Human Acute Respiratory Distress Syndrome Research Articles

Reduced microRNA-744 expression in mast cell-derived exosomes triggers epithelial cell ferroptosis in acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a critical disorder characterized by immune-related damage to epithelial cells; however, its underlying mechanism remains elusive. This study investigated the effects of alterations in microRNA (miRNA) expression in mast cell-derived exosomes on human bronchial epithelial (HBE) cells and ARDS development in cellular and mouse models challenged with lipopolysaccharide. Lipopolysaccharide-treated mast cell-derived exosomes reduced glutathione peroxidase 4 (GPX4) expression and increased long-chain acyl-CoA synthetase 4 (ACSL4), 15-lipoxygenase (ALOX15), and inflammatory mediator levels in HBE cells. miRNA sequencing revealed a reduction in mast cell-derived exosomal miR-744 levels, which was associated with the regulation of ACSL4, ALOX15, and GPX4 expression. This downregulation of exosomal miR-744 expression reduced miR-744 levels and promoted ferroptosis in HBE cells, whereas the experimental upregulation of miR-744 reversed these adverse effects. Down-regulation of miR-744 induced the expression of markers for ferroptosis and inflammation in HBE cells and promoted pulmonary ferroptosis, inflammation, and injury in LPS-stimulated mice. In vivo, treatment with ACSL4, ALOX15, and GPX4 inhibitors mitigated these effects, and experimental miR-744 expression rescued the lipopolysaccharide-induced changes in HBE cells and mouse lungs. Notably, miR-744 levels were reduced in the plasma and exosomes of patients with ARDS. We concluded that decreased mast cell-derived exosomal miR-744 levels trigger epithelial cell ferroptosis, promoting lung inflammation and damage in ARDS. This study provides new mechanistic insights into the development and sustained pulmonary damage associated with ARDS and highlights potential therapeutic strategies.

Bronchoalveolar Lavage and Oleic Acid Two-hit Model for Inducing Acute Respiratory Distress Syndrome in Swine Models.

Acute respiratory distress syndrome (ARDS) is a widespread and often fatal clinical syndrome marked by the acute onset of pulmonary edema and inflammatory-mediated disruptions in alveolar-capillary permeability resulting in impaired gas exchange and tissue oxygenation with subsequent acute respiratory failure that accounts for 10.4% of all intensive care unit admissions worldwide and boasts a mortality rate of 38.5%. The current treatment for ARDS remains largely supportive. This is largely because of the many challenges of achieving a stable and sustainable animal model that recreates the pathophysiology of ARDS experimentally in a controlled setting to allow research to elucidate potential treatments of ARDS moving forward. The bronchoalveolar lavage and oleic acid models are currently the 2 most frequently used experimental models in inducing ARDS in animal models. This study demonstrated that combining them into a "two-hit model" can produce sustained ARDS in swine models per the Horowitz index (PaO2/FiO2 ratio of ≤300 mmHg). Additionally, expected changes in pH, pCO2, lung compliance, cytokines, and tissue histopathology were observed and add to our confidence and reliability that the "two-hit model" produces symptomatic ARDS in a manner very similar to that observed in humans. In conclusion, we demonstrated a viable animal model of human ARDS that is maintained for a prolonged period, suitable for continuous monitoring of the progression, and evaluation of potential future treatments and procedures to reduce patient morbidity and mortality. To carry out this two-hit model, lung injury was induced through a combination of bronchoalveolar lavage and oleic acid administration and the disease process of ARDS is subsequently tracked through clinically relevant parameters such as respiratory mechanics, cytokine response, aretrial blood gas (ABG) changes, and observation of postmortem histopathologic changes. This promising new model has the capacity to successfully replicate human ARDS which is a well-known and notoriously multifactorial pathogenic process to reproduce experimentally for an extended period of time. The "two-hit model" is a viable and appropriate model for the research of novel treatments for ARDS.

