Extracellular vesicles derived from M2-like macrophages alleviate acute lung injury in a miR-709-mediated manner.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is characterised by an uncontrolled inflammatory response, and current treatment strategies have limited efficacy. Although the protective effect of M2-like macrophages (M2φ) and their extracellular vesicles (EVs) has been well-documented in other inflammatory diseases, the role of M2φ-derived EVs (M2φ-EVs) in the pathogenesis of ALI/ARDS remains poorly understood. The present study utilised a mouse model of lipopolysaccharide-induced ALI to first demonstrate a decrease in endogenous M2-like alveolar macrophage-derived EVs. And then, intratracheal instillation of exogenous M2φ-EVs from the mouse alveolar macrophage cell line (MH-S) primarily led to a take up by alveolar macrophages, resulting in reduced lung inflammation and injury. Mechanistically, the M2φ-EVs effectively suppressed the pyroptosis of alveolar macrophages and inhibited the release of excessive cytokines such as IL-6, TNF-α and IL-1ß both in vivo and in vitro, which were closely related to NF-κB/NLRP3 signalling pathway inhibition. Of note, the protective effect of M2φ-EVs was partly mediated by miR-709, as evidenced by the inhibition of miR-709 expression in M2φ-EVs mitigated their protective effect against lipopolysaccharide-induced ALI in mice. In addition, we found that the expression of miR-709 in EVs derived from bronchoalveolar lavage fluid was correlated negatively with disease severity in ARDS patients, indicating its potential as a marker for ARDS severity. Altogether, our study revealed that M2φ-EVs played a protective role in the pathogenesis of ALI/ARDS, partly mediated by miR-709, offering a potential strategy for assessing disease severity and treating ALI/ARDS.

Inhibition of GBP5 activates autophagy to alleviate inflammatory response in LPS-induced lung injury in mice.

BACKGROUND: Pulmonary emphysema is a condition that causes damage to the lung tissue over time. GBP5, as part of the guanylate-binding protein family, is dysregulated in mouse pulmonary emphysema. However, the role of GBP5 in lung inflammation in ARDS remains unveiled. METHODS: To investigate whether GBP5 regulates lung inflammation and autophagy regulation, the study employed a mouse ARDS model and MLE-12 cell culture. Vector transfection was performed for the genetic manipulation of GBP5. Then, RT-qPCR, WB and IHC staining were conducted to assess its transcriptional and expression levels. Histological features of the lung tissue were observed through HE staining. Moreover, ELISA was conducted to evaluate the secretion of inflammatory cytokines, autophagy was assessed by immunofluorescent staining, and MPO activity was determined using a commercial kit. RESULTS: Our study revealed that GBP5 expression was altered in mouse ARDS and LPS-induced MLE-12 cell models. Moreover, the suppression of GBP5 reduced lung inflammation induced by LPS in mice. Conversely, overexpression of GBP5 diminished the inhibitory impact of LPS on ARDS during autophagy, leading to increased inflammation. In the cell line of MLE-12, GBP5 exacerbates LPS-induced inflammation by blocking autophagy. CONCLUSION: The study suggests that GBP5 facilitates lung inflammation and autophagy regulation. Thus, GBP5 could be a potential therapeutic approach for improving ARDS treatment outcomes, but further research is required to validate these findings.

Inhibition the ubiquitination of ENaC and Na,K-ATPase with erythropoietin promotes alveolar fluid clearance in sepsis-induced acute respiratory distress syndrome.

Sepsis-induced acute respiratory distress syndrome (ARDS) causes significant fatalities worldwide and lacks pharmacological intervention. Alveolar fluid clearance (AFC) plays a pivotal role in the remission of ARDS and is markedly impaired in the pathogenesis of ARDS. Here, we demonstrated that erythropoietin could effectively ameliorate lung injury manifestations and lethality, restore lung function and promote AFC in a rat model of lipopolysaccharide (LPS)-induced ARDS. Moreover, it was proven that EPO-induced restoration of AFC occurs through triggering the total protein expression of ENaC and Na,K-ATPase channels, enhancing their protein abundance in the membrane, and suppressing their ubiquitination for degeneration. Mechanistically, the data indicated the possible involvement of EPOR/JAK2/STAT3/SGK1/Nedd4-2 signaling in this process, and the pharmacological inhibition of the pathway markedly eliminated the stimulating effects of EPO on ENaC and Na,K-ATPase, and subsequently reversed the augmentation of AFC by EPO. Consistently, in vitro studies of alveolar epithelial cells paralleled with that EPO upregulated the expression of ENaC and Na,K-ATPase, and patch-clamp studies further demonstrated that EPO substantially strengthened sodium ion currents. Collectively, EPO could effectively promote AFC by improving ENaC and Na,K-ATPase protein expression and abundance in the membrane, dependent on inhibition of ENaC and Na,K-ATPase ubiquitination, and resulting in diminishing LPS-associated lung injuries.

