Down-regulation of the anti-inflammatory protein annexin A1 in cystic fibrosis knock-out mice and patients

Cystic fibrosis is a fatal human genetic disease caused by mutations in the CFTR gene encoding a cAMP-activated chloride channel. It is characterized by abnormal fluid transport across secretory epithelia and chronic inflammation in lung, pancreas, and intestine. Because cystic fibrosis (CF) pathophysiology cannot be explained solely by dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR), we applied a proteomic approach (bidimensional electrophoresis and mass spectrometry) to search for differentially expressed proteins between mice lacking cftr (cftr(tm1Unc), cftr-/-) and controls using colonic crypts from young animals, i.e. prior to the development of intestinal inflammation. By analyzing total proteins separated in the range of pH 6-11, we detected 24 differentially expressed proteins (>2-fold). In this work, we focused on one of these proteins that was absent in two-dimensional gels from cftr-/- mice. This protein spot (molecular mass, 37 kDa; pI 7) was identified by mass spectrometry as annexin A1, an anti-inflammatory protein. Interestingly, annexin A1 was also undetectable in lungs and pancreas of cftr-/- mice, tissues known to express CFTR. Absence of this inhibitory mediator of the host inflammatory response was associated with colonic up-regulation of the proinflammatory cytosolic phospholipase A2. More importantly, annexin A1 was down-regulated in nasal epithelial cells from CF patients bearing homozygous nonsense mutations in the CFTR gene (Y122X, 489delC) and differentially expressed in F508del patients. These results suggest that annexin A1 may be a key protein involved in CF pathogenesis especially in relation to the not well defined field of inflammation in CF. We suggest that decreased expression of annexin A1 contributes to the worsening of the CF phenotype.     

Innervation of pulmonary neuroendocrine cells and neuroepithelial bodies in developing rabbit lung.

We investigated the development of innervation of the pulmonary neuroendocrine cell (PNEC) system composed of single cells and organoid cell clusters, neuroepithelial bodies (NEB) in rabbit fetal and neonatal lungs. To visualize the nerve fibers and their contacts with PNECs/NEBs, we used confocal microscopy and multilabel immunohistochemistry (IHC) with pan-neural marker, synaptic vesicle protein 2 (SV2), and serotonin (5-HT) as markers for PNECs/NEBs, and smooth muscle actin or cytokeratin to identify airway landmarks. The numbers and distribution of PNEC/NEB at different stages of lung development (E16, 18, 21, 26, and P2) and the density of innervation were quantified. First PNECs immunoreactive for 5-HT were identified in primitive airway epithelium at E18 as single cells or as small cell clusters with or without early nerve contacts. At E21 a significant increase in the number of PNECs with formation of early innervated NEB corpuscules was observed. The overall numbers of PNECs/NEBs and the density of mucosal, submucosal, and intercorpuscular innervation increased with progressing gestation and peaked postnatally (P2). At term, the majority of NEBs and single PNECs within airway mucosa possessed neural contacts. Such an extensive and complex innervation of the PNEC system indicates a multifunctional role in developing lung and during neonatal adaptation.     

The porcine lung as a potential model for cystic fibrosis

Airway disease currently causes most of the morbidity and mortality in patients with cystic fibrosis (CF). However, understanding the pathogenesis of CF lung disease and developing novel therapeutic strategies have been hampered by the limitations of current models. Although the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) has been targeted in mice, CF mice fail to develop lung or pancreatic disease like that in humans. In many respects, the anatomy, biochemistry, physiology, size, and genetics of pigs resemble those of humans. Thus pigs with a targeted CFTR gene might provide a good model for CF. Here, we review aspects of porcine airways and lung that are relevant to CF.     

Can correcting the ΔF508-CFTR proteostasis-defect rescue CF lung disease?

Protein homeostasis (proteostasis) generates and maintains individual proteins in their folded and functional-competent states. The components of the cellular proteostasis machinery also dictate the functional lifetime of a protein by constantly regulating its conformation, concentration and subcellular location. The autosomal recessive disease cystic fibrosis (CF) is caused by a proteostasis-defect in CF transmembrane conductance regulator (CFTR). The most common CF mutation leading to this proteostasis-defect is the deletion of a phenylalanine residue at position 508 (ΔF508) of the CFTR protein. This ΔF508-CFTR protein is prone to aberrant folding, increased ER-associated degradation, atypical intracellular trafficking and reduced stability at the apical membrane. This ΔF508-CF proteostasis-defect leads to an obstructive lung disease characterized by impaired ion transport in airway epithelial cells, mucus buildup in air space and chronic airway inflammation. We assess here whether correcting the underlying defect in ΔF508-CFTR protein processing using therapeutic proteostasis regulators can treat chronic CF lung disease. As a proof of concept, recent studies support that the selective modulation of mutant-CFTR proteostasis may offer promising therapies to reverse chronic CF lung disease.     