Mitochondrial function in lungs of rats with different susceptibilities to hyperoxia-induced Acute Respiratory Distress Syndrome (ARDS)

Objective: Rat exposure to hyperoxia is a well-established model of human ARDS. Adult rats exposed to hyperoxia (100% O2) die from respiratory failure within 60-72 hours. However, rats preconditioned by exposure to >95% O2 for 48 hrs followed by a 24-hr “rest period” in room air (H-T) acquire tolerance of the otherwise lethal effects of exposure to 100% O2. In contrast, rats preconditioned by exposure to 60% O2 for 7 days (H-S) become more susceptible to 100% O2. The objective was to evaluate lung tissue mitochondrial bioenergetics in H-T and H-S rats. Methods: Adult rats were exposed to room air (normoxia), >95% O2 for 48 hrs followed by exposure to room air for 24 hrs (H-T), or 60% O2 for 7 days (H-S). Mitochondria were isolated from lung tissue and used to assess mitochondrial bioenergetics. Expressions of electron transport chain complexes were measured in lung tissue homogenate using western blot. Isolated perfused lungs (IPL) were used to determine pulmonary vascular endothelial filtration coeffcient ( Kf) as a measure of pulmonary vascular permeability, and lung tissue mitochondrial membrane potential (ΔΨm). Results: Western blot shows decreased (38%) complex I expression, but increased (70%) complex V expression in H-T lung tissue homogenate compared to normoxia. Complex I expression decreased (43%) in H-S lung tissue homogenate. State 3 oxidative phosphorylation (OxPhos) capacity (Vmax) and respiratory control ratio decreased in mitochondria isolated from H-S lungs. Vmax increased in mitochondria of H-T lungs. Time for ΔΨm repolarization following ADP-stimulated depolarization increased in mitochondria isolated from H-S lungs. IPL studies revealed that tissue ΔΨm is unchanged in H-S and H-T lungs compared to normoxics. Furthermore, complex I plays the dominant role in ΔΨm in H-T and normoxia lungs with no contribution from complex II, whereas complex II has a larger contribution to ΔΨm in H-S lungs than in H-T or normoxia. Kf increased (+178%) in H-S, but not H-T lungs. Discussion: For H-T lungs, decreased complex I expression is countered by increased complex V expression, which could also account for increased Vmax. This along with high tissue glutathione content protects mitochondria from stress such as exposure to 100% O2. For H-S lungs, the effect of decreased complex I expression on lung tissue ΔΨm is countered by a larger contribution from complex II. However, higher dependency of ΔΨm on complex II could lead to higher mitochondrial oxidant production since complex II is the main source of oxidants in rat lungs. This along with decreased Vmax and increased Kf make H-S rats more susceptible to stress such as exposure to 100% O2. These results are clinically relevant since exposure to hyperoxia is a primary therapy for patients with ARDS, and ventilation with 60% O2 is often required for prolonged periods of time, particularly with COVID-19. This study was funded by NHLBI grant 2R15HL129209-03, Department of Veterans Affairs Merit Review Award BX001681, and NSF grant DMS 2153387. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Application of anti-inflammatory treatment in two different ovine Acute Respiratory Distress Syndrome injury models: a preclinical randomized intervention study

Whilst the presence of 2 subphenotypes among the heterogenous Acute Respiratory Distress Syndrome (ARDS) population is becoming clinically accepted, subphenotype-specific treatment efficacy has yet to be prospectively tested. We investigated anti-inflammatory treatment in different ARDS models in sheep, previously shown similarities to human ARDS subphenotypes, in a preclinical, randomized, blinded study. Thirty anesthetized sheep were studied up to 48 h and randomized into: (a) OA: oleic acid (n = 15) and (b) OA-LPS: oleic acid and subsequent lipopolysaccharide (n = 15) to achieve a PaO2/FiO2 ratio of < 150 mmHg. Then, animals were randomly allocated to receive treatment with methylprednisolone or erythromycin or none. Assessed outcomes were oxygenation, pulmonary mechanics, hemodynamics and survival. All animals reached ARDS. Treatment with methylprednisolone, but not erythromycin, provided the highest therapeutic benefit in Ph2 animals, leading to a significant increase in PaO2/FiO2 ratio by reducing pulmonary edema, dead space ventilation and shunt fraction. Animals treated with methylprednisolone displayed a higher survival up to 48 h than all others. In animals treated with erythromycin, there was no treatment benefit regarding assessed physiological parameters and survival in both phenotypes. Treatment with methylprednisolone improves oxygenation and survival, more so in ovine phenotype 2 which resembles the human hyperinflammatory subphenotype.

Acute Respiratory Distress Syndrome: A Review on Animal Models

Acute Respiratory Distress Syndrome (ARDS) is an acute inflammatory lung injury. ARDS occurs when there is disruption in the alveolar-capillary barrier followed by the deposition of protein rich fluid inside the alveoli of the lungs. This protein-rich fluid stops the lungs from filling with enough air, resulting in less oxygen reaching the bloodstream. This depletes the organs of the oxygen that they need for proper functioning. The various clinical disorders that are associated with the development of ARDS include sepsis, pneumonia, aspiration of gastric contents, and major trauma. Human ARDS is not caused by any single event and hence there is no such existing animal model that would completely mimic Human ARDS. This review provides the various methods for insulting ARDS in animal model, along with advantage and disadvantage of each method.

