The ATP-binding cassette subfamily A member 3 (ABCA3) protein on the limiting membrane of lamellar bodies in alveolar type 2 (AT2) cells transports phospholipids required for pulmonary surfactant assembly. ABCA3 deficiency results from biallelic pathogenic variants in ABCA3 and causes progressive neonatal respiratory failure or childhood interstitial lung disease (chILD). Supportive/compassionate care or lung transplantation are the only current definitive treatments for ABCA3 deficiency and progressive respiratory failure. Complementing dysfunctional ABCA3 by gene addition has therapeutic potential. Previous studies show that repairing or complementing ABCA3 in induced pluripotent stem cell (iPSC)-derived AT2 cells rescues lamellar body morphology and surfactant phospholipid composition. Pathogenic variants disrupt ABCA3 function through altered protein trafficking (type 1) or by impaired phospholipid transport (type 2) into lamellar bodies. Here we tested ABCA3 gene complementation using a human pulmonary epithelial cell line (A549) with a genomically silenced ABCA3 locus (ABCA3 KO). From this line, additional cell lines that stably express individual ABCA3 variant cDNA constructs from a single genomic locus were tested: L101P (type 1), E292V (type 2), E690K (type 2), or wild-type ABCA3. Lentiviral-mediated ABCA3 delivery to each cell line partially rescued localization to LAMP3+ vesicles, lamellar body-like structure morphology, and cell proliferation. A functional assay measuring NF-κB signaling suggested that ABCA3 complementation ameliorated aberrant inflammatory signaling in E292V or E690K (type 2) mutant lines, but not in L101P (type 1) or knockout lines. These studies highlight the therapeutic potential of gene addition as well as differences between ABCA3 pathogenic variants that may influence genetic therapy outcomes.

Cystic fibrosis (CF) results from mutations in the CFTR gene, which encodes an anion channel critical for airway physiology. Airway disease is the major cause of morbidity in CF, yet the cellular basis of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl– transport remains incompletely understood. Recent single cell transcriptomic studies revealed that CFTR mRNA expression is concentrated in rare ionocytes. Historically, CFTR in airway epithelia was thought to drive Cl– secretion; however, our previous reports indicate that ionocytes mediate Cl– absorption rather than secretion. The finding raises a fundamental question: which cells drive Cl– secretion in human airways? We hypothesized that CFTR in secretory cells—named for their secretory vesicles—perform Cl– secretion. To test this hypothesis, we engineered n = 6 primary human airway epithelial cultures to selectively deplete ionocytes or secretory cells by disrupting cell signaling essential for their cell type specification. We eliminated ionocytes by CRISPR-mediated disruption of FOXI1 and reduced the number of secretory cells by inhibiting notch signaling with the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Cell populations were quantified by flow cytometry, and CFTR-dependent currents were measured using the transepithelial voltage-clamp technique under conditions favoring apical-to-basolateral Cl– flow (i.e., an apical-to-basolateral [Cl–] gradient, Vt = 0 mV) or active secretion (i.e., symmetrical [Cl–], Vt = 0 mV). Approrate multivariant statistics were applied to each dataset and adjusted for multiple comparisons. FOXI1 disruption (gFOXI1) eliminated ionocytes without affecting other cell types and abolished apical-to-basolateral Cl– flow without affecting active secretion [Igradient (µA*cm–2) control 1.21 ± 0.69 vs. gFOXI1 -0.53 ± 0.83, p = 0.03 and Isc (µA*cm–2) control 12.28 ± 6.53 vs. gFOXI1 12.36 ± 5.56, p = 0.99]. This finding is consistent with our previous reports that ionocytes perform Cl– absorption and inefficiently perform active Cl– secretion. In contrast, DAPT-treated epithelia retained apical-to-basolateral Cl– flow but exhibited markedly reduced active secretion [Igradient (µA*cm–2) control 1.21 ± 0.69 vs. DAPT 1.41 ± 1.18, p = 0.99 and Isc (µA*cm–2), control 12.28 ± 6.53 vs. DAPT 2.82 ± 2.32, p = 0.03]. Flow cytometry revealed DAPT did not decrease ionocytes or the larger population of CEACAM6+ secretory cells but eliminated a subset of MUC5AC+ secretory cells raising the possibility that another rare CFTR-containing cell may oppose CFTR-mediated absorption by ionocytes [ionocytes (% of epithelial cells) control 0.27 ± 0.17 vs. DAPT 0.14 ± 0.10, p = 0.12; secretory cells (% of epithelial cells) control 37.29 ± 21.97 vs. DAPT 32.70 ± 18.95, p = 0.40; and MUC5AC+ (% of epithelial cells), control 0.97 ± 1.88 vs. DAPT 0.49 ± 0.99, p = 0.04].These findings identify secretory cells as a driver of Cl– secretion, while ionocytes mediate absorption. Together, these results suggest a division of labor among airway epithelial cells with CFTR function partitioned between two specialized cell types. The proposed model provides a framework for understanding CF pathogenesis and informing strategies for targeted therapies. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Background: In individuals with cystic fibrosis (CF), respiratory viral infections frequently result in hospitalization and have been linked to secondary bacterial infection and colonization, highlighting viral infections as possible contributors to CF lung disease progression. We hypothesized that expression of antiviral host defense genes is dysregulated in CF airway epithelia. Methods: We infected primary CF and Non-CF airway epithelia with respiratory syncytial virus (RSV) and characterized their responses at 12 hr, 24 hr, 48 hr, 72 hr, and 120 hr post infection (hpi) by RNA sequencing (RNAseq). Results: Our analysis revealed strikingly different gene expression profiles for the CF and Non-CF epithelia over the course of the infection. While both CF and Non-CF cells exhibited an early signature for interferon signaling and antiviral defense pathways, this response was relatively exaggerated and sustained in CF epithelia. We also observed, in both genotypes, a transient downregulation of cilia-associated genes and loss of ciliary activity by 72 hpi. Interestingly, recovery of cilia activity was delayed in the CF epithelia. Conclusions: These findings further our understanding of innate immune dysfunction in the CF airway epithelium and suggest that virus-induced cilia injury may further compromise host defenses in CF airways.

