Alveolar Epithelial Cell Function Research Articles

Aryl Hydrocarbon Receptor Activation in Pulmonary Alveolar Epithelial Cells Limits Inflammation and Preserves Lung Epithelial Cell Integrity.

The aryl hydrocarbon receptor (AHR) is a receptor/transcription factor widely expressed in the lung. The physiological roles of AHR expressed in the alveolar epithelium remain unclear. In this study, we tested the hypothesis that alveolar epithelial AHR activity plays an important role in modulating inflammatory responses and maintaining alveolar integrity during lung injury and repair. AHR is expressed in alveolar epithelial cells (AECs) and is active. AHR activation with the endogenous AHR ligand, FICZ (5,11-dihydroindolo[3,2-b] carbazole-6-carboxaldehyde), significantly suppressed inflammatory cytokine expression in response to inflammatory stimuli in primary murine AECs and in the MLE-15 epithelial cell line. In an LPS model of acute lung injury in mice, coadministration of FICZ with LPS suppressed protein leak, reduced neutrophil accumulation in BAL fluid, and suppressed inflammatory cytokine expression in lung tissue and BAL fluid. Relevant to healing following inflammatory injury, AHR activation suppressed TGF-β-induced expression of genes associated with epithelial-mesenchymal transition. Knockdown of AHR in primary AECs with shRNA or in CRISPR-Cas-9-induced MLE-15 cells resulted in upregulation of α-smooth muscle actin (αSma), Col1a1, and Fn1 and reduced expression of epithelial genes Col4a1 and Sdc1. MLE-15 clones lacking AHR demonstrated accelerated wound closure in a scratch model. AHR activation with FICZ enhanced barrier function (transepithelial electrical resistance) in primary murine AECs and limited decline of transepithelial electrical resistance following inflammatory injury. AHR activation in AECs preserves alveolar integrity by modulating inflammatory cytokine expression while enhancing barrier function and limiting stress-induced expression of mesenchymal genes.

Sirt3-Mediated Opa1 Deacetylation Protects Against Sepsis-Induced Acute Lung Injury by Inhibiting Alveolar Macrophage Pro-Inflammatory Polarization.

Aims: Mitochondrial dynamics in alveolar macrophages (AMs) are associated with sepsis-induced acute lung injury (ALI). In this study, we aimed to investigate whether changes in mitochondrial dynamics could alter the polarization of AMs in sepsis-induced ALI and to explore the regulatory mechanism of mitochondrial dynamics by focusing on sirtuin (SIRT)3-induced optic atrophy protein 1 (OPA1) deacetylation. Results: The AMs of sepsis-induced ALI showed imbalanced mitochondrial dynamics and polarization to the M1 macrophage phenotype. In sepsis, SIRT3 overexpression promotes mitochondrial dynamic equilibrium in AMs. However, 3-(1H-1, 2, 3-triazol-4-yl) pyridine (3TYP)-specific inhibition of SIRT3 increased the mitochondrial dynamic imbalance and pro-inflammatory polarization of AMs and further aggravated sepsis-induced ALI. OPA1 is directly bound to and deacetylated by SIRT3 in AMs. In AMs of sepsis-induced ALI, SIRT3 protein expression was decreased and OPA1 acetylation was increased. OPA1 acetylation at the lysine 792 amino acid residue (OPA1-K792) promotes self-cleavage and is associated with an imbalance in mitochondrial dynamics. However, decreased acetylation of OPA1-K792 reversed the pro-inflammatory polarization of AMs and protected the barrier function of alveolar epithelial cells in sepsis-induced ALI. Innovation: Our study revealed, for the first time, the regulation of mitochondrial dynamics and AM polarization by SIRT3-mediated deacetylation of OPA1 in sepsis-induced ALI, which may serve as an intervention target for precision therapy of the disease. Conclusions: Our data suggest that imbalanced mitochondrial dynamics promote pro-inflammatory polarization of AMs in sepsis-induced ALI and that deacetylation of OPA1 mediated by SIRT3 improves mitochondrial dynamic equilibrium, thereby ameliorating lung injury.

Gut microbes improve prognosis of Klebsiella pneumoniae pulmonary infection through the lung-gut axis.