Hydrogen alleviates impaired lung epithelial barrier in acute respiratory distress syndrome via inhibiting Drp1-mediated mitochondrial fission through the Trx1 pathway.

Acute respiratory distress syndrome (ARDS) is an acute and severe clinical complication lacking effective therapeutic interventions. The disruption of the lung epithelial barrier plays a crucial role in ARDS pathogenesis. Recent studies have proposed the involvement of abnormal mitochondrial dynamics mediated by dynamin-related protein 1 (Drp1) in the mechanism of impaired epithelial barrier in ARDS. Hydrogen is an anti-oxidative stress molecule that regulates mitochondrial function via multiple signaling pathways. Our previous study confirmed that hydrogen modulated oxidative stress and attenuated acute pulmonary edema in ARDS by upregulating thioredoxin 1 (Trx1) expression, but the exact mechanism remains unclear. This study aimed to investigate the effects of hydrogen on mitochondrial dynamics both in vivo and in vitro. Our study revealed that hydrogen inhibited lipopolysaccharide (LPS)-induced phosphorylation of Drp1 (at Ser616), suppressed Drp1-mediated mitochondrial fission, alleviated epithelial tight junction damage and cell apoptosis, and improved the integrity of the epithelial barrier. This process was associated with the upregulation of Trx1 in lung epithelial tissues of ARDS mice by hydrogen. In addition, hydrogen treatment reduced the production of reactive oxygen species in LPS-induced airway epithelial cells (AECs) and increased the mitochondrial membrane potential, indicating that the mitochondrial dysfunction was restored. Then, the expression of tight junction proteins occludin and zonula occludens 1 was upregulated, and apoptosis in AECs was alleviated. Remarkably, the protective effects of hydrogen on the mitochondrial and epithelial barrier were eliminated after applying the Trx1 inhibitor PX-12. The results showed that hydrogen significantly inhibited the cell apoptosis and the disruption of epithelial tight junctions, maintaining the integrity of the epithelial barrier in mice of ARDS. This might be related to the inhibition of Drp1-mediated mitochondrial fission through the Trx1 pathway. The findings of this study provided a new theoretical basis for the application of hydrogen in the clinical treatment of ARDS.

Functional roles of CD26/DPP4 in lipopolysaccharide-induced lung injury.

Acute respiratory distress syndrome (ARDS) is characterized by dysregulated inflammation and increased permeability of lung microvascular cells. CD26/dipeptidyl peptidase-4 (DPP4) is a type II membrane protein that is expressed in several cell types and mediates multiple pleiotropic effects. We previously reported that DPP4 inhibition by sitagliptin attenuates lipopolysaccharide (LPS)-induced lung injury in mice. The current study characterized the functional role of CD26/DPP4 expression in LPS-induced lung injury in mice, isolated alveolar macrophages, and cultured lung endothelial cells. In LPS-induced lung injury, inflammatory responses [bronchoalveolar lavage fluid (BALF) neutrophil numbers and several proinflammatory cytokine levels] were attenuated in Dpp4 knockout (Dpp4 KO) mice. However, multiple assays of alveolar capillary permeability were similar between the Dpp4 KO and wild-type mice. TNF-α and IL-6 production was suppressed in alveolar macrophages isolated from Dpp4 KO mice. In contrast, in cultured mouse lung microvascular endothelial cells (MLMVECs), reduction in CD26/DPP4 expression by siRNA resulted in greater ICAM-1 and IL-6 expression after LPS stimulation. Moreover, the LPS-induced vascular monolayer permeability in vitro was higher in MLMVECs treated with Dpp4 siRNA, suggesting that CD26/DPP4 plays a protective role in endothelial barrier function. In summary, this study demonstrated that genetic deficiency of Dpp4 attenuates inflammatory responses but not permeability in LPS-induced lung injury in mice, potentially through differential functional roles of CD26/DPP4 expression in resident cellular components of the lung. CD26/DPP4 may be a potential therapeutic target for ARDS and warrants further exploration to precisely identify the multiple functional effects of CD26/DPP4 in ARDS pathophysiology.NEW & NOTEWORTHY We aimed to clarify the functional roles of CD26/DPP4 in ARDS pathophysiology using Dpp4-deficient mice and siRNA reduction techniques in cultured lung cells. Our results suggest that CD26/DPP4 expression plays a proinflammatory role in alveolar macrophages while also playing a protective role in the endothelial barrier. Dpp4 genetic deficiency attenuates inflammatory responses but not permeability in LPS-induced lung injury in mice, potentially through differential roles of CD26/DPP4 expression in the resident cellular components of the lung.

