Covid-19 in cystic fibrosis patients compared to the general population: Severity and virus-host cell interactions.

BACKGROUND: People with cystic fibrosis (pwCF) are considered at risk of developing severe forms of respiratory viral infections. We studied the consequences of COVID-19 and virus-host cell interactions in CF vs. non-CF individuals. METHODS: We enrolled CF and non-CF individuals, with /without COVID-like symptoms, who underwent nasopharyngeal swab for detection of SARS-CoV-2. Gene expression was evaluated by RNA sequencing on the same nasopharyngeal swabs. Criteria for COVID-19 severity were hospitalization and requirement or increased need of oxygen therapy. RESULTS: The study included 171 patients (65 pwCF and 106 non-CF individuals). Among them, 10 pwCF (15.4 %) and 43 people without CF (40.6 %) tested positive at RT-PCR. Symptomatic infections were observed in 8 pwCF (with 2 requiring hospitalization) and in 11 individuals without CF (6 requiring hospitalization). Host transcriptomic analysis revealed that genes involved in protein translation, particularly ribosomal components, were downregulated in CF samples irrespective of SARS-CoV-2 status. In SARS-CoV-2 negative individuals, we found a significant difference in genes involved with motile cilia expression and function, which were upregulated in CF samples. Pathway enrichment analysis indicated that interferon signaling in response to SARS-CoV-2 infection was upregulated in both pwCF and non-CF subjects. CONCLUSIONS: COVID-19 does not seem to be more severe in CF, possibly due to factors intrinsic to this population: the lower expression of ribosomal genes may downregulate the protein translation machinery, thus creating an unfavorable environment for viral replication.

CFTR function is impaired in a subset of patients with pancreatitis carrying rare CFTR variants.

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.

Severity of SARS-CoV-2 infection in a hospital population: a clinical comparison across age groups.

BACKGROUND: Children tend to have milder forms of COVID-19 than adults, however post-acute complications have been observed also in the paediatric population. In this study, we compared COVID-19-related outcomes and long-term complications between paediatric and adult patients infected by SARS-CoV-2. METHODS: The study is based on individuals enrolled from October 2020 to June 2021 in the DECO COVID-19 multicentre prospective study supported by the Italian Ministry of Health (COVID-2020-12371781). We included individuals with RT-PCR -confirmed SARS-CoV-2 infection, who were evaluated in the emergency department and/or admitted to COVID-dedicated wards. The severity of SARS-CoV-2 infection was compared across age groups (children/adolescents aged < 18 years, young/middle-aged adults aged 18-64 years and older individuals) through the relative risk (RR) of severe COVID-19. Severity was defined by: 1) hospitalization due to COVID-19 and/or 2) need or supplemental oxygen therapy. RR and corresponding 95% confidence intervals were estimated using log-binomial models. RESULTS: The study included 154 individuals, 84 (54.5%) children/adolescents, 50 (32.5%) young/middle-aged adults and 20 (13%) older adults. Compared to young/middle-aged adults the risk of hospitalization was lower among paediatric patients (RR: 0.49, 95% CI: 0.32-0.75) and higher among older adults (RR: 1.52, 95% CI: 1.12-2.06). The RR of supplemental oxygen was 0.12 (95% CI: 0.05-0.30) among children/adolescents and 1.46 (95% CI: 0.97-2.19) among older adults. Three children developed multisystem inflammatory syndrome (MIS-C), none was admitted to intensive care unit or reported post-acute Covid-19 complications. CONCLUSIONS: Our study confirms that COVID-19 is less severe in children. MIS-C is a rare yet severe complication of SARS-CoV-2 infection in children and its risk factors are presently unknown.

Generation of an induced pluripotent stem cell line (IGGi002A) from nasal cells of a cystic fibrosis patient homozygous for the G542X-CFTR mutation.

Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel defective in cystic fibrosis (CF). Several CFTR mutations are causative of CF, among which G542X is a nonsense mutation introducing a premature stop codon which prevents CFTR protein synthesis. We generated a new iPSC line from nasal cells carrying G542X homozygous mutation for CFTR: IGGi002A. This cell line has normal female karyotype, express pluripotency markers and could differentiate into three germ layers in vitro. This iPSC line may be used for disease modeling (cell differentiation and organoid formation) and development of personalized treatments by genome editing or pharmacological screening.

Distribution of OGTT-Related Variables in Patients with Cystic Fibrosis from Puberty to Adulthood: An Italian Multicenter Study.

BACKGROUND: Insulin secretion and glucose tolerance is annually assessed in patients with cystic fibrosis (PwCF) through oral glucose tolerance tests (OGTTs) as a screening measure for cystic fibrosis-related diabetes. We aimed to describe the distribution and provide reference quartiles of OGTT-related variables in the Italian cystic fibrosis population. METHODS: Cross-sectional study of PwCF receiving care in three Italian cystic fibrosis centers of excellence, from 2016 to 2020. We performed a modified 2-h OGTT protocol (1.75 g/kg, maximum 75 g), sampling at baseline and at 30-min intervals, analyzing plasma glucose, serum insulin, and C-peptide. The modified OGTT allowed for the modeling of ß cell function. For all variables, multivariable quantile regression was performed to estimate the median, the 25th, and 75th percentiles, with age, sex, and pancreatic insufficiency as predictors. RESULTS: We have quantified the deterioration of glucose tolerance and insulin secretion with age according to sex and pancreatic insufficiency, highlighting a deviation from linearity both for patients <10 years and >35 years of age. CONCLUSIONS: References of OGTT variables for PwCF provide a necessary tool to not only identify patients at risk for CFRD or other cystic fibrosis-related complications, but also to evaluate the effects of promising pharmacological therapies.

Lived experiences of people with cystic fibrosis that were not eligible for elexacaftor-tezacaftor-ivacaftor (ETI): A qualitative study.

BACKGROUND: Elexacaftor-tezacaftor-ivacaftor (ETI) represents a significant step forward in cystic fibrosis (CF) care and could change the course of CF lung disease and quality of life for many people with CF (PwCF). However, several PwCF cannot benefit from these modulators because their rare mutations are not eligible for treatment. This study aimed to investigate the lived experiences of PwCF who are not eligible for ETI. METHODS: Data were collected through semi-structured interviews with 13 individuals with CF that were not eligible for ETI. Thematic analysis was used to identify the key themes of their experiences. RESULTS: Two main themes and six subthemes were identified. The first main theme (being deemed ineligible for ETI) had four subthemes (disappointment, information, happiness, and concerns). The second main theme (coping with a life without ETI) had two subthemes (lack of hope and continued hope). CONCLUSIONS: PwCF who are not eligible for ETI experience intense disappointment and conflicting emotions that can influence their decision-making linked to diminishing/renewal hope. Integrated care, including mental health monitoring programs, should be provided to these patients to aid them in overcoming their disappointment and to improve their coping.

Impact of COVID-19 on Lung Disease in People with Cystic Fibrosis: A 6-Month Follow-Up Study on Respiratory Outcomes.

BACKGROUND: The impact of COVID-19 on respiratory outcomes in people with cystic fibrosis (pwCF) has not been clearly characterized. We evaluated changes in respiratory function indicators derived from spirometry and pulmonary exacerbation rates 6 months after SARS-CoV-2 infection. METHODS: This multicentre prospective study was based on pwCF enrolled between October, 2020 and June, 2021 in the DECO COVID-19 project. PwCF complaining of COVID-like symptoms were tested with real-time polymerase chain reaction (RT-PCR) for SARS-CoV-2 on nasopharyngeal swab. Mean changes in respiratory function indicators and time to first episode of pulmonary exacerbation were compared in RT-PCR-positive and RT-PCR-negative patients. Regression models were used to adjust for baseline percent predicted forced expiratory volume in one second (ppFEV1) values, number of comorbidities, and initiation of CFTR modulator therapy during the follow-up. RESULTS: We enrolled 26 pwCF with RT-PCR-confirmed infection and 42 with a RT-PCR-negative test. After 6 months of follow-up, mean ppFEV1 changes were not significantly different between groups (+0.3% in positive vs. +0.2% in negative patients, p = 0.19). The 6-month cumulative probabilities of a first episode of pulmonary exacerbation were: 0.425 among RT-PCR-negative patients and 0.465 among those with a positive test (adjusted hazard ratio: 0.88, 95% CI: 0.44-1.75). CONCLUSIONS: COVID-19 did not appear to negatively influence respiratory outcomes of pwCF at 6 months from infection.

