Alveolar Epithelial Cell Monolayers Research Articles

Effects of nanoparticles on sensitivity to injury and role of autophagy in alveolar epithelial cells

Autophagy, a housekeeping mechanism, is crucial in the maintenance of normal cellular function. Although the involvement of autophagic processes is recognized in numerous diseases, it is unknown how cellular homeostasis might be affected by alterations in available autophagic activity and/or autophagic capacity. In this study, we measured autophagic flux and cellular damage in primary cultured monolayers of rat alveolar epithelial cells (AEC) exposed separately and sequentially to two different autophagy inducers. Rat AEC monolayers were exposed apically for 24 hrs to (1) polystyrene nanoparticles (PNP, 20 nm, carboxylated, near-infrared dye-labeled), (2) tunicamycin (TN, a disruptor of protein synthesis and inducer of the unfolded protein response, at 1-15 μg/mL), or (3) TN (1-15 μg/mL) after 12 hrs of PNP pre-incubation. Release of the cytoplasmic enzyme lactate dehydrogenase (LDH) was used as an indicator of cellular damage and quantified by an LDH assay from Dojindo (Rockville, MD). Autophagic flux was assessed by live cell imaging using confocal microscopy and 0.1 μM DAPRed (Dojindo; Rockville, MD) in the presence (for 1 hr) and absence of 40 μM chloroquine. Serial z sections were collected over the entire cell volume of live single cells to detect DAPRed (autophagosome) fluorescence. PNP were taken up into AEC, where their cytosolic presence induced a gradually increased autophagic flux, reaching a steady state at ~10-24 hrs. When AEC were exposed to TN alone for 24 hrs, autophagy was also activated. Cellular damage in the presence of TN, determined by increased LDH release, revealed dose-dependent injury with LD50 of 8.12 μg/mL over 24 hr TN exposure. When AEC were pre-exposed to PNP for 12 hrs and subsequently exposed to TN, increased LDH release was seen with LD50 of 3.95 μg/mL. In summary, the intracellular presence of PNP taken up from apical fluid of rat AEC monolayers activated autophagy. When cellular protein synthesis was disrupted with TN alone, autophagy was also activated. Furthermore, TN induced dose-dependent cellular damage in AEC. However, after pre-exposure to PNP, TN induced greater cellular damage observed as increased LDH release. These results suggest that autophagic capacity is limited and that pre-induction of autophagy by inhaled PNP makes AEC more susceptible to secondary injury by TN. These findings are consistent with the hypothesis that environmental stressors (e.g., ambient air nanoparticles) exert their harmful effects, at least in part, by reducing available autophagic activity/capacity, thereby causing nanoparticle-exposed AEC to be more susceptible to secondary injury. Funding: NIH; WRMPPF. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ultrasound and Microbubbles for Targeted Drug Delivery to the Lung Endothelium in ARDS: Cellular Mechanisms and Therapeutic Opportunities.

Acute respiratory distress syndrome (ARDS) is characterized by increased permeability of the alveolar–capillary membrane, a thin barrier composed of adjacent monolayers of alveolar epithelial and lung microvascular endothelial cells. This results in pulmonary edema and severe hypoxemia and is a common cause of death after both viral (e.g., SARS-CoV-2) and bacterial pneumonia. The involvement of the lung in ARDS is notoriously heterogeneous, with consolidated and edematous lung abutting aerated, less injured regions. This makes treatment difficult, as most therapeutic approaches preferentially affect the normal lung regions or are distributed indiscriminately to other organs. In this review, we describe the use of thoracic ultrasound and microbubbles (USMB) to deliver therapeutic cargo (drugs, genes) preferentially to severely injured areas of the lung and in particular to the lung endothelium. While USMB has been explored in other organs, it has been under-appreciated in the treatment of lung injury since ultrasound energy is scattered by air. However, this limitation can be harnessed to direct therapy specifically to severely injured lungs. We explore the cellular mechanisms governing USMB and describe various permutations of cargo administration. Lastly, we discuss both the challenges and potential opportunities presented by USMB in the lung as a tool for both therapy and research.

