Apical CFTR Expression in Human Nasal Epithelium Correlates with Lung Disease in Cystic Fibrosis

Introduction

Although most individuals with cystic fibrosis (CF) develop progressive obstructive lung disease, disease severity is highly variable, even for individuals with similar CFTR mutations. Measurements of chloride transport as expression of CFTR function in nasal epithelial cells correlate with pulmonary function and suggest that F508del-CFTR is expressed at the apical membrane. However, an association between quantitative apical CFTR expression in nasal epithelium and CF disease severity is still missing.

Methods and Materials

Nasal epithelial cells from healthy individuals and individuals with CF between 12–18 years were obtained by nasal brushing. Apical CFTR expression was measured by confocal microscopy using CFTR mAb 596. Expression was compared between both groups and expression in CF nasal epithelial cells was associated with standardized pulmonary function (FEV₁%).

Results

The proportion of cells expressing apical CFTR in columnar epithelium is lower in CF compared to non-CF. The apical CFTR expression level was significantly correlated with FEV₁% in F508del homozygous subjects (r = 0.63, p = 0.012).

Conclusion

CFTR expression in nasal epithelial cells is lower in subjects with CF compared to healthy subjects. The proportion of cells expressing F508del-CFTR at the apical membrane is variable between subjects and is positively correlated with FEV₁% in F508del-CFTR homozygous subjects.     

Characterization of Nasal Potential Difference in cftr Knockout and F508del-CFTR Mice

Background

Treatments designed to correct cystic fibrosis transmembrane conductance regulator (CFTR) defects must first be evaluated in preclinical experiments in the mouse model of cystic fibrosis (CF). Mice nasal mucosa mimics the bioelectric defect seen in humans. The use of nasal potential difference (V_TE) to assess ionic transport is a powerful test evaluating the restoration of CFTR function. Nasal V_TE in CF mice must be well characterized for correct interpretation.

Methods

We performed V_TE measurements in large-scale studies of two mouse models of CF—B6;129 cftr knockout and FVB F508del-CFTR—and their respective wild-type (WT) littermates. We assessed the repeatability of the test for cftr knockout mice and defined cutoff points distinguishing between WT and F508del-CFTR mice.

Results

We determined the typical V_TE values for CF and WT mice and demonstrated the existence of residual CFTR activity in F508del-CFTR mice. We characterized intra-animal variability in B6;129 mice and defined the cutoff points for F508del-CFTR chloride secretion rescue. Hyperpolarization of more than -2.15 mV after perfusion with a low-concentration Cl^- solution was considered to indicate a normal response.

Conclusions

These data will make it possible to interpret changes in nasal V_TE in mouse models of CF, in future preclinical studies.     

Characterization of pediatric cystic fibrosis airway epithelial cell cultures at the air-liquid interface obtained by non-invasive nasal cytology brush sampling

Background

In vitro systems of primary cystic fibrosis (CF) airway epithelial cells are an important tool to study molecular and functional features of the native respiratory epithelium. However, undifferentiated CF airway cell cultures grown under submerged conditions do not appropriately represent the physiological situation. A more advanced CF cell culture system based on airway epithelial cells grown at the air-liquid interface (ALI) recapitulates most of the in vivo-like properties but requires the use of invasive sampling methods. In this study, we describe a detailed characterization of fully differentiated primary CF airway epithelial cells obtained by non-invasive nasal brushing of pediatric patients.

Methods

Differentiated cell cultures were evaluated with immunolabelling of markers for ciliated, mucus-secreting and basal cells, and tight junction and CFTR proteins. Epithelial morphology and ultrastructure was examined by histology and transmission electron microscopy. Ciliary beat frequency was investigated by a video-microscopy approach and trans-epithelial electrical resistance was assessed with an epithelial Volt-Ohm meter system. Finally, epithelial permeability was analysed by using a cell layer integrity test and baseline cytokine levels where measured by an enzyme-linked immunosorbent assay.

