Rat Tracheal Epithelial Cells Research Articles

Telocytes protect against lung tissue fibrosis through hexokinase 2-dependent pathway by secreting hepatocyte growth factor.

Pulmonary fibrosis (PF) is one of the common manifestations of end-stage lung disease. Chronic lung failure after lung transplantation is mainly caused by bronchiolitis obliterans syndrome (BOS) and is mainly characterized by lung tissue fibrosis. Pulmonary epithelial-mesenchymal transformation (EMT) is crucial for pulmonary fibrosis. Telocytes (TCs), a new type of mesenchymal cells, play a protective role in various acute injuries. For exploring the anti-pulmonary fibrosis effect of TCs in the BOS model in vitro and the related mechanism, rat tracheal epithelial (RTE) cells were treated with transforming growth factor-β (TGF-β) to simulate lung tissue fibrosis in vitro. The RTE cells were then co-cultured with TCs primarily extracted from rat lung tissue. Western blot, Seahorse XF Analysers and enzyme-linked immunosorbent assay were used to detect the level of EMT and aerobic respiration of RTE cells. Furthermore, anti-hepatocyte growth factor (anti-HGF) antibody was exogenously added to the cultured cells to explore further mechanisms. Moreover, hexokinase 2 (HK2) in RTE cells was knocked down to assess whether it influences the blocking effect of the anti-HGF antibody. TGF-β could induce lung tissue fibrosis in RTE cells in vitro. Nevertheless, TCs co-culture decreased the level of EMT, glucose metabolic indicators (lactate and ATP) and oxygen levels. Furthermore, TCs released hepatocyte growth factor (HGF). Therefore, the exogenous addition of anti-HGF antibody in the co-culture system blocked the anti-lung tissue fibrosis effect. However, HK2 knockdown attenuated the blocking effect of the anti-HGF antibody. In conclusion, TCs can protect against lung tissue fibrosis by releasing HGF, a process dependent on HK2.

GATA6 promotes fibrotic repair of tracheal injury through NLRP3 inflammasome-mediated epithelial pyroptosis

Tracheal injury is a challenging emergency condition that is characterized by the abnormal repair of the trachea. GATA6, a well-established transcription factor, plays a crucial role in tissue injury and epithelial regenerative repair. This study aims to evaluate the role of GATA6 in NF-κB-mediated NLRP3 inflammasome activation and pyroptosis after tracheal injury. Tracheal tissues and serum samples were collected from clinical patients and a rat model of tracheal injury. Upon GATA6 knockdown or overexpression, BEAS-2B and rat tracheal epithelial (RTE) cells were treated with lipopolysaccharides and nigericin before being co-cultured with primary tracheal fibroblasts. The changes of NLRP3 inflammasome activation and pyroptosis and their underlying mechanisms were detected. Additionally, the role of GATA6 downregulation in tracheal injury was verified in rats. GATA6 expression and NLRP3 inflammasome activation were upregulated following tracheal injury in the epithelium of granulation tissues. GATA6 silencing inhibited NLRP3 priming, NLRP3 inflammasome activation, and pyroptosis in BEAS-2B and RTE cells. Mechanistically, GATA6 was determined to have bound to the promoter region of NLRP3 and synergistically upregulated NLRP3 promoter activity with NF-κB. Furthermore, GATA6 overexpression promoted epithelial-mesenchymal transition via modulating the NF-κB/NLRP3 pathway. Epithelial NLRP3 inflammasome activation triggered ECM production in fibroblasts, which was suppressed by GATA6 knockdown and induced by GATA6 overexpression. Finally, the downregulation of GATA6 alleviated NLRP3 inflammasome-mediated pyroptosis induced by tracheal injury in rats, thereby reducing tracheal stenosis, inflammation, and fibrosis. GATA6 promotes fibrotic repair in tracheal injury through NLRP3 inflammasome-mediated epithelial pyroptosis, making it a potential biological therapeutic target for tracheal injury.

