scholarly journals Bioengineered airway epithelial grafts with mucociliary function based on collagen IV- and laminin-containing extracellular matrix scaffolds

2020 ◽  
Vol 55 (6) ◽  
pp. 1901200 ◽  
Author(s):  
Nick J.I. Hamilton ◽  
Dani Do Hyang Lee ◽  
Kate H.C. Gowers ◽  
Colin R. Butler ◽  
Elizabeth F. Maughan ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Airway Epithelium ◽  
Airway Epithelial Cell ◽  
Collagen Iv ◽  
Airway Epithelial ◽  
Cell Adherence ◽  
Bronchial Epithelial ◽  
Mucociliary Function

Current methods to replace damaged upper airway epithelium with exogenous cells are limited. Existing strategies use grafts that lack mucociliary function, leading to infection and the retention of secretions and keratin debris. Strategies that regenerate airway epithelium with mucociliary function are clearly desirable and would enable new treatments for complex airway disease.Here, we investigated the influence of the extracellular matrix (ECM) on airway epithelial cell adherence, proliferation and mucociliary function in the context of bioengineered mucosal grafts. In vitro, primary human bronchial epithelial cells (HBECs) adhered most readily to collagen IV. Biological, biomimetic and synthetic scaffolds were compared in terms of their ECM protein content and airway epithelial cell adherence.Collagen IV and laminin were preserved on the surface of decellularised dermis and epithelial cell attachment to decellularised dermis was greater than to the biomimetic or synthetic alternatives tested. Blocking epithelial integrin α2 led to decreased adherence to collagen IV and to decellularised dermis scaffolds. At air–liquid interface (ALI), bronchial epithelial cells cultured on decellularised dermis scaffolds formed a differentiated respiratory epithelium with mucociliary function. Using in vivo chick chorioallantoic membrane (CAM), rabbit airway and immunocompromised mouse models, we showed short-term preservation of the cell layer following transplantation.Our results demonstrate the feasibility of generating HBEC grafts on clinically applicable decellularised dermis scaffolds and identify matrix proteins and integrins important for this process. The long-term survivability of pre-differentiated epithelia and the relative merits of this approach against transplanting basal cells should be assessed further in pre-clinical airway transplantation models.

scholarly journals Multiple TLRs activate EGFR via a signaling cascade to produce innate immune responses in airway epithelium

2008 ◽  
Vol 294 (6) ◽  
pp. L1068-L1075 ◽  
Author(s):  
Jonathan L. Koff ◽  
Matt X. G. Shao ◽  
Iris F. Ueki ◽  
Jay A. Nadel
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Immune Responses ◽  
Nadph Oxidase ◽  
Airway Epithelium ◽  
Airway Epithelial Cell ◽  
Innate Immune ◽  
Signaling Cascade ◽  
Airway Epithelial ◽  
Innate Immune Responses

Toll-like receptors (TLRs) are critical for the recognition of inhaled pathogens that deposit on the airway epithelial surface. The epithelial response to pathogens includes signaling cascades that activate the EGF receptor (EGFR). We hypothesized that TLRs communicate with EGFR via epithelial signaling to produce certain innate immune responses. Airway epithelium expresses the highest levels of TLR2, TLR3, TLR5, and TLR6, and here we found that ligands for these TLRs increased IL-8 and VEGF production in normal human bronchial epithelial cells. These effects were prevented by treatment with a selective inhibitor of EGFR phosphorylation (AG-1478), a metalloprotease (MP) inhibitor, a reactive oxygen species (ROS) scavenger, and an NADPH oxidase inhibitor. In an airway epithelial cell line (NCI-H292), TNF-α-converting enzyme (TACE) small interfering RNA (siRNA) was used to confirm that TACE is the MP involved in TLR ligand-induced IL-8 and VEGF production. We show that transforming growth factor (TGF)-α is the EGFR ligand in this signaling cascade by using TGF-α neutralizing antibody and by showing that epithelial production of TGF-α occurs in response to TLR ligands. Dual oxidase 1 (Duox1) siRNA was used to confirm that Duox1 is the NADPH oxidase involved in TLR ligand-induced IL-8 and VEGF production. We conclude that multiple TLR ligands induce airway epithelial cell production of IL-8 and VEGF via a Duox1→ ROS→ TACE→ TGF-α→ EGFR phosphorylation pathway. These results show for the first time that multiple TLRs in airway epithelial cells produce innate immune responses by activating EGFR via an epithelial cell signaling cascade.

