Journal of Cystic Fibrosis

BackgroundPatients with cystic fibrosis (CF) are characterised by abnormalities of the intestinal tract relating to gut motility and physiological issues, with daily symptoms of disease including abdominal pain, flatulence, bloating, and constipation. With improvements in respiratory outcomes, a shift in disease manifestations has highlighted the prevalence of the gastrointestinal-related problems associated with CF, yet most therapies currently in clinical use for the gut symptoms of CF have been repurposed from other disease indications and have not been developed with a knowledge of the mechanisms underpinning gastrointestinal disease in CF. Increased attention towards the role of intestinal inflammation and microbial dysbiosis in the CF population warrants a comprehensive knowledge of these aspects alongside the increased luminal fat content, dysmotility, and small intestinal bacterial overgrowth (SIBO) resultant of the primary consequences of CFTR dysfunction (disrupted fluid secretion and pancreatic insufficiency), and how they contribute towards the intestinal complications of CF disease. Methods and Study DesignWe will conduct a systematic review to comprehensively address our current understanding of the primary consequences of CFTR dysfunction, and their subsequent secondary effects that contribute towards the disruption of gut motility, health, and associated symptoms in the CF intestine. Databases searched will include PubMed, CINAHL, MEDLINE and the Cochrane library from 1939 until a specified date of last search, alongside clinical trial databases for ongoing studies. Search strategies will include various terminology that relates to the primary mechanistic defects of CF, postulated secondary effects of such defects, and symptoms experienced in patients. A full search strategy is outlined in appendix B. One reviewer will apply an inclusion criterion to obtained abstracts. Following agreement from a second reviewer, full-text articles will be sought, and data will be extracted from relevant articles. Disagreements will be resolved with a third reviewer. The quality of data will be assessed by the GRADE criteria. Data will be used to present a narrative, and where possible, quantitative synthesis. DiscussionThis systematic review will discuss our current understanding of the underpinning mechanisms of the persisting abnormalities in gut health and motility within CF, addressing potential intricate relationships that further contribute to disease progression within the intestinal tract. Furthermore, we will identify current gaps in the literature to propose directions for future research. A comprehensive understanding of these aspects in relation to intestinal abnormalities will aid future clinical directions.

BackgroundCystic fibrosis (CF) research has increasingly focused on understanding the extra-pulmonary manifestations of CF, including on the gastrointestinal (GI) system. The effect of cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies outside the lungs is also a topic of research interest and both are key research priorities. However, significant evidence gaps persist in understanding the complex pathophysiology of CFTR dysfunction in the GI tract, and the treatment of these GI problems. Inconsistencies in outcome reporting may contribute towards these evidence gaps, and a standardised approach to outcome reporting may help to address this. This systematic rapid review aims to identify and catalogue the range of outcome measurement instruments (OMIs) and associated endpoints currently used in CF GI research. MethodsThis PROSPERO-registered review (CRD42021281961) was conducted following Cochrane Rapid Reviews Methods Group and COMET initiative guidance. Comprehensive searches were performed in MEDLINE, EMBASE, PubMed, Cochrane Library, and ongoing clinical trials databases, covering an 11-year period (August 2013 to November 2024). Screening and data extraction were carried out using Covidence online software. ResultsA total of 1,541 studies were identified, of which 193 met inclusion criteria. These studies collectively used 246 distinct OMIs, of which 172 (70%) were employed in only one study. The OMIs identified were grouped into 14 sub-domains representing key areas of GI research in CF, which were subsequently mapped to 11 of the 38 outcome domains in the taxonomy proposed by the COMET Initiative. The identified outcomes spanned a diverse range of mechanistic and patient-centred measures, reflecting the complexity of GI disease in CF. ConclusionsCurrent research into the GI tract in CF uses a heterogeneous array of OMIs, with limited standardisation. This highlights both the complexity of CFTR dysfunction within the GI tract, requiring a wide scope of OMIs to address this, as well the variability and potential inefficiency in current outcome reporting practices. To advance our understanding of CF pathophysiology in the GI tract, a standardised approach to outcome reporting is needed. Our findings support the development of a core outcome set to promote reporting consistency and improve comparability across studies in CF GI research.