BACKGROUND:Treatments available to prevent progression of virus-induced lung diseases, including coronavirus disease 2019 (COVID-19) are of limited benefit once respiratory failure occurs. The efficacy of approved and emerging cytokine signalling-modulating antibodies is variable and is affected by disease course and patient-specific inflammation patterns. Therefore, understanding the role of inflammation on the viral infectious cycle is critical for effective use of cytokine-modulating agents. METHODS:The role of the type 2 cytokine IL-13 on SARS-CoV-2 binding/entry, replication, and host response was investigated in primary HAE cells in vitro and in a model of mouse-adapted SARS-CoV-2 infection in vivo using single-cell and bulk RNA-sequencing approaches. Additionally, the responses were quantified using immunofluorescence, histopathology, immunohistochemistry and LC-MS/MS assays. FINDINGS:IL-13 protected airway epithelial cells from SARS-CoV-2 infection in vitro by decreasing the abundance of ACE2-expressing ciliated cells rather than by neutralisation in the airway surface liquid or by interferon-mediated antiviral effects. In contrast, IL-13 worsened disease severity in mice; the effects were mediated by eicosanoid signalling and were abolished in mice deficient in the phospholipase A2 enzyme PLA2G2D. INTERPRETATION:IL-13-induced inflammation differentially affects multiple steps of COVID-19 pathogenesis. IL-13-induced inflammation may be protective against initial SARS-CoV-2 airway epithelial infection; however, it enhances disease progression in vivo. Blockade of IL-13 and/or eicosanoid signalling may be protective against progression to severe respiratory virus-induced lung diseases. FUNDING:Carver Trust COVID-19 Grant; CF Foundation Iowa RDP; NIH 1R01HL163024; K01HL140261; NIH R01AI129269; NIH P01AI060699; NIH Grant P30 DK-54759; Cystic Fibrosis Foundation PEZZUL20A1-KB; Stead Family Foundation.

