A novel mechanoeffector role of fibroblast S100A4 in myofibroblast transdifferentiation and fibrosis.

Fibroblast to myofibroblast transdifferentiation mediates numerous fibrotic disorders, such as idiopathic pulmonary fibrosis (IPF). We have previously demonstrated that non-muscle myosin II (NMII) is activated in response to fibrotic lung extracellular matrix, thereby mediating myofibroblast transdifferentiation. NMII-A is known to interact with the calcium-binding protein S100A4, but the mechanism by which S100A4 regulates fibrotic disorders is unclear. In this study, we show that fibroblast S100A4 is a calcium-dependent, mechanoeffector protein that is uniquely sensitive to pathophysiologic-range lung stiffness (8-25 kPa) and thereby mediates myofibroblast transdifferentiation. Re-expression of endogenous fibroblast S100A4 rescues the myofibroblastic phenotype in S100A4 KO fibroblasts. Analysis of NMII-A/actin dynamics reveals that S100A4 mediates the unraveling and redistribution of peripheral actomyosin to a central location, resulting in a contractile myofibroblast. Furthermore, S100A4 loss protects against murine in vivo pulmonary fibrosis, and S100A4 expression is dysregulated in IPF. Our data reveal a novel mechanosensor/effector role for endogenous fibroblast S100A4 in inducing cytoskeletal redistribution in fibrotic disorders such as IPF.

TRPV4 Regulates the Macrophage Metabolic Response to Limit Sepsis-induced Lung Injury.

Sepsis is a systemic inflammatory response that requires effective macrophage metabolic functions to resolve ongoing inflammation. Previous work showed that the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), mediates macrophage phagocytosis and cytokine production in response to lung infection. Here, we show that TRPV4 regulates glycolysis in a stiffness-dependent manner by augmenting macrophage glucose uptake by GLUT1. In addition, TRPV4 is required for LPS-induced phagolysosome maturation in a GLUT1-dependent manner. In a cecal slurry mouse model of sepsis, TRPV4 regulates sepsis-induced glycolysis as measured by BAL fluid (BALF) lactate and sepsis-induced lung injury as measured by BALF total protein and lung compliance. TRPV4 is necessary for bacterial clearance in the peritoneum to limit sepsis-induced lung injury. It is interesting that BALF lactate is increased in patients with sepsis compared with healthy control participants, supporting the relevance of lung cell glycolysis to human sepsis. These data show that macrophage TRPV4 is required for glucose uptake through GLUT1 for effective phagolysosome maturation to limit sepsis-induced lung injury. Our work presents TRPV4 as a potential target to protect the lung from injury in sepsis.

Antipsychotic Use for ICU Delirium and Associated Inflammatory Markers.

Purpose: We sought to evaluate critically ill patients with delirium to evaluate inflammatory cytokine production and delirium progression and the role of antipsychotics. Materials and Methods: Adult critically ill patients with confirmed delirium according to a positive CAM-ICU score were included and IL-6 and IL-8 levels were trended for 24 h in this single-center, prospective, observational cohort study. Results: A total of 23 patients were consented and had blood samples drawn for inclusion. There was no difference in IL-6 and IL-8 levels at baseline, 4 to 8 h, and 22 to 28 h after enrollment when comparing patients based on antipsychotic exposure. We identified 2 patient clusters based on age, APACHE III, need for mechanical ventilation, and concomitant infection. In cluster 1, 5 (33.3%) patients received antipsychotics versus 5 (62.5%) patients in cluster 2 (P = .18). Patients in cluster 1 had more co-inflammatory conditions (P < .0001), yet numerically lower baseline IL-6 (P = .18) and IL-8 levels (P = .80) compared to cluster 2. Patients in cluster 1 had a greater median number of delirium-free days compared to cluster 2 (17.0 vs 6.0 days; P = .05). Conclusions: In critically ill patients with delirium, IL-6 and IL-8 levels were variable and antipsychotics were not associated with improvements in delirium or inflammatory markers.

Adaptive exhaustion during prolonged intermittent hypoxia causes dysregulated skeletal muscle protein homeostasis.

