scholarly journals Paracellular Pathway-Mediated Mycoplasma hyopneumoniae Migration across Porcine Airway Epithelial Barrier under Air-Liquid Interface Conditions

2020 ◽  
Vol 88 (10) ◽  
Author(s):  
Haiyan Wang ◽  
Zhenzhen Zhang ◽  
Xing Xie ◽  
Beibei Liu ◽  
Yanna Wei ◽  
...  
Keyword(s):  
Mycoplasma Hyopneumoniae ◽  
Liquid Interface ◽  
Respiratory Pathogen ◽  
Epithelial Barrier ◽  
Airway Epithelial ◽  
Migration Ability ◽  
Content Type ◽  
Air Liquid Interface

ABSTRACT Mycoplasma hyopneumoniae is an important respiratory pathogen of pigs that causes persistent and secondary infections. However, the mechanisms by which this occurs are unclear. In this study, we established air-liquid interface culture systems for pig bronchial epithelial cells (ALI-PBECs) that were comparable to the conditions in the native bronchus in vivo. We used this ALI-PBECs model to study the infection and migration characteristics of M. hyopneumoniae in vitro. Based on the results, we confirmed that M. hyopneumoniae was able to adhere to ALI-PBECs and disrupt mucociliary function. Importantly, M. hyopneumoniae could migrate to the basolateral chamber through the paracellular route but not the transcellular pathway, and this was achieved by reversibly disrupting tight junctions (TJs) and increasing the permeability and damaging the integrity of the epithelial barrier. We examined the migration ability of M. hyopneumoniae using an ALI-PBECs model for the first time. The disruption of the epithelial barrier allowed M. hyopneumoniae to migrate to the basolateral chamber through the paracellular route, which may be related to immune evasion, extrapulmonary dissemination, and persistent infection of M. hyopneumoniae.

scholarly journals The CULTEX RFS: A Comprehensive Technical Approach for theIn VitroExposure of Airway Epithelial Cells to the Particulate Matter at the Air-Liquid Interface

2013 ◽  
Vol 2013 ◽  
pp. 1-15 ◽  
Author(s):  
Michaela Aufderheide ◽  
Beat Halter ◽  
Niklas Möhle ◽  
Dieter Hochrainer
Keyword(s):  
A549 Cells ◽  
Airway Epithelial Cells ◽  
Liquid Interface ◽  
Alternative Methods ◽  
Airway Epithelial ◽  
Nanosized Particles ◽  
Dose Dependent Decrease ◽  
Air Liquid Interface

The EU Regulation on Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) demands the implementation of alternative methods for analyzing the hazardous effects of chemicals including particulate formulations. In the field of inhalation toxicology, a variety ofin vitromodels have been developed for such studies. To simulate thein vivosituation, an adequate exposure device is necessary for the direct exposure of cultivated lung cells at the air-liquid interface (ALI). The CULTEX RFS fulfills these requirements and has been optimized for the exposure of cells to atomized suspensions, gases, and volatile compounds as well as micro- and nanosized particles. This study provides information on the construction and functional aspects of the exposure device. By using the Computational Fluid Dynamics (CFD) analysis, the technical design was optimized to realize a stable, reproducible, and homogeneous deposition of particles. The efficiency of the exposure procedure is demonstrated by exposing A549 cells dose dependently to lactose monohydrate, copper(II) sulfate, copper(II) oxide, and micro- and nanoparticles. All copper compounds induced cytotoxic effects, most pronounced for soluble copper(II) sulfate. Micro- and nanosized copper(II) oxide also showed a dose-dependent decrease in the cell viability, whereby the nanosized particles decreased the metabolic activity of the cells more severely.

Passive siRNA transfection method for gene knockdown in air-liquid interface airway epithelial cell cultures

2021 ◽  
Author(s):  
Colleen M Bartman ◽  
Kimberly E Stelzig ◽  
David R Linden ◽  
Y. S. Prakash ◽  
Sergio E Chiarella
Keyword(s):  
Epithelial Cell ◽  
Liquid Interface ◽  
Airway Epithelial ◽  
Loss Of Function ◽  
Sirna Transfection ◽  
Simple Method ◽  
Primary Epithelial Cell ◽  
Transfection Protocol ◽  
Air Liquid Interface

Differentiation of human bronchial epithelial cells (HBEs) in air-liquid interface (ALI) cultures recapitulates organotypic modeling of the in vivo environment. Although ALI cultures are invaluable for studying the respiratory epithelial barrier, loss-of-function studies are limited by potentially cytotoxic reagents in classical transfection methods, the length of the differentiation protocol, and the number of primary epithelial cell passages. Here, we present the efficacy and utility of a simple method for siRNA transfection of HBEs in ALI cultures that does not require potentially cytotoxic transfection reagents and does not detrimentally alter the physiology of HBEs during the differentiation process. This transfection protocol introduces a reproducible and efficient method for loss-of-function studies in HBE ALI cultures that can be leveraged for modeling the respiratory system and airway diseases.