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.

Characteristics of Passive Solute Transport across Primary Rat Alveolar Epithelial Cell Monolayers.

Primary rat alveolar epithelial cell monolayers (RAECM) were grown without (type I cell-like phenotype, RAECM-I) or with (type II cell-like phenotype, RAECM-II) keratinocyte growth factor to assess passive transport of 11 hydrophilic solutes. We estimated apparent permeability (Papp) in the absence/presence of calcium chelator EGTA to determine the effects of perturbing tight junctions on “equivalent” pores. Papp across RAECM-I and -II in the absence of EGTA are similar and decrease as solute size increases. We modeled Papp of the hydrophilic solutes across RAECM-I/-II as taking place via heterogeneous populations of equivalent pores comprised of small (0.41/0.32 nm radius) and large (9.88/11.56 nm radius) pores, respectively. Total equivalent pore area is dominated by small equivalent pores (99.92–99.97%). The number of small and large equivalent pores in RAECM-I was 8.55 and 1.29 times greater, respectively, than those in RAECM-II. With EGTA, the large pore radius in RAECM-I/-II increased by 1.58/4.34 times and the small equivalent pore radius increased by 1.84/1.90 times, respectively. These results indicate that passive diffusion of hydrophilic solutes across an alveolar epithelium occurs via small and large equivalent pores, reflecting interactions of transmembrane proteins expressed in intercellular tight junctions of alveolar epithelial cells.

Affinity of Mycobacterium tuberculosis strains for M059K microglial cells after migration through A549 alveolar epithelium.

Tuberculosis (TB) remains a major threat worldwide while central nervous system TB (CNS-TB) is one of the most severe forms of extrapulmonary TB. CNS-TB develops as a secondary infection during the hematogenous spread of Mycobacterium tuberculosis (M. tuberculosis) from the lungs to the CNS. Factors influencing the dissemination of the bacilli to the CNS have not been studied extensively. This study evaluated the transmigration ability through the alveolar epithelium and adhesion and invasion capacity of glial cells of M. tuberculosis strains of varying drug susceptibility and genotype profiles using an in vitro co-culture model. A549 alveolar epithelial cells and M059K glial cells were co-cultured in a Transwell plate with A549 cells cultured in the upper chamber and M059K glial cells in the lower chamber. A549 epithelial cells were infected with F15/LAM4/KZN (susceptible, MDR, XDR), Beijing (susceptible, XDR), F11 (susceptible), F28 (MDR), and H37Rv strains of M. tuberculosis. The transmigration of an A549 monolayer and subsequent adhesion and invasion rates of M059K cells were established. The susceptible and XDR variants of the F15/LAM4/KZN strain transmigrate the alveolar epithelial cell monolayer more efficiently than the MDR variant. The Beijing-XDR variant showed a high transmigration rate, while the susceptible variant showed no transmigration ability. Similar to the MDR F15/LAM4/KZN, the F28 and F11 strains showed a low dissemination ability. The bacteria were still capable to adhere to M059K glial cells after passage through the A549 cells. We conclude that M. tuberculosis isolates that passed through a monolayer of A549 alveolar epithelium by transcellular migration can still adhere to M059K glial cells. There is no genetic link between resistance and transmigration.

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.

Modulating the Barrier Function of Human Alveolar Epithelial (hAELVi) Cell Monolayers as a Model of Inflammation.

The incidence of inflammatory lung diseases such as acute respiratory distress syndrome (ARDS) remains an important problem, particularly in the present time with the Covid-19 pandemic. However, an adequate in vitro test system to monitor the barrier function of the alveolar epithelium during inflammation and for assessing anti-inflammatory drugs is urgently needed. Therefore, we treated human Alveolar Epithelial Lentivirus-immortalised cells (hAELVi cells) with the pro-inflammatory cytokines TNF-α (25 ng/ml) and IFN-γ (30 ng/ml), in the presence or absence of hydrocortisone (HC). While TNF-α and IFN-γ are known to reduce epithelial barrier properties, HC could be expected to protect the barrier function and result in an anti-inflammatory effect. We investigated the impact of anti-inflammatory/inflammatory treatment on transepithelial electrical resistance (TEER) and the apparent permeability coefficient (Papp) of the low permeability marker sodium fluorescein (NaFlu). After incubating hAELVi cells for 48 hours with a combination of TNF-α and IFN-γ, there was a significant decrease in TEER and a significant increase in the Papp. The presence of HC maintained the TEER values and barrier properties, so that no significant Papp change was observed. By using hAELVi cells to study anti-inflammatory drugs in vitro, the need for animal experiments could be reduced and pulmonary drug development accelerated.

