ABSTRACT
BACKGROUND: Many affected by pancreatitis harbor rare variants of the cystic fibrosis (CF) gene, CFTR, which encodes an epithelial chloride/bicarbonate channel. We investigated CFTR function and the effect of CFTR modulator drugs in pancreatitis patients carrying CFTR variants. METHODS: Next-generation sequencing was performed to identify CFTR variants. Sweat tests and nasal potential difference (NPD) assays were performed to assess CFTR function in vivo. Intestinal current measurement (ICM) was performed on rectal biopsies. Patient-derived intestinal epithelial monolayers were used to evaluate chloride and bicarbonate transport and the effects of a CFTR modulator combination: elexacaftor, tezacaftor and ivacaftor (ETI). RESULTS: Of 32 pancreatitis patients carrying CFTR variants, three had CF-causing mutations on both alleles and yielded CF-typical sweat test, NPD and ICM results. Fourteen subjects showed a more modest elevation in sweat chloride levels, including three that were provisionally diagnosed with CF. ICM indicated impaired CFTR function in nine out of 17 non-CF subjects tested. This group of nine included five carrying a wild type CFTR allele. In epithelial monolayers, a reduction in CFTR-dependent chloride transport was found in six out of 14 subjects tested, whereas bicarbonate secretion was reduced in only one individual. In epithelial monolayers of four of these six subjects, ETI improved CFTR function. CONCLUSIONS: CFTR function is impaired in a subset of pancreatitis patients carrying CFTR variants. Mutations outside the CFTR locus may contribute to the anion transport defect. Bioassays on patient-derived intestinal tissue and organoids can be used to detect such defects and to assess the effect of CFTR modulators.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator , Cystic Fibrosis , Pancreatitis , Humans , Bicarbonates/metabolism , Chlorides , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Mutation , Pancreatitis/genetics , Pancreatitis/metabolism , QuinolonesABSTRACT
BACKGROUND: Loss of CFTR-dependent anion and fluid secretion in the ducts of the exocrine pancreas is thought to contribute to the development of pancreatitis, but little is known about the impact of inflammation on ductal CFTR function. Here we used adult stem cell-derived cell cultures (organoids) obtained from porcine pancreas to evaluate the effects of pro-inflammatory cytokines on CFTR function. METHODS: Organoids were cultured from porcine pancreas and used to prepare ductal epithelial monolayers. Monolayers were characterized by immunocytochemistry. Epithelial bicarbonate and chloride secretion, and the effect of IL-1ß, IL-6, IFN-γ, and TNF-α on CFTR function was assessed by electrophysiology. RESULTS: Immunolocalization of ductal markers, including CFTR, keratin 7, and zonula occludens 1, demonstrated that organoid-derived cells formed a highly polarized epithelium. Stimulation by secretin or VIP triggered CFTR-dependent anion secretion across epithelial monolayers, whereas purinergic receptor stimulation by UTP, elicited CFTR-independent anion secretion. Most of the anion secretory response was attributable to bicarbonate transport. The combination of IL-1ß, IL-6, IFN-γ, and TNF-α markedly enhanced CFTR expression and anion secretion across ductal epithelial monolayers, whereas these cytokines had little effect when tested separately. Although TNF-α triggered apoptotic signaling, epithelial barrier function was not significantly affected by cytokine exposure. CONCLUSIONS: Pro-inflammatory cytokines enhance CFTR-dependent anion secretion across pancreatic ductal epithelium. We propose that up-regulation of CFTR in the early stages of the inflammatory response, may serve to promote the removal of pathogenic stimuli from the ductal tree, and limit tissue injury.
Subject(s)
Bicarbonates , Cytokines , Swine , Animals , Tumor Necrosis Factor-alpha , Cystic Fibrosis Transmembrane Conductance Regulator , Interleukin-6 , EpitheliumABSTRACT
Biliary disorders can lead to life-threatening disease and are also a challenging complication of liver transplantation. As there are limited treatment options, tissue engineered bile ducts could be employed to replace or repair damaged bile ducts. We explored how these constructs can be created by seeding hepatobiliary LGR5+ organoids onto tissue-specific scaffold. For this, we decellularized discarded human extrahepatic bile ducts (EBD) that we recellularized with organoids of different origin, that is, liver biopsies, extrahepatic bile duct biopsies, and bile samples. Here, we demonstrate efficient decellularization of EBD tissue. Recellularization of the EBD extracellular matrix (ECM) with the organoids of extrahepatic origin (EBD tissue and bile derived organoids) showed more profound repopulation of the ductal ECM when compared with liver tissue (intrahepatic bile duct) derived organoids. The bile duct constructs that were repopulated with extrahepatic organoids expressed mature cholangiocyte-markers and had increased electrical resistance, indicating restoration of the barrier function. Therefore, the organoids of extrahepatic sources are identified to be the optimal candidate for the development of personalized tissue engineered EBD constructs.