The gut microbiota plays a vital role in the development of sepsis and in protecting against pneumonia. Previous studies have demonstrated the existence of the gut-lung axis and the interaction between the gut and the lung, which is related to the prognosis of critically ill patients; however, most of these studies focused on chronic lung diseases and influenza virus infections. The purpose of this study was to investigate the effect of faecal microbiota transplantation (FMT) on Klebsiella pneumoniae-related pulmonary infection via the gut-lung axis and to compare the effects of FMT with those of traditional antibiotics to identify new therapeutic strategies. We divided the mice into six groups: the blank control (PBS), pneumonia-derived sepsis (KP), pneumonia-derived sepsis + antibiotic (KP + PIP), pneumonia-derived sepsis + faecal microbiota transplantation(KP + FMT), antibiotic treatment control (KP+PIP+PBS), and pneumonia-derived sepsis+ antibiotic + faecal microbiota transplantation (KP + PIP + FMT) groups to compare the survival of mice, lung injury, inflammation response, airway barrier function and the intestinal flora, metabolites and drug resistance genes in each group. Alterations in specific intestinal flora can occur in the gut of patients with pneumonia-derived sepsis caused by Klebsiella pneumoniae. Compared with those in the faecal microbiota transplantation group, the antibiotic treatment group had lower levels of proinflammatory factors and higher levels of anti-inflammatory factors but less amelioration of lung pathology and improvement of airway epithelial barrier function. Additionally, the increase in opportunistic pathogens and drug resistance-related genes in the gut of mice was accompanied by decreased production of favourable fatty acids such as acetic acid, propionic acid, butyric acid, decanoic acid, and secondary bile acids such as chenodeoxycholic acid 3-sulfate, isodeoxycholic acid, taurodeoxycholic acid, and 3-dehydrocholic acid; the levels of these metabolites were restored by faecal microbiota transplantation. Faecal microbiota transplantation after antibiotic treatment can gradually ameliorate gut microbiota disorder caused by antibiotic treatment and reduce the number of drug resistance genes induced by antibiotics. In contrast to direct antibiotic treatment, faecal microbiota transplantation improves the prognosis of mice with pneumonia-derived sepsis caused by Klebsiella pneumoniae by improving the structure of the intestinal flora and increasing the level of beneficial metabolites, fatty acids and secondary bile acids, thereby reducing systemic inflammation, repairing the barrier function of alveolar epithelial cells, and alleviating pathological damage to the lungs. The combination of antibiotics with faecal microbiota transplantation significantly alleviates intestinal microbiota disorder, reduces the selection for drug resistance genes caused by antibiotics, and mitigates lung lesions; these effects are superior to those following antibiotic monotherapy.

Repair and regeneration of the alveolar epithelium in lung injury.

Considerable progress has been made in understanding the function of alveolar epithelial cells in a quiescent state and regeneration mechanism after lung injury. Lung injury occurs commonly from severe viral and bacterial infections, inhalation lung injury, and indirect injury sepsis. A series of pathological mechanisms caused by excessive injury, such as apoptosis, autophagy, senescence, and ferroptosis, have been studied. Recovery from lung injury requires the integrity of the alveolar epithelial cell barrier and the realization of gas exchange function. Regeneration mechanisms include the participation of epithelial progenitor cells and various niche cells involving several signaling pathways and proteins. While alveoli are damaged, alveolar type II (AT2) cells proliferate and differentiate into alveolar type I (AT1) cells to repair the damaged alveolar epithelial layer. Alveolar epithelial cells are surrounded by various cells, such as fibroblasts, endothelial cells, and various immune cells, which affect the proliferation and differentiation of AT2 cells through paracrine during alveolar regeneration. Besides, airway epithelial cells also contribute to the repair and regeneration process of alveolar epithelium. In this review, we mainly discuss the participation of epithelial progenitor cells and various niche cells involving several signaling pathways and transcription factors.

Mechanisms of Alveolar Type 2 epithelial cell death during acute lung injury.