Results

Pediatric CF nasal cultures grown at the ALI showed a differentiation into a pseudostratified epithelium with a mucociliary phenotype. Also, immunofluorescence analysis revealed the presence of ciliated, mucus-secreting and basal cells and tight junctions. CFTR protein expression was observed in CF (F508del/F508del) and healthy cultures and baseline interleukin (IL)-8 and IL-6 release were similar in control and CF ALI cultures. The ciliary beat frequency was 9.67 Hz and the differentiated pediatric CF epithelium was found to be functionally tight.

Conclusion

In summary, primary pediatric CF nasal epithelial cell cultures grown at the ALI showed full differentiation into ciliated, mucus-producing and basal cells, which adequately reflect the in vivo properties of the human respiratory epithelium.

     

Accumulation of metals in GOLD4 COPD lungs is associated with decreased CFTR levels

Background

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that primarily resides in airway epithelial cells. Decreased CFTR expression and/or function lead to impaired airway surface liquid (ASL) volume homeostasis, resulting in accumulation of mucus, reduced clearance of bacteria, and chronic infection and inflammation.

Methods

Expression of CFTR and the cigarette smoke metal content were assessed in lung samples of controls and COPD patients with established GOLD stage 4. CFTR protein and mRNA were quantified by immunohistochemistry and quantitative RT-PCR, respectively. Metals present in lung samples were quantified by ICP-AES. The effect of cigarette smoke on down-regulation of CFTR expression and function was assessed using primary human airway epithelial cells. The role of leading metal(s) found in lung samples of GOLD 4 COPD patients involved in the alteration of CFTR was confirmed by exposing human bronchial epithelial cells 16HBE14o- to metal-depleted cigarette smoke extracts.

Results

We found that CFTR expression is reduced in the lungs of GOLD 4 COPD patients, especially in bronchial epithelial cells. Assessment of metals present in lung samples revealed that cadmium and manganese were significantly higher in GOLD 4 COPD patients when compared to control smokers (GOLD 0). Primary human airway epithelial cells exposed to cigarette smoke resulted in decreased expression of CFTR protein and reduced airway surface liquid height. 16HBE14o-cells exposed to cigarette smoke also exhibited reduced levels of CFTR protein and mRNA. Removal and/or addition of metals to cigarette smoke extracts before exposure established their role in decrease of CFTR in airway epithelial cells.

Conclusions

CFTR expression is reduced in the lungs of patients with severe COPD. This effect is associated with the accumulation of cadmium and manganese suggesting a role for these metals in the pathogenesis of COPD.     

Impact of heterozygote CFTR Mutations in COPD patients with Chronic Bronchitis

Background

Cigarette smoking causes Chronic Obstructive Pulmonary Disease (COPD), the 3rd leading cause of death in the U.S. CFTR ion transport dysfunction has been implicated in COPD pathogenesis, and is associated with chronic bronchitis. However, susceptibility to smoke induced lung injury is variable and the underlying genetic contributors remain unclear. We hypothesized that presence of CFTR mutation heterozygosity may alter susceptibility to cigarette smoke induced CFTR dysfunction. Consequently, COPD patients with chronic bronchitis may have a higher rate of CFTR mutations compared to the general population.

Methods

Primary human bronchial epithelial cells derived from F508del CFTR heterozygotes and mice with (CFTR+/-) and without (CFTR+/+) CFTR heterozygosity were exposed to whole cigarette smoke (WCS); CFTR-dependent ion transport was assessed by Ussing chamber electrophysiology and nasal potential difference measurements, respectively. Caucasians with COPD and chronic bronchitis, age 40 to 80 with FEV₁/FVC < 0.70 and FEV₁ < 60% predicted, were selected for genetic analysis from participants in the NIH COPD Clinical Research Network’s Azithromycin for Prevention of Exacerbations of COPD in comparison to 32,900 Caucasian women who underwent prenatal genetic testing. Genetic analysis involved an allele-specific genotyping of 89 CFTR mutations.