Protective Effects of Theaflavins and Epigallocatechin Gallate against ZnO-NP-Induced Cell Apoptosis In Vitro

Zinc oxide nanoparticles (ZnO-NPs) are commonly used in various commercial applications, causing toxic effects on organisms and destroying biodiversity, but information about their protective approaches remains unknown. This study aims to evaluate the protective effects of theaflavins (TFs) and epigallocatechin gallate (EGCG) against ZnO-NP-induced cytotoxicity in rat tracheal epithelial (RTE) cells. Herein, RTE cells were exposed to 100 μg/L ZnO-NPs for 12 h, then treated with 0, 10, 100, and 1000 μg/L TFs or EGCG for another 12 h; subsequently, oxidative stress, inflammation, and apoptosis analyses were conducted. Relative to the control groups, TFs and EGCG treatment significantly inhibited the levels of reactive oxygen species and malondialdehyde content. Exposure to 1000 μg/L TFs or EGCG treatment downregulated cytochrome C gene expression levels by 59.10% and 77.27%; Caspase 3 gene expression by 50.03% and 60.01%; Caspase 8 gene expression by 45.11% and 55.57%; and Caspase 9 gene expression by 51.33% and 66.67%, respectively. Meanwhile, interleukin 1β and interleukin 6, tumor necrosis factor-α, and the other inflammatory chemokines such as C-C motif chemokine 2 and C-X-C motif chemokine 8 expression were all gradually rescued after the addition of TFs or EGCG. These results imply that TFs or EGCG possibly ameliorated ZnO-NPs-induced toxicity through antiapoptotic, antioxidant, and anti-inflammatory effects. This study provides novel approaches which mitigate the emerging nanoparticle pollutant toxicity in organisms, which may potentially slow down the destruction of biodiversity.

Curcumin-Loaded mPEG-PLGA Nanoparticles Attenuates the Apoptosis and Corticosteroid Resistance Induced by Cigarette Smoke Extract.

The present study was aim to prepare curcumin-loaded methoxypolyethylene-glycols-poly (lactic-co-glycolic acid) nanoparticles (Cur-mPEG-PLGA-NPs) and investigate curcumin’s effect on reversing corticosteroid resistance induced by cigarette smoke extract (CSE) in rat tracheal epithelial (RTE) cells. The Cur-mPEG-PLGA-NPs were spherical, regular in shape with smooth surfaces, and well distributed and Cur-mPEG-PLGA-NP suspensions had good water solubility and presented prolonged release. Furthermore, we found that Cur-mPEG-PLGA-NPs were internalized more than curcumin into the cells and significantly alleviated apoptosis in RTE cells. In addition, 10% CSE reduced the maximal inhibition percentage and increased the half-inhibitory concentration of budesonide (BUD) on IL-8 secretion, and curcumin restored the efficacy of BUD inhibition. BUD in combination with Cur-mPEG-PLGA-NPs showed higher inhibitory rates for LPS- and CSE-induced IL-8 secretion than that in combination with curcumin. Moverover, the relative expression levels of HDAC2 was reduced after CSE exposure and curcumin could improve HDAC2 expression and reverse CSE-induced corticosteroid resistance. Curcumin in high concentration and Cur-mPEG-PLGA-NPs restored HDAC2 levels in RTE cells and thus Cur-mPEG-PCL-NPs have higher biological activity than curcumin.

MiR-21-5p in Macrophage-Derived Exosomes Targets Smad7 to Promote Epithelial Mesenchymal Transition of Airway Epithelial Cells.

BackgroundAsthma is usually associated with airway inflammation and airway remodeling. Epithelial mesenchymal transition (EMT) often occurs in airway remodeling. The purpose of this study is to identify the effect of miR-21-5p and Smad7 signaling pathway in macrophage-derived exosomes on EMT of airway epithelial cells.MethodsHE staining and Masson staining were used to verify the successful establishment of the asthma model. The levels of epithelial cell adhesion factor and stromal cell markers were detected by Western blot. The levels of miR-21-5p were detected by qRT-PCR. The expression of miR-21-5p in lung tissue was further verified by fluorescence in situ hybridization (FISH). Exosome morphology was observed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Luciferase reporter assay was applied to analyze the interaction of miR-21-5p with Smad7.ResultsThe expression of miR-21-5p was upregulated in macrophages of rats in vivo with OVA-induced asthma. In vitro cultured alveolar macrophages stimulated by LPS could secrete exosomes with high levels of miR-21-5p. The exosome-derived miR-21-5p promotes EMT in rat tracheal epithelial cells through TGFβ1/Smad signaling pathway by downregulating Smad7. This process can be blocked by miR-21-5p inhibitor.ConclusionRat alveolar macrophages produced high levels of miR-21-5p-containing exosomes, which transported miR-21-5p to tracheal epithelial cells, thus promoting EMT through TGF-β1/Smad signaling pathway by targeting Smad7.