Nocturnal hypoxaemia, which is common in chronic obstructive pulmonary disease (COPD) patients, is associated with skeletal muscle loss or sarcopenia, which contributes to adverse clinical outcomes. In COPD, we have defined this as prolonged intermittent hypoxia (PIH) because the duration of hypoxia in skeletal muscle occurs through the duration of sleep followed by normoxia during the day, in contrast to recurrent brief hypoxic episodes during obstructive sleep apnoea (OSA). Adaptive cellular responses to PIH are not known. Responses to PIH induced by three cycles of 8 h hypoxia followed by 16 h normoxia were compared to those during chronic hypoxia (CH) or normoxia for 72 h in murine C2C12 and human inducible pluripotent stem cell-derived differentiated myotubes. RNA sequencing followed by downstream analyses were complemented by experimental validation of responses that included both unique and shared perturbations in ribosomal and mitochondrial function during PIH and CH. A sarcopenic phenotype characterized by decreased myotube diameter and protein synthesis, and increased phosphorylation of eIF2α (Ser51) by eIF2α kinase, and of GCN-2 (general controlled non-derepressed-2), occurred during both PIH and CH. Mitochondrial oxidative dysfunction, disrupted supercomplex assembly, lower activity of Complexes I, III, IV and V, and reduced intermediary metabolite concentrations occurred during PIH and CH. Decreased mitochondrial fission occurred during CH. Physiological relevance was established in skeletal muscle of mice with COPD that had increased phosphorylation of eIF2α, lower protein synthesis and mitochondrial oxidative dysfunction. Molecular and metabolic responses with PIH suggest an adaptive exhaustion with failure to restore homeostasis during normoxia. KEY POINTS: Sarcopenia or skeletal muscle loss is one of the most frequent complications that contributes to mortality and morbidity in patients with chronic obstructive pulmonary disease (COPD). Unlike chronic hypoxia, prolonged intermittent hypoxia is a frequent, underappreciated and clinically relevant model of hypoxia in patients with COPD. We developed a novel, in vitro myotube model of prolonged intermittent hypoxia with molecular and metabolic perturbations, mitochondrial oxidative dysfunction, and consequent sarcopenic phenotype. In vivo studies in skeletal muscle from a mouse model of COPD shared responses with our myotube model, establishing the pathophysiological relevance of our studies. These data lay the foundation for translational studies in human COPD to target prolonged, nocturnal hypoxaemia to prevent sarcopenia in these patients.

Vasopressin Response and Clinical Trajectory in Septic Shock Patients.

BACKGROUND: In septic shock, vasopressors aim to improve tissue perfusion and prevent persistent organ dysfunction, a characteristic of chronic critical illness (CCI). Adjunctive vasopressin is often used to decrease catecholamine dosage, but the association of vasopressin response with subsequent patient outcomes is unclear. We hypothesized vasopressin response is associated with favorable clinical trajectory. METHODS: We included patients with septic shock receiving vasopressin as a catecholamine adjunct in this retrospective cohort study. We defined vasopressin response as a lowering of the catecholamine dose required to maintain mean arterial pressure ≥65 mm Hg, 6 h after vasopressin initiation. Clinical trajectories were adjudicated as early death (ED; death before day 14), CCI (ICU stay ≥14 days with persistent organ dysfunction), or rapid recovery (RR; not meeting ED or CCI criteria). Trajectories were placed on an ordinal scale with ED the worst outcome, CCI next, and RR the best outcome. The association of vasopressin response with clinical trajectory was assessed with multivariable ordinal logistic regression. RESULTS: In total 938 patients were included; 426 (45.4%) were vasopressin responders. The most frequent trajectory was ED (49.8%), 29.7% developed CCI, and 20.5% had rapid recovery. In survivors to ICU day 14 (those without ED), 59.2% had CCI and 40.8% experienced RR. Compared with vasopressin non-responders, vasopressin responders less frequently experienced ED (42.5% vs. 55.9%) and more frequently experienced RR (24.6% vs. 17.0%; P < 0.01). After controlling for confounders, vasopressin response was independently associated with higher odds of developing a better clinical trajectory (OR 1.63; 95% CI 1.26-2.10). Medical patients most frequently developed ED and survivors more commonly developed CCI than RR; surgical patients developed the three trajectories with similar frequency (P < 0.01). CONCLUSIONS: Vasopressin responsive status was associated with improved clinical trajectory in septic shock patients. Early vasopressin response is a potential novel prognostic marker for short-term clinical trajectory.

TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching.

