Bob J. Scholte

A functional CFTR assay using primary cystic fibrosis intestinal organoids

We recently established conditions allowing for long-term expansion of epithelial organoids from intestine, recapitulating essential features of the in vivo tissue architecture. Here we apply this technology to study primary intestinal organoids of people suffering from cystic fibrosis, a disease caused by mutations in CFTR, encoding cystic fibrosis transmembrane conductance regulator. Forskolin induces rapid swelling of organoids derived from healthy controls or wild-type mice, but this effect is strongly reduced in organoids of subjects with cystic fibrosis or in mice carrying the Cftr F508del mutation and is absent in Cftr-deficient organoids. This pattern is phenocopied by CFTR-specific inhibitors. Forskolin-induced swelling of in vitro–expanded human control and cystic fibrosis organoids corresponds quantitatively with forskolin-induced anion currents in freshly excised ex vivo rectal biopsies. Function of the CFTR F508del mutant protein is restored by incubation at low temperature, as well as by CFTR-restoring compounds. This relatively simple and robust assay will facilitate diagnosis, functional studies, drug development and personalized medicine approaches in cystic fibrosis.

Baculovirus Infection of Nondividing Mammalian Cells: Mechanisms of Entry and Nuclear Transport of Capsids

ABSTRACT We have studied the infection pathway of Autographa californica multinuclear polyhedrosis virus (baculovirus) in mammalian cells. By titration with a baculovirus containing a green fluorescent protein cassette, we found that several, but not all, mammalian cell types can be infected efficiently. In contrast to previous suggestions, our data show that the asialoglycoprotein receptor is not required for efficient infection. We demonstrate for the first time that this baculovirus can infect nondividing mammalian cells, which implies that the baculovirus is able to transport its genome across the nuclear membrane of mammalian cells. Our data further show that the virus enters via endocytosis, followed by an acid-induced fusion event, which releases the nucleocapsid into the cytoplasm. Cytochalasin D strongly reduces the infection efficiency but not the delivery of nucleocapsids to the cytoplasm, suggesting involvement of actin filaments in cytoplasmic transport of the capsids. Electron microscopic analysis shows the cigar-shaped nucleocapsids located at nuclear pores of nondividing cells. Under these conditions, we observed the viral genome, major capsid protein, and electron-dense capsids inside the nucleus. This suggests that the nucleocapsid is transported through the nuclear pore. This mode of transport seems different from viruses with large spherical capsids, such as herpes simplex virus and adenovirus, which are disassembled before nuclear transport of the genome. The implications for the application of baculovirus or its capsid proteins in gene therapy are discussed.

Mouse models of cystic fibrosis: phenotypic analysis and research applications

Genetically modified mice have been studied for more than fifteen years as models of cystic fibrosis (CF). The large amount of experimental data generated illuminates the complex multi-organ pathology of CF and raises new questions relevant to human disease. CF mice have also been used to test experimental therapies prior to clinical trials. This review recapitulates the major phenotypic traits of CF mice and highlights important new findings including aberrant alveolar macrophages, bone and cartilage abnormalities and abnormal bioactive lipid metabolism. Novel data are presented on the intestinal and nasal physiology of F508del-CFTR CF mice backcrossed onto different genetic backgrounds. Caveats, and sources of variability including age, gender and animal husbandry, are discussed. Interspecies differences limit comparison of lung pathology in CF mice to the human disease. The recent development of genetically modified pigs and ferrets heralds the application of more advanced animal models to CF research and drug development.

A delta F508 mutation in mouse cystic fibrosis transmembrane conductance regulator results in a temperature-sensitive processing defect in vivo.

The most prevalent mutation (delta F508) in cystic fibrosis patients inhibits maturation and transfer to the plasma membrane of the mutant cystic fibrosis transmembrane conductance regulator (CFTR). We have analyzed the properties of a delta F508 CFTR mouse model, which we described recently. We show that the mRNA levels of mutant CFTR are normal in all tissues examined. Therefore the reduced mRNA levels reported in two similar models may be related to their intronic transcription units. Maturation of mutant CFTR was greatly reduced in freshly excised oviduct, compared with normal. Accumulation of mutant CFTR antigen in the apical region of jejunum crypt enterocytes was not observed, in contrast to normal mice. In cultured gallbladder epithelial cells from delta F508 mice, CFTR chloride channel activity could be detected at only two percent of the normal frequency. However, in mutant cells that were grown at reduced temperature the channel frequency increased to over sixteen percent of the normal level at that temperature. The biophysical characteristics of the mutant channel were not significantly different from normal. In homozygous delta F508 mice we did not observe a significant effect of genetic background on the level of residual chloride channel activity, as determined by the size of the forskolin response in Ussing chamber experiments. Our data show that like its human homologue, mouse delta F508-CFTR is a temperature sensitive processing mutant. The delta F508 mouse is therefore a valid in vivo model of human delta F508-CFTR. It may help us to elucidate the processing pathways of complex membrane proteins. Moreover, it may facilitate the discovery of new approaches towards therapy of cystic fibrosis.

