Human Acute Respiratory Distress Syndrome Research Articles

Breath analysis for detection and trajectory monitoring of acute respiratory distress syndrome in swine.

Despite the enormous impact on human health, acute respiratory distress syndrome (ARDS) is poorly defined, and its timely diagnosis is difficult, as is tracking the course of the syndrome. The objective of this pilot study was to explore the utility of breath collection and analysis methodologies to detect ARDS through changes in the volatile organic compound (VOC) profiles present in breath. Five male Yorkshire mix swine were studied and ARDS was induced using both direct and indirect lung injury. An automated portable gas chromatography device developed in-house was used for point of care breath analysis and to monitor swine breath hourly, starting from initiation of the experiment until the development of ARDS, which was adjudicated based on the Berlin criteria at the breath sampling points and confirmed by lung biopsy at the end of the experiment. A total of 67 breath samples (chromatograms) were collected and analysed. Through machine learning, principal component analysis and linear discrimination analysis, seven VOC biomarkers were identified that distinguished ARDS. These represent seven of the nine biomarkers found in our breath analysis study of human ARDS, corroborating our findings. We also demonstrated that breath analysis detects changes 1–6 h earlier than the clinical adjudication based on the Berlin criteria. The findings provide proof of concept that breath analysis can be used to identify early changes associated with ARDS pathogenesis in swine. Its clinical application could provide intensive care clinicians with a noninvasive diagnostic tool for early detection and continuous monitoring of ARDS.

A novel swine model of the acute respiratory distress syndrome using clinically relevant injury exposures.

To date, existing animal models of the acute respiratory distress syndrome (ARDS) have failed to translate preclinical discoveries into effective pharmacotherapy or diagnostic biomarkers. To address this translational gap, we developed a high‐fidelity swine model of ARDS utilizing clinically relevant lung injury exposures. Fourteen male swine were anesthetized, mechanically ventilated, and surgically instrumented for hemodynamic monitoring, blood, and tissue sampling. Animals were allocated to one of three groups: (1) Indirect lung injury only: animals were inoculated by direct injection of Escherichia coli into the kidney parenchyma, provoking systemic inflammation and distributive shock physiology; (2) Direct lung injury only: animals received volutrauma, hyperoxia, and bronchoscope‐delivered gastric particles; (3) Combined indirect and direct lung injury: animals were administered both above‐described indirect and direct lung injury exposures. Animals were monitored for up to 12 h, with serial collection of physiologic data, blood samples, and radiographic imaging. Lung tissue was acquired postmortem for pathological examination. In contrast to indirect lung injury only and direct lung injury only groups, animals in the combined indirect and direct lung injury group exhibited all of the physiological, radiographic, and histopathologic hallmarks of human ARDS: impaired gas exchange (mean PaO2/FiO2 ratio 124.8 ± 63.8), diffuse bilateral opacities on chest radiographs, and extensive pathologic evidence of diffuse alveolar damage. Our novel porcine model of ARDS, built on clinically relevant lung injury exposures, faithfully recapitulates the physiologic, radiographic, and histopathologic features of human ARDS and fills a crucial gap in the translational study of human lung injury.

Upregulation of IGSF3 expression by iron chelation leads to the inhibition of epithelial cell proliferation

