CF Airway Research Articles

Clinical characteristics of Pseudomonas and Aspergillus co-infected cystic fibrosis patients: A UK registry study

BackgroundPseudomonas aeruginosa (Pa) and Aspergillus species (Asp) are the most common bacterial and fungal organisms respectively in CF airways. Our aim was to examine impacts of Asp infection and Pa/Asp co-infection. MethodsPatients on the UK CF Registry in 2016 were grouped into: absent (Pa-), intermittent (Pai) or chronic Pa (Pac), each with Asp positive (Asp+) or negative (Asp-). Primary outcome was best percentage predicted FEV1 (ppFEV1) that year. Secondary outcomes were intravenous (IV) antibiotic courses, growth (height, weight, BMI) and additional disease complications. Associations between outcomes and infection-status were assessed using regression models adjusting for significant confounders (age, sex, Phe508del homozygosity and CF-related diabetes (CFRD)). Results9,270 patients were included (median age 19 [IQR 9-30] years, 54% male, 50% Phe508del/F508del). 4,142 patients (45%) isolated Pa, 1,460 (16%) Asp. Pa-/Asp+ subjects had an adjusted ppFEV1 that was 5.9% lower than Pa-/Asp- (p < 0.0001). In patients with Pai or Pac, there was no additional impact of Asp on ppFEV1. However, there was a higher probability that Pac/Asp+ patients had required IV antibiotics than Pac/Asp- group (OR 1.23 [1.03-1.48]). Low BMI, ABPA, CF-liver disease and CFRD were all more frequent with Asp alone than Pa-/Asp-, though not more common in Pac/Asp+ than Pac/Asp-. ConclusionsCo-infection with Pa and Asp was not associated with reduced lung function compared with Pa alone, but was associated with additional use of IV antibiotics. Asp infection itself is associated with several important indicators of disease severity. Longitudinal analyses should explore the impact of co-infection on disease progression.

IRE1α Is a Therapeutic Target for Cystic Fibrosis Airway Inflammation.

New anti-inflammatory treatments are needed for CF airway disease. Studies have implicated the endoplasmic reticulum stress transducer inositol requiring enzyme 1α (IRE1α) in CF airway inflammation. The activation of IRE1α promotes activation of its cytoplasmic kinase and RNase, resulting in mRNA splicing of X-box binding protein-1 (XBP-1s), a transcription factor required for cytokine production. We tested whether IRE1α kinase and RNase inhibition decreases cytokine production induced by the exposure of primary cultures of homozygous F508del CF human bronchial epithelia (HBE) to supernatant of mucopurulent material (SMM) from CF airways. We evaluated whether IRE1α expression is increased in freshly isolated and native CF HBE, and couples with increased XBP-1s levels. A FRET assay confirmed binding of the IRE1α kinase and RNase inhibitor, KIRA6, to the IRE1α kinase. F508del HBE cultures were exposed to SMM with or without KIRA6, and we evaluated the mRNA levels of XBP-1s, IL-6, and IL-8, and the secretion of IL-6 and IL-8. IRE1α mRNA levels were up-regulated in freshly isolated CF vs. normal HBE and coupled to increased XBP-1s mRNA levels. SMM increased XBP-1s, IL-6, and IL-8 mRNA levels and up-regulated IL-6 and IL-8 secretion, and KIRA6 blunted these responses in a dose-dependent manner. Moreover, a triple combination of CFTR modulators currently used in the clinic had no effect on SMM-increased XBP-1s levels coupled with increased cytokine production in presence or absence of KIRA6. These findings indicate that IRE1α mediates cytokine production in CF airways. Small molecule IRE1α kinase inhibitors that allosterically reduce RNase-dependent XBP-1s may represent a new therapeutic strategy for CF airway inflammation.

CFTR Correctors and Antioxidants Partially Normalize Lipid Imbalance but not Abnormal Basal Inflammatory Cytokine Profile in CF Bronchial Epithelial Cells.