Mechanical cell-matrix interactions can drive the innate immune responses to infection; however, the molecular underpinnings of these responses remain elusive. This study was undertaken to understand the molecular mechanism by which the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), alters the in vivo response to lung infection. For the first time, to our knowledge, we show that TRPV4 protects the lung from injury upon intratracheal Pseudomonas aeruginosa in mice. TRPV4 functions to enhance macrophage bacterial clearance and downregulate proinflammatory cytokine secretion. TRPV4 mediates these effects through a novel mechanism of molecular switching of LPS signaling from predominant activation of the MAPK, JNK, to that of p38. This is accomplished through the activation of the master regulator of inflammation, dual-specificity phosphatase 1. Further, TRPV4's modulation of the LPS signal is mechanosensitive in that both upstream activation of p38 and its downstream biological consequences depend on pathophysiological range extracellular matrix stiffness. We further show the importance of TRPV4 on LPS-induced activation of macrophages from healthy human controls. These data are the first, to our knowledge, to demonstrate new roles for macrophage TRPV4 in regulating innate immunity in a mechanosensitive manner through the modulation of dual-specificity phosphatase 1 expression to mediate MAPK activation switching.

RSV infection potentiates TRPV1-mediated calcium transport in bronchial epithelium of asthmatic children.

The transient receptor potential vanilloid 1 (TRPV1) channel is expressed in human bronchial epithelium (HBE), where it transduces Ca2+ in response to airborne irritants. TRPV1 activation results in bronchoconstriction, cough, and mucus production, and may therefore contribute to the pathophysiology of obstructive airway disease. Since children with asthma face the greatest risk of developing virus-induced airway obstruction, we hypothesized that changes in TRPV1 expression, localization, and function in the airway epithelium may play a role in bronchiolitis and asthma in childhood. We sought to measure TRPV1 protein expression, localization, and function in HBE cells from children with versus without asthma, both at baseline and after RSV infection. We determined changes in TRPV1 protein expression, subcellular localization, and function both at baseline and after RSV infection in primary HBE cells from normal children and children with asthma. Basal TRPV1 protein expression was higher in HBE from children with versus without asthma and primarily localized to plasma membranes (PMs). During RSV infection, TRPV1 protein increased more in the PM of asthmatic HBE as compared with nonasthmatic cells. TRPV1-mediated increase in intracellular Ca2+ was greater in RSV-infected asthmatic cells, but this increase was attenuated when extracellular Ca2+ was removed. Nerve growth factor (NGF) recapitulated the effect of RSV on TRPV1 activation in HBE cells. Our data suggest that children with asthma have intrinsically hyperreactive airways due in part to higher TRPV1-mediated Ca2+ influx across epithelial membranes, and this abnormality is further exacerbated by NGF overexpression during RSV infection driving additional Ca2+ from intracellular stores.

Choice of antifibrotic medication and disease severity predict weight loss in idiopathic pulmonary fibrosis.

PURPOSE: Although safety and tolerability of approved antifibrotics has been reported extensively, little is known about their effects on weight. We analyzed predictors of weight change after one year of uninterrupted antifibrotic therapy in patients followed at our institution's interstitial lung disease clinic. METHODS/RESULTS: We identified 80 patients on antifibrotic therapy (44 pirfenidone/36 nintedanib) with at least one year of follow-up and no therapy interruptions. Thirty-five patients (44%) lost more than 5% of their baseline body weight, and 11 (19%) lost more than 10%. A higher proportion of patients on nintedanib experienced a clinically significant weight loss (>5%) versus pirfenidone (61% vs 30%, p = 0.005). Univariate and multivariate analyses identified nintedanib therapy and a higher composite physiologic index (CPI) as predictors of weight loss. CONCLUSIONS: Weight loss is common among IPF patients on antifibrotic therapy. Nintedanib therapy and more advanced disease were identified as predictors of weight loss in this population.

TRPV4 Mechanosensitive Ion Channel Regulates Lipopolysaccharide-Stimulated Macrophage Phagocytosis.

Macrophage phagocytosis of particles and pathogens is an essential aspect of innate host defense. Phagocytic function requires cytoskeletal rearrangements that depend on the interaction between macrophage surface receptors, particulates/pathogens, and the extracellular matrix. In the present study we determine the role of a mechanosensitive ion channel, transient receptor potential vanilloid 4 (TRPV4), in integrating the LPS and matrix stiffness signals to control macrophage phenotypic change for host defense and resolution from lung injury. We demonstrate that active TRPV4 mediates LPS-stimulated murine macrophage phagocytosis of nonopsonized particles (Escherichia coli) in vitro and opsonized particles (IgG-coated latex beads) in vitro and in vivo in intact mice. Intriguingly, matrix stiffness in the range seen in inflamed or fibrotic lung is required to sensitize the TRPV4 channel to mediate the LPS-induced increment in macrophage phagocytosis. Furthermore, TRPV4 is required for the LPS induction of anti-inflammatory/proresolution cytokines. These findings suggest that signaling through TRPV4, triggered by changes in extracellular matrix stiffness, cooperates with LPS-induced signals to mediate macrophage phagocytic function and lung injury resolution. These mechanisms are likely to be important in regulating macrophage function in the context of pulmonary infection and fibrosis.

