Alveolar Epithelial Barrier Research Articles

COVID-19 ARDS: a review of imaging features and overview of mechanical ventilation and its complications.

The first cluster of cases of COVID-19 pneumonia was reported on December 31, 2019. Since then, this disease has spread rapidly across the world, and as of September 17, 2021, there are 226,844,344 cases of COVID-19 worldwide with 4,666,334 deaths related to COVID-19. While most COVID-19 cases are mild, some cases are severe with patients developing acute respiratory distress syndrome (ARDS). The pathophysiology of ARDS includes damage to the alveolar epithelium that leads to increased permeability of the alveolar epithelial barrier causing hyaline membrane formation, interstitial edema, and alveolar edema that results in severe hypoxia. Patients with COVID-19 ARDS are supported by non-invasive or invasive mechanical ventilation with an aim to improve oxygenation and maintain adequate blood oxygen levels. Increased intra-alveolar pressure while on mechanical ventilation may lead to alveolar rupture and thus barotrauma-related injuries such as lung tension cysts, pulmonary interstitial emphysema (PIE), pneumomediastinum, pneumopericardium, and pneumothorax. Recent studies have shown that the rate of barotrauma-related events is higher in patients with COVID-19 ARDS compared to patients with ARDS secondary to other etiologies. Radiologists should be aware of the imaging features of COVID-19 ARDS as well as the complications of mechanical ventilation. This educational manuscript will review the features of COVID-19 ARDS, discuss imaging of patients on mechanical ventilation, and review the imaging features of complications related to mechanical ventilation, including ventilator-associated lung injuries.

Understanding the biological impact of industrial engineered nanomaterials upon the alveolar epithelial barrier in vitro using occupational and in vivo relevant concentrations

Understanding the biological impact of industrial engineered nanomaterials upon the alveolar epithelial barrier in vitro using occupational and in vivo relevant concentrations

Hypercapnia Induces Inositol-Requiring Enzyme 1α-Driven Endoplasmic Reticulum-associated Degradation of the Na,K-ATPase β-Subunit.

Acute respiratory distress syndrome is often associated with elevated levels of CO2 (hypercapnia) and impaired alveolar fluid clearance. Misfolding of the Na,K-ATPase (NKA), a key molecule involved in both alveolar epithelial barrier tightness and resolution of alveolar edema, in the endoplasmic reticulum (ER) may decrease plasma membrane abundance of the transporter. Here, we investigated how hypercapnia affects the NKA β-subunit (NKA-β) in the ER. Exposing murine precision-cut lung slices and human alveolar epithelial A549 cells to elevated CO2 levels led to a rapid decrease of NKA-β abundance in the ER and at the cell surface. Knockdown of ER mannosidase α class 1B member 1 and ER degradation-enhancing α-mannosidase like protein 1 by siRNA or treatment with the mannosidase α class 1B member 1 inhibitor kifunensine rescued loss of NKA-β in the ER, suggesting ER-associated degradation (ERAD) of the enzyme. Furthermore, hypercapnia activated the unfolded protein response by promoting phosphorylation of inositol-requiring enzyme 1α (IRE1α), and treatment with an siRNA against IRE1α prevented the decrease of NKA-β in the ER. Of note, the hypercapnia-induced phosphorylation of IRE1α was triggered by a Ca2+-dependent mechanism. In addition, inhibition of the inositol trisphosphate receptor decreased phosphorylation levels of IRE1α in precision-cut lung slices and A549 cells, suggesting that Ca2+ efflux from the ER might be responsible for IRE1α activation and ERAD of NKA-β. In conclusion, here we provide evidence that hypercapnia attenuates maturation of the regulatory subunit of NKA by activating IRE1α and promoting ERAD, which may contribute to impaired alveolar epithelial integrity in patients with acute respiratory distress syndrome and hypercapnia.

