scholarly journals Flagellin-stimulated Cl− secretion and innate immune responses in airway epithelia: role for p38

2008 ◽  
Vol 295 (4) ◽  
pp. L531-L542 ◽  
Author(s):  
Beate Illek ◽  
Zhu Fu ◽  
Christian Schwarzer ◽  
Tina Banzon ◽  
Stephen Jalickee ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Lucifer Yellow ◽  
Innate Immune ◽  
Airway Epithelial ◽  
Airway Epithelia ◽  
Anion Secretion ◽  
Epithelial Cultures ◽  
Serous Cell ◽  
Tight Junction Permeability ◽  
Well Differentiated

Activation of an innate immune response in airway epithelia by the human pathogen Pseudomonas aeruginosa requires bacterial expression of flagellin. Addition of flagellin (10−7 M) to airway epithelial cell monolayers (Calu-3, airway serous cell-like) increased Cl− secretion ( ICl) beginning after 3–10 min, reaching a plateau after 20–45 min at Δ ICl = 15–50 μA/cm2. Similar, although 10-fold smaller, responses were observed in well-differentiated bronchial epithelial cultures. Flagellin stimulated ICl in the presence of maximally stimulating doses of the purinergic agonist ATP, but had no effects following forskolin. IL-1β (produced by both epithelia and neutrophils during infections) stimulated ICl similar to flagellin. Flagellin-, IL-1β-, ATP-, and forskolin-stimulated ICl were inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) blockers GlyH101, CFTRinh172, and glibenclamide. Neither flagellin nor IL-1β altered transepithelial fluxes of membrane-impermeant dextran (10 kDa) or lucifer yellow (mol wt = 457), but both activated p38, NF-κB, and IL-8 secretion. Blockers of p38 (SB-202190 and SB-203580) reduced flagellin- and IL-1β-stimulated ICl by 33–50% but had smaller effects on IL-8 and NF-κB. It is concluded that: 1) flagellin and IL-1β activated p38, NF-κB, IL-8, and CFTR-dependent anion secretion without altering tight junction permeability; 2) p38 played a role in regulating ICl and IL-8 but not NF-κB; and 3) p38 was more important in flagellin- than IL-1β-stimulated responses. During P. aeruginosa infections, flagellin and IL-1β are expected to increase CFTR-dependent ion and fluid flow into and bacterial clearance from the airways. In cystic fibrosis, the secretory response would be absent, but activation of p38, NF-κB, and IL-8 would persist.

scholarly journals Alpha-1 antitrypsin inhibits TMPRSS2 protease activity and SARS-CoV-2 infection

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lukas Wettstein ◽  
Tatjana Weil ◽  
Carina Conzelmann ◽  
Janis A. Müller ◽  
Rüdiger Groß ◽  
...  
Keyword(s):  
Serine Protease ◽  
Immune Defense ◽  
Innate Immune ◽  
Respiratory Pathogen ◽  
The Novel ◽  
Airway Epithelial ◽  
Protein Library ◽  
Epithelial Cultures ◽  
Alpha 1 Antitrypsin ◽  
Novel Coronavirus

AbstractSARS-CoV-2 is a respiratory pathogen and primarily infects the airway epithelium. As our knowledge about innate immune factors of the respiratory tract against SARS-CoV-2 is limited, we generated and screened a peptide/protein library derived from bronchoalveolar lavage for inhibitors of SARS-CoV-2 spike-driven entry. Analysis of antiviral fractions revealed the presence of α1-antitrypsin (α1AT), a highly abundant circulating serine protease inhibitor. Here, we report that α1AT inhibits SARS-CoV-2 entry at physiological concentrations and suppresses viral replication in cell lines and primary cells including human airway epithelial cultures. We further demonstrate that α1AT binds and inactivates the serine protease TMPRSS2, which enzymatically primes the SARS-CoV-2 spike protein for membrane fusion. Thus, the acute phase protein α1AT is an inhibitor of TMPRSS2 and SARS-CoV-2 entry, and may play an important role in the innate immune defense against the novel coronavirus. Our findings suggest that repurposing of α1AT-containing drugs has prospects for the therapy of COVID-19.

Innate immune response in CF airway epithelia: hyperinflammatory?

