We then tested translation to humans by investigating the effects of a single nebulised dose of GSK1995057 in healthy humans (n=37) in a randomised controlled clinical trial in which subjects were subsequently exposed to inhaled endotoxin. Results Selective inhibition of TNFR1 signalling potently inhibited cytokine and neutrophil adhesion molecule expression in activated HMVEC-L monolayers in vitro (P<0.01 and P<0.001, respectively), and also significantly attenuated inflammation and signs of lung injury in non-human primates (P<0.01 in all cases). In a randomised, placebo-controlled trial of nebulised GSK1995057 in 37 healthy humans challenged with a low dose of inhaled endotoxin, treatment with GSK1995057 attenuated pulmonary neutrophilia, inflammatory cytokine release (P<0.01 in all cases) and signs of endothelial injury (P<0.05) in bronchoalveolar lavage and serum samples. Conclusion These data support the potential for pulmonary delivery of a selective TNFR1 dAb as a novel therapeutic approach for the prevention of acute respiratory distress syndrome. Trial registration number ClinicalTrials.gov NCT01587807.

T3 Mitochondrial transfer is an important mechanism by which Mesenchymal Stromal Cells (MSC) facilitate macrophage phagocytosis in the in vitro and in vivo models of Acute Respiratory Distress Syndrome (ARDS)

Background ARDS remains a major cause of respiratory failure in critically ill patients with no specific therapy. MSC based cell therapy is a promising candidate and is being used in clinical trials for ARDS. However the mechanisms of MSC effect in lung injury are not very well understood. Islam et al., 2012 showed mitochondrial transfer from MSC to alveolar epithelial cells was protective in the mouse model of LPS induced pneumonia. Pathophysiology of ARDS is underpinned by dysregulated inflammation and pulmonary macrophages are key cellular mediators of the lung immune response. This study was undertaken to test if MSC could transfer their mitochondria to macrophages and to investigate the effects of MSC mitochondria transfer on macrophage function in the in vivo and in vitro models of ARDS.Abstract T3 Figure 1 Mitochondrial transfer from MSC to macrophages can enhance macrophage phagocytic activity in vivo. (A) MSC use tunnelling nano tubules (TNT) structures to transfer mitochondria (arrows). MSC were pre-stained with MitoTracker Red before co-culture with macrophages, 6 hr later sides were fixed and stained for(blue) to visualise macrophages. Almost allpositive cells demonstrate acquisition of red mitochondria from MSC. (B) In the in vivo model, MSC (MitoTracker)-treated mice BALF was taken and alveolar macrophages assessed for phagocytic activity using fluorescent E.coli bioparticles by flow cytometry. Macrophages that had acquired MSC mitochondria showed a higher phagocytic index in comparison to those without. This was assessed by an increase in Mean fluorescence Intensity (MFI). Some of the materials employed in this work were provided by the Texaz A&M Health Science Centre College of Medicine Institute for Regenerative Medicine at Scott and White through a grant from NCRR of the NIH, Grant #P40RR017447 Methods In vivo studies were performed using a mouse model of E.coli pneumonia induced ARDS. C56BL/6 mice were infected with E.coli, human bone marrow-derived MSC or PBS instilled intra-nasally 4 h after infection. For in vitro studies primary human monocyte-derived macrophages (MDM) were infected with E.coli and co-cultured with MSC in contact. MSC mitochondria were pre-stained with MitoTracker Red and MDM stained for CD45 expression. Double positive cells were visualised with confocal microscopy and quantified using flow cytometry. Phagocytosis was assessed using fluorescent E.coli bioparticles by flow cytometry. Results When co-cultured with MSC >90% of MDMs acquired MitoRed fluorescence, indicating mitochondrial transfer from BM-MSC. Confocal imaging revealed presence of Mito-Red positive tunnelling nanotubules (TNTs) formed by MSC. In vivo >78% of CD11chi/F4–80+ alveolar macrophages retained MSC mitochondria at 24 hr post infection. Alveolar macrophages that had acquired MSC mitochondria had a significantly higher phagocytic index compared to those without suggesting enhancement of phagocytic capacity. Inhibition of TNT formation in MSC resulted in decreased transfer to macrophages by 60%, coupled with significant abrogation of MSC effect on macrophage phagocytosis in vitro and anti-microbial effect seen with MSC in vivo. Conclusions Our findings suggest that anti-microbial activity of macrophages is enhanced at least partially by transfer of BM-MSC mitochondria through TNTs, representing an important mechanism of MSC effect in ARDS. Supported by: MRC MR/L017229/1. Reference 1 Islam MN, Das SR, Emin MT, et al. Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury. Nat Med. 2012;18:759–65