Noncanonical role for Ku70/80 in the prevention of allergic airway inflammation via maintenance of airway epithelial cell organelle homeostasis

2020 ◽  
Vol 319 (4) ◽  
pp. L728-L741
Author(s):  
Rakhshinda Rehman ◽  
Vijay Elakkya Vijayakumar ◽  
Ashish Jaiswal ◽  
Vaibhav Jain ◽  
Shravani Mukherjee ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Er Stress ◽  
Airway Inflammation ◽  
Allergic Airway Inflammation ◽  
Cell Organelle ◽  
Continuous Exposure ◽  
Airway Epithelial ◽  
Bronchial Epithelial ◽  
Ku Proteins

Airway epithelial homeostasis is under constant threat due to continuous exposure to the external environment, and abnormally robust sensitivity to external stimuli is critical to the development of airway diseases, including asthma. Ku is a key nonhomologous end-joining DNA repair protein with diverse cellular functions such as VDJ recombination and telomere length maintenance. Here, we show a novel function of Ku in alleviating features of allergic airway inflammation via the regulation of mitochondrial and endoplasmic reticulum (ER) stress. We first determined that airway epithelial cells derived from both asthmatic lungs and murine asthma models demonstrate increased expression of 8-hydroxy-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage. Ku protein expression was dramatically reduced in the bronchial epithelium of patients with asthma as well as in human bronchial epithelial cells exposed to oxidative stress. Knockdown of Ku70 or Ku80 in naïve mice elicited mitochondrial collapse or ER stress, leading to bronchial epithelial cell apoptosis and spontaneous development of asthma-like features, including airway hyperresponsiveness, airway inflammation, and subepithelial fibrosis. These findings demonstrate an essential noncanonical role for Ku proteins in asthma pathogenesis, likely via maintenance of organelle homeostasis. This novel function of Ku proteins may also be important in other disease processes associated with organelle stress.

scholarly journals FOXO3a regulates rhinovirus-induced innate immune responses in airway epithelial cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Joao Gimenes-Junior ◽  
Nicole Owuar ◽  
Hymavathi Reddy Vari ◽  
Wuyan Li ◽  
Nathaniel Xander ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Lung Inflammation ◽  
Inflammatory Cytokine ◽  
Airway Epithelial Cell ◽  
Critical Role ◽  
Airway Epithelial Cells ◽  
Type I ◽  
Airway Epithelial ◽  
Antiviral Responses

AbstractForkhead transcription factor class O (FOXO)3a, which plays a critical role in a wide variety of cellular processes, was also found to regulate cell-type-specific antiviral responses. Airway epithelial cells express FOXO3a and play an important role in clearing rhinovirus (RV) by mounting antiviral type I and type III interferon (IFN) responses. To elucidate the role of FOXO3a in regulating antiviral responses, we generated airway epithelial cell-specific Foxo3a knockout (Scga1b1-Foxo3a−/−) mice and a stable FOXO3a knockout human airway epithelial cell line. Compared to wild-type, Scga1b1-Foxo3a−/− mice show reduced IFN-α, IFN-β, IFN-λ2/3 in response to challenge with RV or double-stranded (ds)RNA mimic, Poly Inosinic-polycytidylic acid (Poly I:C) indicating defective dsRNA receptor signaling. RV-infected Scga1b1-Foxo3a−/− mice also show viral persistence, enhanced lung inflammation and elevated pro-inflammatory cytokine levels. FOXO3a K/O airway epithelial cells show attenuated IFN responses to RV infection and this was associated with conformational change in mitochondrial antiviral signaling protein (MAVS) but not with a reduction in the expression of dsRNA receptors under unstimulated conditions. Pretreatment with MitoTEMPO, a mitochondrial-specific antioxidant corrects MAVS conformation and restores antiviral IFN responses to subsequent RV infection in FOXO3a K/O cells. Inhibition of oxidative stress also reduces pro-inflammatory cytokine responses to RV in FOXO3a K/O cells. Together, our results indicate that FOXO3a plays a critical role in regulating antiviral responses as well as limiting pro-inflammatory cytokine expression. Based on these results, we conclude that FOXO3a contributes to optimal viral clearance and prevents excessive lung inflammation following RV infection.

