In a newborn pig cystic fibrosis (CF) model, the ability of gland-containing airways to fight infection was affected by at least two major host-defense defects: impaired mucociliary transport and a ...lower airway surface liquid (ASL) pH. In the gland-containing airways, the ASL pH is balanced by CFTR (CF transmembrane conductance regulator) and ATP12A, which, respectively, control HCO
transport and proton secretion. We found that, although porcine small airway tissue expressed lower amounts of ATP12A, the ASL of epithelial cultures from CF distal small airways (diameter < 200 μm) were nevertheless more acidic (compared with non-CF airways). Therefore, we hypothesized that gland-containing airways and small airways control acidification using distinct mechanisms. Our microarray data suggested that small airway epithelia mediate proton secretion via ATP6V0D2, an isoform of the V0 d subunit of the H
-translocating plasma membrane V-type ATPase. Immunofluorescence of small airways verified the expression of the V0 d2 subunit isoform at the apical surface of Muc5B
secretory cells, but not ciliated cells. Inhibiting the V-type ATPase with bafilomycin A1 elevated the ASL pH of small airway cultures, in the presence or absence of HCO
, and decreased ASL viscosity. These data suggest that, unlike large airways, which are acidified by ATP12A activity, small airways are acidified by V-type ATPase, thus identifying V-type ATPase as a novel therapeutic target for small airway diseases.
Gene transfer to airway epithelia is the most direct approach for treating the progressive lung disease associated with cystic fibrosis. However, the transduction efficiency is poor when viral ...vectors are applied to the mucosal surface. We reported previously that gene transfer via the apical surface of human airway epithelia in vitro was improved by formulating vectors with ethyleneglycol-bis-(2-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA) in a hypotonic buffer. First, we investigated the mechanism for this enhancement. When 100-nm fluorescent beads were applied to the apical surface in the presence of EGTA, paracellular deposition of the particles was noted. Transmission electron microscopy verified that the epithelial junction complex was disrupted under these conditions. The Ca(2+) chelators EGTA, 1,2-bis (2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), and ethylenediaminetetraacetic acid all caused a rapid, reversible drop in transepithelial resistance and facilitated gene transfer with retrovirus or adenovirus in vitro. When Ca(2+) chelators were applied to rabbit tracheal epithelia or human nasal epithelia in vivo, the transepithelial voltage decreased, and amiloride sensitivity was lost, suggesting that epithelial junctions opened. Importantly, this novel formulation enhanced both retroviral- and adenoviral-mediated gene transfer to rabbit tracheal epithelia in vivo. This technique may have applications for vector or drug delivery to airway epithelia and other polarized cells.
The Na/K ATPase localizes within the basolateral membrane of most epithelia, where Na/K ATPase activity mediates Na+ absorption. The Na/K ATPase requires its alpha subunit for ion transport and its ...beta subunit for proper trafficking to the plasma membrane. The non‐essential gamma subunit of the Na/K ATPase modifies Na/K ATPase activity. Datasets within the NIH Gene Expression Omnibus indicate that FXYD3, a Na/K ATPase gamma subunit, is highly expressed by human airway epithelia. However, how FXYD3 affects airway epithelial function remains unstudied. In addition to binding the Na/K ATPase, FXYD3 can bind and modify H/K ATPases in heterologous expression systems. The H/K ATPase ATP12A acidifies human airway surface liquid. Therefore, we evaluated the role of FXYD3 in both acid secretion and Na+ absorption across human airway epithelia. Single cell RNA sequencing revealed high FXYD3 expression for all the airway cell types, and immunocytochemistry revealed FXYD3 expression within the basolateral membrane of airway epithelia. Consistent with basolateral FXYD3 localization, the airway surface liquid pH, established in part by apical H/K ATPase, was unaffected by siRNA‐mediated knockdown of FXYD3. FXYD3 knockdown decreased amiloride‐sensitive short‐circuit currents (~20% in NaCl and ~40% in NaGluconate solutions), a finding that is consistent with FXYD3 increasing Na/K ATPase activity. Consequently, fluid absorption across airway epithelia was reduced by ~20% with FXYD3 knockdown. FXYD3 may be required to efficiently return the airway surface liquid to homeostatic volumes after fluid secretion into the proximal airways.