Diffuse alveolar epithelial cell death occurs extensively during acute lung injury(ALI). Due to the limited proliferative capacity of alveolar type 1 epithelial (AT1)cells, the differentiation and regenerative capacity of alveolar type 2 epithelial (AT2)cells are required to restore the barrier function of alveolar epithelial cells(AECs). However, during lung injury, AT1 cells are particularly susceptible to injury, and ATII cells die in the presence of severe or certain types of injury. This disruption ultimately results in a hindrance to the ability of AT2 cells to proliferate and differentiate into AT1 cells in time to repair the extensively damaged AECs. Therefore, understanding the mechanism of injury death of AT2 cells may be beneficial to reverse the above situation. This article reviews the main death modes of AT2 cells, including apoptosis, necrosis, necroptosis, pyroptosis, autophagic cell death, and ferroptosis. It compares the various forms of death, showing that various cell injury death modes have unique action mechanisms and partially overlapping pathways. Studying the mechanism of AT2 cell death is helpful in screening and analyzing the target pathway of alveolar epithelial cell barrier function recovery. It opens up new ideas and strategies for preventing and treating ALI.

Roles of lipid metabolism and its regulatory mechanism in idiopathic pulmonary fibrosis: A Review.

Idiopathic pulmonary fibrosis is a progressive lung disease of unknown etiology characterized by distorted distal lung architecture, inflammation, and fibrosis. Several lung cell types, including alveolar epithelial cells and fibroblasts, have been implicated in the development and progression of fibrosis. However, the pathogenesis of idiopathic pulmonary fibrosis is still incompletely understood. The latest research has found that dysregulation of lipid metabolism plays an important role in idiopathic pulmonary fibrosis. The changes in the synthesis and activity of fatty acids, cholesterol and other lipids seriously affect the regenerative function of alveolar epithelial cells and promote the transformation of fibroblasts into myofibroblasts. Mitochondrial function is the key to regulating the metabolic needs of a variety of cells, including alveolar epithelial cells. Sirtuins located in mitochondria are essential to maintain mitochondrial function and cellular metabolic homeostasis. Sirtuins can maintain normal lipid metabolism by regulating respiratory enzyme activity, resisting oxidative stress, and protecting mitochondrial function. In this review, we aimed to discuss the difference between normal and idiopathic pulmonary fibrosis lungs in terms of lipid metabolism. Additionally, we highlight recent breakthroughs on the effect of abnormal lipid metabolism on idiopathic pulmonary fibrosis, including the effects of sirtuins. Idiopathic pulmonary fibrosis has its high mortality and limited therapeutic options; therefore, we believe that this review will help to develop a new therapeutic direction from the aspect of lipid metabolism in idiopathic pulmonary fibrosis.

Promising Therapeutic Effects of Embryonic Stem Cells-Origin Mesenchymal Stem Cells in Experimental Pulmonary Fibrosis Models: Immunomodulatory and Anti-Apoptotic Mechanisms.

Interstitial lung disease (ILD) involves persistent inflammation and fibrosis, leading to respiratory failure and even death. Adult tissue-derived mesenchymal stem cells (MSCs) show potential in ILD therapeutics but obtaining an adequate quantity of cells for drug application is difficult. Daewoong Pharmaceutical's MSCs (DW-MSCs) derived from embryonic stem cells sustain a high proliferative capacity following long-term culture and expansion. The aim of this study was to investigate the therapeutic potential of DW-MSCs in experimental mouse models of ILD. DW-MSCs were expanded up to 12 passages for in vivo application in bleomycin-induced pulmonary fibrosis and collagen-induced connective tissue disease-ILD mouse models. We assessed lung inflammation and fibrosis, lung tissue immune cells, fibrosis-related gene/protein expression, apoptosis and mitochondrial function of alveolar epithelial cells, and mitochondrial transfer ability. Intravenous administration of DW-MSCs consistently improved lung fibrosis and reduced inflammatory and fibrotic markers expression in both models across various disease stages. The therapeutic effect of DW-MSCs was comparable to that following daily oral administration of nintedanib or pirfenidone. Mechanistically, DW-MSCs exhibited immunomodulatory effects by reducing the number of B cells during the early phase and increasing the ratio of Tregs to Th17 cells during the late phase of bleomycin-induced pulmonary fibrosis. Furthermore, DW-MSCs exhibited anti-apoptotic effects, increased cell viability, and improved mitochondrial respiration in alveolar epithelial cells by transferring their mitochondria to alveolar epithelial cells. Our findings indicate the strong potential of DW-MSCs in the treatment of ILD owing to their high efficacy and immunomodulatory and anti-apoptotic effects.

