I. ABSTRACT Background Pulmonary arterial hypertension (PAH) is a debilitating cardiopulmonary disease characterized by progressive remodeling of the pulmonary vasculature. Pathologic transforming growth factor-β (TGF-β) signaling is an essential driver of vascular remodeling in PAH. While global inhibitors of TGF-β exist, their clinical application is limited by systemic adverse effects. Therefore, a critically unmet need in PAH is to identify pulmonary vascular-specific regulators of the TGF-β axis, which would selectively enhance clinical efficacy while minimizing adverse effects. As the clinical care of PAH largely promotes vasodilation, and only one FDA-approved agent targets vascular remodeling, this study aimed to identify selective, therapeutically targetable regulators of the TGF-β axis in the PAH pulmonary vasculature. Methods CD248 was identified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics in human lungs. CD248 levels were assessed across human, rat, and mouse lung tissues using western blotting, RTqPCR, and/or immunofluorescence techniques. CD248-null (CD248 -/- ) mice were used to study the contribution of CD248 to hypoxia-sugen (H/S)-induced PAH. The mechanistic role of CD248 in PAH vascular remodeling and TGF-β signaling was assessed by genetic (siRNA knockdown; overexpression) and pharmacologic (Ontuxizumab) manipulation of primary human pulmonary vascular cells. Results LC-MS/MS proteomics coupled with pathway enrichment analysis of human lung tissue identified CD248 as a putative mediator of vascular remodeling that is elevated in PAH lungs. CD248 was elevated in PAH pulmonary artery smooth muscle cells (PASMCs) across human, rat, and mouse lung tissue. CD248 -/- mice were protected from H/S-induced elevations in right ventricular (RV) systolic pressure (RVSP), RV hypertrophy, and pulmonary artery muscularization. CD248 knock-down reduced cell proliferation and migration of primary PAH PASMCs. CD248 was essential for phospho-activation of TGF-β receptor I (TβRI) at S165 and canonical phosphorylation of SMAD3 at S423/425. CD248 loss blunted TGF-β-induced gene expression (FN1, Col1α1, α-SMA) and activated expression of the vasoprotective matrix metalloprotease, MMP-8. Mechanistically, CD248 interacted with and enhanced de novo phosphorylation and stability of TβRI, blocking its ubiquitin-mediated proteasomal degradation. Ontuxizumab promoted TβRI instability and attenuated the production of FN1, Col1α1, and α-SMA in primary PAH PASMCs. Conclusions This work identifies CD248 as a previously unrecognized co-activator of TβRI in PAH. As CD248 is largely quiescent in most adult tissues yet pathologically upregulated in the PAH pulmonary vasculature, this study supports the potential of anti-CD248 therapy as a novel pulmonary vascular-specific alternative to systemic TGF-β inhibition.

Background: The Cystic Fibrosis Foundation (CFF) recognizes exercise as a critical part of managing cystic fibrosis (CF). This becomes even more important in the era of highly effective modulator therapy (HEMT) due to many people with cystic fibrosis (pwCF) having decreased symptom burden and a newfound ability to tolerate exercise better. Our single-center pilot study was designed to assess the implementation of a remotely delivered, individualized, and comprehensive exercise program for pwCF. We aimed to determine the feasibility, safety and acceptance of this intervention. Methods: PwCF ≥ 18 years old were recruited and consented at the University of Alabama in Birmingham in 2022 and 2023. Basic fitness was assessed for each participant, and an individualized exercise prescription was prepared for each participant, who was expected to exercise three times weekly on a remote basis with the exercise physiologist for 12 consecutive weeks. Subjects were reassessed at 4 and 7 months for post-exercise evaluation. Patient demographics and clinical parameters, including exacerbation rate, FEV1 percent predicted, 6-min walk test (6MWT), and modified shuttle test (MST) were collected. Questionnaire data from the CFQ-R, PRAISE, and IPAQ were also recorded. The study was registered with ClinicalTrials.gov (NCT04680403) and was submitted on 17 December 2020. Results: Our goal was to enroll 12 participants over the 2-year study period. We were able to recruit nine people for the study, with four participants finishing the program. From the 36 sessions offered over the 12-week program, participants completed an average of 15 sessions. Clinical outcome data was observed, including lung function and exacerbation frequency, but not statistically analyzed due to the small sample size. Conclusions: Implementation of an individualized telehealth-based exercise program for pwCF was well received by participants, safe, and appreciated by the participants. Recruitment and adherence were challenging, which was partially due to the ongoing pandemic. Follow-up studies are needed to assess whether improvements in reducing the amount or supervision of weekly exercise sessions and/or extending the total time might help with adherence.