Background/Significance We recently demonstrated that the Immunoglobulin Superfamily-3 (IGSF3), a transmembrane tetraspanin interacting protein, is required for scratch wound repair, cell migration, and barrier function, suggesting a potential key role in lung injury repair. However, the role of IGSF3 in epithelial cell proliferation remain unknown. Furthermore, it is unclear how IGSF3 expression is affected in acute respiratory distress syndrome (ARDS), characterized by hypoxia, loss of lung epithelial barrier, and insufficient cell repair. We hypothesized that hypoxia alters IGSF3 expression and that IGSF3 is mechanistically involved in the effects of hypoxia on human bronchial epithelial cell proliferation. Methods To mimic hypoxia, we exposed human bronchial epithelial cells (Beas2B) submerged in cell culture media to iron chelator deferoxamine (DFO, 100uM, 16-24hr). Proliferation was measured using CCK8 assay. We stably knocked down IGSF3 in Beasw2B using CRISPR-CAS9. Tetraspanin enriched microdomains (TEMs) were isolated using density gradient ultracentrifugation. Single cell RNA-seq (scRNA-seq) was performed on deidentified human ARDS (n=1) and non-diseased (n=3) lungs. Results DFO caused a significant decrease in epithelial cell proliferation (by~ 25%, p<0.001) concomitant to a markedly increased IGSF3 gene and protein expression (by 2- and 2.5-fold, respectively, p<0.01) that was prevented by treatment with the HIF-1a inhibitor, KCF72 (p<0.01). IGSF3-deficient Beas2B exhibited increased cell proliferation at baseline (by~20-50% vs control cells, 24hr; p<0.01) and following treatment with DFO (by ~ 200% vs DFO-treated control cells; 24hr, p<0.01). Compared to non-diseased lungs, scRNA-seq of ARDS lungs revealed marked enrichment of IGSF3 in basal epithelial cell. Interestingly, a similar pattern was observed in Tspan5, an IGSF3 binding tetraspanin induced by iron chelation and in CD147, a hypoxia-inducible Immuhnoglobulin Superfamily protein (Basigin, EMMPRIN). We confirmed that IGSF3 co-localized with Tspan5 within TEMs in Beas2B and, interestingly found that both proteins co-localized with CD147. Conclusions IGSF3 may be induced by hypoxia in lung epithelial cells such as basal cells where it may inhibit cell proliferation and therefore repair. Interestingly, loss of IGSF3 has also a detrimental effect on processes involved in repair such as cell migration and barrier function. This suggests that IGSF3 homeostasis is important in epithelial cell function, possibly through interactions with membrane complexes such as tetraspanins and other transmembrane proteins such as the matrix-regulating CD147.

Enhancing Extracellular Adenosine Levels Restores Barrier Function in Acute Lung Injury Through Expression of Focal Adhesion Proteins.

Background: Acute respiratory distress syndrome (ARDS) is a clinical presentation of acute lung injury (ALI) with often fatal lung complication. Adenosine, a nucleoside generated following cellular stress provides protective effects in acute injury. The levels of extracellular adenosine can be depleted by equilibrative nucleoside transporters (ENTs). ENT inhibition by pharmaceutical agent dipyridamole promotes extracellular adenosine accumulation and is protective in ARDS. However, the therapeutic potential of dipyridamole in acute lung injury has not yet been evaluated. Methods: Adenosine acts on three adenosine receptors, the adenosine A1 (Adora1), A2a (Adora2a), the A2b (Adora2b) or the adenosine A3 (Adora 3) receptor. Accumulation of adenosine is usually required to stimulate the low-affinity Adora2b receptor. In order to investigate the effect of adenosine accumulation and the contribution of epithelial-specific ENT2 or adora2b expression in experimental ALI, dipyridamole, and epithelial specific ENT2 or Adora2b deficient mice were utilized. MLE12 cells were used to probe downstream Adora2b signaling. Adenosine receptors, transporters, and targets were determined in ARDS lungs. Results: ENT2 is mainly expressed in alveolar epithelial cells and is negatively regulated by hypoxia following tissue injury. Enhancing adenosine levels with ENT1/ENT2 inhibitor dipyridamole at a time when bleomycin-induced ALI was present, reduced further injury. Mice pretreated with the ADORA2B agonist BAY 60-6583 were protected from bleomycin-induced ALI by reducing vascular leakage (558.6 ± 50.4 vs. 379.9 ± 70.4, p < 0.05), total bronchoalveolar lavage fluid cell numbers (17.9 ± 1.8 to 13.4 ± 1.4 e4, p < 0.05), and neutrophil infiltration (6.42 ± 0.25 vs. 3.94 ± 0.29, p < 0.05). While mice lacking Adora2b in AECs were no longer protected by dipyridamole. We also identified occludin and focal adhesion kinase as downstream targets of ADORA2B, thus providing a novel mechanism for adenosine-mediated barrier protection. Similarly, we also observed similar enhanced ADORA2B (3.33 ± 0.67 to 16.12 ± 5.89, p < 0.05) and decreased occludin (81.2 ± 0.3 to 13.3 ± 0.4, p < 0.05) levels in human Acute respiratory distress syndrome lungs. Conclusion: We have highlighted a role of dipyridamole and adenosine signaling in preventing or treating ALI and identified Ent2 and Adora2b as key mediators in important for the resolution of ALI.