Subject(s)
Bile Ducts, Extrahepatic/chemistry , Epithelial Cells/metabolism , Extracellular Matrix/chemistry , Organoids/metabolism , Tissue Engineering , Tissue Scaffolds/chemistry , Epithelial Cells/cytology , Humans , Organoids/cytologyABSTRACT
Cystic fibrosis (CF) is caused by mutations in the gene encoding the CFTR anion channel. Loss of CFTR function in pancreatic, biliary and intestinal epithelia, severely affects gastrointestinal function. Transcriptome analysis indicated the activation of an innate and adaptive immune response in the distal small intestine of Cftr null mice. Inflammation was associated with differential regulation of numerous genes involved in the transport and metabolism of nutrients and, particularly, lipids, that are targeted by ligand-dependent nuclear receptors and/or HNF4α. Among the most strongly down-regulated genes are the FXR targets Fgf15 and Nr0b2, the PPARα target Pdk4, and the PXR target Ces2a, whereas expression of the CF modifier gene Slc6a14 was strongly increased. Most changes in gene expression were reversed by bacterial containment. Our data suggest that the gut microbiota has a pervasive effect on gene expression in CF mice, affecting enterocyte maturation, lipid metabolism, and nutrient absorption in CF.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Intestine, Small/metabolism , Transcriptome , Amino Acid Transport Systems/genetics , Amino Acid Transport Systems/metabolism , Animals , Carboxylesterase/genetics , Carboxylesterase/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/deficiency , Down-Regulation , Female , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/metabolism , Gastrointestinal Microbiome , Gene Deletion , Hepatocyte Nuclear Factor 4/genetics , Hepatocyte Nuclear Factor 4/metabolism , Immunity, Innate , Intestine, Small/immunology , Intestine, Small/microbiology , Male , Mice , Pyruvate Dehydrogenase Acetyl-Transferring Kinase/genetics , Pyruvate Dehydrogenase Acetyl-Transferring Kinase/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolismABSTRACT
Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood death from diarrhea and the leading cause of Traveler's diarrhea. E. coli heat-stable enterotoxin (ST) is a major virulence factor of ETEC and inhibits the brush border Na/H exchanger NHE3 in producing diarrhea. NHE3 regulation involves multiprotein signaling complexes that form on its COOH terminus. In this study, the hypothesis was tested that ST signals via members of the Na/H exchanger regulatory factor (NHERF) family of scaffolding proteins, NHERF2, which had been previously shown to have a role, and now with concentration on a role for NHERF3. Two models were used: mouse small intestine and Caco-2/BBe cells. In both models, ST rapidly increased intracellular cGMP, inhibited NHE3 activity, and caused a quantitatively similar decrease in apical expression of NHE3. The transport effects were NHERF3 and NHERF2 dependent. Also, mutation of the COOH-terminal amino acids of NHERF3 supported that NHERF3-NHERF2 heterodimerization was likely to account for this dual dependence. The ST increase in cGMP in both models was partially dependent on NHERF3. The intracellular signaling pathways by which ST-cGMP inhibits NHE3 were different in mouse jejunum (activation of cGMP kinase II, cGKII) and Caco-2 cells, which do not express cGKII (elevation of intracellular Ca2+ concentration [Ca2+]i). The ST elevation of [Ca2+]i was from intracellular stores and was dependent on NHERF3-NHERF2. This study shows that intracellular signaling in the same diarrheal model in multiple cell types may be different; this has implications for therapeutic strategies, which often assume that models have similar signaling mechanisms.
