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Inflammation is a key driver of cystic fibrosis (CF) lung disease, not addressed by current standard care. Improved understanding of the mechanisms leading to aberrant inflammation may assist the development of effective anti-inflammatory therapy. Single-cell RNA sequencing (scRNA-seq) allows profiling of cell composition and function at previously unprecedented resolution. Herein, we seek to use multimodal single-cell analysis to comprehensively define immune cell phenotypes, proportions and functional characteristics in preschool children with CF. We analyzed 42,658 cells from bronchoalveolar lavage of 11 preschool children with CF and a healthy control using scRNA-seq and parallel assessment of 154 cell surface proteins. Validation of cell types identified by scRNA-seq was achieved by assessment of samples by spectral flow cytometry. Analysis of transcriptome expression and cell surface protein expression, combined with functional pathway analysis, revealed 41 immune and epithelial cell populations in BAL. Spectral flow cytometry analysis of over 256,000 cells from a subset of the same patients revealed high correlation in major cell type proportions across the two technologies. Macrophages consisted of 13 functionally distinct sub populations, including previously undescribed populations enriched for markers of vesicle production and regulatory/repair functions. Other novel cell populations included CD4 T cells expressing inflammatory IFNα/β and NFκB signalling genes. Our work provides a comprehensive cellular analysis of the pediatric lower airway in preschool children with CF, reveals novel cell types and provides a reference for investigation of inflammation in early life CF.

Abstract Aminoglycoside antibiotics are lifesaving medicines, crucial for the treatment of chronic or drug resistant infections. However, aminoglycosides are toxic to the sensory hair cells in the inner ear. As a result, aminoglycoside-treated individuals can develop permanent hearing loss and vestibular impairment. There is considerable evidence that reactive oxygen species (ROS) production and the subsequent phosphorylation of c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38) drives apoptosis in aminoglycoside-treated hair cells. However, treatment strategies that directly inhibit ROS, JNK, or P38 are limited by the importance of these molecules for normal cellular function. Alternatively, the upstream regulator apoptosis signal-regulating kinase 1 (ASK1/MAP3K5) is a key mediator of ROS-induced JNK and P38 activation under pathologic but not homeostatic conditions. We investigated ASK1 as a mediator of drug-induced hair cell death using cochlear explants from Ask1 knockout mice, demonstrating that Ask1 deficiency attenuates neomycin-induced hair cell death. We then evaluated pharmacological inhibition of ASK1 with GS-444217 as a potential otoprotective therapy. GS-444217 significantly attenuated hair cell death in neomycin-treated explants but did not impact aminoglycoside efficacy against P. aeruginosa in the broth dilution test. Overall, we provide significant pre-clinical evidence that ASK1 inhibition represents a novel strategy for preventing aminoglycoside ototoxicity. Key messages ASK1 is an upstream, redox-sensitive regulator of P38 and JNK, which are known mediators of hair cell death. Ask1 knockout does not affect hair cell development in vivo, but significantly reduces aminoglycoside-induced hair cell death in vitro. A small-molecule inhibitor of ASK1 attenuates neomycin-induced hair cell death, and does not impact antibiotic efficacy in vitro. ASK1 may be a novel molecular target for preventing aminoglycoside-induced hearing loss.

This protocol describes the collection, processing, cryopreservation and thawing of pediatric bronchoalveolar lavage (BAL) samples for downstream single cell analysis (including flow cytometry, cell sorting, and single cell transcriptomics).

RATIONALE: Despite encouraging reports suggesting that inhaled nitric oxide (iNO) appear to improve outcome in hypoxemic term and near term infants by improving oxygenation and reducing need for ECMO, the long-term benefits of iNO remain unclear. This study aimed to compare lung function at approximately 1 year in infants who were and were not randomly allocated to iNO as part of their neonatal management for severe respiratory failure at birth. Furthermore, results were compared to lung function of healthy infants. METHODS: Maximal expiratory flow at functional residual capacity (V'maxFRC) was measured at approximately 1 year of age (corrected for any prematurity) in survivors of the INNOVO trial. Results were expressed as Z-scores, adjusted for sex and body size, based on data from healthy controls using identical techniques. RESULTS: Technically satisfactory results were obtained in 30 infants (53% < 34 weeks gestation), 19 of whom were randomized to receive iNO V'maxFRC. Z-score was significantly reduced in infants with prior respiratory failure, whether or not they had been allocated to iNO (mean (SD) Z-score: -2.0 (1.2) and -2.6 (1.1), respectively, 95% CI difference; iNO vs. no iNO: -0.3; 1.6, P = 0.2). There was significant respiratory morbidity in both groups during the first year of life. CONCLUSIONS: These results suggest that airway function remains reduced at 1 year of age following severe respiratory failure at birth, whether or not iNO is administered.

