Df McAuley

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

S94 Tumour Necrosis Factor receptor 1 inhibition using a novel inhaled human antibody reduces inflammation in a human model of lung injury induced by inhaled lipopolysaccharide; a randomised placebo-controlled clinical trial

Introduction and Objectives Tumour Necrosis Factor receptor 1 (TNFR1) transduces the pro-inflammatory activity of TNF-α, whereas signalling through TNFR2 may contribute to tissue repair. Attenuation of TNFR1 signalling, whilst simultaneously preserving the effects of TNFR2 signalling, may be beneficial in management of acute lung injury (ALI). GSK1995057 is a novel, fully human antibody fragment (domain antibody) that selectively binds TNFR1 and antagonises signalling of TNF-α via TNFR1. The aim of this clinical study was to investigate the effect of nebulised GSK1995057 on pulmonary and systemic inflammation and cell injury in an in vivo human model of lung injury induced by inhaled lipopolysaccharide (LPS). Methods Healthy subjects were enrolled in a double-blind, placebo-controlled study and randomised to nebulised GSK1995057 or placebo (1:1) administered 1 hour prior to LPS inhalation. Measurements were performed in bronchoalveolar lavage (BAL) fluid obtained at 6 hours after LPS challenge (7 hours after dosing) and in serum obtained over 24 hours post dosing of GSK1995057. The primary endpoint was BAL neutrophil count at 6 hours post LPS exposure. Data are geometric mean (95% CI). Results Thirty-seven healthy subjects were enrolled. One subject in the placebo group was excluded from the analysis of BAL markers as the BAL was technically poor. Pre-treatment with inhaled GSK1995057 significantly reduced pulmonary and systemic markers of inflammation. In addition, there was a reduction in pulmonary vWF reflecting reduced endothelial cell injury/activation (Table 1). The prevalence of LPS-induced clinical symptoms (e.g. fever, nausea) was also lower in GSK1995057 treated subjects compared with placebo treated subjects. There were no serious adverse events related to study drug. Abstract S94 Table 1. Effect of GSK1995057 on markers of pulmonary and systemic inflammation. BALF Placebo(n = 18) GSK1995057(n = 18) % reduction P-value PMN (104 cells/ml) 6.5 (4.5, 9.4) 4.5 (2.89, 6.89)3.8 (2.8, 5.3)* 31(41)† 0.17(0.03)† IL-1b(pg/ml) 6.8(4.3, 10.4) 1.4 (1.0, 2.1) 79 <0.0001 IL-6 (pg/ml) 386.1(277.5, 537.4) 169.4 (111.2, 257.9) 56 0.003 IL-8 (pg/ml) 332.1(254.6, 433.2) 117.5 (82.5, 167.54) 65 <0.0001 MIP1alpha (pg/ml) 133.7(87.2, 205.1) 15.6 (8.3, 29.1) 88 <0.0001 MCP-1 (pg/ml) 799.4 (591.6, 1080.2) 159.5(101.9, 249.5) 80 <0.0001 vWF (ng/ml) 12.8(9.2, 17.9) 8.1(6.1, 10.8) 37 0.04 Serum Placebo (n = 19) GSK1995057 (n = 18) % reduction P-value CRP (µg/ml)** 55.2(31.0, 98.4) 12.0 (6.6, 21.8) 78 0.0007 OSM (pg/ml)*** 20.7(13.4, 32.0) 7.5(4.8, 11.7) 64 0.002 * PMN data with subject classified as biological outlier (>3 x inter quartile range outside the upper quartile) removed. † % Reduction and statistical significance for BALPMN data with biological outlier excluded. ** CRP data taken at 24h post GSK1995057 dosing. Data are adjusted means with baseline and time effects considered. *** OSM data taken at 6h post LPS inhalation. Data are adjusted means with baseline and time effects considered. Conclusion This is the first report that inhalation of a novel human antibody fragment directed against the TNFR1 receptor attenuates mechanisms implicated in the pathophysiology of ALI. GSK1995057 may be a potential therapy for ALI. ClinicalTrials.gov identifier: NCT01587807. This work was funded by GlaxoSmithKline.

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.

Mesenchymal stromal cells and the acute respiratory distress syndrome (ARDS): challenges for clinical application.