Subject(s)
Bacterial Toxins/pharmacology , Enterotoxins/pharmacology , Escherichia coli Proteins/pharmacology , Membrane Proteins/drug effects , Sodium-Hydrogen Exchanger 3/drug effects , Animals , Caco-2 Cells , Cyclic GMP/metabolism , Diarrhea/chemically induced , Escherichia coli/drug effects , Humans , Mice, TransgenicABSTRACT
The guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase II (cGKII) serine/threonine kinase relays signaling through guanylyl cyclase C (GCC) to control intestinal fluid homeostasis. Here, we report the discovery of small-molecule inhibitors of cGKII. These inhibitors were imidazole-aminopyrimidines, which blocked recombinant human cGKII at submicromolar concentrations but exhibited comparatively little activity toward the phylogenetically related protein kinases cGKI and cAMP-dependent protein kinase (PKA). Whereas aminopyrimidyl motifs are common in protein kinase inhibitors, molecular modeling of these imidazole-aminopyrimidines in the ATP-binding pocket of cGKII indicated an unconventional binding mode that directs their amine substituent into a narrow pocket delineated by hydrophobic residues of the hinge and the αC-helix. Crucially, this set of residues included the Leu-530 gatekeeper, which is not conserved in cGKI and PKA. In intestinal organoids, these compounds blocked cGKII-dependent phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). In mouse small intestinal tissue, cGKII inhibition significantly attenuated the anion secretory response provoked by the GCC-activating bacterial heat-stable toxin (STa), a frequent cause of infectious secretory diarrhea. In contrast, both PKA-dependent VASP phosphorylation and intestinal anion secretion were unaffected by treatment with these compounds, whereas experiments with T84 cells indicated that they weakly inhibit the activity of cAMP-hydrolyzing phosphodiesterases. As these protein kinase inhibitors are the first to display selective inhibition of cGKII, they may expedite research on cGMP signaling and may aid future development of therapeutics for managing diarrheal disease and other pathogenic syndromes that involve cGKII.
Subject(s)
Cyclic GMP-Dependent Protein Kinase Type II/antagonists & inhibitors , Cyclic GMP/metabolism , Intestines/physiology , Protein Kinase Inhibitors/pharmacology , Small Molecule Libraries/pharmacology , Amino Acid Sequence , Animals , Cell Adhesion Molecules/metabolism , Cells, Cultured , Crystallography, X-Ray , Humans , Intestines/drug effects , Mice , Microfilament Proteins/metabolism , Models, Molecular , Phosphoproteins/metabolism , Protein Conformation , Sequence Homology , Signal TransductionABSTRACT
BACKGROUND: By definition, effect of synonymous single-nucleotide variants (SNVs) on protein folding and function are neutral, as they alter the codon and not the encoded amino acid. Recent examples indicate tissue-specific and transfer RNA (tRNA)-dependent effects of some genetic variations arguing against neutrality of synonymous SNVs for protein biogenesis. RESULTS: We performed systematic analysis of tRNA abunandance across in various models used in cystic fibrosis (CF) research and drug development, including Fischer rat thyroid (FRT) cells, patient-derived primary human bronchial epithelia (HBE) from lung biopsies, primary human nasal epithelia (HNE) from nasal curettage, intestinal organoids, and airway progenitor-directed differentiation of human induced pluripotent stem cells (iPSCs). These were compared to an immortalized CF bronchial cell model (CFBE41o-) and two widely used laboratory cell lines, HeLa and HEK293. We discovered that specific synonymous SNVs exhibited differential effects which correlated with variable concentrations of cognate tRNAs. CONCLUSIONS: Our results highlight ways in which the presence of synonymous SNVs may alter local kinetics of mRNA translation; and thus, impact protein biogenesis and function. This effect is likely to influence results from mechansistic analysis and/or drug screeining efforts, and establishes importance of cereful model system selection based on genetic variation profile.
Subject(s)
Oligonucleotide Array Sequence Analysis/methods , Polymorphism, Single Nucleotide , RNA, Transfer/genetics , Codon/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Genotype , HEK293 Cells , HeLa Cells , Humans , PhenotypeABSTRACT
Cross-species comparative studies have highlighted differences between human and mouse cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial Cl- channel defective in cystic fibrosis (CF). Here, we compare the impact of the most common CF mutation F508del on the function of human and mouse CFTR heterologously expressed in mammalian cells and their response to CFTR modulators using the iodide efflux and patch-clamp techniques. Once delivered to the plasma membrane, human F508del-CFTR exhibited a severe gating defect characterized by infrequent channel openings and was thermally unstable, deactivating within minutes at 37°C. By contrast, the F508del mutation was without effect on the gating pattern of mouse CFTR, and channel activity demonstrated thermostability at 37°C. Strikingly, at all concentrations tested, the clinically approved CFTR potentiator ivacaftor was without effect on the mouse F508del-CFTR Cl- channel. Moreover, eight CFTR potentiators, including ivacaftor, failed to generate CFTR-mediated iodide efflux from CHO cells expressing mouse F508del-CFTR. However, they all produced CFTR-mediated iodide efflux with human F508del-CFTR-expressing CHO cells, while fifteen CFTR correctors rescued the plasma membrane expression of both human and mouse F508del-CFTR. Interestingly, the CFTR potentiator genistein enhanced CFTR-mediated iodide efflux from CHO cells expressing either human or mouse F508del-CFTR, whereas it only potentiated human F508del-CFTR Cl- channels in cell-free membrane patches, suggesting that its action on mouse F508del-CFTR is indirect. Thus, the F508del mutation has distinct effects on human and mouse CFTR Cl- channels.
