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Preterm birth rates are increasing and now account for >11% of global births. Simultaneously, advances in neonatal care have led to increased survival of lower gestation neonates. A complication of preterm birth, and the biggest determinant of survival, is lung and airway immaturity. After preterm birth, the immature respiratory system is exposed to pro-inflammatory stimuli like injury from resuscitation and oxygen toxicity. The airway epithelium, the physical barrier between insults and the airways, is particularly vulnerable to injury. If epithelial barrier integrity cannot be restored rapidly following damage (i.e. via aberrant repair), the respiratory system is left unprotected, increasing the risk of infection, inflammation and tissue damage. Altered epithelial repair may play an important role in the ongoing respiratory health problems experienced by preterm survivors, including severe respiratory infections throughout early life, or low and declining lung function [1–3]. Deficits are further exacerbated in those with bronchopulmonary dysplasia (BPD). The mechanisms contributing to ongoing respiratory problems are currently unknown, although probably begin in early life. Until now, understanding the role of the preterm epithelial barrier has been limited by a lack of appropriate cellular models. Our study aimed to assess the reparative capacity of the airway epithelium in survivors of preterm birth and its association with early life outcomes, with the hypothesis that preterm airway epithelial cells have an abnormal repair mechanism., Nasal epithelial cells from very preterm infants have a functional defect in their ability to repair beyond the first year of life, and failed repair may be associated with antenatal steroid exposure https://bit.ly/39OFJs7

Introduction: COVID-19 is responsible for a global pandemic that is complicated by acute lung injury, and death The virus SARS-CoV-2 requires the ACE2 receptor and serine proteases to enter airway epithelial cells We sought to determine what factors were associated with levels of ACE2 expression and how this related to people with asthma and chronic obstructive pulmonary disease (COPD) Methods: We obtained airway epithelial cells from 146 people, aged 2 to 89 from Perth and Newcastle The Newcastle cohort were enriched with people with asthma and COPD Gene expression for ACE2 and other genes potentially associated with SARS-CoV-2 cell entry were assessed by quantitative PCR and immunohistochemistry Results: Increased gene expression for ACE2 from lower airway epithelial cells was associated with older age (p=0 02) and male sex (p=0 03), but not packet years smoked When we compared gene expression between adults with asthma, COPD and healthy controls, mean ACE2 was lower in asthma p=0 01 (Figure) Gene expression for Furin a protease that facilitates viral endocytosis was also lower in asthma (p=0 02), while ADAM-17, a disintegrin that has been shown to cleave ACE2 from the surface was increased p=0 02 We confirmed that ACE2 protein expression was lower in endobronchial biopsies from people with asthma Conclusions: Increased ACE2 expression is seen in older people and males While people with asthma appear to have reduced expression Altered ACE2 expression in the lower airway may be an important factor in virus tropism to the lower airway

Introduction:2000 different mutations have been reported in patients with Cystic Fibrosis (CF) and occur in all CFTR exons and introns. Some mutations are not amenable to current therapies, and new drugs must be developed. Antisense oligonucleotides (AO) are synthetic nucleic acid analogues whose binding to pre-mRNA modulates exon selection. We hypothesise that skipping in frame CFTR exon 15 in patients with intra-exonic mutations such as p.Phe861Leufsx3 studied here, the induced isoform may retain residual function. Methods: AO sequences were initially optimised using 2’-O-Methyl modified bases on a phosphorothioate backbone (2OMe) and transfected into monolayer primary non-CF and p.Phe861Leufsx3/p.Phe508del CF airway epithelial cells. The most effective 2OMe AO sequence was re-synthesised as the clinically validated phosphorodiamidate morpholino (PMO) chemistry. Monolayer transfections were repeated and modification of protein was detected by western blot analysis. Residual CFTR function was measured using Ussing Chamber. Results: The 2OMe sequence produced 50% skipping in p.Phe861Leufsx3/p.Phe508del CF airway epithelial cells The PMO induced skipping of exon 15 from non-CF (49%) and p.Phe861Leufsx3/p.Phe508del CF (88%) cells after 7 days in culture. Western blot was then used to determine the effect on the induced CFTR protein. In addition, airway epithelial cells from children with CF prior to and following AO treatment were mucocillary differentiated at the air-liquid interface for 28 days and CFTR function assessed using Ussing chamber. Conclusion: Exon 15 can be skipped from CFTR in airway epithelial cells. We propose that exon skipping to remove disease-causing mutations in selected in-frame exons can improve CFTR function.