Osteopontin: A Novel Therapeutic Target for Respiratory Diseases.

Osteopontin (OPN) is a multifunctional phosphorylated protein that is involved in physiological and pathological events. Emerging evidence suggests that OPN also plays a critical role in the pathogenesis of respiratory diseases. OPN can be produced and secreted by various cell types in lungs and overexpression of OPN has been found in acute lung injury/acute respiratory distress syndrome (ALI/ARDS), pulmonary hypertension (PH), pulmonary fibrosis diseases, lung cancer, lung infection, chronic obstructive pulmonary disease (COPD), and asthma. OPN exerts diverse effects on the inflammatory response, immune cell activation, fibrosis and tissue remodeling, and tumorigenesis of these respiratory diseases, and genetic and pharmacological moudulation of OPN exerts therapeutic effects in the treatment of respiratory diseases. In this review, we summarize the recent evidence of multifaceted roles and underlying mechanisms of OPN in these respiratory diseases, and targeting OPN appears to be a potential therapeutic intervention for these diseases.

Hippophae rhamnoides Prevents Oleic Acid-Induced Acute Respiratory Distress Syndrome by Releasing Acetylcholinesterase Activity and Mitigation of Angiotensin-Converting Enzyme Level.

Hippophae rhamnoides exhibit a wide variety of medicinal and pharmacological effects. The present study aims to determine the role of ethanol extract of H. rhamnoides on oleic acid (OA)-induced acute respiratory distress syndrome (ARDS) in rats. Male rats were randomly divided into the following groups: (I) Control, (II) OA, and (III) OA+H. rhamnoides. H. rhamnoides extract (500 mg/kg) was given orally for 2 weeks before OA in Group III. Levels of total antioxidant capacity, total oxidant status (TOS), myeloperoxidase (MPO), mitogen-activated protein kinase (MAPK), acetylcholinesterase (AChE), and angiotensin-converting enzyme (ACE) were quantified by enzyme-linked immunosorbent assay (ELISA). Real time quantitative polymerase chain reaction was utilized to evaluate the expression of nuclear factor kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and matrix metalloproteinase 2 (MMP2). Also, Caspase-3 immunostaining and expression were performed to evaluate apoptosis. Compared with the OA group, there was a significantly decrease in the levels of MPO, TOS, MAPK, and ACE and in the expression of NF-κB, TNF-α, IL-6, MMP2, and Caspase-3 in the H. rhamnoides administration group. Moreover, the activity of AChE and level of TAS were substantially higher in the H. rhamnoides administration compared with the OA group. The findings in the study suggest that the protective effect of H. rhamnoides pretreatment may act through inhibition of the ACE activity, releasing AChE, regulation of inflammatory cytokine levels, and suppression of apoptotic process in ARDS.

IL-22 Binding Protein Controls IL-22-Driven Bleomycin-Induced Lung Injury.

The high mortality rates of acute lung injury and acute respiratory distress syndrome challenge the field to identify biomarkers and factors that can be exploited for therapeutic approaches. IL-22 is a cytokine that has antibacterial and reparative properties in the lung. However, it also can exacerbate inflammation and requires tight control by the extracellular inhibitory protein known as IL-22 binding protein (IL-22BP) (Il22ra2). This study showed the necessity of IL-22BP in controlling and preventing acute lung injury using IL-22BP knockout mice (Il22ra2-/-) in the bleomycin model of acute lung injury/acute respiratory distress syndrome. Il22ra2-/- mice had greater sensitivity (weight loss and death) and pulmonary inflammation in the acute phase (first 7 days) of the injury compared with wild-type C57Bl/6 controls. The inflammation was driven by excess IL-22 production, inducing the influx of pathogenic IL-17A+ γδ T cells to the lung. Interestingly, this inflammation was initiated in part by the noncanonical IL-22 signaling to macrophages, which express the IL-22 receptor (Il22ra1) in vivo after bleomycin challenge. This study further showed that IL-22 receptor alpha-1+ macrophages can be stimulated by IL-22 to produce a number of IL-17-inducing cytokines such as IL-1ß, IL-6, and transforming growth factor-ß1. Together, the results suggest that IL-22BP prevents IL-22 signaling to macrophages and reduces bleomycin-mediated lung injury.