Subject(s)
Base Sequence , Chloride Channel Agonists/pharmacology , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Sequence Deletion , Adenosine Triphosphate/metabolism , Aminophenols/pharmacology , Aminopyridines/pharmacology , Animals , Benzodioxoles/pharmacology , CHO Cells , Colforsin/pharmacology , Cricetulus , Cyclic AMP-Dependent Protein Kinases/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Gene Expression , Genistein/pharmacology , Ion Transport/drug effects , Mice , NIH 3T3 Cells , Patch-Clamp Techniques , Protein Stability , Quinolones/pharmacology , Species Specificity , Temperature , TransgenesABSTRACT
One of the cardinal symptoms of intestinal inflammation is diarrhea. Acute intestinal inflammation is associated with inhibition of ion absorption and increased secretion, along with fluid leakage due to epithelial injury and changes in permeability. However, in the chronic situation, a downregulation of both absorptive and secretory transport has been reported. We investigated how experimental colitis reduces cAMP levels in intestinal epithelial cells through modulation of adenylyl cyclases (AC). Primary colonic epithelial cells obtained from rats with trinitrobenzenesulfonic acid colitis and non-colitic controls were analyzed for AC expression by RT-qPCR and Western blot, following a preliminary microarray analysis. AC6 and AC5 were found to be expressed in colonocytes, and downregulated by inflammation, with the former exhibiting considerably higher mRNA levels in both cases. To test the hypothesis that inflammatory cytokines may account for this effect, Caco 2 cells were treated with IL-1ß, TNF-α, or IFN-γ. All three cytokines inhibited forskolin evoked short-circuit currents in Ussing chambers and lowered intracellular cAMP, but failed to alter AC6 mRNA levels. AC5/AC6 expression was however inhibited in mouse jejunal organoids treated with IFN-γ and TNF-α, but not IL-1ß. Gene knockdown of AC6 resulted in a significant decrease of ion secretion in T84 cells. We conclude that the disturbances in ion secretion observed in rat TNBS colitis are associated with low intracellular levels of cAMP in the epithelium, which may be explained in part by the downregulation of AC5/AC6 expression by proinflammatory cytokines.
Subject(s)
Adenylyl Cyclases/metabolism , Colitis/metabolism , Intestinal Secretions , Adenylyl Cyclases/genetics , Animals , Caco-2 Cells , Cells, Cultured , Cyclic AMP/metabolism , Cytokines/pharmacology , Enterocytes/drug effects , Enterocytes/metabolism , Female , HEK293 Cells , Humans , Ion Transport , Jejunum/cytology , Jejunum/drug effects , Jejunum/metabolism , Mice , Rats , Rats, WistarABSTRACT
Forskolin-induced swelling (FIS) of intestinal organoids from individuals with cystic fibrosis (CF) measures function of the cystic fibrosis transmembrane conductance regulator (CFTR), the protein mutated in CF.We investigated whether FIS corresponds with clinical outcome parameters and biomarkers of CFTR function in 34 infants diagnosed with CF. Relationships with FIS were studied for indicators of pulmonary and gastrointestinal disease.Children with low FIS had higher levels of immunoreactive trypsinogen (p=0.030) and pancreatitis-associated protein (p=0.039), more often had pancreatic insufficiency (p<0.001), had more abnormalities on chest computed tomography (p=0.049), and had lower z-scores for maximal expiratory flow at functional residual capacity (p=0.033) when compared to children with high FIS values. FIS significantly correlated with sweat chloride concentration (SCC) and intestinal current measurement (ICM) (r=â-0.82 and r=0.70, respectively; both p<0.001). Individual assessment of SCC, ICM and FIS suggested that FIS can help to classify individual disease severity.Thus, stratification by FIS identified subgroups that differed in pulmonary and gastrointestinal outcome parameters. FIS of intestinal organoids correlated well with established CFTR-dependent biomarkers such as SCC and ICM, and performed adequately at group and individual level in this proof-of-concept study.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/diagnosis , Exocrine Pancreatic Insufficiency/diagnosis , Organoids/pathology , Biomarkers/metabolism , Chlorides/metabolism , Cystic Fibrosis/complications , Female , Humans , Infant , Ion Transport , Linear Models , Male , Proof of Concept Study , Severity of Illness IndexABSTRACT
Identification of Lgr5 as the intestinal stem cell marker as well as the growth factors necessary to replicate adult intestinal stem cell division has led to the establishment of the methods to generate "indefinite" ex vivo primary intestinal epithelial cultures, termed "mini-intestines." Primary cultures developed from isolated intestinal crypts or stem cells (termed enteroids/colonoids) and from inducible pluripotent stem cells (termed intestinal organoids) are being applied to study human intestinal physiology and pathophysiology with great expectations for translational applications, including regenerative medicine. Here we discuss the physiologic properties of these cultures, their current use in understanding diarrhea-causing host-pathogen interactions, and potential future applications.