In contemporary reports, approximately 30% of patients with acute respiratory distress syndrome (ARDS) will die1 and up to 70% of survivors have persistent significant disability.2 Several interventions which limit injurious ventilation have been shown to reduce mortality, primarily: low-tidal volume ventilation,3 prone positioning,4 early neuromuscular blockade5 and possibly extracorporeal membrane oxygenation (ECMO).6 To date, no intervention targeted at the underlying pathophysiological process has been shown to be beneficial, including recent studies of β-agonists and statins.7 ,8 Given the failure of pharmacological interventions in ARDS, increasing interest has been shown in the potential of mesenchymal stromal cells (MSCs). While the complex actions of MSCs are not yet fully understood, they appear to attenuate lung injury via three broad mechanisms. First, they have an immunomodulatory ability, influencing both innate and adaptive immunity. This is achieved by the secretion of anti-inflammatory soluble factors, including interleukin 10 (IL-10), IL-1 receptor antagonist and prostaglandin E2.9 Second, MSCs directly augment the host response to sepsis. LL-37 is a peptide secreted by MSCs which has direct antimicrobial properties and has previously been shown to increase bacterial clearance in an Escherichia coli model of lung injury after the intratracheal administration of MSCs.10 Third, MSCs play an important role in the repair and regeneration of lung tissue following injury. This ability appears to be mediated by the secretion of several growth factors, including vascular endothelial growth factor and keratinocyte growth factor.9 In this edition of Thorax , Devaney et al …

S95 Exploiting the immunoregulatory role of Siglec-E via sialic acid-functionalised nanoparticles as a novel approach for the treatment of acute lung injury

Acute Lung Injury (ALI) is a life-threatening disorder underpinned by dysregulated inflammatory cascades, with resultant injury to lung architecture. Currently, provision of supportive care represents the mainstay of treatment for ALI and novel anti-inflammatory therapeutic strategies are urgently required. We have developed a polymeric nanoconstruct surface-functionalised with sialic acid targeting moieties (SNP), exploiting the anti-inflammatory effects arising from the targeted engagement of Siglec-E receptors on activated macrophages, with potential therapeutic utility in ALI. Polylactic-co-glycolic acid (PLGA) nanoparticles of uniform size distribution (approximately 150nm in diameter) were synthesised in accordance with a salting-out formulation. Intratracheal instillation of 20μg lipopolysaccharide (LPS) was utilised as a model of ALI in C57BL/6 mice, co-administered with 1μg SNP or non-functionalised nanoparticles (NP). Bronchoalveolar lavage (BALF) samples were collected 24 hours after treatment for analysis by enzyme-linked immunosorbent assay (ELISA). As exemplified in Figure 1., intratracheal instillation of SNP significantly attenuated BALF levels of pro-inflammatory TNFα and IL-6 cytokines, in addition to the neutrophil chemoattractant KC. Moreover, BALF differential cell counts revealed a decrease in neutrophil numbers upon treatment with SNP under LPS-induced pro-inflammatory conditions. Further analyses addressing the therapeutic utility of SNP have been undertaken, including lung wet/dry ratios, histology and toxicological evaluation, with promising outcomes. Abstract S95 Figure 1. Therapeutic efficacy of SNP in a murine model of LPS-induced ALI (* p<0.05, **p<0.001, ***p<0.001 compared to LPS control, as established by one-way ANOVA and Tuley post-hoc test). This research clearly demonstrates the ability of SNP to diminish the inflammatory response in a murine model of LPS-induced ALI. Considering that chemoattractants and cytokines are key mediators in the pathogenesis of ALI, these results substantiate the credibility of this nanoscaffold as a therapy for ALI. Ultimately, we aim to progress this modality to a human setting, specifically analysing its effects on alveolar macrophages isolated from human volunteers, before advancing to a human ex vivo lung perfusion model.

S79 Potential diagnostic significance of neutrophil proteases in ventilator-associated pneumonia