Background: The role of complement in the host response to respiratory viral infections is debated, with evidence suggesting it can either promote an effective immune response, or induce excessive inflammation and acute lung injury. During the COVID-19 pandemic, complement was implicated in disease pathobiology and complement C5 inhibition was granted emergency use approval in severe cases. Currently, virulent strains of both SARS-CoV-2 and influenza still result in thousands of deaths annually, and new variants which could produce another pandemic event are an ever present threat. Thus, it is vitally important that we understand the mechanisms by which these respiratory viruses induce severe disease and identify new therapeutic options for the future. Methods: We intranasally inoculated Balb/c WT, C3-/+, C3-/- mice with lethal doses of mouse-adapted SARS-CoV-2 (SARS2-N501YMA30) and PR8 influenza (5,000 PFU and 100 PFU, respectively) to determine whether varying degrees of complement expression altered the observed disease phenotype. Mice were monitored for weight loss and survival and euthanized on days 2 and 4 post-infection for sample collection. Results: We found that both viruses induced a significant complement response in the lungs of wild-type mice by lung homogenate western blot for C3, C9, and FB. Interestingly, we found that mouse genotype directly influenced severity of illness, with homozygous mice having the best outcome and wild-type mice the worst, based on differences in weight-loss (p > 0.05) and survival (p < 0.05). Conclusion: This data confirms that complement contributes to the pathobiology in mouse models of both SARS-CoV-2 and influenza pneumonia, suggesting that the mechanisms by which complement drives pathogenesis is conserved across various human pathologic respiratory viruses. This data will serve to support future research investigating the specific mechanisms by which complement influences the host response to these infections, and complement inhibition as a broad spectrum treatment option for severe viral pneumonia.

In vitro studies and observational human disease data suggest the complement system contributes to SARS-CoV-2 pathogenesis, although how complement dysregulation develops in severe COVID-19 is unknown. Here, using a mouse-adapted SARS-CoV-2 virus (SARS2-N501YMA30) and a mouse model of COVID-19, we identify significant serologic and pulmonary complement activation post-infection. We observed C3 activation in airway and alveolar epithelia, and pulmonary vascular endothelia. Our evidence suggests the alternative pathway is the primary route of complement activation, however, components of both the alternative and classical pathways are produced locally by respiratory epithelial cells following infection, and increased in primary cultures of human airway epithelia following cytokine and SARS-CoV-2 exposure. This tissue-specific complement response appears to precede lung injury and inflammation. Our results suggest that complement activation is a defining feature of severe COVID-19 in mice, agreeing with previous publications, and provide the basis for further investigation into the role of complement in COVID-19.

Rationale: COPA syndrome is an autosomal dominant systemic inflammatory disease, which includes interstitial lung disease (ILD) as a major clinical feature. This syndrome is caused by mutations in the COPA gene, leading to dysfunction in the coatomer protein complex I subunit and resulting in immune hyperactivation, such as aberrant activation of the STING pathway. However, the specific role of COPA gene mutations in lung epithelial cells and their contribution to ILD development in COPA syndrome remain unclear. This study aims to elucidate this link.Methods: Single-cell RNA sequencing was performed on explanted lung tissue from a patient with COPA syndrome carrying the COPA E241K mutation. The findings were compared with data from healthy lung and idiopathic pulmonary fibrosis (IPF) samples. Additionally, conditional transgenic mouse models were developed to induce Cre-mediated cell-type-specific conditional overexpression of either wild-type COPA (COPA-WT) or mutant COPA (COPA-MUT). Lung tissue morphology over time was analyzed, as well as sensitivity to bleomycin-induced fibrosis in these mice. To further investigate the cellular effects of COPA mutations, COPA-MUT was overexpressed in HEK293T cells via lentiviral transduction, and gene expression profiles were assessed.Results: Single-cell analysis of lung tissue from the COPA syndrome patient revealed a lower proportion of alveolar epithelial cells compared to healthy and IPF samples. Type II alveolar epithelial cells exhibited reduced expression of surfactant genes but showed upregulation of markers related to cellular senescence and the unfolded protein response (UPR)/integrated stress response (ISR) pathways, indicating a potential role in fibrosis. Although Shh-Cre COPA-MUT mice did not develop spontaneous fibrosis, they exhibited thickened alveolar walls. Compared to Shh-Cre COPA-WT mice, Shh-Cre COPA-MUT mice had increased mortality and greater fibrosis after bleomycin administration. Furthermore, HEK293T cells overexpressing COPA-MUT displayed upregulated genes associated with the UPR/ISR pathways.Conclusions: The presence of COPA gene mutations in lung epithelial cells may contribute to lung tissue remodeling and the pathogenesis of ILD in COPA syndrome, potentially through mechanisms involving upregulation of the UPR/ISR pathways.