Alcohol Decreases the Barrier Function of Airway Epithelial cells through the TGFß1 Pathway

Alcohol use disorder affects over 14 million adults and causes 88,000 deaths each year in the United States. Chronic alcohol use significantly increases people's risk of developing lung infections and acute respiratory distress syndrome (ARDS). This increased sensitivity to injury is a condition known as alcoholic lung syndrome, and it is caused by dysfunction of the lung immune system and alveolar epithelial barrier. However, little is known about how alcohol impacts epithelial cells in the conducting airway, i.e. the trachea and bronchi, which are the first line of defense against infectious pathogens in the lung. We hypothesize that alcohol exposure causes a decrease in barrier function of airway epithelial cells through a TGFß1-dependent mechanism. To test this, we cultured primary airway epithelial cells isolated from rats and humans on Transwell permeable supports and differentiated them in vitro at air liquid interface (ALI). We treated cells in vitro with 60mM or 100mM ethanol changed daily for 2 weeks and measured barrier function by transepithelial electrical resistance (TER). We found that ethanol caused a decrease in barrier function compared to control cells. However, cells treated with ethanol and a TGFß1 inhibitor had barrier function similar to control cells. Additionally, we cultured primary airway epithelial cells isolated from healthy and alcoholic patients on Transwell permeable supports and differentiated them in vitro to determine if in vivo exposure caused long-term changes to tight junction protein levels. It is known that the transmembrane protein junctional adhesion molecule A (JAM-A) regulates barrier function by controlling the paracellular flow of small molecules as part of the tight junction barrier. Our results show in response to alcohol exposure, cells have lower JAM-A protein expression compared to healthy cells. Rat airway cells exposed to ethanol in vitro also show a decrease in JAM-A at both the RNA and protein level. Together, these results indicate that chronic alcohol exposure detrimentally alters the conducting airway epithelial cell barrier potentially through the loss of JAM-A. Future directions include determining if other tight junction proteins are altered by alcohol and whether the alcohol-induced reduction in JAM-A is sufficient to cause barrier dysfunction in airway cells.

Biokinetic Modeling of Nanoparticle Interactions with Lung Alveolar Epithelial Cells

Many studies on the health effects of inhaled ambient and engineered nanomaterials (ENMs) have provided information on nanoparticle toxicity and translocation across airway and alveolar epithelial barriers. Various inhaled ENMs (e.g., gold and iridium nanoparticles) have been reported to partially cross the air-blood barrier in the lung, enter the circulation and distribute into several end organs, including heart, liver, spleen and kidney. Using an in vitro primary rat alveolar epithelial cell (AEC) monolayer model, we reported transport rates of relatively nontoxic polystyrene nanoparticles (PNPs), which appear to be taken up via nonendocytic processes into AECs. PNPs internalized into cytoplasm then trigger autophagy, followed by delivery of PNPs from autophagosomes into lysosomes, from where PNPs can be exocytosed. We used the data from these in vitro experiments to perform biokinetic modeling that incorporates the processes associated with internalization and intracellular distribution of PNPs, autophagy, lysosomal exocytosis of PNPs, and several putative mechanisms of action that extend our previous understanding of AEC processing of PNPs. Results indicate that entry of PNPs into AECs, subsequent activation of autophagy by cytosolic PNPs, accumulation of PNPs in lysosomes, and lysosomal exocytosis are interwoven by complex proposed regulatory mechanisms. These findings are consistent with the presence of intracellular interactions that provide potential new insights into how inhaled nanoparticles may be mechanistically linked to the development of acute and chronic lung injury.

Inhibiting Connexin43/Zonula Occludens‐1 Interactions Improves Alcoholic Lung Barrier Function in vivo