Lipidomic Profiling of Bronchoalveolar Lavage Fluid Extracellular Vesicles Indicates Their Involvement in Lipopolysaccharide-Induced Acute Lung Injury

Emerging data support the pivotal role of extracellular vesicles (EVs) in normal cellular physiology and disease conditions. However, despite their abundance, there is much less information about the lipid mediators carried in EVs, especially in the context of acute lung injury (ALI). Our data demonstrate that C57BL/6 mice subjected to intranasal Escherichia coli lipopolysaccharide (LPS)-induced ALI release, a higher number of EVs into the alveolar space, compared to saline-treated controls. EVs released during ALI originated from alveolar epithelial cells, macrophages, and neutrophils and carry a diverse array of lipid mediators derived from ω-3 and ω-6 polyunsaturated fatty acids (PUFA). The eicosanoids in EVs correlated with cellular levels of arachidonic acid, expression of cytosolic phospholipase A2, cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome epoxygenase p450 proteins in pulmonary macrophages. Furthermore, EVs from LPS-toll-like receptor 4 knockout (TLR4^-/-) mice contained significantly lower amounts of COX and LOX catalyzed eicosanoids and ω-3 PUFA metabolites. More importantly, EVs from LPS-treated wild-type mice increased TNF-α release by macrophages and reduced alveolar epithelial monolayer barrier integrity compared to EVs from LPS-treated TLR4^−/− mice. In summary, our study demonstrates for the first time that the EV carried PUFA metabolite profile in part depends on the inflammatory status of the lung macrophages and modulates pulmonary macrophage and alveolar epithelial cell function during LPS-induced ALI.

Research progress of extracellular vesicle microRNA in acute lung injury

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by the destruction of the barrier function of alveolar epithelial cells and capillary endothelial cells and the recruitment of inflammatory cells, which leads to alveolar and interstitial edema, hyaline membrane formation and inflammatory infiltration of the lungs, etc. The mechanism is not completely defined. The current treatment plan focuses on comprehensive treatments such as ventilator support treatment, fluid management, and nutritional support, but the prognosis is still poor. Studies have shown that extracellular vesicle microRNA (miRNA) from different sources participate in regulating the function of epithelial cells, endothelial cells and phagocytes in different ways, thus aggravating or improving ALI, and have diagnostic, differential diagnosis and the therapeutic value. In this article, the mechanism, diagnostic and differerntial value of extracellular vesicle miRNA from different sources in ALI and the therapy of extracellular vesicle miRNA from stem cell in ALI are reviewed.

Transient receptor potential vanilloid 4 (TRPV4) channels are essential for alveolar epithelial cell function

Ischemia‐reperfusion‐induced edema formation as one of the most common and significant causes of morbidity and mortality after lung transplantation can be mimicked ex‐vivo in isolated perfused mouse lungs (IPL). Transient receptor potential vanilloid 4 (TRPV4) is a non‐selective cation channel expressed in lung endothelium where increased channel activity has been shown to compromise endothelial barrier function. Here we show enhanced edema formation in TRPV4‐deficient (TRPV4−/−) IPL compared to wild‐type (WT) controls in response to ischemia and reperfusion, indicating a protective role of TRPV4 to maintain the alveolar epithelial barrier. By immunohistochemistry or mRNA profiling, we detected TRPV4 in bronchial epithelium, alveolar type I (ATI) and alveolar type II (ATII) cells. Genetic ablation of TRPV4 resulted in reduced expression and plasma membrane insertion of the water conducting aquaporin‐5 (AQP‐5) channel in ATI cells compared to WT mice. Analysis of isolated primary TRPV4‐deficient ATII cells revealed a reduced expression of surfactant protein C (SP‐C), which is essential for decreasing surface tension and alveolar fluid homeostasis. The TRPV4 activator GSK1016790A induced increases in current densities only in WT but not in TRPV4−/− ATII cells. Although the ability of TRPV4−/− ATII cells to differentiate to ATI cells was unchanged, epithelial mesenchymal transition (EMT) of TRPV4−/− ATII cells was impaired and migration and cell barrier function of TRPV4−/− ATI cells was reduced. Moreover, TRPV4−/− lungs of adult mice developed significantly larger mean chord lengths and altered lung function compared to WT lungs. Therefore, our data highlight novel essential functions of TRPV4 channels in alveolar epithelial cells and in the protection from edema formation.Support or Funding InformationThis study was funded by the Deutsche Forschungsgemeinschaft (TRR 152 (AD, GKC, CG), GRK 2338 (AD, CG)) and the German Center for Lung Research (DZL) (AD, MB, NW, TG).Phenotypes in alveolar epithelial cells after ablation of TRPV4. See text for details.Figure 1