A deficiency in cystic fibrosis transmembrane conductance regulator (CFTR) function in CF leads to chronic lung disease. CF is associated with abnormalities in fatty acids, ceramides, and cholesterol, their relationship with CF lung pathology is not completely understood. Therefore, we examined the impact of CFTR deficiency on lipid metabolism and pro-inflammatory signaling in airway epithelium using mass spectrometric, protein array. We observed a striking imbalance in fatty acid and ceramide metabolism, associated with chronic oxidative stress under basal conditions in CF mouse lung and well-differentiated bronchial epithelial cell cultures of CFTR knock out pig and CF patients. Cell-autonomous features of all three CF models included high ratios of ω-6- to ω-3-polyunsaturated fatty acids and of long- to very long-chain ceramide species (LCC/VLCC), reduced levels of total ceramides and ceramide precursors. In addition to the retinoic acid analog fenretinide, the anti-oxidants glutathione (GSH) and deferoxamine partially corrected the lipid profile indicating that oxidative stress may promote the lipid abnormalities. CFTR-targeted modulators reduced the lipid imbalance and oxidative stress, confirming the CFTR dependence of lipid ratios. However, despite functional correction of CF cells up to 60% of non-CF in Ussing chamber experiments, a 72-h triple compound treatment (elexacaftor/tezacaftor/ivacaftor surrogate) did not completely normalize lipid imbalance or oxidative stress.Protein array analysis revealed differential expression and shedding of cytokines and growth factors from CF epithelial cells compared to non-CF cells, consistent with sterile inflammation and tissue remodeling under basal conditions, including enhanced secretion of the neutrophil activator CXCL5, and the T-cell activator CCL17. However, treatment with antioxidants or CFTR modulators that mimic the approved combination therapies, ivacaftor/lumacaftor and ivacaftor/tezacaftor/elexacaftor, did not effectively suppress the inflammatory phenotype.We propose that CFTR deficiency causes oxidative stress in CF airway epithelium, affecting multiple bioactive lipid metabolic pathways, which likely play a role in CF lung disease progression. A combination of anti-oxidant, anti-inflammatory and CFTR targeted therapeutics may be required for full correction of the CF phenotype.

Comparative Analysis of Clinical Parameters and Sputum Biomarkers in Establishing the Relevance of Filamentous Fungi in Cystic Fibrosis.

BackgroundThe relationship between fungal culture (FC) positivity and airway inflammation in CF is largely unknown. Identifying the clinical significance of filamentous fungi in CF using both clinical parameters and biomarkers may change our antimicrobial therapeutic strategies.ObjectivesTo investigate the clinical characteristics and airway biomarker profile in relation to the detection of filamentous fungi in respiratory samples obtained from CF patients.MethodsA prospective cohort study over 24 months, including children and adults with CF. Participants provided sputum and/or bronchoalveolar lavage samples, which underwent processing for bacterial and fungal culture, leukocyte differential cell count and biomarker analysis for neutrophil elastase (NE), interleukin-8 (IL-8), galactomannan and tumor necrosis factor receptor type 2 (TNF-R2). We performed FC using neat sputum plugs, an approach shown to be more sensitive compared to routine laboratory testing.ResultsSixty-one patients provided 76 respiratory samples (72 sputum and 4 BAL). Median age was 17 years (range 6 months–59 years). FC positivity was noted in 49% of the cohort. FC positivity was greater during pulmonary exacerbation compared to the stable state (67 versus 50%). Participants aged 5–30 years had a lower FEV1 within the FC positive group. A significant association between FC positivity and non-tuberculosis mycobacterial (NTM) culture was observed on non-parametric testing (p = 0.022) and regression analysis (p = 0.007). Exposure to indoor mold was a predictor for FC positivity (p = 0.047). There was a trend towards increased lung clearance index (LCI), bronchiectasis and intravenous antibiotic use in the FC positive group. There was no significant difference in biomarkers between FC positive and negative patients.Conclusion Aspergillus. fumigatus is the commonest filamentous fungi cultured from CF airways. We found no difference in the airway biomarker profile between FC positive and negative patients. The role of galactomannan and TNFR2 as fungal specific biomarkers in CF remains uncertain. FC positivity is associated with a lower FEV1 in younger patients, a lower LCI, NTM positivity, bronchiectasis, and intravenous antibiotic exposure. Larger trials are needed to determine the role of galactomannan and TNF-R2 as potential fungal biomarkers in CF.

Update on Respiratory Fungal Infections in Cystic Fibrosis Lung Disease and after Lung Transplantation.