Multiple breath washout (MBW) testing with SF6 gas mixture is routinely used to assess ventilation distribution in infants. It is currently unknown whether SF6 changes tidal breathing parameters during MBW in infants. We investigated if SF6 does change tidal breathing parameters in infants and whether a separate tidal breathing trace prior to MBW testing is necessary.Tidal breathing during MBW was compared to standard tidal breathing in room air in healthy infants (n = 38), preterm infants (n = 41), and infants with cystic fibrosis (n = 41). Outcomes included inspiratory and expiratory times (TI and TE ), time to peak tidal inspiratory and expiratory flow (tPTIF and tPTEF), tidal volume (VT ), respiratory rate (f), and minute ventilation (VE ).Breath times were all significantly increased for both healthy (TE : -0.0790 [-0.10566, -0.05217]; mean difference [95% confidence intervals]) and CF (-0.109 [-0.15235, -0.06607]) infants during the MBW wash-in (P 0.001). Healthy infants and those with CF showed decreased f during MBW wash-in (P 0.001); however, no change in VT, resulting in a decreased VE (0.154 (0.086, 0.222) and 0.128 (0.069, 0.186) for healthy and CF infants, respectively, P 0.001). Preterm infants experienced a decreased VE during both wash-in (0.134 [0.061, 0.207]; P 0.001) and wash-out phases of MBW (P 0.05).There are differences in tidal breathing parameters during MBW testing with SF6 in infants. It is, therefore, important to measure a separate tidal breathing trace in room air, prior to MBW testing to ensure rigour of tidal breath indices derived from analysis.

Risk of infection with Pseudomonas aeruginosa in cystic fibrosis (CF) may be associated with environmental factors.To determine whether residential location is associated with risk of first acquisition of P. aeruginosa.We performed bronchoalveolar lavage and upper airway cultures in children newly diagnosed with CF to identify infection with P. aeruginosa during infancy and early childhood. Children were assessed according to their residence in a regional or metropolitan area. Multilocus sequence typing was used to determine P. aeruginosa genotype. An environmental questionnaire was also administered.A total of 105 of 120 (87.5%) infants diagnosed with CF were included in this study. Diagnosis in 65 infants (61.9%) followed newborn screening at mean age of 4.6 weeks. Sixty subjects (57.1%) were homozygous ΔF508, and 47 (44.8%) were female. Fifty-five (52.3%) infants were regional, of whom 26 (47.3%), compared with 9 of 50 (18.0%) metropolitan children, acquired infection with P. aeruginosa (odds ratio, 4.084; 95% confidence interval, 1.55-11.30). Age at acquisition was similar (regional: median, 2.31 yr; range, 0.27-5.96 yr; metropolitan: median, 3.10 yr, range, 0.89-3.70 yr). Strain typing identified P. aeruginosa genotypes often encountered in different ecological settings and little evidence of cross-infection. Ninety questionnaires (85.7%) were completed. Those who acquired P. aeruginosa were more likely to be living in a household that used water sprinkler systems (P = 0.032), but no differences were identified to explain increased risk of acquisition of P. aeruginosa in regional children.Geographical difference in residence of children with CF was associated with increased risk of first acquisition of P. aeruginosa, usually with strains associated with the environment rather than with cross-infection.

Staphylococcus aureus: is a common bacterial organism infecting children with cystic fibrosis (CF). Emerging evidence suggests early lower airway infection with this organism in young children with CF results in the deterioration of lung function, poorer nutrition parameters and heightens the airway inflammatory response. Despite contributing significantly to the burden of early lung disease among this group, there are ongoing controversies in the management of S. aureus infection, and gaps in our understanding of exactly how this organism causes lung disease. To reduce the morbidity and mortality of early infection ongoing research is needed to: (i) understand the early host immune response that enables this pathogen to reside within the CF lung; (ii) determine if there are organism specific factors that are associated with CF lung disease; and (iii) clarify the utility of anti-staphylococcal antibiotic prophylaxis and/or eradication in the treatment of this patient population.

Though baseline lung function as measured by spirometry in children with cystic fibrosis (CF) has improved, the annual rate of decline has not changed significantly during the critical period of adolescence. The aim of this study was to describe factors associated with longitudinal decline in lung function in a contemporary cohort of children with CF.Best annual lung function data from children attending the CF service of the Royal Children's Hospital Melbourne were reviewed to determine rate of decline in FEV(1) up until time of transfer to an adult center. Mixed models were used to determine the influence of age, sex, genotype, newborn screening, respiratory hospitalization, CF related diabetes mellitus (CFRD), pancreatic insufficiency, Pseudomonas aeruginosa (PsA) infection, and body mass index (BMI) on lung function decline.Longitudinal lung function data (range 5-20 years) were obtained for 98 patients with CF (55 male). Overall, the annual rate of decline in FEV(1) % predicted for the entire cohort was 1.4% per annum though the greatest rate of FEV1 decline was seen during adolescence (2.6%). Increasing age, homozygous ΔF508 genotype, CFRD, mucoid PsA infection, pancreatic insufficiency and respiratory hospitalizations were all significant predictors of FEV1 decline.FEV(1) declines at its sharpest rate during adolescence even in the presence of newborn screening. Genotype, increasing age, CFRD, PsA infection, pancreatic insufficiency and a greater number of respiratory hospitalizations are all associated with an increased rate of lung function decline in Australian children and adolescents with cystic fibrosis.