Neuronal developmental marker FORSE-1 identifies a putative progenitor of the pulmonary neuroendocrine cell lineage during lung development.

The FORSE-1 (forebrain-surface-embryonic) monoclonal antibody (MAb) recognizes a carbohydrate cell surface epitope related to the Lewis-X (LeX) and stage-specific embryonic antigens (SSEAs). In the developing CNS, the FORSE-1 epitope is believed to serve as a marker of progenitor cells. We studied the expression of the FORSE-1 epitope in pulmonary neuroendocrine cells (PNECs) and related neuroepithelial bodies (NEBs), cell types implicated in paracrine regulation of lung development. We used dual immunolabeling to identify PNECs/NEBs in tissue sections from developing rabbit fetal lungs and corresponding primary lung cell cultures. During the early stage (E16), the FORSE-1 MAb labeled primitive airway epithelium, whereas serotonin (5HT) immunoreactivity, a marker of PNEC/NEB differentiation, was negative. After E18, FORSE-1 labeling became restricted to PNECs and NEBs, identified by co-expression with 5HT, then decreased coincident with an increase in 5HT. Expression of the FORSE-1 epitope correlated inversely with 5HT expression in PNEC/NEB cells. FORSE-1 immunoreactivity correlated with cell proliferation assessed by BrdU labeling. Downregulation of the FORSE-1 epitope correlated with maturation of PNECs/NEBs. The presence of few FORSE-1/5HT-positive cells in postnatal lung suggests retention of progenitors. The FORSE-1 epitope was associated with a high molecular weight (286 kD) glycoprotein that decreased with increasing gestational age, as demonstrated by immunoblotting. Overall expression of SSEA-1, -3, and -4 antigens was similar to FORSE-1/5HT, although the former was preferentially localized to neurite-like processes. Because the role of the FORSE-1 epitope in the CNS probably involves cell adhesion and differentiation, we propose a similar function in developing lung. The demonstration of LeX/SSEA antigen expression in the PNEC/NEB cell lineage underscores the importance of these cells in developing lung. Furthermore, the FORSE-1 antigen may identify committed progenitors of the PNEC/NEB cell system.     

Neurochemical pattern of the complex innervation of neuroepithelial bodies in mouse lungs

As best characterized for rats, it is clear that pulmonary neuroepithelial bodies (NEBs) are contacted by a plethora of nerve fiber populations, suggesting that they represent an extensive group of multifunctional intraepithelial airway receptors. Because of the importance of genetically modified mice for functional studies, and the current lack of data, the main aim of the present study was to achieve a detailed analysis of the origin and neurochemical properties of nerve terminals associated with NEBs in mouse lungs. Antibodies against known selective markers for sensory and motor nerve terminals in rat lungs were used on lungs from control and vagotomized mice of two different strains, i.e., Swiss and C57-Bl6. NEB cells were visualized by antibodies against either the general neuroendocrine marker protein gene-product 9.5 (PGP9.5) or calcitonin gene-related peptide (CGRP). Thorough immunohistochemical examination of NEB cells showed that some of these NEB cells also exhibit calbindin D-28 k (CB) and vesicular acetylcholine transporter (VAChT) immunoreactivity (IR). Mouse pulmonary NEBs were found to receive intraepithelial nerve terminals of at least two different populations of myelinated vagal afferents: (1) Immunoreactive (ir) for vesicular glutamate transporters (VGLUTs) and CB; (2) expressing P2X₂ and P2X₃ ATP receptors. CGRP IR was seen in varicose vagal nerve fibers and in delicate non-vagal fibers, both in close proximity to NEBs. VAChT immunostaining showed very weak IR in the NEB-related intraepithelial vagal sensory nerve terminals. nNOS- or VIP-ir nerve terminals could be observed at the base of pulmonary NEBs. While a single NEB can be contacted by multiple nerve fiber populations, it was clear that none of the so far characterized nerve fiber populations contacts all pulmonary NEBs. The present study revealed that mouse lungs harbor several populations of nerve terminals that may selectively contact NEBs. Although at present the physiological significance of the innervation pattern of NEBs remains enigmatic, it is likely that NEBs are receptor–effector end-organs that may host complex and/or multiple functional properties in normal airways. The neurochemical information on the innervation of NEBs in mouse lungs gathered in the present study will be essential for the interpretation of upcoming functional data and for the study of transgenic mice.     