Matrix-driven Myosin II Mediates the Pro-fibrotic Fibroblast Phenotype.

Pro-fibrotic mesenchymal cells are known to be the key effector cells of fibroproliferative disease, but the specific matrix signals and the induced cellular responses that drive the fibrogenic phenotype remain to be elucidated. The key mediators of the fibroblast fibrogenic phenotype were characterized using a novel assay system that measures fibroblast behavior in response to actual normal and fibrotic lung tissue. Using this system, we demonstrate that normal lung promotes fibroblast motility and polarization, while fibrotic lung immobilizes the fibroblast and promotes myofibroblast differentiation. These context-specific phenotypes are surprisingly both mediated by myosin II. The role of myosin II is supported by the observation of an increase in myosin phosphorylation and a change in intracellular distribution in fibroblasts on fibrotic lung, as compared with normal lung. Moreover, loss of myosin II activity has opposing effects on protrusive activity in fibroblasts on normal and fibrotic lung. Loss of myosin II also selectively inhibits myofibroblast differentiation in fibroblasts on fibrotic lung. Importantly, these findings are recapitulated by varying the matrix stiffness of polyacrylamide gels in the range of normal and fibrotic lung tissue. Comparison of the effects of myosin inhibition on lung tissue with that of polyacrylamide gels suggests that matrix fiber organization drives the fibroblast phenotype under conditions of normal/soft lung, while matrix stiffness drives the phenotype under conditions of fibrotic/stiff lung. This work defines novel roles for myosin II as a key regulatory effector molecule of the pro-fibrotic phenotype, in response to biophysical properties of the matrix.

Activation of heat shock response augments fibroblast growth factor-1 expression in wounded lung epithelium.

We previously showed that coincident exposure to heat shock (HS; 42°C for 2 h) and TNF-α synergistically induces apoptosis in mouse lung epithelium. We extended this work by analyzing HS effects on human lung epithelial responses to clinically relevant injury. Cotreatment with TNF-α and HS induced little caspase-3 and poly(ADP-ribose) polymerase cleavage in human small airway epithelial cells, A549 cells, and BEAS2B cells. Scratch wound closure rates almost doubled when A549 and BEAS2B cells and air-liquid interface cultures of human bronchial epithelial cells were heat shocked immediately after wounding. Microarray, qRT-PCR, and immunoblotting showed fibroblast growth factor 1 (FGF1) to be synergistically induced by HS and wounding. Enhanced FGF1 expression in HS/wounded A549 was blocked by inhibitors of p38 MAPK (SB203580) or HS factor (HSF)-1 (KNK-437) and in HSF1 knockout BEAS2B cells. PCR demonstrated FGF1 to be expressed from the two most distal promoters in wounded/HS cells. Wound closure in HS A549 and BEAS2B cells was reduced by FGF receptor-1/3 inhibition (SU-5402) or FGF1 depletion. Exogenous FGF1 accelerated A549 wound closure in the absence but not presence of HS. In the presence of exogenous FGF1, HS slowed wound closure, suggesting that it increases FGF1 expression but impairs FGF1-stimulated wound closure. Frozen sections from normal and idiopathic pulmonary fibrosis (IPF) lung were analyzed for FGF1 and HSP70 by immunofluorescence confocal microscopy and qRT-PCR. FGF1 and HSP70 mRNA levels were 7.5- and 5.9-fold higher in IPF than normal lung, and the proteins colocalized to fibroblastic foci in IPF lung. We conclude that HS signaling may have an important impact on gene expression contributing to lung injury, healing, and fibrosis.