Chronic alcohol consumption is well known to contribute to an array of health complications and increases the incidence and severity of acute respiratory distress syndrome (ARDS), an acute lung injury marked by severe pulmonary edema and impairment of normal gas exchange. Despite advancements in understanding its pathogenesis, the mortality rate for ARDS remains over 40% and there are currently no effective pharmacological treatments for ARDS. Chronic alcohol consumption contributes to the pathogenesis of ARDS by altering the tight junction composition of the pulmonary epithelium which is essential to maintain normal barrier function. In this study, we investigate the use of a pharmacological peptide, aCT1, which mimics the cytoplasmic carboxy-terminal PDZ binding motif of the gap junction protein Connexin43 (Cx43) to disrupt endogenous Cx43 from binding scaffold proteins, including the tight junction protein Zonula Occludens-1 (ZO-1). By inhibiting ZO-1 binding to Cx43, we hypothesize that more ZO-1 is available to associate with tight junction proteins, including claudins, to enhance epithelial barrier function. We have previously shown that chronic alcohol use exacerbates acute endotoxin-mediated lung injury by compromising both the alveolar and pulmonary capillary epithelial barriers. Here, we tested whether treatment with aCT1 strengthens alveolar barrier function and mitigates the effects of alcohol-mediated impairment of tight junctions. Male C57BL/6J mice were pair-fed 20% ethanol or water for 14 weeks to establish a chronic alcohol model in vivo. Mice were sedated and given an intratracheal instillation of 5mg/kg of the bacterial endotoxin lipopolysaccharide (LPS) derived from E. coli O55:B5 to induce a second, direct insult in the alcohol-primed lung to impact the lung epithelium. Mice were administered via nebulizer 1h post-LPS instillation either 5mg/kg of the aCT1 peptide or a scrambled control. Evans Blue (EB) dye was injected into mouse tail veins 24h after LPS instillation. To measure alveolar flooding, bronchoalveolar lavage fluid (BAL) was collected 1h later and EB in BAL was measured and normalized to serum EB. We found that administration of aCT1 decreased the leakage of EB dye from the blood into the alveolar air space in both water-fed and alcohol-fed mice, thus improving lung barrier function. This improvement in pulmonary leak suggests that aCT1 has the capacity to mitigate ARDS. Further investigation will determine whether aCT1 has a positive impact on short-term mortality rates due to endotoxemia and whether it can improve pulmonary barrier dysfunction due to other causes of acute lung injury.

Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.

Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.

The History and Mystery of Alveolar Epithelial Type II Cells: Focus on Their Physiologic and Pathologic Role in Lung.

Alveolar type II (ATII) cells are a key structure of the distal lung epithelium, where they exert their innate immune response and serve as progenitors of alveolar type I (ATI) cells, contributing to alveolar epithelial repair and regeneration. In the healthy lung, ATII cells coordinate the host defense mechanisms, not only generating a restrictive alveolar epithelial barrier, but also orchestrating host defense mechanisms and secreting surfactant proteins, which are important in lung protection against pathogen exposure. Moreover, surfactant proteins help to maintain homeostasis in the distal lung and reduce surface tension at the pulmonary air–liquid interface, thereby preventing atelectasis and reducing the work of breathing. ATII cells may also contribute to the fibroproliferative reaction by secreting growth factors and proinflammatory molecules after damage. Indeed, various acute and chronic diseases are associated with intensive inflammation. These include oedema, acute respiratory distress syndrome, fibrosis and numerous interstitial lung diseases, and are characterized by hyperplastic ATII cells which are considered an essential part of the epithelialization process and, consequently, wound healing. The aim of this review is that of revising the physiologic and pathologic role ATII cells play in pulmonary diseases, as, despite what has been learnt in the last few decades of research, the origin, phenotypic regulation and crosstalk of these cells still remain, in part, a mystery.

BMSC-derived exosomes ameliorate sulfur mustard-induced acute lung injury by regulating the GPRC5A-YAP axis.