Background and aimsGastrointestinal symptoms remain common in adults with cystic fibrosis (CF) despite cystic fibrosis transmembrane conductance regulator modulator use, suggesting persistent and heterogeneous gut dysfunction. This prospective observational study tests the hypothesis that distinct gut symptom phenotypes in CF can be observed and linked to underlying mechanisms. MethodsAdults from three UK CF centres completed the Gastrointestinal Symptom Rating Scale, Patient Assessment of Constipation Symptoms and a bowel-habit questionnaire. Latent class analysis using an ordinal logistic model was applied to 36 symptom indicators. Associations between phenotypes and demographic, clinical, and treatment variables were examined using generalised linear models. ResultsThree hundred participants completed questionnaires (54% male; median 31 years). We identified four symptom phenotypes: mild; moderate-constipation predominant; moderate-diarrhoea predominant and severe. The severe phenotype was associated with gastroesophageal reflux (RRR 2.86; 95%CI: 1.30-6.31; p=0.009), distal intestinal obstruction syndrome (RRR 2.46; 95%CI: 1.04-5.81; p=0.04), proton pump inhibitor (RRR 3.29; 95%CI 1.39-7.74; p=0.007), and laxative use (RRR 6.13; 95%CI 2.54-14.84; p<0.001). CF-related liver disease was associated with both moderate-constipation and diarrhoea phenotypes, respectively (RRR 2.08; 95%CI 1.13-3.81; p=0.018; RRR 2.11; 95%CI 1.03-4.29; p=0.04). There was a lower likelihood of long-term oral antibiotic use in the moderate-constipation phenotype (RRR 0.53; 95%CI 0.3-0.92; p=0.025) and moderate-diarrhoea phenotype (RRR 0.46; 95%CI 0.24-0.91; p=0.025). ConclusionsFour distinct symptom phenotypes were identified, independent of demographics and pancreatic status, but associated with specific complications and medication profiles. These phenotypes provide a framework for mechanistic studies within the GRAMPUS-CF cohort and precision management of CF-related gut disease.

BackgroundCFTR modulators have transformed cystic fibrosis (CF) treatment, but individual responses vary even among patients with identical CFTR genotypes. This underscores the need for predictive biomarkers to optimize therapeutic selection. MethodsWe evaluated 24 paediatric patients homozygous for F508del-CFTR, assessing lung function (FEV1pp) and sweat chloride (SC) before and after CFTR modulator therapy. Whole-gene sequencing was utilised to identify CFTR and pharmacogene variants. Patient-derived human nasal epithelial cells (HNECs) were expanded and differentiated at the air-liquid interface to assess CFTR function via ion transport ({Delta}Isc). ResultsClinical responses varied widely. Twelve participants changed modulators during the study. Sequencing identified 231 additional CFTR variants and pharmacogene polymorphisms, but none correlated with response variability. However, a significant linear relationship emerged between {Delta}Isc and FEV1pp improvement in patients with baseline FEV1pp <90 (R{superscript 2} = 0.651, p = 0.001) and SC reduction (R{superscript 2} = 0.535, p = 0.004). Receiver operating characteristic (ROC) analysis demonstrated high predictive accuracy for SC reduction (AUC = 0.88) and combined FEV1pp/SC response in patients with baseline FEV1pp <90 (AUC = 1.00). Exploratory analysis confirmed that {Delta}Isc predicts FEV1pp changes, modulated by baseline lung function and CFTR modulator type. ConclusionPatient-derived differentiated HNEC cultures serve as a robust predictive tool for CFTR modulator response in paediatric CF patients. Their integration into clinical practice can enhance personalised treatment strategies, minimising ineffective therapy use and improving CF patient outcomes with precision medicine. What is already known on this topicO_LICFTR modulators significantly improve clinical outcomes in people with cystic fibrosis (CF), yet individual responses vary, even among those with identical CFTR genotypes. C_LIO_LIPatient-derived human nasal epithelial cell (HNEC) models have emerged as promising tools to predict CFTR modulator response. However, existing studies have primarily focused on adult and adolescent populations, leaving a gap in personalised treatment strategies for younger children with CF. C_LIO_LIThe relationship between CFTR sequence variations, pharmacogene heterogeneity, and modulator response in paediatric patients has not been extensively explored. C_LI What this study addsO_LIThis study demonstrates a strong correlation between in vitro CFTR function ({Delta}Isc) and clinical improvements in FEV1pp and sweat chloride in children and adolescents with CF. C_LIO_LIWhole-gene sequencing identified 231 additional CFTR variants, yet none were associated with CFTR modulator response, suggesting that genotype alone does not fully explain treatment variability. C_LIO_LIWhile some trends between pharmacogene activity and treatment response were observed, no strong evidence supports pharmacogene profiling as a standalone predictor of CFTR modulator efficacy in paediatric patients. C_LIO_LIDifferentiated-HNEC cultures consistently predicted clinical response across multiple CFTR modulator regimens, reinforcing their value for preclinical drug screening. C_LI How this study might affect research, practice, or policyO_LIOur findings support the integration of differentiated-HNEC models into clinical practice to personalise CFTR modulator selection, reducing ineffective treatments and improving patient outcomes. C_LIO_LIThe study underscores the need for additional clinical endpoints beyond FEV1pp to assess respiratory function in individuals with preserved lung function (FEV1pp > 90%). C_LI