Integrin α10β1-selected mesenchymal stem cells reduced hypercoagulopathy in a porcine model of acute respiratory distress syndrome

Mesenchymal stem cells (MSCs) have been studied for their potential benefits in treating acute respiratory distress syndrome (ARDS) and have reported mild effects when trialed within human clinical trials. MSCs have been investigated in preclinical models with efficacy when administered at the time of lung injury. Human integrin α10β1-selected adipose tissue-derived MSCs (integrin α10β1-MSCs) have shown immunomodulatory and regenerative effects in various disease models. We hypothesized that integrin α10β1 selected-MSCs can be used to treat a sepsis-induced ARDS in a porcine model when administering cells after established injury rather than simultaneously. This was hypothesized to reflect a clinical picture of treatment with MSCs in human ARDS. 12 pigs were randomized to the treated or placebo-controlled group prior to the induction of mild to moderate ARDS via lipopolysaccharide administration. The treated group received 5 × 106 cells/kg integrin α10β1-selected MSCs and both groups were followed for 12 h. ARDS was confirmed with blood gases and retrospectively with histological changes. After intervention, the treated group showed decreased need for inotropic support, fewer signs of histopathological lung injury including less alveolar wall thickening and reduction of the hypercoagulative disease state. The MSC treatment was not associated with adverse events over the monitoring period. This provides new opportunities to investigate integrin α10β1-selected MSCs as a treatment for a disease which does not yet have any definitive therapeutic options.

Experimental Model to Evaluate Resolution of Pneumonia.

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to high morbidity and mortality. Currently, there are no pre-clinical models that recapitulate the complexity of human ARDS. However, infectious models of pneumonia (PNA) can replicate the main pathophysiological features of ARDS. Here, we describe a model of PNA induced by the intratracheal instillation of live Streptococcus pneumoniae and Klebsiella pneumoniae in C57BL6 mice. In order to evaluate and characterize the model, after inducing injury, we carried out serial measurements of body weight and bronchoalveolar lavage (BAL) for measuring markers of lung injury. Additionally, we harvested lungs for cell count and differentials, BAL protein quantification, cytospin, bacterial colony-forming unit counts, and histology. Lastly, high dimensional flow cytometry was performed. We propose this model as a tool to understand the immune landscape during the early and late resolution phases of lung injury.

Lumican is elevated in the lung in human and experimental acute respiratory distress syndrome and promotes early fibrotic responses to lung injury

BackgroundFibroproliferative repair starts early in the inflammatory phase of acute respiratory distress syndrome (ARDS) and indicates a poor prognosis. Lumican, a small leucine-rich proteoglycan, is implicated in homeostasis and fibrogenesis, but its role in ARDS is unclear.MethodsBronchoalveolar lavage fluid (BALF) samples were obtained from ARDS patients (n = 55) enrolled within 24 h of diagnosis and mechanically ventilated (n = 20) and spontaneously breathing (n = 29) control subjects. Lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse models were intratracheally administered an adeno-associated virus (AAV) vector expressing lumican shRNA. Primary human lung fibroblasts (HLF) and small airway epithelial cells (SAECs) were cultured with tumour necrosis factor (TNF)-α or lumican. Luminex/ELISA, histochemistry/immunohistochemistry, immunofluorescence microscopy, quantitative real-time PCR, and western blotting were performed.ResultsLumican levels were significantly higher in the BALF of ARDS patients than in that of ventilated or spontaneously breathing controls (both p < 0.0001); they were correlated with the PaO2/FiO2 ratio and levels of proinflammatory cytokines (interleukin-6, interleukin-8, and TNF-α) and profibrotic factors (fibronectin, alpha-1 type I collagen [COL1A1], and alpha-1 type III collagen [COL3A1]). Lumican expression was enhanced in the alveolar walls and airway epithelium in the ALI mouse model. Murine lumican levels were also linked to proinflammatory and profibrotic cytokine levels in the BALF. In vitro, TNF-α induced the synthesis and secretion of lumican in HLF. In turn, lumican increased the expression of alpha-smooth muscle actin (α-SMA), COL1A1, and COL3A1 in HLF, upregulated α-SMA and COL3A1, downregulated E-cadherin, and caused spindle-shaped morphological changes in SAECs. Moreover, increased ERK phosphorylation and Slug were noted in both HLF and SAECs treated with lumican. In vivo, AAV-mediated knockdown of lumican inhibited the pulmonary production of fibronectin and COL3A1 and alleviated lung fibrotic lesions in LPS-challenged mice.ConclusionsPulmonary lumican levels were increased early in human and experimental ARDS and linked to disease severity and inflammatory fibrotic processes. Lumican triggers the transdifferentiation of lung fibroblasts into myofibroblasts and epithelial-mesenchymal transition in SAECs, possibly via the ERK/Slug pathway. Knockdown of pulmonary lumican attenuated extracellular matrix deposition in ALI mice. Overall, lumican promotes fibrotic responses in the early phase of ARDS, suggesting its potential as a therapeutic target.