Cystic fibrosis is the most common autosomal-recessive metabolic disease in the Western world. Impaired trans-membrane chloride transport via the cystic fibrosis transmembrane conductance regulator (CFTR) protein causes thickened body fluids. In the respiratory system, this leads to chronic suppurative cough and recurrent pulmonary infective exacerbations, resulting in progressive lung damage and respiratory failure. Whilst the impact of bacterial infections on CF lung disease has long been recognized, our understanding of pulmonary mycosis is less clear. The range and detection rates of fungal taxa isolated from CF airway samples are expanding, however, in the absence of consensus criteria and univocal treatment protocols for most respiratory fungal conditions, interpretation of laboratory reports and the decision to treat remain challenging. In this review, we give an overview on fungal airway infections in CF and CF-lung transplant recipients and focus on the most common fungal taxa detected in CF, Aspergillus fumigatus, Candida spp., Scedosporium apiospermum complex, Lomentospora species, and Exophiala dermatitidis, their clinical presentations, common treatments and prophylactic strategies, and clinical challenges from a physician’s point of view.

Secretory Cells Dominate Airway CFTR Expression and Function in Human Airway Superficial Epithelia.

Rationale: Identification of the specific cell types expressing CFTR (cystic fibrosis [CF] transmembrane conductance regulator) is required for precision medicine therapies for CF. However, a full characterization of CFTR expression in normal human airway epithelia is missing. Objectives: To identify the cell types that contribute to CFTR expression and function within the proximal-distal axis of the normal human lung. Methods: Single-cell RNA (scRNA) sequencing (scRNA-seq) was performed on freshly isolated human large and small airway epithelial cells. scRNA in situ hybridization (ISH) and single-cell qRT-PCR were performed for validation. In vitro culture systems correlated CFTR function with cell types. Lentiviruses were used for cell type-specific transduction of wild-type CFTR in CF cells. Measurements and Main Results: scRNA-seq identified secretory cells as dominating CFTR expression in normal human large and, particularly, small airway superficial epithelia, followed by basal cells. Ionocytes expressed the highest CFTR levels but were rare, whereas the expression in ciliated cells was infrequent and low. scRNA ISH and single-cell qRT-PCR confirmed the scRNA-seq findings. CF lungs exhibited distributions of CFTR and ionocytes similar to those of normal control subjects. CFTR mediated Cl- secretion in cultures tracked secretory cell, but not ionocyte, densities. Furthermore, the nucleotide-purinergic regulatory system that controls CFTR-mediated hydration was associated with secretory cells and not with ionocytes. Lentiviral transduction of wild-type CFTR produced CFTR-mediated Cl- secretion in CF airway secretory cells but not in ciliated cells. Conclusions: Secretory cells dominate CFTR expression and function in human airway superficial epithelia. CFTR therapies may need to restore CFTR function to multiple cell types, with a focus on secretory cells.

Coinfection with Pseudomonas aeruginosa and Aspergillus fumigatus in cystic fibrosis.

Cystic fibrosis (CF) lung disease is characterised by mucus stasis, chronic infection and inflammation, causing progressive structural lung disease and eventual respiratory failure. CF airways are inhabited by an ecologically diverse polymicrobial environment with vast potential for interspecies interactions, which may be a contributing factor to disease progression. Pseudomonas aeruginosa and Aspergillus fumigatus are the most common bacterial and fungal species present in CF airways respectively and coinfection results in a worse disease phenotype. In this review we examine existing expert knowledge of chronic co-infection with P.aeruginosa and A. fumigatus in CF patients. We summarise the mechanisms of interaction and evaluate the clinical and inflammatory impacts of this co-infection. P. aeruginosa inhibits A. fumigatus through multiple mechanisms: phenazine secretion, iron competition, quorum sensing and through diffusible small molecules. A.fumigatus reciprocates inhibition through gliotoxin release and phenotypic adaptations enabling evasion of P. aeruginosa inhibition. Volatile organic compounds secreted by P.aeruginosa stimulate A. fumigatus growth, while A. fumigatus stimulates P. aeruginosa production of cytotoxic elastase. A complex bi-directional relationship exists between P. aeruginosa and A.fumigatus, exhibiting both mutually antagonistic and cooperative facets. Cross-sectional data indicate a worsened disease state in coinfected patients; however, robust longitudinal studies are required to derive causality and to determine whether interspecies interaction contributes to disease progression.