1BackgroundWhile cystic fibrosis is caused by loss-of-function variants in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), other modifier genes have been shown to associate with disease severity. Co-expression of modifiers with CFTR in normal tissue indicates a cooperative relationship and suggests the potential for compensation in the presence of CFTR dysfunction. We examined the co-expression relationships with CFTR in the lung using single cell RNA sequencing to pinpoint cell types and their modifiers involved in the forced expiratory volume in 1 second (FEV1)-based cystic fibrosis lung phenotype and support target cell-type prioritization for therapy 1. MethodsSmartSeq2 single cell RNA sequencing data from non-cystic fibrosis lung tissue was used for evaluation of co-expression with CFTR and modifier genes. Zero-inflated negative binomial model was used to formally test the co-expression association. 10X Chromium based single cell RNA sequencing data from both cystic fibrosis and non-cystic fibrosis studies were assessed graphically to confirm conclusions from the SmartSeq2 primary analysis. ResultsDifferentiating basal, club and alveolar epithelial type 2 cells were found to have high proportions of cells expressing CFTR as well as the greatest number of significant co-expression relationships with the modifiers. In particular, among alveolar epithelial type 2 cells, we observed a strong co-expression trio relationship between CFTR, SLC6A14 and SLC26A9 (p < 0.05). ConclusionsCFTR-modifier gene co-expression suggests basal, club and alveolar epithelial type 2 cells show coordinated expression. Alveolar epithelial type 2 cells showed strong co-expression evidence with two of the most established cystic fibrosis modifier genes.

BackgroundCystic fibrosis (CF) is a genetic disorder in which the respiratory system gets clogged with mucus leads to progressive lung damage. There is no known cure for CF but several treatments to manage symptoms and reduce complications. Vitamin D deficiency is common in CF associated with increased infection and inflammation. This systematic review and meta-analysis will evaluate the effectiveness of vitamin D treatment in reducing respiratory tract infection and inflammation in patients with CF. MethodsRandomized and quasi-randomised studies in CF patients with control groups will be identified. The antibacterial activity of vitamin D supplementation will help in reducing respiratory tract infection and inflammation in CF. Overall effects of vitamin D in terms of infection and inflammatory markers such as C-reactive protein, inflammatory cytokine interleukin (IL)-6, IL-8, IL-17, IL-23, antimicrobial peptide (LL-37), lung function defined by forced expiratory volume in 1 second (FEV1) %, other assessed respiratory parameters will be calculated using random-effect models. Study quality will be assessed using RoB 2 - A revised Cochrane Risk of Bias tool for randomised trials. The overall quality of evidence for each outcome will be summarised according to the Grading of Recommendations Assessment, Development, and Evaluations (GRADE) framework.

Cystic fibrosis (CF) is the most prevalent serious inherited disease in Australia, imposing significant health risks. CF is characterised by chronic lung inflammation and recurrent pulmonary infections that increase morbidity and premature mortality rates. The emergence of antimicrobial resistance (AMR) places further challenges on the treatment and management of CF, necessitating research into alternative strategies for treatment of bacterial infections. Bacteriophage therapy, involving bacterial-specific viruses, is a potential avenue for AMR infections in patients with CF. Existing literature supports the feasibility of phage therapy in CF but there has been a gap in investigating attitudes of the CF community including affected individuals and their caregivers, regarding phage therapy. Understanding perspectives and needs of the CF community is essential for successful implementation and acceptance of novel therapies including phage therapy. We conducted a survey that encompasses responses from 112 consumers from across Australia, comprising people living with CF (38.4%), parents of affected children (49.6%), carers (6.4%), and family members (3%). The findings showed a significant reliance on antibiotics with 51.4% requiring oral, 43.4% nebulised, and 11.4% intravenous (IV) antibiotics within the preceding 2 weeks. Respondents highlighted the availability of new treatments, duration of hospitalisations and costs associated with treatment as important priorities to address. Despite an awareness of phage therapy among 62.4% of respondents, 86.4% expressed interest in obtaining more information, primarily from medical staff (66.7%). Notably, 96.0% of respondents expressed willingness to participate in phage therapy trials. The results of this survey highlighted the CF communitys strong interest in advanced therapeutic approaches, specifically phage therapy. The findings reveal a notable recognition and acceptance of phage therapy as a viable treatment option for pulmonary infections associated with CF.