Effects of corticosteroid-induced apoptosis on airway epithelial wound closure in vitro

2006 ◽  
Vol 291 (4) ◽  
pp. L794-L801 ◽  
Author(s):  
Delbert R. Dorscheid ◽  
Benjamin J. Patchell ◽  
Oscar Estrada ◽  
Bertha Marroquin ◽  
Roberta Tse ◽  
...  
Keyword(s):  
Cell Migration ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Untreated Control ◽  
Wound Closure ◽  
Airway Epithelial Cell ◽  
Airway Epithelial ◽  
Wound Area ◽  
Epithelial Cell Migration

Damage to the airway epithelium is common in asthma. Corticosteroids induce apoptosis in and suppress proliferation of airway epithelial cells in culture. Whether apoptosis contributes to impaired epithelial cell repair after injury is not known. We examined whether corticosteroids would impair epithelial cell migration in an in vitro model of wound closure. Wounds (∼0.5–1.3 mm2) were created in cultured 1HAEo−human airway epithelial cell monolayers, after which cells were treated with up to 10 μM dexamethasone or budesonide for 24 h. Cultured cells were pretreated for 24 or 48 h with dexamethasone to observe the effect of long-term exposure on wound closure. After 12 h, the remaining wound area in monolayers pretreated for 48 h with 10 μM dexamethasone was 43 ± 18% vs. 10 ± 8% for untreated control monolayers. The addition of either corticosteroid immediately after injury did not slow closure significantly. After 12 h the remaining wound area in monolayers treated with 10 μM budesonide was 39 ± 4% vs. 43 ± 3% for untreated control monolayers. The proportion of apoptotic epithelial cells as measured by terminal deoxynucleotidyltransferase-mediated dUTP biotin nick end labeling both at and away from the wound edge was higher in monolayers treated with budesonide compared with controls. However, wound closure in the apoptosis-resistant 1HAEo−.Bcl-2+cell line was not different after dexamethasone treatment. We demonstrate that corticosteroid treatment before mechanical wounding impairs airway epithelial cell migration. The addition of corticosteroids after injury does not slow migration, despite their ability to induce apoptosis in these cells.

scholarly journals Prostasin, a membrane-anchored serine peptidase, regulates sodium currents in JME/CF15 cells, a cystic fibrosis airway epithelial cell line

2004 ◽  
Vol 287 (5) ◽  
pp. L928-L935 ◽  
Author(s):  
Zhenyue Tong ◽  
Beate Illek ◽  
Vikash J. Bhagwandin ◽  
George M. Verghese ◽  
George H. Caughey
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Airway Epithelial Cell ◽  
Epithelial Cell Line ◽  
Airway Epithelial ◽  
Ussing Chambers ◽  
Serine Peptidase ◽  
Cystic Fibrosis Airway ◽  
Interfering Rna