MiR-584 and miR-146 are candidate biomarkers for acute respiratory distress syndrome.

MicroRNAs (miRNAs/miRs) have important roles in inflammation and infections, which are common manifestations of acute respiratory distress syndrome (ARDS). The present study aimed to assess whether serum miRNAs are potential diagnostic biomarkers for human ARDS. For this, two sets of serum samples from healthy individuals and patients with ARDS were analysed by high-throughput sequencing to identify differentially expressed genes in ARDS. A total of 679 valid sequences were identified as differentially expressed (P<0.05). Of these, five differentially expressed miRNAs were subjected to reverse transcription-quantitative PCR validation. Finally, two miRNAs (miR-584 and miR-146a) were successfully verified. These two miRNAs were significantly downregulated in the serum of patients with ARDS. Gene Ontology annotations and Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that their target transcripts were implicated in a broad range of biological processes and various metabolic pathways, including involvement in the regulation of various inflammatory factors. The present study provided a framework for understanding the molecular mechanisms of ARDS and suggested that miR-584 and miR-146a are associated with ARDS and may be potential therapeutic targets.

SP-D Serum Levels Reveal Distinct Epithelial Damage in Direct Human ARDS.

Acute respiratory distress syndrome (ARDS) is a heterogeneous syndrome with multiple underlying diseases. Particularly epithelial damage results from direct (e.g., pneumonia) rather than indirect lung injury (e.g., nonpulmonary sepsis), which is more likely associated with endothelial damage. Hence, targeting ARDS patients based on their molecular phenotypes is a promising approach to improve outcome. With regard to distinct inflammatory responses and subsequent lung damage in direct ARDS due to the causing pathogen, we quantified markers of epithelial and endothelial damage and pro-inflammatory cytokines in patients with ARDS triggered by bacterial, viral, and atypical pathogen pneumonia or indirect ARDS. The serum levels of interleukin-6 (IL-6) and interleukin-8 (IL-8), lung epithelial injury markers surfactant protein D (SP-D), and soluble receptor for advanced glycation end-products (sRAGE) as well as endothelial injury marker angiopoietin-2 (Ang-2) from 49 patients with distinct types of ARDS were analyzed by multiplex immunoassay. Epithelial damage marker SP-D was significantly higher in direct ARDS caused by viral and atypical pathogens in contrast to ARDS caused by typical bacterial pneumonia and nonpulmonary sepsis. In contrast, sRAGE levels did not differ due to the causing pathogen. Patients with atypical pathogen pneumonia related ARDS showed significantly lower Ang-2 levels compared to patients with viral and indirect ARDS. Patients with viral and atypical pneumonia related ARDS possessed significantly lower serum IL-6 levels compared to bacterial pneumonia related ARDS and IL-6 levels in atypical pneumonia related ARDS were significantly lower than in indirect ARDS. Current findings report a potential difference in ARDS biomarkers due to the underlying disease and pathogen.

Comparison of direct and indirect models of early induced acute lung injury

BackgroundThe animal experimental counterpart of human acute respiratory distress syndrome (ARDS) is acute lung injury (ALI). Most models of ALI involve reproducing the clinical risk factors associated with human ARDS, such as sepsis or acid aspiration; however, none of these models fully replicates human ARDS.AimTo compare different experimental animal models of ALI, based on direct or indirect mechanisms of lung injury, to characterize a model which more closely could reproduce the acute phase of human ARDS.Materials and methodsAdult male Sprague-Dawley rats were subjected to intratracheal instillations of (1) HCl to mimic aspiration of gastric contents; (2) lipopolysaccharide (LPS) to mimic bacterial infection; (3) HCl followed by LPS to mimic aspiration of gastric contents with bacterial superinfection; or (4) cecal ligation and puncture (CLP) to induce peritonitis and mimic sepsis. Rats were sacrificed 24 h after instillations or 24 h after CLP.ResultsAt 24 h, rats instilled with LPS or HCl-LPS had increased lung permeability, alveolar neutrophilic recruitment and inflammatory markers (GRO/KC, TNF-α, MCP-1, IL-1β, IL-6). Rats receiving only HCl or subjected to CLP had no evidence of lung injury.ConclusionsRat models of ALI induced directly by LPS or HCl-LPS more closely reproduced the acute phase of human ARDS than the CLP model of indirectly induced ALI.

