Assessment of CFTR Dysfunction and Responsiveness to CFTR Modulators in COPD Bronchial Air‐Liquid Interface Cultures

1. Introduction

Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disorder, with more than two thirds of patients exhibiting chronic bronchitis, defined by a persistent productive cough for at least three months over two consecutive years. This phenotype is associated with accelerated lung function decline and increased exacerbation frequency (Kim et al., 2011; Seemungal et al., 2000). Pathologically, COPD with chronic bronchitis is characterised by persistent airway inflammation, with increased neutrophils, macrophages, and lymphocytes, alongside mucus hypersecretion, goblet cell hyperplasia, airway remodeling, and microbial colonization (Di Stefano et al., 1998; Hill et al., 2000; Simpson et al., 2016, 2013). More recently advanced CT imaging has shown that patients with COPD have airway occluding mucus plugs that are associated with worse symptoms, lung function and an increased risk of exacerbations (Lin et al., 2026). A subset of patients also develop bronchiectasis, reflecting more severe airway structural damage (Martinez-Garcia and Miravitlles, 2017).

These pathological features overlap with those observed in cystic fibrosis (CF), a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial anion channel that conducts Cl^- and HCO₃^-. CFTR plays a central role in regulating airway surface liquid (ASL) volume and composition, and its dysfunction leads to impaired mucociliary clearance, mucus stasis, chronic inflammation, and persistent infection, which are central features of chronic airway disease (Frizzell and Hanrahan, 2012). The development of CFTR modulators, including potentiators and correctors, has transformed CF care by partially restoring CFTR function, improving clinical outcomes (Rowe et al., 2014; Van Goor et al., 2011, 2009).

Consistent with this, there is increasing recognition that CFTR dysfunction may also contribute to non-CF airway diseases, including COPD. Cigarette smoke, the principal risk factor for COPD, has been shown to induce acquired CFTR dysfunction in individuals without CFTR mutations. Both in vitro and in vivo studies demonstrate that cigarette smoke reduces CFTR-mediated ion transport through multiple mechanisms, including channel internalization, altered gating, and reduced protein stability (Cantin et al., 2006; Clunes et al., 2012; Cohen et al., 2009; Courville et al., 2014; Dransfield et al., 2013; Kreindler et al., 2005; Raju et al., 2013; Sloane et al., 2012; Teerapuncharoen et al., 2019). This acquired dysfunction has been implicated in mucus dehydration and defective mucociliary clearance, and increased susceptibility to infection in COPD.

These observations have led to the hypothesis that restoring CFTR function could ameliorate key pathological features of COPD. However, while preclinical studies report improvements in ion transport and mucus properties following CFTR activation, clinical outcomes have been inconsistent (Kaza et al., 2022). Early pilot studies suggested that ivacaftor (VX-770), a CFTR potentiator that enhances channel gating, may augment CFTR activity and improve selected symptoms in COPD (Solomon et al., 2016), but larger trials have shown acceptable safety without significant improvement in lung function (Vijaykumar et al., 2025). Similarly, icenticaftor, another CFTR potentiator, has shown modest symptomatic benefit without consistent FEV₁ improvement (Martinez et al., 2023). Collectively, these findings suggest that CFTR dysfunction in COPD is mechanistically distinct from CF and may not be fully corrected by current modulators.

A major limitation in the field is that most mechanistic studies on CFTR dysfunction in COPD rely on acute cigarette smoke extract (CSE) exposure models, which fail to recapitulate the chronic, heterogeneous, and patient-specific nature of the disease. Moreover, CFTR function has not been comprehensively characterised in patient-derived primary bronchial epithelial cells differentiated at air-liquid interface (ALI), where intrinsic disease phenotypes are preserved. As a result, it remains unclear whether CFTR dysfunction in COPD airway epithelium is driven by cumulative smoking exposures or reflects additional intrinsic, disease-specific mechanisms.

Here, we address this gap using conditionally reprogrammed primary bronchial epithelial cells (CRpBECs) derived from COPD donors and differentiated at ALI without exogenous smoke exposure. We hypothesized that if cigarette smoke is the dominant driver of acquired CFTR dysfunction, CFTR functional impairment would scale with cumulative smoking exposure. Conversely, a lack of such association would indicate the presence of intrinsic disease-specific defects.