Base editing could correct nonsense mutations that cause cystic fibrosis (CF), but clinical development is limited by the lack of delivery methods that efficiently breach the barriers presented by airway epithelia. Here, we present a novel amphiphilic shuttle peptide based on the previously reported S10 peptide that substantially improved base editor ribonucleoprotein (RNP) delivery. Studies of the S10 secondary structure revealed that the alpha-helix formed by the endosomal leakage domain (ELD), but not the cell penetrating peptide (CPP), was functionally important for delivery. By isolating and extending the ELD, we created a novel shuttle peptide, termed S237. While S237 achieved lower delivery of green fluorescent protein, it outperformed S10 at Cas9 RNP delivery to cultured human airway epithelial cells and to pig airway epithelia in vivo, possibly due to its lower net charge. In well-differentiated primary human airway epithelial cell cultures, S237 achieved a 4.6-fold increase in base editor RNP delivery, correcting up to 9.4% of the cystic fibrosis transmembrane conductance regulator (CFTR) R553X allele and restoring CFTR channel function close to non-CF levels. These findings deepen the understanding of peptide-mediated delivery and offer a translational approach for base editor RNP delivery for CF airway disease.

Mutations in more than 50 different genes cause primary ciliary dyskinesia (PCD) by disrupting the activity of motile cilia that facilitate mucociliary transport (MCT). Knowledge of PCD has come from studies identifying disease-causing mutations, characterizing structural cilia abnormalities, finding genotype-phenotype relationships, and studying the cell biology of cilia. Despite these important findings, we still lack effective treatments and people with PCD have significant pulmonary impairment. As with many other diseases, a better understanding of pathogenic mechanisms may lead to effective treatments. To pursue disease mechanisms, we used CRISPR-Cas9 to develop a PCD pig with a disrupted DNAI1 gene. PCD pig airway cilia lacked the outer dynein arm and had impaired beating. MCT was impaired under both baseline conditions and after cholinergic stimulation in PCD pigs. Neonatal PCD pigs developed neonatal respiratory distress with evidence of atelectasis, air trapping, and airway mucus obstruction. Despite airway mucus accumulation, lung bacterial counts were similar between neonatal wild-type and PCD pigs. Sinonasal disease was present in all neonatal PCD pigs. Older PCD pigs developed worsening airway mucus obstruction, inflammation, and bacterial infection. This pig model closely mimics the disease phenotype seen in people with PCD and can be used to better understand the pathophysiology of PCD airway disease.