Introduction and Objectives The clinical diagnosis of ventilator-associated pneumonia (VAP) remains notoriously difficult, as several non-infective conditions mimic VAP. Microbiological confirmation of the diagnosis using conventional cultures typically takes 48–72 h. Identification of molecules measurable within a short time frame and closely associated with microbiologically confirmed VAP is therefore highly desirable. VAP is associated with significant influx of activated neutrophils into the alveolar space. We postulated that extracellular neutrophil proteases in bronchoalveolar lavage fluid (BALF) may reliably identify VAP in suspected cases. Methods Fifty-four intubated and mechanically ventilated patients in the intensive care unit developed clinically suspected VAP and were recruited. Bronchoalveolar lavage (BAL) was performed using a standardised protocol. An aliquot of BALF was sent to the diagnostic microbiology laboratory for quantitative culture, with confirmation of VAP defined as growth of a pathogen(s) at >104 colony forming units/ml. Remaining BALF was centrifuged. The following neutrophil-specific proteases were assayed in cell-free BALF supernatant—matrix metalloproteinase (MMP)-8 and MMP-9 by Luminex assay, and human neutrophil elastase (HNE) by enzyme-linked immunosorbent assay. Urea was simultaneously measured in serum and BALF, and used to correct for the dilution of epithelial lining induced by BAL. Receiver operating characteristic (ROC) curves were constructed and optimal specificity and sensitivity for each marker calculated. Results Eleven patients (20%) had confirmed VAP. For HNE (cut off 670ng/ml) the ROC area under curve (AUC) was 0.87 (p<0.0001), sensitivity 93%, specificity 79%. For MMP-8 (13 ng/ml), ROC AUC was 0.81 (p<0.005), sensitivity 91%, specificity 63%. For MMP-9 (22 ng/ml), ROC AUC was 0.79 (p<0.005), sensitivity 82%, specificity 63%. Conclusions Neutrophil proteases are strongly associated with confirmed infection in cases of suspected VAP. The values for HNE, in particular, compare extremely favourably with any previously published equivalent values. These data suggest that neutrophil protease concentrations in BALF deserve further attention as potentially diagnostic markers for VAP. They further suggest that neutrophil proteases, inappropriately released into the extracellular space, may contribute to the pathophysiology of VAP.

S104 Monocyte influx accompanies the early neutrophilic inflammation seen in bronchoalveolar lavage fluid following lipopolysaccharide inhalation

Introduction Acute lung injury (ALI) has a mortality rate of over 30%, with no proven pharmacological treatment. Inhalation of lipopolysaccharide (LPS) in healthy volunteers induces transient inflammation resembling that found in patients with ALI. Inhaled LPS causes neutrophilia that is detectable in bronchoalveolar lavage fluid (BALF) and blood, but its effect on BALF and blood monocyte populations is not well established. Methods 12 healthy volunteers were recruited and randomly allocated to receive either 60 μg of inhaled LPS or saline (n=6 each arm). Clinical parameters, including temperature, and any reported symptoms were recorded. Full blood counts were taken at baseline and 2, 4, 6, 8 and 24 h post-inhalation. BAL was performed at 8 h. BALF cell populations were analysed morphologically using cytospins and cytometrically by flow cytometry after staining for cell surface markers (alveolar macrophages: CD163, CD206, CCR5; neutrophils/monocytes: HLA-DR, CD14, CD16). Results 4 LPS volunteers developed pyrexia, two reported cough and one myalgia. The mean maximal increment in temperature was significantly greater in the LPS arm (p=0.047). Compared to saline inhalation, LPS caused a peripheral blood neutrophilia (p=0.006) that was evident from 4 h and greatest at 8 h. There was no significant difference in peripheral blood monocyte counts between treatment arms at any point measured (p=0.87). Although mean total alveolar macrophage numbers were similar between the two groups, their relative proportion in the LPS volunteers was significantly reduced due to the expansion in neutrophil and monocyte populations. Flow cytometry revealed a 24-fold expansion of the neutrophil population following LPS (in parallel with morphological data). These neutrophils were distinguishable by HLA-DR-/CD14-/CD16+ staining. There was a concomitant similar rise in the population of HLA-DR+/CD14+/CD16- ‘classical’ monocytes. Further analysis of these monocytes revealed that macrophage cell surface marker expression was absent. Conclusion Morphological analysis of BAL fluid in previous LPS inhalation studies has consistently suggested that there is no change in the monocyte population. Using flow cytometry enables a more detailed analysis. This study is the first to clearly demonstrate that an early expansion in the monocyte population accompanies the neutrophil influx seen in BALF 8 h following inhalation of LPS.