Regulator of G protein signaling protein 6 alleviates acute lung injury by inhibiting inflammation and promoting cell self-renewal in mice.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a disease with high mortality and morbidity. Regulator of G protein signaling protein 6 (RGS6), identified as a tumor suppressor gene, has received increasing attention owing to its close relationship with oxidative stress and inflammation. However, the association between ARDS and RGS6 has not been reported. METHODS: Congruously regulated G protein-coupled receptor (GPCR)-related genes and differentially expressed genes (DEGs) in an acute lung injury (ALI) model were identified, and functional enrichment analysis was conducted. In an in vivo study, the effects of RGS6 knockout were studied in a mouse model of ALI induced by lipopolysaccharide (LPS). HE staining, ELISA, and immunohistochemistry were used to evaluate pathological changes and the degree of inflammation. In vitro, qRT‒PCR, immunofluorescence staining, and western blotting were used to determine the dynamic changes in RGS6 expression in cells. The RGS6 overexpression plasmid was constructed for transfection. qRT‒PCR was used to assess proinflammatory factors transcription. Western blotting and flow cytometry were used to evaluate apoptosis and reactive oxygen species (ROS) production. Organoid culture was used to assess the stemness and self-renewal capacity of alveolar epithelial type II cells (AEC2s). RESULTS: A total of 110 congruously regulated genes (61 congruously upregulated and 49 congruously downregulated genes) were identified among GPCR-related genes and DEGs in the ALI model. RGS6 was downregulated in vivo and in vitro in the ALI model. RGS6 was expressed in the cytoplasm and accumulated in the nucleus after LPS stimulation. Compared with the control group, we found higher mortality, more pronounced body weight changes, more serious pulmonary edema and pathological damage, and more neutrophil infiltration in the RGS6 knockout group upon LPS stimulation in vivo. Moreover, AEC2s loss was significantly increased upon RGS6 knockout. Organoid culture assays showed slower alveolar organoid formation, fewer alveolar organoids, and impaired development of new structures after passaging upon RGS6 knockout. In addition, RGS6 overexpression decreased ROS production as well as proinflammatory factor transcription in macrophages and decreased apoptosis in epithelial cells. CONCLUSIONS: RGS6 plays a protective role in ALI not only in early inflammatory responses but also in endogenous lung stem cell regeneration.

Human Placental Mesenchymal Stem Cells and Derived Extracellular Vesicles Ameliorate Lung Injury in Acute Respiratory Distress Syndrome Murine Model.

This study investigates the therapeutic potential of human placental mesenchymal stem cells (P-MSCs) and their extracellular vesicles (EVs) in a murine model of acute respiratory distress syndrome (ARDS), a condition with growing relevance due to its association with severe COVID-19. We induced ARDS-like lung injury in mice using intranasal LPS instillation and evaluated histological changes, neutrophil accumulation via immunohistochemistry, bronchoalveolar lavage fluid cell count, total protein, and cytokine concentration, as well as lung gene expression changes at three time points: 24, 72, and 168 h. We found that both P-MSCs and EV treatments reduced the histological evidence of lung injury, decreased neutrophil infiltration, and improved alveolar barrier integrity. Analyses of cytokines and gene expression revealed that both treatments accelerated inflammation resolution in lung tissue. Biodistribution studies indicated negligible cell engraftment, suggesting that intraperitoneal P-MSC therapy functions mostly through soluble factors. Overall, both P-MSC and EV therapy ameliorated LPS-induced lung injury. Notably, at the tested dose, EV therapy was more effective than P-MSCs in reducing most aspects of lung injury.

Morphofunctional Characteristics of Lung Macrophages in Rats with Acute Respiratory Distress Syndrome.

