TRPV2 modulates mechanically Induced ATP Release from Human bronchial epithelial cells.

Repetitive bouts of coughing expose the large airways to significant cycles of shear stress. This leads to the release of alarmins and the tussive agent adenosine triphosphate (ATP) which may be modulated by the activity of ion channels present in the human airway. This study aimed to investigate the role of the transient receptor potential subfamily vanilloid member 2 (TRPV2) channel in mechanically induced ATP release from primary bronchial epithelial cells (PBECs).PBECs were obtained from individuals undergoing bronchoscopy. They were cultured in vitro and exposed to mechanical stress in the form of compressive and fluid shear stress (CFSS) or fluid shear stress (FSS) alone at various intensities. ATP release was measured using a luciferin-luciferase assay. Functional TRPV2 protein expression in human PBECs was investigated by confocal calcium imaging. The role of TRPV2 inhibition on FSS-induced ATP release was investigated using the TRPV2 inhibitor tranilast or siRNA knockdown of TRPV2. TRPV2 protein expression in human lung tissue was also determined by immunohistochemistry.ATP release was significantly increased in PBECs subjected to CFSS compared with control (unstimulated) PBECs (N = 3, ***P < 0.001). PBECs expressed functional TRPV2 channels. TRPV2 protein was also detected in fixed human lung tissue. ATP release from FFS stimulated PBECs was decreased by the TRPV2 inhibitor tranilast (N = 3, **P < 0.01) (vehicle: 159 ± 17.49 nM, tranilast: 25.08 ± 5.1 nM) or by TRPV2 siRNA knockdown (N = 3, *P < 0.05) (vehicle: 197 ± 24.52 nM, siRNA: 119 ± 26.85 nM).In conclusion, TRPV2 is expressed in the human airway and modulates ATP release from mechanically stimulated PBECs.

Vascular leak in sepsis: physiological basis and potential therapeutic advances.

Sepsis is a life-threatening condition characterised by endothelial barrier dysfunction and impairment of normal microcirculatory function, resulting in a state of hypoperfusion and tissue oedema. No specific pharmacological therapies are currently used to attenuate microvascular injury. Given the prominent role of endothelial breakdown and microcirculatory dysfunction in sepsis, there is a need for effective strategies to protect the endothelium. In this review we will discuss key mechanisms and putative therapeutic agents relevant to endothelial barrier function.

Patient stratification using plasma cytokines and their regulators in sepsis: relationship to outcomes, treatment effect and leucocyte transcriptomic subphenotypes.

RATIONALE: Heterogeneity of the host response within sepsis, acute respiratory distress syndrome (ARDS) and more widely critical illness, limits discovery and targeting of immunomodulatory therapies. Clustering approaches using clinical and circulating biomarkers have defined hyper-inflammatory and hypo-inflammatory subphenotypes in ARDS associated with differential treatment response. It is unknown if similar subphenotypes exist in sepsis populations where leucocyte transcriptomic-defined subphenotypes have been reported. OBJECTIVES: We investigated whether inflammatory clusters based on cytokine protein abundance were seen in sepsis, and the relationships with previously described transcriptomic subphenotypes. METHODS: Hierarchical cluster and latent class analysis were applied to an observational study (UK Genomic Advances in Sepsis (GAinS)) (n=124 patients) and two clinical trial datasets (VANISH, n=155 and LeoPARDS, n=484) in which the plasma protein abundance of 65, 21, 11 circulating cytokines, cytokine receptors and regulators were quantified. Clinical features, outcomes, response to trial treatments and assignment to transcriptomic subphenotypes were compared between inflammatory clusters. MEASUREMENTS AND MAIN RESULTS: We identified two (UK GAinS, VANISH) or three (LeoPARDS) inflammatory clusters. A group with high levels of pro-inflammatory and anti-inflammatory cytokines was seen that was associated with worse organ dysfunction and survival. No interaction between inflammatory clusters and trial treatment response was found. We found variable overlap of inflammatory clusters and leucocyte transcriptomic subphenotypes. CONCLUSIONS: These findings demonstrate that differences in response at the level of cytokine biology show clustering related to severity, but not treatment response, and may provide complementary information to transcriptomic sepsis subphenotypes. TRIAL REGISTRATION NUMBER: ISRCTN20769191, ISRCTN12776039.