High CO2 Levels Impair Lung Wound Healing.

Delayed lung repair leads to alveolopleural fistulae, which are a major cause of morbidity after lung resections. We have reported that intrapleural hypercapnia is associated with delayed lung repair after lung resection. Here, we provide new evidence that hypercapnia delays wound closure of both large airway and alveolar epithelial cell monolayers because of inhibition of epithelial cell migration. Cell migration and airway epithelial wound closure were dependent on Rac1-GTPase activation, which was suppressed by hypercapnia directly through the upregulation of AMP kinase and indirectly through inhibition of injury-induced NF-κB-mediated CXCL12 (pleural CXC motif chemokine 12) release, respectively. Both these pathways were independently suppressed, because dominant negative AMP kinase rescued the effects of hypercapnia on Rac1-GTPase in uninjured resting cells, whereas proteasomal inhibition reversed the NF-κB-mediated CXCL12 release during injury. Constitutive overexpression of Rac1-GTPase rescued the effects of hypercapnia on both pathways as well as on wound healing. Similarly, exogenous recombinant CXCL12 reversed the effects of hypercapnia through Rac1-GTPase activation by its receptor, CXCR4. Moreover, CXCL12 transgenic murine recipients of orthotopic tracheal transplantation were protected from hypercapnia-induced inhibition of tracheal epithelial cell migration and wound repair. In patients undergoing lobectomy, we found inverse correlation between intrapleural carbon dioxide and pleural CXCL12 levels as well as between CXCL12 levels and alveolopleural leak. Accordingly, we provide first evidence that high carbon dioxide levels impair lung repair by inhibiting epithelial cell migration through two distinct pathways, which can be restored by recombinant CXCL12.

Evidence for Nanoparticle-Induced Lysosomal Dysfunction in Lung Adenocarcinoma (A549) Cells.

Background: Polystyrene nanoparticles (PNP) are taken up by primary rat alveolar epithelial cell monolayers (RAECM) in a time-, dose-, and size-dependent manner without involving endocytosis. Internalized PNP in RAECM activate autophagy, are delivered to lysosomes, and undergo [Ca2+]-dependent exocytosis. In this study, we explored nanoparticle (NP) interactions with A549 cells. Methods: After exposure to PNP or ambient pollution particles (PM0.2), live single A549 cells were studied using confocal laser scanning microscopy. PNP uptake and egress were investigated and activation of autophagy was confirmed by immunolabeling with LC3-II and LC3-GFP transduction/colocalization with PNP. Mitochondrial membrane potential, mitophagy, and lysosomal membrane permeability (LMP) were assessed in the presence/absence of apical nanoparticle (NP) exposure. Results: PNP uptake into A549 cells decreased in the presence of cytochalasin D, an inhibitor of macropinocytosis. PNP egress was not affected by increased cytosolic [Ca2+]. Autophagy activation was indicated by increased LC3 expression and LC3-GFP colocalization with PNP. Increased LMP was observed following PNP or PM0.2 exposure. Mitochondrial membrane potential was unchanged and mitophagy was not detected after NP exposure. Conclusions: Interactions between NP and A549 cells involve complex cellular processes leading to lysosomal dysfunction, which may provide opportunities for improved nanoparticle-based therapeutic approaches to lung cancer management.