2006 ◽  
Vol 291 (2) ◽  
pp. C218-C230 ◽  
Author(s):  
Terry E. Machen
Keyword(s):  
Immune Response ◽  
Epithelial Cells ◽  
Innate Immune Response ◽  
Cellular Signaling ◽  
Basolateral Membrane ◽  
Airway Epithelial Cells ◽  
Innate Immune ◽  
Airway Epithelial ◽  
Airway Epithelia ◽  
Intracellular Ca

The lack of functional cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in the apical membranes of CF airway epithelial cells abolishes cAMP-stimulated anion transport, and bacteria, eventually including Pseudomonas aeruginosa, bind to and accumulate in the mucus. Flagellin released from P. aeruginosa triggers airway epithelial Toll-like receptor 5 and subsequent NF-κB signaling and production and release of proinflammatory cytokines that recruit neutrophils to the infected region. This response has been termed hyperinflammatory because so many neutrophils accumulate; a response that damages CF lung tissue. We first review the contradictory data both for and against the idea that epithelial cells exhibit larger-than-normal proinflammatory signaling in CF compared with non-CF cells and then review proposals that might explain how reduced CFTR function could activate such proinflammatory signaling. It is concluded that apparent exaggerated innate immune response of CF airway epithelial cells may have resulted not from direct effects of CFTR on cellular signaling or inflammatory mediator production but from indirect effects resulting from the absence of CFTRs apical membrane channel function. Thus, loss of Cl−, HCO3−, and glutathione secretion may lead to reduced volume and increased acidification and oxidation of the airway surface liquid. These changes concentrate proinflammatory mediators, reduce mucociliary clearance of bacteria and subsequently activate cellular signaling. Loss of apical CFTR will also hyperpolarize basolateral membrane potentials, potentially leading to increases in cytosolic [Ca2+], intracellular Ca2+, and NF-κB signaling. This hyperinflammatory effect of CF on intracellular Ca2+and NF-κB signaling would be most prominently expressed during exposure to both P. aeruginosa and also endocrine, paracrine, or nervous agonists that activate Ca2+signaling in the airway epithelia.

Enteroviral 3C protease activates the human NLRP1 inflammasome in airway epithelia

Science ◽  
2020 ◽  
Vol 370 (6521) ◽  
pp. eaay2002 ◽  
Author(s):  
Kim S. Robinson ◽  
Daniel Eng Thiam Teo ◽  
Kai Sen Tan ◽  
Gee Ann Toh ◽  
Hsiao Hui Ong ◽  
...  
Keyword(s):  
Inflammatory Diseases ◽  
Airway Epithelial Cells ◽  
Innate Immune ◽  
Inflammasome Activation ◽  
Airway Epithelial ◽  
Airway Epithelia ◽  
Nlrp1 Inflammasome ◽  
Terminal Fragment ◽  
Human Immune ◽  
Sensor Proteins

Immune sensor proteins are critical to the function of the human innate immune system. The full repertoire of cognate triggers for human immune sensors is not fully understood. Here, we report that human NACHT, LRR, and PYD domains-containing protein 1 (NLRP1) is activated by 3C proteases (3Cpros) of enteroviruses, such as human rhinovirus (HRV). 3Cpros directly cleave human NLRP1 at a single site between Glu130 and Gly131. This cleavage triggers N-glycine–mediated degradation of the autoinhibitory NLRP1 N-terminal fragment via the cullinZER1/ZYG11B complex, which liberates the activating C-terminal fragment. Infection of primary human airway epithelial cells by live human HRV triggers NLRP1-dependent inflammasome activation and interleukin-18 secretion. Our findings establish 3Cpros as a pathogen-derived trigger for the human NLRP1 inflammasome and suggest that NLRP1 may contribute to inflammatory diseases of the airway.

scholarly journals Epinephrine stimulation of anion secretion in the Calu-3 serous cell model