Results

Exposure to WCS caused a pronounced reduction in CFTR activity in both CFTR (+/+) cells and F508del CFTR (+/-) cells; however, neither the degree of decrement (44.7% wild-type vs. 53.5% F508del heterozygous, P = NS) nor the residual CFTR activity were altered by CFTR heterozygosity. Similarly, WCS caused a marked reduction in CFTR activity measured by NPD in both wild type and CFTR heterozygous mice, but the severity of decrement (91.1% wild type vs. 47.7% CF heterozygous, P = NS) and the residual activity were not significantly affected by CFTR genetic status. Five of 127 (3.9%) COPD patients with chronic bronchitis were heterozygous for CFTR mutations which was not significantly different from controls (4.5%) (P = NS).

Conclusions

The magnitude of WCS induced reductions in CFTR activity was not affected by the presence of CFTR mutation heterozygosity. CFTR mutations do not increase the risk of COPD with chronic bronchitis. CFTR dysfunction due to smoking is primarily an acquired phenomenon and is not affected by the presence of congenital CFTR mutations.     

Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis

Background

Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis.

Methods

Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age.

Results

Membrane cholesterol measurements are elevated in both R117H and ΔF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTR_inh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility.

Conclusions

The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content.     

Influence of the cystic fibrosis transmembrane conductance regulator on expression of lipid metabolism-related genes in dendritic cells

Background

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Infections of the respiratory tract are a hallmark in CF. The host immune responses in CF are not adequate to eradicate pathogens, such as P. aeruginosa. Dendritic cells (DC) are crucial in initiation and regulation of immune responses. Changes in DC function could contribute to abnormal immune responses on multiple levels. The role of DC in CF lung disease remains unknown.

Methods

This study investigated the expression of CFTR gene in bone marrow-derived DC. We compared the differentiation and maturation profile of DC from CF and wild type (WT) mice. We analyzed the gene expression levels in DC from naive CF and WT mice or following P. aeruginosa infection.

Results

CFTR is expressed in DC with lower level compared to lung tissue. DC from CF mice showed a delayed in the early phase of differentiation. Gene expression analysis in DC generated from naive CF and WT mice revealed decreased expression of Caveolin-1 (Cav1), a membrane lipid raft protein, in the CF DC compared to WT DC. Consistently, protein and activity levels of the sterol regulatory element binding protein (SREBP), a negative regulator of Cav1 expression, were increased in CF DC. Following exposure to P. aeruginosa, expression of 3β-hydroxysterol-Δ7 reductase (Dhcr7) and stearoyl-CoA desaturase 2 (Scd2), two enzymes involved in the lipid metabolism that are also regulated by SREBP, was less decreased in the CF DC compared to WT DC.

Conclusion

These results suggest that CFTR dysfunction in DC affects factors involved in membrane structure and lipid-metabolism, which may contribute to the abnormal inflammatory and immune response characteristic of CF.     

5’-adenosine monophosphate mediated cooling treatment enhances ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) stability in vivo

Background

Gene mutations that produce misprocessed proteins are linked to many human disorders. Interestingly, some misprocessed proteins retained their biological function when stabilized by low temperature treatment of cultured cells in vitro. Here we investigate whether low temperature treatment in vivo can rescue misfolded proteins by applying 5’-AMP mediated whole body cooling to a Cystic Fibrosis (CF) mouse model carrying a mutant cystic fibrosis transmembrane conductance regulator (CFTR) with a deletion of the phenylalanine residue in position 508 (ΔF508-CFTR). Low temperature treatment of cultured cells was previously shown to be able to alleviate the processing defect of ΔF508-CFTR, enhancing its plasma membrane localization and its function in mediating chloride ion transport.

Results

Here, we report that whole body cooling enhanced the retention of ΔF508-CFTR in intestinal epithelial cells. Functional analysis based on β-adrenergic dependent salivary secretion and post-natal mortality rate revealed a moderate but significant improvement in treated compared with untreated CF mice.

