Protective role of CFTR during fungal infection of cystic fibrosis bronchial epithelial cells with Aspergillus fumigatus.

Lung infection with the fungus Aspergillus fumigatus (Af) is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function. We established a fungal epithelial co-culture model to examine the impact of Af infection on CF bronchial epithelial barrier function using Af strains 10AF and AF293-GFP, and the CFBE41o- cell line homozygous for the F508del mutation with (CF+CFTR) and without (CF) normal CFTR expression. Following exposure of the epithelial surface to Af conidia, formation of germlings (early stages of fungal growth) was detected after 9-12 hours and hyphae (mature fungal growth) after 12-24 hours. During fungal morphogenesis, bronchial epithelial cells showed signs of damage including rounding, and partial detachment after 24 hours. Fluorescently labeled conidia were internalized after 6 hours and more internalized conidia were observed in CF compared to CF+CFTR cells. Infection of the apical surface with 10AF conidia, germlings, or hyphae was performed to determine growth stage-specific effects on tight junction protein zona occludens protein 1 (ZO-1) expression and transepithelial electrical resistance (TER). In response to infection with conidia or germlings, epithelial barrier function degraded time-dependently (based on ZO-1 immunofluorescence and TER) with a delayed onset in CF+CFTR cell monolayers and required viable fungi and apical application. Infection with hyphae caused an earlier onset and faster rate of decline in TER compared to conidia and germlings. Gliotoxin, a major Af virulence factor, caused a rapid decline in TER and induced a transient chloride secretory response in CF+CFTR but not CF cells. Our findings suggest growth and internalization of Af result in deleterious effects on bronchial epithelial barrier function that occurred more rapidly in the absence of CFTR. Bronchial epithelial barrier breakdown was time-dependent and morphotype-specific and mimicked by acute administration of gliotoxin. Our study also suggests a protective role for CFTR by turning on CFTR-dependent chloride transport in response to gliotoxin, a mechanism that will support mucociliary clearance, and could delay the loss of epithelial integrity during fungal development in vivo.

Role of Dual Oxidases in Ventilator-induced Lung Injury.

Positive-pressure ventilation results in ventilator-induced lung injury, and few therapeutic modalities have been successful at limiting the degree of injury to the lungs. Understanding the primary drivers of ventilator-induced lung injury will aid in the development of specific treatments to ameliorate the progression of this syndrome. There are conflicting data for the role of neutrophils in acute respiratory distress syndrome pathogenesis. Here, we specifically examined the importance of neutrophils as a primary driver of ventilator-induced lung injury in a mouse model known to have impaired ability to recruit neutrophils in previous models of inflammation. We exposed Duoxa+/+ and Duoxa-/- mice to low- or high-tidal volume ventilation with or without positive end-expiratory pressure (PEEP) and recruitment maneuvers for 4 hours. Absolute neutrophils in BAL fluid were significantly reduced in Duoxa-/- mice compared with Duoxa+/+ mice (6.7 cells/µl; 16.4 cells/µl; P = 0.003), consistent with our hypothesis that neutrophil translocation across the capillary endothelium is reduced in the absence of DUOX1 or DUOX2 in response to ventilator-induced lung injury. Reduced lung neutrophilia was not associated with a reduction in overall lung injury in this study, suggesting that neutrophils do not play an important role in early features of acute lung injury. Surprisingly, Duoxa-/- mice exhibited significant hypoxemia, as measured by the arterial oxygen tension/fraction of inspired oxygen ratio and arterial oxygen content, which was out of proportion with that seen in the Duoxa+/+ mice (141, 257, P = 0.012). These findings suggest a role for dual oxidases to limit physiologic impairment during early ventilator-induced lung injury.

Functional Profiling of CFTR-Directed Therapeutics Using Pediatric Patient-Derived Nasal Epithelial Cell Models.