Human mesenchymal stromal cells inhibit Mycobacterium avium replication in clinically relevant models of lung infection.

INTRODUCTION: Novel therapeutic strategies are urgently needed for Mycobacterium avium complex pulmonary disease (MAC-PD). Human mesenchymal stromal cells (MSCs) can directly inhibit MAC growth, but their effect on intracellular bacilli is unknown. We investigated the ability of human MSCs to reduce bacterial replication and inflammation in MAC-infected macrophages and in a murine model of MAC-PD. METHODS: Human monocyte-derived macrophages (MDMs) were infected with M. avium Chester strain and treated with human bone marrow-derived MSCs. Intracellular and extracellular colony-forming units (CFUs) were counted at 72 hours. Six-week-old female balb/c mice were infected by nebulisation of M. avium Chester. Mice were treated with 1×106 intravenous human MSCs or saline control at 21 and 28 days post-infection. Lungs, liver and spleen were harvested 42 days post-infection for bacterial counts. Cytokines were quantified by ELISA. RESULTS: MSCs reduced intracellular bacteria in MDMs over 72 hours (median 35% reduction, p=0.027). MSC treatment increased extracellular concentrations of prostaglandin E2 (PGE2) (median 10.1-fold rise, p=0.002) and reduced tumour necrosis factor-α (median 28% reduction, p=0.025). Blocking MSC PGE2 production by cyclo-oxygenase-2 (COX-2) inhibition with celecoxib abrogated the antimicrobial effect, while this was restored by adding exogenous PGE2. MSC-treated mice had lower pulmonary CFUs (median 18% reduction, p=0.012), but no significant change in spleen or liver CFUs compared with controls. CONCLUSION: MSCs can modulate inflammation and reduce intracellular M. avium growth in human macrophages via COX-2/PGE2 signalling and inhibit pulmonary bacterial replication in a murine model of chronic MAC-PD.

Cell-based Therapies for Acute Respiratory Distress Syndrome: Where Are We Now?

There is considerable interest in the potential for cell-based therapies, particularly mesenchymal stromal cells (MSCs) and their products, as a therapy for acute respiratory distress syndrome (ARDS). MSCs exert effects via diverse mechanisms including reducing excessive inflammation by modulating neutrophil, macrophage and T-cell function, decreasing pulmonary permeability and lung edema, and promoting tissue repair. Clinical studies indicate that MSCs are safe and well tolerated, with promising therapeutic benefits in specific clinical settings, leading to regulatory approvals of MSCs for specific indications in some countries.This perspective reassesses the therapeutic potential of MSC-based therapies for ARDS given insights from recent cell therapy trials in both COVID-19 and in 'classic' ARDS, and discusses studies in graft-vs.-host disease, one of the few licensed indications for MSC therapies. We identify important unknowns in the current literature, address challenges to clinical translation, and propose an approach to facilitate assessment of the therapeutic promise of MSC-based therapies for ARDS.

The Effect of Lower Tidal Volume Ventilation Facilitated by Extracorporeal Carbon Dioxide Removal Compared With Conventional Lung Protective Ventilation on Cardiac Function.