S133 A core outcome set for mechanical ventilation trials: the covent study

There is inconsistency in the selection and measurement of outcomes in clinical trials of mechanically ventilated critically ill patients.1 This presents challenges when comparing trials, and particularly when undertaking meta-analysis of trial data. A core outcome set (COS) is a minimum set of standardised outcomes that should be reported in every trial of a specific intervention. We therefore aimed to develop a COS for use in future trials where the aim of the intervention is to modify the duration of mechanical ventilation. Mixed consensus methods were used to develop this COS. A large, international, online Delphi study was followed by 2 consensus webinars with representatives from the Delphi panel and additional input from a separate patient representative group teleconference. Participants were recruited via international trials groups, critical care societies, charities and associations. Additional researchers were identified through a PubMed search. Participants represented 4 main stakeholder groups; patients, clinicians, researchers and industry. The study was conducted between December 2015 and October 2016. The Delphi ran over 3 rounds; Round 1 included 24 outcomes obtained from a systematic review. During this round a further 23 outcomes were proposed and added by participants. Numbers of participants completing each round were 200, 178 and 161 respectively. A total of 19 outcomes gained consensus through the Delphi process and were discussed at the consensus webinars and the patient teleconference. The outcomes in the final COS were agreed across all 3 meetings and included mortality, health-related quality of life, duration of mechanical ventilation, reintubation, length of stay and successful extubation (Table 1). Using robust consensus methodology this COS has been developed for use in all trials and systematic reviews evaluating interventions that may reduce the duration of mechanical ventilation. Agreement now needs to be reached on how these outcomes should be measured and defined. Reference Blackwood B, Clarke M, McAuley DF, McGuigan PJ, Marshall JC, Rose L. How outcomes are defined in clinical trials of mechanically ventilated adults and children. American Journal of Respiratory and Critical Care Medicine 2014, Apr 15;189(8):886. Abstract S133 Table 1 Core outcomes agreed at each consensus meeting and the final COS Webinar 1 Webinar2 Teleconference Final COS Mortality Mortality Mortality Mortality HRQOL HRQOL HRQOL HRQOL Duration IMV Duration IMV Duration IMV Duration IMV Reintubation Reintubation Reintubation Reintubation Length of Stay Length of Stay Length of Stay Length of Stay Successful Extubation Successful Extubation Successful Extubation Successful Extubation Delirium Survival Survival Pulmonary Complications Pulmonary Complications Key: HRQOL, health related quality of life; IMV, invasive mechanical ventilation

S67 Vitamin D deficiency drives pulmonary inflammation in a human model of the acute respiratory distress syndrome induced by inhaled lipopolysaccharide in healthy volunteers

The acute respiratory distress syndrome (ARDS) is characterised by exaggerated alveolar inflammation. Vitamin D deficiency in an LPS induced murine model of ARDS results in exaggerated alveolar inflammation. However the role of vitamin D deficiency in pulmonary inflammation in humans is unclear. We hypothesised that in healthy volunteers with vitamin D deficiency, pulmonary inflammation would be increased following LPS inhalation. Methods Healthy volunteers inhaled 50 micrograms of LPS and six hours later underwent bronchoalveolar lavage for measurement of cytokines. Plasma was collected at baseline and one day post LPS inhalation for measurement of vitamin D. Results 28 participants were included. The mean age of volunteers was 26.2 +/- 5.5 years. All 28 patients were vitamin D deficient (plasma levels <50 nmol/l), with 89% (25/28) patients having severe vitamin D deficiency (<25 nmol/l). Vitamin D levels were significantly higher after LPS inhalation (p < 0.002). Levels of IL-1β in BALF were significantly higher in those with severe deficiency than those with mild/moderate deficiency (Figure 1; p = 0.04). Levels of IL-6, IL-8 or TNF-α did not differ between groups.Abstract S67 Figure 1 Bronchoalveolar lavage fluid (BALF) levels of IL-1 beta were significantly elevated in volunteers with severe plasma vitamin D deficiency (<25 nmol/l) compared to those with mild or moderate deficiency (25–50 nmol/l) Conclusions Vitamin D deficiency was highly prevalent in this population of healthy volunteers. The rise in vitamin D levels post LPS exposure may represent mobilisation of vitamin D from fat stores during inflammation though vitamin D metabolism and kinetics are complex and may differ in healthy volunteers and the critically ill. Severe deficiency correlated with increased alveolar inflammation.

0995. Aspirin reduces neutrophilic pulmonary inflammation in a human model of acute respiratory distress syndrome induced by inhaled lipopolysaccharide

Acute respiratory distress syndrome (ARDS) is characterized by damage to the alveolar epithelial-endothelial barrier resulting in neutrophil influx and pulmonary oedema. The activation of platelet and secondary capture of neutrophils may play an important role in propagation of inflammation in ARDS [1]. Various animal studies have shown that aspirin therapy reduces pulmonary oedema and development of lung injury [2]. In observational studies, patients on aspirin therapy prior to hospital admission had a reduced incidence of ARDS [3]. By acetylating cyclooxygenase, aspirin inhibits platelet aggregation and generates anti-inflammatory molecules which modulate neutrophilic inflammation [4].