scholarly journals The role of secreted Hsp90α in HDM-induced asthmatic airway epithelial barrier dysfunction

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Cuiping Ye ◽  
Chaowen Huang ◽  
Mengchen Zou ◽  
Yahui Hu ◽  
Lishan Luo ◽  
...  
Keyword(s):  
Barrier Function ◽  
Epithelial Barrier ◽  
Airway Epithelial ◽  
Barrier Dysfunction ◽  
Epithelial Barrier Function ◽  
Asthmatic Airway ◽  
Epithelial Barrier Dysfunction

Abstract Background The dysfunction of airway epithelial barrier is closely related to the pathogenesis of asthma. Secreted Hsp90α participates in inflammation and Hsp90 inhibitor protects endothelial dysfunction. In the current study, we aimed to explore the role of secreted Hsp90α in asthmatic airway epithelial barrier function. Methods Male BALB/c mice were sensitized and challenged with HDM to generate asthma model. The 16HBE and Hsp90α-knockdown cells were cultured and treated according to the experiment requirements. Transepithelial Electric Resistance (TEER) and permeability of epithelial layer in vitro, distribution and expression of junction proteins both in vivo and in vitro were used to evaluate the epithelial barrier function. Western Blot was used to evaluate the expression of junction proteins and phosphorylated AKT in cells and lung tissues while ELISA were used to evaluate the Hsp90α expression and cytokines release in the lung homogenate. Results HDM resulted in a dysfunction of airway epithelial barrier both in vivo and in vitro, paralleled with the increased expression and release of Hsp90α. All of which were rescued in Hsp90α-knockdown cells or co-administration of 1G6-D7. Furthermore, either 1G6-D7 or PI3K inhibitor LY294002 suppressed the significant phosphorylation of AKT, which caused by secreted and recombinant Hsp90α, resulting in the restoration of epithelial barrier function. Conclusions Secreted Hsp90α medicates HDM-induced asthmatic airway epithelial barrier dysfunction via PI3K/AKT pathway, indicating that anti-secreted Hsp90α therapy might be a potential treatment to asthma in future.

scholarly journals Redundant Catalases Detoxify Phagocyte Reactive Oxygen and Facilitate Histoplasma capsulatum Pathogenesis

2013 ◽  
Vol 81 (7) ◽  
pp. 2334-2346 ◽  
Author(s):  
Eric D. Holbrook ◽  
Katherine A. Smolnycki ◽  
Brian H. Youseff ◽  
Chad A. Rappleye
Keyword(s):  
Immune System ◽  
Innate Immune System ◽  
Histoplasma Capsulatum ◽  
Reactive Oxygen ◽  
Innate Immune ◽  
Respiratory Pathogen ◽  
Human Neutrophils ◽  
Content Type

ABSTRACTHistoplasma capsulatumis a respiratory pathogen that infects phagocytic cells. The mechanisms allowingHistoplasmato overcome toxic reactive oxygen molecules produced by the innate immune system are an integral part ofHistoplasma's ability to survive during infection. To probe the contribution ofHistoplasmacatalases in oxidative stress defense, we created and analyzed the virulence defects of mutants lacking CatB and CatP, which are responsible for extracellular and intracellular catalase activities, respectively. Both CatB and CatP protectedHistoplasmafrom peroxide challengein vitroand from antimicrobial reactive oxygen produced by human neutrophils and activated macrophages. Optimal protection required both catalases, as the survival of a double mutant lacking both CatB and CatP was lower than that of single-catalase-deficient cells. Although CatB contributed to reactive oxygen species defensesin vitro, CatB was dispensable for lung infection and extrapulmonary disseminationin vivo. Loss of CatB from a strain also lacking superoxide dismutase (Sod3) did not further reduce the survival ofHistoplasmayeasts. Nevertheless, some catalase function was required for pathogenesis since simultaneous loss of both CatB and CatP attenuatedHistoplasmavirulencein vivo. These results demonstrate thatHistoplasma's dual catalases comprise a system that enablesHistoplasmato efficiently overcome the reactive oxygen produced by the innate immune system.

scholarly journals Filamentous Bacteriophage Produced by Pseudomonas aeruginosa Alters the Inflammatory Response and Promotes Noninvasive Infection In Vivo