S80 Mesenchymal stromal cells (MSC) modulate human macrophages in acute respiratory distress syndrome (ARDS) via secretion of extracellular vesicles (EV) which enhance oxidative phosphorylation and regulate jak/stat signalling

Background ARDS remains a major cause of respiratory failure in critically ill patients with no effective treatment. MSC are a promising candidate for therapy. However the mechanisms of MSC effects in lung injury are not well understood. We have recently shown that alveolar macrophages are critical cellular mediators of the therapeutic effect of MSC in the mouse model of E.coli pneumonia.1 Here we focused on the paracrine effect of MSC on macrophage polarisation and intracellular signalling. Methods Primary human macrophages were co-cultured with human bone marrow derived-MSC, without contact, at a 5:1 ratio, MSC-conditioned medium (CM) or EV with or without LPS or bronchoalveolar lavage fluid (BALF) from ARDS patients. A phospho-kinase array was performed on lysates for analysis of signalling cascades. Levels of pSTAT, Suppressor of Cytokine Signalling (SOCS) 1 and 3 proteins were tested by Western blot. Cell metabolism was investigated using Seahorse technology. Results Cytokine and surface marker expression show that MSC promote an M2-like macrophage phenotype with enhanced phagocytic activity. MSC-CM enhanced mitochondrial respiration in macrophages and oligomycin inhibited the effect of MSC-CM on cytokine secretion and phagocytosis, suggesting that MSC-CM induced a metabolic switch to oxidative phosphorylation, characteristic of M2 macrophages. Consistently with the M2 phenotype, MSC induced a high SOCS1:SOCS3 protein expression ratio, accompanied with activation of STAT6 and inhibition of STAT1 phosphorylation. MSC effects were reversed by anti-CD44 antibody (important for internalisation of MSC-derived EV) suggesting that EV in MSC-CM are mediators of their effect. Importantly, adoptive transfer of EV-treated alveolar macrophages conferred protection in the mild model of murine LPS-induced pneumonia. EV contents responsible for these effects are currently being investigated. Conclusion MSC promote M2-like macrophage polarisation via secretion of EV. This effect is associated with enhanced oxidative phosphorylation and altered JAK/STAT signalling, potentially regulated by differential expression of SOCS1 and 3 proteins. Reference Jackson MV, Morrison TJ, Doherty DF, et al. Mitochondrial Transfer via Tunnelling Nanotubes (TNT) is an important mechanism by which mesenchymal stem cells enhance macrophage phagocytosis in the in vitro and in vivo models of ARDS. Stem Cells 2016;34(8):2210–23.

S63 Human Mesenchymal Stromal Cell (hMSC) regulation of human macrophages in in vitro models of the Acute Respiratory Distress Syndrome (ARDS)