Disease-modifying treatment of chemical threat agent-induced acute lung injury.

Acute respiratory distress syndrome (ARDS) is a highly morbid lung pathology induced by exposure to chemical warfare agents, including vesicants, phosgene, chlorine, and ricin. In this review, we describe the pathology associated with the development of ARDS in humans and experimental models of acute lung injury following animal exposure to these high-priority threat agents. Potential future approaches to disease-modifying treatment used in preclinical animal studies, including antioxidants, anti-inflammatories, biologics, and mesenchymal stem cells, are also described. As respiratory pathologies, including ARDS, are the major cause of morbidity and mortality following exposure to chemical threat agents, understanding mechanisms of disease pathogenesis is key to the development of efficacious therapeutics beyond the primary intervention principle, which remains mechanical ventilation.

Bronchoalveolar Lavage and Oleic Acid-Injection in Pigs as a Double-Hit Model for Acute Respiratory Distress Syndrome (ARDS).

The treatment of ARDS continues to pose major challenges for intensive care physicians in the 21st century with mortality rates still reaching up to 50% in severe cases. Further research efforts are needed to better understand the complex pathophysiology of this disease. There are different well-established animal models to induce acute lung injury but none has been able to adequately mimic the complex pathomechanisms of ARDS. The most crucial factor for the development of this condition is the damage to the alveolar capillary unit. The combination of two well-established lung injury models allow us to mimic in more detail the underlying pathomechanism. Bronchoalveolar lavage (BAL) leads to surfactant depletion as well as alveolar collapse. The repeated instillation of fluid volumes causes subsequent hypoxemia. Surfactant depletion is a key factor of ARDS in humans. BAL is often combined with other lung injury approaches, but not with a second hit followed by oleic acid injection (OAI) yet. Oleic acid injection leads to severely impaired gas exchange, a deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The OAI mimics most of the expected effects of ARDS consisting of extended inflammation of lung tissue with an increase of alveolar leakage and gas exchange impairment. A disadvantage of the combination of different models is the difficulty to determine the influence to the lung injury caused by BAL alone, OAI alone or both together. The model presented in this report represents the combination of BAL and OAI as a new double-hit lung injury model. This new model is easy to implement and an alternative to study different therapeutic approaches in ARDS in the future.

Circulating fibrocytes traffic to the lung in murine acute lung injury and predict outcomes in human acute respiratory distress syndrome: a pilot study

BackgroundFibrosis is an integral component of the pathogenesis of acute lung injury and is associated with poor outcomes in patients with acute respiratory distress syndrome (ARDS). Fibrocytes are bone marrow-derived cells that traffic to injured tissues and contribute to fibrosis; hence their concentration in the peripheral blood has the potential to serve as a biomarker of lung fibrogenesis. We therefore sought to test the hypothesis that the concentration and phenotype of circulating fibrocytes in patients with ARDS predicts clinical outcomes.MethodsFor the animal studies, C57Bl/6 mice were infected with experimental Klebsiella pneumoniae in a model of acute lung injury; one-way ANOVA was used to compare multiple groups and two-way ANOVA was used to compare two groups over time. For the human study, 42 subjects with ARDS and 12 subjects with pneumonia (without ARDS) were compared to healthy controls. Chi-squared or Fisher’s exact test were used to compare binary outcomes. Survival data was expressed using a Kaplan-Meier curve and compared by log-rank test. Univariable and multivariable logistic regression were used to predict death.ResultsIn mice with acute lung injury caused by Klebsiella pneumonia, there was a time-dependent increase in lung soluble collagen that correlated with sequential expansion of fibrocytes in the bone marrow, blood, and then lung compartments. Correspondingly, when compared via cross-sectional analysis, the initial concentration of blood fibrocytes was elevated in human subjects with ARDS or pneumonia as compared to healthy controls. In addition, fibrocytes from subjects with ARDS displayed an activated phenotype and on serial measurements, exhibited intermittent episodes of markedly elevated concentration over a median of 1 week. A peak concentration of circulating fibrocytes above a threshold of > 4.8 × 106 cells/mL cells correlated with mortality that was independent of age, ratio of arterial oxygen concentration to the fraction of inspired oxygen, and vasopressor requirement.ConclusionsCirculating fibrocytes increase in a murine model of acute lung injury and elevation in the number of these cells above a certain threshold is correlated with mortality in human ARDS. Therefore, these cells may provide a useful and easily measured biomarker to predict outcomes in these patients.