Wood Smoke Disrupts Alveolar Epithelial Barrier Function through a p44/42 MAPK Signaling Pathway

BackgroundWood smoke (WS) contains many of the same toxic compounds as those found in cigarette smoke (CS). Although CS is a leading cause of respiratory disease, limited studies investigating the mechanisms involved in WS mediated respiratory disease have been undertaken. Exposure to WS particulate reduces epithelial cell viability and barrier function. Both CS and WS induce MAPK signaling and contribute to airway remodeling. With an increase in the size, duration, and number of forest fires annually, there is a need for a better understanding with regards to the acute and chronic effects of WS on respiratory health. Therefore, we undertook studies to evaluate the role of WS exposure on the function and remodeling of the alveolar epithelium.MethodsBirch wood was combusted and the smoke bubbled into PBS. The pH pf the resulting solution was adjusted to 7.1 and was considered 100% WS solution. Monolayers of A549 alveolar epithelial cells were exposed to increasing concentrations of WS for up to 48 hours. Cell viability was determined using MTT and trypan blue exclusion. Epithelial barrier function was monitored via electrical cell‐substrate impedance sensing (ECIS). Adhesion junctional structure was observed by immunofluorescent staining for E‐cadherin. Western blot was used to assess activation of MAPK signaling pathways.ResultsConcentrations of WS up to 10% had no effect on cell viability. WS promoted a concentration dependent reduction in epithelial barrier function after 24 hours which was maintained out to 48 hours. Cells not exposed to WS demonstrated distinct cobblestone E‐cadherin staining. Exposure to 2.5% WS diminished the cobblestone patterning after 24 hours and was completely lost after exposure to 5.0% WS. The wound healing capacity of cells after WS exposure was assessed using a scratch assay. After 48 hours, cells not exposed to WS had closed the scratched area by >50% whereas those cells exposed to 10% WS had closed the area by <20%. Exposure to WS induced phosphorylation of the p44/42, but not p38, MAPK signaling pathway. Pre‐treatment with the MAPK inhibitor U0126 prevented WS mediated epithelial barrier dysfunction. Additionally, U0126 preserved E‐cadherin cobblestone patterning even after WS exposure and had no effect on cell viability.ConclusionsExposure to WS disrupts alveolar epithelial adhesion that is partially regulated through the p44/42 MAPK signaling pathway. Pharmacological inhibition of p44/42 phosphorylation preserved epithelial barrier function, which may be as a result of maintained E‐cadherin‐mediated cell adhesion.Support or Funding InformationGrant in aide funding was provided by provided CIHR (DJG). MRZ and CTT are recipients of CIHR Fellowships. MRZ also received a MSFHR Trainee Award. CC is a Canada Research Chair and holds the Astra‐Zeneca Chair in Occupational and Environmental Lung Disease at the University of British Columbia.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance.

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

Alveolar epithelial cell processing of nanoparticles activates autophagy and lysosomal exocytosis.

Using confocal microscopy, we quantitatively assessed uptake, processing, and egress of near-infrared (NIR)-labeled carboxylated polystyrene nanoparticles (PNP) in live alveolar epithelial cells (AEC) during interactions with primary rat AEC monolayers (RAECM). PNP fluorescence intensity (content) and colocalization with intracellular vesicles in a cell were determined over the entire cell volume via z stacking. Isotropic cuvette-based microfluorimetry was used to determine PNP concentration ([PNP]) from anisotropic measurements of PNP content assessed by confocal microscopy. Results showed that PNP uptake kinetics and steady-state intracellular content decreased as diameter increased from 20 to 200 nm. For 20-nm PNP, uptake rate and steady-state intracellular content increased with increased apical [PNP] but were unaffected by inhibition of endocytic pathways. Intracellular PNP increasingly colocalized with autophagosomes and/or lysosomes over time. PNP egress exhibited fast Ca2+ concentration-dependent release and a slower diffusion-like process. Inhibition of microtubule polymerization curtailed rapid PNP egress, resulting in elevated vesicular and intracellular PNP content. Interference with autophagosome formation led to slower PNP uptake and markedly decreased steady-state intracellular content. At steady state, cytosolic [PNP] was higher than apical [PNP], and vesicular [PNP] (~80% of intracellular PNP content) exceeded both cytosolic and intracellular [PNP]. These data are consistent with the following hypotheses: 1) autophagic processing of nanoparticles is essential for maintenance of AEC integrity; 2) altered autophagy and/or lysosomal exocytosis may lead to AEC injury; and 3) intracellular [PNP] in AEC can be regulated, suggesting strategies for enhancement of nanoparticle-driven AEC gene/drug delivery and/or amelioration of AEC nanoparticle-related cellular toxicity.