Subject(s)
Adult Stem Cells/metabolism , Antigens, Differentiation/metabolism , Diarrhea , Intestinal Mucosa , Intestines , Organoids , Receptors, G-Protein-Coupled/metabolism , Adult Stem Cells/pathology , Diarrhea/metabolism , Diarrhea/pathology , Diarrhea/physiopathology , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Intestinal Mucosa/metabolism , Intestines/pathology , Intestines/physiopathology , Organoids/metabolism , Organoids/pathology , Organoids/physiopathologyABSTRACT
Progressive familial intrahepatic cholestasis type 1 (PFIC1) is caused by mutations in the gene encoding the phospholipid flippase ATP8B1. Apart from severe cholestatic liver disease, many PFIC1 patients develop extrahepatic symptoms characteristic of cystic fibrosis (CF), such as pulmonary infection, sweat gland dysfunction and failure to thrive. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel essential for epithelial fluid transport. Previously it was shown that CFTR transcript levels were strongly reduced in livers of PFIC1 patients. Here we have investigated the hypothesis that ATP8B1 is important for proper CFTR expression and function. We analyzed CFTR expression in ATP8B1-depleted intestinal and pulmonary epithelial cell lines and assessed CFTR function by measuring short-circuit currents across transwell-grown ATP8B1-depleted intestinal T84 cells and by a genetically-encoded fluorescent chloride sensor. In addition, we studied CFTR surface expression upon induction of CFTR transcription. We show that CFTR protein levels are strongly reduced in the apical membrane of human ATP8B1-depleted intestinal and pulmonary epithelial cell lines, a phenotype that coincided with reduced CFTR activity. Apical membrane insertion upon induction of ectopically-expressed CFTR was strongly impaired in ATP8B1-depleted cells. We conclude that ATP8B1 is essential for correct apical localization of CFTR in human intestinal and pulmonary epithelial cells, and that impaired CFTR localization underlies some of the extrahepatic phenotypes observed in ATP8B1 deficiency.
Subject(s)
Adenosine Triphosphatases/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Phospholipid Transfer Proteins/metabolism , Phospholipids/metabolism , Animals , Cell Line, Tumor , Cell Membrane/metabolism , Chlorides/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Epithelial Cells/metabolism , Humans , Intestinal Mucosa/metabolism , Ion Channel Gating , Lung/cytology , Male , Mice, Inbred C57BL , Mice, Mutant Strains , Protein Transport , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
OBJECTIVES: Hepatobiliary complications are a leading cause of morbidity and mortality in cystic fibrosis (CF) patients. Knowledge of the underlying pathological aspects and optimal clinical management is, however, sorely lacking. METHODS: We provide a summary of the lectures given by international speakers at the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) monothematic conference on cystic fibrosis-related liver disease (CFLD) held in Paris in January 2016, to discuss the status of our current knowledge of liver disease in CF patients, to define the critical areas that need to be addressed, and to resolve actions to elucidate relevant mechanisms of disease to optimise future therapeutic options. CONCLUSIONS: The need for a universal consensus on the definition of CFLD to clarify disease stage and to identify relevant biomarkers to assess disease severity was highlighted. A deeper understanding of the pathophysiology and prognostic factors for the long-term evolution of CFLD is fundamental to move forward and has a strong bearing on identifying potential treatments. Novel experimental models and new treatment options under investigation are discussed and offer hope for the near future of CFLD.