IgA autoantibodies directed against self DNA are elevated in cystic fibrosis and associated with more severe lung dysfunction

Although extracellular host DNA (ecDNA) levels in CF airways were linked to airflow obstruction and recombinant DNAse therapy is beneficial for CF patients, it remains incompletely understood whether ecDNA also leads to an autoimmune response. Here we hypothesized that chronic presence of DNA in CF airways triggers the production of autoantibodies targeting host human DNA. We measured the levels of IgA autoantibodies recognising host double-stranded (ds) DNA in the blood and sputum samples of CF patients and only sera of controls subjects and patients suffering from rheumatoid arthritis and systemic lupus erythematosus (SLE) that served as non-CF, autoimmune disease cohorts. We found that concentrations of anti-dsDNA IgA, but not IgG, autoantibodies in the circulation were significantly elevated in adult CF patients compared to age-matched, control subjects. Systemic levels of anti-dsDNA IgA antibodies negatively correlated with FEV1% predicted, a measure of lung function, in CF patients. Anti-dsDNA IgA autoantibodies were also detected in CF sputa but sputum levels did not correlate with the degree of airway obstruction or sputum levels of DNA. We also found elevated autoantibody levels in CF children as 76.5% of CF patients younger than 10 years and 87.5% of CF patients 10–21 years had higher blood anti-dsDNA IgA levels than the highest value found in healthy control adults. Overall, our results detect elevated systemic anti-dsDNA IgA autoantibody levels in CF adults, teenagers and young children. We speculate that the appearance of an autoimmune response against host DNA in CF is an early event potentially contributing to disease pathogenesis. Highlights CF serum contains elevated levels of anti-dsDNA IgA, but not anti-dsDNA IgG, autoantibodies Anti-dsDNA IgA autoantibody levels in serum correlate with airflow obstruction in CF Anti-dsDNA IgA autoantibodies are detected in CF sputum but do not correlate with airflow obstruction Anti-dsDNA IgA autoantibodies are also elevated in the blood of the majority of CF toddlers and youth

Designing Clinical Trials for Anti-Inflammatory Therapies in Cystic Fibrosis.

The inflammatory response in the CF airway begins early in the disease process and becomes persistent through life in most patients. Inflammation, which is predominantly neutrophilic, worsens airway obstruction and plays a critical role in the development of structural lung damage. While cystic fibrosis transmembrane regulator modulators will likely have a dramatic impact on the trajectory of CF lung disease over the coming years, addressing other important aspects of lung disease such as inflammation will nevertheless remain a priority. Considering the central role of neutrophils and their products in the inflammatory response, potential therapies should ultimately affect neutrophils and their products. The ideal anti-inflammatory therapy would exert a dual effect on the pro-inflammatory and pro-resolution arms of the inflammatory cascade, both of which contribute to dysregulated inflammation in CF. This review outlines the key factors to be considered in the design of clinical trials evaluating anti-inflammatory therapies in CF. Important lessons have been learned from previous clinical trials in this area and choosing the right efficacy endpoints is key to the success of any anti-inflammatory drug development program. Identifying and validating non-invasive biomarkers, novel imaging techniques and sensitive lung function tests capable of monitoring disease activity and therapeutic response are important areas of research and will be useful for the design of future anti-inflammatory drug trials.

Immune response of polarized cystic fibrosis airway epithelial cells infected with Influenza A virus

BackgroundCystic fibrosis (CF), a genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is characterized by dysfunction of the immune response in the airway epithelium that leads to prolonged infection, colonization and exacerbated inflammation. In this study, we determined the gene expression profile of airway epithelial cells knockdown for CFTR (CFTR KD) in response to bacterial and viral challenges. MethodsIn a first approach, polarized CFTR KD and their control counterpart (CFTR CTL) cells were stimulated with P. aeruginosa-derived virulence factor flagellin. Next, we developed a model of Influenza A virus (IAV) infection in CTL and CFTR KD polarized cells. mRNA was collected for transcriptome analysis. ResultsBeside the expected pro-inflammatory response, Gene Set Enrichment Analysis highlighted key molecular pathways and players involved in IAV and anti-viral interferon signaling. Although IAV replication was similar in both cell types, multiplex gene expression analysis revealed changes of key immune genes dependent on time of infection that were found to be CFTR-dependent and/or IAV-dependent. Interferons are key signaling proteins/cytokines in the antibacterial and antiviral response. To evaluate their impact on the altered gene expression profile in CFTR responses to pathogens, we measured transcriptome changes after exposure to Type I-, Type II- and Type III-interferons. ConclusionsOur findings reveal target genes in understanding the defective immune response in the CF airway epithelium in the context of viral infection. Information provided in this study would be useful to understand the dysfunctional immune response of the CF airway epithelium during infection.

CFTR Modulator Therapy Enhances Peripheral Blood Monocyte Contributions to Immune Responses in People With Cystic Fibrosis.