Chronic Alcohol Significantly Affects Pulmonary Function Both at Baseline and in Response to Endotoxemia

Acute respiratory distress syndrome (ARDS) is an inflammatory disease characterized by significant pulmonary edema and pathologic alveolar flooding due to disrupted fluid homeostasis and increased alveolar permeability. ARDS accounts for 10% of global ICU admissions annually and carries a significant mortality risk. Chronic alcohol abuse exacerbates acute lung injury and is an independent risk factor to develop ARDS. Alveolar epithelial permeability is regulated by claudin‐family proteins that are incorporated into structures known as tight junctions. We have found that chronic alcohol negatively impacts alveolar barrier function in vitro by changing claudin expression and integration into tight junctions. Defining the molecular mechanisms responsible for the increased incidence of ARDS seen with chronic alcohol abuse is critical to understand the pathology of ARDS. In this study we used an Evans Blue permeability assay to assess changes in alveolar and vasculature permeability due to alcohol in combination with endotoxin as a ‘second‐hit’ to mimic the effects of pneumonia and sepsis on the lung. We also assessed the effects of alcohol and endotoxemia injury on pulmonary barrier function comparing intraperitoneal (IP) and intratracheal (IT) administration. Consistent with previous reports, we found that in alcohol‐fed mice, the alveolar barrier was impaired by both IP and IT endotoxin, allowing Evans Blue to permeate into the airspaces. However, protein content of airspaces was significantly higher in lungs treated with IT endotoxin as compared with IP which we attribute to a heightened inflammatory response in response to endotoxin. We also assessed whether the alcoholic lung is more susceptible to pulmonary edema due to increased fluid pressure from the interstitium (pulmonary wedge pressure). Alcohol‐fed mice showed increased susceptibility to alveolar leak when subjected to increased fluid pressure as compared with water‐fed controls. To test if alcohol‐induced increases in permeability affect pulmonary function and normal gas exchange, arterial oxygen saturation was measured and we found that there was a statistically significant decrease in blood oxygenation in alcohol‐fed mice at baseline as compared with water‐fed controls (97.3 ± 0.3 (n=4) vs 99.3 ± 1 (n=6)) predisposing them to hypoxia. Additionally, we have previously shown that primary alveolar cells cultured from alcohol‐fed rats continue to demonstrate defects in barrier function even after differentiation. We then performed an alcohol withdrawal experiment in mice and found a persistent effect on permeability even after 6 weeks without dietary alcohol. These results suggest alcohol has long lasting effects on the cellular biology and barrier function of alveolar epithelial cells. Current studies are focusing on targeting the tight junction protein Claudin‐5 as a potential mediator of alcoholic lung syndrome, using a specific antagonist peptide to rescue alcohol‐induced barrier dysfunction in vivo.Support or Funding InformationThis work was supported by R01‐AA025854, R01‐HL137112 (M.K.) and K12‐GM000680 (P.S.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of Host and Bacterial Contributions to Lung Barrier Dysfunction Following Co-infection with 2009 Pandemic Influenza and Methicillin Resistant Staphylococcus aureus.