Interactions of Inhaled Nanoparticles with Rat Alveolar Epithelial Cell Monolayers

RationaleTo understand the contribution to lung pathogenesis of ambient ultrafine air pollution particles, knowledge of basic mechanisms underlying how inhaled nanoparticles interact with alveolar epithelial cells (AEC) is needed. We have reported that polystyrene nanoparticles (PNP) at the apical surface of primary rat alveolar AEC monolayers (RAECM) are taken up in a manner dependent on exposure time, dose and particle size via a non‐endocytic ‘diffusional’ process, induce autophagy, and exit via both fast intracellular calcium‐dependent lysosomal exocytosis and a slower (‘diffusional’) process. In this study, we further investigated intracellular fate of PNP.MethodsRAECM were apically exposed to near‐infrared labeled PNP (80 μg/mL; carboxylated; 20 nm). AEC were subjected to confocal imaging throughout the volume of the cells. In each z plane, total intracellular fluorescence intensity as well as vesicular and cytosolic PNP were determined and normalized to total intracellular PNP (PNPic). Volumes of endosomal, autophagosomal and lysosomal compartments, and colocalization of PNP in cytosolic vs vesicular compartments, were assessed using an early endosome marker (Rab5) tagged with green fluorescent protein (GFP), quinacrine, microtubule‐associated protein 1 light chain 3 (LC3B)‐GFP, and Magic Red (a lysosomal marker). [PNPic] was then determined by an isotropic method (cuvette‐based microfluorometry) utilizing AEC lysates as a function of time. [PNP] in vesicles ([PNPv]) and cytosol ([PNPc]) were estimated from the respective fractions of compartmental volumes and co‐localized PNP in each compartment.ResultsAfter 12 hr, [PNPic] reached a plateau of 1.84 mg/mL. Distribution of intracellular PNP was ~80% in intracellular vesicles (primarily lysosomes) and ~20% in cytosol at steady state, equivalent to 8.86 mg/ml and 0.58 mg/mL for [PNPv] and [PNPc], respectively. About 3% of total intracellular PNP was observed in Rab5‐GFP positive early endosomes by 3 hr after PNP exposure, but inhibition of classical endocytosis pathways did not alter PNP uptake kinetics. In contrast, blockade of autophagosome formation by 3‐methyladenine (3MA) markedly reduced PNP uptake and decreased steady state [PNPic] by ~75% with no detectable vesicular PNP. Inhibition of autophagosome‐lysosome fusion by bafilomycin lowered steady state PNPic by ~20% compared to control, with ~81% in cytosol and ~19% in vesicles.SummaryIn AEC exposed to PNP, [PNPv] exceeds both [PNPc] and [PNPic], with [PNPc]>>apical [PNP]. Cytosolic presence of PNP triggers autophagosome formation, leading to PNP accumulation in lysosomes. Inhibition of autophagy leads to decreased PNP entry and [PNPic]. These data suggest that intracellular processing of PNP is regulable, offering possible approaches to amelioration of inhaled nanoparticle‐related cytotoxicity in AEC.Support or Funding InformationFunding: NIH; Will Rogers Institute; Hastings and Whittier FoundationsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protection of Meconium-Induced Lung Epithelial Injury by Protease Inhibitors