Characterization of wild-type and deltaF508 cystic fibrosis transmembrane regulator in human respiratory epithelia

Previous studies in native tissues have produced conflicting data on the localization and metabolic fate of WT and deltaF508 cystic fibrosis transmembrane regulator (CFTR) in the lung. Combining immunocytochemical and biochemical studies utilizing new high-affinity CFTR mAbs with ion transport assays, we examined both 1) the cell type and region specific expression of CFTR in normal airways and 2) the metabolic fate of deltaF508 CFTR and associated ERM proteins in the cystic fibrosis lung. Studies of lungs from a large number of normal subjects revealed that WT CFTR protein localized to the apical membrane of ciliated cells within the superficial epithelium and gland ducts. In contrast, other cell types in the superficial, gland acinar, and alveolar epithelia expressed little WT CFTR protein. No deltaF508 CFTR mature protein or function could be detected in airway specimens freshly excised from a large number of deltaF508 homozygous subjects, despite an intact ERM complex. In sum, our data demonstrate that WT CFTR is predominantly expressed in ciliated cells, and deltaF508 CFTR pathogenesis in native tissues, like heterologous cells, reflects loss of normal protein processing.     

The CFTR and ENaC debate: how important is ENaC in CF lung disease?

Cystic fibrosis (CF) is caused by the loss of the cystic fibrosis transmembrane conductance regulator (CFTR) function and results in a respiratory phenotype that is characterized by dehydrated mucus and bacterial infections that affect CF patients throughout their lives. Much of the morbidity and mortality in CF results from a failure to clear bacteria from the lungs. What causes the defect in the bacterial clearance in the CF lung has been the subject of an ongoing debate. Here we discuss the arguments for and against the role of the epithelial sodium channel, ENaC, in the development of CF lung disease.     

Animal models of chronic lung infection with Pseudomonas aeruginosa: useful tools for cystic fibrosis studies

Cystic fibrosis (CF) is caused by a defect in the transmembrane conductance regulator (CFTR) protein that functions as a chloride channel. Dysfunction of the CFTR protein results in salty sweat, pancreatic insufficiency, intestinal obstruction, male infertility and severe pulmonary disease. In most patients with CF life expectancy is limited due to a progressive loss of functional lung tissue. Early in life a persistent neutrophylic inflammation can be demonstrated in the airways. The cause of this inflammation, the role of CFTR and the cause of lung morbidity by different CF-specific bacteria, mostly Pseudomonas aeruginosa, are not well understood. The lack of an appropriate animal model with multi-organ pathology having the characteristics of the human form of CF has hampered our understanding of the pathobiology and chronic lung infections of the disease for many years. This review summarizes the main characteristics of CF and focuses on several available animal models that have been frequently used in CF research. A better understanding of the chronic lung infection caused particularly by P. aeruginosa, the pathophysiology of lung inflammation and the pathogenesis of lung disease necessitates animal models to understand CF, and to develop and improve treatment.     

Cystic fibrosis: a disorder with defective autophagy

The accumulation of misfolded and/or ubiquitinated protein aggregates with a perturbation of autophagy has been described in several human pathologies. A sequestration of misfolded cystic: fibrosis transmembrane conductance regulator (CFTR) and cross-linked PPARγ has been observed in airway epithelia of cystic fibrosis (CF) patients. CF airways are also characterized by chronic inflammation, pro-oxidative environment and increased transglutaminase 2 (TG2) levels. We showed that defective CFTR drives autophagy inhibition through reactive oxygen species (ROS)-TG2- mediated aggresome sequestration of the Beclin 1 interactome. Rescuing Beclin 1 at the level of the endoplasmic reticulum and autophagy favors clearance of aggresomes, improves CFTR trafficking and ameliorates CF lung inflammation both in vitro and in vivo. Therefore, rescuing autophagy interrupts the vicious cycle linking defective CFTR and lung inflammation and may pave the way to the development of a novel class of drugs for the treatment of CF.     