2014 ◽  
Vol 306 (10) ◽  
pp. L937-L946 ◽  
Author(s):  
Amiraj Banga ◽  
Stephanie Flaig ◽  
Shanta Lewis ◽  
Seth Winfree ◽  
Bonnie L. Blazer-Yost
Keyword(s):  
Adrenergic Receptor ◽  
Mucociliary Clearance ◽  
Cell Model ◽  
Transmembrane Protein ◽  
Intracellular Camp ◽  
Anion Secretion ◽  
Dual Channel ◽  
Submucosal Glands ◽  
Serous Cell ◽  
Well Differentiated

Calu-3 is a well-differentiated human bronchial cell line with the characteristics of the serous cells of airway submucosal glands. The submucosal glands play a major role in mucociliary clearance because they secrete electrolytes that facilitate airway hydration. Given the significance of both long- and short-term β-adrenergic receptor agonists in the treatment of respiratory diseases, it is important to determine the role of these receptors and their ligands in normal physiological function. The present studies were designed to characterize the effect of epinephrine, the naturally occurring β-adrenergic receptor agonist, on electrolyte transport of the airway serous cells. Interestingly, epinephrine stimulated two anion secretory channels, the cystic fibrosis transmembrane conductance regulator and a Ca2+-activated Cl− channel, with the characteristics of transmembrane protein 16A, thereby potentially altering mucociliary clearance via multiple channels. Consistent with the dual channel activation, epinephrine treatment resulted in increases in both intracellular cAMP and Ca2+. Furthermore, the present results extend previous reports indicating that the two anion channels are functionally linked.

scholarly journals Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2

2004 ◽  
Vol 287 (2) ◽  
pp. L428-L437 ◽  
Author(s):  
Hong Peng Jia ◽  
Joel N. Kline ◽  
Andrea Penisten ◽  
Michael A. Apicella ◽  
Theresa L. Gioannini ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Airway Epithelial Cells ◽  
Response To Treatment ◽  
Airway Epithelial ◽  
Airway Epithelia ◽  
Human Airway ◽  
Tnf Α ◽  
Pulmonary Macrophages ◽  
Well Differentiated ◽  
Human Airway Epithelia

The expression of inducible antimicrobial peptides, such as human β-defensin-2 (HBD-2) by epithelia, comprises a component of innate pulmonary defenses. We hypothesized that HBD-2 induction in airway epithelia is linked to pattern recognition receptors such as the Toll-like receptors (TLRs). We found that primary cultures of well-differentiated human airway epithelia express the mRNA for TLR-4, but little or no MD-2 mRNA, and display little HBD-2 expression in response to treatment with purified endotoxin ± LPS binding protein (LBP) and soluble CD14. Expression of endogenous MD-2 by transduction of airway epithelial cells with an adenoviral vector encoding MD-2 or extracellular addition of recombinant MD-2 both increased the responses of airway epithelia to endotoxin + LBP and sCD14 by >100-fold, as measured by NF-κB-luciferase activity and HBD-2 mRNA expression. MD-2 mRNA could be induced in airway epithelia by exposure of these cells to specific bacterial or host products (e.g., killed Haemophilus influenzae, the P6 outer membrane protein from H. influenzae, or TNF-α + IFN-γ). These findings suggest that MD-2, either coexpressed with TLR-4 or secreted when produced in excess of TLR-4 from neighboring cells, is required for airway epithelia to respond sensitively to endotoxin. The regulation of MD-2 expression in airway epithelia and pulmonary macrophages may serve as a means to modify endotoxin responsiveness in the airway.

scholarly journals Infection by Toxoplasma gondii, a severe parasite in neonates and AIDS patients, causes impaired anion secretion in airway epithelia

2015 ◽  
Vol 112 (14) ◽  
pp. 4435-4440 ◽  
Author(s):  
Hong-Mei Guo ◽  
Jiang-Mei Gao ◽  
Yu-Li Luo ◽  
Yan-Zi Wen ◽  
Yi-Lin Zhang ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Toxoplasma Gondii ◽  
Infected Mouse ◽  
Inflammatory Responses ◽  
Short Circuit ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Airway Epithelia ◽  
Anion Secretion ◽  
Cystic Fibrosis Transmembrane

