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Background: Both latent class analysis (LCA) assignment based upon a panel of plasma biomarkers and interleukin-18 (IL-18) plasma level have been shown to predict prognosis and treatment response in Acute Respiratory Distress Syndrome (ARDS). Interleukin-18 is a measure of inflammasome activation and plays a distinct role in inflammation that is not captured by the biomarkers used in LCA assignments. We hypothesized that elevated IL-18 would provide additive prognostic and therapeutic information to previously published LCA assignments in ARDS, identifying additional “high-risk” patients not captured by LCA who could be eligible for inclusion in future precision medicine-focused trials. Methods: IL-18 and a panel of protein markers used for LCA had been previously measured in plasma from 683/745 patients in the Statins for Acutely Injured Lungs from Sepsis (SAILS) and 511/540 patients in the Hydroxymethylglutaryl-CoA reductase inhibition with simvastatin in Acute lung injury to Reduce Pulmonary dysfunction (HARP-2) trials. We tested the association between high IL-18 (>800 pg/mL) and LCA class assignment using McNemar’s test and evaluated the association of each subgrouping as well as treatment with 60-day mortality using Fisher’s exact test. We assessed 60-day mortality in each combination (high/low IL-18, hypo-/hyper-inflammatory LCA class, and treatment/placebo) using Kaplan-Meier survival analysis. We evaluated the correlation between the log2 transformed IL-18 level and LCA biomarkers using Pearson’s correlation coefficient. Results: 33% of patients in SAILS and HARP-2 were discordant by IL-18 level and LCA class. Elevated IL-18 identified a high-risk group of individuals previously classified as hypo-inflammatory by LCA in both SAILS (OR 3.3, 95% CI 1.8-6.1, p of 0.17-0.47. Conclusions: High Plasma IL-18 level provides additional prognostic information to LCA sub-phenotypes in two large ARDS cohorts.

Respiratory infections are a leading cause of mortality worldwide. Most of the research on the underlying disease mechanisms is based on cell culture, organoid, or surrogate animal models. Although these provide important insights, they have limitations. Cell culture models fail to recapitulate cellular interactions in the lung and animal models often do not permit high-throughput analysis of drugs or pathogen isolates; hence, there is a need for improved, scalable models. Precision-cut lung slices (PCLS), small, uniform tissue slices generated from animal or human lungs are increasingly recognized and employed as an ex vivo organotypic model. PCLS retain remarkable cellular complexity and the architecture of the lung, providing a platform to investigate respiratory pathogens in a near-native environment. Here, we review the generation and features of PCLS, their use to investigate the pathogenesis of viral and bacterial pathogens, and highlight their potential to advance respiratory infection research in the future.

Background Interleukin (IL)-18 is a marker of inflammasome activation, and high baseline plasma IL-18 is associated with increased mortality in patients with sepsis-induced ARDS. The aim of this analysis was to determine if simvastatin was associated with benefit in patients with ARDS and high plasma IL-18. Methods In this secondary analysis of the HARP-2 study, we compared 28-day mortality and response to simvastatin according to baseline plasma IL-18 using cox proportional hazards analysis. Separately, monocyte-derived macrophages from healthy volunteers were pre-incubated with simvastatin or rosuvastatin before stimulation with ATP and LPS, and the effect on secreted IL-18 and IL-1β compared. Results 511 patients from HARP-2 had available data. High baseline plasma IL-18 (≥ 800 pg/ml) was associated with increased 28-day mortality (high IL-18 30.6% vs. low IL-18 17.5%; HR 1.89 [95% CI 1.30–2.73]; p = 0.001). Allocation to simvastatin in patients with high baseline plasma IL-18 was associated with a lower probability of 28-day mortality compared with placebo (24.0% vs 36.8%; p = 0.01). Finally, simvastatin, but not rosuvastatin, reduced stimulated macrophage secretion of IL-18 and IL-1β. Conclusion In patients with high baseline plasma IL-18, simvastatin is associated with a higher probability of survival, and this effect may be due to reduced inflammasome activation. These data suggest that baseline plasma IL-18 may allow a personalised treatment approach by identifying patients with ARDS who could benefit from simvastatin therapy.

