CF Airway Research Articles

Gene expression responses of CF airway epithelial cells exposed to elexacaftor/tezacaftor/ivacaftor (ETI) suggest benefits beyond improved CFTR channel function.

Gene expression responses by CF AEC exposed to ETI suggest that in addition to improving CFTR channel function, ETI is likely to increase resistance to bacterial infection by increasing levels of beta defensin 1 (hBD-1). ETI may also reduce lung damage by suppressing MMP10, and reduce airway inflammation by repressing proinflammatory cytokine secretion by AEC cells.

Impact of high-dose cholecalciferol (vitamin D3) and inulin prebiotic on intestinal and airway microbiota in adults with cystic fibrosis: A 2 × 2 randomized, placebo-controlled, double-blind pilot study

BackgroundCystic fibrosis (CF) is a multi-organ disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Individuals with CF often have gastrointestinal (GI) dysbiosis due to chronic inflammation and antibiotic use. Previous studies suggested a role for vitamin D in reversing the GI dysbiosis found in CF. ObjectiveTo explore the potential role of a combination of high-dose oral cholecalciferol (vitamin D3) and fermentable dietary fiber, inulin, to impact bacterial composition, richness, and diversity of intestinal and airway microbiota in adults with CF. MethodsThis was a 2 × 2 factorial, double-blinded, placebo-controlled, randomized, pilot clinical trial in which adults with CF received oral cholecalciferol (vitamin D3) (50,000 IU/week) and/or inulin (12 g/day) for 12 weeks. Thus, there were 4 study groups (n = 10 subjects per group); 1) placebo 2) vitamin D3 3) inulin 4) vitamin D3 plus inulin. Stool and sputum samples were collected at baseline (just before) and after the intervention and were analysed using 16S ribosomal RNA gene sequencing for gut and airway microbiota composition. Statistical analyses assessed alpha and beta diversity to evaluate microbial community changes. ResultsOf a total of 254 screened participants, 40 eligible participants were randomized to one of the 4 treatment arms. Participants receiving vitamin D3 plus inulin exhibited greater changes in microbiome indexes in both intestinal and airway relative to those in the other study groups. Specific taxonomic changes supported the potential beneficial influence of this combination to mitigate both intestinal and airway dysbiosis in adults with CF. ConclusionThis pilot study established that the combination of oral vitamin D3 and the prebiotic inulin was well tolerated over 12 weeks in adults with CF and altered gut and airway bacterial communities. Future research appear warranted to define clinical outcomes and the role of microbiota changes therein with this approach.

Beyond Borders of the Cell: How Extracellular Vesicles Shape COVID-19 for People with Cystic Fibrosis.

The interaction between extracellular vesicles (EVs) and SARS-CoV-2, the virus causing COVID-19, especially in people with cystic fibrosis (PwCF) is insufficiently studied. EVs are small membrane-bound particles involved in cell-cell communications in different physiological and pathological conditions, including inflammation and infection. The CF airway cells release EVs that differ from those released by healthy cells and may play an intriguing role in regulating the inflammatory response to SARS-CoV-2. On the one hand, EVs may activate neutrophils and exacerbate inflammation. On the other hand, EVs may block IL-6, a pro-inflammatory cytokine associated with severe COVID-19, and protect PwCF from adverse outcomes. EVs are regulated by TGF-β signaling, essential in different disease states, including COVID-19. Here, we review the knowledge, identify the gaps in understanding, and suggest future research directions to elucidate the role of EVs in PwCF during COVID-19.

An in vitro medium for modeling gut dysbiosis associated with cystic fibrosis.