Sulfur mustard (SM) is a highly toxic chemical warfare agent that causes acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS). There are no effective therapeutic treatments or antidotes available currently to counteract its toxic effects. Our previous study shows that bone marrow-derived mesenchymal stromal cells (BMSCs) could exert therapeutic effects against SM-induced lung injury. In this study, we explored the therapeutic potential of BMSC-derived exosomes (BMSC-Exs) against ALI and the underlying mechanisms. ALI was induced in mice by injection of SM (30 mg/kg, sc) at their medial and dorsal surfaces. BMSC-Exs (20 μg/kg in 200 μL PBS, iv) were injected for a 5-day period after SM exposure. We showed that BMSC-Exs administration caused a protective effect against pulmonary edema. Using a lung epithelial cell barrier model, BMSC-Exs (10, 20, 40 μg) dose-dependently inhibited SM-induced cell apoptosis and promoted the recovery of epithelial barrier function by facilitating the expression and relocalization of junction proteins (E-cadherin, claudin-1, occludin, and ZO-1). We further demonstrated that BMSC-Exs protected against apoptosis and promoted the restoration of barrier function against SM through upregulating G protein-coupled receptor family C group 5 type A (GPRC5A), a retinoic acid target gene predominately expressed in the epithelial cells of the lung. Knockdown of GPRC5A reduced the antiapoptotic and barrier regeneration abilities of BMSC-Exs and diminished their therapeutic effects in vitro and in vivo. BMSC-Exs-caused upregulation of GPRC5A promoted the expression of Bcl-2 and junction proteins via regulating the YAP pathway. In summary, BMSC-Exs treatment exerts protective effects against SM-induced ALI by promoting alveolar epithelialbarrier repair and maybe an alternative approach to stem cell-based therapy.

Quercetin: a savior of alveolar barrier integrity under hypoxic microenvironment

ABSTRACT High altitude pulmonary edema (HAPE) is generally characterized by the loss of alveolar epithelial barrier integrity. The current study was undertaken to assess the noninvasive approaches of HAPE diagnosis and to evaluate the prophylactic potential of quercetin in preventing alveolar junction impairments. Male SD rats fed with quercetin 1 h prior to hypoxia (7,620 m, for 6 h) were selected. PET/CT imaging was performed to visualize the lung uptake of 18F-FDG in animals under hypoxia. Further, oxidant status, catalase activity, hematological & blood gas parameters were evaluated. Moreover, tight junction (TJ) proteins (ZO-1, JAM-C, Claudin-4, and occludin) expression analysis was accomplished using immune-blotting. The structural differences in lung epithelia were noted by TEM imaging. Quercetin prophylaxis has significantly reduced the FDG uptake in rat lungs under hypoxia. It has also dramatically alleviated the protein oxidation followed by an elevation in catalase activity in the lungs under hypoxia. The TJ protein expression in the lungs has also been restored to normal upon quercetin pre-treatment. Concomitantly, the quercetin preconditioning has elicited the stable blood gas and hematological parameters under hypoxia. The observations from TEM imaging have also implicated the normal lung epithelial structures in the quercetin pretreated animals under hypoxia. Quercetin prophylaxis has significantly restored alveolar epithelium integrity by abating oxidative stress in the lungs under hypoxia. Abbreviations: CT- Computed Tomography 18F-FDG- Fluorodeoxyglucose (18FHAPE- High Altitude Pulmonary EdemaHb- HemoglobinHCT- HematocritHCO3 – BicarbonateJAM- Junctional Adhesion MoleculeKBq- Killo BecquerelPaO2 - Partial pressure of arterial oxygenPaCO2 - Partial pressure of arterial carbon di-oxidePET- Positron Emission TomographyRBC- Red Blood CorpusclesSD- Sprague DawleyTJ- Tight JunctionsTEM- Transmission Electron MicroscopyWBC- White Blood CorpusclesZO- Zona Occludin.

MitoQ alleviates LPS-mediated acute lung injury through regulating Nrf2/Drp1 pathway