An inflammatory Signature of Glucose Impairment in Cystic Fibrosis.

Objective and Design: Cystic fibrosis-related diabetes (CFRD) is a severe complication associated with increased morbidity and mortality in cystic fibrosis (CF) patients. Extensive inflammatory state in CF leads to pancreas damage and insulin resistance with consequent altered glucose tolerance and CFRD development. The aim of the present study was to identify circulating levels of inflammatory markers specifically associated with impaired glucose tolerance (IGT) and overt CFRD in a sample of young adults with CF. Materials and Methods: Sixty-four CF outpatients, without evident active pulmonary exacerbation, infectious and autoimmune diseases, were enrolled in the study and the levels of 45 inflammatory serum mediators were measured through x magnetic bead panel multiplex technology. Results: Serum levels of PDGF-AA, CCL20/MIP3α, IFNα, CCL11/eotaxin, CXCL1/GROα, GMCSF, B7H1/PDL1, IL13, IL7, VEGF, and TGFα were all significantly (p<0.05) elevated in patients according to glycemic status and directly correlated with glycated hemoglobin and C-reactive protein levels. Conclusion: Our findings suggest that increased levels of specific circulating inflammatory mediators are directly associated with impaired glucose tolerance in CF patients, thus, potentially implicating them in CFRD pathogenesis and warranting larger longitudinal studies to validate their monitoring as predictor of CFRD onset.

Profiling the response to lumacaftor-ivacaftor in children with cystic between fibrosis and new insight from a French-Italian real-life cohort.

INTRODUCTION: Clinical trials for CFTR modulators consider mean changes of clinical status at the cohort level, and thus fail to assess the heterogeneity of the response. We aimed to study the different response profiles to lumacaftor-ivacaftor according to age in children with cystic fibrosis (CF). METHODS: A mathematical framework, including principal component analysis, data clustering, and data completion, was applied to a multicenter cohort of 112 children aged 6-18 years, treated with lumacaftor-ivacaftor. Studied parameters at baseline and 6 months included body mass index (BMI), number of days of antibiotics (ATB), Sweat test (ST), forced expiratory volume in 1 s expressed in percentage predicted (ppFEV1 ), forced vital capacity (ppFVC), and forced expiratory flow at 25%-75% of FVC (ppFEF25-75 ). RESULTS: Change in ppFEV1 was the most significant parameter in characterizing response heterogeneity among the 12-18-year-old patients. Patients with minimal changes in ppFEV1 were further separated by change in BMI and ATB course. In the 6-12-year-old children both BMI and ppFEV1 evolution were the most relevant. ST change was not associated with a clinical response. CONCLUSIONS: Change in ppFEV1 , BMI, and ATB course are the most relevant outcomes to discriminate clinical response profiles in children treated with lumacaftor-ivacaftor. Prepubertal and pubertal children display different response profiles.

Clinical outcomes of a large cohort of individuals with the F508del/5T;TG12 CFTR genotype.