BackgroundCFTR modulators decrease some etiologies of CF airway inflammation; however, data indicate that non-resolving airway infection and inflammation persist in individuals with CF and chronic bacterial infections. Thus, identification of therapies that diminish airway inflammation without allowing unrestrained bacterial growth remains a critical research goal. Novel strategies for combatting deleterious airway inflammation in the CFTR modulator era require better understanding of cellular contributions to chronic CF airway disease, and how inflammatory cells change after initiation of CFTR modulator therapy. Peripheral blood monocytes, which traffic to the CF airway, can develop both pro-inflammatory and inflammation-resolving phenotypes, represent intriguing cellular targets for focused therapies. This therapeutic approach, however, requires a more detailed knowledge of CF monocyte cellular programming and phenotypes.Material and MethodsIn order to characterize the inflammatory phenotype of CF monocytes, and how these cells change after initiation of CFTR modulator therapy, we studied adults (n=10) with CF, chronic airway infections, and the CFTR-R117H mutations before and 7 days after initiation of ivacaftor. Transcriptomes of freshly isolated blood monocytes were interrogated by RNA-sequencing (RNA-seq) followed by pathway-based analyses. Plasma concentrations of cytokines and chemokines were evaluated by multiplex ELISA.ResultsRNAseq identified approximately 50 monocyte genes for which basal expression was significantly changed in all 10 subjects after 7 days of ivacaftor. Of these, the majority were increased in expression post ivacaftor, including many genes traditionally associated with enhanced inflammation and immune responses. Pathway analyses confirmed that transcriptional programs were overwhelmingly up-regulated in monocytes after 7 days of ivacaftor, including biological modules associated with immunity, cell cycle, oxidative phosphorylation, and the unfolded protein response. Ivacaftor increased plasma concentrations of CXCL2, a neutrophil chemokine secreted by monocytes and macrophages, and CCL2, a monocyte chemokine.ConclusionsOur results demonstrate that ivacaftor causes acute changes in blood monocyte transcriptional profiles and plasma chemokines, and suggest that increased monocyte inflammatory signals and changes in myeloid cell trafficking may contribute to changes in airway inflammation in people taking CFTR modulators. To our knowledge, this is the first report investigating the transcriptomic response of circulating blood monocytes in CF subjects treated with a CFTR modulator.

UPR modulation of host immunity by Pseudomonas aeruginosa in cystic fibrosis.

Cystic fibrosis (CF) is a progressive multiorgan autosomal recessive disease with devastating impact on the lungs caused by derangements of the CF transmembrane conductance regulator (CFTR) gene. Morbidity and mortality are caused by the triad of impaired mucociliary clearance, microbial infections and chronic inflammation. Pseudomonas aeruginosa is the main respiratory pathogen in individuals with CF infecting most patients in later stages. Despite its recognized clinical impact, molecular mechanisms that underlie P. aeruginosa pathogenesis and the host response to P. aeruginosa infection remain incompletely understood. The nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) γ (PPARγ), has shown to be reduced in CF airways. In the present study, we sought to investigate the upstream mechanisms repressing PPARγ expression and its impact on airway epithelial host defense. Endoplasmic reticulum-stress (ER-stress) triggered unfolded protein response (UPR) activated by misfolded CFTR and P. aeruginosa infection contributed to attenuated expression of PPARγ. Specifically, the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway led to the enhanced expression of the CCAAT-enhancer-binding-protein homologous protein (CHOP). CHOP induction led to the repression of PPARγ expression. Mechanistically, we showed that CHOP induction mediated PPARγ attenuation, impacted the innate immune function of normal and ∆F508 primary airway epithelial cells by reducing expression of antimicrobial peptide (AMP) and paraoxanse-2 (PON-2), as well as enhancing IL-8 expression. Furthermore, mitochondrial reactive oxygen species production (mt-ROS) and ER-stress positive feedforward loop also dysregulated mitochondrial bioenergetics. Additionally, our findings implicate that PPARγ agonist pioglitazone (PIO) has beneficial effect on the host at the multicellular level ranging from host defense to mitochondrial re-energization.

Targeting IgG Autoantibodies for Improved Cytotoxicity of Bactericidal Permeability Increasing Protein in Cystic Fibrosis.

