Ion channels, chloride channels, calcium signaling, drug discovery, cystic fibrosis
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Scopus Publications
Scopus Publications
Evaluation of ATP12A and NFKBIZ as potential markers of inflammatory status in cystic fibrosis airway epithelial cells Caterina Allegretta, Daniela Guidone, Silvia Boscia, Laura Pisano, Silvia Ricci, et al. Inflammation Research, 2026 Background People with Cystic Fibrosis (pwCF) are prone to bacterial lung infections with P. aeruginosa , which have been linked to chronic inflammation in the lung. Although the highly effective CFTR modulator therapy (Elexacaftor-Tezacaftor-Ivacaftor, ETI) has dramatically improved respiratory outcomes in pwCF, airway inflammation and bacterial colonization persist in the upper and lower respiratory tracts. Methods We investigated the effect of ETI in both plasma and fresh primary nasal epithelial (HNE) cells obtained from pwCF pre- and post-three months of ETI treatment. Given that inflammation has been shown to upregulate NFKBIZ and the ATP12A proton pump, we measured their levels in fresh HNE cells and in cultured HNE cells exposed to clinical exoproducts (EXO) of P. aeruginosa or other inflammatory stimuli. Results ELISA analysis revealed a significant reduction of IL-6, IL-8, and IL-17C in both plasma and HNE cells after ETI treatment. NFKBIZ and ATP12A expression was increased after infection and inflammatory stimuli in CF bronchial epithelial (CFBE) and HNE cells, and this increase was reduced by Dimethyl-Fumarate, an anti-inflammatory drug. Conclusions These preclinical studies, using patient-derived tissues, suggest that NFKBIZ and ATP12A may play a relevant role in the pathophysiology and inflammatory response of the CF airway epithelium.
Functional correction of the untreatable CFTR 1717-1G>A mutation through mRNA- and sgRNA-optimized base editing Alessandro Umbach, Annalisa Santini, Mattijs Bulcaen, Daniela Guidone, Giulia Maule, et al. Science Translational Medicine, 2026 The 1717-1G>A is a prevalent splicing mutation causing cystic fibrosis (CF) for which no pharmacological treatments have been approved. This mutation disrupts a canonical 3′ AG splice acceptor site in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene, leading to severe RNA missplicing, which prevents the correct synthesis of the encoded protein. In this study, we developed an adenine base editing (ABE) strategy to efficiently correct the 1717-1G>A mutation. By using the ABE9 base editor with the protospacer adjacent motif–relaxed Streptococcus pyogenes clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) variant SpRY, we obtained up to 30% editing with limited bystander effects in a human embryonic kidney (HEK) 293–based cellular model. Through systematic optimizations of the ABE system, delivered by electroporation of base editor messenger RNA (mRNA) and single guide RNA (sgRNA), we demonstrated genetic repair of the 1717-1G>A mutation in airway epithelial cells and intestinal organoids derived from people with CF. Functional analysis was performed by measuring short-circuit current in air-liquid interface (ALI) culture and by assessing forskolin-induced swelling (FIS) in intestinal organoids, which revealed restoration of CFTR channel activity. These results highlight SpRY-ABE9 as a potential genome editing strategy to permanently correct the CFTR 1717-1G>A mutation and restore CFTR function.
Esc peptides and derivatives potentiate the activity of CFTR with gating defects and display antipseudomonal activity in cystic fibrosis-like lung disease Loretta Ferrera, Floriana Cappiello, Arianna Venturini, Hexin Lu, Bruno Casciaro, et al. Cellular and Molecular Life Sciences, 2025 Cystic fibrosis (CF) is a rare disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), a chloride channel with an important role in the airways. Despite the clinical efficacy of present modulators in restoring the activity of defective CFTR, there are patients who show persistent pulmonary infections, mainly due to Pseudomonas aeruginosa. Recently, we reported an unprecedented property of antimicrobial peptides i.e. Esc peptides, which consists in their ability to act as potentiators of CFTR carrying the most common mutation (the loss of phenylalanine 508) affecting protein folding, trafficking and gating. In this work, by electrophysiology experiments and computational studies, the capability of these peptides and de-novo designed analogs was demonstrated to recover the function of other mutated forms of CFTR which severely affect the channel gating (G551D and G1349D). This is presumably due to direct interaction of the peptides with the nucleotide binding domains (NBDs) of CFTR, followed by a novel local phenomenon consisting in distancing residues located at the cytosolic side of the NBDs interface, thus stabilizing the open conformation of the pore at its cytosolic end. The most promising peptides for the dual antimicrobial and CFTR potentiator activities were also shown to display antipseudomonal activity in conditions mimicking the CF pulmonary ion transport and mucus obstruction, with a higher efficacy than the clinically used colistin. These studies should assist in development of novel drugs for lung pathology in CF, with dual CFTR potentiator and large spectrum antibiotic activities.