Nocturnal Hypoxemia Is Associated with Sarcopenia in COPD Patients.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide. Our previous studies have identified that nocturnal hypoxemia causes skeletal muscle loss (i.e. sarcopenia) in in vitro models of COPD. RATIONALE: We aimed to extend our preclinical mechanistic findings by analyzing a large sleep registry to determine whether nocturnal hypoxemia is associated with sarcopenia in COPD patients. METHODS: Sleep studies from COPD patients (n=479) and control subjects without COPD (n=275) were analyzed. Patients with obstructive sleep apnea (OSA), as defined by apnea hypopnea index >5, were excluded. Pectoralis muscle cross sectional area (PMcsa) was quantified using CT scans performed within one year of the sleep study. We defined sarcopenia as less than the lowest 20% residuals for PMcsa of controls, which was adjusted for age, BMI, and stratified by sex. Youden's optimal cutpoint criteria was used to predict sarcopenia based on mean oxygen saturation (mean SaO2) during sleep. Additional measures of nocturnal hypoxemia were analyzed. Pectoralis muscle index (PMI) was defined as PMcsa normalized to BMI. RESULTS: On average, COPD males had 16.6% lower PMI than control males (1.41+0.44 vs 1.69+0.56 cm2/BMI, p<0.001), while COPD females had 9.4% lower PMI than control females (0.96+0.27 vs 1.06+0.33 cm2/BMI, p<0.001). COPD males with nocturnal hypoxemia had a 9.5% decrease in PMI versus COPD with normal O2 (1.33+0.39 vs 1.47+0.46 cm2/BMI, p<0.05), and 23.6% decrease compared to controls (1.33+0.39 vs 1.74+0.56 cm2/BMI, p<0.001). COPD females with nocturnal hypoxemia had a 11.2% decrease versus COPD with normal O2 (0.87+0.26 vs 0.98+0.28 cm2/BMI, p<0.05), and 17.9% decrease compared to controls (0.87+0.26 vs 1.06+0.33 cm2/BMI, p<0.001). These findings were largely replicated using multiple measures of nocturnal hypoxemia. CONCLUSIONS: We defined sarcopenia in the pectoralis muscle using residuals that take into account age, BMI, and sex. We found that COPD patients have lower PMI than non-COPD patients, and that nocturnal hypoxemia was associated with an additional decrease in the PMI of COPD patients. Additional prospective analyses are needed to determine a protective threshold of oxygen saturation to prevent or reverse sarcopenia due to nocturnal hypoxemia in COPD.

Forecasting disease trajectories in critical illness: comparison of probabilistic dynamic systems to static models to predict patient status in the intensive care unit.

OBJECTIVE: Conventional prediction models fail to integrate the constantly evolving nature of critical illness. Alternative modelling approaches to study dynamic changes in critical illness progression are needed. We compare static risk prediction models to dynamic probabilistic models in early critical illness. DESIGN: We developed models to simulate disease trajectories of critically ill COVID-19 patients across different disease states. Eighty per cent of cases were randomly assigned to a training and 20% of the cases were used as a validation cohort. Conventional risk prediction models were developed to analyse different disease states for critically ill patients for the first 7 days of intensive care unit (ICU) stay. Daily disease state transitions were modelled using a series of multivariable, multinomial logistic regression models. A probabilistic dynamic systems modelling approach was used to predict disease trajectory over the first 7 days of an ICU admission. Forecast accuracy was assessed and simulated patient clinical trajectories were developed through our algorithm. SETTING AND PARTICIPANTS: We retrospectively studied patients admitted to a Cleveland Clinic Healthcare System in Ohio, for the treatment of COVID-19 from March 2020 to December 2022. RESULTS: 5241 patients were included in the analysis. For ICU days 2-7, the static (conventional) modelling approach, the accuracy of the models steadily decreased as a function of time, with area under the curve (AUC) for each health state below 0.8. But the dynamic forecasting approach improved its ability to predict as a function of time. AUC for the dynamic forecasting approach were all above 0.90 for ICU days 4-7 for all states. CONCLUSION: We demonstrated that modelling critical care outcomes as a dynamic system improved the forecasting accuracy of the disease state. Our model accurately identified different disease conditions and trajectories, with a <10% misclassification rate over the first week of critical illness.

Effect of Ethanol-exposure on Innate Immune Response in Sepsis.

Alcohol use disorder, reported by one in eight critically ill patients, is a risk factor for death in sepsis patients. Sepsis, the leading cause of death kills over 270,000 patients in the United States alone and remains without targeted therapy. Immune response in sepsis transitions from an early hyper-inflammation to persistent inflammation and immunosuppression and multiple organ dysfunction during late sepsis. Innate immunity is the first line of defense against pathogen invasion. Ethanol exposure is known to impair innate and adaptive immune response and bacterial clearance in sepsis patients. Specifically, ethanol-exposure is known to modulate every aspect of innate immune response with and without sepsis. Multiple molecular mechanisms are implicated in causing dysregulated immune response in ethanol-exposure with sepsis, but targeted treatments have remained elusive. In this manuscript, we outline the effects of ethanol-exposure on various innate immune cell types in general and during sepsis.