In this study COPD-derived cultures exhibited impaired epithelial barrier integrity, defective mucociliary differentiation, and reduced CFTR and CaCC-mediated chloride transport despite preserved CFTR expression. Residual CFTR activity was detectable but showed only modest and heterogeneous response to CFTR potentiators. These findings provide a functional characterization of ion transport abnormalities in COPD airway epithelium and highlight the limited and variable responsiveness to current CFTR-targeted therapies.

2. Materials and Methods

Human pBECs procurement and processing

The study was performed in accordance with approvals from the Hunter New England Area Health Service Ethics Committee (05/08/10/3.09) and the University of Newcastle (Newcastle, NSW, Australia) Safety Committee (R5/2017). Human pBECs were obtained from healthy controls and COPD individuals (n = 7 donors for each group) by endobronchial brushing during fibre-optic bronchoscopy (Wark et al., 2005). COPD donors were stage II or III as defined by the Global Initiative for Obstructive Lung Disease (GOLD) guidelines (Gómez and Rodriguez-Roisin, 2002). Written informed consent was obtained from all study participants prior to the collection of samples. Patient-derived pBECs were expanded using standard BEGM media (BEGM;Lonza) and cryopreserved for later use.

NIH/3T3 feeder cell culture and irradiation

NIH/3T3 mouse embryonic fibroblast cells were cultured in DMEM (Sigma D5796) supplemented with 10% FBS and 1% (v/v) penicillin/streptomycin (Life Technologies, Australia). At 80-90% confluency, cells were trypsinized, resuspended in fresh culture media and subsequently exposed to 30 Gy γ-irradiation (RS 2000 X-Ray irradiator, RAD SOURCE). Irradiated NIH/3T3 cells were seeded into collagen I (PureCol; Advanced Biomatrix 5005) coated flasks at a 1:1 ratio with pBECs as previously described .

Conditional reprogramming (CR) cell expansion culture/co-culture

Cryopreserved pBECs were revived and expanded in BEGM media. To establish CRpBECs, Passage 1 pBECs were co-cultured with an equal amount of irradiated NIH/3T3 feeder cells in Collagen I-coated flasks in F-media containing ROCK inhibitor Y-27632 as described previously (Awatade et al., 2023). After reaching 50% confluency, cells were gradually weaned off Y-27632, and no Y-27632 added to culture media after 85% confluency (Awatade et al., 2023). Cells were differentially trypsinized using a Trypsin/EDTA reagent pack (Lonza). Cell count and cell viability were obtained using the trypan-blue method.

Air-liquid interface (ALI) cultures

CRpBECs were seeded on collagen I coated 24-well Transwell membranes (Sigma CLS3470) at a density of 1.5x10⁵ cells/insert and cultured in bronchial epithelial base medium and Dulbecco’s modified eagle medium BEBM:DMEM (50:50) differentiation media as described previously (Awatade et al., 2023). Apical media was removed once cells were confluent after 3-5 days to establish air-liquid interface culture conditions. Media in the basal compartment was changed every second day for 28 days. Phosphate-buffered saline (PBS, no Ca²⁺ and Mg²⁺) wash of the apical compartment was performed once a week at 37 °C to remove excess mucus.

Transepithelial electrical resistance (TEER) measurements

TEER measurements were performed at days 7, 14, 21 and 28 of ALI cultures using EVOM2 Voltohmmeter and STX2 electrodes (World Precision Instruments, Sarasota, USA). TEER values (expressed as Ω.cm²) were calculated by subtracting the values of the blank reference insert (no cells) and then corrected to the surface area of the inserts (0.33 cm²).