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
Patrick R. Secor ◽  
Lia A. Michaels ◽  
Kate S. Smigiel ◽  
Maryam G. Rohani ◽  
Laura K. Jennings ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Inflammatory Response ◽  
Chronic Infection ◽  
Bacterial Invasion ◽  
Airway Epithelial ◽  
Content Type ◽  
Epithelial Cultures ◽  
Opportunistic Human Pathogen

ABSTRACT Pseudomonas aeruginosa is an important opportunistic human pathogen that lives in biofilm-like cell aggregates at sites of chronic infection, such as those that occur in the lungs of patients with cystic fibrosis and nonhealing ulcers. During growth in a biofilm, P. aeruginosa dramatically increases the production of filamentous Pf bacteriophage (Pf phage). Previous work indicated that when in vivo Pf phage production was inhibited, P. aeruginosa was less virulent. However, it is not clear how the production of abundant quantities of Pf phage similar to those produced by biofilms under in vitro conditions affects pathogenesis. Here, using a murine pneumonia model, we show that the production of biofilm-relevant amounts of Pf phage prevents the dissemination of P. aeruginosa from the lung. Furthermore, filamentous phage promoted bacterial adhesion to mucin and inhibited bacterial invasion of airway epithelial cultures, suggesting that Pf phage traps P. aeruginosa within the lung. The in vivo production of Pf phage was also associated with reduced lung injury, reduced neutrophil recruitment, and lower cytokine levels. Additionally, when producing Pf phage, P. aeruginosa was less prone to phagocytosis by macrophages than bacteria not producing Pf phage. Collectively, these data suggest that filamentous Pf phage alters the progression of the inflammatory response and promotes phenotypes typically associated with chronic infection.

scholarly journals Parametric Optimization of an Air–Liquid Interface System for Flow-Through Inhalation Exposure to Nanoparticles: Assessing Dosimetry and Intracellular Uptake of CeO2 Nanoparticles

Nanomaterials ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2369 ◽  
Author(s):  
Lars Leibrock ◽  
Harald Jungnickel ◽  
Jutta Tentschert ◽  
Aaron Katz ◽  
Blaza Toman ◽  
...  
Keyword(s):  
Co2 Concentration ◽  
Liquid Interface ◽  
In Vivo Studies ◽  
Inhalation Toxicity ◽  
Exposure System ◽  
Inhalation Study ◽  
Flow Through ◽  
Air Liquid Interface

Air–liquid interface (ALI) systems have been widely used in recent years to investigate the inhalation toxicity of many gaseous compounds, chemicals, and nanomaterials and represent an emerging and promising in vitro method to supplement in vivo studies. ALI exposure reflects the physiological conditions of the deep lung more closely to subacute in vivo inhalation scenarios compared to submerged exposure. The comparability of the toxicological results obtained from in vivo and in vitro inhalation data is still challenging. The robustness of ALI exposure scenarios is not yet well understood, but critical for the potential standardization of these methods. We report a cause-and-effect (C&E) analysis of a flow through ALI exposure system. The influence of five different instrumental and physiological parameters affecting cell viability and exposure parameters of a human lung cell line in vitro (exposure duration, relative humidity, temperature, CO2 concentration and flow rate) was investigated. After exposing lung epithelia cells to a CeO2 nanoparticle (NP) aerosol, intracellular CeO2 concentrations reached values similar to those found in a recent subacute rat inhalation study in vivo. This is the first study showing that the NP concentration reached in vitro using a flow through ALI system were the same as those in an in vivo study.

scholarly journals HDM-induced Atg5 Mediates TSLP Expressing to Disrupt Epithelial Barrier

2021 ◽  
Author(s):  
Changhui Yu ◽  
Zicong Zhou ◽  
Shixiu Liang ◽  
Zili Zhou ◽  
Jieyi Liu ◽  
...  
Keyword(s):  
Epithelial Barrier ◽  
Dependent Manner ◽  
Airway Epithelial ◽  
Mice Model ◽  
Rt Pcr ◽  
Intracellular Process ◽  
Self Protection ◽  
One Way Anova

Abstract Background: Asthma is a complex and heterogeneous disease. Autophagy, process of self-protection in cells, is an intracellular process when cells are being attacked by certain stress. Our team focused upon the disruption of airways epithelial barrier in asthma, and we interested in whether autophagy played a key role in asthma. Methods: 400U HDM was used to treat HBECs and established asthmatic mice model. Western blotting, RT-PCR and immunofluorescence were mainly used to detect autophagy process in vivo and in vitro. One way ANOVA and Mann Whitney test were used for statistic. Results: After treated with HDM, expression of LC3ab increased in vivo and in vitro. Using Rapamycin, 3-MA and Chloroquine to treat HBECs, then we surprisingly found that HDM disrupts epithelial barrier through incomplete autophagy. To find out the connection between asthma and autophagy, we chose known autophagy related genes to determine the association between autophagy and disruption of airway epithelial barrier. Atg5 and atg12 were chosen because these two genes varied upon the time dependent manner. Knocked down the expression of atg5 or atg12 by siRNA, the expression of TSLP, which can induce the disruption of airway epithelial barrier, remarkably reduced. Conclusions: These results demonstrated that HDM induced inflammatory in airway epithelium through autophagy, and then knocked down autophagy related genes alleviated the inflammatory in HBECs.