Prostasin is a tryptic peptidase expressed in prostate, kidney, lung, and airway. Mammalian prostasins are related to Xenopus channel-activating protease, which stimulates epithelial Na+ channel (ENaC) activity in frogs. In human epithelia, prostasin is one of several membrane peptidases proposed to regulate ENaC. This study tests the hypothesis that prostasin can regulate ENaC in cystic fibrosis epithelia in which excessive Na+ uptake contributes to salt and water imbalance. We show that prostasin mRNA and protein are strongly expressed by human airway epithelial cell lines, including immortalized JME/CF15 nasal epithelial cells homozygous for the ΔF508 cystic fibrosis mutation. Epithelial cells transfected with vectors encoding recombinant soluble prostasin secrete active, tryptic peptidase that is highly sensitive to inactivation by aprotinin. When studied as monolayers in Ussing chambers, JME/CF15 cells exhibit amiloride-sensitive, transepithelial Na+ currents that are markedly diminished by aprotinin, suggesting regulation by serine-class peptidases. Overproduction of membrane-anchored prostasin in transfected JME/CF15 cells does not augment Na+ currents, and trypsin-induced increases are small, suggesting that baseline serine peptidase-dependent ENaC activation is maximal in these cells. To probe prostasin’s involvement in basal ENaC activity, we silenced expression of prostasin using short interfering RNA targeting of prostasin mRNA’s 3′-untranslated region. This drops ENaC currents to 26 ± 9% of baseline. These data predict that prostasin is a major regulator of ENaC-mediated Na+ current in ΔF508 cystic fibrosis epithelia and suggest that airway prostasin is a target for therapeutic inhibition to normalize ion current in cystic fibrosis airway.

Paraoxonase-2 deficiency enhancesPseudomonas aeruginosaquorum sensing in murine tracheal epithelia

2007 ◽  
Vol 292 (4) ◽  
pp. L852-L860 ◽  
Author(s):  
David A. Stoltz ◽  
Egon A. Ozer ◽  
Carey J. Ng ◽  
Janet M. Yu ◽  
Srinivasa T. Reddy ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Quorum Sensing ◽  
Airway Epithelial Cell ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Airway Epithelia ◽  
Tracheal Epithelial Cells ◽  
Human Airway ◽  
Tracheal Epithelial

Pseudomonas aeruginosa is an important cause of nosocomial infections and is frequently present in the airways of cystic fibrosis patients. Quorum sensing mediates P. aeruginosa's virulence and biofilm formation through density-dependent interbacterial signaling with autoinducers. N-3-oxododecanoyl homoserine lactone (3OC12-HSL) is the major autoinducer in P. aeruginosa. We have previously shown that human airway epithelia and paraoxonases (PONs) degrade 3OC12-HSL. This study investigated the role of PON1, PON2, and PON3 in airway epithelial cell inactivation of 3OC12-HSL. All three PONs were present in murine tracheal epithelial cells, with PON2 and PON3 expressed at the highest levels. Lysates of tracheal epithelial cells from PON2, but not PON1 or PON3, knockout mice had impaired 3OC12-HSL inactivation compared with wild-type mice. In contrast, PON1-, PON2-, or PON3-targeted deletions did not affect 3OC12-HSL degradation by intact epithelia. Overexpression of PON2 enhanced 3OC12-HSL degradation by human airway epithelial cell lysates but not by intact epithelia. Finally, using a quorum-sensing reporter strain of P. aeruginosa, we found that quorum sensing was enhanced in PON2-deficient airway epithelia. In summary, these results show that loss of PON2 impairs 3OC12-HSL degradation by airway epithelial cells and suggests that diffusion of 3OC12-HSL into the airway cells can be the rate-limiting step for degradation of the molecule.