Introduction/Aim: Children with Cystic Fibrosis (CF) typically exhibit prolonged and severe symptoms during rhinovirus (RV) infection compared to healthy children. Here, we studied the host-rhinovirus interaction signature after infection, integrating two omics: (1) Whole transcriptome sequencing (WTS) to determine the viral load and host’s gene expression, and (2) metabolomics to profile the metabolites associated with the viral infection of primary tracheal epithelial cells obtained from healthy children (H) and those with CF. Methods: WTS and hydrophilic interaction liquid chromatography (HILIC) coupled to mass spectrometry, were used to identify the differences in transcripts/metabolites and RV coverage produced by H (3.9 ± 1.5 years; n=8) and CF (2.6 ± 1.8 years; n=8; all p.Phe508del/p.Phe508del) primary epithelial cells at 24 hours post-infection with human rhinovirus 1B. Univariate and multivariate analyses were then performed to identify the infection hallmark. Results: RV coverage in uninfected cells (Mock) was less than 0.5X, whereas, infected cells presented 44.4X and 101.6X of RV in children with and without CF respectively. Global RV infection was associated with 14 metabolites and 1713 genes differential expressed. From these, several metabolic pathways were found dysregulated including inositol metabolism and glycerophospholipid biosynthesis. Conclusion: Metabolic host-derived pathways associated with RV infection were identified. Although functional analysis is still required, these pathways could be used as potential biomarkers of RV infection in CF. Future analysis will help to understand whether these compounds can be targeted for antiviral purposes.

Introduction and Aim: Biodiesel (BD) is promoted as a sustainable replacement for commercial mineral diesel. As BD exhaust components change depending on the source oil, the aim of this project was to compare the effects of exposure to exhaust generated by the combustion of diesel (D) and BD made from different source oils. Methods: Primary human airway epithelial cells (n=8, from children aged 2.6-3.4yrs) were exposed for 1 hour to diluted exhaust from an engine combusting D or BD (from soy, canola, tallow, palm and waste cooking oil (WCO)). Exhaust characteristics were analysed and health impacts including viability and inflammatory mediator release were assessed. Results: Exhaust gas composition varied significantly between fuels with soy BD containing the highest levels of NOx and CO2. Particle size spectra also differed between exhaust types with all BD exhausts displaying peaks of varying intensity in the ultrafine particle size ( Exposure to WCO exhaust resulted in significant cell death (p Conclusion: The majority of BD exhausts contained more and smaller particles and more toxic gases compared with D and the health impacts varied between BD types with WCO, canola and tallow BD causing the worst health effects. Grant: ARC-DP170104346.

Introduction: Airway epithelial cells (AECs) line the airway to create a protective barrier between the lung and the external environment. Very preterm birth ( Methods: Nasal brushings were used to collect AECs from infants born very preterm (n=35, 24-31.7wks gestation, 1.07-1.22yrs corrected at sampling). Control samples were provided by children born at term (n=6, >37wks gestation, 2.4-6.5yrs at sampling). Cultured AECs were scratch wounded and repair tracked for 72 hours (IncuCyte ZOOM®, Essen Bioscience). Wound closure was then assessed for correlations with neonatal factors including gestation, birthweight, duration of respiratory support, and steroid exposure. Results: Term AECs achieved complete repair within 60 hours. Repair in successfully cultured preterm AECs (n=22) was significantly altered and fell into three categories; delayed but complete repair (>80% n=5), significant but incomplete closure (50-80% n=6) and incomplete closure (20-50% n=10). Neonatal factors did not predict altered wound repair, though infants born to mothers completing a course of antenatal steroids (n=13) exhibited significantly worse repair (p=0.017, 47.30% vs 74.02%). Conclusion: Data confirm that preterm infants have an intrinsic functional defect in their airway reparative capacity. Exposure to antenatal steroids may further alter repair and raises questions about the long-term impact of antenatal steroid use.