In silico, in vitro and ex vivo characterization of cystic fibrosis transmembrane conductance regulator pathogenic variants localized in the fourth intracellular loop and their rescue by modulators Emanuela Pesce, Valeria Tomati, Valeria Capurro, Mariateresa Lena, Cristina Pastorino, et al. British Journal of Pharmacology, 2025 Background and PurposeCystic fibrosis (CF) is due to loss‐of‐function variants of the CF transmembrane conductance regulator (CFTR) channel. The most effective treatment for people with CF carrying the F508del mutation is the triple combination of elexacaftor–tezacaftor–ivacaftor (ETI). ETI can correct the underlying defect(s) in other CFTR mutants. The use of disease‐relevant predictive models such as patient‐derived human nasal epithelial cells allow to investigate the response to CFTR modulators of specific genotypes, possibly supporting patients' access to treatment.Experimental ApproachUsing computational, biochemical and functional methodologies, a detailed analysis of selected variants in the intracellular loop 4 (ICL4) to understand their impact on CFTR structure and function.Key ResultsMutations affecting L1065, R1066 and L1077 compromise structural stability of CFTR. Analyses of single variants expressed heterologously in immortalized bronchial cells showed that, upon ETI, rescued activity for both L1065P and R1066C was close to 50% of the wild‐type CFTR activity. Biochemical studies of ICL4 variants expression pattern in CFBE41o‐cells, following treatment for 24 h, demonstrate the appearance of the mature, fully glycosylated band, with no changes in the immature band. Finally, our study provides evidence in primary nasal cells from a cohort of people with CF that L1065P and R1066C can be effectively rescued by ETI up to 25%–45% of the activity measured in non‐CF epithelia.Conclusion and ImplicationsAlthough the observed rescue for L1065P and R1066C was smaller than that of the F508del, it should fall in a range predicted, by various studies, to provide a clinical benefit.
Sodium-Coupled Monocarboxylate Absorption in the Airway Epithelium Is Facilitated by the SLC5A8 Co-Transporter Anita Guequen, Bárbara Tapia‐Balladares, Tábata Apablaza, Daniela Guidone, Nátali Cárcamo‐Lemus, et al. Acta Physiologica, 2025 AimAmino acids, sugars, short‐chain fatty acids (SCFA), vitamins, and other small molecules compose the extracellular metabolome on the airway lumen surface, but how the airway epithelium deals with these molecules has not been deeply studied. Due to the broad spectrum of metabolites transported by SLC5A8 and SLC5A12, we aim to determine if they are functionally expressed and participate in the absorption of Na+, short‐chain fatty acids, and monocarboxylates in mouse and human airway epithelium.MethodsTracheas isolated from male or female mice and human bronchial epithelial cells (HBECs) were used for electrophysiological studies in the Ussing chamber and to detect members of the SLC16 family by RT‐PCR and bulk RNAseq. Additionally, cell lines expressing the human and murine SLC5A8 transporter were employed for uptake studies using a fluorescent lactate probe.ResultsWe showed for the first time that human and murine airway epithelium express a functional SLC5A8 transporter, facilitating the absorption of glucose metabolites and SCFAs. The Na+‐coupled monocarboxylate transport was not additive with ENaC‐mediated Na+ absorption in mouse trachea. We observed that valproate acts as an inhibitor of the murine but not of the human SLC5A8 transporter.ConclusionsOur results demonstrate that several metabolites derived from bacterial and cellular metabolism can be transported from the airway lumen into the epithelial cells, participating in a homeostatic relation of the tissue with its environment.