Functional profiling of CFTR-directed therapeutics offers the potential to provide significant benefits to young people with cystic fibrosis (CF). However, the development of 2D airway epithelial cell models for individual response tests in CF children remains a central task. The objective of this study was to determine the utility of EpiXTM technology for expansion of nasal epithelial cells for use in electrophysiological CFTR function measurements. An initial harvest of as few as 20,000 cells was sufficient to expand up to 50 million cells that were used to generate air-liquid interface (ALI) cultures for ion transport studies with the Ussing assay. CFTR function was assessed by measuring responses to forskolin and the CFTR potentiator VX-770 (ivacaftor) in ALI cultures generated from passage 3 and 4 cells. Short-circuit current (Isc) measurements of blocked CFTR currents (ΔICFTRinh) discriminated CFTR function between healthy control (wild type, WT) and patients with intermediate (F508del/R117H-7T: 56% WT) and severe (F508del/F508del: 12% WT) CF disease. For the mixed genotypes, CFTR activity for F508del/c.850dupA was 12% WT, R334W/406-1G>A was 24% WT, and CFTRdele2,3(21 kb)/CFTRdele2,3(21 kb) was 9% WT. The CFTR correctors VX-809 (lumacaftor) and VX-661 (tezacaftor) significantly increased CFTR currents for F508del/R117H to 73 and 67% WT, respectively. Cultures with the large deletion mutation CFTRdele2,3(21 kb) unexpectedly responded to VX-661 treatment (20% WT). Amiloride-sensitive sodium currents were robust and ranged between 20-80 µA/cm2 depending on the subject. In addition to characterizing the electrophysiological profile of mutant CFTR activity in cultures for five genotypes, our study exemplifies the promising paradigm of bed-to-bench side cooperation and personalized medicine.

Differential Chloride Secretory Capacity in Transepithelial Ion Transport Properties in Chronic Rhinosinusitis.

BACKGROUND: Epithelial ion transport regulates hydration of airway mucosal surfaces, and thus promotes effective mucociliary clearance (MCC). Decreased transepithelial Cl- transport may contribute to epithelial dysfunction by abrogating MCC and increasing mucus viscosity in chronic rhinosinusitis (CRS). The objective of the current study is to evaluate Cl- channel transport properties from cultures of human sinonasal epithelia. METHODS: Human sinonasal epithelia (HSNE) from patients undergoing sinus surgery were cultured at an air-liquid interface to confluence and full differentiation. The epithelial monolayers were mounted in Ussing Chambers to investigate pharmacological manipulation of ion transport. Epithelial Na+ channel (via Amiloride), CFTR (via forskolin), and Ca2+-activated Cl- channel (CaCC, via UTP) transport were investigated among three different patient groups: Control, CRS and CRS with polyposis. CFTR mRNA levels were evaluated with quantitative RT-PCR. RESULTS: HSNE cultures from 18 patients (Control = 9, CRS = 6, CRS with polyposis = 3) were evaluated in 142 experiments. Summary data from the 18 patients demonstrated that stimulated CFTR-mediated anion transport (Δ ISC) was significantly lower with CRS (7.58+/-2.24 µA/cm2) compared to control (25.86+/-3.44 µA/cm2) and CRS with polyposis (20.16+/-4.0 µA/cm2) (p = 0.004). No statistically significant difference was found for CaCC anion transport between groups (p = 0.39). Significantly decreased mRNA (relative expression) was noted in CRS cultures (CRS = 40.83+/-1.76 vs. control = 116.2+/-24.27, p = 0.03). CONCLUSIONS: A substantial decrease in the Cl- secretory capacity of HSNE monolayers was demonstrated in CRS subjects. Data suggest that CFTR may contribute more to abnormal ion transport in CRS than CaCC.

CFTR modulator theratyping: Current status, gaps and future directions.

BACKGROUND: New drugs that improve the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein with discreet disease-causing variants have been successfully developed for cystic fibrosis (CF) patients. Preclinical model systems have played a critical role in this process, and have the potential to inform researchers and CF healthcare providers regarding the nature of defects in rare CFTR variants, and to potentially support use of modulator therapies in new populations. METHODS: The Cystic Fibrosis Foundation (CFF) assembled a workshop of international experts to discuss the use of preclinical model systems to examine the nature of CF-causing variants in CFTR and the role of in vitro CFTR modulator testing to inform in vivo modulator use. The theme of the workshop was centered on CFTR theratyping, a term that encompasses the use of CFTR modulators to define defects in CFTR in vitro, with application to both common and rare CFTR variants. RESULTS: Several preclinical model systems were identified in various stages of maturity, ranging from the expression of CFTR variant cDNA in stable cell lines to examination of cells derived from CF patients, including the gastrointestinal tract, the respiratory tree, and the blood. Common themes included the ongoing need for standardization, validation, and defining the predictive capacity of data derived from model systems to estimate clinical outcomes from modulator-treated CF patients. CONCLUSIONS: CFTR modulator theratyping is a novel and rapidly evolving field that has the potential to identify rare CFTR variants that are responsive to approved drugs or drugs in development.