Isotype-specific activation of cystic fibrosis transmembrane conductance regulator-chloride channels by cGMP-dependent protein kinase II

Type II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type Iα cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl− channel in excised, inside-out membrane patches from NIH-3T3 fibroblasts and from a rat intestinal cell line (IEC-CF7) stably expressing recombinant CFTR. In both cell models, in the presence of cGMP and ATP, cGKII was found to mimic the effect of the catalytic subunit of cAMP-dependent protein kinase (cAK) on opening CFTR-Cl− channels, albeit with different kinetics (2-3-min lag time, reduced rate of activation). By contrast, cGKI or a monomeric cGKI catalytic fragment was incapable of opening CFTR-Cl− channels and also failed to potentiate cGKII activation of the channels. The cAK activation but not the cGKII activation was blocked by a cAK inhibitor peptide. The slow activation by cGKII could not be ascribed to counteracting protein phosphatases, since neither calyculin A, a potent inhibitor of phosphatase 1 and 2A, nor ATPγS (adenosine 5′-O-(thiotriphosphate)), producing stable thiophosphorylation, was able to enhance the activation kinetics. Channels preactivated by cGKII closed instantaneously upon removal of ATP and kinase but reopened in the presence of ATP alone. Paradoxically, immunoprecipitated CFTR or CF-2, a cloned R domain fragment of CFTR (amino acids 645-835) could be phosphorylated to a similar extent with only minor kinetic differences by both isotypes of cGK. Phosphopeptide maps of CF-2 and CFTR, however, revealed very subtle differences in site-specificity between the cGK isoforms. These results indicate that cGKII, in contrast to cGKIα, is a potential activator of chloride transport in CFTR-expressing cell types.

cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl− secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels was analyzed after expression of cGK II or cGK Iβ in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I− efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Iβ or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Iβ enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e.g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Iβ nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.

Correction of the ΔPhe508 Cystic Fibrosis Transmembrane Conductance Regulator Trafficking Defect by the Bioavailable Compound Glafenine

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated anion channel expressed in epithelial cells. The most common mutation ΔPhe508 leads to protein misfolding, retention by the endoplasmic reticulum, and degradation. One promising therapeutic approach is to identify drugs that have been developed for other indications but that also correct the CFTR trafficking defect, thereby exploiting their known safety and bioavailability in humans and reducing the time required for clinical development. We have screened approved, marketed, and off-patent drugs with known safety and bioavailability using a ΔPhe508-CFTR trafficking assay. Among the confirmed hits was glafenine, an anthranilic acid derivative with analgesic properties. Its ability to correct the misprocessing of CFTR was confirmed by in vitro and in vivo studies using a concentration that is achieved clinically in plasma (10 μM). Glafenine increased the surface expression of ΔPhe508-CFTR in baby hamster kidney (BHK) cells to ∼40% of that observed for wild-type CFTR, comparable with the known CFTR corrector 4-cyclohexyloxy-2-{1-[4-(4-methoxybenzensulfonyl)-piperazin-1-yl]-ethyl}-quinazoline (VRT-325). Partial correction was confirmed by the appearance of mature CFTR in Western blots and by two assays of halide permeability in unpolarized BHK and human embryonic kidney cells. Incubating polarized CFBE41o− monolayers and intestines isolated from ΔPhe508-CFTR mice (treated ex vivo) with glafenine increased the short-circuit current (Isc) response to forskolin + genistein, and this effect was abolished by 10 μM CFTRinh172. In vivo treatment with glafenine also partially restored total salivary secretion. We conclude that the discovery of glafenine as a CFTR corrector validates the approach of investigating existing drugs for the treatment of CF, although localized delivery or further medicinal chemistry may be needed to reduce side effects.