Lipopolysaccharide (LPS) has been known to cause alveolar epithelial cell (AEC) apoptosis and barrier breakdown that characterize acute lung injury (ALI) and acute respiratory distress syndrome. We aimed to investigate whether mitoquinone (MitoQ), a mitochondria-targeted antioxidant, could alleviate LPS-induced AEC damage in ALI and its underlying mechanisms. In vitro studies in AEC A549 cell line, we noted that LPS could induce dynamin-related protein 1 (Drp1)-mediated mitochondrial fission, AEC apoptosis and barrier breakdown, which could be reversed with MitoQ and mitochondrial division inhibitor 1 treatment. Moreover, the protective role of MitoQ was attenuated with Drp1 overexpression. Nuclear factor E2-related factor 2 (Nrf2) downregulation could block the effect of MitoQ by decreasing the expression of Nrf2 target genes in LPS-treated AEC, such as heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1). Nrf2 gene knockdown in LPS-treated A549 cells prevented the protective effect of MitoQ from decreasing Drp1-mediated mitochondrial fission, AEC apoptosis and barrier breakdown. The lung protective effect of MitoQ by regulating the Drp1-mediated mitochondrial fission, AEC apoptosis and barrier breakdown was further confirmed in vivo with LPS-induced ALI mouse model. Additionally, the protective effect of MitoQ was inhibited by Nrf2 inhibitor ML385. We therefore conclude that MitoQ exerts ALI-protective effects by preventing Nrf2/Drp1-mediated mitochondrial fission, AEC apoptosis as well as barrier breakdown.

The Na+, K+-ATPase β1 subunit regulates epithelial tight junctions via MRCKα.

An intact lung epithelial barrier is essential for lung homeostasis. The Na+, K+-ATPase (NKA), primarily serving as an ion transporter, also regulates epithelial barrier function via modulation of tight junctions. However, the underlying mechanism is not well understood. Here, we show that overexpression of the NKA β1 subunit upregulates the expression of tight junction proteins, leading to increased alveolar epithelial barrier function by an ion transport–independent mechanism. Using IP and mass spectrometry, we identified a number of unknown protein interactions of the β1 subunit, including a top candidate, myotonic dystrophy kinase–related cdc42-binding kinase α (MRCKα), which is a protein kinase known to regulate peripheral actin formation. Using a doxycycline-inducible gene expression system, we demonstrated that MRCKα and its downstream activation of myosin light chain is required for the regulation of alveolar barrier function by the NKA β1 subunit. Importantly, MRCKα is expressed in both human airways and alveoli and has reduced expression in patients with acute respiratory distress syndrome (ARDS), a lung illness that can be caused by multiple direct and indirect insults, including the infection of influenza virus and SARS-CoV-2. Our results have elucidated a potentially novel mechanism by which NKA regulates epithelial tight junctions and have identified potential drug targets for treating ARDS and other pulmonary diseases that are caused by barrier dysfunction.

Metabolic Glycoengineering Enables the Ultrastructural Visualization of Sialic Acids in the Glycocalyx of the Alveolar Epithelial Cell Line hAELVi.

The glycocalyx—a plethora of sugars forming a dense layer that covers the cell membrane—is commonly found on the epithelial surface of lumen forming tissue. New glycocalyx specific properties have been defined for various organs in the last decade. However, in the lung alveolar epithelium, its structure and functions remain almost completely unexplored. This is partly due to the lack of physiologically relevant, cost effective in vitro models. As the glycocalyx is an essential but neglected part of the alveolar epithelial barrier, understanding its properties holds the promise to enhance the pulmonary administration of drugs and delivery of nanoparticles. Here, using air-liquid-interface (ALI) cell culture, we focus on combining metabolic glycoengineering with glycan specific electron and confocal microscopy to visualize the glycocalyx of a recently immortalized human alveolar epithelial cell line (hAELVi). For this purpose, we applied different bioorthogonal labeling approaches to visualize sialic acid—an amino sugar that provides negative charge to the lung epithelial glycocalyx—using both fluorescence and gold-nanoparticle labeling. Further, we compared mild chemical fixing/freeze substitution and standard cytochemical electron microscopy embedding protocols for their capacity of contrasting the glycocalyx. In our study, we established hAELVi cells as a convenient model for investigating human alveolar epithelial glycocalyx. Transmission electron microscopy revealed hAELVi cells to develop ultrastructural features reminiscent of alveolar epithelial type II cells (ATII). Further, we visualized extracellular uni- and multilamellar membranous structures in direct proximity to the glycocalyx at ultrastructural level, indicating putative interactions. The lamellar membranes were able to form structures of higher organization, and we report sialic acid to be present within those. In conclusion, combining metabolite specific glycoengineering with ultrastructural localization presents an innovative method with high potential to depict the molecular distribution of individual components of the alveolar epithelial glycocalyx and its interaction partners.