OBJECTIVES: Lower tidal volume ventilation (targeting 3 mL/kg predicted body weight, PBW) facilitated by extracorporeal carbon dioxide removal (ECCO2R) has been investigated as a potential therapy for acute hypoxemic respiratory failure (AHRF) in the pRotective vEntilation with veno-venouS lung assisT in respiratory failure (REST) trial. We investigated the effect of this strategy on cardiac function, and in particular the right ventricle. DESIGN: Substudy of the REST trial. SETTING: Nine U.K. ICUs. PATIENTS: Patients with AHRF (Pao2/Fio2 < 150 mm Hg [20 kPa]). INTERVENTION: Transthoracic echocardiography and N-terminal pro-B-type natriuretic peptide (NT-proBNP) measurements were collected at baseline and postrandomization in patients randomized to ECCO2R or usual care. MEASUREMENTS: The primary outcome measures were a difference in tricuspid annular plane systolic excursion (TAPSE) on postrandomization echocardiogram and difference in NT-proBNP postrandomization. RESULTS: There were 21 patients included in the echocardiography cohort (ECCO2R, n = 13; usual care, n = 8). Patient characteristics were similar in both groups at baseline. Median (interquartile range) tidal volumes were lower in the ECCO2R group compared with the usual care group postrandomization; 3.6 (3.1-4.2) mL/kg PBW versus 5.2 (4.9-5.7) mL/kg PBW, respectively (p = 0.01). There was no difference in the primary outcome measure of mean (sd) TAPSE in the ECCO2R and usual care groups postrandomization; 21.3 (5.4) mm versus 20.1 (3.2) mm, respectively (p = 0.60). There were 75 patients included in the NT-proBNP cohort (ECCO2R, n = 36; usual care, n = 39). Patient characteristics were similar in both groups at baseline. Median (interquartile range [IQR]) tidal volumes were lower in the ECCO2R group than the usual care group postrandomization; 3.8 (3.3-4.2) mL/kg PBW versus 6.7 (5.8-8.1) mL/kg PBW, respectively (p < 0.0001). There was no difference in median (IQR) NT-proBNP postrandomization; 1121 (241-5370) pg/mL versus 1393 (723-4332) pg/mL in the ECCO2R and usual care groups, respectively (p = 0.30). CONCLUSIONS: In patients with AHRF, a reduction in tidal volume facilitated by ECCO2R, did not modify cardiac function.

Elevated ferritin, mediated by IL-18 is associated with systemic inflammation and mortality in acute respiratory distress syndrome (ARDS).

BACKGROUND: Inflammatory subphenotypes have been identified in acute respiratory distress syndrome (ARDS). Hyperferritinaemia in sepsis is associated with hyperinflammation, worse clinical outcomes, and may predict benefit with immunomodulation. Our aim was to determine if raised ferritin identified a subphenotype in patients with ARDS. METHODS: Baseline plasma ferritin concentrations were measured in patients with ARDS from two randomised controlled trials of simvastatin (Hydroxymethylglutaryl-CoA Reductase Inhibition with Simvastatin in Acute Lung Injury to Reduce Pulmonary Dysfunction-2 (HARP-2); discovery cohort, UK) and neuromuscular blockade (ROSE; validation cohort, USA). Results were analysed using a logistic regression model with restricted cubic splines, to determine the ferritin threshold associated with 28-day mortality. RESULTS: Ferritin was measured in 511 patients from HARP-2 (95% of patients enrolled) and 847 patients (84% of patients enrolled) from ROSE. Ferritin was consistently associated with 28-day mortality in both studies and following a meta-analysis, a log-fold increase in ferritin was associated with an OR 1.71 (95% CI 1.01 to 2.90) for 28-day mortality. Patients with ferritin >1380 ng/mL (HARP-2 28%, ROSE 24%) had a significantly higher 28-day mortality and fewer ventilator-free days in both studies. Mediation analysis, including confounders (acute physiology and chronic health evaluation-II score and ARDS aetiology) demonstrated a statistically significant contribution of interleukin (IL)-18 as an intermediate pathway between ferritin and mortality. CONCLUSIONS: Ferritin is a clinically useful biomarker in ARDS and is associated with worse patient outcomes. These results provide support for prospective interventional trials of immunomodulatory agents targeting IL-18 in this hyperferritinaemic subgroup of patients with ARDS.

Simvastatin in Critically Ill Patients with Covid-19.

BACKGROUND: The efficacy of simvastatin in critically ill patients with coronavirus disease 2019 (Covid-19) is unclear. METHODS: In an ongoing international, multifactorial, adaptive platform, randomized, controlled trial, we evaluated simvastatin (80 mg daily) as compared with no statin (control) in critically ill patients with Covid-19 who were not receiving statins at baseline. The primary outcome was respiratory and cardiovascular organ support-free days, assessed on an ordinal scale combining in-hospital death (assigned a value of -1) and days free of organ support through day 21 in survivors; the analyis used a Bayesian hierarchical ordinal model. The adaptive design included prespecified statistical stopping criteria for superiority (>99% posterior probability that the odds ratio was >1) and futility (>95% posterior probability that the odds ratio was <1.2). RESULTS: Enrollment began on October 28, 2020. On January 8, 2023, enrollment was closed on the basis of a low anticipated likelihood that prespecified stopping criteria would be met as Covid-19 cases decreased. The final analysis included 2684 critically ill patients. The median number of organ support-free days was 11 (interquartile range, -1 to 17) in the simvastatin group and 7 (interquartile range, -1 to 16) in the control group; the posterior median adjusted odds ratio was 1.15 (95% credible interval, 0.98 to 1.34) for simvastatin as compared with control, yielding a 95.9% posterior probability of superiority. At 90 days, the hazard ratio for survival was 1.12 (95% credible interval, 0.95 to 1.32), yielding a 91.9% posterior probability of superiority of simvastatin. The results of secondary analyses were consistent with those of the primary analysis. Serious adverse events, such as elevated levels of liver enzymes and creatine kinase, were reported more frequently with simvastatin than with control. CONCLUSIONS: Although recruitment was stopped because cases had decreased, among critically ill patients with Covid-19, simvastatin did not meet the prespecified criteria for superiority to control. (REMAP-CAP ClinicalTrials.gov number, NCT02735707.).

