A fibroblast-dependent TGFß1/sFRP2 noncanonical Wnt signaling axis promotes epithelial metaplasia in idiopathic pulmonary fibrosis.

Reciprocal interactions between alveolar fibroblasts and epithelial cells are crucial for lung homeostasis, injury repair, and fibrogenesis, but underlying mechanisms remain unclear. To investigate, we administered the fibroblast-selective TGFß1 signaling inhibitor, epigallocatechin gallate (EGCG), to Interstitial Lung Disease (ILD) patients undergoing diagnostic lung biopsy and conducted single-cell RNA sequencing on spare tissue. Biopsies from untreated patients showed higher fibroblast TGFß1 signaling compared to non-disease donor or end-stage ILD tissues. In vivo, EGCG downregulated TGFß1 signaling and several pro-inflammatory and stress pathways in biopsy samples. Notably, EGCG reduced fibroblast secreted frizzle-like receptor protein 2 (sFRP2), an unrecognized TGFß1 fibroblast target gene induced near type II alveolar epithelial cells (AEC2s) in situ. Using AEC2-fibroblast coculture organoids and precision cut lung slices (PCLS) from non-diseased donors, we found TGFß1 signaling promotes a spread AEC2 KRT17+ basaloid state, whereupon sFRP2 then activates a mature Krt5+ basal cell program. Wnt-receptor Frizzled 5 (Fzd5) expression and downstream calcineurin signaling were required for sFRP2-induced nuclear NFATc3 accumulation and KRT5 expression. These findings highlight stage-specific TGFß1 signaling in ILD, the therapeutic potential of EGCG in reducing IPF-related transcriptional changes, and identify TGFß1-non-canonical Wnt pathway crosstalk via sFRP2 as a novel mechanism for dysfunctional epithelial signaling in Idiopathic Pulmonary Fibrosis/ILD.

NF-κB inhibitor alpha controls SARS-CoV-2 infection in ACE2-overexpressing human airway organoids.

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor (ACE2-OE) that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. Interferon-lambda was upregulated in cells with low-level infection while the NF-kB inhibitor alpha gene (encoding IkBa) was consistently upregulated in infected cells, and its expression positively correlated with infection levels. Confocal microscopy showed more IkBa expression in infected than bystander cells, but found concurrent nuclear translocation of NF-kB that IkBa usually prevents. Overexpressing a nondegradable IkBa mutant reduced NF-kB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and underscore that the strength of the NF-kB feedback loop in infected cells controls viral replication.

Vagal-α7 nicotinic acetylcholine receptor signaling exacerbates influenza severity by promoting lung epithelial cell infection.

The vagus nerve circuit, operating through the alpha-7 nicotinic acetylcholine receptor (α7 nAChR), regulates the inflammatory response by influencing immune cells. However, the role of vagal-α7 nAChR signaling in influenza virus infection is unclear. In particular, does vagal-α7 nAChR signaling impact the infection of alveolar epithelial cells (AECs), the primary target cells of influenza virus? Here, we demonstrated a distinct role of α7 nAChR in type II AECs compared to its role in immune cells during influenza infection. We found that deletion of Chrna7 (encoding gene of α7 nAChR) in type II AECs or disruption of vagal circuits reduced lung influenza infection and protected mice from influenza-induced lung injury. We further unveiled that activation of α7 nAChR enhanced influenza infection through PTP1B-NEDD4L-ASK1-p38MAPK pathway. Mechanistically, activation of α7 nAChR signaling decreased p38MAPK phosphorylation during infection, facilitating the nuclear export of influenza viral ribonucleoproteins and thereby promoting infection. Taken together, our findings reveal a mechanism mediated by vagal-α7 nAChR signaling that promotes influenza viral infection and exacerbates disease severity. Targeting vagal-α7 nAChR signaling may offer novel strategies for combating influenza virus infections.