BACKGROUND: In recent years, patients with cystic fibrosis (CF) conductance regulator (CFTR) variant poly(T) sequences have been increasingly reported with a wide spectrum of clinical severity. We describe the long-term clinical outcomes and progression to a CF diagnosis over time in a large Italian cohort of patients carrying the CFTR F508del/5T;TG12 genotype. METHODS: A retrospective analysis of subjects from 10 CF centres in Italy with the F508del/5T;TG12 genotype was performed. Demographic, clinical, microbiological, and biochemical data, as well as information about the follow-ups and complications of the enroled patients, were collected. RESULTS: A total of 129 subjects (54 females; median age: 15.0 years, range: 0-58 years; 59 older than 18 years) were included. In terms of initial diagnoses, 30 were CF (23.3%), 41 were CFTR-related disorder (CFTR-RD) (31.7%), and 58 were CF transmembrane conductance regulator-related metabolic syndrome/cystic fibrosis screen positive, inconclusive diagnosis (CRMS/CFSPID) (45.0%). After a median follow-up of 6.7 years (range 0.2-25 years), 15 patients progressed to CF, bringing the total number of CF diagnoses to 45/129 (34.9%). Most of these patients had mild lung diseases with pancreatic sufficiency and a low prevalence of CF-related complications. CONCLUSIONS: At the end of the study, 34.9% of subjects with the CFTR F508del/5T;TG12 genotype were diagnosed with CF. We suggest including patients with the F508del/5T;TG12 genotype in long-term follow-ups.

Theratyping of the Rare CFTR Variants E193K and R334W in Rectal Organoid-Derived Epithelial Monolayers.

BACKGROUND: The effect of presently available CFTR modulator combinations, such as elexacaftor (ELX), tezacaftor (TEZ), and ivacaftor (IVA), on rare CFTR alleles is often unknown. Several assays have been developed, such as forskolin-induced swelling (FIS), to evaluate the rescue of such uncommon CFTR alleles both by established and novel modulators in patient-derived primary cell cultures (organoids). Presently, we assessed the CFTR-mediated electrical current across rectal organoid-derived epithelial monolayers. This technique, which allows separate measurement of CFTR-dependent chloride or bicarbonate transport, was used to assess the effect of ELX/TEZ/IVA on two rare CFTR variants. METHODS: Intestinal organoid cultures were established from rectal biopsies of CF patients carrying the rare missense mutations E193K or R334W paired with F508del. The effect of the CFTR modulator combination ELX/TEZ/IVA on CFTR-mediated Cl- and HCO3- secretion was assessed in organoid-derived intestinal epithelial monolayers. Non-CF organoids were used for comparison. Clinical biomarkers (sweat chloride, FEV1) were monitored in patients receiving modulator therapy. RESULTS: ELX/TEZ/IVA markedly enhanced CFTR-mediated bicarbonate and chloride transport across intestinal epithelium of both patients. Consistent with the rescue of CFTR function in cultured intestinal cells, ELX/TEZ/IVA therapy improved biomarkers of CFTR function in the R334W/F508del patient. CONCLUSIONS: Current measurements in organoid-derived intestinal monolayers can readily be used to monitor CFTR-dependent epithelial Cl- and HCO3- transport. This technique can be explored to assess the functional consequences of rare CFTR mutations and the efficacy of CFTR modulators. We propose that this functional CFTR assay may guide personalized medicine in patients with CF-like clinical manifestations as well as in those carrying rare CFTR mutations.

Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection.