A comprehensive morphofunctional study of the lungs and alveolar macrophages was carried out in Sprague-Dawley rats with acute respiratory distress syndrome (n=10) induced by intratracheal administration of E. coli LPS 0111:B4 in a dose of 15 mg/kg. On the first day after LPS administration, bronchopneumonia was observed in the lungs, the number of macrophages of the bone marrow origin and the number of M1 macrophages with the proinflammatory phenotype in the bronchoalveolar lavage increased, the expression of proinflammatory cytokines increased and the expression of anti-inflammatory cytokines decreased, which was accompanied by an increase in LPS and C-reactive protein in the blood serum. The revealed changes correspond to the development of acute respiratory distress syndrome in humans, and the decrease in the number of macrophages in the lungs and their predominant polarization to the M1-proinflammatory phenotype substantiate the use of cell therapy with reprogrammed M2 macrophages.

Aerosol Delivery of a Novel Recombinant Modified Superoxide Dismutase Protein Reduces Oxidant Injury and Attenuates Escherichia coli Induced Lung Injury in Rats.

Background: Acute respiratory distress syndrome (ARDS) is a life-threatening respiratory failure syndrome with diverse etiologies characterized by increased permeability of alveolar-capillary membranes, pulmonary edema, and acute onset hypoxemia. During the ARDS acute phase, neutrophil infiltration into the alveolar space results in uncontrolled release of reactive oxygen species (ROS) and proteases, overwhelming antioxidant defenses and causing alveolar epithelial and lung endothelial injury. Objectives: To investigate the therapeutic potential of a novel recombinant human Cu-Zn-superoxide dismutase (SOD) fusion protein in protecting against ROS injury and for aerosolized SOD delivery to treat Escherichia coli induced ARDS. Methods: Fusion proteins incorporating human Cu-Zn-SOD (hSOD1), with (pep1-hSOD1-his) and without (hSOD1-his) a fused hyaluronic acid-binding peptide, were expressed in E. coli. Purified proteins were evaluated in in vitro assays with human bronchial epithelial cells and through aerosolized delivery to the lung of an E. coli-induced ARDS rat model. Results: SOD proteins exhibited high SOD activity in vitro and protected bronchial epithelial cells from oxidative damage. hSOD1-his and pep1-hSOD1-his retained SOD activity postnebulization and exhibited no adverse effects in the rat. Pep1-hSOD1-his administered through instillation or nebulization to the lung of an E. coli-induced pneumonia rat improved arterial oxygenation and lactate levels compared to vehicle after 48 hours. Static lung compliance was improved when the pep1-hSOD1-his protein was delivered by instillation. White cell infiltration to the lung was significantly reduced by aerosolized delivery of protein, and reduction of cytokine-induced neutrophil chemoattractant-1, interferon-gamma, and interleukin 6 pro-inflammatory cytokine concentrations in bronchoalveolar lavage was observed. Conclusions: Aerosol delivery of a novel recombinant modified SOD protein reduces oxidant injury and attenuates E. coli induced lung injury in rats. The results provide a strong basis for further investigation of the therapeutic potential of hSOD1 in the treatment of ARDS.

Synthetic surfactant with a combined SP-B and SP-C analogue is efficient in rabbit models of adult and neonatal respiratory distress syndrome.

Respiratory distress syndrome (RDS) in premature infants is caused by insufficient amounts of endogenous lung surfactant and is efficiently treated with replacement therapy using animal-derived surfactant preparations. On the other hand, adult/acute RDS (ARDS) occurs secondary to for example, sepsis, aspiration of gastric contents, and multitrauma and is caused by alveolar endothelial damage, leakage of plasma components into the airspaces and inhibition of surfactant activity. Instillation of surfactant preparations in ARDS has so far resulted in very limited treatment effects, partly due to inactivation of the delivered surfactants in the airspace. Here, we develop a combined surfactant protein B (SP-B) and SP-C peptide analogue (Combo) that can be efficiently expressed and purified from Escherichia coli without any solubility or purification tag. NMR spectroscopy shows that Combo peptide forms α-helices both in organic solvents and in lipid micelles, which coincide with the helical regions described for the isolated SP-B and SP-C parts. Artificial Combo surfactant composed of synthetic dipalmitoylphosphatidylcholine:palmitoyloleoylphosphatidylglycerol, 1:1, mixed with 3 weights % relative to total phospholipids of Combo peptide efficiently improves tidal volumes and lung gas volumes at end-expiration in a premature rabbit fetus model of RDS. Combo surfactant also improves oxygenation and respiratory parameters and lowers cytokine release in an acid instillation-induced ARDS adult rabbit model. Combo surfactant is markedly more resistant to inhibition by albumin and fibrinogen than a natural-derived surfactant in clinical use for the treatment of RDS. These features of Combo surfactant make it attractive for the development of novel therapies against human ARDS.