Transepithelial ion transport assay

Differentiated CRpBECs ALI cultures (28-30 days) were mounted in circulating Ussing chambers (Physiologic Instruments VCC MC8 multichannel voltage/current clamp). Short-circuit currents (I_sc) were measured under voltage-clamp conditions, with at least three transwells analyzed per donor per condition. For I_sc recordings, cells were bathed in 5ml of 37 °C Krebs-bicarbonate-Ringer containing (mM): 115 NaCl, 25 NaHCO₃, 2.4 K₂HPO₄, 0.4 KH₂PO₄, 1.2 CaCl₂, 1.2 MgCl₂ and 10 glucose, pH 7.4, which was continuously gassed with 95% O₂-5% CO₂. After 15min of stable baseline I_sc recording, cells were sequentially treated with pharmacological compounds: (1) 100µM amiloride (apical), (2) 5µM VX-770 or 5µM G1837 or 5µM Icenticaftor (apical), (3) 10µM forskolin and 100µM IBMX (apical and basal), (4) 30µM CFTR_inh-172 (apical) and (5) 100µM ATP (apical). Data analyses were performed using Acquire and Analyze software (v2.3, Physiologic Instruments).

Cilia beating frequency and active area measurements

Live imaging of cilia beating in CRpBECs ALI cultures (frequency 3-20 Hz) was performed using a Nikon eclipse Ti2 microscope (Nikon, Japan) connected to a high-speed digital video recorder and Video Savant 4.0 software. Images were captured at 300 frames per second (fps) and a minimum of 512 frames were acquired. Five fields of view were captured at random for each donor per insert. Data analysis for median cilia beat frequency were performed using the CiliaFA plugin (Smith et al., 2012) together with free open-source Fiji-ImageJ software (v1.53, Image J, US). Ciliary active area was calculated using the in-built ‘Stack difference’ analysis in Fiji-Image J software by highlighting areas of ciliary motion for each 512-frame file. Thresholding was then applied identically to all projected images and active areas measured. Results from each sample are the mean of 5 fields of view.

Whole-mount immunolabelling and fluorescent microscopy

CRpBECs ALI cultures were fixed in 4% paraformaldehyde for 15min followed by storage in PBS containing 50mM Glycine at 4 °C as previously described (Reid et al., 2020). Membranes were permeabilized with 0.1% v/v Triton-X 100, blocked with 10% v/v Goat serum in PBS and incubated with antibodies against acetylated tubulin (T7451, Sigma) and ZO-1 (33-9100, Invitrogen) overnight at 4 °C. After washing with PBS, membranes were incubated with anti-mouse Alexafluor 594 secondary antibody (8890, Cell signaling technology, USA). Membranes were mounted with Fluoromount-G containing DAPI (00-4959-52, Invitrogen, USA). A minimum of 3 images were captured per donor at random using Nikon Eclipse DS-Qi2 fitted with CoolLED box (pE-300). Images were then processed using ImageJ software (National Institute of Health, Bethesda, MD).

RNA extraction, cDNA synthesis, and quantitative PCR

Total RNA was extracted from CRpBECs ALI cultures lysed with RLT buffer containing β-mercaptoethnaol (QIAGEN). RNA was extracted using RNeasy Mini Kits (QIAGEN, Germany) according to manufacturer’s instructions. RNA quality and quantity were measured using a nano-drop 2000 spectrophotometer (Thermo Scientific). RNA (200ng) was reverse transcribed to cDNA using high-capacity cDNA reverse transcription kits (Thermo Scientific). qPCR was performed using a QuanStudio 6 as per manufacturer’s instructions using TaqMan gene expression assays (ThermoFisher Scientific, Australia) and normalized to the ribosomal RNA (18s) housekeeping gene (Table S1). Relative gene expression was calculated using the 2^-ΔΔCt method (where Ct is the threshold cycle) as described previously (Hsu et al., 2017).

Statistical Analysis

Data are represented as dot plots with mean ± standard error of the mean (SEM) except for qPCR data which are represented with mean ± standard deviation (SD). For statistical analysis, unpaired t test, Mann Whitney t test, and paired Wilcoxon tests were used as appropriate. Correlation between smoking exposure (pack-years) and CFTR function (ΔIsc) was assessed using Pearson’s correlation coefficient (two-tailed), with 95% confidence intervals calculated using Fisher’s z-transformation. Statistical analysis was performed with GraphPad Prism software (v9.3.1, San Diego, CA). A p-value <0.05 was considered statistically significant.