Mycobacterium abscessus is the etiological agent of severe pulmonary infections in vulnerable patients, such as those with cystic fibrosis (CF), where it represents a relevant cause of morbidity and mortality. Treatment of pulmonary infections caused by M. abscessus remains extremely difficult, as this species is resistant to most classes of antibiotics, including macrolides, aminoglycosides, rifamycins, tetracyclines, and ß-lactams. Here, we show that apoptotic body like liposomes loaded with phosphatidylinositol 5-phosphate (ABL/PI5P) enhance the antimycobacterial response, both in macrophages from healthy donors exposed to pharmacological inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) and in macrophages from CF patients, by enhancing phagosome acidification and reactive oxygen species (ROS) production. The treatment with liposomes of wild-type as well as CF mice, intratracheally infected with M. abscessus, resulted in about a 2-log reduction of pulmonary mycobacterial burden and a significant reduction of macrophages and neutrophils in bronchoalveolar lavage fluid (BALF). Finally, the combination treatment with ABL/PI5P and amikacin, to specifically target intracellular and extracellular bacilli, resulted in a further significant reduction of both pulmonary mycobacterial burden and inflammatory response in comparison with the single treatments. These results offer the conceptual basis for a novel therapeutic regimen based on antibiotic and bioactive liposomes, used as a combined host- and pathogen-directed therapeutic strategy, aimed at the control of M. abscessus infection, and of related immunopathogenic responses, for which therapeutic options are still limited. IMPORTANCE Mycobacterium abscessus is an opportunistic pathogen intrinsically resistant to many antibiotics, frequently linked to chronic pulmonary infections, and representing a relevant cause of morbidity and mortality, especially in immunocompromised patients, such as those affected by cystic fibrosis. M. abscessus-caused pulmonary infection treatment is extremely difficult due to its high toxicity and long-lasting regimen with life-impairing side effects and the scarce availability of new antibiotics approved for human use. In this context, there is an urgent need for the development of an alternative therapeutic strategy that aims at improving the current management of patients affected by chronic M. abscessus infections. Our data support the therapeutic value of a combined host- and pathogen-directed therapy as a promising approach, as an alternative to single treatments, to simultaneously target intracellular and extracellular pathogens and improve the clinical management of patients infected with multidrug-resistant pathogens such as M. abscessus.

Clinical course and risk factors for severe COVID-19 among Italian patients with cystic fibrosis: a study within the Italian Cystic Fibrosis Society.

PURPOSE: To describe the clinical course of COVID-19 in patients with cystic fibrosis (CF) and to identify risk factors for severe COVID-19. METHODS: We conducted a prospective study within the Italian CF Society. CF centers collected baseline and follow-up data of patients with virologically confirmed SARS-CoV-2 infection between March 2020 and June 2021. Odds ratios (ORs) for severe SARS-CoV-2 (as defined by hospital admission) were estimated by logistic regression models. RESULTS: The study included 236 patients with positive molecular test for SARS-CoV-2. Six patients died, 43 patients were admitted to hospital, 4 admitted to intensive care unit. Pancreatic insufficiency was associated with increased risk of severe COVID-19 (OR 4.04, 95% CI 1.52; 10.8). After adjusting for age and pancreatic insufficiency, forced expiratory volume in one second (FEVp) < 40% (OR 4.54, 95% CI 1.56; 13.2), oxygen therapy (OR 12.3, 95% CI 2.91-51.7), underweight (OR 2.92, 95% CI 1.12; 7.57), organ transplantation (OR 7.31, 95% CI 2.59; 20.7), diabetes (OR 2.67, 95% CI 1.23; 5.80) and liver disease (OR 3.67, 95% CI 1.77; 7.59) were associated with increased risk of severe COVID-19, while use of dornase alfa was associated with a reduced risk (OR 0.34, 95% CI 0.13-0.88). No significant changes were observed in FEVp from baseline to a median follow-up of 2 months (median difference: 0, interquartile range: - 4; 5, P = 0.62). CONCLUSION: Clinical features indicative of severe form of CF are associated with increased risk of COVID-19 hospitalization. SARS-CoV-2 infected patients do not experience a deterioration of respiratory function.

Liposomes Loaded With Phosphatidylinositol 5-Phosphate Improve the Antimicrobial Response to Pseudomonas aeruginosa in Impaired Macrophages From Cystic Fibrosis Patients and Limit Airway Inflammatory Response.

