Longitudinal changes in the cystic fibrosis airway microbiota with time and treatment.

BACKGROUND: Whether there is any benefit in integrating culture-independent molecular analysis of the lower airway microbiota of people with cystic fibrosis into clinical care is unclear. This study determined the longitudinal trajectory of the microbiota and if there were microbiota characteristics that corresponded with response to treatment or predicted a future pulmonary exacerbation. METHODS: At least one sputum sample was collected from 149 participants enrolled in this prospective longitudinal multi-centre study and total bacterial density and microbiota community measurements were determined and compared with clinical parameters. RESULTS: In 114 participants with paired samples when clinically stable, ∼8 months apart, the microbiota remained conserved between timepoints, regardless of whether participants received acute intravenous antibiotic treatment or not. In 62 participants, who presented with an acute exacerbation, a decrease in community richness correlated best with patient response to antibiotic treatment. Analysis of baseline samples from 30 participants who exacerbated within 4 months of their stable sample being collected and 72 participants who remained stable throughout the study showed that community characteristics such as lower richness at baseline may be predictive of an exacerbation in addition to several clinical parameters. However, lasso regression analysis indicated that only lung function (p = 0.014) was associated with a future exacerbation. CONCLUSIONS: The airway microbiota remains stable over periods <1 year with modest shifts related to treatment apparent which might provide some additional insights to patient-level measurements.

Anaerobic bacteria cultured from cystic fibrosis airways correlate to milder disease: a multisite study.

Anaerobic and aerobic bacteria were quantitated in respiratory samples across three cystic fibrosis (CF) centres using extended culture methods. Subjects aged 1-69 years who were clinically stable provided sputum (n=200) or bronchoalveolar lavage (n=55). 18 anaerobic and 39 aerobic genera were cultured from 59% and 95% of samples, respectively; 16 out of 57 genera had a ≥5% prevalence across centres.Analyses of microbial communities using co-occurrence networks in sputum samples showed groupings of oral, including anaerobic, bacteria, whereas typical CF pathogens formed distinct entities. Pseudomonas was associated with worse nutrition and F508del genotype, whereas anaerobe prevalence was positively associated with pancreatic sufficiency, better nutrition and better lung function. A higher total anaerobe/total aerobe CFU ratio was associated with pancreatic sufficiency and better nutrition. Subjects grouped by factor analysis who had relative dominance of anaerobes over aerobes had milder disease compared with a Pseudomonas-dominated group with similar proportions of subjects that were homozygous for F508del.In summary, anaerobic bacteria occurred at an early age. In sputum-producing subjects anaerobic bacteria were associated with milder disease, suggesting that targeted eradication of anaerobes may not be warranted in sputum-producing CF subjects.

Neutrophil Membrane Cholesterol Content is a Key Factor in Cystic Fibrosis Lung Disease.

BACKGROUND: Identification of mechanisms promoting neutrophil trafficking to the lungs of patients with cystic fibrosis (CF) is a challenge for next generation therapeutics. Cholesterol, a structural component of neutrophil plasma membranes influences cell adhesion, a key step in transmigration. The effect of chronic inflammation on neutrophil membrane cholesterol content in patients with CF (PWCF) remains unclear. To address this we examined neutrophils of PWCF to evaluate the cause and consequence of altered membrane cholesterol and identified the effects of lung transplantation and ion channel potentiator therapy on the cellular mechanisms responsible for perturbed membrane cholesterol and increased cell adhesion. METHODOLOGY: PWCF homozygous for the ΔF508 mutation or heterozygous for the G551D mutation were recruited (n=48). Membrane protein expression was investigated by mass spectrometry. The effect of lung transplantation or ivacaftor therapy was assessed by ELISAs, and calcium fluorometric and µ-calpain assays. FINDINGS: Membranes of CF neutrophils contain less cholesterol, yet increased integrin CD11b expression, and respond to inflammatory induced endoplasmic reticulum (ER) stress by activating µ-calpain. In vivo and in vitro, increased µ-calpain activity resulted in proteolysis of the membrane cholesterol trafficking protein caveolin-1. The critical role of caveolin-1 for adequate membrane cholesterol content was confirmed in caveolin-1 knock-out mice. Lung transplant therapy or treatment of PWCF with ivacaftor, reduced levels of circulating inflammatory mediators and actuated increased caveolin-1 and membrane cholesterol, with concurrent normalized neutrophil adhesion. INTERPRETATION: Results demonstrate an auxiliary benefit of lung transplant and potentiator therapy, evident by a reduction in circulating inflammation and controlled neutrophil adhesion.