The effects of autophagy in rat tracheal epithelial cells induced by silver nanoparticles.

The massive use of silver nanoparticles (AgNPs) is potentially harmful to exposed humans. Although previous studies have found that AgNPs can induce cell autophagy, few studies have focused on the toxic pathways and mechanisms of autophagy induced by AgNPs in rat respiratory epithelial (RTE) cells. In this study, RTE cells were exposed to two kinds of AgNPs in vitro to ascertain the influence of mTOR-autophagy pathway-associated protein expression, including Beclin1, LC3B, Atg5, and Atg7. After exposure to different sizes and concentrations of AgNPs for 12 h, the uptake of silver in RTE cells reached 0.45 μg/L to 1.11 μg/L, indicating that AgNPs can enter RTE cells, leading to toxic effects. Our study found that this toxic effect was related to autophagy caused by ROS accumulation that was mediated by the mTOR pathway. With increasing AgNP exposure concentrations, the expression of p-mTOR was significantly downregulated, and expression of the autophagy-related proteins Beclin1, LC3B, Atg5, and Atg7 was significantly increased in RTE cells in all exposed groups. At a concentration of 1000 μg/L, the expression of LC3BII/LC3BI in all exposed groups was 24.49 times and 12.71 times that of the control, and the expression of Atg7 in all exposed groups was 23.21 times and 13.21 times that of the control. The upregulation of autophagy-related proteins in the AgNP-10 nm exposure groups was greater than that of the AgNP-100 nm exposure group. In summary, the mTOR pathway mediates AgNP-induced autophagy in RTE cells, which leads to damage to the respiratory system barrier and human health risks. This study can facilitate the development of prevention and intervention policies against adverse consequences induced by AgNPs.

A Novel G542X CFTR Rat Model of Cystic Fibrosis Is Sensitive to Nonsense Mediated Decay.

Nonsense mutations that lead to the insertion of a premature termination codon (PTC) in the cystic fibrosis transmembrane conductance regulator (CFTR) transcript affect 11% of patients with cystic fibrosis (CF) worldwide and are associated with severe disease phenotype. While CF rat models have contributed significantly to our understanding of CF disease pathogenesis, there are currently no rat models available for studying CF nonsense mutations. Here we created and characterized the first homozygous CF rat model that bears the CFTR G542X nonsense mutation in the endogenous locus using CRISPR/Cas9 gene editing. In addition to displaying severe CF manifestations and developmental defects such as reduced growth, abnormal tooth enamel, and intestinal obstruction, CFTR G542X knockin rats demonstrated an absence of CFTR function in tracheal and intestinal sections as assessed by nasal potential difference and transepithelial short-circuit current measurements. Reduced CFTR mRNA levels in the model further suggested sensitivity to nonsense-mediated decay, a pathway elicited by the presence of PTCs that degrades the PTC-bearing transcripts and thus further diminishes the level of CFTR protein. Although functional restoration of CFTR was observed in G542X rat tracheal epithelial cells in response to single readthrough agent therapy, therapeutic efficacy was not observed in G542X knockin rats in vivo. The G542X rat model provides an invaluable tool for the identification and in vivo validation of potential therapies for CFTR nonsense mutations.

More serious autophagy can be induced by ZnO nanoparticles than single-walled carbon nanotubes in rat tracheal epithelial cells.