Mitochondria of lung venular capillaries mediate lung-liver cross talk in pneumonia.

Failure of the lung's endothelial barrier underlies lung injury, which causes the high mortality acute respiratory distress syndrome (ARDS). Multiple organ failure predisposes to the mortality, but mechanisms are poorly understood. Here, we show that mitochondrial uncoupling protein 2 (UCP2), a component of the mitochondrial inner membrane, plays a role in the barrier failure. Subsequent lung-liver cross talk mediated by neutrophil activation causes liver congestion. We intranasally instilled lipopolysaccharide (LPS). Then, we viewed the lung endothelium by real-time confocal imaging of the isolated, blood-perfused mouse lung. LPS caused alveolar-capillary transfer of reactive oxygen species and mitochondrial depolarization in lung venular capillaries. The mitochondrial depolarization was inhibited by transfection of alveolar Catalase and vascular knockdown of UCP2. LPS instillation caused lung injury as indicated by increases in bronchoalveolar lavage (BAL) protein content and extravascular lung water. LPS or Pseudomonas aeruginosa instillation also caused liver congestion, quantified by liver hemoglobin and plasma aspartate aminotransferase (AST) increases. Genetic inhibition of vascular UCP2 prevented both lung injury and liver congestion. Antibody-mediated neutrophil depletion blocked the liver responses, but not lung injury. Knockdown of lung vascular UCP2 mitigated P. aeruginosa-induced mortality. Together, these data suggest a mechanism in which bacterial pneumonia induces oxidative signaling to lung venular capillaries, known sites of inflammatory signaling in the lung microvasculature, depolarizing venular mitochondria. Successive activation of neutrophils induces liver congestion. We conclude that oxidant-induced UCP2 expression in lung venular capillaries causes a mechanistic sequence leading to liver congestion and mortality. Lung vascular UCP2 may present a therapeutic target in ARDS.NEW & NOTEWORTHY We report that mitochondrial injury in lung venular capillaries underlies barrier failure in pneumonia, and venular capillary uncoupling protein 2 (UCP2) causes neutrophil-mediated liver congestion. Using in situ imaging, we found that epithelial-endothelial transfer of H2O2 activates UCP2, depolarizing mitochondria in venular capillaries. The conceptual advance from our findings is that mitochondrial depolarization in lung capillaries mediates liver cross talk through circulating neutrophils. Pharmacologic blockade of UCP2 could be a therapeutic strategy for lung injury.

Advancements in understanding the role of microRnas in regulating macrophage polarization during acute lung injury.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a critical and life-threatening illness that causes severe dyspnea, and respiratory distress and is often caused by a variety of direct or indirect factors that damage the alveolar epithelium and capillary endothelial cells, leading to inflammation factors and macrophage infiltration. Macrophages play a crucial role in the progression of ALI/ARDS, exhibiting different polarized forms at different stages of the disease that control the disease outcome. MicroRNAs (miRNA) are conserved, endogenous, short non-coding RNAs composed of 18-25 nucleotides that serve as potential markers for many diseases and are involved in various biological processes, including cell proliferation, apoptosis, and differentiation. In this review, we provide a brief overview of miRNA expression in ALI/ARDS and summarize recent research on the mechanism and pathways by which miRNAs respond to macrophage polarization, inflammation, and apoptosis. The characteristics of each pathway are also summarized to provide a comprehensive understanding of the role of miRNAs in regulating macrophage polarization during ALI/ARDS.

Umbilical cord mesenchymal stem cells overexpressing CXCR7 facilitate treatment of ARDS-associated pulmonary fibrosis via inhibition of Notch/Jag1 mediated by the Wnt/ß-catenin pathway.