Introduction:Cystic fibrosis (CF) is an autosomal recessive disorder, which manifests in many organ systems including the lungs. Chronic inflammation is a hallmark of CF lung disease leading to bronchiectasis and lung function decline. This is worsened by airway colonization and recurrent infections due to opportunistic pathogens such as Pseudomonas aeruginosa [PA], but the crosstalk between host-bronchial epithelium and immune system has been under characterized. Extracellular vesicles have been found to mediate intercellular crosstalk in different lung diseases and EVs have been shown to be increased in the bronchoalveolar fluid of CF patients. We hypothesize that EVs from PA-infected CF bronchial epithelial cells can modulate pro-inflammatory cytokines and neutrophil migration and activation in an autocrine and paracrine manner. Methods:CF bronchial epithelial cells (CFBEs) and control bronchial epithelial cells (16HBEs) were infected with PA for 24 hours followed by EV isolation, which were used to treat uninfected CFBE and 16HBEs to assess expression and secretion of pro-inflammatory markers. In addition, the effects of EVs on neutrophil migration and activation were determined as well as the role of CFTR deficiency by using CFTR modulator therapy (Elexacaftor/Tezacaftor/Ivacaftor). Results:EVs derived from PA infected CFBEs (EVpPAs) increased IL-6, IL-8, and TNFα expression and neutrophil activation in CFBEs but not in 16HBEs. Interestingly, the effect of EVpPAs on inflammation was not attenuated by pre-treatment with ETI. Discussion:EVs from the PA-infected CF bronchial epithelium seem to facilitate an autocrine and paracrine pro-inflammatory response that is not attenuated by ETI treatment, suggesting a novel contribution of EVs to the chronic inflammatory phenotype observed in the PA-infected CF lung.

The lung is a unique organ because of its continuous exposure to the external environment, which requires a complex and tightly regulated immune network [...]

Vanazacaftor-Tezacaftor-Deutivacaftor (VTD) is a novel cystic fibrosis transmembrane receptor (CFTR) modulator therapy. This development added another highly effective modulator therapy to the market as an alternative to the standard of care, Elexacaftor-Tezacaftor-Ivacaftor (ETI). Phase III trial data comparing VTD and ETI demonstrated non-inferiority in outcomes and similar adverse effect profile and rates. Here we present a case series demonstrating known and potentially unknown side effects, specific to VTD. From January 2025 to June 2025, 38 out of 400 patients at our adult CF center, were switched from ETI to VTD. Six of those patients experienced potential VTD related adverse events. Liver function testing (LFT) elevations were seen in 2/6 cases, worsening pulmonary symptoms in 3/6 cases, dermatologic manifestations in 2/6 cases, hypertension in 1/6 cases, neuropsychiatric symptoms in 2/6 cases, and gastrointestinal symptoms in 2/6 cases. Notably, one case led to hospitalization, with development of a severe rash, thrombocytopenia, and LFT elevations. In each case, all symptoms and laboratory manifestations resolved within days after switching back to ETI. This case series demonstrates both previously recognized and unrecognized adverse effects of VTD in a “real-world population.” The majority of our patients’ presenting symptoms were described in phase III clinical trials, including LFT elevations, pulmonary exacerbations, rash, and neuropsychiatric symptoms. On the contrary, thrombocytopenia, gastrointestinal manifestations, and hypertension have not been described thus far with VTD. Further research and more robust monitoring are needed to determine the overall incidence and risk of lesser-known side effects of novel CFTR modulators.

The population of adults with cystic fibrosis (CF) exceeds that of the pediatric CF population, and life expectancy for people with CF (PwCF) continues to improve, resulting in an adult CF population with chronic extrapulmonary comorbid conditions, including CF-related diabetes (CFRD), a common complication affecting roughly 50% of PwCF, as well as mental health disorders, including depression and generalized anxiety. These comorbidities have been strongly associated with increased risk of progressive lung disease, impaired quality of life, and increased risk of acute exacerbations requiring healthcare utilization. These risks disproportionately affect PwCF who are from ethnic minorities and are often genetically ineligible for novel treatments. Social determinants of health (SDOH) are related to and contribute to health disparities in PwCF. To reduce the impact of SDOH on health-related disparities in this population, PwCF needs greater awareness of associated factors to develop interventions that can improve care. This narrative review critically synthesizes the available literature on the complex intersections of social determinants of health, mental health, and concomitant CFRD that affect health outcomes for PwCF.