3. Results

Donor characteristics define a clinically relevant COPD cohort enriched for chronic bronchitis phenotype

Clinical characteristics of healthy controls and COPD donors are summarized in Table 1. COPD donors were significantly older than healthy controls (70 ± 7.4 vs 54 ± 13.8 years, p = 0.026) and had a substantial cumulative smoking history (53.3 ± 38.9 pack-years), whereas controls were non-smokers. Consistent with moderate-to-severe airflow limitation, COPD donors exhibited markedly reduced lung function compared to controls, including FEV1 (% predicted: 44.3 ± 6.8 vs 90.1 ± 8.3, p = 0.0006), FVC (% predicted: 68.6 ± 14.0 vs 96.6 ± 11.1, p = 0.004), and FEV₁/FVC ratio (51.8 ± 7.5 vs 73.2 ± 6.4, p = 0.001).

Symptom burden, assessed by the COPD Assessment Test (CAT), was elevated in COPD donors (total CAT: 15.9 ± 2.7), with prominent cough (CAT1: 2.43 ± 0.53) and sputum production (CAT2: 2.14 ± 0.89). Based on combined CAT1+CAT2 scores (>2), the majority of COPD donors were consistent with a chronic bronchitis phenotype. None of the COPD patients had bronchiectasis.

Together, these data establish a clinically well-characterised COPD cohort with features relevant to airway mucus dysfunction, providing a robust framework for interpreting epithelial functional phenotypes.

COPD airway epithelial cultures exhibit preserved differentiation but reduced barrier function

Primary CRpBECs from healthy controls and COPD donors formed well-differentiated, pseudostratified epithelium following 28 days of ALI culture. Both groups displayed appropriate epithelial organization and mucociliary differentiation, indicating successful culture establishment.

Epithelial barrier integrity assessed by TEER, displayed inter-individual variability in both groups. COPD cultures exhibited lower TEER values compared to healthy controls (308.9 ± 63.79 vs 506.8 ± 104.5 Ω.cm²), although this did not reach statistical significance (p = 0.137) (Figure 1A). The TEER values for healthy controls cultures ranged from 295 to 1094 Ω.cm² while those in COPD donors ranged from 143.1 to 549.5 Ω.cm².

Despite this reduction, tight junction organization remained intact, as demonstrated by comparable ZO-1 (tight junction protein) localisation between groups (Figure S1A). These findings indicate that while epithelial barrier function is modestly reduced in COPD cultures, overall structural integrity is preserved. Given that epithelial barrier integrity and differentiation are closely linked, we next assessed whether cellular composition was also altered in COPD cultures.

Lower abundance of multiciliated cells in COPD CRpBECs ALI cultures

We then examined whether the epithelial differentiation profiles were also altered. Cell markers were assessed by qPCR. No disparity was observed in the expression of the club cells marker (SCGB1A1) (Figure 1B). However, there was a trend towards lower expression of FOXJ1 (Figure 1C), a marker of ciliogenesis, SPDEF (Figure 1D), a marker of goblet cells and MUC5AC (Figure 1E) a marker of mucus secretory cells in COPD ALI cultures when compared to healthy controls cultures.

Notably, expression of the proinflammatory cytokine IL-6 was significantly increased in COPD cultures compared to controls (p = 0.009) (Figure 1F), indicating a heightened inflammatory state.

Immunofluorescence analysis further revealed a marked reduction in ciliated cell abundance in COPD cultures. While healthy control cultures exhibited evenly distributed ciliated cells, COPD cultures showed sparse and clustered ciliation (anti-acetylated tubulin, red, Figure 1G-H, Figure S2 A-C). Quantification confirmed an approximately 4-fold reduction in active ciliated area in COPD cultures (27.42 ± 4.85 vs 6.65 ± 1.86%; p = 0.0006) (Figure 1I).

Despite this reduction in ciliated cell abundance, ciliary beat frequency (CBF) was comparable between groups (COPD: 12.36 ± 1.48 Hz vs controls: 11.56 ± 1.08 Hz), with substantial inter-individual variability observed in both cohorts (Figure 1J).