Testing oxygenated microbubbles via intraperitoneal and intrathoracic routes on a large pig model of LPS‐induced acute respiratory distress syndrome

With a mortality rate of 46% before the onset of COVID‐19, acute respiratory distress syndrome (ARDS) affected 200,000 people in the US, causing 75,000 deaths. Mortality rates in COVID‐19 ARDS patients are currently at 39%. Extrapulmonary support for ARDS aims to supplement mechanical ventilation by providing life‐sustaining oxygen to the patient. A new rapid‐onset, human‐sized pig ARDS model in a porcine intensive care unit (ICU) was developed. The pigs were nebulized intratracheally with a high dose (4 mg/kg) of the endotoxin lipopolysaccharide (LPS) over a 2 h duration to induce rapid‐onset moderate‐to‐severe ARDS. They were then catheterized to monitor vitals and to evaluate the therapeutic effect of oxygenated microbubble (OMB) therapy delivered by intrathoracic (IT) or intraperitoneal (IP) administration. Post‐LPS administration, the PaO2 value dropped below 70 mmHg, the PaO2/FiO2 ratio dropped below 200 mmHg, and the heart rate increased, indicating rapidly developing (within 4 h) moderate‐to‐severe ARDS with tachycardia. The SpO2 and PaO2 of these LPS‐injured pigs did not show significant improvement after OMB administration, as they did in our previous studies of the therapy on small animal models of ARDS injury. Furthermore, pigs receiving OMB or saline infusions had slightly lower survival than their ARDS counterparts. The OMB administration did not induce a statistically significant or clinically relevant therapeutic effect in this model; instead, both saline and OMB infusion appeared to lower survival rates slightly. This result is significant because it contradicts positive results from our previous small animal studies and places a limit on the efficacy of such treatments for larger animals under more severe respiratory distress. While OMB did not prove efficacious in this rapid‐onset ARDS pig model, it may retain potential as a novel therapy for the usual presentation of ARDS in humans, which develops and progresses over days to weeks.

Tumor Progression Locus 2 Protects against Acute Respiratory Distress Syndrome in Influenza A Virus-Infected Mice.

ABSTRACTExcessive inflammation in patients with severe influenza disease may lead to acute lung injury that results in acute respiratory distress syndrome (ARDS). ARDS is associated with alveolar damage and pulmonary edema that severely impair gas exchange, leading to hypoxia. With no existing FDA-approved treatment for ARDS, it is important to understand the factors that lead to virus-induced ARDS development to improve prevention, diagnosis, and treatment. We have previously shown that mice deficient in the serine-threonine mitogen-activated protein kinase, Tpl2 (MAP3K8 or COT), succumb to infection with a typically low-pathogenicity strain of influenza A virus (IAV; HKX31, H3N2 [x31]). The goal of the current study was to evaluate influenza A virus-infected Tpl2−/− mice clinically and histopathologically to gain insight into the disease mechanism. We hypothesized that Tpl2−/− mice succumb to IAV infection due to development of ARDS-like disease and pulmonary dysfunction. We observed prominent signs of alveolar septal necrosis, hyaline membranes, pleuritis, edema, and higher lactate dehydrogenase (LDH) levels in the lungs of IAV-infected Tpl2−/− mice compared to wild-type (WT) mice from 7 to 9 days postinfection (dpi). Notably, WT mice showed signs of regenerating epithelium, indicative of repair and recovery, that were reduced in Tpl2−/− mice. Furthermore, biomarkers associated with human ARDS cases were upregulated in Tpl2−/− mice at 7 dpi, demonstrating an ARDS-like phenotype in Tpl2−/− mice in response to IAV infection.IMPORTANCE This study demonstrates the protective role of the serine-threonine mitogen-activated protein kinase, Tpl2, in influenza virus pathogenesis and reveals that host Tpl2 deficiency is sufficient to convert a low-pathogenicity influenza A virus infection into severe influenza disease that resembles ARDS, both histopathologically and transcriptionally. The IAV-infected Tpl2−/− mouse thereby represents a novel murine model for studying ARDS-like disease that could improve our understanding of this aggressive disease and assist in the design of better diagnostics and treatments.