Sodium-coupled neutral amino acid transporter SNAT2 counteracts cardiogenic pulmonary edema by driving alveolar fluid clearance.

The constant transport of ions across the alveolar epithelial barrier regulates alveolar fluid homeostasis. Dysregulation or inhibition of Na+ transport causes fluid accumulation in the distal airspaces resulting in impaired gas exchange and respiratory failure. Previous studies have primarily focused on the critical role of amiloride-sensitive epithelial sodium channel (ENaC) in alveolar fluid clearance (AFC), yet activation of ENaC failed to attenuate pulmonary edema in clinical trials. Since 40% of AFC is amiloride-insensitive, Na+ channels/transporters other than ENaC such as Na+-coupled neutral amino acid transporters (SNATs) may provide novel therapeutic targets. Here, we identified a key role for SNAT2 (SLC38A2) in AFC and pulmonary edema resolution. In isolated perfused mouse and rat lungs, pharmacological inhibition of SNATs by HgCl2 and α-methylaminoisobutyric acid (MeAIB) impaired AFC. Quantitative RT-PCR identified SNAT2 as the highest expressed System A transporter in pulmonary epithelial cells. Pharmacological inhibition or siRNA-mediated knockdown of SNAT2 reduced transport of l-alanine across pulmonary epithelial cells. Homozygous Slc38a2-/- mice were subviable and died shortly after birth with severe cyanosis. Isolated lungs of Slc38a2+/- mice developed higher wet-to-dry weight ratios (W/D) as compared to wild type (WT) in response to hydrostatic stress. Similarly, W/D ratios were increased in Slc38a2+/- mice as compared to controls in an acid-induced lung injury model. Our results identify SNAT2 as a functional transporter for Na+ and neutral amino acids in pulmonary epithelial cells with a relevant role in AFC and the resolution of lung edema. Activation of SNAT2 may provide a new therapeutic strategy to counteract and/or reverse pulmonary edema.

Interleukin-38 ameliorates poly(I:C) induced lung inflammation: therapeutic implications in respiratory viral infections

Interleukin-38 has recently been shown to have anti-inflammatory properties in lung inflammatory diseases. However, the effects of IL-38 in viral pneumonia remains unknown. In the present study, we demonstrate that circulating IL-38 concentrations together with IL-36α increased significantly in influenza and COVID-19 patients, and the level of IL-38 and IL-36α correlated negatively and positively with disease severity and inflammation, respectively. In the co-cultured human respiratory epithelial cells with macrophages to mimic lung microenvironment in vitro, IL-38 was able to alleviate inflammatory responses by inhibiting poly(I:C)-induced overproduction of pro-inflammatory cytokines and chemokines through intracellular STAT1, STAT3, p38 MAPK, ERK1/2, MEK, and NF-κB signaling pathways. Intriguingly, transcriptomic profiling revealed that IL-38 targeted genes were associated with the host innate immune response to virus. We also found that IL-38 counteracts the biological processes induced by IL-36α in the co-culture. Furthermore, the administration of recombinant IL-38 could mitigate poly I:C-induced lung injury, with reduced early accumulation of neutrophils and macrophages in bronchoalveolar lavage fluid, activation of lymphocytes, production of pro-inflammatory cytokines and chemokines and permeability of the alveolar-epithelial barrier. Taken together, our study indicates that IL-38 plays a crucial role in protection from exaggerated pulmonary inflammation during poly(I:C)-induced pneumonia, thereby providing the basis of a novel therapeutic target for respiratory viral infections.

Mesenchymal stromal cell extracellular vesicles rescue mitochondrial dysfunction and improve barrier integrity in clinically relevant models of ARDS.