Elevated Plasma Interleukin-18 Identifies High-Risk Acute Respiratory Distress Syndrome Patients not Distinguished by Prior Latent Class Analyses Using Traditional Inflammatory Cytokines: A Retrospective Analysis of Two Randomized Clinical Trials.

OBJECTIVES: Interleukin-18 (IL-18) plasma level and latent class analysis (LCA) have separately been shown to predict prognosis and treatment response in acute respiratory distress syndrome (ARDS). IL-18 is a measure of inflammasome activation, a pathway potentially distinct from inflammation captured by biomarkers defining previously published LCA classes. We hypothesized that elevated IL-18 would identify distinct "high-risk" patients not captured by prior LCA classifications. DESIGN: Statins for acutely injured lungs from sepsis (SAILS) and hydroxymethylglutaryl-CoA reductase inhibition with simvastatin in acute lung injury to reduce pulmonary dysfunction trial (HARP-2) are two large randomized, controlled trials in ARDS in which both LCA assignments and IL-18 levels were shown to predict mortality. We first evaluated the overlap between high IL-18 levels (≥ 800 pg/mL) with prior LCA class assignments using McNemar's test and then tested the correlation between IL-18 and LCA biomarkers using Pearson's exact test on log-2 transformed values. Our primary analysis was the association of IL-18 level with 60-day mortality in the hypoinflammatory LCA class, which was assessed using the Fisher exact test and Cox proportional hazards modeling adjusting for age, Acute Physiology and Chronic Health Evaluation score, and gender. Secondary analyses included the association of IL-18 and LCA with mortality within each IL-18/LCA subgroup. SETTING: Secondary analysis of two multicenter, randomized controlled clinical trials of ARDS patients. SUBJECTS: Six hundred eighty-three patients in SAILS and 511 patients in HARP-2. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We found that 33% of patients in SAILS and HARP-2 were discordant by IL-18 level and LCA class. We further found that IL-18 level was only modestly correlated (0.17-0.47) with cytokines used in the LCA assignment. A substantial subset of individuals classified as hypoinflammatory by LCA (14% of SAILS and 43% of HARP-2) were classified as high risk by elevated IL-18. These individuals were at high risk for mortality in both SAILS (42% 60-d mortality, odds ratio [OR] 3.3; 95% CI, 1.8-6.1; p < 0.001) and HARP-2 (27% 60-d mortality, OR 2.1; 95% CI, 1.2-3.8; p = 0.009). CONCLUSIONS: Plasma IL-18 level provides important additional prognostic information to LCA subphenotypes defined largely by traditional inflammatory biomarkers in two large ARDS cohorts.

Repair of Acute Respiratory Distress Syndrome in COVID-19 by Stromal Cells (REALIST-COVID Trial): A Multicenter, Randomized, Controlled Clinical Trial.