Single cell RNA sequencing reveals novel inflammatory mechanisms of human acute respiratory distress syndrome

Abstract The acute respiratory distress syndrome (ARDS) is a common etiology of acute respiratory failure that results from overwhelming pulmonary inflammation. Since prior bulk RNA sequence analyses have provided limited insights into ARDS pathogenesis, we used the approach of single cell RNA sequencing (scRNA-seq) to probe ARDS at a higher resolution. Towards this end, scRNA-seq data of peripheral blood mononuclear cells (PBMCs) of patients with pneumonia and sepsis with early ARDS were compared to that of sepsis patients who did not develop ARDS. We detected a previously unrecognized reduced frequency of NK cells in the PBMCs of ARDS patients, which also showed complete loss of IFNGR1 expression. Monocyte clusters from ARDS patients revealed multiple distinguishing characteristics in comparison to monocytes from patients without ARDS. This included a pro-inflammatory signature with up-regulation of multiple type I IFN-induced genes, especially in CD16+ cells. To generate an ARDS risk score, we identified 29 differentially expressed genes in the monocytes of these patients, 17 of which showed a similar profile in cells of patients in independent cohorts. Genes with increased expression in monocytes that may have relevance in ARDS pathogenesis include RAB11A, known to inhibit neutrophil efferocytosis, ATP2B1, a calcium pump that exports Ca2+ implicated in endothelial barrier disruption, and SPARC, associated with processing of pro-collagen to collagen. Together, our findings identify molecules that are likely involved in ARDS pathogenesis that may inform biomarker and therapeutic development.

Lessons learned in acute respiratory distress syndrome from the animal laboratory.

Since the description of the acute respiratory distress syndrome (ARDS) in 1967, investigators have struggled to reproduce the syndrome in the animal laboratory. While several different models of experimental acute lung injury (ALI) have been developed, none completely capture the inciting etiologies, initial inflammation, heterogeneity, and resolution of human ARDS. This potentially has contributed to the poor translation of potential therapeutics between animal ALI models and human ARDS. It was only recently that standardized criteria were suggested for what makes an ALI model comparable to human ARDS. Nevertheless, despite model heterogeneity, these models have contributed substantially to our understanding of the syndrome. From the initial studies identifying the risks of mechanical ventilation to the identification of potentially targetable inflammatory mediators, to modern studies focusing on regional heterogeneity and novel molecular pathways, animal models continue to inform our understanding of ARDS. This review will cover several major lessons learned from animal models of ALI, and provide some direction for future studies in this field.

Challenges and perspectives in porcine model of acute lung injury using oleic acid

The oleic acid (OA) models of lung injury try to simulate the findings of human Acute Respiratory Distress Syndrome (ARDS). However, these models are difficult to replicate because they vary in terms of animals species, OA doses, time for establishment of lung injury, different observation periods and settings of mechanical ventilation. The objective of this study was to evaluate a protocol of administration of OA in lung injury model, challenges in its development and its effects on respiratory mechanics, hemodynamic changes, histology, gas exchange and mortality. We then submitted ten Large White pigs to acute lung injury through intravenous infusion of acid oleic in the pulmonary artery. The mortality of the model was 50%, due to an intense hemodynamic instability during OA administration, even with early use of vasoactive drugs. Three animals required additional doses of OA to achieve criteria for acute lung injury. Histology showed findings consistent with acute lung injury. However, more pulmonary edema was observed in lower segments than in upper segments of both lungs (p = 0.01). IL-6 and IL-8 were significantly increased compared to normal lungs (p < 0.05), and IL-6 showed higher levels in upper segments compared to lower segments (p = 0.03). Positive cells for Caspase 3 were present in all samples, localized mainly in respiratory epithelial cells and macrophages. In conclusion, this model shows histological findings of acute lung injury and inflammatory response similar to those of clinical ARDS, it presents high mortality, inconsistent reproducibility and hardly controlled hemodynamic instability.