Effect of inspired air conditions on exercise-induced bronchoconstriction and urinary CC16 levels in athletes

2011 ◽  
Vol 111 (4) ◽  
pp. 1059-1065 ◽  
Author(s):  
C. Bolger ◽  
E. Tufvesson ◽  
S. D. Anderson ◽  
G. Devereux ◽  
J. G. Ayres ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Relative Humidity ◽  
Airway Epithelium ◽  
Cell Injury ◽  
Airway Epithelial Cell ◽  
Airway Epithelial ◽  
Maximal Aerobic Capacity ◽  
Humid Air ◽  
Exercise Tests ◽  
Exercise Induced

Injury to the airway epithelium has been proposed as a key susceptibility factor for exercise-induced bronchoconstriction (EIB). Our goals were to establish whether airway epithelial cell injury occurs during EIB in athletes and whether inhalation of warm humid air inhibits this injury. Twenty-one young male athletes (10 with a history of EIB) performed two 8-min exercise tests near maximal aerobic capacity in cold dry (4°C, 37% relative humidity) and warm humid (25°C, 94% relative humidity) air on separate days. Postexercise changes in urinary CC16 were used as a biomarker of airway epithelial cell perturbation and injury. Bronchoconstriction occurred in eight athletes in the cold dry environment and was completely blocked by inhalation of warm humid air [maximal fall in forced expiratory volume in 1 s = 18.1 ± 2.1% (SD) in cold dry air and 1.7 ± 0.8% in warm humid air, P < 0.01]. Exercise caused an increase in urinary excretion of CC16 in all subjects ( P < 0.001), but this rise in CC16 was blunted following inhalation of warm humid air [median CC16 increase pre- to postchallenge = 1.91 and 0.35 ng/μmol in cold dry and warm humid air, respectively, in athletes with EIB ( P = 0.017) and 1.68 and 0.48 ng/μmol in cold dry and warm humid air, respectively, in athletes without EIB ( P = 0.002)]. The results indicate that exercise hyperpnea transiently disrupts the airway epithelium of all athletes (not only in those with EIB) and that inhalation of warm moist air limits airway epithelial cell perturbation and injury.

scholarly journals The antimicrobial peptide S100A8/A9 produced by airway epithelium functions as a potent and direct regulator of macrophage phenotype and function

2021 ◽  
pp. 2002732
Author(s):  
Wioletta Skronska-Wasek ◽  
Sibel Durlanik ◽  
Huy Quang Le ◽  
Victoria Schroeder ◽  
Kerstin Kitt ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Airway Epithelium ◽  
Airway Epithelial Cell ◽  
Macrophage Phenotype ◽  
Airway Epithelial ◽  
Phagocytic Capacity ◽  
Macrophage Phagocytosis ◽  
Depth Analysis ◽  
And Function

Elevated counts of alveolar macrophages and attenuated phagocytic capacity are associated with COPD. Factors governing macrophage phagocytosis are poorly understood. In this study we aimed to compare the influence of airway epithelial cell secretions from COPD and nonCOPD subjects on macrophage phagocytic activity, and the role of antimicrobial peptides (AMPs).Supernatants from nonCOPD and COPD small airway epithelial cell (SAEC) cultures exposed to Haemophilus influenzae (NTHi), were applied to human monocyte-derived macrophages (MDM) for assessment of their influence on phagocytosis. SAECs were analysed for changes in AMP expression by qRT-PCR and the influence of select AMPs on macrophage phenotype and function was assessed by flow cytometry and metabolic activity assay.Secretions from the apical and basolateral surface of NTHi-exposed SAECs from nonCOPD donors elicited superior phagocytic capacity of MDMs. Moreover, NTHi exposure led to a rapid increase in expression of a range of AMPs by nonCOPD SAECs, but this response was delayed in COPD SAECs. We demonstrate that treatment with AMPs β-defensin 2 and S100A8/A9 improved the phagocytic capacity of MDMs. In depth analysis of S100A8/A9 influence of MDMs revealed a role of this AMP in macrophage phenotype and function. Furthermore, we show that the expression of S100A8 and S100A9 is directly regulated by WNT/β-catenin signalling activity, a known deregulated pathway in COPD.In conclusion, for the first time, we demonstrate that airway epithelium from COPD subjects has a reduced capacity to support the phagocytic function of macrophages in response to acute NTHi exposure, and we identify the WNT/β-catenin signalling-modulated and epithelium-derived S100A8/A9 as a potent regulator of macrophage phenotype and function.