BACKGROUND:Therapeutic advancements utilizing modulators of the cystic fibrosis transmembrane conductance regulator (CFTR) have revolutionized the treatment of people with cystic fibrosis (pwCF). Elexacaftor-Tezacaftor-Ivacaftor (ETI) is a highly effective modulator therapy and has been shown to improve health outcomes in people with CF (PwCF). Due to these therapeutic advancements, many pwCF are getting older, but little is known regarding the safety and efficacy of ETI in pwCF at a more advanced age. OBJECTIVES:We aimed to determine the effect of ETI on clinical outcomes in older pwCF. DESIGN:This study was a single-center, retrospective analysis of pwCF who received open-label ETI following FDA approval and were over the age of 40 at the time of ETI initiation. METHODS:Data were obtained from the electronic medical record from a large CF center in the United States of America between November 2019 and January 2021, including body mass index (BMI), lung function as % predicted FEV1 before ETI initiation, and approximately 3 months and one a follow-up visit within 9-15 months post-ETI initiation. The exacerbation frequency over 12 months was recorded before and after ETI initiation. RESULTS:Forty-two patients met the inclusion criteria. Mean age at time of ETI initiation was 47.9, 23 patients (54.8%) were male, and 11 (26.2%) were homozygous for the F508del mutation. Linear mixed effects models suggest a monthly increase of 0.24 (95% CI 0.08-0.41, p = 0.003) for ppFEV1 and 0.03 (95% CI 0.002-0.06, p = 0.036) for BMI post-ETI, resulting in a 2.96 (95% CI 0.98-4.95) increase in ppFEV1 and 0.39 (95% CI 0.03-0.76) increase in BMI approximately 1-year post-ETI. In addition, a significant decline in pulmonary exacerbations was seen in the year following ETI initiation (1.5 ± 1.3 exacerbations/year prior vs 0.5 ± 0.7 exacerbations/year post; p < 0.0001). CONCLUSION:Treatment with ETI in this unique cohort of pwCF was safe. Whereas ETI affected BMI in a subtle way, initiation of ETI was associated with stabilization of lung disease with a significant but moderate increase in lung function and a decline in the number of exacerbations in the follow-up period. Longer and larger studies will be needed to analyze the effect of ETI on an aging CF population.

Cystic fibrosis (CF) is a genetic disorder that causes chronic airway disease, leading to accelerated aging, inflammation, and susceptibility to opportunistic infections. Pseudomonas aeruginosa (PA) is prevalent in the CF lung, often multidrug-resistant, and affecting lung function decline, highlighting a need for alternative treatment strategies, such as host-directed therapeutics. The PA metabolite pyocyanin can induce senescence-associated β-galactosidase (SA-βgal), a lysosomal cell senescence marker. However, whether PA can induce cell senescence and therefore affect immune functions has not been studied. Using established airway epithelial culture models, we observed that PA infection increased SA-βgal activity and proinflammatory cytokines without increasing a comprehensive set of cell senescence markers. PA infection also resulted in lysosomal neutralization and decreased lysosome biogenesis, with the latter attenuated by rapamycin (RAPA), a senolytic and an established inhibitor of mTOR signaling. In conclusion, our study implies that PA infection does not induce cell senescence in the CF bronchial epithelium but disturbs lysosomal homeostasis, which can be attenuated by RAPA. These findings highlight lysosomal health as a promising therapeutic target in CF airways, especially as patients live longer yet remain susceptible to recurrent airway infections.

i. ABSTRACT Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by augmented transforming growth factor-β (TGF-β) signaling leading to excessive extracellular matrix (ECM) deposition. The fibroblast-to-myofibroblast-transition (FMT) and metabolic reprogramming of lung fibroblasts (HLFs) are essential to IPF pathogenesis, yet the connection between nutrient metabolism and fibrogenesis remains poorly defined. The O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a nutrient-sensitive enzyme that adds O-GlcNAc moieties to substrates. We previously showed that loss of OGT reverses bleomycin-induced pulmonary fibrosis in mice. Here, using unbiased kinomics, we show that pharmacologic inhibition of OGT suppressed non-canonical TGF-β-induced mitogen-activated protein kinase (MAPK) signaling. Molecular confirmation revealed that TGF-β-induced phosphorylation of p38, but not ERK or JNK, was reduced by OGT blockade. Furthermore, p38 itself was O-GlcNAc-modified, which enhanced its phosphorylation and promoted downstream phosphorylation of the NADPH oxidase subunit, p47 phox . Inhibition of OGT, p38, or p47 phox reduced reactive oxygen species (ROS) in HLFs, revealing a previously unknown role of OGT-p38-p47 phox signaling in ROS production. Collectively, this work establishes that O-GlcNAc-modified p38 enhances p47 phox -dependent H 2 O 2 production. Highlights Using PamChip STK arrays, we show that OGT inhibition causes broad kinomic remodeling, including suppression of non-canonical TGF-β MAPKs and multiple CDKs. OGT blockade selectively attenuates p38 phosphorylation, despite TGF-β-induced substrate redundancy with ERK and JNK. We provide evidence that p38 MAPK undergoes O-GlcNAcylation in human lung fibroblasts, a modification not previously reported. The study identifies a new signaling axis where O-GlcNAc modification of p38 modulates the phosphorylation of p47 phox , therefore regulating NOX-dependent H 2 O 2 production. Blocking OGT or inhibiting p38/p47 phox dramatically reduces TGF-β-driven H 2 O 2 production in human lung fibroblasts. Graphical Abstract