The gut physiology of pediatric and adult persons with cystic fibrosis (pwCF) is altered relative to healthy persons. The CF gut is characterized, in part, as having excess mucus, increased fat content, acidic pH, increased inflammation, increased antibiotic perturbation, and the potential for increased oxygen availability. These physiological differences shift nutritional availability and the local environment for intestinal microbes, thus likely driving significant changes in microbial metabolism, colonization, and competition with other microbes. The impact of any specific change in this physiological landscape is difficult to parse using human or animal studies. Thus, we have developed a novel culture medium representative of the CF gut environment, inclusive of all the aforementioned features. This medium, called CF-MiPro, maintains CF gut microbiome communities, while significantly shifting nonCF gut microbiome communities toward a CF-like microbial profile, characterized by low Bacteroidetes and high Proteobacteria abundance. This medium is able to maintain this culture composition for up to 5 days of passage. Additionally, microbial communities passaged in CF-MiPro produce significantly less immunomodulatory short-chain fatty acids (SCFA), including propionate and butyrate, than communities passaged in MiPro, a culture medium representative of healthy gut physiology, confirming not only a shift in microbial composition but also altered community function. Our results support the potential for this in vitro culture medium as a new tool for the study of CF gut dysbiosis. IMPORTANCE Cystic fibrosis is an autosomal recessive disease that disrupts ion transport at mucosal surfaces, leading to mucus accumulation and altered physiology of both the lungs and the intestines, among other organs, with the resulting altered environment contributing to an imbalance of microbial communities. Culture media representative of the CF airway have been developed and validated; however, no such medium exists for modeling the CF intestine. Here, we develop and validate a first-generation culture medium inclusive of features that are altered in the CF colon. Our findings suggest this novel medium, called CF-MiPro, as a maintenance medium for CF gut microbiome samples and a flexible tool for studying key drivers of CF-associated gut dysbiosis.

Divergent Pseudomonas aeruginosa LpxO enzymes perform site-specific lipid A 2-hydroxylation.

Pseudomonas aeruginosa can survive in a myriad of environments, partially due to modifications of its lipid A, the membrane anchor of lipopolysaccharide. We previously demonstrated that divergent late acyltransferase paralogs, HtrB1 and HtrB2, add acyloxyacyl laurate to lipid A 2- and 2'-acyl chains, respectively. The genome of P. aeruginosa also has genes which encode two dioxygenase enzymes, LpxO1 and LpxO2, that individually hydroxylate a specific secondary laurate. LpxO1 acts on the 2'-acyloxyacyl laurate (added by HtrB2), whereas LpxO2 acts on the 2-acyloxyacyl laurate (added by HtrB1) in a site-specific manner. Furthermore, while both enzyme pairs are evolutionarily linked, phylogenomic analysis suggests the LpxO1/HtrB2 enzyme pair as being of ancestral origin, present throughout the Pseudomonas lineage, whereas the LpxO2/HtrB1 enzyme pair likely arose via horizontal gene transfer and has been retained in P. aeruginosa over time. Using a murine pulmonary infection model, we showed that both LpxO1 and LpxO2 enzymes are functional in vivo, as direct analysis of in vivo lipid A structure from bronchoalveolar lavage fluid revealed 2-hydroxylated lipid A. Gene expression analysis reveals increased lpxO2 but unchanged lpxO1 expression in vivo, suggesting differential regulation of these enzymes during infection. We also demonstrate that loss-of-function mutations arise in lpxO1 and lpxO2 during chronic lung infection in people with cystic fibrosis (CF), indicating a potential role for pathogenesis and airway adaptation. Collectively, our study characterizes lipid A 2-hydroxylation during P. aeruginosa airway infection that is regulated by two distinct lipid A dioxygenase enzymes.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that causes severe infection in hospitalized and chronically ill individuals. During infection, P. aeruginosa undergoes adaptive changes to evade host defenses and therapeutic interventions, increasing mortality and morbidity. Lipid A structural alteration is one such change that P. aeruginosa isolates undergo during chronic lung infection in CF. Investigating genetic drivers of this lipid A structural variation is crucial in understanding P. aeruginosa adaptation during infection. Here, we describe two lipid A dioxygenases with acyl-chain site specificity, each with different evolutionary origins. Further, we show that loss of function in these enzymes occurs in CF clinical isolates, suggesting a potential pathoadaptive phenotype. Studying these bacterial adaptations provides insight into selection pressures of the CF airway on P. aeruginosa phenotypes that persist during chronic infection. Understanding these adaptive changes may ultimately provide clinicians better control over bacterial populations during chronic infection.