In people with cystic fibrosis (PWCF), inflammation with concurrent infection occurs from a young age and significantly influences lung disease progression. Studies indicate that neutrophils are important effector cells in the pathogenesis of CF and in the development of anti-neutrophil cytoplasmic autoantibodies (ANCA). ANCA specific for bactericidal permeability increasing protein (BPI-ANCA) are detected in people with CF, and correlate with infection with Pseudomonas aeruginosa. The aim of this study was to determine the signaling mechanism leading to increased BPI release by CF neutrophils, while identifying IgG class BPI-ANCA in CF airways samples as the cause for impaired antimicrobial activity of BPI against P. aeruginosa. Plasma and/or bronchoalveolar lavage fluid (BAL) was collected from PWCF (n = 40), CF receiving ivacaftor therapy (n = 10), non-CF patient cohorts (n = 7) and healthy controls (n = 38). Plasma and BAL BPI and BPI-ANCA were measured by ELISA and GTP-bound Rac2 detected using an in vitro assay. The antibacterial effect of all treatments tested was determined by colony forming units enumeration. Levels of BPI are significantly increased in plasma (p = 0.007) and BALF (p < 0.0001) of PWCF. The signaling mechanism leading to increased degranulation and exocytosis of BPI by CF neutrophils (p = 0.02) involved enhancement of Rac2 GTP-loading (p = 0.03). The full-length BPI protein was detectable in all CF BAL samples and patients displayed ANCA with BPI specificity. IgG class autoantibodies were purified from CF BAL complexed to BPI (n=5), with IgG autoantibody cross-linking of antigen preventing BPI induced P. aeruginosa killing (p < 0.0001). Results indicate that the immune-mediated diminished antimicrobial defense, attributed to anti-BPI-IgG, necessitates the formation of a drug/immune complex intermediate that can maintain cytotoxic effects of BPI towards Gram-negative pathogens, with the potential to transform the current treatment of CF airways disease.

Ivacaftor or lumacaftor/ivacaftor treatment does not alter the core CF airway epithelial gene response to rhinovirus

BackgroundAberrant responses by the cystic fibrosis airway epithelium during viral infection may underly the clinical observations. Whether CFTR modulators affect antiviral responses by CF epithelia is presently unknown. We tested the hypothesis that treatment of CF epithelial cells with ivacaftor (Iva) or ivacaftor/lumacaftor (Iva/Lum) would improve control of rhinovirus infection. MethodsNineteen CF epithelial cultures (10 homozygous for p.Phe508del as CFTR Class 2, 9 p.Phe508del/p.Gly551Asp as Class 3) were infected with rhinovirus 1B at multiplicity of infection 12 for 24 h. Culture RNA and supernatants were harvested to assess gene and protein expression respectively. ResultsRNA-seq analysis comparing rhinovirus infected cultures to control identified 796 and 629 differentially expressed genes for Class 2 and Class 3, respectively. This gene response was highly conserved when cells were treated with CFTR modulators and were predicted to be driven by the same interferon-pathway transcriptional regulators (IFNA, IFNL1, IFNG, IRF7, STAT1). Direct comparisons between treated and untreated infected cultures did not yield any differentially expressed genes for Class 3 and only 68 genes for Class 2. Changes were predominantly related to regulators of lipid metabolism and inflammation, aspects of epithelial biology known to be dysregulated in CF. In addition, CFTR modulators did not affect viral copy number, or levels of pro-inflammatory cytokines produced post-infection. ConclusionsThough long-term clinical data is not yet available, results presented here suggest that first generation CFTR modulators do not interfere with core airway epithelial responses to rhinovirus infection. Future work should investigate the latest triple modulation therapies.

The CF airway environment affects neutrophil antibacterial effector mechanisms: phagocytosis and NET formation

Abstract Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the epithelial chloride channel CFTR. Lung disease is the main cause of mortality and morbidity in CF. CF airways are characterized by chronic, mainly neutrophil-mediated inflammation and persistent bacterial infections. Despite their increased presence in the CF lung, airway neutrophils are unable to clear respiratory pathogens like Pseudomonas aeruginosa and Staphylococcus aureus. We hypothesized that the CF airway environment affects two antibacterial effector mechanisms of neutrophils: phagocytosis and neutrophil extracellular trap (NETs) formation. While neutrophils are known to kill P. aeruginosa and S. aureus mainly by phagocytosis-mediated intracellular killing, NET-mediated extracellular killing remains relatively inefficient against these pathogens. To examine this in CF, blood PMNs from healthy donors were incubated in sputum supernatants from CF patients to mimic the CF airway environment. Using this in vitro model, different markers and functions of neutrophils were measured by flow cytometry. Phagocytosis of fluorescently-labelled P. aeruginosa was significantly inhibited by CF sputum treatment. Surface expression of CD33, a myeloid-derived suppressor cell marker known to inhibit phagocytosis, was increased following CF sputum treatment. CF sputum exposure slightly increased both of the NET-specific markers studied: intracellular levels of citrullinated histone H3 and surface expression of gasdermin-D. These results suggest that the CF airway environment contributes to the inability of neutrophils to eliminate bacteria while promoting the release of dangerous, tissue-damaging neutrophil cargo via NET formation.