Frontiers in Research on Cystic Fibrosis: Understanding Its Molecular and Chemical Basis and Relationship to the Pathogenesis of the Disease

In recent years a new family of transport proteins called ABC transporters has emerged. One member of this novel family, called CFTR (cystic fibrosis transmembrane conductance regulator), has received special attention because of its association with the disease cystic fibrosis (CF). This is an inherited disorder affecting about 1 in 2000 Caucasians by impairing epithelial ion transport, particularly that of chloride. Death may occur in severe cases because of chronic lung infections, especially by Pseudomonas aeruginosa, which cause a slow decline in pulmonary function. The prospects of ameliorating the symptoms of CF and even curing the disease were greatly heightened in 1989 following the cloning of the CFTR gene and the discovery that the mutation (F508), which causes most cases of CF, is localized within a putative ATP binding/ATP hydrolysis domain. The purpose of this introductory review in this minireview series is to summarize what we and others have learned during the past eight years about the structure and function of the first nucleotide binding domain (NBF1 or NBD1) of the CFTR protein and the effect thereon of disease-causing mutations. The relationship of these new findings to the pathogenesis of CF is also discussed.     

Pseudomonas aeruginosa pyocyanin inactivates lung epithelial vacuolar ATPase-dependent cystic fibrosis transmembrane conductance regulator expression and localization

Pseudomonas aeruginosa (PA) is a major pathogen causing morbidity and ultimately mortality in patients afflicted with cystic fibrosis (CF) lung disease. One important virulence factor, pyocyanin (PCN), is a blue, redox-active compound that is secreted in such copious amounts by PA in the CF lungs that it determines the colour of expectorated sputum. In this study, we discovered that physiological concentrations of PCN inactivate the airway epithelial vacuolar ATPase, resulting in reduced expression and trafficking of the cystic fibrosis transmembrane conductance regulator in cultured lung and primary nasal epithelial cells. Our study supports the notion that PCN contributes significantly to the pathogenesis of CF and other bronchiectasis patients infected by PA.     

Reduced number of CFTR molecules in erythrocyte plasma membrane of cystic fibrosis patients

Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator). The most frequent mutation, DeltaF508, results in protein misfolding and, as a consequence, prevents CFTR from reaching its final location at the cell surface. CFTR is expressed in various cell types including red blood cells. The functional role of CFTR in erythrocytes is still unclear. Since the number of CFTR copies in a single erythrocyte of healthy donors and CF patients with a homozygous DeltaF508 mutation is unknown, we counted CFTR, localized in erythrocyte plasma membrane, at the single molecule level. A novel experimental approach combining atomic force microscopy with quantum-dot-labeled anti-CFTR antibodies, used as topographic surface markers, was employed to detect individual CFTR molecules. Analysis of erythrocyte plasma membranes taken from healthy donors and CF patients with a homozygous DeltaF508 mutation reveals mean (SEM) values of 698 (12.8) (n=542) and 172 (3.8) (n=538) CFTR molecules per red blood cell, respectively. We conclude that erythrocytes reflect the CFTR status of the organism and that quantification of CFTR in a blood sample could be useful in the diagnosis of CFTR related diseases.     

GABAergic signaling in the pulmonary neuroepithelial body microenvironment: functional imaging in GAD67-GFP mice

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates, but has also been reported in multiple cell types outside the CNS. A GABAergic system has been proposed in neuroepithelial bodies (NEBs) in monkey lungs. Pulmonary NEBs are known as complex intraepithelial sensory airway receptors and are part of the NEB microenvironment. Aim of the present study was to unravel a GABAergic signaling system in the NEB microenvironment in mouse lungs, enabling the use of genetically modified animals for future functional studies. Immunostaining of mouse lungs revealed that glutamic acid decarboxylase 65/67 (GAD65/67), a rate-limiting enzyme in the biosynthesis of GABA, and the vesicular GABA transporter (VGAT) were exclusively expressed in NEB cells. In GAD67-green fluorescent protein (GFP) knock-in mice, all pulmonary NEBs appeared to express GFP. For confocal live cell imaging, ex vivo vibratome lung slices of GAD67-GFP mice can be directly loaded with fluorescent functional probes, e.g. a red-fluorescent calcium dye, without the necessity of time-consuming prior live visualization of NEBs. RT-PCR of the NEB microenvironment obtained by laser microdissection revealed the presence of both GABA_A and GABA_B (R1 and R2) receptors, which was confirmed by immunostaining. In conclusion, the present study not only revealed the presence of a GABAergic signaling pathway, but also the very selective expression of GFP in pulmonary NEBs in a GAD67-GFP mouse model. Different proof of concept experiments have clearly shown that adoption of the GAD67-GFP mouse model will certainly boost future functional imaging and gene expression analysis of the mouse NEB microenvironment.     