Cystic fibrosis airway inflammation enables elexacaftor/tezacaftor/ivacaftor-mediated rescue of N1303K CFTR mutation.

Rescue of N1303K CFTR by highly effective modulator therapy (HEMT) is enabled by CF airway inflammation. These findings suggest that evaluation of HEMT for rare CFTR mutations must be performed under inflammatory conditions relevant to CF airways. https://bit.ly/3tTcoJE.

Infection, Allergy, and Inflammation: The Role of Aspergillus fumigatus in Cystic Fibrosis.

Aspergillus fumigatus (Af) is a mold frequently detected in airway samples from people with cystic fibrosis (pwCF). Abnormal airway mucus may allow Af to germinate, resulting in airway infection or an allergic response. While Af is known to increase morbidity in pwCF, individual responses and the degree of impact on lung disease vary. Improved approaches to diagnosis, treatment, and prevention of Af, particularly the persistent Af infection, are needed. This update highlights our current understanding of Af pathophysiology in the CF airway, the effects of Af on pwCF, and areas of research needed to improve clinical outcomes.

Airway infections as a risk factor for Pseudomonas aeruginosa acquisition and chronic colonisation in children with cystic fibrosis

BackgroundPseudomonas aeruginosa (Pa) infection is detrimental to people with cystic fibrosis (pwCF). Several clinical and genetic factors predispose to early Pa infections. However, the role of earlier infections with other pathogens on the risk of Pa infection in paediatric pwCF remains unknown. MethodsUsing Kaplan–Meier method, we computed the cumulative incidences of bacterial and fungal initial acquisition (IA) and chronic colonisation (CC) in 1,231 French pwCF under 18 years of age for methicillin-susceptible and resistant Staphylococcus aureus (MSSA and MRSA), Stenotrophomonas maltophilia, Haemophilus influenzae, Achromobacter xylosoxidans, and Aspergillus species. Previous infections were analysed as Pa-IA and Pa-CC risk factors using Cox regression models. ResultsBy 2 years of age, 65.5% pwCF had experienced at least one bacterial or fungal IA, and 27.9% had experienced at least one CC. The median age of Pa-IA was 5.1 years, and Pa-CC was present in 25% pwCF by 14.7 years. While 50% acquired MSSA at 2.1 years, 50% progressed to chronic MSSA colonisation at 8.4 years. At 7.9 and 9.7 years, 25% pwCF were infected by S. maltophilia and Aspergillus spp., respectively. The risk of Pa-IA and Pa-CC increased with IAs of all other species, with hazard ratios (HR) up to 2.19 (95% Confidence interval (CI) 1.18–4.07). The risk of Pa-IA increased with the number of previous bacterial/fungal IAs (HR=1.89, 95% CI 1.57–2.28), with a 16% increase per additional pathogen; same trend was noted for Pa-CC. ConclusionsThis study establishes that the microbial community in CF airways can modulate Pa occurrence. At the dawn of targeted therapies, it paves the way for characterizing future trends and evolution of infections.

Detection of bile acids in bronchoalveolar lavage fluid defines the inflammatory and microbial landscape of the lower airways in infants with cystic fibrosis