Metal oxide nanoparticles and carbon nanoparticles, as common nanoparticles (NPs), can cause autophagy in certain cells, which will lead to biohealth risk issues. This study determined the difference in autophagy induced by zinc oxide nanoparticles (ZnO NPs) and single-walled carbon nanotubes (SWCNTs) in respiratory epithelial cells. ICP-OES results showed that NPs uptake as well as the intercellular contents of particles affected cytotoxicity in a dose-dependent manner. ZnO NPs-30 nm had a distinct green dot structure representing autophagy, the SWCNTs exposure group had a few green light spots at a concentration of 10 μg/L. The ROS content of the ZnO NP-30 nm exposure group had the greatest increase at a concentration of 1000 μg/L, which was 2.5 times higher than that of the control, the SWCNTs exposure group showed a 2.2-fold increase. A slight downregulation of p-mTOR was detected, and the ZnO NPs-30 nm treatment group had the significant downregulation rate. The gene and protein expression levels of Beclin-1 and LC3B were upregulated as the exposure concentration increased. The protein expression of Beclin-1 and LC3B in the 1000 μg/L ZnO NPs-30 nm exposure group were 5.21 times and 4.12 times that of the control, respectively. The mRNA expression of Beclin-1 and LC3B in the 1000 μg/L ZnO NPs-30 nm exposure group were 5.04 times and 3.61 times that of the control, respectively. At any concentration, the effect of ZnO NPs-30 nm was greater than that of the SWCNTs. Interaction and crosstalk analysis showed that exposure to ZnO NPs-30 nm caused autophagy through the aggregation of undegraded autophagosomes, whereas SWCNTs exposure induced diminished intercellular oxidative stress to inhibit autophagy. Therefore, this study demonstrated that the effects of autophagy induced by ZnO NPs-30 nm and SWCNTs were different. The health risks of ZnO-30 nm NPs are higher than those of SWCNTs.

Development of a system for the detection of the inflammatory response induced by airborne fine particulate matter in rat tracheal epithelial cells

Exposure to airborne particulate matter (PM) is related to the increased risk of several diseases, including chronic and allergic rhinitis. We have previously shown that atmospheric endotoxin level was positively associated with the number of emergency department visits for asthma even after adjusting for meteorological factors, suggestive of the significant association between atmospheric endotoxin level and asthma exacerbation. Whether atmospheric endotoxin level is related to inflammatory response induction is, however, unclear. Here, we established stable cell lines to determine the promoter activity of the genes encoding pro-inflammatory cytokines such as tumor necrosis factor alpha, interleukin 6 (IL6), and IL33 by transfection of each reporter plasmid into rat tracheal epithelial EGV-4 T cells. These cells could measure the inflammatory response induced by endotoxin treatment more easily, rapidly, and sensitively than the conventional system using immunodetection assays. Furthermore, we revealed a relationship between atmospheric endotoxin level and inflammatory response induction. Thus, the system established herein may serve as a promising tool to monitor inflammatory response induced upon PM exposure.

Development of a system for the detection of the inflammatory response induced by airborne fine particulate matter in rat tracheal epithelial cells

Exposure to airborne particulate matter (PM) is related to the increased risk of several diseases, including chronic and allergic rhinitis. We have previously shown that atmospheric endotoxin level was positively associated with the number of emergency department visits for asthma even after adjusting for meteorological factors, suggestive of the significant association between atmospheric endotoxin level and asthma exacerbation. Whether atmospheric endotoxin level is related to inflammatory response induction is, however, unclear. Here, we established stable cell lines to determine the promoter activity of the genes encoding pro-inflammatory cytokines such as tumor necrosis factor alpha, interleukin 6 (IL6), and IL33 by transfection of each reporter plasmid into rat tracheal epithelial EGV-4 T cells. These cells could measure the inflammatory response induced by endotoxin treatment more easily, rapidly, and sensitively than the conventional system using immunodetection assays. Furthermore, we revealed a relationship between atmospheric endotoxin level and inflammatory response induction. Thus, the system established herein may serve as a promising tool to monitor inflammatory response induced upon PM exposure.

Mechanisms of silver nanoparticles-induced cytotoxicity and apoptosis in rat tracheal epithelial cells.