There is an urgent need for new antimicrobial strategies for treating complex infections and emerging pathogens. Human mesenchymal stromal cells (MSCs) are adult multipotent cells with antimicrobial properties, mediated through direct bactericidal activity and modulation of host innate and adaptive immune cells. More than 30 in vivo studies have reported on the use of human MSCs for the treatment of infectious diseases, with many more studies of animal MSCs in same-species models of infection. MSCs demonstrate potent antimicrobial effects against the major classes of human pathogens (bacteria, viruses, fungi, and parasites) across a wide range of infection models. Mechanistic studies have yielded important insight into their immunomodulatory and bactericidal activity, which can be enhanced through various forms of preconditioning. MSCs are being investigated in over 80 clinical trials for difficult-to-treat infectious diseases, including sepsis and pulmonary, intra-abdominal, cutaneous, and viral infections. Completed trials consistently report MSCs to be safe and well tolerated, with signals of efficacy against some infectious diseases. Although significant obstacles must be overcome to produce a standardized, affordable, clinical-grade cell therapy, these studies suggest that MSCs may have particular potential as an adjunct therapy in complex or resistant infections.

Background Oxygen therapy is routinely administered to mechanically ventilated patients. However, there remains uncertainty about the optimal oxygen titration target in patients with the acute respiratory distress syndrome (ARDS). Methods Prospectively identified adult patients meeting the Berlin definition of ARDS between 1st January 2014 and 13th December 2016 were analyzed. Oxygen exposure variables were collected at 6-hourly intervals. The primary exposure was the average time-weighted partial pressure of arterial oxygen (PaO2) calculated over a maximum of 7 days from meeting ARDS criteria. The primary outcome was ICU mortality. Univariable and multivariable logistic regression analyses were performed to assess the impact of exposure variables on clinical outcomes. Results are presented as odds ratio [95% confidence interval]. Results 202 patients were included in the final analysis. Overall ICU mortality was 31%. The average time-weighted PaO2 during the first 7 days of ARDS was similar between non-survivors and survivors (11.3 kPa [10.2, 12.5] (84.8 mmHg [76.5, 93.8]) vs. 11.9 kPa [10.9, 12.6] (89.3 mmHg [81.8, 94.5]); p = 0.08). In univariable and multivariable analysis, average time-weighted PaO2 demonstrated a U-shaped relationship with ICU mortality. There was a similar relationship identified with hospital mortality. Conclusions In patients with ARDS, the predicted probability of both ICU and hospital mortality was lowest when the average time-weighted PaO2 was between 12.5 and 14 kPa (93.8–105.0 mmHg), suggesting this is a reasonable oxygenation target for clinicians to aim for.

Alveolar epithelial–capillary barrier disruption is a hallmark of acute respiratory distress syndrome (ARDS). Contribution of mitochondrial dysfunction to the compromised alveolar-capillary barrier in ARDS remains unclear. Mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) are considered as a cell-free therapy for ARDS. Mitochondrial transfer was shown to be important for the therapeutic effects of MSCs and MSC-EVs. Here we investigated the contribution of mitochondrial dysfunction to the injury of alveolar epithelial and endothelial barriers in ARDS and the ability of MSC-EVs to modulate alveolar–capillary barrier integrity through mitochondrial transfer. Primary human small airway epithelial and pulmonary microvascular endothelial cells and human precision cut lung slices (PCLSs) were stimulated with endotoxin or plasma samples from patients with ARDS and treated with MSC-EVs, barrier properties and mitochondrial functions were evaluated. Lipopolysaccharide (LPS)-injured mice were treated with MSC-EVs and degree of lung injury and mitochondrial respiration of the lung tissue were assessed. Inflammatory stimulation resulted in increased permeability coupled with pronounced mitochondrial dysfunction in both types of primary cells and PCLSs. Extracellular vesicles derived from normal MSCs restored barrier integrity and normal levels of oxidative phosphorylation while an extracellular vesicles preparation which did not contain mitochondria was not effective. In vivo, presence of mitochondria was critical for extracellular vesicles ability to reduce lung injury and restore mitochondrial respiration in the lung tissue. In the ARDS environment, MSC-EVs improve alveolar–capillary barrier properties through restoration of mitochondrial functions at least partially via mitochondrial transfer., This study demonstrates that mitochondrial dysfunction is an important mechanism of ARDS pathogenesis. Mitochondrial transfer is crucial for the ability of MSC extracellular vesicles to restore integrity of the alveolar–capillary barrier. https://bit.ly/2JuqoCY