Characterisation of I37R - a novel mutation in the lasso motif of ABC-transporter CFTR, a chloride channel - was conducted by theratyping using CFTR potentiators which increase channel gating activity and correctors which repair protein trafficking defects. I37R-CFTR function was characterised using intestinal current measurements (ICM) in rectal biopsies, forskolin-induced swelling (FIS) in intestinal organoids and short circuit current measurements (Isc) in organoid-derived monolayers from an individual with I37R/F508del CFTR genotype. We demonstrated that the I37R-CFTR mutation results in a residual function defect amenable to treatment with potentiators and type III, but not to type I, correctors. Molecular dynamics of I37R-CFTR using an extended model of the phosphorylated, ATP-bound human CFTR identified an altered lasso motif conformation which results in an unfavourable strengthening of the interactions between the lasso motif, the regulatory (R) domain and the transmembrane domain two (TMD2). In conclusion, structural and functional characterisation of the I37R-CFTR mutation increases understanding of CFTR channel regulation and provides a potential pathway to access CFTR modulator treatments for individuals with CF caused by ultra-rare CFTR mutations.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although many people with CF (pwCF) are treated using CFTR modulators, some are non-responsive due to their genotype or other uncharacterized reasons. Autologous airway stem cell therapies, in which the CFTR cDNA has been replaced, may enable a durable therapy for all pwCF. Previously, CRISPR-Cas9 with two AAVs was used to sequentially insert two halves of the CFTR cDNA and an enrichment cassette into the CFTR locus. However, the editing efficiency was <10% and required enrichment to restore CFTR function. Further improvement in gene insertion may enhance cell therapy production. To improve CFTR cDNA insertion in human airway basal stem cells (ABCs), we evaluated the use of the small molecules AZD7648 and ART558 which inhibit non-homologous end joining (NHEJ) and micro-homology mediated end joining (MMEJ). Adding AZD7648 alone improved gene insertion by 2-3-fold. Adding both ART558 and AZD7648 improved gene insertion but induced toxicity. ABCs edited in the presence of AZD7648 produced differentiated airway epithelial sheets with restored CFTR function after enrichment. Adding AZD7648 did not increase off-target editing. Further studies are necessary to validate if AZD7648 treatment enriches cells with oncogenic mutations.

Cystic Fibrosis (CF) is a life-shortening disease that is caused by mutations in the CFTR gene, a gene that is expressed in multiple organs. There are several primary tissue models of CF disease, including nasal epithelial cultures and rectal organoids, that are effective in reporting the potential efficacy of mutation-targeted therapies called CFTR modulators. However, there is the well-documented variation in tissue dependent, therapeutic response amongst CF patients, even those with the same CF-causing mutation. Hence, there is an interest in developing strategies for benchmarking therapeutic efficacy in different organs relative to isogenic controls. In this study, we evaluated the CFTR chloride channel response to the highly effective CFTR modulator: Trikafta, in CF patient specific, iPSC-derived colonic and airway cultures relative to mutation-corrected (non-CF) tissues from that same individual. We measured pharmacological rescue in both tissues, but interestingly, Trikafta treatment resulted in different levels of functional rescue in the two tissues relative to the relevant isogenic control. This proof-of-concept study lays the groundwork for future comparisons of patient-specific CF therapeutic responses in both pulmonary and extra-pulmonary systems.