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation.

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic findings similar to human acute respiratory distress syndrome (ARDS). The resulting alveolar edema, alveolar-capillary barrier damage, and leukocyte infiltration predominantly affect the left lung, preserving the right lung as an uninjured control and allowing animals to survive. This model of self-limited acute lung injury enables investigation of tissue resolution mechanisms, such as macrophage efferocytosis of apoptotic neutrophils and restitution of alveolar-capillary barrier integrity. This model has helped identify important roles for resolution agonists, including specialized pro-resolving mediators (SPMs), providing a foundation for the development of new therapeutic approaches for patients with ARDS.

Necrosis Rather Than Apoptosis is the Dominant form of Alveolar Epithelial Cell Death in Lipopolysaccharide-Induced Experimental Acute Respiratory Distress Syndrome Model.

Alveolar epithelial cell (AEC) death, which is classified as apoptosis or necrosis, plays a critical role in the pathogenesis of acute respiratory distress syndrome (ARDS). In addition to apoptosis, some types of necrosis are known to be molecularly regulated, and both apoptosis and necrosis can be therapeutic targets for diseases. However, the relative contribution of apoptosis and necrosis to AEC death during ARDS has not been elucidated. Here, we evaluated which type of AEC death is dominant and whether regulated necrosis is involved in lipopolysaccharide (LPS)-induced lung injury, an experimental ARDS model. In the bronchoalveolar lavage fluid from the LPS-induced lung injury mice, both the levels of cytokeratin 18-M65 antigen (a marker of total epithelial cell death) and cytokeratin 18-M30 antigen (an epithelial apoptosis marker) were increased. The M30/M65 ratio, which is an indicator of the proportion of apoptosis to total epithelial cell death, was significantly lower than that in healthy controls. In addition, the number of propidium iodide-positive, membrane-disrupted cells was significantly higher than the number of TUNEL-positive apoptotic cells in the lung sections of lung injury mice. Activated neutrophils seemed to mediate AEC death. Finally, we demonstrated that necroptosis, a regulated necrosis pathway, is involved in AEC death during LPS-induced lung injury. These results indicate that necrosis including necroptosis, rather than apoptosis, is the dominant type of AEC death in LPS-induced lung injury. Although further studies investigating human ARDS subjects are necessary, targeting necrosis including its regulated forms might represent a more efficient approach to protecting the alveolar epithelial barrier during ARDS.

IL4Rα Signaling Abrogates Hypoxic Neutrophil Survival and Limits Acute Lung Injury Responses In Vivo.

Rationale: Acute respiratory distress syndrome is defined by the presence of systemic hypoxia and consequent on disordered neutrophilic inflammation. Local mechanisms limiting the duration and magnitude of this neutrophilic response remain poorly understood.Objectives: To test the hypothesis that during acute lung inflammation tissue production of proresolution type 2 cytokines (IL-4 and IL-13) dampens the proinflammatory effects of hypoxia through suppression of HIF-1α (hypoxia-inducible factor-1α)-mediated neutrophil adaptation, resulting in resolution of lung injury.Methods: Neutrophil activation of IL4Ra (IL-4 receptor α) signaling pathways was explored ex vivo in human acute respiratory distress syndrome patient samples, in vitro after the culture of human peripheral blood neutrophils with recombinant IL-4 under conditions of hypoxia, and in vivo through the study of IL4Ra-deficient neutrophils in competitive chimera models and wild-type mice treated with IL-4.Measurements and Main Results: IL-4 was elevated in human BAL from patients with acute respiratory distress syndrome, and its receptor was identified on patient blood neutrophils. Treatment of human neutrophils with IL-4 suppressed HIF-1α–dependent hypoxic survival and limited proinflammatory transcriptional responses. Increased neutrophil apoptosis in hypoxia, also observed with IL-13, required active STAT signaling, and was dependent on expression of the oxygen-sensing prolyl hydroxylase PHD2. In vivo, IL-4Ra–deficient neutrophils had a survival advantage within a hypoxic inflamed niche; in contrast, inflamed lung treatment with IL-4 accelerated resolution through increased neutrophil apoptosis.Conclusions: We describe an important interaction whereby IL4Rα-dependent type 2 cytokine signaling can directly inhibit hypoxic neutrophil survival in tissues and promote resolution of neutrophil-mediated acute lung injury.