Collectively, these findings demonstrate that COPD airway epithelium is characterised by altered cellular composition most notably reduced ciliation alongside increased inflammatory signaling, despite preserved overall differentiation. As epithelial differentiation state can directly influence ion transport activity, we next evaluated functional ion channel activity in these cultures.

COPD ALI cultures have impaired CFTR- and CaCC-mediated chloride channel transport

Transepithelial ion transport in healthy controls and COPD ALI cultures was assessed by measuring amiloride-inhibited sodium channel (ENaC) currents, forskolin (Fsk)/IBMX stimulated and CFTR_inh-172-inhibited CFTR currents and ATP-induced calcium-activated chloride channel (CaCC) currents (Figure 2A-B). Some heterogeneity was observed in all ion channel measurements in both healthy controls and COPD cultures (Figure 2C-F). No difference in the inhibition of sodium channel (ENaC) currents by amiloride was observed between healthy controls and COPD cultures (ΔI_sc-Amiloride -7.67 ± 1.90 vs -7.78 ± 1.55 µA/cm², respectively, p = 0.966) (Figure 2A-C), indicating preserved sodium absorption.

CFTR-mediated Cl^- secretion was assessed using cAMP agonist Fsk and phosphodiesterase inhibitor IBMX, followed by CFTR specific inhibitor CFTR_Inh-172. COPD cultures exhibited significantly impaired Fsk/IBMX-induced currents, approximately 2.5 times lower than healthy controls cultures (ΔI_sc-Fsk/IBMX 6.64 ± 2.10 vs 16.55 ± 1.58 µA/cm², p = 0.008, Figure 2A-B, D). A similar finding was observed in CFTR-inhibited currents, whereby COPD cultures displayed significantly lower CFTR_inh-172 inhibited currents compared to healthy-control cultures (ΔI_{sc-CFTR-inh-172} -10.93 ± 1.48 vs -19.60 ± 1.94 µA/cm², p = 0.008, Figure 2A-B, E).

In addition to CFTR dysfunction, ATP-induced calcium-activated chloride channel (CaCC) currents were also significantly reduced in COPD cultures, approximately 2-fold lower than healthy-control cultures (ΔI_sc-ATP 1.24 ± 0.22 vs 2.43 ± 0.42 µA/cm², p = 0.025, Figure 2A-B, F).

Importantly, CFTR mRNA expression did not differ between healthy controls and COPD cultures (Figure 2G), indicating that the observed defect is functional rather than transcriptional.

Together, these results demonstrate a coordinated impairment in epithelial chloride secretion in COPD airway epithelium, affecting both CFTR-dependent and alternative chloride transport pathways.

CFTR potentiators partially restore CFTR function in COPD airway epithelium with marked inter-donor variability

Given the presence of residual CFTR activity despite functional impairment, we next investigated whether pharmacological potentiation could restore CFTR function in COPD cultures. CFTR potentiators VX-770, GLPG1837, and Icenticaftor were tested in selected COPD donors representing high (D4 and D7) and low (D5 and D6) baseline CFTR activity.

All potentiators produced variable, donor-dependent increase in CFTR-mediated currents. Treatment with VX-770 in COPD donors 4, 5, and 7 induced modest improvement in CFTR restoration compared to DMSO (vehicle), ranging between 0.87 to 4.54 µA/cm². However, this difference did not reach statistical significance (Figure 3 A, B, D, Supp Figure S3 A, B, D). Treatment with potentiator G1837 in COPD donors 5 and 7, increased Fsk/IBMX currents by 1.00 and 12.03 µA/cm² respectively when compared to DMSO (Figure 3 B, D, Supp Figure S3 B, D). Treatment with Icenticaftor in donors 5, 6, and 7 increased CFTR currents compared to DMSO, ranging between 0.52 and 10.71 µA/cm² (Figure 3 B, C, D, Supp Figure S3 B, C, D).

Overall, these findings indicate that while individual donors displayed evidence of improved CFTR function in response to one or more potentiators, the effects were modest, highly variable, and did not reach statistically significance across all samples regardless of baseline CFTR activity. This is consistent with the presence of residual functional CFTR at the epithelial surface, which serves as the foundation for potentiators action.