Rationale: Mesenchymal stromal cells (MSCs) may modulate inflammation, promoting repair in coronavirus disease (COVID-19)-related acute respiratory distress syndrome (ARDS). Objectives: We investigated the safety and efficacy of ORBCEL-C (CD362 [cluster of differentiation 362]-enriched, umbilical cord-derived MSCs) in COVID-19-related ARDS. Methods: In this multicenter, randomized, double-blind, allocation-concealed, placebo-controlled trial (NCT03042143), patients with moderate to severe COVID-19-related ARDS were randomized to receive ORBCEL-C (400 million cells) or placebo (Plasma-Lyte 148). The primary safety and efficacy outcomes were the incidence of serious adverse events and oxygenation index at Day 7, respectively. Secondary outcomes included respiratory compliance, driving pressure, PaO2:FiO2 ratio, and Sequential Organ Failure Assessment score. Clinical outcomes relating to duration of ventilation, lengths of ICU and hospital stays, and mortality were collected. Long-term follow-up included diagnosis of interstitial lung disease at 1 year and significant medical events and mortality at 2 years. Transcriptomic analysis was performed on whole blood at Days 0, 4, and 7. Measurements and Main Results: Sixty participants were recruited (final analysis: n = 30 received ORBCEL-C, n = 29 received placebo; 1 participant in the placebo group withdrew consent). Six serious adverse events occurred in the ORBCEL-C group and three in the placebo group (risk ratio, 2.9 [95% confidence interval, 0.6-13.2]; P = 0.25). Day 7 mean (SD) oxygenation index did not differ (ORBCEL-C, 98.3 [57.2] cm H2O/kPa; placebo, 96.6 [67.3] cm H2O/kPa). There were no differences in secondary surrogate outcomes or in mortality at Day 28, Day 90, 1 year, or 2 years. There was no difference in the prevalence of interstitial lung disease at 1 year or significant medical events up to 2 years. ORBCEL-C modulated the peripheral blood transcriptome. Conclusion: ORBCEL-C MSCs were safe in subjects with moderate to severe COVID-19-related ARDS but did not improve surrogates of pulmonary organ dysfunction.

Effect of total cholesterol and statin therapy on mortality in ARDS patients: a secondary analysis of the SAILS and HARP-2 trials.

BACKGROUND: Two acute respiratory distress syndrome (ARDS) trials showed no benefit for statin therapy, though secondary analyses suggest inflammatory subphenotypes may have a differential response to simvastatin. Statin medications decrease cholesterol levels, and low cholesterol has been associated with increased mortality in critical illness. We hypothesized that patients with ARDS and sepsis with low cholesterol could be harmed by statins. METHODS: Secondary analysis of patients with ARDS and sepsis from two multicenter trials. We measured total cholesterol from frozen plasma samples obtained at enrollment in Statins for Acutely Injured Lungs from Sepsis (SAILS) and Simvastatin in the Acute Respiratory Distress Syndrome (HARP-2) trials, which randomized subjects with ARDS to rosuvastatin versus placebo and simvastatin versus placebo, respectively, for up to 28 days. We compared the lowest cholesterol quartile (< 69 mg/dL in SAILS, < 44 mg/dL in HARP-2) versus all other quartiles for association with 60-day mortality and medication effect. Fisher's exact test, logistic regression, and Cox Proportional Hazards were used to assess mortality. RESULTS: There were 678 subjects with cholesterol measured in SAILS and 509 subjects in HARP-2, of whom 384 had sepsis. Median cholesterol at enrollment was 97 mg/dL in both SAILS and HARP-2. Low cholesterol was associated with higher APACHE III and shock prevalence in SAILS, and higher Sequential Organ Failure Assessment score and vasopressor use in HARP-2. Importantly, the effect of statins differed in these trials. In SAILS, patients with low cholesterol who received rosuvastatin were more likely to die (odds ratio (OR) 2.23, 95% confidence interval (95% CI) 1.06-4.77, p = 0.02; interaction p = 0.02). In contrast, in HARP-2, low cholesterol patients had lower mortality if randomized to simvastatin, though this did not reach statistical significance in the smaller cohort (OR 0.44, 95% CI 0.17-1.07, p = 0.06; interaction p = 0.22). CONCLUSIONS: Cholesterol levels are low in two cohorts with sepsis-related ARDS, and those in the lowest cholesterol quartile are sicker. Despite the very low levels of cholesterol, simvastatin therapy seems safe and may reduce mortality in this group, though rosuvastatin was associated with harm.

Mesenchymal stromal cells-derived extracellular vesicles reprogramme macrophages in ARDS models through the miR-181a-5p-PTEN-pSTAT5-SOCS1 axis.