Conclusions

Our findings demonstrate that temperature sensitive processing of mutant proteins can be responsive to low temperature treatment in vivo.     

The ΔF508-CFTR mutation inhibits wild-type CFTR processing and function when co-expressed in human airway epithelia and in mouse nasal mucosa

Background

Rescue or correction of CFTR function in native epithelia is the ultimate goal of CF therapeutics development. Wild-type (WT) CFTR introduction and replacement is also of particular interest. Such therapies may be complicated by possible CFTR self-assembly into an oligomer or multimer.

Results

Surprisingly, functional CFTR assays in native airway epithelia showed that the most common CFTR mutant, ??F508-CFTR (??F-CFTR), inhibits WT-CFTR when both forms are co-expressed. To examine more mechanistically, both forms of CFTR were transfected transiently in varying amounts into IB3-1 CF human airway epithelial cells and HEK-293 human embryonic kidney cells null for endogenous CFTR protein expression. Increasing amounts of ??F-CFTR inhibited WT-CFTR protein processing and function in CF human airway epithelial cells but not in heterologous HEK-293 cells. Stably expressed ??F-CFTR in clones of the non-CF human airway epithelial cell line, CALU-3, also showed reduction in cAMP-stimulated anion secretion and in WT-CFTR processing. An ultimate test of this dominant negative-like effect of ??F-CFTR on WT-CFTR was the parallel study of two different CF mouse models: the ??F-CFTR mouse and the bitransgenic CFTR mouse corrected in the gut but null in the lung and airways. WT/??F heterozygotes had an intermediate phenotype with regard to CFTR agonist responses in in vivo nasal potential difference (NPD) recordings and in Ussing chamber recordings of short-circuit current (ISC) in vitro on primary tracheal epithelial cells isolated from the same mice. In contrast, CFTR bitransgenic +/? heterozygotes had no difference in their responses versus +/+ wild-type mice.

Conclusions

Taken altogether, these data suggest that ??F-CFTR and WT-CFTR co-assemble into an oligomeric macromolecular complex in native epithelia and share protein processing machinery and regulation at the level of the endoplasmic reticulum (ER). As a consequence, ??F-CFTR slows WT-CFTR protein processing and limits its expression and function in the apical membrane of native airway epithelia. Implications of these data for the relative health of CF heterozygous carriers, for CFTR protein processing in native airway epithelia, and for the relative efficacy of different CF therapeutic approaches is significant and is discussed.     

Deprived TLR9 Expression in Apparently Healthy Nasal Mucosa Might Trigger Polyp-Growth in Chronic Rhinosinusitis Patients

Background

The origin of nasal polyps in chronic rhinosinusitis is unknown, but the role of viral infections in polyp growth is clinically well established. Toll-like receptors (TLRs) have recently emerged as key players in our local airway defense against microbes. Among these, TLR9 has gained special interest in viral diseases. Many studies on chronic rhinosinusitis with nasal polyps (CRSwNP) compare polyp tissue with nasal mucosa from polyp-free individuals. Knowledge about changes in the turbinate tissue bordering the polyp tissue is limited.

Objectives

To analyse the role of TLR9 mediated microbial defense in tissue bordering the polyp.

Methods

Nasal polyps and turbinate tissue from 11 patients with CRSwNP and turbinate tissue from 11 healthy controls in total were used. Five biopsies from either group were analysed immediately with flow cytometry regarding receptor expression and 6 biopsies were used for in vitro stimulation with a TLR9 agonist, CpG. Cytokine release was analysed using Luminex. Eight patients with CRSwNP in total were intranasally challenged with CpG/placebo 24 hours before surgery and the biopsies were collected and analysed as above.

Results

TLR9 expression was detected on turbinate epithelial cells from healthy controls and polyp epithelial cells from patients, whereas TLR9 was absent in turbinate epithelial cells from patients. CpG stimulation increased the percentage cells expressing TLR9 and decreased percentage cells expressing VEGFR2 in turbinate tissue from patients. After CpG stimulation the elevated levels of IL-6, G-CSF and MIP-1β in the turbinate tissue from patients were reduced towards the levels demonstrated in healthy controls.