Neutrophil Effector Responses to Cystic Fibrosis Clinical Isolates of Staphylococcus aureus and Pseudomonas aeruginosa

Abstract Cystic fibrosis (CF) airway disease is characterized by chronic microbial infections and infiltration of polymorphonuclear neutrophil granulocytes (PMNs). In CF, PMNs are unable to clear lung pathogens resulting in lung damage. Neutrophil extracellular traps (NETs) and superoxide production represent defense mechanisms of PMNs to kill extracellular pathogens. NETs and superoxide in the CF lung, however, seem unable to clear Pseudomonas aeruginosa and Staphylococcus aureus, the two main lung pathogens in CF. Our goal of this study was to determine antimicrobial functions of PMNs to S. aureus and P. aeruginosa CF isolates and to observe how the CF airway environment influences them. Human PMNs were isolated from healthy donors. Isolates of S. aureus were obtained from adult CF patients: four strains were methicillin-resistant (MRSA), four were methicillin-sensitive S. aureus (MSSA), and ten CF isolates of P. aeruginosa. PMN effector functions were measured: killing was assessed by bacterial growth assay, NET formation was measured by Sytox fluorescence and superoxide was monitored by Diogenes chemiluminescence. Results were subjected to paired t-test, significant if p&amp;lt;0.05. PMNs from healthy volunteers were able to kill all CF isolates. The extent of killing varied among strains, however, P. aeruginosa was killed more efficiently by blood PMNs than S. aureus clinical isolates. The enhanced killing of P. aeruginosa is correlated with enhanced NET release and superoxide production compared to S. aureus. Killing of all isolates was inhibited by CF sputum pretreatment. Overall, the results show that CF bacterial clinical isolates stimulate PMN effector responses that are impaired by the CF airway environment.

Total bacterial load, inflammation, and structural lung disease in paediatric cystic fibrosis

BackgroundCystic fibrosis (CF) is characterised by reduced airway clearance, microbial accumulation, inflammation, and lung function decline. Certain bacterial species may contribute disproportionately to worsening lung disease. However, the relative importance of these microorganisms compared to the absolute abundance of all bacteria is uncertain. We aimed to identify the characteristics of lower airway microbiology that best reflect CF airway inflammation and disease in children. MethodsAnalysis was performed on bronchoalveolar lavage (BAL) fluid from 78 participants of the Australasian CF Bronchoalveolar Lavage (ACFBAL) clinical trial, aged 4.5–5.5 years. Universal bacterial quantitative PCR (qPCR), species-specific qPCR, and 16S rRNA gene sequencing were performed on DNA extracts to determine total bacterial load, species-specific load and taxa relative abundance. Quantification of pre-specified pathogens was performed by culture-based methods. Bacteriological data were related to neutrophil counts, interleukin-8, lung function, and two computed-tomography based measures, CF-CT (as the primary measure) and PRAGMA. ResultsOf all bacteriological measures assessed, total bacterial load determined by qPCR correlated most strongly with structural disease (CF-CT total score, rs=0.30, P=0.0095). Specifically, total bacterial load correlated with bronchiectasis, airway wall thickening, mucus plugging and parenchymal disease sub-scores. In contrast, culture-based quantification, microbiota-derived measures, and pathogen-specific qPCR-based quantification were weakly associated with total CF-CT. Regression analyses supported correlation findings, with total bacterial load explaining the greatest variance in total CF-CT (R2=0.097, P=0.0061). Correlations with PRAGMA score were comparable to CF-CT total score. ConclusionsWithin the ACFBAL trial, culture-independent quantification of total bacteria provided the most clinically-informative bacteriological measure in 5-year-old CF patients.

