Transcriptomic analysis of primary nasal epithelial cells reveals altered interferon signalling in preterm birth survivors at one year of age.

Introduction: Many survivors of preterm birth (<37 weeks gestation) have lifelong respiratory deficits, the drivers of which remain unknown. Influencers of pathophysiological outcomes are often detectable at the gene level and pinpointing these differences can help guide targeted research and interventions. This study provides the first transcriptomic analysis of primary nasal airway epithelial cells in survivors of preterm birth at approximately 1 year of age. Methods: Nasal airway epithelial brushings were collected, and primary cell cultures established from term (>37 weeks gestation) and very preterm participants (≤32 weeks gestation). Ex vivo RNA was collected from brushings with sufficient cell numbers and in vitro RNA was extracted from cultured cells, with bulk RNA sequencing performed on both the sample types. Differential gene expression was assessed using the limma-trend pipeline and pathway enrichment identified using Reactome and GO analysis. To corroborate gene expression data, cytokine concentrations were measured in cell culture supernatant. Results: Transcriptomic analysis to compare term and preterm cells revealed 2,321 genes differentially expressed in ex vivo samples and 865 genes differentially expressed in cultured basal cell samples. Over one third of differentially expressed genes were related to host immunity, with interferon signalling pathways dominating the pathway enrichment analysis and IRF1 identified as a hub gene. Corroboration of disrupted interferon release showed that concentrations of IFN-α2 were below measurable limits in term samples but elevated in preterm samples [19.4 (76.7) pg/ml/µg protein, p = 0.03]. IFN-γ production was significantly higher in preterm samples [3.3 (1.5) vs. 9.4 (17.7) pg/ml/µg protein; p = 0.01] as was IFN-ß [7.8 (2.5) vs. 13.6 (19.5) pg/ml/µg protein, p = 0.01]. Conclusion: Host immunity may be compromised in the preterm nasal airway epithelium in early life. Altered immune responses may lead to cycles of repeated infections, causing persistent inflammation and tissue damage which can have significant impacts on long-term respiratory function.

Biodiesel feedstock determines exhaust toxicity in 20% biodiesel: 80% mineral diesel blends.

To address climate change concerns, and reduce the carbon footprint caused by fossil fuel use, it is likely that blend ratios of renewable biodiesel with commercial mineral diesel fuel will steadily increase, resulting in biodiesel use becoming more widespread. Exhaust toxicity of unblended biodiesels changes depending on feedstock type, however the effect of feedstock on blended fuels is less well known. The aim of this study was to assess the impact of biodiesel feedstock on exhaust toxicity of 20% blended biodiesel fuels (B20). Primary human airway epithelial cells were exposed to exhaust diluted 1/15 with air from an engine running on conventional ultra-low sulfur diesel (ULSD) or 20% blends of soy, canola, waste cooking oil (WCO), tallow, palm or cottonseed biodiesel in diesel. Physico-chemical exhaust properties were compared between fuels and the post-exposure effect of exhaust on cellular viability and media release was assessed 24 h later. Exhaust properties changed significantly between all fuels with cottonseed B20 being the most different to both ULSD and its respective unblended biodiesel. Exposure to palm B20 resulted in significantly decreased cellular viability (96.3 ± 1.7%; p < 0.01) whereas exposure to soy B20 generated the greatest number of changes in mediator release (including IL-6, IL-8 and TNF-α, p < 0.05) when compared to air exposed controls, with palm B20 and tallow B20 closely following. In contrast, canola B20 and WCO B20 were the least toxic with only mediators G-CSF and TNF-α being significantly increased. Therefore, exposure to palm B20, soy B20 and tallow B20 were found to be the most toxic and exposure to canola B20 and WCO B20 the least. The top three most toxic and the bottom three least toxic B20 fuels are consistent with their unblended counterparts, suggesting that feedstock type greatly impacts exhaust toxicity, even when biodiesel only comprises 20% of the fuel.

Toxicity of different biodiesel exhausts in primary human airway epithelial cells grown at air-liquid interface.

Biodiesel is created through the transesterification of fats/oils and its usage is increasing worldwide as global warming concerns increase. Biodiesel fuel properties change depending on the feedstock used to create it. The aim of this study was to assess the different toxicological properties of biodiesel exhausts created from different feedstocks using a complex 3D air-liquid interface (ALI) model that mimics the human airway. Primary human airway epithelial cells were grown at ALI until full differentiation was achieved. Cells were then exposed to 1/20 diluted exhaust from an engine running on Diesel (ULSD), pure or 20% blended Canola biodiesel and pure or 20% blended Tallow biodiesel, or Air for control. Exhaust was analysed for various physio-chemical properties and 24-h after exposure, ALI cultures were assessed for permeability, protein release and mediator response. All measured exhaust components were within industry safety standards. ULSD contained the highest concentrations of various combustion gases. We found no differences in terms of particle characteristics for any of the tested exhausts, likely due to the high dilution used. Exposure to Tallow B100 and B20 induced increased permeability in the ALI culture and the greatest increase in mediator response in both the apical and basal compartments. In contrast, Canola B100 and B20 did not impact permeability and induced the smallest mediator response. All exhausts but Canola B20 induced increased protein release, indicating epithelial damage. Despite the concentrations of all exhausts used in this study meeting industry safety regulations, we found significant toxic effects. Tallow biodiesel was found to be the most toxic of the tested fuels and Canola the least, both for blended and pure biodiesel fuels. This suggests that the feedstock biodiesel is made from is crucial for the resulting health effects of exhaust exposure, even when not comprising the majority of fuel composition.