BackgroundCystic fibrosis (CF) has profoundly changed since the introduction of CF Transmembrane Conductance Regulator modulator therapies (CFTRmt), a class of medications that improve function of the CFTR protein encoded by certain CF-causing gene mutations. Amongst these, the triple combination therapy elexacaftor-tezacaftor-ivacaftor (ETI) has been the most impactful and widely used to date. Given chronic respiratory infection and concomitant inflammation is the leading cause of morbidity and early mortality for the majority in CF, what is not certain are the long-term effects of ETI therapy on the respiratory microbiota and pathogens imbedded within. Here we assessed the long-term effects of ETI CFTRmt over 3-years on the respiratory microbiota of a multi-centre cohort of 276 adults with CF (awCF) from 6 CF centres in the UK, USA, and Canada, and compared to a non-CF healthy cohort. ResultsWe determined that respiratory microbiota characteristics (diversity, dominance, and composition) became decreasingly like those of awCF pre-ETI and remodelled to align more with the healthy cohort, where canonical CF pathogens increasingly became less ecologically important in terms of their distributions and abundances across awCF with increased duration on therapy. However, the on-ETI microbiota was impeded from becoming fully healthy due to continued antibiotic exposure and irreversible lung damage experienced by awCF. Specifically, we found that azithromycin, an antibiotic widely used principally for its immunomodulatory benefits, had adverse effects on the respiratory microbiota nullifying the observed positive effects of ETI treatment. When administered alongside ETI-therapy, the use of azithromycin maintained a pre-ETI microbiota dysbiosis and enabled enhanced persistence of emblematic CF pathogens. ConclusionsThe highly anticipated introduction of ETI CFTRmt has greatly changed the course of CF for many people living with this inherited disease. Here we find that ETI CFTRmt enabled positive remodelling of the respiratory microbiota towards a healthy-like state. However, azithromycin impeded total remodelling, making it an ideal candidate for evaluation for discontinuation in the CFTRmt era. While traditional pathogens become less ecologically important the potential evolution and emergence of virulent strains should be investigated. Additionally, the impacts and implications of ETI therapy on the understudied fungal microbiota should also be explored.

Chronic inflammation dominates disease pathogenesis in Cystic Fibrosis (CF) and there is a need to characterise CF immunity. Whole blood cultures offer a cost-effective and non-invasive approach to investigate immune responses within the host environment. Here we used whole blood cultures to investigate the differentiation potential of monocytes (CD45+CD14+ cells) in CF (N=10) and controls (N=8) in the presence and absence of exogenous macrophage-colony stimulatory factor (M-CSF) or granulocyte-macrophage (GM)-CSF with and without interleukin (IL)-4. In CF and control cultures, CD45+CD14+ cells upregulated HLA-DR expression in all instances, and increased CD206 in the presence of GM-CSF with and without IL-4, and CD209 in the presence of GM-CSF and IL-4. In CF, we consistently observed reduced upregulation of CD206 in response to GM-CSF and a positive correlation between CD206 expression and lung function (FEV1). This was unique to cultured monocytes, and not seen with any other marker. These results highlight the potential of whole blood cultures to reveal cellular characteristics in differentiating monocytes related to clinical parameters that could guide the identification of novel biomarkers in CF.