There is no pharmacologic treatment for ARDS. Platelets play an important role in the pathophysiology of ARDS. Preclinical, observational, and clinically relevant models of ARDS indicate aspirin as a potential therapeutic option.Is enteral aspirin (75 mg, once daily) safe and effective in improving surrogate outcomes in adult patients with ARDS?This randomized, double-blind (patient and investigator), allocation-concealed, placebo-controlled phase 2 trial was conducted in five UK ICUs. Patients fulfilling the Berlin definition of ARDS were randomly assigned at a 1:1 ratio to receive enteral aspirin (75 mg) or placebo, for a maximum of 14 days, using a computer-generated randomization schedule, with variable block size, stratified by vasopressor requirement. The primary end point was oxygenation index (OI) on day 7. Secondary outcomes included safety parameters and other respiratory physiological markers. Analyses were by intention to treat.The trial was stopped early, due to slow recruitment, after 49 of a planned 60 patients were recruited. Twenty-four patients were allocated to aspirin and 25 to placebo. There was no significant difference in day 7 OI [aspirin group: unadjusted mean, 54.4 (SD 26.8); placebo group: 42.4 (SD 25); mean difference, 12.0; 95% CI, -6.1 to 30.1; P = .19]. Aspirin did not significantly impact the secondary outcomes. There was no difference in the number of adverse events between the groups (13 in each; OR, 1.04; 95% CI, 0.56-1.94; P = .56).Aspirin was well tolerated but did not improve OI or other physiological outcomes; a larger trial is not feasible in its current design.ClinicalTrials.gov; No.: NCT02326350; URL: www.gov.

Rationale: Mechanical ventilation is a mainstay of intensive care but contributes to the mortality of patients through ventilator induced lung injury. Extracellular Cyclophilin A is an emerging inflammatory mediator and metalloproteinase inducer, and the gene responsible for its expression has recently been linked to COVID-19 infection. Objectives: Here we explore the involvement of extracellular Cyclophilin A in the pathophysiology of ventilator-induced lung injury. Methods: Mice were ventilated with low or high tidal volume for up to 3 hours, with or without blockade of extracellular Cyclophilin A signalling, and lung injury and inflammation were evaluated. Human primary alveolar epithelial cells were exposed to in vitro stretch to explore the cellular source of extracellular Cyclophilin A, and Cyclophilin A levels were measured in bronchoalveolar lavage fluid from acute respiratory distress syndrome patients, to evaluate clinical relevance. Measurements and Main Results: High tidal volume ventilation in mice provoked a rapid increase in soluble Cyclophilin A levels in the alveolar space, but not plasma. In vivo ventilation and in vitro stretch experiments indicated alveolar epithelium as the likely major source. In vivo blockade of extracellular Cyclophilin A signalling substantially attenuated physiological dysfunction, macrophage activation and matrix metalloproteinases. Finally, we found that patients with acute respiratory distress syndrome showed markedly elevated levels of extracellular Cyclophilin A within bronchoalveolar lavage. Conclusions: Cyclophilin A is upregulated within the lungs of injuriously ventilated mice (and critically ill patients), where it plays a significant role in lung injury. Extracellular Cyclophilin A represents an exciting novel target for pharmacological intervention.