Subject(s)
Cystic Fibrosis/complications , Liver Diseases/etiology , Biomarkers/metabolism , Cystic Fibrosis/diagnosis , Cystic Fibrosis/metabolism , Cystic Fibrosis/therapy , Gastrointestinal Agents/therapeutic use , Humans , Liver Diseases/diagnosis , Liver Diseases/metabolism , Liver Diseases/therapy , Liver Transplantation , Pancreas Transplantation , PrognosisABSTRACT
RATIONALE: Gene therapy holds promise for a curative mutation-independent treatment applicable to all patients with cystic fibrosis (CF). The various viral vector-based clinical trials conducted in the past have demonstrated safety and tolerance of different vectors, but none have led to a clear and persistent clinical benefit. Recent clinical breakthroughs in recombinant adeno-associated viral vector (rAAV)-based gene therapy encouraged us to reexplore an rAAV approach for CF. OBJECTIVES: We evaluated the preclinical potential of rAAV gene therapy for CF to restore chloride and fluid secretion in two complementary models: intestinal organoids derived from subjects with CF and a CF mouse model, an important milestone toward the development of a clinical rAAV candidate for CF gene therapy. METHODS: We engineered an rAAV vector containing a truncated CF transmembrane conductance regulator (CFTRΔR) combined with a short promoter (CMV173) to ensure optimal gene expression. A rescue in chloride and fluid secretion after rAAV-CFTRΔR treatment was assessed by forskolin-induced swelling in CF transmembrane conductance regulator (CFTR)-deficient organoids and by nasal potential differences in ΔF508 mice. MEASUREMENTS AND MAIN RESULTS: rAAV-CFTRΔR transduction of human CFTR-deficient organoids resulted in forskolin-induced swelling, indicating a restoration of CFTR function. Nasal potential differences demonstrated a clear response to low chloride and forskolin perfusion in most rAAV-CFTRΔR-treated CF mice. CONCLUSIONS: Our study provides robust evidence that rAAV-mediated gene transfer of a truncated CFTR functionally rescues the CF phenotype across the nasal mucosa of CF mice and in patient-derived organoids. These results underscore the clinical potential of rAAV-CFTRΔR in offering a cure for all patients with CF in the future.
Subject(s)
Cystic Fibrosis/therapy , Dependovirus , Genetic Therapy/methods , Genetic Vectors , Intestines , Organoids , Animals , Body Fluids/metabolism , Chloride Channels/genetics , Chlorides/metabolism , Colforsin/pharmacology , Cystic Fibrosis/genetics , Disease Models, Animal , Gene Transfer Techniques , Genotype , HeLa Cells , Humans , Mice , Organoids/metabolism , Transduction, GeneticABSTRACT
Diarrhea is one of the most common adverse side effects observed in â¼7% of individuals consuming Food and Drug Administration (FDA)-approved drugs. The mechanism of how these drugs alter fluid secretion in the gut and induce diarrhea is not clearly understood. Several drugs are either substrates or inhibitors of multidrug resistance protein 4 (MRP4), such as the anti-colon cancer drug irinotecan and an anti-retroviral used to treat HIV infection, 3'-azido-3'-deoxythymidine (AZT). These drugs activate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated fluid secretion by inhibiting MRP4-mediated cAMP efflux. Binding of drugs to MRP4 augments the formation of MRP4-CFTR-containing macromolecular complexes that is mediated via scaffolding protein PDZK1. Importantly, HIV patients on AZT treatment demonstrate augmented MRP4-CFTR complex formation in the colon, which defines a novel paradigm of drug-induced diarrhea.