Hog barn dust extract augments lymphocyte adhesion to human airway epithelial cells

2004 ◽  
Vol 96 (5) ◽  
pp. 1738-1744 ◽  
Author(s):  
T. Mathisen ◽  
S. G. Von Essen ◽  
T. A. Wyatt ◽  
D. J. Romberger
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Function ◽  
Airway Epithelial Cell ◽  
Airway Epithelial Cells ◽  
Intercellular Adhesion Molecule ◽  
Airway Epithelial ◽  
Intercellular Adhesion Molecule 1 ◽  
Lymphocyte Adhesion ◽  
Dust Extract

The dust of hog confinement facilities induces airway inflammation. Mechanisms by which this dust modulates inflammation are not completely defined, although it is clear that exposure to dust can modulate both epithelial cell and inflammatory cell function. In this work, we demonstrate that airway epithelial cell (BEAS-2B) treatment with hog barn dust extract (HDE) results in augmentation of peripheral blood lymphocyte adhesion to epithelial cell cultures in vitro. The augmentation of lymphocyte adhesion to epithelial cells is dependent on the concentration of HDE and time of HDE exposure, with twofold increases observed by 3 h and maintained at 24 h. Similar results are seen with primary human bronchial epithelial cells in culture. Lymphocyte adhesion to epithelial cells is inhibited in a concentration-dependent fashion by the treatment of epithelial cells with antibody to intercellular adhesion molecule-1 (ICAM-1). In addition, HDE exposure of epithelial cells results in an approximate twofold increase in ICAM-1 expression as determined by flow cytometry analysis. Pretreatment of epithelial cells with a protein kinase C-α (PKC-α) inhibitor, Gö-6976, also inhibited subsequent lymphocyte adhesion to HDE-exposed epithelial cells. These data suggest that airway epithelial cell HDE exposure enhances subsequent lymphocyte adhesion to epithelial cells that is mediated in part by HDE modulation of ICAM-1 expression and PKC-α.

Glucocorticoid regulation of GM-CSF: evidence for transcriptional mechanisms in airway epithelial cells

1998 ◽  
Vol 275 (2) ◽  
pp. L372-L378 ◽  
Author(s):  
Karissa K. Adkins ◽  
Tricia D. Levan ◽  
Roger L. Miesfeld ◽  
John W. Bloom
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Line ◽  
Bronchial Epithelial Cell ◽  
Airway Epithelial Cells ◽  
Epithelial Cell Line ◽  
Airway Epithelial ◽  
Dexamethasone Treatment ◽  
Bronchial Epithelial ◽  
Gm Csf

Inflammation plays a central role in the pathogenesis of asthma. Glucocorticoids are first-line anti-inflammatory therapy in the treatment of asthma and are effective inhibitors of inflammatory cytokines. Clinical data demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF) production by airway epithelial cells may be an important target of inhaled glucocorticoid therapy. We examined the regulatory mechanisms of GM-CSF expression by interleukin-1β (IL-1β) and the synthetic glucocorticoid dexamethasone in the BEAS-2B human bronchial epithelial cell line. IL-1β stimulation resulted in a 15-fold induction of GM-CSF protein, which was associated with a corresponding 47-fold maximal induction of GM-CSF mRNA levels. Treatment with the transcriptional inhibitor actinomycin D before IL-1β stimulation completely abolished induction of GM-CSF mRNA, whereas incubation with cycloheximide had no effect. Taken together, these data demonstrate that IL-1β induction of GM-CSF is mediated through transcriptional mechanisms. Dexamethasone treatment of BEAS-2B cells produced an 80% inhibition of IL-1β-induced GM-CSF protein and a 51% inhibition of GM-CSF mRNA. GM-CSF mRNA was rapidly degraded in these cells, and dexamethasone treatment did not significantly affect this decay rate. We conclude that, in the BEAS-2B bronchial epithelial cell line, IL-1β induction and dexamethasone repression of GM-CSF expression are mediated predominantly through transcriptional mechanisms.

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