During the immediate response to an inhaled allergen, there is an increase in the paracellular permeability of the airway epithelium.1 Histamine is an important agonist released during the immediate ...response to inhaled allergen. We hypothesized that histamine would increase human airway epithelial paracellular permeability and that it would do this by interrupting E-cadherin-based cell adhesion. Histamine, applied to the basolateral surface, increased the paracellular permeability of cultured human airway epithelia, and this effect of histamine was blocked by the histamine receptor antagonist promethazine. ECV304 cells express a histamine receptor, N-cadherin, and elements of the tight junction, including claudins, but they do not express E-cadherin. Histamine increased the paracellular permeability of ECV304 cells transfected with a vector and expressing E-cadherin but not ECV304 cells expressing lac-Z in the same vector. L cells do not express the histamine receptor, cadherins, or claudins. Histamine decreased adhesion of L cells expressing the human histamine receptor and E-cadherin to an E-cadherin-Fc fusion protein. Histamine did not alter the adhesion to the E-cadherin fusion protein of L cells expressing either the histamine receptor or E-cadherin alone. When applied to the apical surface, adenovirus poorly infects airway epithelial cells because its receptor, CAR, is restricted to the basolateral surface of the cells. When histamine was applied to the basolateral surface of airway epithelial cells, infection of the cells by adenovirus increased by approximately one log. This effect of histamine was also blocked by promethazine. Histamine increases airway paracellular permeability and increases susceptibility of airway epithelial cells to infection by adenovirus by interrupting E-cadherin adhesion.
Abstract Objective The study aimed to describe the patterns and density of early tracheal colonization among intubated patients and to correlate colonization status with levels of antimicrobial ...peptides and inflammatory cytokines. Design The was a prospective cohort study. Setting The study was conducted in medical and cardiovascular intensive care units of a tertiary referral hospital. Patients Seventy-four adult patients admitted between March 2003 and May 2006 were recruited for the study. Interventions Tracheal aspirates were collected daily for the first 4 days of intubation using standardized, sterile technique and sent for quantitative culture and cytokines, lactoferrin and lysozyme measurements. Measurements and Main Results The mean acute physiology and chronic health evaluation (APACHE II) score in this cohort was 24 ± 7. Proportion of subjects colonized by any microorganism increased over the first 4 days of intubation (47%, 60%, 70%, 70%, P = .08), but density of colonization for bacteria or yeast did not change significantly. No known risk factors predicted tracheal colonization on day 1 of intubation. Several patterns of colonization were observed (persistent, transient, new colonization, and clearance of initial colonization).The most common organisms cultured were Candida albicans and coagulase-negative Staphylococcus . Levels of cytokines, lactoferrin, or lysozyme did not change over time and were not correlated with tracheal colonization status. Four subjects (6%) had ventilator-associated pneumonia. Conclusions The density of tracheal colonization did not change significantly over the first 4 days of intubation in medical intensive care unit patients. There was no correlation between tracheal colonization and the levels of antimicrobial peptides or cytokines. Several different patterns of colonization may have to be considered while planning interventions to reduce airway colonization.
Na/K ATPase activity is essential for ion transport across epithelia. FXYD3, a γ subunit of the Na/K ATPase, is expressed in the airway, but its function remains undetermined. Single-cell RNA ...sequencing and immunohistochemistry revealed that FXYD3 localizes within the basolateral membrane of all airway epithelial cells. To study FXYD3 function, we reduced
expression using siRNA. After permeabilizing the apical membrane with nystatin, epithelia pretreated with
-targeting siRNA had lower ouabain-sensitive short-circuit currents than control epithelia.
-targeting siRNA also reduced amiloride-sensitive short-circuit currents and liquid absorption across intact epithelia. These data are consistent with FXYD3 facilitating Na
and liquid absorption. FXYD3 may be needed to maintain the high rates of Na
and fluid absorption observed for airway and other FXYD3-expressing epithelia.