Increased expression of cystic fibrosis transmembrane conductance regulator in rat liver after common bile duct ligation

Liver disease associated with cystic fibrosis (CF) has been increasingly diagnosed and recognized as one of the major causes of death in CF during recent years. The autosomal-recessive disorder of CF results from mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) that encodes the CFTR protein. Due to its existence and multifunction in biliary epithelial, over- or less-expression of CFTR in the liver may play an important role in the development of CF liver disease (CFLD). The aim of current study is to investigate the expression of CFTR in the liver of common bile duct ligated (BDL) rats. After BDL, there was an increase in the abundance of CFTR mRNA and protein. Immunohistochemical staining also demonstrated an increased intensity of CFTR staining in the liver tissue section. In conclusion, there is an increased expression of CFTR in the liver after common BDL.     

Selective visualisation of neuroepithelial bodies in vibratome slices of living lung by 4-Di-2-ASP in various animal species

Pulmonary neuroepithelial bodies (NEBs) are extensively innervated organoid groups of neuroendocrine cells that lie in the epithelium of intrapulmonary airways. Our present understanding of the morphology of NEBs is comprehensive, but direct physiological studies have so far been challenging because the extremely diffuse distribution of NEBs makes them inaccessible in vivo and because a reliable in vitro model is lacking. Our aim has been to optimise an in vitro method based on vibratome slices of living lungs, a model that includes NEBs, the surrounding tissues and at least part of their complex innervation. This in vitro model offers satisfactory access to pulmonary NEBs, provided that they can be differentiated from other tissue elements. The model was first optimised for living rat lung slices. Neutral red staining, reported to stain rabbit NEBs, proved unsuccessful in rat slices. On the other hand, the styryl pyridinium dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP), showed brightly fluorescent cell groups, reminiscent of NEBs, in the airway epithelium of living lung slices from rat. In addition, nerve fibres innervating the NEBs were labelled. The reliable and specific labelling of pulmonary NEBs by 4-Di-2-ASP was corroborated by immunostaining for protein gene-product 9.5. Live cell imaging and propidium iodide staining further established the acceptable viability of 4-Di-2-ASP-labelled NEB cells in lung slices, even over long periods. Importantly, the in vitro model and 4-Di-2-ASP staining procedure for pulmonary NEBs appeared to be equally reproducible in mouse, hamster and rabbit lungs. Diverse immunocytochemical procedures could be applied to the lung slices providing an opportunity to combine physiological and functional morphological studies. Such an integrated approach offers additional possibilities for elucidating the function(s) of pulmonary NEBs in health and disease. This work was supported by the following research grants: Fund for Scientific Research Flanders (G.0155.01 to D.A.), NOI-BOF (to D.A.) and BOF-RUCA Small Projects (KPO2 to D.A., I.B. and F.V.M.) from the University of Antwerp.     

Myeloperoxidase promoter polymorphism -463G is associated with more severe clinical expression of cystic fibrosis pulmonary disease

The severity of cystic fibrosis (CF) pulmonary disease is not directly related to CFTR genotype but depends upon several parameters, including neutrophil-dominated inflammation. Identification of agents modulating inflammation constitutes a relevant goal. Myeloperoxidase (MPO) is involved in both microbicidal and proinflammatory neutrophil activities. The aim of this study was to evaluate whether the -463GA MPO promoter polymorphism is linked to clinical severity of CF-associated pulmonary inflammation. This polymorphism significantly affects the level of MPO gene expression in leukocytes and the G allele is more expressing than the A allele. We show that MPO genotype significantly influences the severity of pulmonary disease in early stages, prior to the development of chronic lung infections, with GG genotype being associated with more severe CF disease. Our findings indicate that the level of MPO gene expression influences the CF pathogenesis, presumably reflecting cellular damage by MPO-generated oxidants or other activity of MPO in airway inflammation.     

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.