We aimed to determine whether myeloperoxidase (MPO) is the main peroxidase present in the airways of children with cystic fibrosis (CF) and to assess which oxidants it produces and whether they are associated with clinical features of CF. Children with CF (n=54) and without CF (n=16) underwent bronchoscopy and bronchoalveolar lavage (BAL) for assessment of pulmonary infection and inflammation. BAL fluid was analyzed for MPO, halogenated tyrosines as markers of hypohalous acids, thiocyanate, and protein carbonyls. MPO was the only peroxidase detected in BAL samples from children with CF and its concentration was markedly higher than in controls. Levels of 3-chlorotyrosine and 3-bromotyrosine in proteins were higher in the CF group. They correlated with neutrophils and MPO. The concentration of thiocyanate in BAL samples was below 1μM. Protein carbonyl levels correlated with MPO and halogenated tyrosines in patients with CF. Levels of MPO and halogenated tyrosines were higher in children with infections, especially Pseudomonas aeruginosa, and in the presence of respiratory symptoms. They also correlated with the Kanga clinical score. Our findings suggest that MPO produces hypobromous acid as well as hypochlorous acid in the airways of children with CF and that these oxidants are involved in the early pathogenesis of CF.

The promise of newborn screening (NBS) for cystic fibrosis (CF) has not been fully realized, and the extent of improvement in respiratory outcomes is unclear. We hypothesized that significant lung disease was present at diagnosis.To determine the extent of lung disease in a geographically defined population of infants with CF diagnosed after detection by NBS.Fifty-seven infants (median age, 3.6 mo) with CF underwent bronchoalveolar lavage and chest computed tomography (CT) using a three-slice inspiratory and expiratory protocol.Despite the absence of respiratory symptoms in 48 (84.2%) of infants, a substantial proportion had lung disease with bacterial infection detected in 12 (21.1%), including Staphylococcus aureus (n = 4) and Pseudomonas aeruginosa (n = 3); neutrophilic inflammation (41. 4 x 10(3) cells/ml representing 18.7% of total cell count); proinflammatory cytokines, with 44 (77.2%) having detectable IL-8; and 17 (29.8%) having detectable free neutrophil elastase activity. Inflammation was increased in those with infection and respiratory symptoms; however, the majority of those infected were asymptomatic. Radiologic evidence of structural lung disease was common, with 46 (80.7%) having an abnormal CT; 11 (18.6%) had bronchial dilatation, 27 (45.0%) had bronchial wall thickening, and 40 (66.7%) had gas trapping. On multivariate analysis, free neutrophil elastase activity was associated with structural lung disease. Most children with structural lung disease had no clinically apparent lung disease.These data support the need for full evaluation in infancy and argue for new treatment strategies, especially those targeting neutrophilic inflammation, if the promise of NBS for CF is to be realized.

Progressive lung damage in cystic fibrosis (CF) starts in infancy, and early detection may aid preventative strategies.To measure lung function in infants with CF diagnosed by newborn screening and describe its association with pulmonary infection and inflammation.Infants with CF (n = 68, 6 weeks to 30 months of age) and healthy infants without CF (n = 49) were studied. Forced vital capacity, FEV(0.5), and forced expiratory flows at 75% of exhaled vital capacity (FEF(75)) were measured using the raised-volume rapid thoracoabdominal compression technique. Forty-eight hours later, infants with CF had bronchoalveolar lavage (BAL) for assessment of pulmonary infection and inflammation.In the CF group, the deficit in FEV(0.5) z score increased by -0.77 (95% confidence interval, -1.14 to -0.41; P0.001) with each year of age. The mean FEV(0.5) z score did not differ between infants with CF and healthy control subjects less than 6 months of age (-0.06 and 0.02, respectively; P = 0.87). However, the mean FEV(0.5) z score was lower by 1.15 in infants with CF who were older than 6 months of age compared with healthy infants (P0.001). FVC and FEF(75) followed a similar pattern. Pulmonary infection and inflammation in BAL samples did not explain the lung function results.Lung function, measured by forced expiration, is normal in infants with CF at the time of diagnosis by newborn screening but is diminished in older infants. These findings suggest that in CF the optimal timing of therapeutic interventions aimed at preserving lung function may be within the first 6 months of life.