Nonbronchoscopic Bronchoalveolar Lavage Improves Respiratory Culture Accuracy in Critically Ill Patients.

OBJECTIVES: Diagnosis of pneumonia is challenging in critically ill, intubated patients due to limited diagnostic modalities. Endotracheal aspirate (EA) cultures are standard of care in many ICUs; however, frequent EA contamination leads to unnecessary antibiotic use. Nonbronchoscopic bronchoalveolar lavage (NBBL) obtains sterile, alveolar cultures, avoiding contamination. However, paired NBBL and EA sampling in the setting of a lack of gold standard for airway culture is a novel approach to improve culture accuracy and limit antibiotic use in the critically ill patients. DESIGN: We designed a pilot study to test respiratory culture accuracy between EA and NBBL. Adult, intubated patients with suspected pneumonia received concurrent EA and NBBL cultures by registered respiratory therapists. Respiratory culture microbiology, cell counts, and antibiotic prescribing practices were examined. SETTING: We performed a prospective pilot study at the Cleveland Clinic Main Campus Medical ICU in Cleveland, Ohio for 22 months from May 2021 through March 2023. PATIENTS OR SUBJECTS: Three hundred forty mechanically ventilated patients with suspected pneumonia were screened. Two hundred fifty-seven patients were excluded for severe hypoxia (Fio2 ≥ 80% or positive end-expiratory pressure ≥ 12 cm H2O), coagulopathy, platelets less than 50,000, hemodynamic instability as determined by the treating team, and COVID-19 infection to prevent aerosolization of the virus. INTERVENTIONS: All 83 eligible patients were enrolled and underwent concurrent EA and NBBL. MEASUREMENTS AND MAIN RESULTS: More EA cultures (42.17%) were positive than concurrent NBBL cultures (26.51%, p = 0.049), indicating EA contamination. The odds of EA contamination increased by eight-fold 24 hours after intubation. EA was also more likely to be contaminated with oral flora when compared with NBBL cultures. There was a trend toward decreased antibiotic use in patients with positive EA cultures if paired with a negative NBBL culture. Alveolar immune cell populations were recovered from NBBL samples, indicating successful alveolar sampling. There were no major complications from NBBL. CONCLUSIONS: NBBL is more accurate than EA for respiratory cultures in critically ill, intubated patients. NBBL provides a safe and effective technique to sample the alveolar space for both clinical and research purposes.

Design and rationale of the CHILL phase II trial of hypothermia and neuromuscular blockade for acute respiratory distress syndrome.

The Cooling to Help Injured Lungs (CHILL) trial is an open label, two group, parallel design multicenter, randomized phase IIB clinical trial assessing the efficacy and safety of targeted temperature management with combined external cooling and neuromuscular blockade to block shivering in patients with early moderate-severe acute respiratory distress syndrome (ARDS). This report provides the background and rationale for the clinical trial and outlines the methods using the Consolidated Standards of Reporting Trials guidelines. Key design challenges include: [1] protocolizing important co-interventions; [2] incorporation of patients with COVID-19 as the cause of ARDS; [3] inability to blind the investigators; and [4] ability to obtain timely informed consent from patients or legally authorized representatives early in the disease process. Results of the Reevaluation of Systemic Early Neuromuscular Blockade (ROSE) trial informed the decision to mandate sedation and neuromuscular blockade only in the group assigned to therapeutic hypothermia and proceed without this mandate in the control group assigned to a usual temperature management protocol. Previous trials conducted in National Heart, Lung, and Blood Institute ARDS Clinical Trials (ARDSNet) and Prevention and Early Treatment of Acute Lung Injury (PETAL) Networks informed ventilator management, ventilation liberation and fluid management protocols. Since ARDS due to COVID-19 is a common cause of ARDS during pandemic surges and shares many features with ARDS from other causes, patients with ARDS due to COVID-19 are included. Finally, a stepwise approach to obtaining informed consent prior to documenting critical hypoxemia was adopted to facilitate enrollment and reduce the number of candidates excluded because eligibility time window expiration.