Background Currently there is no effective therapy which targets the mechanisms underlying the development of ARDS. MSCs present a promising candidate therapy and are being tested in clinical trials for ARDS however their mechanisms of effect in ARDS are not fully understood. Since the alveolar macrophage is key to orchestrating the alveolar inflammatory response, it was hypothesised that hMSCs induce an anti-inflammatory M2-like phenotype in human macrophages. The aim of this study therefore was to determine the effect of MSCs on macrophage phenotype and function and to elucidate the mechanisms of these effects. Methods Using an in vitro non-contact co-culture system, human MSCs and human monocyte-derived-macrophages (MDMs) were stimulated with E.coli lipopolysaccharide (LPS). Cytokine and marker expression profiles were examined using ELISAs, multiplex and flow cytometry. Phagocytic capacity of MDMs was measured using fluorescent E.coli bioparticles by flow cytometry. For additional clinical relevance, the ARDS microenvironment was mimicked by using bronchoalveolar lavage fluid (BALF) obtained from patients with ARDS to examine the effect of MSCs. Results MSCs suppress the production of both pro-inflammatory and anti-inflammatory cytokines by MDMs stimulated with LPS. MSCs increase expression of M2 markers CD163 and CD206 and have no effect on M1 markers CD80 and ICAM-1. Importantly, in spite of the immunosuppressive effect on macrophages, MSCs increase their phagocytic capacity. MSC effects on cytokine secretion and marker expression were maintained in the presence of BALF from patients with ARDS (Figure 1).Abstract S63 Figure 1 MSCs decrease secretion of pro-inflammatory cytokines TNF-α (A) and IL-8 (B) and increase expression of M2 macrophage marker CD206 (C) by MDMs stimulated with BALF from non-septic (NS) or septic (S) patients of ARDS. (A + B, n = 3–7, Kruskal Wallis *p < 0.05) (C, n = 4, ANOVA *p < 0.05) Conclusions Human bone marrow-derived MSCs induce an M2-like phenotype and suppress cytokine secretion in primary human MDMs stimulated with LPS or ARDS patient BALF. Importantly, these effects are coupled with augmentation of macrophage phagocytosis which may be important in the clearance of bacteria and apoptotic cells. Uncovering the paracrine mechanisms responsible for the MSC effects on human macrophages remain the focus of ongoing work. Supported by MRC MR/L017229/1, Department of Employment and Learning. Some of the materials employed in this work were provided by the Texas A&M Health Science Centre College of Medicine Institute for Regenerative Medicine at Scott and White through a grant from NCRR of the NIH, Grant # P40RR017447.

Identifying associations between diabetes and acute respiratory distress syndrome in patients with acute hypoxemic respiratory failure: an analysis of the LUNG SAFE database

BackgroundDiabetes mellitus is a common co-existing disease in the critically ill. Diabetes mellitus may reduce the risk of acute respiratory distress syndrome (ARDS), but data from previous studies are conflicting. The objective of this study was to evaluate associations between pre-existing diabetes mellitus and ARDS in critically ill patients with acute hypoxemic respiratory failure (AHRF).MethodsAn ancillary analysis of a global, multi-centre prospective observational study (LUNG SAFE) was undertaken. LUNG SAFE evaluated all patients admitted to an intensive care unit (ICU) over a 4-week period, that required mechanical ventilation and met AHRF criteria. Patients who had their AHRF fully explained by cardiac failure were excluded. Important clinical characteristics were included in a stepwise selection approach (forward and backward selection combined with a significance level of 0.05) to identify a set of independent variables associated with having ARDS at any time, developing ARDS (defined as ARDS occurring after day 2 from meeting AHRF criteria) and with hospital mortality. Furthermore, propensity score analysis was undertaken to account for the differences in baseline characteristics between patients with and without diabetes mellitus, and the association between diabetes mellitus and outcomes of interest was assessed on matched samples.ResultsOf the 4107 patients with AHRF included in this study, 3022 (73.6%) patients fulfilled ARDS criteria at admission or developed ARDS during their ICU stay. Diabetes mellitus was a pre-existing co-morbidity in 913 patients (22.2% of patients with AHRF). In multivariable analysis, there was no association between diabetes mellitus and having ARDS (OR 0.93 (0.78–1.11); p = 0.39), developing ARDS late (OR 0.79 (0.54–1.15); p = 0.22), or hospital mortality in patients with ARDS (1.15 (0.93–1.42); p = 0.19). In a matched sample of patients, there was no association between diabetes mellitus and outcomes of interest.ConclusionsIn a large, global observational study of patients with AHRF, no association was found between diabetes mellitus and having ARDS, developing ARDS, or outcomes from ARDS.Trial registrationNCT02010073. Registered on 12 December 2013.