Two enzymes, protein phosphatase 2A and atypical protein kinase C, are associated with the tight junction and regulate its function. For example, phosphorylation of the tight junction protein ...occludin is required for its incorporation into the junction. The association of a kinase and phosphatase with the tight junction suggests that a balance between their activities exists and is required for normal tight junction function. This hypothesis predicts that loss of epithelial integrity may disrupt this balance in such a way as to facilitate restoration of epithelial integrity. Our previous data have shown that apically localized growth factors segregate from their basolaterally localized erbB receptors. Loss of epithelial integrity allows ligand access to the basolateral membrane where it immediately binds to and activates erbB receptors. We found that activation of erbB1 leads to phosphorylation of protein phosphatase 2A, inhibiting its activity. Importantly, this phosphorylation event was dependent on factors in the overlying airway surface liquid; washing away this liquid prevented phosphorylation. erbB1-mediated inhibition of phosphatase activity would shift the balance in favor of the kinase such that tight junction proteins would regain their phosphorylation, allowing for their incorporation into the junction complex. This mechanism provides a rapid means of sensing the loss of epithelial integrity and subsequently restoring barrier function.
Adeno-associated virus (AAV) has been investigated to transfer the cystic fibrosis transmembrane conductance regulator (CFTR) to airways. Inhaled AAV2-CFTR in people with cystic fibrosis (CF) is ...safe, but inefficient. In vitro, AAV2 transduction of human airway epithelia on the apical (luminal) side is inefficient, but efficient basolaterally. We previously selected AAV2.5T, a novel capsid that apically transduces CF human airway epithelia and efficiently restores CFTR function. We hypothesize the AAV receptor (AAVR) is basolaterally localized, and that AAV2.5T utilizes an alternative apical receptor. We found AAVR in human airway epithelia by western blot and RNA-Seq analyses. Using immunocytochemistry we did not find endogenous AAVR at membranes but overexpression localized AAVR to the basolateral membrane, where it preferentially increased transduction. Anti-AAVR antibodies blocked transduction by AAV2 from the basolateral side but not AAV2.5T from the apical side, suggesting a unique apical receptor. Finally, we found infection by AAV2 but not AAV2.5T was blocked by CRISPR knockout of AAVR in cell lines. Our data suggest the absence of apical AAVR is rate limiting for AAV2, and efficient transduction by AAV2.5T is accomplished using an AAVR independent pathway. Our findings inform the development of gene therapy for CF, and AAV vectors in general.
Gene transfer could represent an important advance in the treatment of both genetic and acquired diseases. Cationic lipid-mediated gene transfer have advantages over viral gene transfer because they ...are non-immunogenic, they are easy to produce and they are not oncogenic. The major limitation of cationic lipid-mediated gene transfer is its inefficiency. The development of new cationic lipids, and the combination of a cationic lipid with a neutral lipid has conferred improved efficiency to cationic lipid-mediated gene transfer. The mechanism of gene transfer involves entry into the cell via endocytosis, escape from the endocytic compartment, uncomplexing the DNA from the lipids, and transport of DNA to the nucleus. In vivo gene therapy trials for cancer and cystic fibrosis, have shown promising results both in safety and efficacy.
Goblet cell metaplasia, a disabling hallmark of chronic lung disease, lacks curative treatments at present. To identify novel therapeutic targets for goblet cell metaplasia, we studied the ...transcriptional response profile of IL-13-exposed primary human airway epithelia in vitro and asthmatic airway epithelia in vivo. A perturbation-response profile connectivity approach identified geldanamycin, an inhibitor of heat shock protein 90 (HSP90) as a candidate therapeutic target. Our experiments confirmed that geldanamycin and other HSP90 inhibitors prevented IL-13-induced goblet cell metaplasia in vitro and in vivo. Geldanamycin also reverted established goblet cell metaplasia. Geldanamycin did not induce goblet cell death, nor did it solely block mucin synthesis or IL-13 receptor-proximal signaling. Geldanamycin affected the transcriptome of airway cells when exposed to IL-13, but not when exposed to vehicle. We hypothesized that the mechanism of action probably involves TGF-β, ERBB, or EHF, which would predict that geldanamycin would also revert IL-17-induced goblet cell metaplasia, a prediction confirmed by our experiments. Our findings suggest that persistent airway goblet cell metaplasia requires HSP90 activity and that HSP90 inhibitors will revert goblet cell metaplasia, despite active upstream inflammatory signaling. Moreover, HSP90 inhibitors may be a therapeutic option for airway diseases with goblet cell metaplasia of unknown mechanism.
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