The airway epithelia initiate and modulate the inflammatory responses to various pathogens. The cystic fibrosis transmembrane conductance regulator-mediated Cl− secretion system plays a key role in mucociliary clearance of inhaled pathogens. We have explored the effects of Toxoplasma gondii, an opportunistic intracellular protozoan parasite, on Cl− secretion of the mouse tracheal epithelia. In this study, ATP-induced Cl− secretion indicated the presence of a biphasic short-circuit current (Isc) response, which was mediated by a Ca2+-activated Cl− channel (CaCC) and the cystic fibrosis transmembrane conductance regulator. However, the ATP-evoked Cl− secretion in T. gondii-infected mouse tracheal epithelia and the elevation of [Ca2+]i in T. gondii-infected human airway epithelial cells were suppressed. Quantitative reverse transcription–PCR revealed that the mRNA expression level of the P2Y2 receptor (P2Y2-R) increased significantly in T. gondii-infected mouse tracheal cells. This revealed the influence that pathological changes in P2Y2-R had on the downstream signal, suggesting that P2Y2-R was involved in the mechanism underlying T. gondii infection in airways. These results link T. gondii infection as well as other pathogen infections to Cl− secretion, via P2Y2-R, which may provide new insights for the treatment of pneumonia caused by pathogens including T. gondii.

scholarly journals Inhibition of ATP hydrolysis restores airway surface liquid production in cystic fibrosis airway epithelia

2020 ◽  
Vol 318 (2) ◽  
pp. L356-L365 ◽  
Author(s):  
Catharina van Heusden ◽  
Brian Button ◽  
Wayne H. Anderson ◽  
Agathe Ceppe ◽  
Lisa C. Morton ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Steady State ◽  
Atp Hydrolysis ◽  
Fluid Secretion ◽  
Pathological Feature ◽  
Airway Surface Liquid ◽  
Airway Epithelial ◽  
Airway Epithelia ◽  
Atp Levels ◽  
Surface Liquid

Airway surface dehydration is a pathological feature of cystic fibrosis (CF) lung disease. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated Cl− channel controlled in part by the adenosine A2B receptor. An alternative CFTR-independent mechanism of fluid secretion is regulated by ATP via the P2Y2 receptor (P2Y2R) that activates Ca2+-regulated Cl− channels (CaCC/TMEM16) and inhibits Na+ absorption. However, due to rapid ATP hydrolysis, steady-state ATP levels in CF airway surface liquid (ASL) are inadequate to maintain P2Y2R-mediated fluid secretion. Therefore, inhibiting airway epithelial ecto-ATPases to increase ASL ATP levels constitutes a strategy to restore airway surface hydration in CF. Using [γ32P]ATP as radiotracer, we assessed the effect of a series of ATPase inhibitory compounds on the stability of physiologically occurring ATP concentrations. We identified the polyoxometalate [Co4(H2O)2(PW9O34)2]10− (POM-5) as the most potent and effective ecto-ATPase inhibitor in CF airway epithelial cells. POM-5 caused long-lasting inhibition of ATP hydrolysis in airway epithelia, which was reversible upon removal of the inhibitor. Importantly, POM-5 markedly enhanced steady-state levels of released ATP, promoting increased ASL volume in CF cell surfaces. These results provide proof of concept for ecto-ATPase inhibitors as therapeutic agents to restore hydration of CF airway surfaces. As a test of this notion, cell-free sputum supernatants from CF subjects were studied and found to have abnormally elevated ATPase activity, which was markedly inhibited by POM-5.

scholarly journals Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner

2017 ◽  
Vol 312 (5) ◽  
pp. L688-L702 ◽  
Author(s):  
Samuel A. Molina ◽  
Hannah K. Moriarty ◽  
Daniel T. Infield ◽  
Barry R. Imhoff ◽  
Rachel J. Vance ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Small Molecules ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Airway Epithelia ◽  
Human Airway ◽  
Airway Cells