Assessment and management of diarrhea following VEGF receptor TKI treatment in patients with ovarian cancer.

Angiogenesis is a proven clinical target for the treatment of advanced epithelial ovarian cancer. Vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKIs) offer patients potential new treatment regimens as they can be given as monotherapy, in combination with poly(ADP-ribose) polymerase (PARP) inhibitors, or with and following cytotoxic chemotherapy. If VEGFR-TKIs are licensed for use in ovarian cancer, patients will require prompt and effective management of adverse events, including diarrhea, to optimize compliance and benefit. As diarrhea is one of the most prevalent toxicities of this class of drug, it is important to consider the potential causes, be they disease related (bowel obstruction), treatment related (VEGFR-TKI-related or infective/neutropenic septic diarrhea when patients are receiving cytotoxic chemotherapy combined with VEGFR inhibitor treatment), or incurred through diet. Here, we provide an overview of the possible mechanisms responsible for VEGFR-TKI-induced diarrhea. We review potential interventions that can help in the management of diarrhea induced by VEGFR-TKIs, when used in combination or as single agents, and we provide a diarrhea treatment algorithm to serve as a clinical reference point for the management of diarrhea in patients with ovarian cancer treated with a VEGFR-TKI in combination with chemotherapy or PARP inhibitors, or as monotherapy.

Impaired PGE2-stimulated Cl- and HCO3- secretion contributes to cystic fibrosis airway disease.

BACKGROUND: Airway mucociliary clearance (MCC) is an important defense mechanism against pulmonary infections and is compromised in cystic fibrosis (CF). Cl- and HCO3- epithelial transport are integral to MCC. During pulmonary infections prostaglandin E2 (PGE2) production is abundant. AIM: To determine the effect of PGE2 on airway Cl- and HCO3- secretion and MCC in normal and CF airways. METHODS: We examined PGE2 stimulated MCC, Cl- and HCO3- secretion using ferret trachea, human bronchial epithelial cell cultures (CFBE41o- with wildtype CFTR (CFBE41 WT) or homozygous F508del CFTR (CFBE41 CF) and human normal bronchial submucosal gland cell line (Calu-3) in Ussing chambers with or without pH-stat. RESULTS: PGE2 stimulated MCC in a dose-dependent manner and was partially impaired by CFTRinh-172. PGE2-stimulated Cl- current in ferret trachea was partially inhibited by CFTRinh-172, with niflumic acid eliminating the residual current. CFBE41 WT cell monolayers produced a robust Cl- and HCO3- secretory response to PGE2, both of which were completely inhibited by CFTRinh-172. CFBE41 CF cells exhibited no response to PGE2. In Calu-3 cells, PGE2 stimulated Cl- and HCO3- secretion. Cl- secretion was partially inhibited by CFTRinh-172, with additional inhibition by niflumic acid. HCO3- secretion was completely inhibited by CFTRinh-172. CONCLUSIONS: PGE2 stimulates bronchotracheal MCC and this response is decreased in CF. In CF airway, PGE2-stimulated Cl- and HCO3- conductance is impaired and may contribute to decreased MCC. There remains a CFTR-independent Cl- current in submucosal glands, which if exploited, could represent a means of improving airway Cl- secretion and MCC in CF.

Effect of Vaccine-Elicited Antibodies on Colonization of Neisseria meningitidis Serogroup B and C Strains in a Human Bronchial Epithelial Cell Culture Model.