BackgroundCystic Fibrosis (CF) is a lethal genetic condition affecting over 100,000 people worldwide, characterised by multi-organ dysfunction and a progressive lethal lung disease. The disease occurs due to faulty cystic fibrosis transmembrane conductance regulator (CFTR) ion channels effecting flow of chloride, bicarbonate and water out of cells. This causes thick mucus with repeated bacterial infections, systemic inflammation and a decrease in lung function. CFTR modulator therapies have shown variable improvements in lung function and reduction in exacerbation frequency. Basal cells within the lung act as a stem cell for repair following injury and can repopulate the epithelial layer. This process is dysfunctional in CF causing progressive damage. Spontaneous lung repair is well described but not well characterised. Nothing is known about the effects of CFTR modulator therapy on these cells, but this could be of major consequence for people with CF (pwCF). AimsTo determine the effects of CFTR modulator therapy on the activity of CF basal cells and relate this to progenitor function and to study the effects of CFTR modulators on systemic inflammation and clinical outcomes. MethodsClinical information, blood and nasal brushes were obtained from pwCF prior to commencing modulator therapy and at multiple time points up until 1 year of treatment. 10 pwCF were recruited to undertake thoracic CT scans pre-treatment and at 1 year of therapy. Nasal samples were used to isolate basal cells and serum to study systemic markers of inflammation. RNA sequencing of basal cells was undertaken by Ilumina Novoseq to a depth of 20 million read pairs and gene ontology analysis was performed. Functional assays of basal cell activity were carried out. Proteomic analysis and ELISAs were undertaken to determine changes in inflammatory cytokines within the serum across the first year of treatment. Quantitative results were generated by Lung Quantification (LungQ) analysis with qualitative reports from independent radiologists. Results were compared with clinical outcomes. Results110 pwCF were recruited in total who commenced a commercially available CFTR modulator therapy. Serum samples were collected from 77pwCF, nasal brushes obtained from 40 pwCF and 10 completed their CT scans following 1 year of highly effective CFTR modulator therapy. Systemic IL-6, CRP and calprotectin (a biomarker of CF exacerbation) were all significantly reduced with highly effective CFTR modulator treatment. Clinical results were in keeping with those seen in published CFTR modulator clinical trials with improvement in lung function, weight, and exacerbation frequency. Subjective improvements were seen in all 10 CT scans following 1 year of modulator therapy. Significant reductions were seen in airway wall thickening and reduction in thoracic lymphadenopathy were also observed. Basal cell RNA sequencing showed that the relative expression of 2570 genes were significantly different following treatment with CFTR modulators. Ontology analysis showed enrichment in multiple pathways including cilliagenesis and Notch signalling, a key pathway in lung tissue development and homeostasis. Functional assays exhibited a deficit in repair mechanisms of the CF basal cell compared to healthy controls, and reduction in progenitor function. ConclusionsAlthough CFTR modulators improve multiple clinical and radiological outcomes, they also have impacts on basal cell function. There are however, limited impacts on systemic inflammation and more work is needed in this area to understand the disease process.

BackgroundElexacaftor/Tezacaftor/Ivacaftor (ETI) has significantly improved clinical outcomes for people with cystic fibrosis (pwCF), but the molecular effects on airway inflammation remains incompletely understood. This study aimed to characterise longitudinal changes in airway inflammation and sputum proteomes following ETI treatment, and to correlate proteomic shifts to changes in inflammatory cytokines. Patients and methodsSputum from pwCF (n=30) were collected before start of ETI and after 3 and 9-12 months of treatment. Sputum from healthy control subjects (n=7) were included for comparison. Samples were analysed for total proteome content using data independent acquisition liquid chromatography tandem mass spectrometry (DIA LC-MS/MS), and inflammatory cytokines using Mesoscale assays. Protein expression trends were analysed using k-means clustering, and correlations between airway proteomes and inflammatory cytokines were performed using Pearson correlation and enrichment analysis. ResultsETI therapy resulted in significant changes in the airway proteome, mainly related to decreased neutrophil degranulation and an increase in anti-proteases. Levels of IL-1{beta}, IL-8, and TNF decreased with ETI therapy, which correlated with proteins involved in neutrophil degranulation. In contrast, IL-6 levels increased and correlated with proteins involved in O-glycosylation of mucins. Despite these improvements, proteomic and cytokine profiles remained distinct from healthy controls after 9-12 months. ConclusionETI leads to broad shifts in airway protein expression in pwCF with reduced neutrophilic inflammation and restored protease/antiprotease balance. Despite these changes, there is still increased airway inflammation compared to healthy control sputum. This dataset provides a valuable resource for further exploration of CF airway biology under ETI therapy.

Cystic fibrosis (CF) is caused by defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. CFTR controls chloride (Cl-) and bicarbonate (HCO3-) transport into the Airway Surface Liquid (ASL).We investigated the impact of F508del-CFTR correction on HCO3- secretion by studying transepithelial HCO3- fluxes. HCO3- secretion was measured by pH-stat techniquein primary human respiratory epithelial cells from healthy subjects (WT) and people with CF (pwCF)carrying at least oneF508del variant.Its changes after CFTR modulation by the triple combination VX445/661/770 and in the context of TNF-+IL-17 induced inflammation were related to ASL pH and transcriptionnal levels of CFTRand other HCO3- transporters ofairway epithelia such asSLC26A4 (Pendrin), SLC26A9 and NBCe1. CFTR-mediated HCO3-secretion was not detected in F508del primary human respiratory epithelial cells. It was rescued up to [~] 80% of the WT levelby VX-445/661/770. In contrast,TNF-+IL-17 normalized transepithelial HCO3-transportand ASL acidic pH. This was related to anincrease in SLC26A4 and CFTR transcript levels.VX-445/661/770 induced an increase in pH only in the context of inflammation.Effects on HCO3- transport werenot differentbetween F508del homozygous and F508del heterozygous CF airway epithelia. Our studies show that correction of F508del-CFTRHCO3- is not sufficient to buffer acidic ASL and that inflammation is a key regulator of HCO3-secretion in CF airways. Prediction of the response to CFTR modulators by theratyping should take into account airway inflammation.