ARDS, first described in 1967, is the commonest form of acute severe hypoxemic respiratory failure. Despite considerable advances in our knowledge regarding the pathophysiology of ARDS, insights into the biologic mechanisms of lung injury and repair, and advances in supportive care, particularly ventilatory management, there remains no effective pharmacological therapy for this syndrome. Hospital mortality at 40% remains unacceptably high underlining the need to continue to develop and test therapies for this devastating clinical condition. The purpose of the review is to critically appraise the current status of promising emerging pharmacological therapies for patients with ARDS and potential impact of these and other emerging therapies for COVID-19-induced ARDS. We focus on drugs that: (1) modulate the immune response, both via pleiotropic mechanisms and via specific pathway blockade effects, (2) modify epithelial and channel function, (3) target endothelial and vascular dysfunction, (4) have anticoagulant effects, and (5) enhance ARDS resolution. We also critically assess drugs that demonstrate potential in emerging reports from clinical studies in patients with COVID-19-induced ARDS. Several therapies show promise in earlier and later phase clinical testing, while a growing pipeline of therapies is in preclinical testing. The history of unsuccessful clinical trials of promising therapies underlines the challenges to successful translation. Given this, attention has been focused on the potential to identify biologically homogenous subtypes within ARDS, to enable us to target more specific therapies 'precision medicines.' It is hoped that the substantial number of studies globally investigating potential therapies for COVID-19 will lead to the rapid identification of effective therapies to reduce the mortality and morbidity of this devastating form of ARDS.

BackgroundUsing latent class analysis (LCA) in five randomised controlled trial (RCT) cohorts, two distinct phenotypes of acute respiratory distress syndrome (ARDS) have been identified: hypoinflammatory and hyperinflammatory. The phenotypes are associated with differential outcomes and treatment response. The objective of this study was to develop parsimonious models for phenotype identification that could be accurate and feasible to use in the clinical setting.MethodsIn this retrospective study, three RCT cohorts from the National Lung, Heart, and Blood Institute ARDS Network (ARMA, ALVEOLI, and FACTT) were used as the derivation dataset (n=2022), from which the machine learning and logistic regression classifer models were derived, and a fourth (SAILS; n=715) from the same network was used as the validation test set. LCA-derived phenotypes in all of these cohorts served as the reference standard. Machine-learning algorithms (random forest, bootstrapped aggregating, and least absolute shrinkage and selection operator) were used to select a maximum of six important classifier variables, which were then used to develop nested logistic regression models. Only cases with complete biomarker data in the derivation dataset were used for variable selection. The best logistic regression models based on parsimony and predictive accuracy were then evaluated in the validation test set. Finally, the models' prognostic validity was tested in two external ARDS clinical trial datasets (START and HARP-2) by assessing mortality at days 28, 60, and 90 and ventilator-free days to day 28.FindingsThe six most important classifier variables were interleukin (IL)-8, IL-6, protein C, soluble tumour necrosis factor receptor 1, bicarbonate, and vasopressor use. From the nested models, three-variable (IL-8, bicarbonate, and protein C) and four-variable (3-variable plus vasopressor use) models were adjudicated to be the best performing. In the validation test set, both models showed good accuracy (AUC 0·94 [95% CI 0·92-0·95] for the three-variable model and 0·95 [95% CI 0·93-0·96] for the four-variable model) against LCA classifications. As with LCA-derived phenotypes, the hyperinflammatory phenotype as identified by the classifier model was associated with higher mortality at day 90 (87 [39%] of 223 patients vs 112 [23%] of 492 patients; p