Inter- and Intra-donor variability in bone marrow-derived mesenchymal stromal cells: implications for clinical applications.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are attractive as a therapeutic modality in multiple disease conditions characterized by inflammation and vascular compromise. Logistically they are advantageous because they can be isolated from adult tissue sources, such as bone marrow (BM). The phase 2a START clinical trial determined BM-MSCs to be safe in patients with moderate-to-severe acute respiratory distress syndrome (ARDS). Herein, we examine a subset of the clinical doses of MSCs generated for the phase 2a START trial from three unique donors (1-3), where one of the donors' donated BM on two separate occasions (donor 3 and 3W). METHODS: The main objective of this study was to correlate properties of the cells from the four lots with plasma biomarkers from treated patients and relevant to ARDS outcomes. To do this we evaluated MSC donor lots for (i) post-thaw viability, (ii) growth kinetics, (iii) metabolism, (iv) surface marker expression, (v) protein expression, (vi) immunomodulatory ability and (vii) their functional effects on regulating endothelial cell permeability. RESULTS: MSC-specific marker expression and protection of thrombin-challenged endothelial barrier permeability was similar among all four donor lots. Inter and intra-donor variability was observed in all the other in vitro assays. Furthermore, patient plasma ANG-2 and protein C levels at 6 hours post-transfusion were correlated to cell viability in an inter- and intra-donor dependent manner. CONCLUSIONS: These findings highlight the potential of donor dependent (inter-) and collection dependent (intra-) effects in patient biomarker expression.

Incidence of acute kidney injury and attributive mortality in acute respiratory distress syndrome randomized trials.

PURPOSE: The development of acute kidney injury (AKI) after the acute respiratory distress syndrome (ARDS) reduces the chance of organ recovery and survival. The purpose of this study was to examine the AKI rate and attributable mortality in ARDS patients. METHODS: We performed an individual patient-data analysis including 10 multicenter randomized controlled trials conducted over 20 years. We employed a Super Learner ensemble technique, including a time-dependent analysis, to estimate the adjusted risk of AKI. We calculated the mortality attributable to AKI using an inverse probability of treatment weighting estimator integrated with the Super Learner. RESULTS: There were 5148 patients included in this study. The overall incidence of AKI was 43.7% (n = 2251). The adjusted risk of AKI ranged from 38.8% (95% confidence interval [CI], 35.7 to 41.9%) in ARMA, to 55.8% in ROSE (95% CI, 51.9 to 59.6%). 37.1% recovered rapidly from AKI, with a significantly lower recovery rate in recent trials (P < 0.001). The 90-day excess in mortality attributable to AKI was 15.4% (95% CI, 12.8 to 17.9%). It decreased from 25.4% in ARMA (95% CI, 18.7 to 32%), to 11.8% in FACTT (95% CI, 5.5 to 18%) and then remained rather stable over time. The 90-day overall excess in mortality attributable to acute kidney disease was 28.4% (95% CI, 25.3 to 31.5%). CONCLUSIONS: The incidence of AKI appears to be stable over time in patients with ARDS enrolled in randomized trials. The development of AKI remains a significant contributing factor to mortality. These estimates are essential for designing future clinical trials for AKI prevention or treatment.

Early trajectories of virological and immunological biomarkers and clinical outcomes in patients admitted to hospital for COVID-19: an international, prospective cohort study.