Background Chronic pulmonary conditions such as asthma and chronic obstructive pulmonary disease increase the risk of morbidity and mortality during infection with the Middle East respiratory syndrome coronavirus (MERS-CoV). We hypothesized that individuals with such comorbidities are more susceptible to MERS-CoV infection due to increased expression of its receptor, dipeptidyl peptidase 4 (DPP4).Methods We modeled chronic airway disease by treating primary human airway epithelia with the Th2 cytokine interleukin 13 (IL-13), examining how this affected DPP4 protein levels with MERS-CoV entry and replication.Results IL-13 exposure for 3 days led to greater DPP4 protein abundance, while a 21-day treatment raised DPP4 levels and caused goblet cell metaplasia. Surprisingly, despite this increase in receptor availability, MERS-CoV entry and replication were not significantly affected by IL-13 treatment.Conclusions Our results suggest that greater DPP4 abundance is likely not the primary mechanism leading to increased MERS severity in the setting of Th2 inflammation. Transcriptional profiling analysis highlighted the complexity of IL-13-induced changes in airway epithelia, including altered expression of genes involved in innate immunity, antiviral responses, and maintenance of the extracellular mucus barrier. These data suggest that additional factors likely interact with DPP4 abundance to determine MERS-CoV infection outcomes. Stimulating airway epithelia with interleukin 13 increased DPP4, the receptor for Middle East respiratory syndrome coronavirus (MERS-CoV). However, this failed to increase MERS-CoV infection, suggesting that elevated airway epithelial DPP4 cannot explain increased risk of severe MERS in chronic airway disease.

The volume and composition of airway surface liquid (ASL) is regulated by liquid secretion and absorption across airway epithelia, controlling the pH, solute concentration, and biophysical properties of ASL in health and disease. Here, we developed a method integrating explanted tracheal tissue with a micro-machined device (referred to as "ex vivo trachea-chip") to study the dynamic properties of ASL volume regulation. The ex vivo trachea-chip allows real-time measurement of ASL transport (J(v)) with intact airway anatomic structures, environmental control, high-resolution, and enhanced experimental throughput. Applying this technology to freshly excised tissue we observed ASL absorption under basal conditions. The apical application of amiloride, an inhibitor of airway epithelial sodium channels (ENaC), reduced airway liquid absorption. Furthermore, the basolateral addition of NPPB, a Cl- channel inhibitor, reduced the basal rate of ASL absorption, implicating a role for basolateral Cl- channels in ASL volume regulation. When tissues were treated with apical amiloride and basolateral methacholine, a cholinergic agonist that stimulates secretion from airway submucosal glands, the net airway surface liquid production shifted from absorption to secretion. This ex vivo trachea-chip provides a new tool to investigate ASL transport dynamics in pulmonary disease states and may aid the development of new therapies targeting ASL regulation.

The airway surface liquid (ASL) plays a crucial role in lung defense mechanisms, and its composition and volume are regulated by the airway epithelium. The cystic fibrosis transmembrane conductance regulator (CFTR) is abundantly expressed in a rare airway epithelial cell type called an ionocyte. Recently, we demonstrated that ionocytes can increase liquid absorption through apical CFTR and basolateral barttin/chloride channels, while airway secretory cells mediate liquid secretion through apical CFTR channels and basolateral NKCC1 transporters. Th2-driven (IL-4/IL-13) airway diseases, such as asthma, cause goblet cell metaplasia, accompanied by increased mucus production and airway secretions. In this study, we investigate the effect of IL-13 on chloride and liquid transport performed by ionocytes. IL-13 treatment of human airway epithelia was associated with reduced epithelial liquid absorption rates and increased ASL volume. Additionally, IL-13 treatment reduced the abundance of CFTR-positive ionocytes and increased the abundance of CFTR-positive secretory cells. Increasing ionocyte abundance attenuated liquid secretion caused by IL-13. Finally, CFTR-positive ionocytes were less common in asthma and chronic obstructive pulmonary disease and were associated with airflow obstruction. Our findings suggest that loss of CFTR in ionocytes contributes to the liquid secretion observed in IL-13-mediated airway diseases.

Pulmonary ionocytes express high levels of cystic fibrosis transmembrane conductance regulator (CFTR) channels. When studied using the short-circuit current technique, ionocytes produce CFTR-dependent short-circuit currents consistent with Cl- secretion. However, when studied without a voltage clamp, data indicate that ionocytes absorb Cl-. In this review, we resolve these seemingly conflicting findings by considering the different transepithelial voltages and the resultant movement of Cl- during short circuit and physiological open-circuit conditions. This analysis indicates that behavior under short-circuit conditions cannot be directly extrapolated to infer behavior under physiologic conditions. Finally, we discuss the potential role of basolateral Cl- channels in controlling absorption and secretion in ionocytes.