CFTR dysfunction is independent of cumulative smoking exposure

We next assessed whether cumulative cigarette exposure (pack-years) was associated with CFTR-mediated chloride transport in COPD donors. No correlation was observed between pack-years and CFTR function (Spearman r = 0.12, 95% CI -0.70 to 0.80, p = 0.80, Figure 4), indicating that smoking burden alone does not predict the extent of CFTR dysfunction. Notably, donors with high smoking exposure exhibited divergent CFTR activity, with some individuals demonstrating markedly impaired function (e.g., D2), while others retained relatively high CFTR activity (e.g., D7). These findings highlight substantial inter-donor variability and suggest that factors beyond smoking exposure contribute to CFTR dysfunction in COPD.

4. Discussion

COPD is characterised by airway mucus obstruction, chronic inflammation, and recurrent infection—features that overlap with CF and have led to increasing recognition of acquired CFTR dysfunction in COPD. While this has generated interest in repurposing CFTR-targeted therapies, most experimental models rely on exogenous CSE, which incompletely recapitulates disease complexity. Here, using CRpBECs differentiated at ALI, we demonstrate that COPD airway epithelium exhibits intrinsic abnormalities in epithelial differentiation, barrier integrity, and ion transport, including reduced CFTR and CaCC function, independent of acute smoke exposure.

Intrinsic epithelial dysfunction in COPD

Although COPD and healthy control cultures both formed a mucociliary epithelium, COPD-derived cultures exhibited reduced barrier integrity (lower TEER), decreased expression of key differentiation markers (FOXJ1, SPDEF, and MUC5AC), and a reduced abundance of multiciliated cells, consistent with previous reports (Aghapour et al., 2018; Carlier et al., 2021; Guo-Parke et al., 2022; Haswell et al., 2010; Park et al., 2021; Schamberger et al., 2015; Shaykhiev et al., 2011). Notably, these abnormalities were observed in the absence of acute exogenous smoke exposure, indicating that COPD airway epithelium retains intrinsic structural and functional defects. The reduction in multiciliated cells may further limit effective ion transport at the tissue level, linking epithelial differentiation state to functional CFTR capacity. Given that mature multiciliated cells serve as a major site of apical anion transport (Collin et al., 2021; Sears et al., 2015), their depletion is likely to reduce overall tissue-level CFTR function (Boucher, 2007) and contribute to impaired airway surface homeostasis.

Acquired CFTR dysfunction in COPD is independent of transcriptional and smoking burden

A central finding of this study is that CFTR-mediated chloride secretion is markedly reduced in COPD cultures despite preserved CFTR mRNA expression, supporting a post-transcriptional mechanism of dysfunction. This is consistent with prior human and animal studies (Tilley et al., 2016; Kaza et al., 2022) and aligns with established mechanisms of acquired CFTR dysfunction, including altered channel gating, internalisation, and reduced protein stability (Clunes et al., 2012; Marklew et al., 2019; Raju et al., 2017). Inflammatory processes may further contribute, as neutrophil-derived proteases have been shown to degrade CFTR in the COPD airway (Le Gars et al., 2013).

Importantly, we observed no correlation between cumulative smoking exposure (pack-years) and CFTR function, with individuals of similar smoking histories exhibiting markedly different levels of CFTR activity. This lack of association indicates that CFTR dysfunction in COPD is not a simple dose-dependent consequence of cigarette smoke exposure but instead reflects differential susceptibility and additional disease-specific mechanisms. Consistent with this, acquired CFTR dysfunction is not universal in COPD and some studies have shown it to be present in only a subset of individuals, including up to ~40% of smokers (Dransfield et al., 2022; Mall et al., 2023). Together, these findings support a model in which CFTR dysfunction arises from convergent environmental, inflammatory, and epithelial remodeling processes rather than smoking exposure alone.

Divergent regulation of epithelial ion channels in COPD

Beyond CFTR, we identified a reduction in calcium-activated chloride channel (CaCC) activity in COPD cultures, potentially reflecting impaired calcium signaling and altered expression of regulators such as ORAI3 (ORAI—Calcium release activated calcium modulator 3), as previously shown by (Petit et al., 2019). In contrast, ENaC activity was not significantly altered in COPD cultures, diverging from observation in CSE-based models (Cantin et al., 2006; Kreindler et al., 2005; Tyrrell et al., 2023). This discrepancy highlights important differences between acute exposure systems and patient-derived epithelial models and underscores the value of primary cell systems in capturing disease-relevant physiology.