scholarly journals Disruption of the airway epithelial barrier in a murine model of respiratory syncytial virus infection

2019 ◽  
Vol 316 (2) ◽  
pp. L358-L368 ◽  
Author(s):  
Carrie C. Smallcombe ◽  
Debra T. Linfield ◽  
Terri J. Harford ◽  
Vladimir Bokun ◽  
Andrei I. Ivanov ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Epithelial Cells ◽  
Tight Junction ◽  
Epithelial Barrier ◽  
Airway Epithelial ◽  
Barrier Dysfunction ◽  
Rsv Infection ◽  
Syncytial Virus

Respiratory syncytial virus (RSV) is a major cause of hospitalization for infants and young children worldwide. RSV is known to infect epithelial cells and increase the permeability of model airway epithelial monolayers in vitro. We hypothesized that RSV infection also induces airway barrier dysfunction in vivo. C57BL/6 mice were intranasally inoculated with RSV, and on day 4 post-inoculation were examined for viral replication, lung inflammation, and barrier integrity as well as the structure and molecular composition of epithelial junctions. In parallel, primary mouse tracheal epithelial cells (mTEC) were cultured for in vitro studies. RSV-infected mice lost weight and showed significant peribronchial inflammation compared with noninfected controls and UV-inactivated RSV-inoculated animals. RSV infection increased the permeability of the airway epithelial barrier and altered the molecular composition of epithelial tight junctions. The observed RSV-induced barrier disruption was accompanied by decreased expression of several tight-junction proteins and accumulation of cleaved extracellular fragments of E-cadherin in bronchoalveolar lavage and mTEC supernatants. Similarly, in vitro RSV infection of mTEC monolayers resulted in enhanced permeability and disruption of tight-junction structure. Furthermore, incubation of mTEC monolayers with a recombinant fragment of E-cadherin caused tight-junction disassembly. Taken together, these data indicate that RSV infection leads to airway barrier dysfunction in vivo, mediated by either decreased expression or cleavage of junctional proteins. Our observations provide further insights into the pathophysiology of RSV infection and provide a rationale for development of barrier-protecting agents to alleviate the pathogenic effects of RSV infection.

scholarly journals Causes and Biophysical Consequences of Cellulose Production byPseudomonas fluorescensSBW25 at the Air-Liquid Interface

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Maxime Ardré ◽  
Djinthana Dufour ◽  
Paul B. Rainey
Keyword(s):  
Liquid Interface ◽  
Water Soluble ◽  
Diguanylate Cyclase ◽  
Content Type ◽  
Environmental Signals ◽  
Fluid Instabilities ◽  
Rayleigh Taylor Instability ◽  
Air Liquid Interface ◽  
Surface Production

ABSTRACTCellulose-overproducing wrinkly spreader mutants ofPseudomonas fluorescensSBW25 have been the focus of much investigation, but conditions promoting the production of cellulose in ancestral strain SBW25 and its effects and consequences have escaped in-depth investigation through lack of anin vitrophenotype. Here, using a custom-built device, we reveal that in static broth microcosms, ancestral SBW25 encounters environmental signals at the air-liquid interface that activate, via three diguanylate cyclase-encoding pathways (Wsp, Aws, and Mws), production of cellulose. Secretion of the polymer at the meniscus leads to modification of the environment and growth of numerous microcolonies that extend from the surface. Accumulation of cellulose and associated microbial growth leads to Rayleigh-Taylor instability resulting in bioconvection and rapid transport of water-soluble products over tens of millimeters. Drawing upon data, we built a mathematical model that recapitulates experimental results and captures the interactions between biological, chemical and physical processes.IMPORTANCEThis work reveals a hitherto unrecognized behavior that manifests at the air-liquid interface that depends on production of cellulose and hints at undiscovered dimensions to bacterial life at surfaces. Additionally, the study links activation of known diguanylate cyclase-encoding pathways to cellulose expression and to signals encountered at the meniscus. Further significance stems from recognition of the consequences of fluid instabilities arising from surface production of cellulose for transport of water-soluble products over large distances.

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