Identification and Modulation of Microenvironment Is Crucial for Effective Mesenchymal Stromal Cell Therapy in Acute Lung Injury.

Rationale: There are controversial reports on applications of mesenchymal stromal cells (MSCs) in patients with acute respiratory distress syndrome (ARDS). Objectives: We hypothesized that lung microenvironment was the main determinant of beneficial versus detrimental effects of MSCs during ARDS. Methods: Lung proteome was profiled in three models of injury induced by acid instillation and/or mechanical ventilation in mice. Human gene of glutathione peroxidase-1 was delivered before MSC administration; or MSCs carrying human gene of IL-10 or hepatocyte growth factor were administered after lung injury. An inhibitory cocktail against IL-6, fibronectin, and oxidative stress was used in in vitro studies using human small airway epithelial cells and human MSCs after exposure to plasma of patients with ARDS. Measurements and Main Results: Distinct proteomic profiles were observed in three lung injury models. Administration of MSCs protected lung from ventilator-induced injury, whereas it worsened acid-primed lung injuries associated with fibrotic development in lung environment that had high levels of IL-6 and fibronectin along with low antioxidant capacity. Correction of microenvironment with glutathione peroxidase-1, or treatment with MSCs carrying human gene of IL-10 or hepatocyte growth factor after acid-primed injury, reversed the detrimental effects of native MSCs. Proteomic profiles obtained in the mouse models were also similarly observed in human ARDS. Treatment with the inhibitory cocktail in samples of patients with ARDS retained protective effects of MSCs in small airway epithelial cells. Conclusions: MSCs can be beneficial or detrimental depending on microenvironment at the time of administration. Identification of potential beneficiaries seems to be crucial to guide MSC therapy in ARDS.

IL-1R signaling enhances neutrophil extracellular traps (NETs) formation and leads to increase lung permeability and hypoxemia in LPS/mechanical ventilation-induced acute lung injury mouse model

Abstract Although neutrophils extracellular traps (NETs) play a role in host defense against invading microorganisms, NETs alone play a role in pathogenesis of human acute respiratory distress syndrome (ARDS) either with or without infection. However, the mechanism of NET formation and its functional implications in alveolar space during acute lung injury are not less understood. We created a two-hit lung injury mouse model using intratracheal LPS (0.2 mg/kg), followed by 2.5 hours high-volume mechanical ventilation (30 ml/kg). We observed significant hypoxemia along with increased alveolar leakage, neutrophil infiltration, and NET formation using this model. Neutrophil depletion using (Ly6G[1A8]) monoclonal antibody inhibited these effects. Intriguingly, Il1r1−/− mice were significantly protected from hypoxemia and alveolar leakage despite similar alveolar neutrophil infiltration. We also found that Il1r1−/− mice had reduced levels of cell-free DNA and MPO-DNA complexes in bronchoalveolar lavage fluid (BALF), suggesting that NETs were involved in inducing lung vascular permeability and that IL-1R signaling was involved in inducing NET formation in alveolar space. Inhibition of NET formation via Cl-amidine or removal of NET DNA via DNase I led to reduced hypoxemia and alveolar leakage despite unaltered neutrophil infiltration. Additionally we found that recombinant IL-1β treatment of mouse neutrophils induces NETs, and this was inhibited by IL-1R antagonist (Anakinra). Taken together, we have found that IL-1R signaling-induced NETs play a critical role in development of lung injury, and may offer a new target for therapeutics.

Neutrophil Extracellular Traps Are Elevated in Patients with Pneumonia-related Acute Respiratory Distress Syndrome.