Capsular polysaccharide-protein conjugate vaccines protect individuals from invasive disease and decrease carriage, which reduces spread of the organism in the population. In contrast, antibodies elicited by plain polysaccharide or protein antigen-based meningococcal (Men) vaccines have little or no effect on decreasing carriage. In this study, we investigated the mechanism by which vaccine-induced human immunoglobulin G (IgG) antibodies affect colonization by meningococcal serogroup B (MenB) or C (MenC) strains using a human bronchial epithelial cell culture model (16HBE14o-). Fluorescence microscopy showed that bacteria colonizing the apical side of 16HBE14o- monolayers had decreased capsular polysaccharide on the bacterial surface that resulted from shedding the capsule and not decreased production of polysaccharide. Capsular polysaccharide shedding depended on the presence of 16HBE14o- cells and bacteria but not direct adherence of the bacteria to the cells. Treatment of bacteria and cells with postimmunization MenC-conjugate IgG or murine anti-MenB polysaccharide monoclonal antibodies (MAbs) inhibited capsule shedding, microcolony dispersal, and invasion of the 16HBE14o- cell monolayer. In contrast, the IgG responses elicited by immunization with MenC polysaccharide (PS), MenB outer membrane vesicle (OMV)-based, or factor H binding protein (FHbp)-based vaccines were not different than preimmune IgG or no-treatment response. The results provide new insights on the mechanism by which high-avidity anticapsular antibodies elicited by polysaccharide-conjugate vaccines affect meningococcal colonization. The data also suggest that any effect on colonization by IgG elicited by OMV- or FHbp-based vaccines may involve a different mechanism.

In vivo and in vitro ivacaftor response in cystic fibrosis patients with residual CFTR function: N-of-1 studies.

RATIONALE: Ivacaftor, a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator, decreases sweat chloride concentration, and improves pulmonary function in 6% of cystic fibrosis (CF) patients with specific CFTR mutations. Ivacaftor increases chloride transport in many other CFTR mutations in non-human cells, if CFTR is in the epithelium. Some CF patients have CFTR in the epithelium with residual CFTR function. The effect of ivacaftor in these patients is unknown. METHODS: This was a series of randomized, crossover N-of-1 trials of ivacaftor and placebo in CF patients ≥8 years old with potential residual CFTR function (intermediate sweat chloride concentration, pancreatic sufficient, or mild bronchiectasis on chest CT). Human nasal epithelium (HNE) was obtained via nasal brushing and cultured. Sweat chloride concentration change was the in vivo outcome. Chloride current change in HNE cultures with ivacaftor was the in vitro outcome. RESULTS: Three subjects had decreased sweat chloride concentration (-14.8 to -40.8 mmol/L, P < 0.01). Two subjects had unchanged sweat chloride concentration. Two subjects had increased sweat chloride concentration (+23.8 and +27.3 mmol/L, P < 0.001); both were heterozygous for A455E and pancreatic sufficient. Only subjects with decreased sweat chloride concentration had increased chloride current in HNE cultures. CONCLUSIONS: Some CF patients with residual CFTR function have decreased sweat chloride concentration with ivacaftor. Increased chloride current in HNE cultures among subjects with decreased sweat chloride concentrations may predict clinical response to ivacaftor. Ivacaftor can increase sweat chloride concentration in certain mutations with unclear clinical effect. Pediatr Pulmonol. 2017;52:472-479. © 2017 Wiley Periodicals, Inc.

TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs.

Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ~100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways.

Expression and function of the epithelial sodium channel δ-subunit in human respiratory epithelial cells in vitro.

Using human airway epithelial cell lines (i.e. NCI-H441 and Calu-3) as well as human alveolar epithelial type I-like (ATI) cells in primary culture, we studied the contribution of the epithelial sodium channel δ-subunit (δ-ENaC) to transepithelial sodium transport in human lung in vitro. Endogenous δ-ENaC protein was present in all three cell types tested; however, protein abundance was low, and no expression was detected in the apical cell membrane of these cells. Similarly, known modulators of δ-ENaC activity, such as capsazepine and icilin (activators) and Evans blue (inhibitor), did not show effects on short-circuit current (I SC), suggesting that δ-ENaC is not involved in the modulation of transcellular sodium absorption in NCI-H441 cell monolayers. Over-expression of δ-ENaC in NCI-H441 cells resulted in detectable protein expression in the apical cell membrane, as well as capsazepine and icilin-stimulated increases in I SC that were effectively blocked by Evans blue and that were consistent with δ-ENaC activation and inhibition, respectively. Consequently, these observations suggest that δ-ENaC expression is low in NCI-H441, Calu-3, and ATI cells and does not contribute to transepithelial sodium absorption.