BackgroundCystic Fibrosis (CF) is a genetic condition characterized by neutrophilic inflammation and recurrent infection of the airways. How these processes are initiated and perpetuated in CF remains largely unknown. We have demonstrated a link between the intestinal microbiota-related metabolites bile acids (BA) and inflammation in the bronchoalveolar lavage fluid (BALF) from children with stable CF lung disease. To establish if BA indicate early pathological processes in CF lung disease, we combined targeted mass spectrometry and amplicon sequencing-based microbial characterization of 121 BALF specimens collected from 12-month old infants with CF enrolled in the COMBAT-CF study, a multicentre randomized placebo-controlled clinical trial comparing azithromycin versus placebo. We evaluated whether detection of BA in BALF is associated with the establishment of the inflammatory and microbial landscape of early CF lung disease, and whether azithromycin, a motilin agonist that has been demonstrated to reduce aspiration of gastric contents, alters the odds of detecting BA in BALF. We also explored how different prophylactic antibiotics regimens impact the early life BALF microbiota.ResultsDetection of BA in BALF was strongly associated with biomarkers of airway inflammation, more exacerbation episodes during the first year of life, increased use of oral antibiotics with prolonged treatment periods, a higher degree of structural lung damage, and distinct microbial profiles. Treatment with azithromycin, a motilin agonist, which has been reported to reduce aspiration of gastric contents, did not reduce the odds of detecting BA in BALF. Culture and molecular methods showed that azithromycin does not alter bacterial load or diversity in BALF. Conversely, penicillin-type prophylaxis reduced the odds of detecting BAs in BALF, which was associated with elevated levels of circulating biomarkers of cholestasis. We also observed that environmental factors such as penicillin-type prophylaxis or BAs detection were linked to distinct early microbial communities of the CF airways, which were associated with different inflammatory landscapes but not with structural lung damage.ConclusionsDetection of BA in BALF portend early pathological events in CF lung disease. Benefits early in life associated with azithromycin are not linked to its antimicrobial properties.AFd3CvqLfCjKZ9n4H93NFTVideo

WS18.06 The impact of cytomegalovirus on airway epithelial gene expression

Introduction: The pathogenicity of cytomegalovirus (CMV) has predominantly been recognized in immunosuppressed populations & newborns. However, a recent study in persons with CF observed that CMV seropositive individuals died from lung disease 8 years sooner than those who were seronegative. Further, CMV DNA was identified in those individuals’ sputum (Parkins 2019, ERJ). This observation aligns with increasing evidence associating CMV infection with worse outcomes in many inflammatory conditions, and a higher overall mortality. To date, models of CMV infection of human bronchial epithelial (HBE) cells have not been established, limiting our ability to study its impact in CF airways.

WS14.03 Combined therapeutic strategies for the restoration of airway epithelial integrity and function in cystic fibrosis

Background: In CF airways, dysfunctional Cl– secretion and mucociliary clearance favor bacterial infections contributing to progressive lung damage and respiratory failure, the first cause of morbidity and mortality. We thus devote our efforts to identify novel therapeutic strategies to promote CF lung repair. We previously discovered that the capability of CF airway epithelia to repair is less efficient, than in healthy controls, due to the CFTR defect and bacterial infections, in particular with P. aeruginosa (PA) and S. aureus (SA). Our work also unveiled that CFTR modulators enhance airway repair, but both CFTR rescue and repair improvement are dampened by infection.

WS18.02 ETI triple therapy shows sustained, progressive normalisation of airway cytokine and antiprotease balance and systemic inflammation over one year of treatment

Objectives: Pulmonary morbidity in cystic fibrosis comprises reduced lung function with chronic infection and pronounced neutrophilia with substantial burden of proteases and inflammatory mediators. Highly effective modulator therapy with elexacaftor/tezacaftor/ivacaftor improves lung function in those with eligible genotypes. In this context, we sought to understand the influence of ETI therapy on the inflammatory milieu in the CF airway.