The apical mucus layer alters the pharmacological properties of the airway epithelium Daniela Guidone, Martina de Santis, Emanuela Pesce, Valeria Capurro, Nicoletta Pedemonte, et al. Journal of Physiology, 2025 Electrogenic transepithelial ion transport can be measured with the short‐circuit current technique. Such experiments are frequently used to evaluate the activity of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP‐activated chloride channel that is defective in cystic fibrosis, one of the most frequent genetic diseases. Typically, CFTR activity is estimated from the effect of CFTRinh‐172, a selective CFTR inhibitor. Unexpectedly, we found that CFTRinh‐172, in addition to PPQ‐102, another CFTR inhibitor, caused only partial inhibition of CFTR function, particularly in epithelia in pro‐inflammatory conditions, which are characterized by abundant mucus secretion. We hypothesized that the mucus layer was responsible for the poor activity of CFTR inhibitors. Therefore, we treated the epithelial surface with the reducing agent dithiothreitol to remove mucus. Removal of mucus, confirmed by immunofluorescence, resulted in highly enhanced sensitivity of CFTR to pharmacological inhibition. Our results show that the mucus layer represents an important barrier whose presence limits the activity of pharmacological agents. This is particularly relevant for CFTR and the evaluation of therapeutic approaches for correction of the basic defect in cystic fibrosis. imageKey points Activity of the cAMP‐activated cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel can be evaluated by measuring the inhibition elicited by the selective blockers CFTRinh‐172 and PPQ‐102. In short‐circuit current recordings on human airway epithelia, CFTR inhibitors had only a partial effect on cAMP‐dependent chloride secretion, suggesting the possible contribution of other ion channels. The mucus layer covering the epithelial surface was removed with the reducing agent dithiothreitol. Treatment of epithelia with dithiothreitol markedly improved the efficacy of CFTR inhibitors. The partial effect of CFTR inhibitors might be explained by the presence of the mucus layer acting as a barrier.
Distinct Responses of Cystic Fibrosis Epithelial Cells to SARS-CoV-2 and Influenza A Virus Isabel Pagani, Arianna Venturini, Valeria Capurro, Alessandro Nonis, Silvia Ghezzi, et al. American Journal of Respiratory Cell and Molecular Biology, 2025 The coronavirus disease (COVID-19) pandemic has underscored the impact of viral infections on individuals with cystic fibrosis (CF). Initial observations suggested lower COVID-19 rates among CF populations; however, subsequent clinical data have presented a more complex scenario. This study aimed to investigate how bronchial epithelial cells from individuals with and without CF, including various CFTR (CF transmembrane conductance regulator) mutations, respond to in vitro infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and SARS-CoV. Comparisons with the influenza A virus (IAV) were included based on evidence that patients with CF experience heightened morbidity from IAV infection. Our findings showed that CF epithelial cells exhibited reduced replication of SARS-CoV-2, regardless of the type of CFTR mutation or SARS-CoV-2 variant, as well as the original 2003 SARS-CoV. In contrast, these cells displayed more efficient IAV replication than non-CF cells. Interestingly, the reduced susceptibility to SARS-CoV-2 in CF was not linked to the expression of ACE2 (angiotensin-converting enzyme 2) receptor or to CFTR dysfunction, as pharmacological treatments to restore CFTR function did not normalize the viral response. Both SARS-CoV-2 infection and CFTR function influenced the concentrations of certain cytokines and chemokines, although these effects were not correlated. Overall, this study reveals a unique viral response in CF epithelial cells, characterized by reduced replication for some viruses like SARS-CoV-2, while showing increased susceptibility to others, such as IAV. This research offers a new perspective on CF and viral interactions, emphasizing the need for further investigation into the mechanisms underlying these differences.
The role of pendrin in the airways: Links with asthma and copd Kenji Izuhara, Shoichi Suzuki, Charity Nofziger, Masahiro Ogawa, Shoichiro Ohta, et al. Role of Pendrin in Health and Disease Molecular and Functional Aspects of the Slc26a4 Anion Exchanger, 2017
CFTR pharmacology Olga Zegarra-Moran, Luis J. V. Galietta Cellular and Molecular Life Sciences, 2017
Correction of delF508-CFTR activity with benzo(c)quinolizinium compounds through facilitation of its processing in cystic fibrosis airway cells Journal of Cell Science, 2001
Molecular genetics. Research and clinical activity Gaslini, 2001