Earlier work form this laboratory showed that exposure of alveolar epithelial cells (AECs) to meconium caused significant cell detachment and that meconium-induced detachment of cells was prevented by a protease inhibitor cocktail. Therefore, it was hypothesized that protease inhibitors might protect AEC monolayers against meconium-induced collapse of epithelial barrier function both in vitro and in vivo. To investigate this theory in vitro, albumin flux was measured across cultured, confluent monolayers of human type II derived cell line A549 on microporous filter inserts. Human meconium was collected from seven healthy full-term neonates and the samples were pooled and diluted prior to analysis. Exposure of AECs to 5% human meconium increased albumin flux across the cultured AEC monolayers, but the increase was significantly blocked by protease inhibitors (P<0.001). In C57/BL6 mice, intratracheal instillation of 5% human meconium increased the passage of Evans Blue Dye (EBD) from the vascular compartment into the alveolar spaces, measured in bronchoalveolar lavage (BAL) fluid after intravenous injection of EBD. Moreover, intratrachial coinstillation of protease inhibitors prevented the meconium-induced increase in EBD passage into BAL fluid (P<0.01). The data presented herein clearly demonstrate that protease inhibitors protect AEC barrier function against meconium-induced injury, and suggest the future possibility of using protease inhibitors in the treatment of meconium aspiration syndrome.

Oligopeptide Transport in Rat Lung Alveolar Epithelial Cells is Mediated by Pept2.

Studies were conducted in primary cultured rat alveolar epithelial cell monolayers to characterize peptide transporter expression and function. Freshly isolated rat lung alveolar epithelial cells were purified and cultured on permeable support with and without keratinocyte growth factor (KGF). Messenger RNA and protein expression of Pept1 and Pept2 in alveolar epithelial type I- and type II-like cell monolayers (±KGF, resp.) were examined by RT-PCR and Western blotting. 3H-Glycyl-sarcosine (3H-gly-sar) transmonolayer flux and intracellular accumulation were evaluated in both cell types. RT-PCR showed expression of Pept2, but not Pept1, mRNA in both cell types. Western blot analysis revealed presence of Pept2 protein in type II-like cells, and less in type I-like cells. Bi-directional transmonolayer 3H-gly-sar flux lacked asymmetry in transport in both types of cells. Uptake of 3H-gly-sar from apical fluid of type II-like cells was 7-fold greater than that from basolateral fluid, while no significant differences were observed from apical vs. basolateral fluid of type I-like cells. This study confirms the absence of Pept1 from rat lung alveolar epithelium in vitro. Functional Pept2 expression in type II-like cell monolayers suggests its involvement in oligopeptide lung disposition, and offers rationale for therapeutic development of di/tripeptides, peptidomimetics employing pulmonary drug delivery.

Integrated Stress Response Mediates Epithelial Injury in Mechanical Ventilation.

Ventilator-induced lung injury (VILI) is a severe complication of mechanical ventilation that can lead to acute respiratory distress syndrome. VILI is characterized by damage to the epithelial barrier with subsequent pulmonary edema and profound hypoxia. Available lung-protective ventilator strategies offer only a modest benefit in preventing VILI because they cannot impede alveolar overdistension and concomitant epithelial barrier dysfunction in the inflamed lung regions. There are currently no effective biochemical therapies to mitigate injury to the alveolar epithelium. We hypothesize that alveolar stretch activates the integrated stress response (ISR) pathway and that the chemical inhibition of this pathway mitigates alveolar barrier disruption during stretch and mechanical ventilation. Using our established rat primary type I-like alveolar epithelial cell monolayer stretch model and in vivo rat mechanical ventilation that mimics the alveolar overdistension seen in acute respiratory distress syndrome, we studied epithelial responses to mechanical stress. Our studies revealed that the ISR signaling pathway is a key modulator of epithelial permeability. We show that prolonged epithelial stretch and injurious mechanical ventilation activate the ISR, leading to increased alveolar permeability, cell death, and proinflammatory signaling. Chemical inhibition of protein kinase RNA-like endoplasmic reticulum kinase, an upstream regulator of the pathway, resulted in decreased injury signaling and improved barrier function after prolonged cyclic stretch and injurious mechanical ventilation. Our results provide new evidence that therapeutic targeting of the ISR can mitigate VILI.

Gene expression patterns induced at different stages of rhinovirus infection in human alveolar epithelial cells.