BACKGROUND: Serial measurement of virological and immunological biomarkers in patients admitted to hospital with COVID-19 can give valuable insight into the pathogenic roles of viral replication and immune dysregulation. We aimed to characterise biomarker trajectories and their associations with clinical outcomes. METHODS: In this international, prospective cohort study, patients admitted to hospital with COVID-19 and enrolled in the Therapeutics for Inpatients with COVID-19 platform trial within the Accelerating COVID-19 Therapeutic Interventions and Vaccines programme between Aug 5, 2020 and Sept 30, 2021 were included. Participants were included from 108 sites in Denmark, Greece, Poland, Singapore, Spain, Switzerland, Uganda, the UK, and the USA, and randomised to placebo or one of four neutralising monoclonal antibodies: bamlanivimab (Aug 5 to Oct 13, 2020), sotrovimab (Dec 16, 2020, to March 1, 2021), amubarvimab-romlusevimab (Dec 16, 2020, to March 1, 2021), and tixagevimab-cilgavimab (Feb 10 to Sept 30, 2021). This trial included an analysis of 2149 participants with plasma nucleocapsid antigen, anti-nucleocapsid antibody, C-reactive protein (CRP), IL-6, and D-dimer measured at baseline and day 1, day 3, and day 5 of enrolment. Day-90 follow-up status was available for 1790 participants. Biomarker trajectories were evaluated for associations with baseline characteristics, a 7-day pulmonary ordinal outcome, 90-day mortality, and 90-day rate of sustained recovery. FINDINGS: The study included 2149 participants. Participant median age was 57 years (IQR 46-68), 1246 (58·0%) of 2149 participants were male and 903 (42·0%) were female; 1792 (83·4%) had at least one comorbidity, and 1764 (82·1%) were unvaccinated. Mortality to day 90 was 172 (8·0%) of 2149 and 189 (8·8%) participants had sustained recovery. A pattern of less favourable trajectories of low anti-nucleocapsid antibody, high plasma nucleocapsid antigen, and high inflammatory markers over the first 5 days was observed for high-risk baseline clinical characteristics or factors related to SARS-CoV-2 infection. For example, participants with chronic kidney disease demonstrated plasma nucleocapsid antigen 424% higher (95% CI 319-559), CRP 174% higher (150-202), IL-6 173% higher (144-208), D-dimer 149% higher (134-165), and anti-nucleocapsid antibody 39% lower (60-18) to day 5 than those without chronic kidney disease. Participants in the highest quartile for plasma nucleocapsid antigen, CRP, and IL-6 at baseline and day 5 had worse clinical outcomes, including 90-day all-cause mortality (plasma nucleocapsid antigen hazard ratio (HR) 4·50 (95% CI 3·29-6·15), CRP HR 3·37 (2·30-4·94), and IL-6 HR 5·67 (4·12-7·80). This risk persisted for plasma nucleocapsid antigen and CRP after adjustment for baseline biomarker values and other baseline factors. INTERPRETATION: Patients admitted to hospital with less favourable 5-day biomarker trajectories had worse prognosis, suggesting that persistent viral burden might drive inflammation in the pathogenesis of COVID-19, identifying patients that might benefit from escalation of antiviral or anti-inflammatory treatment. FUNDING: US National Institutes of Health.

Acetaminophen for Prevention and Treatment of Organ Dysfunction in Critically Ill Patients With Sepsis: The ASTER Randomized Clinical Trial.

Importance: Acetaminophen (paracetamol) has many pharmacological effects that might be beneficial in sepsis, including inhibition of cell-free hemoglobin-induced oxidation of lipids and other substrates. Objective: To determine whether acetaminophen increases days alive and free of organ dysfunction in sepsis compared with placebo. Design, Setting, and Participants: Phase 2b randomized, double-blind, clinical trial conducted from October 2021 to April 2023 with 90-day follow-up. Adults with sepsis and respiratory or circulatory organ dysfunction were enrolled in the emergency department or intensive care unit of 40 US academic hospitals within 36 hours of presentation. Intervention: Patients were randomized to 1 g of acetaminophen intravenously every 6 hours or placebo for 5 days. Main Outcome and Measures: The primary end point was days alive and free of organ support (mechanical ventilation, vasopressors, and kidney replacement therapy) to day 28. Treatment effect modification was evaluated for acetaminophen by prerandomization plasma cell-free hemoglobin level higher than 10 mg/dL. Results: Of 447 patients enrolled (mean age, 64 [SD, 15] years, 51% female, mean Sequential Organ Failure Assessment [SOFA] score, 5.4 [SD, 2.5]), 227 were randomized to acetaminophen and 220 to placebo. Acetaminophen was safe with no difference in liver enzymes, hypotension, or fluid balance between treatment arms. Days alive and free of organ support to day 28 were not meaningfully different for acetaminophen (20.2 days; 95% CI, 18.8 to 21.6) vs placebo (19.6 days; 95% CI, 18.2 to 21.0; P = .56; difference, 0.6; 95% CI, -1.4 to 2.6). Among 15 secondary outcomes, total, respiratory, and coagulation SOFA scores were significantly lower on days 2 through 4 in the acetaminophen arm as was the rate of development of acute respiratory distress syndrome within 7 days (2.2% vs 8.5% acetaminophen vs placebo; P = .01; difference, -6.3; 95% CI, -10.8 to -1.8). There was no significant interaction between cell-free hemoglobin levels and acetaminophen. Conclusions and Relevance: Intravenous acetaminophen was safe but did not significantly improve days alive and free of organ support in critically ill sepsis patients. Trial Registration: ClinicalTrials.gov Identifier: NCT04291508.