Subject(s)
Cyclic AMP/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Diarrhea/chemically induced , Multidrug Resistance-Associated Proteins/metabolism , Animals , Camptothecin/adverse effects , Camptothecin/analogs & derivatives , Drug Approval , HT29 Cells , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Irinotecan , Mice , Models, Molecular , Multidrug Resistance-Associated Proteins/chemistry , Multidrug Resistance-Associated Proteins/deficiency , Protein Conformation , United States , United States Food and Drug AdministrationABSTRACT
We hypothesized that people with cystic fibrosis (CF) who express CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations associated with residual function may benefit from G-protein coupled receptor (GPCR)-targeting drugs that can activate and enhance CFTR function.We used intestinal organoids to screen a GPCR-modulating compound library and identified ß2-adrenergic receptor agonists as the most potent inducers of CFTR function.ß2-Agonist-induced organoid swelling correlated with the CFTR genotype, and could be induced in homozygous CFTR-F508del organoids and highly differentiated primary CF airway epithelial cells after rescue of CFTR trafficking by small molecules. The in vivo response to treatment with an oral or inhaled ß2-agonist (salbutamol) in CF patients with residual CFTR function was evaluated in a pilot study. 10 subjects with a R117H or A455E mutation were included and showed changes in the nasal potential difference measurement after treatment with oral salbutamol, including a significant improvement of the baseline potential difference of the nasal mucosa (+6.35â mV, p<0.05), suggesting that this treatment might be effective in vivo Furthermore, plasma that was collected after oral salbutamol treatment induced CFTR activation when administered ex vivo to organoids.This proof-of-concept study suggests that organoids can be used to identify drugs that activate CFTR function in vivo and to select route of administration.
Subject(s)
Adrenergic beta-2 Receptor Agonists/pharmacology , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/drug therapy , Cystic Fibrosis/metabolism , Administration, Oral , Albuterol/administration & dosage , Biological Assay , Bronchi/pathology , Cell Line , Cells, Cultured , Chlorides/chemistry , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Drug Evaluation, Preclinical , Epithelial Cells/metabolism , Epithelium/metabolism , Humans , Mutation , Nasal Mucosa/drug effects , Nasal Mucosa/metabolism , Organoids , Pilot Projects , Respiratory System/metabolism , Signal TransductionABSTRACT
Guanylin (GN) and uroguanylin (UGN), through activation of guanylyl cyclase C (GCC), serve to control intestinal fluid homeostasis. Both peptides are produced in the intestinal epithelium, but their cellular origin has not been fully charted. Using quantitative PCR and an improved in situ hybridization technique (RNAscope), we have assessed the expression of GN (Guca2a), UGN (Guca2b), and GCC (Gucy2c) in mouse intestine. In the crypts of Lieberkühn, expression of Guca2a and Guca2b was restricted to cells of secretory lineage, at the crypt's base, and to a region above, previously identified as a common origin of cellular differentiation. In this compartment, comparatively uniform levels of Guca2a and Guca2b expression were observed throughout the length of the gut. In contrast, Guca2a and Guca2b expression in the villus-surface region was more variable, and reflected the distinct, but overlapping expression pattern observed previously. Accordingly, in jejunum and ileum, Guca2a and Guca2b were abundantly expressed by enterocytes, whereas in colon only Guca2a transcript was found in the surface region. In duodenum, only low levels of Guca2b transcript were observed in columnar cells, and Guca2a expression was restricted entirely to cells of the secretory lineage. Gucy2c was shown to be expressed relatively uniformly along the rostrocaudal and crypt-villus axes and was also found in the duodenal glands. Our study reveals novel aspects of the cellular localization of the GCC signaling axis that, apart from its role in the regulation of fluid balance, link it to pH regulation, cell cycle control, and host defense.
Subject(s)
Cell Lineage , Epithelial Cells/cytology , Epithelial Cells/metabolism , Gastrointestinal Hormones/biosynthesis , Intestines/cytology , Natriuretic Peptides/biosynthesis , Animals , Gastrointestinal Hormones/analysis , Gastrointestinal Hormones/genetics , Intestinal Mucosa/metabolism , Mice , Mice, Inbred Strains , Natriuretic Peptides/analysis , Natriuretic Peptides/genetics , Signal TransductionABSTRACT
BACKGROUND: Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectious diarrheal disease. METHODS: Compounds that inhibit STa-induced cyclic guanosine 3',5'-monophosphate (cGMP) production were selected by performing cyclase assays on cells and membranes containing GCC, or the related GCA. The effect of leads on STa/GCC-dependent activation of the cystic fibrosis transmembrane conductance regulator anion channel was investigated in T84 cells, and in porcine and human intestinal tissue. Their effect on STa-provoked fluid transport was assessed in ligated intestinal loops in piglets. RESULTS: Four N-2-(propylamino)-6-phenylpyrimidin-4-one-substituted piperidines were shown to inhibit GCC-mediated cellular cGMP production. The half maximal inhibitory concentrations were ≤ 5 × 10(-7) mol/L, whereas they were >10 times higher for GCA. In T84 monolayers, these leads blocked STa/GCC-dependent, but not forskolin/adenylyl cyclase-dependent, cystic fibrosis transmembrane conductance regulator activity. GCC inhibition reduced STa-provoked anion secretion in pig jejunal tissue, and fluid retention and cGMP levels in STa-exposed loops. These GCC inhibitors blocked STa-provoked anion secretion in rectal biopsy specimens. CONCLUSIONS: We have identified a novel class of GCC inhibitors that may form the basis for development of future therapeutics for (infectious) diarrheal disease.