Human rhinovirus (HRV) is the common virus that causes acute respiratory infection (ARI) and is frequently associated with lower respiratory tract infections (LRTIs). We aimed to investigate whether HRV infection induces a specific gene expression pattern in airway epithelial cells. Alveolar epithelial cell monolayers were infected with HRV species B (HRV-B). RNA was extracted from both supernatants and infected monolayer cells at 6, 12, 24 and 48 hours post infection (hpi) and transcriptional profile was analyzed using Affymetrix GeneChip and the results were subsequently validated using quantitative Real-time PCR method. HRV-B infects alveolar epithelial cells which supports implication of the virus with LRTIs. In total 991 genes were found differentially expressed during the course of infection. Of these, 459 genes were up-regulated whereas 532 genes were down-regulated. Differential gene expression at 6 hpi (187 genes up-regulated vs. 156 down-regulated) were significantly represented by gene ontologies related to the chemokines and inflammatory molecules indicating characteristic of viral infection. The 75 up-regulated genes surpassed the down-regulated genes (35) at 12 hpi and their enriched ontologies fell into discrete functional entities such as regulation of apoptosis, anti-apoptosis, and wound healing. At later time points of 24 and 48 hpi, predominated down-regulated genes were enriched for extracellular matrix proteins and airway remodeling events. Our data provides a comprehensive image of host response to HRV infection. The study suggests the underlying molecular regulatory networks genes which might be involved in pathogenicity of the HRV-B and potential targets for further validations and development of effective treatment.

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.

Knockout Mice Reveal a Major Role for Alveolar Epithelial Type I Cells in Alveolar Fluid Clearance.

Active ion transport by basolateral Na-K-ATPase (Na pump) creates an Na(+) gradient that drives fluid absorption across lung alveolar epithelium. The α1 and β1 subunits are the most highly expressed Na pump subunits in alveolar epithelial cells (AEC). The specific contribution of the β1 subunit and the relative contributions of alveolar epithelial type II (AT2) versus type I (AT1) cells to alveolar fluid clearance (AFC) were investigated using two cell type-specific mouse knockout lines in which the β1 subunit was knocked out in either AT1 cells or both AT1 and AT2 cells. AFC was markedly decreased in both knockout lines, revealing, we believe for the first time, that AT1 cells play a major role in AFC and providing insights into AEC-specific roles in alveolar homeostasis. AEC monolayers derived from knockout mice demonstrated decreased short-circuit current and active Na(+) absorption, consistent with in vivo observations. Neither hyperoxia nor ventilator-induced lung injury increased wet-to-dry lung weight ratios in knockout lungs relative to control lungs. Knockout mice showed increases in Na pump β3 subunit expression and β2-adrenergic receptor expression. These results demonstrate a crucial role for the Na pump β1 subunit in alveolar ion and fluid transport and indicate that both AT1 and AT2 cells make major contributions to these processes and to AFC. Furthermore, they support the feasibility of a general approach to altering alveolar epithelial function in a cell-specific manner that allows direct insights into AT1 versus AT2 cell-specific roles in the lung.

NLRP3 protects alveolar barrier integrity by an inflammasome-independent increase of epithelial cell adherence.

Bacterial pneumonia is a major cause of acute lung injury and acute respiratory distress syndrome, characterized by alveolar barrier disruption. NLRP3 is best known for its ability to form inflammasomes and to regulate IL-1β and IL-18 production in myeloid cells. Here we show that NLRP3 protects the integrity of the alveolar barrier in a mouse model of Streptococcus pneumoniae-induced pneumonia, and ex vivo upon treatment of isolated perfused and ventilated lungs with the purified bacterial toxin, pneumolysin. We reveal that the preserving effect of NLRP3 on the lung barrier is independent of inflammasomes, IL-1β and IL-18. NLRP3 improves the integrity of alveolar epithelial cell monolayers by enhancing cellular adherence. Collectively, our study uncovers a novel function of NLRP3 by demonstrating that it protects epithelial barrier function independently of inflammasomes.