Differential Protein Expression among Two Different Ovine ARDS Phenotypes-A Preclinical Randomized Study.

Despite decades of comprehensive research, Acute Respiratory Distress Syndrome (ARDS) remains a disease with high mortality and morbidity worldwide. The discovery of inflammatory subphenotypes in human ARDS provides a new approach to study the disease. In two different ovine ARDS lung injury models, one induced by additional endotoxin infusion (phenotype 2), mimicking some key features as described in the human hyperinflammatory group, we aim to describe protein expression among the two different ovine models. Nine animals on mechanical ventilation were included in this study and were randomized into (a) phenotype 1, n = 5 (Ph1) and (b) phenotype 2, n = 4 (Ph2). Plasma was collected at baseline, 2, 6, 12, and 24 h. After protein extraction, data-independent SWATH-MS was applied to inspect protein abundance at baseline, 2, 6, 12, and 24 h. Cluster analysis revealed protein patterns emerging over the study observation time, more pronounced by the factor of time than different injury models of ARDS. A protein signature consisting of 33 proteins differentiated among Ph1/2 with high diagnostic accuracy. Applying network analysis, proteins involved in the inflammatory and defense response, complement and coagulation cascade, oxygen binding, and regulation of lipid metabolism were activated over time. Five proteins, namely LUM, CA2, KNG1, AGT, and IGJ, were more expressed in Ph2.

Validation of Messenger Ribonucleic Acid Markers Differentiating Among Human Acute Respiratory Distress Syndrome Subgroups in an Ovine Model of Acute Respiratory Distress Syndrome Phenotypes.

BackgroundThe discovery of biological subphenotypes in acute respiratory distress syndrome (ARDS) might offer a new approach to ARDS in general and possibly targeted treatment, but little is known about the underlying biology yet. To validate our recently described ovine ARDS phenotypes model, we compared a subset of messenger ribonucleic acid (mRNA) markers in leukocytes as reported before to display differential expression between human ARDS subphenotypes to the expression in lung tissue in our ovine ARDS phenotypes model (phenotype 1 (Ph1): hypoinflammatory; phenotype 2 (Ph2): hyperinflammatory).MethodsWe studied 23 anesthetized sheep on mechanical ventilation with observation times between 6 and 24 h. They were randomly allocated to the two phenotypes (n = 14 to Ph1 and n = 9 to Ph2). At study end, lung tissue was harvested and preserved in RNAlater. After tissue homogenization in TRIzol, total RNA was extracted and custom capture and reporter probes designed by NanoString Technologies were used to measure the expression of 14 genes of interest and the 6 housekeeping genes on a nCounter SPRINT profiler.ResultsAmong the 14 mRNA markers, in all animals over all time points, 13 markers showed the same trend in ovine Ph2/Ph1 as previously reported in the MARS cohort: matrix metalloproteinase 8, olfactomedin 4, resistin, G protein-coupled receptor 84, lipocalin 2, ankyrin repeat domain 22, CD177 molecule, and transcobalamin 1 expression was higher in Ph2 and membrane metalloendopeptidase, adhesion G protein-coupled receptor E3, transforming growth factor beta induced, histidine ammonia-lyase, and sulfatase 2 expression was higher in Ph1. These expression patterns could be found when different sources of mRNA – such as blood leukocytes and lung tissue – were compared.ConclusionIn human and ovine ARDS subgroups, similar activated pathways might be involved (e.g., oxidative phosphorylation, NF-κB pathway) that result in specific phenotypes.

Mechanistic Understanding of Lung Inflammation: Recent Advances and Emerging Techniques.