Despite intensive antimicrobial and anti-inflammatory therapies, cystic fibrosis (CF) patients are subjected to chronic infections due to opportunistic pathogens, including multidrug resistant (MDR) Pseudomonas aeruginosa. Macrophages from CF patients show many evidences of reduced phagocytosis in terms of internalization capability, phagosome maturation, and intracellular bacterial killing. In this study, we investigated if apoptotic body-like liposomes (ABLs) loaded with phosphatidylinositol 5-phosphate (PI5P), known to regulate actin dynamics and vesicular trafficking, could restore phagocytic machinery while limiting inflammatory response in in vitro and in vivo models of MDR P. aeruginosa infection. Our results show that the in vitro treatment with ABL carrying PI5P (ABL/PI5P) enhances bacterial uptake, ROS production, phagosome acidification, and intracellular bacterial killing in human monocyte-derived macrophages (MDMs) with pharmacologically inhibited cystic fibrosis transmembrane conductance regulator channel (CFTR), and improve uptake and intracellular killing of MDR P. aeruginosa in CF macrophages with impaired bactericidal activity. Moreover, ABL/PI5P stimulation of CFTR-inhibited MDM infected with MDR P. aeruginosa significantly reduces NF-κB activation and the production of TNF-α, IL-1ß, and IL-6, while increasing IL-10 and TGF-ß levels. The therapeutic efficacy of ABL/PI5P given by pulmonary administration was evaluated in a murine model of chronic infection with MDR P. aeruginosa. The treatment with ABL/PI5P significantly reduces pulmonary neutrophil infiltrate and the levels of KC and MCP-2 cytokines in the lungs, without affecting pulmonary bacterial load. Altogether, these results show that the ABL/PI5P treatment may represent a promising host-directed therapeutic approach to improve the impaired phagocytosis and to limit the potentially tissue-damaging inflammatory response in CF.

Ionocytes and CFTR Chloride Channel Expression in Normal and Cystic Fibrosis Nasal and Bronchial Epithelial Cells.

The airway epithelium contains ionocytes, a rare cell type with high expression of Forkhead Box I1 (FOXI1) transcription factor and Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a chloride channel that is defective in cystic fibrosis (CF). Our aim was to verify if ionocyte development is altered in CF and to investigate the relationship between ionocytes and CFTR-dependent chloride secretion. We collected nasal cells by brushing to determine ionocyte abundance. Nasal and bronchial cells were also expanded in vitro and reprogrammed to differentiated epithelia for morphological and functional studies. We found a relatively high (~3%) ionocyte abundance in ex vivo nasal samples, with no difference between CF and control individuals. In bronchi, ionocytes instead appeared very rarely as previously reported, thus suggesting a possible proximal-distal gradient in human airways. The difference between nasal and bronchial epithelial cells was maintained in culture, which suggests an epigenetic control of ionocyte development. In the differentiation phase of the culture procedure, we used two media that resulted in a different pattern of CFTR expression: confined to ionocytes or more broadly expressed. CFTR function was similar in both conditions, thus indicating that chloride secretion equally occurs irrespective of CFTR expression pattern.

Unravelling druggable signalling networks that control F508del-CFTR proteostasis.

Cystic fibrosis (CF) is caused by mutations in CF transmembrane conductance regulator (CFTR). The most frequent mutation (F508del-CFTR) results in altered proteostasis, that is, in the misfolding and intracellular degradation of the protein. The F508del-CFTR proteostasis machinery and its homeostatic regulation are well studied, while the question whether 'classical' signalling pathways and phosphorylation cascades might control proteostasis remains barely explored. Here, we have unravelled signalling cascades acting selectively on the F508del-CFTR folding-trafficking defects by analysing the mechanisms of action of F508del-CFTR proteostasis regulator drugs through an approach based on transcriptional profiling followed by deconvolution of their gene signatures. Targeting multiple components of these signalling pathways resulted in potent and specific correction of F508del-CFTR proteostasis and in synergy with pharmacochaperones. These results provide new insights into the physiology of cellular proteostasis and a rational basis for developing effective pharmacological correctors of the F508del-CFTR defect.

Ouabain Mimics Low Temperature Rescue of F508del-CFTR in Cystic Fibrosis Epithelial Cells.