Alveolar epithelial–capillary barrier disruption is a hallmark of acute respiratory distress syndrome (ARDS). Contribution of mitochondrial dysfunction to the compromised alveolar-capillary barrier in ARDS remains unclear. Mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) are considered as a cell-free therapy for ARDS. Mitochondrial transfer was shown to be important for the therapeutic effects of MSCs and MSC-EVs. Here we investigated the contribution of mitochondrial dysfunction to the injury of alveolar epithelial and endothelial barriers in ARDS and the ability of MSC-EVs to modulate alveolar–capillary barrier integrity through mitochondrial transfer.Primary human small airway epithelial and pulmonary microvascular endothelial cells and human precision cut lung slices (PCLSs) were stimulated with endotoxin or plasma samples from patients with ARDS and treated with MSC-EVs, barrier properties and mitochondrial functions were evaluated. Lipopolysaccharide (LPS)-injured mice were treated with MSC-EVs and degree of lung injury and mitochondrial respiration of the lung tissue were assessed.Inflammatory stimulation resulted in increased permeability coupled with pronounced mitochondrial dysfunction in both types of primary cells and PCLSs. Extracellular vesicles derived from normal MSCs restored barrier integrity and normal levels of oxidative phosphorylation while an extracellular vesicles preparation which did not contain mitochondria was not effective. In vivo, presence of mitochondria was critical for extracellular vesicles ability to reduce lung injury and restore mitochondrial respiration in the lung tissue.In the ARDS environment, MSC-EVs improve alveolar–capillary barrier properties through restoration of mitochondrial functions at least partially via mitochondrial transfer.

Realtime Transepithelial/Endothelial Electrical Resistance Measurements in Multiple Transwell Culture Inserts

Epithelial and endothelial cells line the surface of the body. Adjacent cells of both epithelial and endothelial cells are connected to each other via intercellular junction called tight junction that regulates the diffusion of molecules and ions between the apical and basolateral side of the body. Quantification of this barrier is important to evaluate the suitability of in vitro cellular barrier models for drug toxicity or transport studies. Transepithelial/endothelial electrical resistance (TEER), a quantitative technique to access electrical resistance of the cellular monolayer during culture has become one of the most popular quantitative techniques to evaluate the integrity of tight junction. The major advantage of the TEER technique over traditional permeability measurements is that it is quick, label-free, and non-invasive. As such, the TEER technique has been widely used to evaluate in vitro models such as the blood-brain barrier, the intestine epithelial barrier, the alveolar epithelial barrier, and the blood-retina barrier.However, the reported normalized TEER values of a specific cell type in transwell inserts are inconsistent between different research groups. A major cause of data variation is the manual technique that is used for measuring TEER. Typically, a “chopstick” like electrode pair is immersed inside a cell cultured semi-porous transwell insert membrane to measure TEER. Due to the variation in the alignment of the electrodes every time a TEER value is measured, the actual values vary significantly between measurements. A second problem with the conventional manual measurement process is time gap between each measurement (typically 12h or 24h) since a more frequent measurement requires frequent opening and closing of cell culture incubator door that can disturb the cells and introduce potential contamination.Thus, to enable consistency in TEER measurement, and allow automated real-time data acquisition in multiple samples, we built an add-on device (used atop EVOM®, a commercially available TEER instrument) consisting of a 3D printed electrode holder, a temperature sensor, and a custom designed data logger. The 3D printed electrode holder allowed data variation to be minimized by allowing perfect alignment of electrodes during measurement. The real-time data acquisition and in-situ temperature measurement allowed detection and correction of data offset during medium replacement process. The concept and device demonstration presented here should enable research labs to minimize data variations allowing them to produce consistent TEER results without having to spend extra time manually measuring multiple samples.

Single-cell analysis identified lung progenitor cells in COVID-19 patients.