The therapeutic efficacy of umbilical cord mesenchymal stem cells (UCMSCs) in acute respiratory distress syndrome (ARDS) is mainly limited by the efficiency of homing of UCMSCs toward tissue damage. C-X-C chemokine receptor type 7 (CXCR7), which is involved in the mobilization of UCMSCs, is only expressed on the surface of a small proportion of UCMSCs. This study examined whether overexpression of CXCR7 in UCMSCs (UCMSCsOE-CXCR7) could improve their homing efficiency, and therefore, improve their effectiveness in fibrosis repair at the site of lung injury caused by ARDS. A lentiviral vector expressing CXCR7 was built and then transfect into UCMSCs. The impacts of CXCR7 expression of the proliferationand homing of UCMSCs were examined in a lipopolysaccharide-induced ARDS mouse model. The potential role and underlying mechanism of CXCR7 were examined by performing scratch assays, transwell assays, and immunoassays. The therapeutic dose and treatment time of UCMSCsOE-CXCR7 were directly proportional to their therapeutic effect on lung injury. In addition, overexpression of CXCR7 increased SDF-1-induced proliferation and migration of lung epithelial cells (Base-2b cells), and upregulation of CXCR7 inhibited α-SMA expression, suggesting that CXCR7 may have a role in alleviating pulmonary fibrosis caused by ARDS. Overexpression of CXCR7 in UCMSCs may improve their therapeutic effect of acute lung injury mouse, The mechanism of fibrosis repair by CXCR7 is inhibition of Jag1 via suppression of the Wnt/ß-catenin pathway under the chemotaxis of SDF-1.

PR1P, a VEGF-stabilizing peptide, reduces injury and inflammation in acute lung injury and ulcerative colitis animal models.

Acute Respiratory Distress Syndrome (ARDS) and Ulcerative Colitis (UC) are each characterized by tissue damage and uncontrolled inflammation. Neutrophils and other inflammatory cells play a primary role in disease progression by acutely responding to direct and indirect insults to tissue injury and by promoting inflammation through secretion of inflammatory cytokines and proteases. Vascular Endothelial Growth Factor (VEGF) is a ubiquitous signaling molecule that plays a key role in maintaining and promoting cell and tissue health, and is dysregulated in both ARDS and UC. Recent evidence suggests a role for VEGF in mediating inflammation, however, the molecular mechanism by which this occurs is not well understood. We recently showed that PR1P, a 12-amino acid peptide that binds to and upregulates VEGF, stabilizes VEGF from degradation by inflammatory proteases such as elastase and plasmin thereby limiting the production of VEGF degradation products (fragmented VEGF (fVEGF)). Here we show that fVEGF is a neutrophil chemoattractant in vitro and that PR1P can be used to reduce neutrophil migration in vitro by preventing the production of fVEGF during VEGF proteolysis. In addition, inhaled PR1P reduced neutrophil migration into airways following injury in three separate murine acute lung injury models including from lipopolysaccharide (LPS), bleomycin and acid. Reduced presence of neutrophils in the airways was associated with decreased pro-inflammatory cytokines (including TNF-α, IL-1ß, IL-6) and Myeloperoxidase (MPO) in broncho-alveolar lavage fluid (BALF). Finally, PR1P prevented weight loss and tissue injury and reduced plasma levels of key inflammatory cytokines IL-1ß and IL-6 in a rat TNBS-induced colitis model. Taken together, our data demonstrate that VEGF and fVEGF may each play separate and pivotal roles in mediating inflammation in ARDS and UC, and that PR1P, by preventing proteolytic degradation of VEGF and the production of fVEGF may represent a novel therapeutic approach to preserve VEGF signaling and inhibit inflammation in acute and chronic inflammatory diseases.

Rab26 promotes macrophage phagocytosis through regulation of MFN2 trafficking to mitochondria.

Acute respiratory distress syndrome (ARDS) is an inflammatory disorder of the lungs caused by bacterial or viral infection. Timely phagocytosis and clearance of pathogens by macrophages are important in controlling inflammation and alleviating ARDS. However, the precise mechanism of macrophage phagocytosis remains to be explored. Here, we show that the expression of Rab26 is increased in Escherichia coli- or Pseudomonas aeruginosa-stimulated bone marrow-derived macrophages. Knocking out Rab26 reduced phagocytosis and bacterial clearance by macrophages. Rab26 interacts with mitochondrial fusion protein mitofusin-2 (MFN2) and affects mitochondrial reactive oxygen species generation by regulating MFN2 transport. The levels of MFN2 in mitochondria were reduced in Rab26-deficient bone marrow-derived macrophages, and the levels of mitochondrial reactive oxygen species and ATP were significantly decreased. Knocking down MFN2 using small interfering RNA resulted in decreased phagocytosis and killing ability of macrophages. Rab26 knockout reduced phagocytosis and bacterial clearance by macrophages in vivo, significantly increased inflammatory factors, aggravated lung tissue damage, and increased mortality in mice. Our results demonstrate that Rab26 regulates phagocytosis and clearance of bacteria by mediating the transport of MFN2 to mitochondria in macrophages, thus alleviating ARDS in mice and potentially in humans.