Influenza viruses are a threat to global public health resulting in ~500,000 deaths each year. Despite an intensive vaccination program, influenza infections remain a recurrent, yet unsolved public health problem. Secondary bacterial infections frequently complicate influenza infections during seasonal outbreaks and pandemics, resulting in increased morbidity and mortality. Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is frequently associated with these co-infections, including the 2009 influenza pandemic. Damage to alveolar epithelium is a major contributor to severe influenza-bacterial co-infections and can result in gas exchange abnormalities, fluid leakage, and respiratory insufficiency. These deleterious manifestations likely involve both pathogen- and host-mediated mechanisms. However, there is a paucity of information regarding the mechanisms (pathogen- and/or host-mediated) underlying influenza-bacterial co-infection pathogenesis. To address this, we characterized the contributions of viral-, bacterial-, and host-mediated factors to the altered structure and function of alveolar epithelial cells during co-infection with a focus on the 2009 pandemic influenza (pdm2009) and MRSA. Here, we characterized pdm2009 and MRSA replication kinetics, temporal host kinome responses, modulation of MRSA virulence factors, and disruption of alveolar barrier integrity in response to pdm2009-MRSA co-infection. Our results suggest that alveolar barrier disruption during co-infection is mediated primarily through host response dysregulation, resulting in loss of alveolar barrier integrity.

Differential effects of Nintedanib and Pirfenidone on lung alveolar epithelial cell function in ex vivo murine and human lung tissue cultures of pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease. Repetitive injury and reprogramming of the lung epithelium are thought to be critical drivers of disease progression, contributing to fibroblast activation, extracellular matrix remodeling, and subsequently loss of lung architecture and function. To date, Pirfenidone and Nintedanib are the only approved drugs known to decelerate disease progression, however, if and how these drugs affect lung epithelial cell function, remains largely unexplored.MethodsWe treated murine and human 3D ex vivo lung tissue cultures (3D-LTCs; generated from precision cut lung slices (PCLS)) as well as primary murine alveolar epithelial type II (pmATII) cells with Pirfenidone or Nintedanib. Murine 3D-LTCs or pmATII cells were derived from the bleomycin model of fibrosis. Early fibrotic changes were induced in human 3D-LTCs by a mixture of profibrotic factors. Epithelial and mesenchymal cell function was determined by qPCR, Western blotting, Immunofluorescent staining, and ELISA.ResultsLow μM concentrations of Nintedanib (1 μM) and mM concentrations of Pirfenidone (2.5 mM) reduced fibrotic gene expression including Collagen 1a1 and Fibronectin in murine and human 3D-LTCs as well as pmATII cells. Notably, Nintedanib stabilized expression of distal lung epithelial cell markers, especially Surfactant Protein C in pmATII cells as well as in murine and human 3D-LTCs.ConclusionsPirfenidone and Nintedanib exhibit distinct effects on murine and human epithelial cells, which might contribute to their anti-fibrotic action. Human 3D-LTCs represent a valuable tool to assess anti-fibrotic mechanisms of potential drugs for the treatment of IPF patients.

A2BAR activation attenuates acute lung injury by inhibiting alveolar epithelial cell apoptosis both in vivo and in vitro.

The epithelial barrier of the lung is destroyed during acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) due to the apoptosis of alveolar epithelial cells (AECs). Therefore, treatments that block AEC apoptosis might be a therapeutic strategy to ameliorate ALI. Based on recent evidence, A2B adenosine receptor (A2BAR) plays an important role in ALI in several different animal models, but its exact function in AECs has not been clarified. We investigated the role of A2BAR in AEC apoptosis in a mouse model of oleic acid (OA)-induced ALI and in hydrogen peroxide (H2O2)-induced AEC (A549 cells and MLE-12 cells) injury. Mice treated with BAY60-6583, a selective A2BAR agonist, showed lower AEC apoptosis rates than mice treated with OA. However, the role of BAY60-6583 in OA-induced ALI was attenuated by a specific blocker of A2BAR, PSB1115. A2BAR activation decreased H2O2-induced cell apoptosis in vitro, as characterized by the translocation of apoptotic proteins, the release of cytochrome c, and the activation of caspase-3 and poly (ADP ribose) polymerase 1 (PARP-1). In addition, apoptosis was required for the phosphorylation of ERK1/2, p38, and JNK. Importantly, compared with cells transfected with the A2BAR-siRNA, an ERK inhibitor or p38 inhibitor exhibited decreased apoptotic ratios and cleaved caspase-9 and cleaved PARP-1 levels, whereas the JNK inhibitor displayed increases in these parameters. In conclusion, A2BAR activation effectively attenuated OA-induced ALI by inhibiting AEC apoptosis and mitigated H2O2-induced AEC injury by suppressing the p38 and ERK1/2-mediated mitochondrial apoptosis pathway.