Biological effects of corticosteroids on pneumococcal pneumonia in Mice-translational significance.

BACKGROUND: Streptococcus pneumoniae is the most common bacterial cause of community acquired pneumonia and the acute respiratory distress syndrome (ARDS). Some clinical trials have demonstrated a beneficial effect of corticosteroid therapy in community acquired pneumonia, COVID-19, and ARDS, but the mechanisms of this benefit remain unclear. The primary objective of this study was to investigate the effects of corticosteroids on the pulmonary biology of pneumococcal pneumonia in a mouse model. A secondary objective was to identify shared transcriptomic features of pneumococcal pneumonia and steroid treatment in the mouse model and clinical samples. METHODS: We carried out comprehensive physiologic, biochemical, and histological analyses in mice to identify the mechanisms of lung injury in Streptococcus pneumoniae with and without adjunctive steroid therapy. We also studied lower respiratory tract gene expression from a cohort of 15 mechanically ventilated patients (10 with Streptococcus pneumoniae and 5 controls) to compare with the transcriptional studies in the mice. RESULTS: In mice with pneumonia, dexamethasone in combination with ceftriaxone reduced (1) pulmonary edema formation, (2) alveolar protein permeability, (3) proinflammatory cytokine release, (4) histopathologic lung injury score, and (5) hypoxemia but did not increase bacterial burden. Transcriptomic analyses identified effects of steroid therapy in mice that were also observed in the clinical samples. CONCLUSIONS: In combination with appropriate antibiotic therapy in mice, treatment of pneumococcal pneumonia with steroid therapy reduced hypoxemia, pulmonary edema, lung permeability, and histologic criteria of lung injury, and also altered inflammatory responses at the protein and gene expression level. The transcriptional studies in patients suggest that the mouse model replicates some of the features of pneumonia in patients with Streptococcus pneumoniae and steroid treatment. Overall, these studies provide evidence for the mechanisms that may explain the beneficial effects of glucocorticoid therapy in patients with community acquired pneumonia from Streptococcus Pneumoniae.

Life-saving effect of pulmonary surfactant in premature babies.

The discovery and replacement of lung surfactant have helped increase survival rates for neonatal respiratory distress syndrome in extremely premature infants.

Neutrophil Reduction Attenuates the Severity of Lung Injury in the Early Phase of Pneumococcal Pneumonia in Mice.

Neutrophils are the first leukocytes to be recruited to sites of inflammation in response to chemotactic factors released by activated macrophages and pulmonary epithelial and endothelial cells in bacterial pneumonia, a common cause of acute respiratory distress syndrome (ARDS). Although neutrophilic inflammation facilitates the elimination of pathogens, neutrophils also may cause bystander tissue injury. Even though neutrophils in alveolar spaces is a key feature of acute lung injury and ARDS especially from pneumonia, their contribution to the pathogenesis of lung injury is uncertain. The goal of this study was to elucidate the role of neutrophils in a clinically relevant model of bacterial pneumonia. We investigated the effect of reducing neutrophils in a mouse model of pneumococcal pneumonia treated with antibiotics. Neutrophils were reduced with anti-Ly6G monoclonal antibody 24 hours before and immediately preceding infection. Mice were inoculated intranasally with Streptococcus pneumoniae and received ceftriaxone 12 hours after bacterial inoculation. Neutrophil reduction in mice treated with ceftriaxone attenuated hypoxemia, alveolar permeability, epithelial injury, pulmonary edema, and inflammatory biomarker release induced by bacterial pneumonia, even though bacterial loads in the distal air spaces of the lung were modestly increased as compared to antibiotic treatment alone. Thus, when appropriate antibiotics are administered, lung injury in the early phase of bacterial pneumonia is mediated in part by neutrophils. In the early phase of bacterial pneumonia, neutrophils contribute to the severity of lung injury, although they also participate in host defense.