Acute respiratory distress syndrome (ARDS) is a life-threatening lung injury characterized by an acute inflammatory response in the lung parenchyma. Hence, it is considered as the most appropriate clinical syndrome to study pathogenic mechanisms of lung inflammation. ARDS is associated with increased morbidity and mortality in the intensive care unit (ICU), while no effective pharmacological treatment exists. It is very important therefore to fully characterize the underlying pathobiology and the related mechanisms, in order to develop novel therapeutic approaches. In vivo and in vitro models are important pre-clinical tools in biological and medical research in the mechanistic and pathological understanding of the majority of diseases. In this review, we will present data from selected experimental models of lung injury/acute lung inflammation, which have been based on clinical disorders that can lead to the development of ARDS and related inflammatory lung processes in humans, including ventilation-induced lung injury (VILI), sepsis, ischemia/reperfusion, smoke, acid aspiration, radiation, transfusion-related acute lung injury (TRALI), influenza, Streptococcus (S.) pneumoniae and coronaviruses infection. Data from the corresponding clinical conditions will also be presented. The mechanisms related to lung inflammation that will be covered are oxidative stress, neutrophil extracellular traps, mitogen-activated protein kinase (MAPK) pathways, surfactant, and water and ion channels. Finally, we will present a brief overview of emerging techniques in the field of omics research that have been applied to ARDS research, encompassing genomics, transcriptomics, proteomics, and metabolomics, which may recognize factors to help stratify ICU patients at risk, predict their prognosis, and possibly, serve as more specific therapeutic targets.

In vivo Molecular Imaging Stratifies Rats with Different Susceptibilities to Hyperoxic Acute Lung Injury

ObjectivesAcute Respiratory Distress Syndrome (ARDS) is one of the most frequent causes of admission to medical intensive care units. It carries a mortality rate &gt;30%, due to lack of early detection tools, inability to identify patients at risk for progression, and limited therapies. Previously, we demonstrated the utility of 99mTc‐hexamethylpropyleneamine oxime (HMPAO) and 99mTc‐duramycin in vivo imaging to detect oxidative stress and endothelial cell death in rats exposed to 100% O2 (hyperoxia) as a model of human ARDS. Pre‐exposure to hyperoxia for 48 hrs followed by 24 hr in room air (H‐T) protects against ARDS in that, when subsequently re‐exposed to hyperoxia, rats are tolerant of and survive the otherwise lethal effects of hyperoxia. Thus, the objective of this study was to evaluate the ability of 99mTc ‐HMPAO and ‐duramycin to track the protection of hyperoxia pre‐exposure and to identify mechanisms involved as potential therapeutic targets.MethodsSprague‐Dawley rats were exposed to either room air, hyperoxia (100% O2) for 60 hr, hyperoxia for 48 hr followed by 24 hr of room air (H‐T), or H‐T followed by 60 hr of hyperoxia (H‐T+60). Rats were anesthetized and imaged using either 99mTc‐HMPAO or 99mTc‐duramycin (37‐74 MBq) (n=4‐6 per group). In vivo scintigraphy images were acquired 20 min post i.v. injection and lung uptake was determined from the images as mean lung activity normalized to mean forelimb blood activity. Rat lung endpoints including wet‐to‐dry weight ratio, volume of pleural effusion in the chest cavity, expression of 3‐nitrotyrosine (3NT), and histological H&amp;E and cleaved caspase 3 positive‐cells (apoptosis marker) were also measured as indices of injury/protection.ResultsLung 99mTc‐HMPAO uptake increased by 200% in hyperoxia‐exposed rats, whereas uptake in H‐T+60 rats was 24% less than in H‐T rats. Lung uptake of 99mTc‐duramycin increased by 167% in hyperoxia‐exposed rats, but uptake in H‐T+60 rats was only 17% greater than H‐T rats. Lung wet/dry weight ratio increased by 21% in hyperoxia‐exposed rats, but by only 15% in H‐T+60 versus HT rats. Similarly, pleural effusion was reduced significantly (66%) in the H‐T+60 rats compared to the hyperoxia rats. 3NT expression, as a measure of oxidative stress, was increased ~300% with hyperoxia, but no significant increase was detected in H‐T+60 rats compared to H‐T. Histological analysis indicates that neutrophilic influx, edema, and apoptotic cells were increased with hyperoxia, but this increase was small or not observed with H‐T+60.ConclusionsLung uptake of 99mTc‐HMPAO and 99mTc‐duramycin, reflecting oxidative stress and cell death, respectively, is reduced significantly in rats preconditioned to tolerate hyperoxia compared to those without the preconditioning. This tolerance is also reflected in lung functional and histological endpoints measured independently. Overall these results suggest the potential utility of 99mTc‐HMPAO and 99mTc‐ duramycin imaging as a tool for identifying those hosts that are more or less susceptible to progressing to severe ARDS following mild symptoms of lung injury.