In a healthy lung, the airway epithelium regulates glucose transport to maintain low glucose concentrations in the airway surface liquid (ASL). However, hyperglycemia and chronic lung diseases, such as cystic fibrosis (CF), can result in increased glucose in bronchial aspirates. People with CF are also at increased risk of lung infections caused by bacterial pathogens, including methicillin-resistant Staphylococcus aureus. Yet, it is not known how increased airway glucose availability affects bacteria in chronic CF lung infections or impacts treatment outcomes. To model the CF airways, we cultured immortalized CF (CFBE41o-) and non-CF (16HBE) human bronchial epithelial cells at the air-liquid interface (ALI). Glucose concentrations in the basolateral media were maintained at 5.5 or 12.5 mM to mimic a normal and hyperglycemic milieu, respectively. We found that glucose concentrations in the ASL of ALI cultures maintained in normal or high glucose mimicked levels measured in breath condensate assays from people with CF and hyperglycemia. Additionally, we found hyperglycemia increased S. aureus aggregation and antibiotic resistance during infection of cells maintained in high glucose compared to normal glucose conditions. Heightened antibiotic resistance was not observed during in vitro growth with elevated glucose. Limiting glucose with 2-deoxyglucose both decreased aggregation and reduced antibiotic resistance back to levels comparable to non-hyperglycemic conditions. These data indicate that hyperglycemia alters S. aureus growth during infection and may reduce efficacy of antibiotic treatment. Glucose restriction is a potential option that could be explored to limit bacterial growth and improve treatment outcomes in chronic airway infections.

The leading cause of death for people with cystic fibrosis (pwCF) continues to be due to respiratory-related illnesses. Both wound repair and immune cell responses are dysregulated in the CF airways, creating a cycle of unresolved injury and perpetuating inflammation. PwCF are predisposed to colonization and infections with opportunistic bacteria like Pseudomonas aeruginosa (Pa), the most common adult pathogen in CF. Pa possesses key virulence factors that can exacerbate chronic inflammation and lung injury. With the approval of highly effective modulator therapies like elexacaftor/tezacaftor/ivacaftor (ETI), pwCF eligible for ETI have seen drastic improvements in lung function and clinical outcomes, including an increased life expectancy. While modulator therapies are improving bronchial epithelial cellular processes in wound repair and some areas of immunity, many of these processes do not reach a non-CF baseline state or have not been thoroughly studied. The effect of modulator therapy on Pa may lead to a reduction in infection, but in more longitudinal studies, there is not always eradication of Pa, and colonization and infection frequency can return to pre-modulator levels over time. Finally, in this review we explore the current state of additional treatments for CF lung disease, independent of CFTR genotype, including anti-inflammatories, phage-therapies, and Pa vaccines.

There are a paucity of data regarding the pharmacokinetics (PK) of elexacaftor (ELX)/tezacaftor(TEZ)/ivacaftor(IVA)(ETI) in pregnant and/or lactating mothers and their offspring. We conducted a PK assessment of ETI and their metabolites in maternal/neonatal/cord blood and breast milk from a cystic fibrosis (CF) carrier mother/affected fetus dyad, collecting specimens at delivery, 1 and 4 weeks after birth. The infant received on-label direct dosing after 1 month of life; all PK samples were collected prior to initiation of neonatal dosing and analyzed using LC-MS/MS. Measured metabolites were IVA-M1, IVA-M6, ELX-M23 and TEZ-M1. The female infant was born at 37 weeks gestation with successful meconium passage on the first day of life. Sweat chloride at 15 days (16 and 17 mmol/L) and immunoreactive trypsinogen (27.5 ng/mL) were normal. Neonatal genetics confirmed F508del/P67L genotype. Parent drug concentrations were measurable in cord blood and capillary heel sticks, indicating they cross the placenta. After delivery, the infant’s only source of modulators was via breast milk. Breast milk concentrations were measured at 1 and 4 weeks of life. Relative to maternal concentrations, ETI and their metabolites were present at lower concentrations. Heel stick specimens revealed undetectable IVA, but ELX and TEZ were below the assay limit. IVA-M1 and IV-M6 concentrations were lower at 1 and 4 weeks relative to delivery. To our knowledge, this is the first report of ETI metabolite concentrations following in utero administration.