Causes and attributable fraction of death from ARDS in inflammatory phenotypes of sepsis.

BACKGROUND: Hypoinflammatory and hyperinflammatory phenotypes have been identified in both Acute Respiratory Distress Syndrome (ARDS) and sepsis. Attributable mortality of ARDS in each phenotype of sepsis is yet to be determined. We aimed to estimate the population attributable fraction of death from ARDS (PAFARDS) in hypoinflammatory and hyperinflammatory sepsis, and to determine the primary cause of death within each phenotype. METHODS: We studied 1737 patients with sepsis from two prospective cohorts. Patients were previously assigned to the hyperinflammatory or hypoinflammatory phenotype using latent class analysis. The PAFARDS in patients with sepsis was estimated separately in the hypo and hyperinflammatory phenotypes. Organ dysfunction, severe comorbidities, and withdrawal of life support were abstracted from the medical record in a subset of patients from the EARLI cohort who died (n = 130/179). Primary cause of death was defined as the organ system that most directly contributed to death or withdrawal of life support. RESULTS: The PAFARDS was 19% (95%CI 10,28%) in hypoinflammatory sepsis and, 14% (95%CI 6,20%) in hyperinflammatory sepsis. Cause of death differed between the two phenotypes (p < 0.001). Respiratory failure was the most common cause of death in hypoinflammatory sepsis, whereas circulatory shock was the most common cause in hyperinflammatory sepsis. Death with severe underlying comorbidities was more frequent in hypoinflammatory sepsis (81% vs. 67%, p = 0.004). CONCLUSIONS: The PAFARDS is modest in both phenotypes whereas primary cause of death among patients with sepsis differed substantially by phenotype. This study identifies challenges in powering future clinical trials to detect changes in mortality outcomes among patients with sepsis and ARDS.

Timing of antibiotic treatment identifies distinct clinical presentations among patients presenting with suspected septic shock.

Objective: Recent clinical guidelines for sepsis management emphasize immediate antibiotic initiation for suspected septic shock. Though hypotension is a high-risk marker of sepsis severity, prior studies have not considered the precise timing of hypotension in relation to antibiotic initiation and how clinical characteristics and outcomes may differ. Our objective was to evaluate antibiotic initiation in relation to hypotension to characterize differences in sepsis presentation and outcomes in patients with suspected septic shock. Methods: Adults presenting to the emergency department (ED) June 2012-December 2018 diagnosed with sepsis (Sepsis-III electronic health record [EHR] criteria) and hypotension (non-resolving for ≥30 min, systolic blood pressure <90 mmHg) within 24 h. We categorized patients who received antibiotics before hypotension ("early"), 0-60 min after ("immediate"), and >60 min after ("late") treatment. Results: Among 2219 patients, 55% received early treatment, 13% immediate, and 32% late. The late subgroup often presented to the ED with hypotension (median 0 min) but received antibiotics a median of 191 min post-ED presentation. Clinical characteristics notable for this subgroup included higher prevalence of heart failure and liver disease (p < 0.05) and later onset of systemic inflammatory response syndrome (SIRS) criteria compared to early/immediate treatment subgroups (median 87 vs. 35 vs. 20 min, p < 0.0001). After adjustment, there was no difference in clinical outcomes among treatment subgroups. Conclusions: There was significant heterogeneity in presentation and timing of antibiotic initiation for suspected septic shock. Patients with later treatment commonly had hypotension on presentation, had more hypotension-associated comorbidities, and developed overt markers of infection (eg, SIRS) later. While these factors likely contribute to delays in clinician recognition of suspected septic shock, it may not impact sepsis outcomes.