Conclusion

Defects in the TLR9 mediated microbial defense in the mucosa adjacent to the anatomic origin of the polyp might explain virus induced polyp growth. CpG stimulation decreased VEGFR2, suggesting a role for CpG in polyp formation. The focus on turbinate tissue in patients with CRSwNP opens new perspectives in CRSwNP-research.     

ABCC1 Is Related to the Protection of the Distal Nephron against Hyperosmolality and High Sodium Environment: Possible Implications for Cancer Chemotherapy

Aims

Glutathione (GSH) plays an important role in protecting cells against oxidative damage. ABCC1 protein transports GSH. Although this protein is largely studied in cancer, due to multidrug resistance phenotype, its role in the tubular cells of the kidney is unknown. The goal of this study was to find out whether ABCC1 has a role in protecting cells from the distal nephron against the stress caused by high medullar osmolality.

Main Methods

MA104 cells were treated with high concentrations of sodium chloride, urea, or both to raise the osmolality of the culture medium. Cell viability was accessed by MTT and trypan blue assays. ABCC1 expression and extrusion of carboxi-fluorescein (CF), a fluorescent ABCC1 substrate, were measured by flow cytometry.

Key Findings

Incubation of MA104 cells in a high sodium concentration medium resulted in changes in cell granularity and altered expression and activity of ABCC1. Urea did not alter ABCC1 expression or activity, but reversed the observed NaCl effects. High sodium concentrations also had a negative effect on cell viability and urea also protected cells against this effect.

Significance

Our findings demonstrate that ABCC1 plays a significant role in the protection of kidney epithelial cells against the stress caused by high sodium environment present in renal medulla.     

RNA interference for CFTR attenuates lung fluid absorption at birth in rats

Background

Small interfering RNA (siRNA) against αENaC (α-subunit of the epithelial Na channel) and CFTR (cystic fibrosis transmembrane conductance regulator) was used to explore ENaC and CTFR function in newborn rat lungs.

Methods

Twenty-four hours after trans-thoracic intrapulmonary (ttip) injection of siRNA-generating plasmid DNA (pSi-0, pSi-4, or pSi-C₂), we measured CFTR and ENaC expression, extravascular lung water, and mortality.

Results

αENaC and CFTR mRNA and protein decreased by ~80% and ~85%, respectively, following αENaC and CFTR silencing. Extravascular lung water and mortality increased after αENaC and CFTR-silencing. In pSi-C₂-transfected isolated DLE cells there were attenuated CFTR mRNA and protein. In pSi-4-transfected DLE cells αENaC mRNA and protein were both reduced. Interestingly, CFTR-silencing also reduced αENaC mRNA and protein. αENaC silencing, on the other hand, only slightly reduced CFTR mRNA and protein.

Conclusion

Thus, ENaC and CFTR are both involved in the fluid secretion to absorption conversion around at birth.     

Electrophysiological evaluation of Cystic Fibrosis Conductance Transmembrane Regulator (CFTR) expression in human monocytes

Background

Cystic fibrosis is caused by mutations of CFTR gene, a protein kinase A-activated anion channel, and is associated to a persistent and excessive chronic lung inflammation, suggesting functional alterations of immune cells. Leukocytes express detectable levels of CFTR but the molecule has not been fully characterized in these cells.

Methods

Freshly isolated monocytes from healthy individuals and CF patients were assessed by protein expression, single cell electrophysiological and membrane depolarization assays.

Results

We recorded chloride currents by patch clamp in healthy monocytes, after the administration of a CFTR stimulus. Currents were sensitive to a specific blocker of the CFTR channel, CFTR_inh-172 and were absent in CF monocytes. Next, we evaluated the effects of ex vivo exposure of monocytes from cystic fibrosis patients carrying the F508del mutation to a chemical corrector, Vertex-325. We found an increase in CFTR expression by confocal microscopy and a recovery of CFTR function by both patch clamp and single cell fluorescence analysis.