Animal Models of Cystic Fibrosis Pathology: Phenotypic Parallels and Divergences.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The resultant characteristic ion transport defect results in decreased mucociliary clearance, bacterial colonisation, and chronic neutrophil-dominated inflammation. Much knowledge surrounding the pathophysiology of the disease has been gained through the generation of animal models, despite inherent limitations in each. The failure of certain mouse models to recapitulate the phenotypic manifestations of human disease has initiated the generation of larger animals in which to study CF, including the pig and the ferret. This review will summarise the basic phenotypes of three animal models and describe the contributions of such animal studies to our current understanding of CF.

Chitinase activation in patients with fungus-associated cystic fibrosis lung disease.

BACKGROUND: Chitinases have recently gained attention in the field of pulmonary diseases, particularly in asthma and chronic obstructive pulmonary disease, but their potential role in patients with cystic fibrosis (CF)-associated lung disease remains unclear. OBJECTIVE: The aim of this study was to assess chitinase activity systemically and in the airways of patients with CF and asthma compared with healthy subjects. Additionally, we assessed factors that regulate chitinase activity within the lungs of patients with CF. METHODS: Chitinase activities were quantified in serum and bronchoalveolar lavage fluid from patients with CF, asthmatic patients, and healthy control subjects. Mechanistically, the role of CF airway proteases and genetic chitinase deficiency was assessed. RESULTS: Chitinase activity was systemically increased in patients with CF compared with that in healthy control subjects and asthmatic patients. Further stratification showed that chitinase activity was enhanced in patients with CF colonized with Candida albicans compared with that in noncolonized patients. CF proteases degraded chitinases in the airway microenvironment of patients with CF. Genetic chitinase deficiency was associated with C albicans colonization in patients with CF. CONCLUSION: Patients with CF have enhanced chitinase activation associated with C albicans colonization. Therefore chitinases might represent a novel biomarker and therapeutic target for CF-associated fungal disease.

Blood basophil activation is a reliable biomarker of allergic bronchopulmonary aspergillosis in cystic fibrosis.

The diagnosis of cystic fibrosis (CF) patients with allergic bronchopulmonary aspergillosis (ABPA) is clinically challenging, due to the absence of an objective biological test. Since blood basophils play a major role in allergic responses, we hypothesised that changes in their surface activation pattern discriminate between CF patients with and without ABPA.We conducted a prospective longitudinal study (Stanford cohort) comparing basophil activation test CD203c levels by flow cytometry before and after activation with Aspergillus fumigatus allergen extract or recombinant Asp f1 in 20 CF patients with ABPA (CF-ABPA) and in two comparison groups: CF patients with A. fumigatus colonisation (AC) but without ABPA (CF-AC; n=13) and CF patients without either AC or ABPA (CF; n=12). Patients were tested every 6 months and when ill with pulmonary exacerbation. We also conducted cross-sectional validation in a separate patient set (Dublin cohort).Basophil CD203c surface expression reliably discriminated CF-ABPA from CF-AC and CF over time. Ex vivo stimulation with A. fumigatus extract or recombinant Asp f1 produced similar results within the Stanford (p<0.0001) and the Dublin cohorts. CF-ABPA patients were likelier to have elevated specific IgE to A. fumigatus and were less frequently co-infected with Staphylococcus aureus.Basophil CD203c upregulation is a suitable diagnostic and stable monitoring biomarker of ABPA in CF.

The basophil surface marker CD203c identifies Aspergillus species sensitization in patients with cystic fibrosis.

BACKGROUND: Colonization by Aspergillus fumigatus in patients with cystic fibrosis (CF) can cause A fumigatus sensitization and/or allergic bronchopulmonary aspergillosis (ABPA), which affects pulmonary function and clinical outcomes. Recent studies show that specific allergens upregulate the surface-expressed basophil marker CD203c in sensitized subjects, a response that can be readily measured by using flow cytometry. OBJECTIVE: We sought to identify A fumigatus sensitization in patients with CF by using the basophil activation test (BAT). METHODS: Patients with CF attending Beaumont Hospital were screened for study inclusion. BAT was used to identify A fumigatus sensitization. Serologic (total and A fumigatus-specific IgE), pulmonary function, and body mass index measurements were performed. RESULTS: The BAT discriminates A fumigatus-sensitized from nonsensitized patients with CF. Persistent isolation of A fumigatus in sputum is a significant risk factor for A fumigatus sensitization. Levels of the A fumigatus-stimulated basophil activation marker CD203c inversely correlated with pulmonary function and body mass index in A fumigatus-sensitized but not nonsensitized patients with CF. Total and A fumigatus-specific IgE, but not IgG, levels are increased in A fumigatus-sensitized patients with CF and ABPA when compared with those in A fumigatus-sensitized and nonsensitized patients with CF without ABPA. Itraconazole treatment did not affect A fumigatus sensitization. CONCLUSION: Combining the BAT with routine serologic testing allows classification of patients with CF into 3 groups: nonsensitized, A fumigatus-sensitized, and ABPA. Accurate and prompt identification of A fumigatus-associated clinical status might allow early and targeted therapeutic intervention, potentially improving clinical outcomes.