Most cases of cystic fibrosis (CF) are caused by the deletion of a single phenylalanine residue at position 508 of the cystic fibrosis transmembrane conductance regulator (CFTR). The mutant F508del-CFTR is retained in the endoplasmic reticulum and degraded, but can be induced by low temperature incubation (29°C) to traffic to the plasma membrane where it functions as a chloride channel. Here we show that, cardiac glycosides, at nanomolar concentrations, can partially correct the trafficking of F508del-CFTR in human CF bronchial epithelial cells (CFBE41o-) and in an F508del-CFTR mouse model. Comparison of the transcriptional profiles obtained with polarized CFBE41o-cells after treatment with ouabain and by low temperature has revealed a striking similarity between the two corrector treatments that is not shared with other correctors. In summary, our study shows a novel function of ouabain and its analogs in the regulation of F508del-CFTR trafficking and suggests that compounds that mimic this low temperature correction of trafficking will provide new avenues for the development of therapeutics for CF.

Decreasing Poly(ADP-Ribose) Polymerase Activity Restores ΔF508 CFTR Trafficking.

Most cystic fibrosis is caused by mutations in CFTR that prevent its trafficking from the ER to the plasma membrane and is associated with exaggerated inflammation, altered metabolism, and diminished responses to oxidative stress. PARP-1 is activated by oxidative stress and causes energy depletion and cell dysfunction. Inhibition of this enzyme protects against excessive inflammation and recent studies have also implicated it in intracellular protein trafficking. We hypothesized that PARP-1 activity is altered in CF and affects trafficking and function of the most common CF mutant ΔF508 CFTR. Indeed, PARP-1 activity was 2.9-fold higher in CF (ΔF508/ΔF508) human bronchial epithelial primary cells than in non-CF cells, and similar results were obtained by comparing CF vs. non-CF bronchial epithelial cell lines (2.5-fold higher in CFBE41o(-) vs. 16HBE14o(-), P < 0.002). A PARP-1 inhibitor (ABT-888, Veliparib) partially restored CFTR channel activity in CFBE41o(-) cells overexpressing ΔF508 CFTR. Similarly, reducing PARP-1 activity by 85% in ileum from transgenic CF mice (Cftr(tm1)Eur) partially rescued ΔF508 CFTR activity to 7% of wild type mouse levels, and similar correction (7.8%) was observed in vivo by measuring salivary secretion. Inhibiting PARP-1 with ABT-888 or siRNA partially restored ΔF508 CFTR trafficking in cell lines, and most ΔF508 CFTR was complex glycosylated when heterologously expressed in PARP-1(-/-) mouse embryonic fibroblasts. Finally, levels of the mature glycoform of CFTR were reduced by peroxynitrite, a strong activator of PARP-1. These results demonstrate that PARP-1 activity is increased in CF, and identify a novel pathway that could be targeted by proteostatic correctors of CFTR trafficking.

Generation of long RNA chains in water.

The synthesis of RNA chains from 3',5'-cAMP and 3',5'-cGMP was observed. The RNA chains formed in water, at moderate temperatures (40-90 degrees C), in the absence of enzymes or inorganic catalysts. As determined by RNase analyses, the bonds formed were canonical 3',5'-phosphodiester bonds. The polymerizations are based on two reactions not previously described: 1) oligomerization of 3', 5'-cGMP to approximately 25-nucleotide-long RNA molecules, and of 3',5'-cAMP to 4- to 8-nucleotide-long molecules. Oligonucleotide A molecules were further extended by reciprocal terminal ligation to yield RNA molecules up to >120 nucleotides long and 2) chain extension by terminal ligation of newly polymerized products of 3',5'-cGMP on preformed oligonucleotides. The enzyme- and template-independent synthesis of long oligomers in water from prebiotically affordable precursors approaches the concept of spontaneous generation of (pre)genetic information.

From formamide to RNA: the roles of formamide and water in the evolution of chemical information.

In pursuing the origin of informational polymers, we followed the assumption that their spontaneous formation could only have occurred: (i) if all the components were present at the same site and in the same reaction, and (ii) if the thermodynamics of the processes involved favored a polymerized over a monomeric state of the precursors. A plausible scenario satisfying both assumptions is provided.