Conclusions

We confirm the expression of functional CFTR in human monocytes and demonstrate that blood monocytes can represent an adequate source of primary cells to assess new therapies and define diagnosis of CF.

General significance

Tests to evaluate CFTR functional abnormalities in CF disease might greatly benefit from the availability of a convenient source of primary cells. This electrophysiological study promotes the use of monocytes as a minimally invasive tool to study and monitor CFTR function in individual patients.     

Nasal Epithelial Cells Can Act as a Physiological Surrogate for Paediatric Asthma Studies

Introduction

Differentiated paediatric epithelial cells can be used to study the role of epithelial cells in asthma. Nasal epithelial cells are easier to obtain and may act as a surrogate for bronchial epithelium in asthma studies. We assessed the suitability of nasal epithelium from asthmatic children to be a surrogate for bronchial epithelium using air-liquid interface cultures.

Methods

Paired nasal and bronchial epithelial cells from asthmatic children (n = 9) were differentiated for 28 days under unstimulated and IL-13-stimulated conditions. Morphological and physiological markers were analysed using immunocytochemistry, transepithelial-electrical-resistance, Quantitative Real-time-PCR, ELISA and multiplex cytokine/chemokine analysis.

Results

Physiologically, nasal epithelial cells from asthmatic children exhibit similar cytokine responses to stimulation with IL-13 compared with paired bronchial epithelial cells. Morphologically however, nasal epithelial cells differed significantly from bronchial epithelial cells from asthmatic patients under unstimulated and IL-13-stimulated conditions. Nasal epithelial cells exhibited lower proliferation/differentiation rates and lower percentages of goblet and ciliated cells when unstimulated, while exhibiting a diminished and varied response to IL-13.

Conclusions

We conclude that morphologically, nasal epithelial cells would not be a suitable surrogate due to a significantly lower rate of proliferation and differentiation of goblet and ciliated cells. Physiologically, nasal epithelial cells respond similarly to exogenous stimulation with IL-13 in cytokine production and could be used as a physiological surrogate in the event that bronchial epithelial cells are not available.     

Diminished levels of nasal S100A7 (psoriasin) in seasonal allergic rhinitis: an effect mediated by Th2 cytokines

Background

S100A7 is an antimicrobial peptide involved in several inflammatory diseases. The aim of the present study was to explore the expression and regulation of S100A7 in seasonal allergic rhinitis (SAR).

Methods

Nasal lavage (NAL) fluid was obtained from healthy controls before and after lipopolysaccharide (LPS) provocation, from SAR patients before and after allergen challenge, and from SAR patients having completed allergen-specific immunotherapy (ASIT). Nasal biopsies, nasal epithelial cells and blood were acquired from healthy donors. The airway epithelial cell line FaDu was used for in vitro experiments. Real-time RT-PCR and immunohistochemistry were used to determine S100A7 expression in nasal tissue and cells. Release of S100A7 in NAL and culture supernatants was measured by ELISA. The function of recombinant S100A7 was explored in epithelial cells, neutrophils and peripheral blood mononuclear cells (PBMC).

Results

Nasal administration of LPS induced S100A7 release in healthy non-allergic subjects. The level of S100A7 was lower in NAL from SAR patients than from healthy controls, and it was further reduced in the SAR group 6 h post allergen provocation. In contrast, ASIT patients displayed higher levels after completed treatment. S100A7 was expressed in the nasal epithelium and in glands, and it was secreted by cultured epithelial cells. Stimulation with IL-4 and histamine repressed the epithelial S100A7 release. Further, recombinant S100A7 induced activation of neutrophils and PBMC.

Conclusions

The present study shows an epithelial expression and excretion of S100A7 in the nose after microbial stimulation. The levels are diminished in rhinitis patients and in the presence of an allergic cytokine milieu, suggesting that the antimicrobial defense is compromised in patients with SAR.