The Role of Short-Chain Fatty Acids, Produced by Anaerobic Bacteria, in the Cystic Fibrosis Airway.

RATIONALE: Anaerobic bacteria are present in large numbers in the airways of people with cystic fibrosis (PWCF). In the gut, anaerobes produce short-chain fatty acids (SCFAs) that modulate immune and inflammatory processes. OBJECTIVES: To investigate the capacity of anaerobes to contribute to cystic fibrosis (CF) airway pathogenesis via SCFAs. METHODS: Samples of 109 PWCF were processed using anaerobic microbiological culture with bacteria present identified by 16S RNA sequencing. SCFA levels in anaerobic supernatants and bronchoalveolar lavage (BAL) were determined by gas chromatography. The mRNA and/or protein expression of two SCFA receptors, GPR41 and GPR43, in CF and non-CF bronchial brushings and 16HBE14o(-) and CFBE41o(-) cells were evaluated using reverse transcription polymerase chain reaction, Western blot analysis, laser scanning cytometry, and confocal microscopy. SCFA-induced IL-8 secretion was monitored by ELISA. MEASUREMENTS AND MAIN RESULTS: Fifty-seven (52.3%) of 109 PWCF were anaerobe positive. Prevalence increased with age, from 33.3% to 57.7% in PWCF younger (n = 24) and older (n = 85) than 6 years of age. All evaluated anaerobes produced millimolar concentrations of SCFAs, including acetic, propionic, and butyric acids. SCFA levels were higher in BAL samples of adults than in those of children. GPR41 levels were elevated in CFBE41o(-) versus 16HBE14o(-) cells; CF versus non-CF bronchial brushings; and 16HBE14o(-) cells after treatment with cystic fibrosis transmembrane conductance regulator inhibitor CFTR(inh)-172, CF BAL, or inducers of endoplasmic reticulum stress. SCFAs induced a dose-dependent and pertussis toxin-sensitive IL-8 response in bronchial epithelial cells, with a higher production of IL-8 in CFBE41o(-) than in 16HBE14o(-) cells. CONCLUSIONS: This study illustrates that SCFAs contribute to excessive production of IL-8 in CF airways colonized with anaerobes via up-regulated GPR41.

Immunoevasive Aspergillus virulence factors.

Individuals with structural lung disease or defective immunity are predisposed to Aspergillus-associated disease. Manifestations range from allergic to cavitary or angio-invasive syndromes. Despite daily spore inhalation, immunocompetence facilitates clearance through initiation of innate and adaptive host responses. These include mechanical barriers, phagocyte activation, antimicrobial peptide release and pattern recognition receptor activation. Adaptive responses include Th1 and Th2 approaches. Understanding Aspergillus virulence mechanisms remains critical to the development of effective research and treatment strategies to counteract the fungi. Major virulence factors relate to fungal structure, protease release and allergens; however, mechanisms utilized to evade immune recognition continue to be important in establishing infection. These include the fungal rodlet layer, dihydroxynaphthalene-melanin, detoxifying systems for reactive oxygen species and toxin release. One major immunoevasive toxin, gliotoxin, plays a key role in mediating Aspergillus-associated colonization in the context of cystic fibrosis. Here, it down-regulates vitamin D receptor expression which following itraconazole therapy is rescued concurrent with decreased Th2 cytokine (IL-5 and IL-13) concentrations in the CF airway. This review focuses on the interaction between Aspergillus pathogenic mechanisms, host immune responses and the immunoevasive strategies employed by the organism during disease states such as that observed in cystic fibrosis.

Aspergillus-associated airway disease, inflammation, and the innate immune response.

Aspergillus moulds exist ubiquitously as spores that are inhaled in large numbers daily. Whilst most are removed by anatomical barriers, disease may occur in certain circumstances. Depending on the underlying state of the human immune system, clinical consequences can ensue ranging from an excessive immune response during allergic bronchopulmonary aspergillosis to the formation of an aspergilloma in the immunocompetent state. The severest infections occur in those who are immunocompromised where invasive pulmonary aspergillosis results in high mortality rates. The diagnosis of Aspergillus-associated pulmonary disease is based on clinical, radiological, and immunological testing. An understanding of the innate and inflammatory consequences of exposure to Aspergillus species is critical in accounting for disease manifestations and preventing sequelae. The major components of the innate immune system involved in recognition and removal of the fungus include phagocytosis, antimicrobial peptide production, and recognition by pattern recognition receptors. The cytokine response is also critical facilitating cell-to-cell communication and promoting the initiation, maintenance, and resolution of the host response. In the following review, we discuss the above areas with a focus on the innate and inflammatory response to airway Aspergillus exposure and how these responses may be modulated for therapeutic benefit.