C2-ceramide influences alveolar epithelial barrier function by downregulating Zo-1, occludin and claudin-4 expression

Noncardiogenic lung edema is a key factor affecting the prognosis of acute lung injury (ALI). Previous studies have been focused on regulatory roles of ceramide on lung vascular endothelial barrier functions and had already identified ceramide as mediator involved in the formation of lung edema. However, the effects of ceramide on lung epithelial barrier were still unknown. This study aimed to investigate the effects of ceramide on the barrier function of alveolar epithelial cells. Primary mouse alveolar type II epithelial cells (AECII) were grown on Transwell polyester membranes to construct monolayer, and stimulated with different concentrations of ceramide. Transepithelial resistance (TER) was measured to assess the epithelial cell permeability. Western blotting and real-time quantitative polymerase chain reaction were used to detect the mRNA and protein levels of tight junction, respectively. After incubation with different concentrations of c2-ceramide, TER of AECII monolayer decreased significantly in a dose-dependent manner. Moreover, expressions of ZO-1, occludin and claudin-4 were significantly reduced by c2-ceramide in the study. This study demonstrated that ceramide could increase alveolar epithelial cell monolayer permeability by downregulation of tight junction proteins. Therefore, modulation of ceramide expression may serve as a new therapeutic approach to treat acute lung injury.

Thyroid hormone inhibits pulmonary fibrosis through enhancement of mitochondrial function in alveolar epithelial cells

Thyroid hormone inhibits pulmonary fibrosis through enhancement of mitochondrial function in alveolar epithelial cells

Macrophage-epithelial paracrine crosstalk inhibits lung edema clearance during influenza infection.

Influenza A viruses (IAV) can cause lung injury and acute respiratory distress syndrome (ARDS), which is characterized by accumulation of excessive fluid (edema) in the alveolar airspaces and leads to hypoxemia and death if not corrected. Clearance of excess edema fluid is driven mostly by the alveolar epithelial Na,K-ATPase and is crucial for survival of patients with ARDS. We therefore investigated whether IAV infection alters Na,K-ATPase expression and function in alveolar epithelial cells (AECs) and the ability of the lung to clear edema. IAV infection reduced Na,K-ATPase in the plasma membrane of human and murine AECs and in distal lung epithelium of infected mice. Moreover, induced Na,K-ATPase improved alveolar fluid clearance (AFC) in IAV-infected mice. We identified a paracrine cell communication network between infected and noninfected AECs and alveolar macrophages that leads to decreased alveolar epithelial Na,K-ATPase function and plasma membrane abundance and inhibition of AFC. We determined that the IAV-induced reduction of Na,K-ATPase is mediated by a host signaling pathway that involves epithelial type I IFN and an IFN-dependent elevation of macrophage TNF-related apoptosis-inducing ligand (TRAIL). Our data reveal that interruption of this cellular crosstalk improves edema resolution, which is of biologic and clinical importance to patients with IAV-induced lung injury.

Alveolar Barrier Function in Alcoholic Lung Syndrome Is Impaired by Tight Junction Destabilization

Alcohol abuse increases the severity of acute respiratory distress syndrome by impairing alveolar barrier function and increasing airspace flooding. We used a rodent model of chronic alcohol ingestion and cultured primary alveolar epithelial cells (AECs) to investigate the influence of alcohol on tight junction formation and function. AECs from alcohol-fed rats had disrupted tight junction morphology and were leakier than control AECs. Alcohol-induced changes to AEC tight junctions were accompanied by increased expression of a claudin family tight junction protein, claudin-5. Claudin-18 also increased in response to alcohol; however, claudin-4 was decreased. Using an adenovector to increase claudin-5 expression by normal AECs mimicked the effects of alcohol on tight junctions, whereas short hairpin interfering RNA targeting to decrease claudin-5 improved tight junction morphology and function of AECs from alcohol-fed rats. We then used stochastic optical reconstruction microscopy to examine interactions in situ between claudin-5, claudin-18, and the tight junction scaffold protein, zonula occludens-1 at a level of 20-nm resolution. Stochastic optical reconstruction microscopy analysis of AECs revealed that alcohol significantly increased colocalization between claudin-5 and claudin-18, and that this was associated with a significant decrease in colocalization between claudin-18 and zonula occludens-1. Thus, increased claudin-5 destabilized integration of claudin-18 into tight junctions. Taken together, these data suggest that increased claudin-5 disrupts alveolar barrier function, and that strategies to target claudin-5 may provide a therapeutic approach to improve barrier function in acute respiratory distress syndrome and the alcoholic lung syndrome.