The implementation of infection models that approximate human disease is essential to understand infections and for testing new therapies before they enter into clinical stages. Rodents are used in most preclinical studies, although the differences between mice and humans have fueled the conclusion that murine studies are unreliable predictors of human outcomes. In this study, we have developed a whole-lung porcine model of infection using the ex vivo lung perfusion (EVLP) system established to recondition human lungs for transplant. As a proof of principle, we provide evidence demonstrating that infection of the porcine EVLP with the human pathogen Klebsiella pneumoniae recapitulates the known features of Klebsiella-triggered pneumonia. Moreover, our data revealed that the porcine EVLP model is useful to reveal features of the virulence of K. pneumoniae, including the manipulation of immune cells. Together, the findings of this study support the utility of the EVLP model using pig lungs as a surrogate host for assessing respiratory infections., The use of animal infection models is essential to understand microbial pathogenesis and to develop and test treatments. Insects and two-dimensional (2D) and 3D tissue models are increasingly being used as surrogates for mammalian models. However, there are concerns about whether these models recapitulate the complexity of host-pathogen interactions. In this study, we developed the ex vivo lung perfusion (EVLP) model of infection using porcine lungs to investigate Klebsiella pneumoniae-triggered pneumonia as a model of respiratory infections. The porcine EVLP model recapitulates features of K. pneumoniae-induced pneumonia lung injury. This model is also useful to assess the pathogenic potential of K. pneumoniae, as we observed that the attenuated Klebsiella capsule mutant strain caused less pathological tissue damage with a concomitant decrease in the bacterial burden compared to that in lungs infected with the wild type. The porcine EVLP model allows assessment of inflammatory responses following infection; similar to the case with the mouse pneumonia model, we observed an increase of il-10 in the lungs infected with the wild type and an increase of ifn-γ in lungs infected with the capsule mutant. This model also allows monitoring of phenotypes at the single-cell level. Wild-type K. pneumoniae skews macrophages toward an M2-like state. In vitro experiments probing pig bone marrow-derived macrophages uncovered the role for the M2 transcriptional factor STAT6 and that Klebsiella-induced il-10 expression is controlled by p38 and extracellular signal-regulated kinase (ERK). Klebsiella-induced macrophage polarization is dependent on the capsule. Together, the findings of this study support the utility of the EVLP model using pig lungs as a platform to investigate the infection biology of respiratory pathogens.

The use of animal infection models is essential to understand microbial pathogenesis and to develop and test treatments. Insects, and 2D and 3D tissue models are increasingly being used as surrogate for mammalian models. However, there are concerns whether these models recapitulate the complexity of host-pathogen interactions. Here, we developed theex vivolung perfusion (EVLP) model of infection using porcine lungs to investigateKlebsiella pneumoniae-triggered pneumonia as model of respiratory infections. The porcine EVLP model recapitulates features ofK. pneumoniae-induced pneumonia lung injury. This model is also useful to assess the pathogenic potential ofK. pneumoniaeas we observed that the attenuatedKlebsiellacapsule mutant strain caused less pathological tissue damage with a concomitant decrease in the bacterial burden compare to lungs infected with the wild type. The porcine EVLP model allows assessment of inflammatory responses following infection; similar to the mouse pneumonia model, we observed an increase ofil-10in the lungs infected with the wild type and an increase ofifn-γin lungs infected with the capsule mutant. This model also allows monitoring phenotypes at the single-cell level. Wild-typeK. pneumoniaeskews macrophages towards an M2-like state. In vitro experiments probing pig bone marrow-derived macrophages uncovered the role of the M2 transcriptional factor STAT6, and thatKlebsiella-inducedil10expression is controlled by p38 and ERK.Klebsiella-induced macrophage polarization is dependent on the capsule. Altogether, this study support the utility of the EVLP model using pig lungs as platform to investigate the infection biology of respiratory pathogens.IMPORTANCEThe implementation of infection models that approximate human disease is essential to understand infections and for testing new therapies before they enter into clinical stages. Rodents are used in most of pre-clinical studies, although the differences between mouse and man have fuelled the conclusion that murine studies are unreliable predictors of human outcomes. Here, we have developed a whole lung porcine model of infection using the establishedex vivolung perfusion (EVLP) system established to re-condition human lungs for transplant. As a proof-of-principle, we provide evidence demonstrating that infection of the porcine EVLP with the human pathogenK. pneumoniaerecapitulates the known features ofKlebsiella-triggered pneumonia. Moreover, our data revealed the porcine EVLP model is useful to reveal features of the virulence ofK. pneumoniaeincluding the manipulation of immune cells. Altogether, this study supports the utility of the EVLP model using pig lungs as surrogate host for assessing respiratory infections.

Background Acute 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/design This 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 registration http://ISRCTN95690673