Silver nanoparticles (AgNPs) are increasingly utilized in a number of applications. This study was designed to investigate AgNPs induced cytotoxicity, oxidative stress and apoptosis in rat tracheal epithelial cells (RTE). The RTE cells were treated with 0, 100 μg/L and 10,000 μg/L of the AgNPs with diameters of 10 nm and 100 nm for 12 hr. The cell inhibition level, apoptosis ratio, reactive oxygen species (ROS), malondialdehyde (MDA) and metallothionein (MT) content were determined. The mRNA expression of cytoc, caspase 3, and caspase 9 was measured by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, we also analyzed the cytoc, caspase 3, pro-caspase 3, caspase 9, and pro-caspase 9 protein expression by western blotting. Electric cell-substrate impedance sensing (ECIS) analysis showed that the growth and proliferation of RTE cells were significantly inhibited in a dose-dependent manner under AgNPs exposure. The cell dynamic changes induced by 10 nm AgNPs were more severe than that of the 100 nm AgNPs exposure group. The intracellular MT, ROS, and MDA content increased when the exposure concentration increased and size reduced, whereas Ca2+-ATPase activity and Na+/K+-ATPase activity changed inversely. The relative expression of protein of cytoc, caspase 3, and caspase 9 were upregulated significantly, which indicated that AgNPs induced apoptosis of RTE cells through the caspase-dependent mitochondrial pathway. Our results demonstrate that AgNPs caused obvious cytotoxicity, oxidative stress, and apoptosis in RTE cells, which promoted the releasing of cytochrome C and pro-apoptotic proteins into the cytoplasm to activate the caspase cascade and finally led to apoptosis.

Pingchuanning decoction attenuates airway inflammation by suppressing autophagy via phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway in rat models of asthma.

Pingchuanning decoction is a well-known traditional Chinese medicinefor the treatment of airway inflammatory diseases, including asthma. However, the potential mechanism by which Pingchuanning decoction contributes to the amelioration of airway inflammation remains unknown. A rat model of asthma was well established by inducing ovalbumin. Lipopolysaccharide-stimulated rat tracheal epithelial (RTE) cells were used as cellular model. Lung histopathology and goblet cell hyperplasia were assessed by hematoxylin-eosin (HE) and periodic acid Schiffstaining, respectively. Total inflammatory cells count and RTE cell apoptosis were analyzed by flow cytometry. The autophagic activities were evaluated by immunohistochemical and immunofluorescence analysis and Western blot analysis of autophagy-related proteins. We also detected the effects of Pingchuanning decoction on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) and high-mobility group box 1 (HMGB1)-mediated toll-like receptor 4 (TLR4)/NF-κB pathways-related proteins and inflammatory cytokines using the Western blot analysis and enzyme-linked immunosorbentassay. Pingchuanning decoction effectively attenuated pulmonary pathology and autophagy. Treatment with Pingchuanning decoction activated PI3K/Akt/mTOR pathway and inhibited HMGB1/TLR4/NF-κB pathway, which could be overturned by LY294002, a PI3K antagonist, or rapamycin (Rapa), an autophagy inducer. Pingchuanning decoction exerted a therapeutic effect on asthma by inhibiting autophagy via PI3K/Akt /mTOR signaling pathway.

Trachea Engineering Using a Centrifugation Method and Mouse-Induced Pluripotent Stem Cells.