RATIONALE: A better understanding of the mechanism of action of mesenchymal stromal cells (MSCs) and their extracellular vesicles (EVs) is needed to support their use as novel therapies for acute respiratory distress syndrome (ARDS). Macrophages are important mediators of ARDS inflammatory response. Suppressor of cytokine signalling (SOCS) proteins are key regulators of the macrophage phenotype switch. We therefore investigated whether SOCS proteins are involved in mediation of the MSC effect on human macrophage reprogramming. METHODS: Human monocyte-derived macrophages (MDMs) were stimulated with lipopolysaccharide (LPS) or plasma samples from patients with ARDS (these samples were previously classified into hypo-inflammatory and hyper-inflammatory phenotype) and treated with MSC conditioned medium (CM) or EVs. Protein expression was measured by Western blot. EV micro RNA (miRNA) content was determined by miRNA sequencing. In vivo: LPS-injured C57BL/6 mice were given EVs isolated from MSCs in which miR-181a had been silenced by miRNA inhibitor or overexpressed using miRNA mimic. RESULTS: EVs were the key component of MSC CM responsible for anti-inflammatory modulation of human macrophages. EVs significantly reduced secretion of tumour necrosis factor-α and interleukin-8 by LPS-stimulated or ARDS plasma-stimulated MDMs and this was dependent on SOCS1. Transfer of miR-181a in EVs downregulated phosphatase and tensin homolog (PTEN) and subsequently activated phosphorylated signal transducer and activator of transcription 5 (pSTAT5) leading to upregulation of SOCS1 in macrophages. In vivo, EVs alleviated lung injury and upregulated pSTAT5 and SOCS1 expression in alveolar macrophages in a miR181-dependent manner. Overexpression of miR-181a in MSCs significantly enhanced therapeutic efficacy of EVs in this model. CONCLUSION: miR-181a-PTEN-pSTAT5-SOCS1 axis is a novel pathway responsible for immunomodulatory effect of MSC EVs in ARDS.

Acute respiratory distress syndrome in adults: diagnosis, outcomes, long-term sequelae, and management.

Acute respiratory distress syndrome (ARDS) is characterised by acute hypoxaemic respiratory failure with bilateral infiltrates on chest imaging, which is not fully explained by cardiac failure or fluid overload. ARDS is defined by the Berlin criteria. In this Series paper the diagnosis, management, outcomes, and long-term sequelae of ARDS are reviewed. Potential limitations of the ARDS definition and evidence that could inform future revisions are considered. Guideline recommendations, evidence, and uncertainties in relation to ARDS management are discussed. The future of ARDS strives towards a precision medicine approach, and the framework of treatable traits in ARDS diagnosis and management is explored.

Baseline plasma IL-18 may predict simvastatin treatment response in patients with ARDS: a secondary analysis of the HARP-2 randomised clinical trial.

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-1ß 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-1ß. 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.

Cathepsin S Contributes to Lung Inflammation in Acute Respiratory Distress Syndrome.

Rationale: Although the cysteine protease cathepsin S has been implicated in the pathogenesis of several inflammatory lung diseases, its role has not been examined in the context of acute respiratory distress syndrome, a condition that still lacks specific and effective pharmacological treatments. Objectives: To characterize the status of cathepsin S in acute lung inflammation and examine the role of cathepsin S in disease pathogenesis. Methods: Human and mouse model BAL fluid samples were analyzed for the presence and activity of cathepsin S and its endogenous inhibitors. Recombinant cathepsin S was instilled directly into the lungs of mice. The effects of cathepsin S knockout and pharmacological inhibition were examined in two models of acute lung injury. Protease-activated receptor-1 antagonism was used to test a possible mechanism for cathepsin S-mediated inflammation. Measurements and Main Results: Pulmonary cathepsin S concentrations and activity were elevated in acute respiratory distress syndrome, a phenotype possibly exacerbated by the loss of the endogenous antiprotease cystatin SN. Direct cathepsin S instillation into the lungs induced key pathologies of acute respiratory distress syndrome, including neutrophilia and alveolar leakage. Conversely, in murine models of acute lung injury, genetic knockdown and prophylactic or therapeutic inhibition of cathepsin S reduced neutrophil recruitment and protein leakage. Cathepsin S may partly mediate its pathogenic effects via protease-activated receptor-1, because antagonism of this receptor abrogated cathepsin S-induced airway inflammation. Conclusions: Cathepsin S contributes to acute lung injury and may represent a novel therapeutic target for acute respiratory distress syndrome.