Air pollutants cause release of hydrogen peroxide and interleukin-8 in a human primary nasal tissue culture model.

BACKGROUND: A component of primary innate defense of the nasal mucosa against inhaled pathogens includes continuous, low-level release of hydrogen peroxide (H2 O2 ) into luminal secretions. Epidemiologically, an association exists between poor air quality and increased prevalence of sinonasal disease. To understand the effects of particulate matter (PM) in nasal mucosa, we studied the release of H2 O2 and interleukin 8 (IL-8) after PM exposure. METHODS: Human nasal specimens were collected from surgery and cultured in serum-free growth medium. Cell integrity and recovery during culture was monitored by lactate dehydrogenase (LDH) release into the medium. Cultures were exposed to PM for 24 hours in the presence/absence of diphenyleneiodonium sulfate (DPI; a nicotinamide adenine dinucleotide phosphate [NADPH] oxidase inhibitor). Luminex cytokine and Amplex-Red H2 O2 assays were performed. RESULTS: LDH levels dropped rapidly within 2 days, indicative of stabilization and cell recovery after harvest. All cultures released H2 O2 into the medium. Exposure to PM (20 µg/cm(2) ) increased H2 O2 levels significantly (94.6 ± 7.7 nM) compared to untreated controls (55.8 ± 4.0 nM; p = 0.001). PM-induced H2 O2 production was partially inhibited by DPI (80.1 ± 3.8nM), indicating that cellular NADPH oxidase may be a primary source of H2 O2 production. Exposure to PM increased IL-8 levels in a dose-dependent fashion (control = 2301 ± 412 MFI; 20 µg/cm(2) = 5002 ± 1327 MFI; 40 µg/cm(2) = 8219 ± 1090 MFI; p = 0.022). CONCLUSION: PM increases the quantity of H2 O2 released by nasal epithelial cells, indicating that PM can contribute to oxidative stress in part by activating a normal cellular defense mechanism. Exposure to PM resulted in elevated IL-8 levels and mucin production in explants. Efforts to reduce airborne PM may lead to reduced H2 O2 and mucin production in sinonasal epithelium.

Thapsigargin blocks Pseudomonas aeruginosa homoserine lactone-induced apoptosis in airway epithelia.

Pseudomonas aeruginosa secretes N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule to regulate gene expression. Micromolar concentrations are found in the airway surface liquid of infected lungs. Exposure of the airway surface to C12 caused a loss of transepithelial resistance within 1 h that was accompanied by disassembly of tight junctions, as indicated by relocation of the tight junction protein zonula occludens 1 from the apical to the basolateral pole and into the cytosol of polarized human airway epithelial cell cultures (Calu-3 and primary tracheal epithelial cells). These effects were blocked by carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone, a pan-caspase blocker, indicating that tight junction disassembly was an early event in C12-triggered apoptosis. Short-duration (10 min) pretreatment of airway epithelial (Calu-3 and JME) cells with 1 µM thapsigargin (Tg), an inhibitor of Ca(2+) uptake into the endoplasmic reticulum (ER), was found to be protective against the C12-induced airway epithelial barrier breakdown and also against other apoptosis-related effects, including shrinkage and fragmentation of nuclei, activation of caspase 3/7 (the executioner caspase in apoptosis), release of ER-targeted redox-sensitive green fluorescent protein into the cytosol, and depolarization of mitochondrial membrane potential. Pretreatment of Calu-3 airway cell monolayers with BAPTA-AM [to buffer cytosolic Ca(2+) concentration (Cacyto)] or Ca(2+)-free solution + BAPTA-AM reduced C12 activation of apoptotic events, suggesting that C12-triggered apoptosis may involve Ca(2+). Because C12 and Tg reduced Ca(2+) concentration in the ER and increased Cacyto, while Tg increased mitochondrial Ca(2+) concentration (Camito) and C12 reduced Camito, it is proposed that Tg may reduce C12-induced apoptosis in host cells not by raising Cacyto, but by preventing C12-induced decreases in Camito.