The outcomes of tracheal transplantation for the treatment of airway stenosis are unsatisfactory. We investigated the feasibility of regeneration of the trachea using a rat decellularized tracheal scaffold and mouse-induced pluripotent stem (iPS) cells for in vivo transplantation. The rat trachea was first decellularized using a detergent/enzymatic treatment method. We successfully established a centrifugation method that can transplant cells onto the luminal surface of the decellularized rat tracheal scaffold circumferentially. Two types of mouse iPS cells were differentiated into definitive endoderm cells and transplanted onto the luminal surface of the decellularized tracheal matrix scaffold using this centrifugation method. For in vivo study, normal rat tracheas, no-cell rat tracheal scaffolds, or rat tracheal scaffolds recellularized with rat tracheal epithelial cells (EGV-4T) were orthotopically transplanted on F344 rats, and rat tracheal scaffolds recellularized with mouse iPS cells were transplanted on F344/NJc1-rnu/rnu rats. Rats transplanted with no-cell scaffolds or scaffolds recellularized with EGV-4T survived for 1 month, although airway stenosis was observed. One of the F344/NJc1-rnu/rnu rats transplanted with rat trachea regenerated using mouse iPS cells survived over 5 weeks. Histological analysis indicated the cause of death was airway stenosis due to colonic cellular proliferation of undifferentiated iPS cells. Re-epithelialization with numerous ciliated epithelial cells was observed in one of the rats transplanted with trachea bioengineered using iPS cells. In this study, we present a simple and efficient tracheal tissue engineering model using a centrifugation method in a small-animal model. Tissue-engineered trachea using decellularized tracheal scaffolds and iPS cells is potentially applicable for tracheal transplantation.

Cellular distribution and function of ion channels involved in transport processes in rat tracheal epithelium.

Transport of water and electrolytes in airway epithelia involves chloride‐selective ion channels, which are controlled either by cytosolic Ca2+ or by cAMP. The contributions of the two pathways to chloride transport differ among vertebrate species. Because rats are becoming more important as animal model for cystic fibrosis, we have examined how Ca2+‐ dependent and cAMP‐ dependent Cl− secretion is organized in the rat tracheal epithelium. We examined the expression of the Ca2+‐gated Cl− channel anoctamin 1 (ANO1), the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel, the epithelial Na+ channel ENaC, and the water channel aquaporin 5 (AQP5) in rat tracheal epithelium. The contribution of ANO1 channels to nucleotide‐stimulated Cl− secretion was determined using the channel blocker Ani9 in short‐circuit current recordings obtained from primary cultures of rat tracheal epithelial cells in Ussing chambers. We found that ANO1, CFTR and AQP5 proteins were expressed in nonciliated cells of the tracheal epithelium, whereas ENaC was expressed in ciliated cells. Among nonciliated cells, ANO1 occurred together with CFTR and Muc5b and, in addition, in a different cell type without CFTR and Muc5b. Bioelectrical studies with the ANO1‐blocker Ani9 indicated that ANO1 mediated the secretory response to the nucleotide uridine‐5′‐triphosphate. Our data demonstrate that, in rat tracheal epithelium, Cl− secretion and Na+ absorption are routed through different cell types, and that ANO1 channels form the molecular basis of Ca2+‐dependent Cl− secretion in this tissue. These characteristic features of Cl−‐dependent secretion reveal similarities and distinct differences to secretory processes in human airways.

Label-free LC-MS/MS shotgun proteomics to investigate the anti-inflammatory effect of rCC16.

Clara cell protein (CC16) is an anti-inflammatory protein, which is expressed in the airway epithelium. It is involved in the development of airway inflammatory diseases, including chronic obstructive pulmonary disease and asthma. However, the exact molecular mechanism underlying its anti-inflammatory action remains to be fully elucidated. The aim of the present study was to define the protein profiles of the anti-inflammatory effect of CC16 in lipopolysaccharide (LPS)-treated rat tracheal epithelial (RTE) cells using shotgun proteomics. Protein extracts were obtained from control RTE cells, RTE cells treated with LPS and RTE cells treated with LPS and recombinant CC16 (rCC16). Subsequent label-free quantification and bioinformatics analyses identified 12 proteins that were differentially expressed in the three treatment groups as a cluster of five distinct groups according to their molecular functions. Five of the twelve proteins were revealed to be associated with the cytoskeleton: Matrix metalloproteinase-9, myosin heavy chain 10, actin-related protein-3 homolog, elongation factor 1-α-1 (EF-1-α-1), and acidic ribosomal phosphoprotein P0. Five of the twelve proteins were associated with cellular proliferation: DNA-dependent protein kinase catalytic subunit, EF-1-α-1, tyrosine 3-monooxygenase, caspase recruitment domain (CARD) protein 12 and adenosylhomocysteinase (SAHH) 3. Three proteins were associated with gene regulation: EF-1-α-1, SAHH 3 and acidic ribosomal phosphoprotein P0. Three proteins were associated with inflammation: Tyrosine 3-monooxygenase, CARD protein 12 and statin-related protein. ATPase (H+-transporting, V1 subunit A, isoform 1) was revealed to be associated with energy metabolism, and uridine diphosphate glycosyltransferase 1 family polypeptide A8 with drug metabolism and detoxification. The identified proteins were further validated using reverse transcription-quantitative polymerase chain reaction. These protein profiles, and their interacting protein network, may facilitate the elucidation of the molecular mechanisms underlying the anti-inflammatory effects of CC16.