BackgroundWe recently demonstrated that the triple combination CFTR modulator therapy elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) improves lung ventilation and airway mucus plugging determined by multiple-breath washout and magnetic resonance imaging in CF patients with at least one F508del allele. However, effects of ELX/TEZ/IVA on viscoelastic properties of airway mucus, chronic airway infection and inflammation have not been studied. The aim of this study was, therefore, to determine the effects of ELX/TEZ/IVA on airway mucus rheology, microbiome and inflammation in CF patients with one or two F508del alleles aged 12 years and older. MethodsIn this prospective observational study, we assessed sputum rheology, the microbiome, inflammation markers and proteome before and 8 to 16 weeks after initiation of ELX/TEZ/IVA. ResultsIn total, 59 patients with CF and at least one F508del allele and 10 healthy controls were enrolled in this study. ELX/TEZ/IVA improved the elastic modulus (G; -6.3 Pa; IQR, -17.9 to 1.2; P<0.01) and viscous modulus (G; -1.6 Pa; IQR, -3.6 to 0.5; P<0.05) of CF sputum. Further, ELX/TEZ/IVA improved the microbiome -diversity (0.6; IQR, 0.0 to 1.2; P<0.001) and decreased the relative abundance of Pseudomonas aeruginosa in CF sputum. ELX/TEZ/IVA also reduced IL-8 (-11.7 ng/ml, IQR, -36.5 to 11.2; P<0.05) and free NE activity (-27.5 {micro}g/ml, IQR, - 64.5 to -3.5; P<0.001), and shifted the CF sputum proteome towards healthy. ConclusionsOur data demonstrate that ELX/TEZ/IVA improves sputum viscoelastic properties, chronic airway infection and inflammation in CF patients with at least one F508del allele, however, without reaching levels close to healthy. Clinical trial registered with www.clinicaltrials.gov (NCT04732910)

IntroductionN1303K is the fourth most frequent Cystic Fibrosis (CF) causing mutation. People with CF (pwCF) clinical status can be improved by Elexacaftor(ELX)/Tezacaftor(TEZ)/Ivacaftor (ETI) combotherapy. We investigated the mechanism underlying N1303K-CFTR rescue. MethodsN1303K-CFTR expression and maturation was evaluated by Western Blot in cell lines and Human Nasal Epithelial Primary Cells (HNECs). Cell surface expression was studied by nanoluciferase complementation assay and TurboID proximity labeling. Functional rescue was tested in vitro by YFP-Based Assay and Short Circuit Current. ResultsCorrection by ELX/TEZ increases N1303K-CFTR amounts, but not its maturation in CFTR-expressing HEK and 16HBEge cell lines and in HNECs. In control conditions, N1303K-CFTR is more distributed at the cell surface and significantly more surface partners are identified in the N1303K-CFTR interactome as compared to F508del-CFTR in HEK cells. ELX/TEZ induces a global stabilization of N1303K-CFTR without favoring its plasma membrane relocation in contrast to F508del-CFTR which is redistributed to the membrane. ETI increases N1303K-CFTR activity in HNECs and can be increased by API co-potentiation with a predicted increase in Forced Expiratory Volume in 1 second (ppFEV1) by respectively 13(2)% and 18%(3). This is consistent with a gain in ppFEV1 reported in pwCF carrying the N1303K mutation and additional improvement by API in a patient. ConclusionThese results support the expansion of ETI approval to N1303K mutation but highlight different mechanisms of action than for F508del.