Introduction: Mixed viral-bacterial aetiology is common in lower respiratory tract infections of children. However the pathogenic mechanisms involved are poorly understood. Using primary airway epithelial cells (AEC) derived from children which were then differentiated by growing at the Air-Liquid Interface (ALI), we infected/inoculated Respiratory Syncytial Virus (RSV) and nontypeable Haemophilus influenzae (NTHi) to assess the interaction between viral infection, NTHi biofilm and AEC. We hypothesised that an interaction occurs between RSV and NTHi manifested by enhanced formation of biofilms following viral infection, and release of planktonic NTHi from established biofilms following introduction of RSV. Methods: Red fluorescent-tagged RSV (A2 strain, rrRSV-BN1, 1x106 pfu) and green fluorescent-tagged NTHi (86-028 NP, 2.5x107 CFU) were inoculated individually, concurrently or sequentially onto fully differentiated AEC grown on Transwell inserts (Corning). Cultures were left for 7 days. Insert surfaces were fixed, excised, mounted and overlayed with coverslips. Five confocal microscopy field-of-views at 200x magnification were obtained (Nikon C2+ system) and 1.2µm Z-stacks thickness used to reconstruct 3-dimensional images. Biomass thickness, surface area, and surface area to biomass ratio were determined using the COMSTAT program. Results: No red fluorescence arising from RSV infection and replication were detected in epithelial cells pre-colonised by NTHi. However when infected earlier with RSV, NTHi were able to colonise and form biofilms on epithelial cells. Conclusions: Prior NTHi colonisation of respiratory epithelium appears to shield against a secondary RSV infection, but not vice versa

Introduction: The invasive nature of accessing bronchial epithelial cells limits the capacity to source and utilise these cells for research purposes. Nasal epithelial cells have been identified as a potential surrogate however, there is conflicting evidence to support this. Aim: To compare baseline transcriptomic differences between nasal and bronchial airway epithelial cells. Methods: Matched Nasal and Bronchial Epithelial cells (NEC and BEC respectively) were obtained via non-bronchoscopic brushings from 32 healthy children (mean age: 5.7 years, 15 males). Total RNA was extracted, sequenced via Illumina and differential gene expression analysis was performed (DESeq2 and Ingenuity Systems). Results: RNA-seq analysis identified 3,500 differentially expressed genes, where 2,388 genes were significantly upregulated, in NEC compared to BEC samples (±2-fold, adjusted p≤0.05). The most enriched pathways in NEC samples were related to; innate immune response (e.g. complement cascade; pattern recognition receptors, scavenger and Fcγ receptors), and metabolic processes (e.g. retinol metabolism, biological oxidations and cytochrome P450). Interestingly, NEC samples expressed genes related to innate immune pathways at lower levels compared to BEC, whereas genes related to metabolic pathways were expressed at significantly higher levels in NEC samples. Conclusions: This is the largest transcriptomic profiling study, to date, of ex vivo nasal and bronchial airway epithelial cells from a healthy paediatric cohort. Data generated here illustrates intrinsic differences between nasal and bronchial airway samples, however functional validation is necessary to confirm these observations.

Background: miRNAs are critical regulators of signalling pathways and have lately been shown to be secreted into exosomes as a way of inter-cell communication (Valadi et al ., Nat Cell Biol, 2007). Previously, we found an increase of miR-17, -144 and -21 in murine lung homogenate in Ovalbumin (OVA) induced allergic airway inflammation (AAI) and in nasal epithelial cells of children with allergic asthma. Objective: We now aimed to decipher the role of the bronchial epithelium in the production and secretion of miRNAs in asthma. Methods: Air-liquid interface cultures of primary normal human bronchial epithelial (NHBE) cells were treated with Interleukin (IL)13 to model early epithelial changes to a T-helper 2 environment. Exoquick-TC (System Biosciences) was used to isolate exosomes and they were quantified by flow cytometry or ELISA for CD63. miRNAs were analysed by qRT-PCR. Results: Initially, miR-17 and -21 levels were increased upon IL13 in the basal compartment of NHBE cells and later on the apical side. Both miRNAs were significantly up-regulated in murine bronchoalveolar lavage of OVA- and house dust mite induced AAI, although total exosome amounts were similar. We were able to detect all miRNAs in exosomes of human nasal washes. Conclusion: NHBE cells secrete exosomes upon IL13 to the apical and basal cell side, which show differential miR-17 and -21 levels. Both exosomal miRNAs were increased in murine BALF in AAI and detectable in human paediatric asthma. This is a first hint that exosomal miRNA patterns are changing during AAI development which could perpetuate an asthmatic response between epithelial cells and expand it to e.g. immune cells.