Treatment of Acute Respiratory Distress Syndrome Caused by COVID-19 with Human Umbilical Cord Mesenchymal Stem Cells.

This study aimed to identify the impact of mesenchymal stem cell transplantation on the safety and clinical outcomes of patients with severe COVID-19. This research focused on how lung functional status, miRNA, and cytokine levels changed following mesenchymal stem cell transplantation in patients with severe COVID-19 pneumonia and their correlation with fibrotic changes in the lung. This study involved 15 patients following conventional anti-viral treatment (Control group) and 13 patients after three consecutive doses of combined treatment with MSC transplantation (MCS group). ELISA was used to measure cytokine levels, real-time qPCR for miRNA expression, and lung computed tomography (CT) imaging to grade fibrosis. Data were collected on the day of patient admission (day 0) and on the 7th, 14th, and 28th days of follow-up. A lung CT assay was performed on weeks 2, 8, 24, and 48 after the beginning of hospitalization. The relationship between levels of biomarkers in peripheral blood and lung function parameters was investigated using correlation analysis. We confirmed that triple MSC transplantation in individuals with severe COVID-19 was safe and did not cause severe adverse reactions. The total score of lung CT between patients from the Control and MSC groups did not differ significantly on weeks 2, 8, and 24 after the beginning of hospitalization. However, on week 48, the CT total score was 12 times lower in patients in the MSC group (p ≤ 0.05) compared to the Control group. In the MSC group, this parameter gradually decreased from week 2 to week 48 of observation, whereas in the Control group, a significant drop was observed up to week 24 and remained unchanged afterward. In our study, MSC therapy improved lymphocyte recovery. The percentage of banded neutrophils in the MSC group was significantly lower in comparison with control patients on day 14. Inflammatory markers such as ESR and CRP decreased more rapidly in the MSC group in comparison to the Control group. The plasma levels of surfactant D, a marker of alveocyte type II damage, decreased after MSC transplantation for four weeks in contrast to patients in the Control group, in whom slight elevations were observed. We first showed that MSC transplantation in severe COVID-19 patients led to the elevation of the plasma levels of IP-10, MIP-1α, G-CSF, and IL-10. However, the plasma levels of inflammatory markers such as IL-6, MCP-1, and RAGE did not differ between groups. MSC transplantation had no impact on the relative expression levels of miR-146a, miR-27a, miR-126, miR-221, miR-21, miR-133, miR-92a-3p, miR-124, and miR-424. In vitro, UC-MSC exhibited an immunomodulatory impact on PBMC, increasing neutrophil activation, phagocytosis, and leukocyte movement, activating early T cell markers, and decreasing effector and senescent effector T cell maturation.

Overexpression of FoxM1 Enhanced the Protective Effect of Bone Marrow-Derived Mesenchymal Stem Cells on Lipopolysaccharide-Induced Acute Lung Injury through the Activation of Wnt/ß-Catenin Signaling.

Background: Mesenchymal stem cell- (MSC-) based cell and gene therapies have made remarkable progress in alleviating acute lung injury/acute respiratory distress syndrome (ALI/ARDS). However, the benefits of Forkhead box protein M1 (FoxM1) gene-modified MSCs in the treatment of ALI have not been studied. Methods: We evaluated the therapeutic effects of FoxM1-modified MSCs in ALI mice induced by lipopolysaccharide (LPS) by quantifying the survival rate, lung weight ratio (wet/dry), and contents of bronchoalveolar lavage fluid. In addition, microcomputed tomography, histopathology, Evans Blue assay, and quantification of apoptosis were performed. We also explored the underlying mechanism by assessing Wnt/ß-catenin signaling following the treatment of mice with FoxM1-modified MSCs utilizing the Wnt/ß-catenin inhibitor XAV-939. Results: Compared with unmodified MSCs, transplantation of FoxM1-modified MSCs improved survival and vascular permeability; reduced total cell counts, leukocyte counts, total protein concentrations, and inflammatory cytokines in BALF; attenuated lung pathological impairments and fibrosis; and inhibited apoptosis in LPS-induced ALI/ARDS mice. Furthermore, FoxM1-modified MSCs maintained vascular integrity during ALI/ARDS by upregulating Wnt/ß-catenin signaling, which was partly reversed via a pathway inhibitor. Conclusion: Overexpression of FoxM1 optimizes the treatment action of MSCs on ALI/ARDS by inhibiting inflammation and apoptosis and restoring vascular integrity partially through Wnt/ß-catenin signaling pathway stimulation.