ObjectivesThe high mortality of severe 2019 novel coronavirus disease (COVID‐19) cases is mainly caused by acute respiratory distress syndrome (ARDS), which is characterized by increased permeability of the alveolar epithelial barriers, pulmonary oedema and consequently inflammatory tissue damage. Some but not all patients showed full functional recovery after the devastating lung damage, and so far there is little knowledge about the lung repair process. We focused on crucial roles of lung progenitor cells in alveolar cell regeneration and epithelial barrier re‐establishment and aimed to uncover a possible mechanism of lung repair after severe SARS‐CoV‐2 infection.Materials and methodsBronchoalveolar lavage fluid (BALF) of COVID‐19 patients was analysed by single‐cell RNA‐sequencing (scRNA‐seq). Transplantation of a single KRT5+ cell‐derived cell population into damaged mouse lung and time‐course scRNA‐seq analysis was performed.ResultsIn severe (or critical) COVID‐19 patients, there is a remarkable expansion of TM4SF1+ and KRT5+ lung progenitor cells. The two distinct populations of progenitor cells could play crucial roles in alveolar cell regeneration and epithelial barrier re‐establishment, respectively. The transplanted KRT5+ progenitors could long‐term engraft into host lung and differentiate into HOPX+ OCLN+ alveolar barrier cell which restored the epithelial barrier and efficiently prevented inflammatory cell infiltration.ConclusionsThis work uncovered the mechanism by which various lung progenitor cells work in concert to prevent and replenish alveoli loss post‐severe SARS‐CoV‐2 infection.

Biokinetic modeling of nanoparticle interactions with lung alveolar epithelial cells: uptake, intracellular processing, and egress.

Studies on health effects of engineered nanomaterials (ENMs) in the lung have provided information on ENM toxicity and translocation across airway and alveolar epithelial barriers. Various inhaled ENMs (e.g., gold and iridium nanoparticles) have been reported to partially cross the air-blood barrier in the lung, enter the vasculature, and distribute in several end organs, including the heart, liver, spleen, and kidney. Using an in vitro primary rat alveolar epithelial cell (AEC) monolayer model, we reported transport rates of relatively nontoxic polystyrene nanoparticles (PNPs), which appear to be taken up via nonendocytic processes into AECs. PNPs internalized into cytoplasm then trigger autophagy, followed by delivery of PNPs from autophagosomes into lysosomes, from where PNPs are exocytosed. We used the data from these experiments to perform biokinetic modeling that incorporates the processes associated with internalization and intracellular distribution of PNPs, autophagy, lysosomal exocytosis of PNPs, and several putative mechanisms of action that extend our previous understanding of AEC processing of PNPs. Results suggest that entry of PNPs into AECs, subsequent activation of autophagy by cytosolic PNPs, accumulation of PNPs in lysosomes, and lysosomal exocytosis are interwoven by proposed regulatory mechanisms.

TRPV4 channels are essential for alveolar epithelial barrier function as protection from lung edema

Ischemia/reperfusion-induced edema (IRE), one of the most significant causes of mortality after lung transplantation, can be mimicked ex vivo in isolated perfused mouse lungs (IPL). Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel studied in endothelium; however, its role in the lung epithelium remains elusive. Here, we show enhanced IRE in TRPV4-deficient (TRPV4–/–) IPL compared with that of WT controls, indicating a protective role of TRPV4 in maintenance of the alveolar epithelial barrier. By immunohistochemistry, mRNA profiling, and electrophysiological characterization, we detected TRPV4 in bronchial epithelium, alveolar epithelial type I (ATI), and alveolar epithelial type II (ATII) cells. Genetic ablation of TRPV4 resulted in reduced expression of the water-conducting aquaporin-5 (AQP-5) channel in ATI cells. Migration of TRPV4–/– ATI cells was reduced, and cell barrier function was impaired. Analysis of isolated primary TRPV4–/– ATII cells revealed a reduced expression of surfactant protein C, and the TRPV4 activator GSK1016790A induced increases in current densities only in WT ATII cells. Moreover, TRPV4–/– lungs of adult mice developed significantly larger mean chord lengths and altered lung function compared with WT lungs. Therefore, our data illustrate essential functions of TRPV4 channels in alveolar epithelial cells and in protection from edema formation.