Subject(s)
Bacterial Toxins/antagonists & inhibitors , Enterotoxins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Escherichia coli Proteins/antagonists & inhibitors , Jejunum/drug effects , Piperidines/pharmacology , Receptors, Guanylate Cyclase-Coupled/antagonists & inhibitors , Receptors, Peptide/antagonists & inhibitors , Adenylyl Cyclases/metabolism , Adult , Animals , Bacterial Toxins/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Diarrhea , Enterotoxigenic Escherichia coli , Enterotoxins/metabolism , Escherichia coli Proteins/metabolism , HeLa Cells , Humans , Jejunum/cytology , Jejunum/metabolism , Models, Biological , Receptors, Enterotoxin , Receptors, Guanylate Cyclase-Coupled/metabolism , Receptors, Peptide/metabolism , Signal Transduction/drug effects , Swine , Young AdultABSTRACT
CONTEXT: Acting through different receptors, natriuretic peptides (atrial natriuretic peptide [ANP], brain type natriuretic peptide [BNP] and C-type natriuretic peptide [CNP]) increase intracellular cGMP, which then stimulates different pathways that activate fluid secretion. OBJECTIVE: We used two-electrode voltage clamping to define the dominant pathway that is employed when natriuretic peptides activate cystic fibrosis transmembrane conductance regulator (CFTR) in the Xenopus oocyte expression system. Natriuretic peptides could activate CFTR by 1) cGMP cross-activation of protein kinase A (PKA), 2) cGMP activation of cGMP-dependent protein kinase II, 3) cGMP inhibition of phosphodiesterase type III (PDE3), or 4) direct activation of CFTR. MATERIALS AND METHODS: cRNA-microinjected Xenopus laevis oocytes were perfused with diverse compounds that examined these pathways of natriuretic peptide signaling. RESULTS AND DISCUSSION: ANP stimulated the shark CFTR (sCFTR)-mediated chloride conductance and this activation was inhibited by H-89, a specific inhibitor of PKA. After co-expression of the CNP receptor (NPR-B), sCFTR became stimulatable by CNP and was similarly inhibited by H-89, pointing to cross-activation of PKA. 8-pCPT-cGMP, a relatively cGKII-selective cGMP, failed to stimulate sCFTR. Another membrane-permeable and non-hydrolyzable analog of cGMP, 8-Br-cGMP, stimulated CFTR only at millimolar concentrations, consistent with cross-activation of PKA. The PDE inhibitors EHNA, rolipram, cilostamide, and amrinone did not significantly increase chloride conductance, arguing against a significant role for PDE2, PDE3 and PDE4 signaling in the oocyte. Sildenafil, a PDE5 inhibitor, caused a partial activation of sCFTR channels and this effect was again inhibited by H-89. CONCLUSION: From these experiments we conclude that in the Xenopus oocyte system, natriuretic peptides, 8-Br-cGMP, and PDE5 inhibitors activate CFTR by cross-activation of PKA.
Subject(s)
Atrial Natriuretic Factor/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Natriuretic Peptide, C-Type/metabolism , Oocytes/metabolism , Animals , Cells, Cultured , Enzyme Activation , Xenopus laevisABSTRACT
Organoids have tremendous therapeutic potential. They were recently defined as a collection of organ-specific cell types, which self-organize through cell-sorting, develop from stem cells, and perform an organ specific function. The ability to study organoid development and growth in culture and manipulate their genetic makeup makes them particularly suitable for studying development, disease, and drug efficacy. Organoids show great promise in personalized medicine. From a single patient biopsy, investigators can make hundreds of organoids with the genetic landscape of the patient of origin. This genetic similarity makes organoids an ideal system in which to test drug efficacy. While many investigators assume human organoids are the ultimate model system, we believe that the generation of epithelial organoids of comparative model organisms has great potential. Many key transport discoveries were made using marine organisms. In this paper, we describe how deriving organoids from the spiny dogfish shark, zebrafish, and killifish can contribute to the fields of comparative biology and disease modeling with future prospects for personalized medicine.