BackgroundDespite significant clinical improvements, there is evidence of persisting airway inflammation in people with cystic fibrosis established on Elexacaftor/tezacaftor/ivacaftor (ETI) therapy. As CF is a multi-system disease, systemic immune profiles can reflect local inflammation within the lungs and other organs. Understanding systemic inflammation after ETI therapy may reveal important translational insights. This study aims to profile systemic inflammatory changes and relate these to the well-documented improvements observed with ETI therapy. MethodsWe conducted a single-centre longitudinal study with 57 CF subjects initiating ETI therapy. All participants were Phe508del homozygous or Phe508del/minimal function. Blood samples were collected pre-ETI and 3-12 months post-therapy initiation. Analyses included mass spectrometry-based proteomics, a multiplex immunoassay, and flow cytometry for peripheral immune cell counts and phenotype. Controls samples were provided by 29 age-matched healthy controls. ResultsSystemic inflammation reduced with ETI therapy; however, the immune profile remained distinct from healthy controls. ETI reduced neutrophil counts and was associated with a more mature, less inflammatory phenotype, as well as a shift toward an immune resolving state associated with increased CD206 expression. Cytokines known to influence neutrophil levels reduced with therapy. Despite ETI therapy, neutrophil and monocyte counts remained elevated compared to healthy controls. There was no obvious association between the ETI-related improvements in systemic inflammation and lung function. ConclusionsPatients with CF show evidence of persisting systemic inflammation despite ETI therapy, this may have long term potentially adverse effects on respiratory and other organ systems.

Staphylococcus aureus (SA) is a common pathogen in children with cystic fibrosis (CF), frequently preceding or coexisting with Pseudomonas aeruginosa (PA). However, the extent to which its phenotypic traits are shaped by transient changes in the airway microenvironment, including coinfection and antibiotic exposure, remains poorly understood. In this study, we characterized 546 SA isolates from a longitudinal cohort of 16 pediatric CF patients over 18 months. Based on clinical records, patients were classified as SA-monoinfected or SA-PA-coinfected. Isolates were analyzed for staphyloxanthin production, hemolytic activity, DNase activity, biofilm formation, resistance to PA. Using principal component and hierarchical cluster analyses, we identified three phenotypic clusters. Isolates from monoinfected patients were predominantly found in a cluster characterized by high pigment and biofilm production and absence of chronic PA infection. In contrast, transient PA detection was associated with a shift toward a cluster displaying reduced pigment and biofilm levels. Additionally, in two out of five monoinfected patients, this shift coincided with ciprofloxacin eradication treatment including inhaled tobramycin or colistin and was accompanied by a significant increase in methicillin-resistant SA (MRSA) isolates. No phenotypic transitions were observed in chronically coinfected patients. Molecular characterization of a set of isolates showed on the one hand heterogeneity in the SA lineages and on the other hand the phenotypic profile was not determined by the genetic background. In vitro experiments showed a higher survival of MRSA over MSSA isolates in presence of both PA and ciprofloxacin. These results suggest that the phenotypic adaptation of SA in pediatric CF airway is driven by microenvironment rather than clonal background, and that transient coinfection and antibiotic exposure can favor MRSA emergence even in early-stage disease.

BackgroundNovel small molecule therapies for cystic fibrosis (CF) are showing promising efficacy and becoming more widely available since recent FDA approval. The newest of these is a triple therapy of Elexacaftor-Tezacaftor-Ivacaftor (ETI, Trikafta(R)). Little is known about how these drugs will affect polymicrobial lung infections, which are the leading cause of morbidity and mortality among people with CF (pwCF). Methodswe analyzed the sputum microbiome and metabolome from pwCF (n=24) before and after ETI therapy using 16S rRNA gene amplicon sequencing and untargeted metabolomics. ResultsThe lung microbiome diversity, particularly its evenness, was increased (p = 0.044) and the microbiome profiles were different between individuals before and after therapy (PERMANOVA F=1.92, p=0.044). Despite these changes, the microbiomes were more similar within an individual than across the sampled population. There were no specific microbial taxa that were different in abundance before and after therapy, but collectively, the log-ratio of anaerobes to classic CF pathogens significantly decreased. The sputum metabolome also showed changes due to ETI. Beta-diversity increased after therapy (PERMANOVA F=4.22, p=0.022) and was characterized by greater variation across subjects while on treatment. This significant difference in the metabolome was driven by a decrease in peptides, amino acids, and metabolites from the kynurenine pathway. Metabolism of the three small molecules that make up ETI was extensive, including previously uncharacterized structural modifications. ConclusionsThis study shows that ETI therapy affects both the microbiome and metabolome of airway mucus. This effect was stronger on sputum biochemistry, which may reflect changing niche spaces for microbial residency in lung mucus as the drugs effects take hold, which then leads to changing microbiology. FundingThis project was funded by a National Institute of Allergy and Infectious Disease Grant R01AI145925