Residual CFTR activity provides a rationale for therapeutic potentiation

Despite reduced function, COPD cultures retained substantial residual CFTR activity (~50–60% of healthy controls), indicating the presence of functional channels at the cell surface and providing a rationale for pharmacological potentiation. CFTR potentiators increase channel open probability and/or conductance (Van Goor et al., 2009), with the potential to improve airway hydration, mucociliary clearance, and host defence (Raju et al., 2017). Consistent with this, indirect CFTR activation has been implicated in the clinical efficacy of agents such as roflumilast (Lambert et al., 2014), suggesting that CFTR modulation may contribute to therapeutic benefit in COPD.

Limited and heterogenous responses to CFTR modulators

Despite this mechanistic rationale, CFTR potentiators (VX-770, GLPG1837, and icenticaftor) produced modest, highly variable, and non-significant improvements in CFTR function across COPD donors. The addition of highly effective triple therapy (elexacaftor/tezacaftor/ivacaftor) conferred no additional benefit. These heterogeneous responses provide a mechanistic explanation for the inconsistent outcomes observed in clinical trials, where ivacaftor and icenticaftor have shown limited or delayed efficacy (Martinez et al., 2023; Vijaykumar et al., 2025).

Several factors likely contribute to this limited responsiveness. First, CFTR dysfunction in COPD is likely to arise from multiple mechanisms. This may include a direct effect on the CFTR protein such as oxidative modification, altered gating, and proteolytic degradation, that may also be inherently less amenable to potentiator-based rescue. Second, reduced apical membrane CFTR density may limit the available substrate for pharmacological activation. Third, pharmacokinetic factors and suboptimal drug exposure in COPD populations may further constrain efficacy.

Mechanistic basis of variability and therapeutic resistance

The marked inter-donor variability observed in this study highlights the heterogeneity of COPD at the molecular level. Cigarette smoke–derived reactive species can induce oxidative and covalent modifications of CFTR, potentially altering channel conformation in ways that are not rescued by potentiators. In parallel, smoke-induced internalization and epithelial remodeling may reduce functional channel availability. These effects are likely compounded by distinct disease endotypes, which differentially influence CFTR dysfunction and drug responsiveness. Notably, tobacco smoke exposure itself may antagonize modulator efficacy, as suggested in CF populations (Baker et al., 2021). Collectively, these findings indicate that CFTR dysfunction in COPD is mechanistically diverse and unlikely to be corrected by a uniform therapeutic strategy.

Therapeutic implications and future directions

Taken together, our data suggest that CFTR potentiators alone are insufficient to restore acquired CFTR dysfunction in COPD. More effective approaches may require combination strategies incorporating CFTR correctors and or amplifiers, or emerging modalities such as CFTR mRNA delivery. Mechanistically informed patient stratification will be critical to identify individuals most likely to benefit from CFTR-targeted interventions.

Limitations

This study is limited by the relatively small donor cohort, which restricts statistical power and may not fully capture the heterogeneity of COPD. In addition, donor genotyping was not performed, preventing assessment of whether observed variability in CFTR function and therapeutic responsiveness is related to underlying genetic variation, including CFTR gene variants. Larger, well-characterised cohorts and in vivo validation studies will be required to confirm the clinical relevance of these findings.

5. Conclusions

In summary, COPD airway epithelial cultures exhibit intrinsic defects in epithelial differentiation, barrier integrity, and ion channel function, including acquired CFTR dysfunction and reduced CaCC activity. Although residual CFTR activity is preserved, pharmacological potentiation results in limited and heterogeneous functional improvement. These findings demonstrate that CFTR dysfunction in COPD is a multifactorial process not solely determined by smoking exposure and provide a mechanistic framework for the limited efficacy of current CFTR modulators. Stratified, mechanism-based therapeutic approaches will be required to achieve meaningful clinical benefit.