Depolarized Mitochondrial Membrane Potential and Protection with Duroquinone in Isolated Perfused Lungs from Rats Exposed to Hyperoxia

ObjectiveDissipation of mitochondrial membrane potential (Δψm) is a hallmark of mitochondrial dysfunction, which plays a key in the pathogenesis of Acute Respiratory Distress Syndrome (ARDS). Our objective was to use a previously developed experimental‐computational approach to estimate tissue Δψm in isolated perfused lungs of rats exposed to hyperoxia as a rat model of human ARDS, and to evaluate the ability of the ubiquinone analog duroquinone (DQ) to reverse any hyperoxia‐induced depolarization of lung Δψm.MethodsRats were exposed to hyperoxia (&gt;95% O2) or normoxia (room air) for 48 hrs, after which lungs were excised and connected to a ventilation‐perfusion system. The experimental protocol consisted of measuring the concentration of the fluorescent dye rhodamine 6G (R6G) during three single‐pass phases: loading, washing, and uncoupling, in which the lungs were perfused with and without R6G, and with the mitochondrial uncoupler FCCP, respectively. For normoxic lungs, the protocol was repeated with 1) rotenone (mitochondrial complex I inhibitor), 2) rotenone and either DQ or its vehicle (DMSO), and 3) rotenone, glutathione (GSH), and either DQ or DMSO added to the perfusate. Hyperoxic lungs were studied with and without DQ and GSH added to the perfusate. A physiologically‐based pharmacokinetic (PBPK) computational model was used to estimate lung Δψm from R6G data.ResultsFigure 1 shows that for hyperoxic lungs, R6G venous effluent concentrations tended to be higher during the loading phase, but significantly lower during the uncoupling phase compared to normoxic lungs, consistent with less uptake of R6G by hyperoxic lungs due to partial depolarization of Δψm. Figure 1 also shows that adding DQ to perfusate during the three phases almost completely reversed differences in R6G concentrations between normoxic and hyperoxic lungs. The PBPK computational model analysis of the R6G data show that exposure to hyperoxia depolarized lung tissue Δψm from ‐140 ± 9 (SE, n = 5) mV to ‐107 ± 9 (n = 5) mV, and complex I inhibition depolarized lung Δψm from ‐140 ± 9 (n = 5) to ‐57 ± 2 (n = 4) mV. Computational model analysis results also demonstrate the efficacy of DQ to fully reverse both rotenone‐induced and hyperoxia‐induced lung Δψm depolarization, consistent with the ability of the lungs to reduce DQ to its hydroquinone form (DQH2), which in turn bypasses mitochondrial complexes I and II to reduce mitochondrial complex III and restores Δψm.ConclusionsThis study demonstrates hyperoxia‐induced Δψm depolarization in isolated perfused lungs, and the utility of this approach for assessing the impact of potential therapies such as exogenous quinones that target mitochondria in isolated perfused lungs.

Extracellular HMGB1 Impairs Macrophage-Mediated Efferocytosis by Suppressing the Rab43-Controlled Cell Surface Transport of CD91.

High-mobility group box 1 (HMGB1) protein can impair phagocyte function by suppressing the macrophage-mediated clearance of apoptotic cells (ACs), thereby delaying inflammation resolution in the lungs and allowing the progression of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the precise mechanism underlying this HMGB1-mediated inhibition of efferocytosis remains unknown. The aim of this study was to determine the effect of HMGB1 on macrophage-mediated efferocytosis. We discovered that HMGB1 prevented efferocytosis by bone marrow-derived macrophages (BMDMs) and suppressed the expression of Ras-related GTP-binding protein 43 (Rab43), a member of the Ras-associated binding (Rab) family. The downregulation of Rab43 expression resulted in impaired clearance of apoptotic thymocytes by BMDMs. Subsequent analysis of HMGB1-treated and Rab43-deficient BMDMs revealed the inhibited transport of cluster of differentiation 91 (CD91), a phagocyte recognition receptor, from the cytoplasm to the cell surface. Notably, Rab43 directly interacted with CD91 to mediate its intercellular trafficking. Furthermore, Rab43 knockout delayed the inflammation resolution and aggravated the lung tissue damage in mice with ALI. Therefore, our results provide evidence that HMGB1 impairs macrophage-mediated efferocytosis and delays inflammation resolution by suppressing the Rab43-regulated anterograde transport of CD91, suggesting that the restoration of Rab43 levels is a promising strategy for attenuating ALI and ARDS in humans.

Update on the Features and Measurements of Experimental Acute Lung Injury in Animals: An Official American Thoracic Society Workshop Report.