In this study, we present a detailed workflow for the isolation, quantitation, and evaluation of mucin proteins. These methods are applicable to a variety of biological, mucin-containing samples from the airways and other mucosal organ systems. While this report focuses on the salivary MUC5B protein from the respiratory system, the presented methodologies can be applied to other mucins, contributing to a broader application of these techniques. We used a simplified isopycnic centrifugation to purify and enrich MUC5B from human saliva. Isolated MUC5B was then subjected to a Bradford protein assay using a bovine submaxillary mucin (BSM) standard, which more accurately reflects the mucin concentration in our samples compared to a bovine serum albumin (BSA) standard. Additionally, we compare the mucin levels following quantitation using agarose polyacrylamide gel electrophoresis. Our findings show a near 2-fold increase in quantitation from the more representative, BSM standard, suggesting its importance for mucin studies. These methods support a wide range of experimental applications looking to assess mucins, thereby contributing to the broader field of mucin studies and advancing our understanding of the implications of mucins in health and disease.•A streamlined, one-step isopycnic ultracentrifugation to isolate MUC5B from human saliva•A Mucin Bradford assay that is modified from existing Bradford assay techniques to better quantitate mucin for mucin studies•An agarose-polyacrylamide gel electrophoresis method used to visualize and confirm the isolation and quantitation of mucin.

BACKGROUND:Exercise is an important part of a healthy lifestyle for people with cystic fibrosis (CF). Exercise testing is also recommended as part of a routine assessment to provide physical activity counseling/exercise prescription. Body composition assessment is also an important assessment that can also guide dietary counseling. Prior studies have found that exercise testing and exercise training programs are underutilized in CF care centers, and no one has assessed the prevalence of body composition assessment. Thus, the purpose of this study was to develop and validate a survey to explore the barriers and facilitators that obstruct or promote the use of exercise and body composition testing in the CF care centers. METHODS:Seventy-nine individuals with expertise in exercise and CF were invited to participate in a Delphi study. They rated the importance and quality of a 106-item survey on exercise and body composition testing and exercise training and shared their feedback. RESULTS:Thirteen subject matter experts agreed to participate in the study, which lasted three rounds, but only six of them were in the final round. Of the 106 original items, 46 were kept with revisions, 58 were revised, 2 items were dropped, and 8 items were added, resulting in a final survey of 114 items. CONCLUSIONS:This Delphi study validated a survey to be used in CF care centers to provide valuable information about the benefits and barriers to exercise testing, exercise training, and body composition assessment in people with CF in the CFTR-modulator era.

Epigenetic modifications are involved in fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), and contribute to the silencing of anti-fibrotic genes. H3K27me3, a key repressive histone mark, is catalysed by the methyltransferase enhancer of Zeste homologue 2 (EZH2), which is regulated by the post-translational modification, O-linked N-Acetylglucosamine (O-GlcNAc). In this study, we explored the effects of O-GlcNAc and EZH2 on the expression of antifibrotic genes, cyclooxygenase-2 (Cox2) and Heme Oxygenase (Homx1). The expression of Cox2 and Hmox1 was examined in primary IPF or non-IPF lung fibroblasts with or without EZH2 inhibitor EZP6438, O-GlcNAc transferase (OGT) inhibitor (OSMI-1) or O-GlcNAcase (OGA) inhibitor (thiamet G). Non-IPF cells were also subjected to TGF-β1 with or without OGT inhibition. The reduced expression of Cox2 and Hmox1 in IPF lung fibroblasts is restored by OGT inhibition. In non-IPF fibroblasts, TGF-β1 treatment reduces Cox2 and Hmox1 expression, which was restored by OGT inhibition. ChIP assays demonstrated that the association of H3K27me3 is reduced at the Cox2 and Hmox1 promoter regions following OGT or EZH2 inhibition. EZH2 levels and stability were decreased by reducing O-GlcNAc. Our study provided a novel mechanism of O-GlcNAc modification in regulating anti-fibrotic genes in lung fibroblasts and in the pathogenesis of IPF.