Rapidly improving ARDS differs clinically and biologically from persistent ARDS.

BACKGROUND: Rapidly improving acute respiratory distress syndrome (RIARDS) is an increasingly appreciated subgroup of ARDS in which hypoxemia improves within 24 h after initiation of mechanical ventilation. Detailed clinical and biological features of RIARDS have not been clearly defined, and it is unknown whether RIARDS is associated with the hypoinflammatory or hyperinflammatory phenotype of ARDS. The purpose of this study was to define the clinical and biological features of RIARDS and its association with inflammatory subphenotypes. METHODS: We analyzed data from 215 patients who met Berlin criteria for ARDS (endotracheally intubated) and were enrolled in a prospective observational cohort conducted at two sites, one tertiary care center and one urban safety net hospital. RIARDS was defined according to previous studies as improvement of hypoxemia defined as (i) PaO2:FiO2 > 300 or (ii) SpO2: FiO2 > 315 on the day following diagnosis of ARDS (day 2) or (iii) unassisted breathing by day 2 and for the next 48 h (defined as absence of endotracheal intubation on day 2 through day 4). Plasma biomarkers were measured on samples collected on the day of study enrollment, and ARDS phenotypes were allocated as previously described. RESULTS: RIARDS accounted for 21% of all ARDS participants. Patients with RIARDS had better clinical outcomes compared to those with persistent ARDS, with lower hospital mortality (13% vs. 57%; p value < 0.001) and more ICU-free days (median 24 vs. 0; p value < 0.001). Plasma levels of interleukin-6, interleukin-8, and plasminogen activator inhibitor-1 were significantly lower among patients with RIARDS. The hypoinflammatory phenotype of ARDS was more common among patients with RIARDS (78% vs. 51% in persistent ARDS; p value = 0.001). CONCLUSIONS: This study identifies a high prevalence of RIARDS in a multicenter observational cohort and confirms the more benign clinical course of these patients. We report the novel finding that RIARDS is characterized by lower concentrations of plasma biomarkers of inflammation compared to persistent ARDS, and that hypoinflammatory ARDS is more prevalent among patients with RIARDS. Identification and exclusion of RIARDS could potentially improve prognostic and predictive enrichment in clinical trials.

Respiratory drive heterogeneity associated with systemic inflammation and vascular permeability in acute respiratory distress syndrome.

BACKGROUND: In acute respiratory distress syndrome (ARDS), respiratory drive often differs among patients with similar clinical characteristics. Readily observable factors like acid-base state, oxygenation, mechanics, and sedation depth do not fully explain drive heterogeneity. This study evaluated the relationship of systemic inflammation and vascular permeability markers with respiratory drive and clinical outcomes in ARDS. METHODS: ARDS patients enrolled in the multicenter EPVent-2 trial with requisite data and plasma biomarkers were included. Neuromuscular blockade recipients were excluded. Respiratory drive was measured as PES0.1, the change in esophageal pressure during the first 0.1 s of inspiratory effort. Plasma angiopoietin-2, interleukin-6, and interleukin-8 were measured concomitantly, and 60-day clinical outcomes evaluated. RESULTS: 54.8% of 124 included patients had detectable respiratory drive (PES0.1 range of 0-5.1 cm H2O). Angiopoietin-2 and interleukin-8, but not interleukin-6, were associated with respiratory drive independently of acid-base, oxygenation, respiratory mechanics, and sedation depth. Sedation depth was not significantly associated with PES0.1 in an unadjusted model, or after adjusting for mechanics and chemoreceptor input. However, upon adding angiopoietin-2, interleukin-6, or interleukin-8 to models, lighter sedation was significantly associated with higher PES0.1. Risk of death was less with moderate drive (PES0.1 of 0.5-2.9 cm H2O) compared to either lower drive (hazard ratio 1.58, 95% CI 0.82-3.05) or higher drive (2.63, 95% CI 1.21-5.70) (p = 0.049). CONCLUSIONS: Among patients with ARDS, systemic inflammatory and vascular permeability markers were independently associated with higher respiratory drive. The heterogeneous response of respiratory drive to varying sedation depth may be explained in part by differences in inflammation and vascular permeability.