Advancements in methods, technology, and our understanding of the pathobiology of lung injury have created the need to update the definition of experimental acute lung injury (ALI). We queried 50 participants with expertise in ALI and acute respiratory distress syndrome using a Delphi method composed of a series of electronic surveys and a virtual workshop. We propose that ALI presents as a “multidimensional entity” characterized by four “domains” that reflect the key pathophysiologic features and underlying biology of human acute respiratory distress syndrome. These domains are 1) histological evidence of tissue injury, 2) alteration of the alveolar–capillary barrier, 3) presence of an inflammatory response, and 4) physiologic dysfunction. For each domain, we present “relevant measurements,” defined as those proposed by at least 30% of respondents. We propose that experimental ALI encompasses a continuum of models ranging from those focusing on gaining specific mechanistic insights to those primarily concerned with preclinical testing of novel therapeutics or interventions. We suggest that mechanistic studies may justifiably focus on a single domain of lung injury, but models must document alterations of at least three of the four domains to qualify as “experimental ALI.” Finally, we propose that a time criterion defining “acute” in ALI remains relevant, but the actual time may vary based on the specific model and the aspect of injury being modeled. The continuum concept of ALI increases the flexibility and applicability of the definition to multiple models while increasing the likelihood of translating preclinical findings to critically ill patients.

Blockade of caspase cascade overcomes malaria-associated acute respiratory distress syndrome in mice

Malaria is an enormous burden on global health that caused 409,000 deaths in 2019. Severe malaria can manifest in the lungs, an illness known as acute respiratory distress syndrome (ARDS). Not much is known about the development of malaria-associated ARDS (MA-ARDS), especially regarding cell death in the lungs. We had previously established a murine model that mimics various human ARDS aspects, such as pulmonary edema, hemorrhages, pleural effusion, and hypoxemia, using DBA/2 mice infected with Plasmodium berghei ANKA. Here, we explored the mechanisms and the involvement of apoptosis in this syndrome. We found that apoptosis contributes to the pathogenesis of MA-ARDS, primarily as facilitators of the alveolar-capillary barrier breakdown. The protection of pulmonary endothelium by inhibiting caspase activation could be a promising therapeutic strategy to prevent the pathogenicity of MA-ARDS. Therefore, intervention in the programmed death cell mechanism could help patients not to develop severe malaria.

Fatal COVID-19 and Non–COVID-19 Acute Respiratory Distress Syndrome Is Associated with Incomplete Alveolar Type 1 Epithelial Cell Differentiation from the Transitional State without Fibrosis

Acute respiratory distress syndrome (ARDS) due to coronavirus disease 2019 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; recovery requires epithelial regeneration. During physiological regeneration in mice, type 2 AECs (AEC2s) proliferate, exit the cell cycle, transiently assume a transitional state, then differentiate into type 1 AECs (AEC1s); in humans, persistence of the transitional state is associated with pulmonary fibrosis. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiological regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or single-cell RNA sequencing revealed that transitional cells in mouse models of physiological regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis.

Syrian hamsters as a model of lung injury with SARS-CoV-2 infection: Pathologic, physiologic, and detailed molecular profiling

The acute respiratory distress syndrome (ARDS) is a common complication of severe COVID-19 (coronavirus disease 2019) caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection. Knowledge of molecular mechanisms driving host responses to SARS-CoV-2 is limited by the lack of reliable preclinical models of COVID-19 that recapitulate human illness. Further, existing COVID-19 animal models are not characterized as models of experimental acute lung injury (ALI) or ARDS. Acknowledging differences in experimental lung injury in animal models and human ARDS, here we systematically evaluate a model of experimental acute lung injury as a result of SARS-CoV-2 infection in Syrian golden hamsters. Following intranasal inoculation, hamsters demonstrate acute SARS-CoV-2 infection, viral pneumonia, and systemic illness but survive infection with clearance of virus. Hamsters exposed to SARS-CoV-2 exhibited key features of experimental ALI, including histologic evidence of lung injury, increased pulmonary permeability, acute inflammation, and hypoxemia. RNA sequencing of lungs indicated upregulation of inflammatory mediators that persisted after infection clearance. Lipidomic analysis demonstrated significant differences in hamster phospholipidome with SARS-CoV-2 infection. Lungs infected with SARS-CoV-2 showed increased apoptosis and ferroptosis. Thus, SARS-CoV-2 infected hamsters exhibit key features of experimental lung injury supporting their use as a preclinical model of COVID-19 ARDS.