Expression of dual oxidases and secreted cytokines in chronic rhinosinusitis.

BACKGROUND: The airway epithelium generates reactive oxygen species (ROS) as a first line of defense. Dual oxidases (DUOX1 and DUOX2) are the H2 O2 -producing isoforms of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family in the airway epithelium. The purpose of this study was to explore the molecular expression, function, and regulation of DUOXs in chronic rhinosinusitis (CRS). METHODS: Human nasal tissue samples and nasal secretions were collected from 3 groups of patients undergoing sinus surgery (normal, n = 7; CRS with polyposis [CRSwP], n = 6; CRS without polyposis [CRSsP], n = 6). Nasal secretions were studied for cytokine and H2 O2 content. Tissue samples were used to determine DUOX mRNA and protein expression. RESULTS: DUOX1 mRNA level (80.7 ± 60.5) was significantly increased in CRSwP compared to normal (2.7 ± 1.2) and CRSsP (2.3 ± 0.5, p = 0.042). DUOX2 mRNA levels were increased in both CRSwP (18.6 ± 9.9) and CRSsP (4.0 ± 1.3) compared to normal (1.1 ± 0.3; p = 0.008). DUOX protein was found in the apical portion of the nasal epithelium and protein expression was increased in CRSwP and CRSsP. H2 O2 production was significantly higher in CRSwP (160.9 ± 59.4 nM) and CRSsP (81.7 ± 5.6 nM) compared to normal (53.5 ± 11.5 nM, p = 0.032). H2 O2 content of nasal secretions correlated tightly with DUOX expression (p < 0.001). Cytokines (eotaxin, monokine-induced by interferon γ [MIG], tumor necrosis factor [TNF]-α, interleukin [IL]-8) showed significantly higher levels in nasal secretions from CRSwP compared to normal (p < 0.05). Levels of eotaxin, MIG, and TNF-α correlated closely with DUOX expression. CONCLUSION: DUOX1 and DUOX2 were identified as factors upregulated in CRS. Close correlations between DUOX expression and H2 O2 release, and correlation between key inflammatory cytokines and DUOX expression, indicate DUOX in the inflammatory response in CRS.

Pseudomonas aeruginosa biofilm-associated homoserine lactone C12 rapidly activates apoptosis in airway epithelia.

Pseudomonas aeruginosa (PA) forms biofilms in lungs of cystic fibrosis (CF) patients, a process regulated by quorum-sensing molecules including N-(3-oxododecanoyl)-l-homoserine lactone (C12). C12 (10-100 µM) rapidly triggered events commonly associated with the intrinsic apoptotic pathway in JME (CF ΔF508CFTR, nasal surface) epithelial cells: depolarization of mitochondrial (mito) membrane potential (Δψ(mito)) and release of cytochrome C (cytoC) from mitos into cytosol and activation of caspases 3/7, 8 and 9. C12 also had novel effects on the endoplasmic reticulum (release of both Ca(2+) and ER-targeted GFP and oxidized contents into the cytosol). Effects began within 5 min and were complete in 1-2 h. C12 caused similar activation of caspases and release of cytoC from mitos in Calu-3 (wtCFTR, bronchial gland) cells, showing that C12-triggered responses occurred similarly in different airway epithelial types. C12 had nearly identical effects on three key aspects of the apoptosis response (caspase 3/7, depolarization of Δψ(mito) and reduction of redox potential in the ER) in JME and CFTR-corrected JME cells (adenoviral expression), showing that CFTR was likely not an important regulator of C12-triggered apoptosis in airway epithelia. Exposure of airway cultures to biofilms from PAO1wt caused depolarization of Δψ(mito) and increases in Ca(cyto) like 10-50 µM C12. In contrast, biofilms from PAO1ΔlasI (C12 deficient) had no effect, suggesting that C12 from P. aeruginosa biofilms may contribute to accumulation of apoptotic cells that cannot be cleared from CF lungs. A model to explain the effects of C12 is proposed.

Vitamin E forms inhibit IL-13/STAT6-induced eotaxin-3 secretion by up-regulation of PAR4, an endogenous inhibitor of atypical PKC in human lung epithelial cells.