Human and rat epithelial cells inactivate extracellular influenza A virus via H2O2-dependent hypothiocyanite

Abstract Influenza A virus (IAV) causes ongoing epidemics characterized by high morbidity and variable mortality in several species including humans and swine. Current intervention strategies incorporate either vaccines with several strains of IAV or antiviral drugs against which the virus is developing resistance. Identifying novel anti-influenza mechanisms of the respiratory innate immune system is a potential source of influenza therapies. The first line of defense against respiratory pathogens is the respiratory epithelium. One of the innate defense mechanisms respiratory epithelial cells use to prevent infection with pathogens such as IAV is an extracellular oxidative burst at the airway surface mediated by lactoperoxidase (LPO), thiocyanate anion (SCN−), and hydrogen peroxide (H2O2) to produce antiviral hypothiocyanite ions. Cellular H2O2 is produced by two isoforms of the NADPH oxidase dual oxidase (DUOX) enzymes, DUOX1 and DUOX2. Our data has shown that differentiated, primary human or rat tracheal epithelial cells supplemented with LPO and SCN− are capable of inactivating IAV in vitro. This system has shown a 4–5 log reduction in viral titers that was eliminated by the addition of catalase (an H2O2 scavenger) demonstrating its H2O2 dependence. Additionally, we have shown that this system is capable of inactivating human-origin, swine-origin, and recombinant IAV strains. These results suggest that by modulating the DUOX system IAV infection could be prevented or at least modulated to improve viral killing thereby reducing virus shedding which could alter IAV infection dynamics within a population.

Application of glutathione to antagonize H2O2-induced oxidative stress in rat tracheal epithelial cells

With increasing industrialization, numerous air pollutants are generated. This research aimed to investigate the effects of inhalation of oxidative pollutants. H2O2 was used to simulate oxidative air pollutants, and glutathione, a reducing agent that is widely distributed in organisms, was used as an antagonist, to protect cells from oxidative stress. H2O2 was diluted using two gradients (0.05 mM, 0.20 mM, 0.80 mM, 3.20 mM and 0.05 mM, 0.10 mM, 0.15 mM, 0.20 mM) and GSH was dissolved at 20 μM. MTT, MDA, ROS, GSH, and TSLP were used as biomarkers to evaluate oxidative stress and possible resulting molecular events. A dose–response relationship was observed between H2O2 concentrations and the above-mentioned biomarkers. Glutathione significantly reduced levels of oxidative stress.

Hypothiocyanite produced by human and rat respiratory epithelial cells inactivates extracellular H1N2 influenza A virus.

Our aim was to study whether an extracellular, oxidative antimicrobial mechanism inherent to tracheal epithelial cells is capable of inactivating influenza H1N2 virus. Epithelial cells were isolated from tracheas of male Sprague-Dawley rats. Both primary human and rat tracheobronchial epithelial cells were differentiated in air-liquid interface cultures. A/swine/Illinois/02860/09 (swH1N2) influenza A virions were added to the apical side of airway cells for 1h in the presence or absence of lactoperoxidase or thiocyanate. Characterization of rat epithelial cells (morphology, Duox expression) occurred via western blotting, PCR, hydrogen peroxide production measurement and histology. The number of viable virions was determined by plaque assays. Statistical difference of the results was analyzed by ANOVA and Tukey's test. Our data show that rat tracheobronchial epithelial cells develop a differentiated, polarized monolayer with high transepithelial electrical resistance, mucin production and expression of dual oxidases. Influenza A virions are inactivated by human and rat epithelial cells via a dual oxidase-, lactoperoxidase- and thiocyanate-dependent mechanism. Differentiated air-liquid interface cultures of rat tracheal epithelial cells provide a novel model to study airway epithelium-influenza interactions. The dual oxidase/lactoperoxidase/thiocyanate extracellular oxidative system producing hypothiocyanite is a fast and potent anti-influenza mechanism inactivating H1N2 viruses prior to infection of the epithelium.