Claudin‐5 increases alveolar permeability in alcoholic lung syndrome by destabilizing claudin‐18/zonula occludens‐1 interactions (716.4)

We found that alcohol abuse increases the severity of acute respiratory distress syndrome (ARDS) by impairing alveolar epithelial cell (AEC) tight junctions. In a rat model of chronic alcohol ingestion, decreased AEC barrier function was accompanied by increased claudin‐5 (cldn‐5) and claudin‐18 (cldn‐18) expression. Immunofluorescence of model type I AECs cultured on Transwell permeable supports showed that paracellular leak in cells from alcohol fed rats was accompanied by disrupted tight junction morphology. We then used Stochastic Optical Reconstruction Microscopy (STORM) which increases the resolution of immunofluorescence to 20 nm. STORM revealed that alcohol caused a significant decrease in co‐localization between AEC cldn‐18 and ZO‐1 along with increased co‐localization between cldn‐18 and cldn‐5. Thus, cldn‐5 binding to cldn‐18 inhibits binding to ZO‐1 which destabilizes tight junctions. We found that shRNA targeting to decrease cldn‐5 expression improved AEC tight junction morphology and function. Conversely, transfecting cells to increase expression of YFP‐tagged cldn‐5 altered tight junction morphology and impaired barrier function. Thus, increased cldn‐5 was necessary and sufficient to inhibit cldn‐18 assembly and function. These data suggest that targeting cldn‐5 may provide a therapeutic strategy to improve barrier function in ARDS.Grant Funding Source: Emory URC, NIH‐NIAAA, NIH‐NHLBI, DAAD

Exogenous Hydrogen Sulfide (H2S) Protects Alveolar Growth in Experimental O2-Induced Neonatal Lung Injury

BackgroundBronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, remains a major health problem. BPD is characterized by impaired alveolar development and complicated by pulmonary hypertension (PHT). Currently there is no specific treatment for BPD. Hydrogen sulfide (H2S), carbon monoxide and nitric oxide (NO), belong to a class of endogenously synthesized gaseous molecules referred to as gasotransmitters. While inhaled NO is already used for the treatment of neonatal PHT and currently tested for the prevention of BPD, H2S has until recently been regarded exclusively as a toxic gas. Recent evidence suggests that endogenous H2S exerts beneficial biological effects, including cytoprotection and vasodilatation. We hypothesized that H2S preserves normal alveolar development and prevents PHT in experimental BPD.MethodsWe took advantage of a recently described slow-releasing H2S donor, GYY4137 (morpholin-4-ium-4-methoxyphenyl(morpholino) phosphinodithioate) to study its lung protective potential in vitro and in vivo.Results In vitro, GYY4137 promoted capillary-like network formation, viability and reduced reactive oxygen species in hyperoxia-exposed human pulmonary artery endothelial cells. GYY4137 also protected mitochondrial function in alveolar epithelial cells. In vivo, GYY4137 preserved and restored normal alveolar growth in rat pups exposed from birth for 2 weeks to hyperoxia. GYY4137 also attenuated PHT as determined by improved pulmonary arterial acceleration time on echo-Doppler, pulmonary artery remodeling and right ventricular hypertrophy. GYY4137 also prevented pulmonary artery smooth muscle cell proliferation.ConclusionsH2S protects from impaired alveolar growth and PHT in experimental O2-induced lung injury. H2S warrants further investigation as a new therapeutic target for alveolar damage and PHT.