Eotaxin-3 (CCL-26), a potent chemokine for eosinophil recruitment and contributing significantly to the pathogenesis of asthma, is secreted by lung epithelial cells in response to T helper 2 cytokines including interleukin 13 (IL-13). Here we showed that vitamin E forms, but not their metabolites, differentially inhibited IL-13-stimulated generation of eotaxin-3 in human lung epithelial A549 cells. The relative inhibitory potency was γ-tocotrienol (γ-TE) (IC50 ~15 µM)>γ-tocopherol, δ-tocopherol (IC50 ~25-50 µM)>α-tocopherol. Consistent with suppression of eotaxin, γ-TE treatment impaired IL-13-induced phosphorylation of STAT6, the key transcription factor for activation of eotaxin expression, and consequently blocked IL-13-stimulated DNA-binding activity of STAT6. In search of the upstream target of γTE by using inhibitor and siRNA approaches, we discovered that the atypical protein kinase C (aPKC) signaling, instead of classical PKC, p38 MAPK, JNK or ERK, played a critical role in IL-13-stimulated eotaxin generation and STAT6 activation. While showing no obvious effect on aPKC expression or phosphorylation, γ-TE treatment resulted in increased expression of prostate-apoptosis-response 4 (PAR4), an endogenous negative regulator of aPKCs. Importantly, γ-TE treatment led to enhanced formation of aPKC/PAR4 complex that is known to reduce aPKC activity via protein-protein crosstalk. Our study demonstrated that γ-TE inhibited IL-13/STAT6-activated eotaxin secretion via up-regulation of PAR4 expression and enhancement of aPKC-PAR4 complex formation. These results support the notion that specific vitamin E forms may be useful anti-asthmatic agents.

Acid and base secretion in freshly excised nasal tissue from cystic fibrosis patients with ΔF508 mutation.

BACKGROUND: Cystic fibrosis (CF) is caused by a misfunctional CF transmembrane conductance regulator (CFTR) protein, which is believed to contributes to the regulation of the airway surface liquid (ASL) pH. This study investigated acid and base secretion in freshly excised human nasal tissues from CF patients homozygous for the ΔF508 mutation. METHODS: Human nasal mucosa was collected during sinus surgery and investigated in Ussing chambers. Mucosal equilibrium pH values and rate of acid and base secretion were determined using the pH-stat technique. RESULTS: The equilibrium pH of nasal epithelia from ΔF508 CF patients with chronic rhinosinusitis (CRS) was pH = 7.08 ± 0.09 and was significantly lower compared to nasal epithelia from CRS patients without CF (pH = 7.33 ± 0.06) and normal subjects (pH = 7.34 ± 0.08, n = 6). The rate of base secretion in CF nasal tissues was 11.8 ± 2.4 nmol · min(−1) · cm(−2), which was significantly lower than normal (57.2 ± 9.2 nmol · min(−1) · cm(−2)). The HCO3(−) secretory rate was further increased by forskolin by 16.1% in normal, but not in CF tissues. CONCLUSION: Our data suggests that CF patients exhibited significantly lower base secretion by the nasal airway epithelium. It is possible that improper regulation of ASL pH in CF may negatively impact the innate host defense system.

A phospholipid-apolipoprotein A-I nanoparticle containing amphotericin B as a drug delivery platform with cell membrane protective properties.

Amphotericin B (AMB), a potent antifungal agent, has been employed as an inhalable therapy for pulmonary fungal infections. We recently described a novel nano-sized delivery vehicle composed of phospholipid (PL) and apolipoprotein A-I, NanoDisk (ND), to which we added AMB as a payload (ND-AMB). The goal of the present study was to evaluate whether ND-AMB, compared to other formulations, preserves lung cell integrity in vitro, as AMB can be toxic to mammalian cells and reduce lung function when inhaled. Epithelial integrity was assessed by measuring K(+) ion flux across a model airway epithelium, Calu-3 cells. In this assay ND-AMB was at least 8-fold less disruptive than AMB/deoxycholate (DOC). Cell viability studies confirmed this observation. Unexpectedly, the ND vehicle restored the integrity of a membrane compromised by prior exposure to AMB. An alternative formulation of ND-AMB containing a high load of AMB per ND was not protective, suggesting that ND with a low ratio of AMB to PL can sequester additional AMB from membranes. ND-AMB also protected HepG2 cells from the cytotoxicity of AMB, as determined by cellular viability and lactate dehydrogenase (LDH) levels. This study suggests that ND-AMB may be safe for administration via inhalation and reveals a unique activity whereby ND-AMB protects lung epithelial membranes from AMB toxicity.