Biological Effects of Corticosteroids on Pneumococcal Pneumonia in Mice and Humans.

Background: Streptococcus pneumoniae is the most common bacterial cause of community acquired pneumonia and the acute respiratory distress syndrome (ARDS). Some clinical trials have demonstrated a beneficial effect of corticosteroid therapy in community acquired pneumonia, COVID-19, and ARDS, but the mechanisms of this benefit remain unclear. The objective of this study was to investigate the effects of corticosteroids on the pulmonary biology of pneumococcal pneumonia in an observational cohort of mechanically ventilated patients and in a mouse model of bacterial pneumonia with Streptococcus pneumoniae. Methods: We studied gene expression with lower respiratory tract transcriptomes from a cohort of mechanically ventilated patients and in mice. We also carried out comprehensive physiologic, biochemical, and histological analyses in mice to identify the mechanisms of lung injury in Streptococcus pneumoniae with and without adjunctive steroid therapy. Results: Transcriptomic analysis identified pleiotropic effects of steroid therapy on the lower respiratory tract in critically ill patients with pneumococcal pneumonia, findings that were reproducible in mice. In mice with pneumonia, dexamethasone in combination with ceftriaxone reduced (1) pulmonary edema formation, (2) alveolar protein permeability, (3) proinflammatory cytokine release, (4) histopathologic lung injury score, and (5) hypoxemia but did not increase bacterial burden. Conclusions: The gene expression studies in patients and in the mice support the clinical relevance of the mouse studies, which replicate several features of pneumococcal pneumonia and steroid therapy in humans. In combination with appropriate antibiotic therapy in mice, treatment of pneumococcal pneumonia with steroid therapy reduced hypoxemia, pulmonary edema, lung permeability, and histologic criteria of lung injury, and also altered inflammatory responses at the protein and gene expression level. The results from these studies provide evidence for the mechanisms that may explain the beneficial effects of glucocorticoid therapy in patients with community acquired pneumonia from Streptococcus Pneumoniae.

Novel subtypes of severe COVID-19 respiratory failure based on biological heterogeneity: a secondary analysis of a randomized controlled trial.

BACKGROUND: Despite evidence associating inflammatory biomarkers with worse outcomes in hospitalized adults with COVID-19, trials of immunomodulatory therapies have met with mixed results, likely due in part to biological heterogeneity of participants. Latent class analysis (LCA) of clinical and protein biomarker data has identified two subtypes of non-COVID acute respiratory distress syndrome (ARDS) with different clinical outcomes and treatment responses. We studied biological heterogeneity and clinical outcomes in a multi-institutional platform randomized controlled trial of adults with severe COVID-19 hypoxemic respiratory failure (I-SPY COVID). METHODS: Clinical and plasma protein biomarker data were analyzed from 400 trial participants enrolled from September 2020 until October 2021 with severe COVID-19 requiring ≥ 6 L/min supplemental oxygen. Seventeen hypothesis-directed protein biomarkers were measured at enrollment using multiplex Luminex panels or single analyte enzyme linked immunoassay methods (ELISA). Biomarkers and clinical variables were used to test for latent subtypes and longitudinal biomarker changes by subtype were explored. A validated parsimonious model using interleukin-8, bicarbonate, and protein C was used for comparison with non-COVID hyper- and hypo-inflammatory ARDS subtypes. RESULTS: Average participant age was 60 ± 14 years; 67% were male, and 28-day mortality was 25%. At trial enrollment, 85% of participants required high flow oxygen or non-invasive ventilation, and 97% were receiving dexamethasone. Several biomarkers of inflammation (IL-6, IL-8, IL-10, sTNFR-1, TREM-1), epithelial injury (sRAGE), and endothelial injury (Ang-1, thrombomodulin) were associated with 28- and 60-day mortality. Two latent subtypes were identified. Subtype 2 (27% of participants) was characterized by persistent derangements in biomarkers of inflammation, endothelial and epithelial injury, and disordered coagulation and had twice the mortality rate compared with Subtype 1. Only one person was classified as hyper-inflammatory using the previously validated non-COVID ARDS model. CONCLUSIONS: We discovered evidence of two novel biological subtypes of severe COVID-19 with significantly different clinical outcomes. These subtypes differed from previously established hyper- and hypo-inflammatory non-COVID subtypes of ARDS. Biological heterogeneity may explain inconsistent findings from trials of hospitalized patients with COVID-19 and guide treatment approaches.