P195 Prospective longitudinal evaluation of the lung clearance index (LCI) in the clinical setting

Lung clearance index (LCI) is recognized of great value as an endpoint for CF clinical trials, particularly among young children. However, there are still gaps in the information required to understand its role in the clinical care setting. We conducted a longitudinal study to evaluate the potential of LCI as a clinical decision tool. We enrolled 55 children with CF (mean age 10.9 + 3.6 years) and followed for 2 years LCI, FEV1 and other clinical parameters during quarterly routine visits. Reported symptoms, findings of exacerbation and changes in therapies were recorded for each visit. LCI was significantly increased from the previous visit in the presence of new respiratory symptoms reported (p = 0.0006), as well as being associated with changes in respiratory regimen (p = 0.0008), but not with prescription for antibiotics (p > 0.2). In contrast, FEV1 did not change significantly under any of these conditions (p > 0.2). A significant increase in LCI was noted for children diagnosed at the visit with a pulmonary exacerbation (1.3 vs 0.005 units, p = 0.04), this was not the case with FEV1 (+0.7 vs –2.4%-pred., p = 0.26). Most exacerbations were mild and treated as an outpatient, only 2 led to hospitalization. In addition, a mild decline in lung function with age during the study period was noted, with an increase of LCI by 0.32 units per year (p = 0.002) and a drop in FEV1 of –1.12%-pred. per year (p = 0.02). The decline in FEV1 was associated with LCI, with a drop of –2.0%-pred./year for every unit of increase in LCI (p < 0.0001). Further, there was a strong correlation between the change in FEV1 between visits and the change in LCI (p < 0.0001). We are conducting additional sensitivity analyses to evaluate cut-offs for minimal clinically important difference (MCID) considerations. We conclude that LCI has high sensitivity to progression of CF airway disease in children with what could be considered as well preserved lung function based on FEV1 criteria alone.

WS14.05 Effect of an agro-based compound (A-bC) on remodelling and regeneration of airway epithelium in cystic fibrosis

The CF airway epithelium (AE) exhibits large areas of remodelling such as basal or goblet cell hyperplasia, ciliated cell absence or epithelial desquamation, therefore leading to impaired mucociliary clearance (MCC). We have previously demonstrated that the regeneration of CF AE leads to a remodelled epithelium, the role of CF cell inflammatory memory in basal cell hyperplasia and increased CF AE height, the involvement of lung tissue inflammation in goblet cell hyperplasia, and the inflammationindependent default of ciliated cell differentiation. The identification of therapies favouring MCC through an improvement of CF AE regeneration is therefore crucial for CF patients. In this perspective, we examined the influence of an agro-based compound (A-bC) obtained by bacterial fermentation of wheat, on the regeneration of the human CF AE.

P155 The impact of elexacaftor/tezacaftor/ivacaftor (ETI) therapy on occurrence of bacterial lung infections in cystic fibrosis patients

Lung disease is the main cause of morbidity and mortality in CF. The introduction of CFTR modulators has led to clinical benefits in CF patients. However, the extent to which these therapies reduce susceptibility to chronic lung infections remains to be elucidated. We aimed to evaluate the impact of ETI on CF airway microbiology after one year of treatment.

P260 Co-culture of Prevotella spp. and Pseudomonas aeruginosa from chronic cystic fibrosis infection in artificial sputum medium

Previous research using agar well diffusion assays revealed that Pseudomonas aeruginosa (PA) isolates from chronic CF infection, grown aerobically and anaerobically, inhibited the growth of other clinically relevant species. This study aimed to determine if this observed inhibition was also exhibited in conditions more physiologically representative of the CF airways.

P069 Effect of Orkambi therapy on the lung microbiota in people with cystic fibrosis (PwCF) over the first 12 months of therapy (ROCK Study)

Objectives: CFTR modulator therapy with Orkambi® (lumacaftor/ivacaftor) reduces the rate of decline of FEV1 in people with cystic fibrosis (PwCF) homozygous for the F508del mutation. However, the effect of Orkambi® on the composition and structure of the CF airway microbiota remains unclear. In this study, we determined the effect of therapy with Orkambi® on lung microbiota composition.