Pseudomonas aeruginosa Homoserine lactone activates store-operated cAMP and cystic fibrosis transmembrane regulator-dependent Cl- secretion by human airway epithelia.

The ubiquitous bacterium Pseudomonas aeruginosa frequently causes hospital-acquired infections. P. aeruginosa also infects the lungs of cystic fibrosis (CF) patients and secretes N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) to regulate bacterial gene expression critical for P. aeruginosa persistence. In addition to its effects as a quorum-sensing gene regulator in P. aeruginosa, 3O-C12 elicits cross-kingdom effects on host cell signaling leading to both pro- or anti-inflammatory effects. We find that in addition to these slow effects mediated through changes in gene expression, 3O-C12 also rapidly increases Cl(-) and fluid secretion in the cystic fibrosis transmembrane regulator (CFTR)-expressing airway epithelia. 3O-C12 does not stimulate Cl(-) secretion in CF cells, suggesting that lactone activates the CFTR. 3O-C12 also appears to directly activate the inositol trisphosphate receptor and release Ca(2+) from the endoplasmic reticulum (ER), lowering [Ca(2+)] in the ER and thereby activating the Ca(2+)-sensitive ER signaling protein STIM1. 3O-C12 increases cytosolic [Ca(2+)] and, strikingly, also cytosolic [cAMP], the known activator of CFTR. Activation of Cl(-) current by 3O-C12 was inhibited by a cAMP antagonist and increased by a phosphodiesterase inhibitor. Finally, a Ca(2+) buffer that lowers [Ca(2+)] in the ER similar to the effect of 3O-C12 also increased cAMP and I(Cl). The results suggest that 3O-C12 stimulates CFTR-dependent Cl(-) and fluid secretion in airway epithelial cells by activating the inositol trisphosphate receptor, thus lowering [Ca(2+)] in the ER and activating STIM1 and store-operated cAMP production. In CF airways, where CFTR is absent, the adaptive ability to rapidly flush the bacteria away is compromised because the lactone cannot affect Cl(-) and fluid secretion.

CFTR and calcium-activated chloride channels in primary cultures of human airway gland cells of serous or mucous phenotype.

Using cell culture models, we have investigated the relative importance of cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels (CaCC) in Cl secretion by mucous and serous cells of human airway glands. In transepithelial recordings in Ussing chambers, the CFTR inhibitor CFTR(inh)-172 abolished 60% of baseline Cl secretion in serous cells and 70% in mucous. Flufenamic acid (FFA), an inhibitor of CaCC, reduced baseline Cl secretion by ∼20% in both cell types. Methacholine and ATP stimulated Cl secretion in both cell types, which was largely blocked by treatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and partially by mucosal FFA or CFTR(inh)-172 with the exception of methacholine responses in mucous cells, which were not blocked by FFA and partially (∼60%) by CFTR(inh)-172. The effects of ionomycin on short-circuit current (I(sc)) were less than those of ATP or methacholine. Forskolin stimulated Cl secretion only if Cl in the mucosal medium was replaced by gluconate. In whole cell patch-clamp studies of single isolated cells, cAMP-induced Cl currents were ∼3-fold greater in serous than mucous cells. Ionomycin-induced Cl currents were 13 times (serous) or 26 times (mucous) greater than those generated by cAMP and were blocked by FFA. In serous cells, mRNA for transmembrane protein 16A (TMEM16A) was ∼10 times more abundant than mRNA for CFTR. In mucous cells it was ∼100 times more abundant. We conclude: 1) serous and mucous cells both make significant contributions to gland fluid secretion; 2) baseline Cl secretion in both cell types is mediated predominantly by CFTR, but CaCC becomes increasingly important after mediator-induced elevations of intracellular Ca; and 3) the high CaCC currents seen in patch-clamp studies and the high TMEM16A expression in intact polarized cells sheets are not reflected in transepithelial current recordings.