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.

Molecular Phenotypes of Acute Respiratory Distress Syndrome in the ROSE Trial Have Differential Outcomes and Gene Expression Patterns That Differ at Baseline and Longitudinally over Time.

Rationale: Two molecular phenotypes have been identified in acute respiratory distress syndrome (ARDS). In the ROSE (Reevaluation of Systemic Early Neuromuscular Blockade) trial of cisatracurium in moderate to severe ARDS, we addressed three unanswered questions: 1) Do the same phenotypes emerge in a more severe ARDS cohort with earlier recruitment; 2) Do phenotypes respond differently to neuromuscular blockade? and 3) What biological pathways most differentiate inflammatory phenotypes?Methods: We performed latent class analysis in ROSE using preenrollment clinical and protein biomarkers. In a subset of patients (n = 134), we sequenced whole-blood RNA using enrollment and Day 2 samples and performed differential gene expression and pathway analyses. Informed by the differential gene expression analysis, we measured additional plasma proteins and evaluated their abundance relative to gene expression amounts.Measurements and Main Results: In ROSE, we identified the hypoinflammatory (60.4%) and hyperinflammatory (39.6%) phenotypes with similar biological and clinical characteristics as prior studies, including higher mortality at Day 90 for the hyperinflammatory phenotype (30.3% vs. 61.6%; P < 0.0001). We observed no treatment interaction between the phenotypes and randomized groups for mortality. The hyperinflammatory phenotype was enriched for genes associated with innate immune response, tissue remodeling, and zinc metabolism at Day 0 and collagen synthesis and neutrophil degranulation at Day 2. Longitudinal changes in gene expression patterns differed dependent on survivorship. For most highly expressed genes, we observed correlations with their corresponding plasma proteins' abundance. However, for the class-defining plasma proteins in the latent class analysis, no correlation was observed with their corresponding genes' expression.Conclusions: The hyperinflammatory and hypoinflammatory phenotypes have different clinical, protein, and dynamic transcriptional characteristics. These findings support the clinical and biological potential of molecular phenotypes to advance precision care in ARDS.

The antibiotic resistance reservoir of the lung microbiome expands with age in a population of critically ill patients.

Antimicrobial resistant lower respiratory tract infections are an increasing public health threat and an important cause of global mortality. The lung microbiome can influence susceptibility of respiratory tract infections and represents an important reservoir for exchange of antimicrobial resistance genes. Studies of the gut microbiome have found an association between age and increasing antimicrobial resistance gene burden, however, corollary studies in the lung microbiome remain absent. We performed an observational study of children and adults with acute respiratory failure admitted to the intensive care unit. From tracheal aspirate RNA sequencing data, we evaluated age-related differences in detectable antimicrobial resistance gene expression in the lung microbiome. Using a multivariable logistic regression model, we find that detection of antimicrobial resistance gene expression was significantly higher in adults compared with children after adjusting for demographic and clinical characteristics. This association remained significant after additionally adjusting for lung bacterial microbiome characteristics, and when modeling age as a continuous variable. The proportion of adults expressing beta-lactam, aminoglycoside, and tetracycline antimicrobial resistance genes was higher compared to children. Together, these findings shape our understanding of the lung resistome in critically ill patients across the lifespan, which may have implications for clinical management and global public health.