Azithromycin reduces exaggerated cytokine production by M1 alveolar macrophages in cystic fibrosis

Macrophages phagocyte pathogenic microorganisms and orchestrate immune responses by producing a variety of inflammatory mediators. The cystic fibrosis (CF) transmembrane conductance regulator chloride channel has been reported to be of pivotal importance for macrophage functions. The exact phenotype and role of macrophages in CF is still unknown. Alveolar and peritoneal macrophages were monitored in CF mice homozygous for the F508 del mutation and in wild-type control animals. Classical (M1) and alternative (M2) macrophage polarization and responses to LPS from Pseudomonas aeruginosa were investigated, and the effect of azithromycin was examined in both cell populations. We show that alveolar macrophage counts were 1.7-fold higher in CF as compared with wild-type mice. The macrophage-related chemokine, chemokine C-C motif ligand (CCL)-2, was found to be at least 10-fold more abundant in the alveolar space of mutant mice. Cell count and CCL-2 protein levels were also increased in the peritoneal cavity of CF mice. Both M1 and M2 macrophage polarization were significantly enhanced in alveolar and peritoneal cells from F508del-CF mice as compared with control animals. LPS-stimulated expression of proinflammatory mediators, such as nitric oxide synthase-2, IL-1beta, and CCL-2, was increased, whereas anti-inflammatory IL-10 expression was decreased in CF macrophages. Azithromycin, added to cell cultures at 1 mg/liter, significantly reduced proinflammatory cytokine expression (IL-1beta, CCL-2, TNF-alpha) in M1-induced CF and wild-type alveolar macrophages. Our findings indicate that CF macrophages are ubiquitously accumulated, and that these cells are polarized toward classical and alternative activation status. Azithromycin down-regulates inflammatory cytokine production by M1-polarized CF alveolar macrophages.

Azithromycin reduces spontaneous and induced inflammation in ΔF508 cystic fibrosis mice

BackgroundInflammation plays a critical role in lung disease development and progression in cystic fibrosis. Azithromycin is used for the treatment of cystic fibrosis lung disease, although its mechanisms of action are poorly understood. We tested the hypothesis that azithromycin modulates lung inflammation in cystic fibrosis mice.MethodsWe monitored cellular and molecular inflammatory markers in lungs of cystic fibrosis mutant mice homozygous for the ΔF508 mutation and their littermate controls, either in baseline conditions or after induction of acute inflammation by intratracheal instillation of lipopolysaccharide from Pseudomonas aeruginosa, which would be independent of interactions of bacteria with epithelial cells. The effect of azithromycin pretreatment (10 mg/kg/day) given by oral administration for 4 weeks was evaluated.ResultsIn naive cystic fibrosis mice, a spontaneous lung inflammation was observed, characterized by macrophage and neutrophil infiltration, and increased intra-luminal content of the pro-inflammatory cytokine macrophage inflammatory protein-2. After induced inflammation, cystic fibrosis mice combined exaggerated cellular infiltration and lower anti-inflammatory interleukin-10 production. In cystic fibrosis mice, azithromycin attenuated cellular infiltration in both baseline and induced inflammatory condition, and inhibited cytokine (tumor necrosis factor-α and macrophage inflammatory protein-2) release in lipopolysaccharide-induced inflammation.ConclusionOur findings further support the concept that inflammatory responses are upregulated in cystic fibrosis. Azithromycin reduces some lung inflammation outcome measures in cystic fibrosis mice. We postulate that some of the benefits of azithromycin treatment in cystic fibrosis patients are due to modulation of lung inflammation.

Gene and cell therapy for cystic fibrosis: From bench to bedside

Clinical trials in cystic fibrosis (CF) patients established proof-of-principle for transfer of the wild-type cystic fibrosis transmembrane conductance regulator (CFTR) gene to airway epithelial cells. However, the limited efficacy of gene transfer vectors as well as extra- and intracellular barriers have prevented the development of a gene therapy-based treatment for CF. Here, we review the use of new viral and nonviral gene therapy vectors, as well as human artificial chromosomes, to overcome barriers to successful CFTR expression. Pre-clinical studies will surely benefit from novel animal models, such as CF pigs and ferrets. Prenatal gene therapy is a potential alternative to gene transfer to fully developed lungs. However, unresolved issues, including the possibility of adverse effects on pre- and postnatal development, the risk of initiating oncogenic or degenerative processes and germ line transmission require further investigation. Finally, we discuss the therapeutic potential of stem cells for CF lung disease.