Neutrophil extracellular traps have been associated with tissue damage. Whether these are involved in the pathogenesis of human acute respiratory distress syndrome (ARDS) and could be a potential therapeutic target is unknown. The authors quantified bronchoalveolar and blood neutrophil extracellular traps in patients with pneumonia-related ARDS and assessed their relationship with ventilator-free days. Immunocompetent patients with pneumonia and moderate or severe ARDS (n = 35) and controls (n = 4) were included in a prospective monocentric study. Neutrophil extracellular trap concentrations were quantified (as DNA-myeloperoxidase complexes) in bronchoalveolar lavage fluid and serum by enzyme-linked immunosorbent assay. The relationship between bronchoalveolar lavage neutrophil extracellular trap concentrations and the primary clinical endpoint (i.e., the number of live ventilator-free days at day 28) was assessed using linear regression analyses. There was no significant relationship between bronchoalveolar lavage neutrophil extracellular trap concentrations and ventilator-free days by multiple regression analysis (β coefficient = 2.40; 95% CI, -2.13 to 6.92; P = 0.288). Neutrophil extracellular trap concentrations were significantly higher in bronchoalveolar lavage than in blood of ARDS patients (median [first to third quartiles]:154 [74 to 1,000] vs. 26 [4 to 68] arbitrary units, difference: -94; 95% CI, -341 to -57; P < 0.0001). Bronchoalveolar concentrations of patients were higher than those of controls (154 [74 to 1,000] vs. 4 [4 to 4] arbitrary units, difference: -150; 95% CI, -996 to -64; P < 0.001) and associated with bronchoalveolar interleukin-8 (Spearman's ρ = 0.42; P = 0.012) and neutrophil concentrations (ρ = 0.57; P < 0.0001). Intensive care unit mortality (12%, n = 2 of 17 vs. 17%, n = 3 of 18; P > 0.99) and the number of ventilator-free days at day 28 (22 [14 to 25] vs. 14 [0 to 21] days; difference: -5; 95% CI, -15 to 0; P = 0.066) did not significantly differ between patients with higher (n = 17) versus lower (n = 18) bronchoalveolar neutrophil extracellular trap concentrations. Bronchoalveolar neutrophil extracellular trap concentration was not significantly associated with mechanical ventilation duration in pneumonia-related ARDS.

Macrophage Polarization Favors Epithelial Repair During Acute Respiratory Distress Syndrome.

Alveolar macrophage polarization and role on alveolar repair during human acute respiratory distress syndrome remain unclear. This study aimed to determine during human acute respiratory distress syndrome: the alveolar macrophage polarization, the effect of alveolar environment on macrophage polarization, and the role of polarized macrophages on epithelial repair. Experimental ex vivo and in vitro investigations. Four ICUs in three teaching hospitals. Thirty-three patients with early moderate-to-severe acute respiratory distress syndrome were enrolled for assessment of the polarization of alveolar macrophages. Polarization of acute respiratory distress syndrome macrophages was studied by flow cytometry and quantitative polymerase chain reaction. Modulation of macrophage polarization was studied in vitro using phenotypic and functional readouts. Macrophage effect on repair was studied using alveolar epithelial cells in wound healing models. Ex vivo, alveolar macrophages from early acute respiratory distress syndrome patients exhibited anti-inflammatory characteristics with high CD163 expression and interleukin-10 production. Accordingly, early acute respiratory distress syndrome-bronchoalveolar lavage fluid drives an acute respiratory distress syndrome-specific anti-inflammatory macrophage polarization in vitro, close to that induced by recombinant interleukin-10. Culture supernatants from macrophages polarized in vitro with acute respiratory distress syndrome-bronchoalveolar lavage fluid or interleukin-10 and ex vivo acute respiratory distress syndrome alveolar macrophages specifically promoted lung epithelial repair. Inhibition of the hepatocyte growth factor pathway in epithelial cells and hepatocyte growth factor production in macrophages both reversed this effect. Finally, hepatocyte growth factor and soluble form of CD163 concentrations expressed relatively to macrophage count were higher in bronchoalveolar lavage fluid from acute respiratory distress syndrome survivors. Early acute respiratory distress syndrome alveolar environment drives an anti-inflammatory macrophage polarization favoring epithelial repair through activation of the hepatocyte growth factor pathway. These results suggest that macrophage polarization may be an important step for epithelial repair and acute respiratory distress syndrome recovery.