Candida glabrata induced infection of rat tracheal epithelial cells is mediated by TLR-2 induced activation of NF-κB

An increasing number of reports identified Candida glabrata (C. glabrata) as the important causative agent of invasive pulmonary fungal infection. However, little is known about immune responses to C. glabrata in rat tracheal epithelial cell (RTEC). Here, the effect of C. glabrata on RTEC and the role of TLR-2 and NF-κB in the immune response were investigated by treatment with TLR-2 siRNA and NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC), respectively. Our results showed that the knockdown of TLR-2 and pretreatment of PDTC led to inhibition of cell proliferation by C. glabrata, further enhanced cells in G0/G1 phases, and promoted C. glabrata -induced apoptosis. C. glabrata infection induced the expression or secretion of TLR-2, NF-κB, TNF-α, and IL-6, and its effect was inhibited by knockdown of TLR-2. Pretreatment with PDTC inhibited the C. glabrata -induced expression of TLR2, and also inhibited the expression of p65 subunit of NF-κB in the first 4 h. Although the expression of p65 subunit at 6 h was elevated compared to baseline, the C. glabrata -induced expression of TNF-α and IL-6 remained attenuated by PDTC pretreatment. Therefore, C. glabrata recognized the TLR-2 in rat tracheal epithelial cell (RTEC), and then activated the transcription factor NF-κB and further promoted the secretion of TNF-α and IL-6 to contribute to the immune response and inflammation.

Recombinant rat CC16 protein inhibits LPS-induced MMP-9 expression via NF-κB pathway in rat tracheal epithelial cells.

Clara cell protein (CC16) is a well-known anti-inflammatory protein secreted by the epithelial Clara cells of the airways. It is involved in the development of airway inflammatory diseases such as chronic obstructive pulmonary disease and asthma. Previous studies suggest that CC16 gene transfer suppresses expression of interleukin (IL)-8 in bronchial epithelial cells. However, its role in the function of these cells during inflammation is not well understood. In this study, we evaluated the effect of CC16 on the expression of matrix metalloproteinase (MMP)-9 in lipopolysaccharide (LPS)-stimulated rat tracheal epithelial cells and its underlying molecular mechanisms. We generated recombinant rat CC16 protein (rCC16) which was bioactive in inhibiting the activity of phospholipase A2. rCC16 inhibited LPS-induced MMP-9 expression at both mRNA and protein levels in a concentration-dependent (0-2 µg/mL) manner, as demonstrated by real time RT-PCR, ELISA, and zymography assays. Gene transcription and DNA binding studies demonstrated that rCC16 suppressed LPS-induced NF-κB activation and its binding of gene promoters as identified by luciferase reporter and gel mobility shift assays, respectively. Western blotting and immunofluorescence staining analyses further revealed that rCC16 concentration dependently inhibited the effects of LPS on nuclear increase and cytosol reduction of NF-κB, on the phosphorylation and reduction of NF-κB inhibitory IκBα, and on p38 MAPK-dependent NF-κB activation by phosphorylation at Ser276 of its p65 subunit. These data indicate that inhibition of LPS-mediated NF-κB activation by rCC16 involves both translocation- and phosphorylation-dependent signaling pathways. When the tracheal epithelial cells were pretreated with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, cellular uptake of rCC16 and its inhibition of LPS-induced NF-κB nuclear translocation and also MMP-9 production were significantly abolished. Taken together, our data suggest that clathrin-mediated uptake of rCC16 suppresses LPS-mediated inflammatory MMP-9 production through inactivation of NF-κB and p38 MAPK pathways in tracheal epithelial cells.