Proteome of airway surface liquid and mucus in newborn wildtype and cystic fibrosis piglets

BackgroundThe respiratory tract is protected from inhaled particles and microbes by mucociliary clearance, mediated by the mucus and the cilia creating a flow to move the mucus cephalad. Submucosal glands secrete linear MUC5B mucin polymers and because they pass through the gland duct before reaching the airway surface, bundled strands of 1000–5000 parallel molecules exit the glands. In contrast, the surface goblet cells secrete both MUC5AC and MUC5B.MethodsWe used mass-spectrometry based proteomic analysis of unstimulated and carbachol stimulated newborn wild-type (WT) and cystic fibrosis transmembrane conductance regulator (CFTR) null (CF) piglet airways to study proteins in the airway surface liquid and mucus, to investigate if levels of MUC5AC and MUC5B were affected by carbachol stimulation and whether the proteins clustered according to function.ResultsProteins in the first four extracted fractions clustered together and the fifth fraction contained the mucus cluster, mucins and other proteins known to associate with mucins, whereas the traditional airway surface liquid proteins clustered to fraction 1–4 and were absent from the mucus fraction. Carbachol stimulation resulted in increased MUC5AC and MUC5B.ConclusionsThese results indicate a distinct separation between proteins in the washable surface liquid and the mucus fraction. In fractions 1–4 from newborn CF piglets an additional cluster containing acute phase proteins was observed, suggesting an early inflammatory response in CF piglets. Alternatively, increased levels of these proteins could indicate altered lung development in the CF piglets. This observation suggests that CF airway disease is present at birth and thus, treatment should commence directly after diagnosis.

Inflammation and Infection in Cystic Fibrosis: Update for the Clinician.

Inflammation and infection play an important role in the pathophysiology of cystic fibrosis, and they are significant causes of morbidity and mortality in CF. The presence of thick mucus in the CF airways predisposes to local hypoxia and promotes infection and inflammation. A vicious cycle of airway obstruction, inflammation, and infection is of critical importance for the progression of the disease, and new data elucidate the different factors that influence it. Recent research has been focused on improving infection and inflammation in addition to correcting the basic gene defect. This review aims to summarize important advances in infection and inflammation as well as the effect of new treatments modulating the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. New approaches to target infection and inflammation are being studied, including gallium, nitric oxide, and phage therapy for infection, along with retinoids and neutrophil elastase inhibitors for inflammation.

Direct Detection of Pseudomonas aeruginosa from Respiratory Samples of Children with Cystic Fibrosis

Introduction: Pseudomonas aeruginosa is the most important cause of lung infection and major cause of morbidity and mortality among Cystic Fibrosis patients. Early detection of P. aeruginosa from clinical samples is a key element in patient management. Appropriate antimicrobial treatment will postpone the transition to chronic lung infection.&#x0D; Methods: Sputum and deep pharyngeal swab samples (DPS) of CF patients were inoculated into conventional agar plates and identified by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry. DNA was extracted with a column based QIAamp DNA mini kit from the samples. Molecular detection was done by using 23S rDNA specific primers with Taqman probe for quantitative PCR (qPCR) and exoA gene specific primers for in house PCR.&#x0D; Results: A total number of 67 sputum and 33 DPS samples were included to the study. Median age of the patients was 6 (2-14) for DPS and 15(7-33) for sputum samples. Detection limit was 103 cfu/ml for qPCR which displayed 94% sensitivity and 93% specificity for sputum; 44% sensitivity and 100% specificity for DPS. In house PCR displayed 94% sensitivity and 93% specificity for sputum; 50% sensitivity and 100% specificity for DPS. Time reqired for conventional method was 48 hours, 6 hours for in- house PCR and 4 hours for qPCR.&#x0D; Conclusion: Although culture is a reliable detection technique, a more rapid and sensitive way to detect P. aeruginosa from CF airway samples is essential. In our study which culture is accepted as a gold standard; PCR results is promising especially for sputum samples. Validation of molecular methods is necessary before implementation in routine laboratories. Clinical implications of discrepant results between culture and PCR detection should be carefully evaluated and combining both approaches considering antibiotic treatment could be reasonable.