Cystic fibrosis-related diabetes is the most common comorbidity associated with cystic fibrosis (CF) and correlates with increased rates of lung function decline. Because glucose is a nutrient present in the airways of patients with bacterial airway infections and because insulin controls glucose metabolism, the effect of insulin on CF airway epithelia was investigated to determine the role of insulin receptors and glucose transport in regulating glucose availability in the airway. The response to insulin by human airway epithelial cells was characterized by quantitative PCR, immunoblot, immunofluorescence, and glucose uptake assays. Phosphatidylinositol 3-kinase/protein kinase B (Akt) signaling and cystic fibrosis transmembrane conductance regulator (CFTR) activity were analyzed by pharmacological and immunoblot assays. We found that normal human primary airway epithelial cells expressed glucose transporter 4 and that application of insulin stimulated cytochalasin B-inhibitable glucose uptake, consistent with a requirement for glucose transporter translocation. Application of insulin to normal primary human airway epithelial cells promoted airway barrier function as demonstrated by increased transepithelial electrical resistance and decreased paracellular flux of small molecules. This provides the first demonstration that airway cells express insulin-regulated glucose transporters that act in concert with tight junctions to form an airway glucose barrier. However, insulin failed to increase glucose uptake or decrease paracellular flux of small molecules in human airway epithelia expressing F508del-CFTR. Insulin stimulation of Akt1 and Akt2 signaling in CF airway cells was diminished compared with that observed in airway cells expressing wild-type CFTR. These results indicate that the airway glucose barrier is regulated by insulin and is dysfunctional in CF.

Basolateral chloride current in human airway epithelia

2007 ◽  
Vol 293 (4) ◽  
pp. L991-L999 ◽  
Author(s):  
Omar A. Itani ◽  
Fred S. Lamb ◽  
James E. Melvin ◽  
Michael J. Welsh
Keyword(s):  
Cystic Fibrosis ◽  
Signaling Pathways ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Airway Epithelia ◽  
Human Airway ◽  
Well Differentiated ◽  
Human Airway Epithelia

Electrolyte transport by airway epithelia regulates the quantity and composition of liquid covering the airways. Previous data indicate that airway epithelia can absorb NaCl. At the apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR) provides a pathway for Cl− absorption. However, the pathways for basolateral Cl− exit are not well understood. Earlier studies, predominantly in cell lines, have reported that the basolateral membrane contains a Cl− conductance. However, the properties have varied substantially in different epithelia. To better understand the basolateral Cl− conductance in airway epithelia, we studied primary cultures of well-differentiated human airway epithelia. The basolateral membrane contained a Cl− current that was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The current-voltage relationship was nearly linear, and the halide selectivity was Cl− > Br− >> I−. Several signaling pathways increased the current, including elevation of cellular levels of cAMP, activation of protein kinase C (PKC), and reduction of pH. In contrast, increasing cell Ca2+ and inducing cell swelling had no effect. The basolateral Cl− current was present in both cystic fibrosis (CF) and non-CF airway epithelia. Likewise, airway epithelia from wild-type mice and mice with disrupted genes for ClC-2 or ClC-3 all showed similar Cl− currents. These data suggest that the basolateral membrane of airway epithelia possesses a Cl− conductance that is not due to CFTR, ClC-2, or ClC-3. Its regulation by cAMP and PKC signaling pathways suggests that coordinated regulation of Cl− conductance in both apical and basolateral membranes may be important in controlling transepithelial Cl− movement.

scholarly journals Inter-subject variation in ACE2 protein expression in human airway epithelia and its relationship to SARS-CoV-2 infection

2021 ◽  
Author(s):  
Kun Li ◽  
Christine Wohlford-Lenane ◽  
Jennifer A Bartlett ◽  
Paul B McCray
Keyword(s):  
Protein Expression ◽  
Receptor Expression ◽  
Interferon Response ◽  
Airway Epithelial ◽  
Human Donor ◽  
Airway Epithelia ◽  
Viral Receptor ◽  
Disease Outcomes ◽  
Age Dependent ◽  
Epithelial Cultures

Abstract SARS-CoV-2 initiates entry into airway epithelia by binding its receptor, ACE2. To explore whether inter-individual variation in ACE2 abundance contributes to variability in COVID-19 outcomes, we measured ACE2 protein abundance in primary airway epithelial cultures derived from 58 human donor lungs. We found no evidence for sex- or age-dependent differences in ACE2 protein expression. Further, we found that variations in ACE2 abundance had minimal effects on viral replication and induction of the interferon response in airway epithelia infected with SARS-CoV-2. Our results highlight the relative importance of additional host factors, beyond viral receptor expression, in determining COVID-19 lung disease outcomes.

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