Sexual dimorphism in the microbiology of the CF ‘Gender Gap’: Estrogen modulation of Pseudomonas aeruginosa virulence

There is increasing evidence for sexual dimorphism of estrogen (E2) actions in the exacerbation of lung function, infection and inflammation in females with cystic fibrosis - the so-called “CF gender gap”. The effects of estrogen on virulence factors that enhance P. aeruginosa persistence in CF lung epithelium were investigated by phenotypic and chemical assays in various PsA clinical isolates and laboratory strains in isolation or in co-culture with normal (Nuli-1) and CF dPhe508-CFTR (CuFi-1) human bronchial epithelial cell lines. Estrogen (E2, 10 nM) significantly increased secretion of the virulence factor pyocyanin by 80% in PsA early infection isolates from female CF patients and by 280% in late infection PsA isolates. Estrogen also increased the swarming motility by up to 50% in all PsA isolates and strains tested in 0.5% agar. A significant increase of 110% in the twitching motility of all PsA isolates and strains tested was also observed with estrogen treatment. Treatment with E2 increased biofilm formation of P. aeruginosa PsAO1 which became more adherent to, and invasive into, normal and CF bronchial epithelial cells. The selective estrogen receptor modulators (SERMs), Tamoxifen and ICI 182780 inhibited P. aeruginosa motility. The potency of various steroid hormones to stimulate motility of P. aeruginosa was in the order; estradiol ≫ estrone > E3 estriol ≥ testosterone ≥ progesterone ≫ aldosterone, cortisol. Estrogen was also shown to reduce ciliary beat intensity in CF bronchial epithelium which would further exacerbate PsA trapping and virulence in the CF airways. In conclusion, we have demonstrated for the first time that estrogen exacerbates P. aeruginosa virulence factors and enhances bacterial interactions with CF bronchial epithelium which can be inhibited by tamoxifen. Our work suggests that SERMs could be used as an adjuvant treatment to reduce estrogen-induced P. aeruginosa infections and associated lung exacerbations in females with CF.

Airway Pressure Gradient May Decrease the Beating Amplitude of Cilia

Motile cilia reside on the surface of the epithelial layer of the lungs and facilitates the clearance of mucus in the airways. Bordering the epithelial layer and surrounding cilia is the periciliary liquid (PCL) that lubricates the epithelial layer. In the present work, we propose a novel approach to study how changes in biomechanics affect the physiological functioning of cilia in healthy subjects and in patients with CF, COPD and primary ciliary dyskinesia (PCD). In particular, we investigate the response of cilia to different local pressure gradient during gaseous exchange. We hypothesize that the airway pressure gradient that occur during inhalation and exhalation may displace mucus and PCL and exert pressure on cilia. Therefore, cilia must be able to withstand the forces created by the airway pressure gradient, otherwise the magnitude of its efficient strokes and its rate of mucociliary clearance would decrease. We develop a computational model of the airways to quantify the effect of airway pressure gradient on cilia dynamics. In the model, cilia are represented as elastic solids, PCL and mucus is represented as fluids with different densities and viscosities. The simulation results show that in diseases such as PCD, where there exist changes in ciliary structure, the airway pressure gradient may affect the effective stroke of cilia and decrease the rate of mucociliary clearance. Simulation results predict that the average stress experienced by cilia varies exponential with the number of cilia shed from CF and COPD airways.

Restoration of F508-del Function by Transcomplementation: The Partners Meet in the Endoplasmic Reticulum.

Because of the small size of adeno-associated virus, AAV, the cystic fibrosis conductance regulator, CFTR, cDNA is too large to fit within AAV and must be truncated. We report here on two truncated versions of CFTR, which, when inserted into AAV1 and used to infect airway cells, rescue F508-del CFTR via transcomplementation. The purpose of this study is to shed light on where in the cell transcomplementation occurs and how it results in close association between the endogenous F508-del and truncated CFTR. We treated CF airway cells (CFBE41o-) with AAV2/1 (AAV2 inverted terminal repeats/AAV1 capsid) containing truncated forms of CFTR, ∆264 and ∆27-264 CFTR, who can restore the function of F508-del by transcomplementation. We addressed the aims of the study using a combination of confocal microscopy and short circuit currents measurements. For the latter, CF bronchial epithelial cells (CFBE) were grown on permeable supports. We show that both F508del and the truncation mutants colocalize in the ER and that both the rescued F508-del and the transcomplementing mutants reach the plasma membrane together. There was significant fluorescence resonance energy transfer (FRET) between F508-del and the transcomplementing mutants within the endoplasmic reticulum (ER), suggesting that transcomplementation occurs through a bimolecular interaction. We found that transcomplementation could increase the Isc in CFBE41o- cells stably expressing additional wt-CFTR or F508-del and in parental CFBE41o- cells expressing endogenous levels of F508-del. We conclude that the functional rescue of F508-del by transcomplementation occurs via a bimolecular interaction that most likely begins in the ER and continues at the plasma membrane. These results come at an opportune time for developing a gene therapy for CF and offer new treatment options for a wide range of CF patients.