Fuel feedstock determines biodiesel exhaust toxicity in a human airway epithelial cell exposure model.

BACKGROUND: Biodiesel is promoted as a sustainable replacement for commercial diesel. Biodiesel fuel and exhaust properties change depending on the base feedstock oil/fat used during creation. The aims of this study were, for the first time, to compare the exhaust exposure health impacts of a wide range of biodiesels made from different feedstocks and relate these effects with the corresponding exhaust characteristics. METHOD: Primary airway epithelial cells were exposed to diluted exhaust from an engine running on conventional diesel and biodiesel made from Soy, Canola, Waste Cooking Oil, Tallow, Palm and Cottonseed. Exhaust properties and cellular viability and mediator release were analysed post exposure. RESULTS: The exhaust physico-chemistry of Tallow biodiesel was the most different to diesel as well as the most toxic, with exposure resulting in significantly decreased cellular viability (95.8 ± 6.5%) and increased release of several immune mediators including IL-6 (+223.11 ± 368.83 pg/mL) and IL-8 (+1516.17 ± 2908.79 pg/mL) above Air controls. In contrast Canola biodiesel was the least toxic with exposure only increasing TNF-α (4.91 ± 8.61). CONCLUSION: This study, which investigated the toxic effects for the largest range of biodiesels, shows that exposure to different exhausts results in a spectrum of toxic effects in vitro when combusted under identical conditions.

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

BACKGROUND: Aberrant 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. METHODS: Nineteen 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. RESULTS: RNA-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. CONCLUSIONS: Though 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.

Aberrant cell migration contributes to defective airway epithelial repair in childhood wheeze.

Abnormal wound repair has been observed in the airway epithelium of patients with chronic respiratory diseases, including asthma. Therapies focusing on repairing vulnerable airways, particularly in early life, present a potentially novel treatment strategy. We report defective lower airway epithelial cell repair to strongly associate with common pre-school-aged and school-aged wheezing phenotypes, characterized by aberrant migration patterns and reduced integrin α5ß1 expression. Next generation sequencing identified the PI3K/Akt pathway as the top upstream transcriptional regulator of integrin α5ß1, where Akt activation enhanced repair and integrin α5ß1 expression in primary cultures from children with wheeze. Conversely, inhibition of PI3K/Akt signaling in primary cultures from children without wheeze reduced α5ß1 expression and attenuated repair. Importantly, the FDA-approved drug celecoxib - and its non-COX2-inhibiting analogue, dimethyl-celecoxib - stimulated the PI3K/Akt-integrin α5ß1 axis and restored airway epithelial repair in cells from children with wheeze. When compared with published clinical data sets, the identified transcriptomic signature was also associated with viral-induced wheeze exacerbations highlighting the clinical potential of such therapy. Collectively, these results identify airway epithelial restitution via targeting the PI3K-integrin α5ß1 axis as a potentially novel therapeutic avenue for childhood wheeze and asthma. We propose that the next step in the therapeutic development process should be a proof-of-concept clinical trial, since relevant animal models to test the crucial underlying premise are unavailable.

Soy Biodiesel Exhaust is More Toxic than Mineral Diesel Exhaust in Primary Human Airway Epithelial Cells.

As global biodiesel production increases, there are concerns over the potential health impact of exposure to the exhaust, particularly in regard to young children who are at high risk because of their continuing lung development. Using human airway epithelial cells obtained from young children, we compared the effects of exposure to exhaust generated by a diesel engine with Euro V/VI emission controls running on conventional diesel (ultra-low-sulfur mineral diesel, ULSD), soy biodiesel (B100), or a 20% blend of soy biodiesel with diesel (B20). The exhaust output of biodiesel was found to contain significantly more respiratory irritants, including NOx, CO, and CO2, and a larger overall particle mass. Exposure to biodiesel exhaust resulted in significantly greater cell death and a greater release of immune mediators compared to both air controls and ULSD exhaust. These results have concerning implications for potential global health impacts, particularly for the pediatric population.

Preterm birth: Born too soon for the developing airway epithelium?

Birth prior to term interrupts the normal development of the respiratory system and consequently results in poor respiratory outcomes that persist throughout childhood. The mechanisms underpinning these poor respiratory outcomes are not well understood, but intrinsic abnormalities within the airway epithelium may be a contributing factor. Current evidence suggests that the airway epithelium is both structurally and functionally abnormal after preterm birth, with reports of epithelial thickening and goblet cell hyperplasia in addition to increased inflammation and apoptosis in the neonatal intensive care unit. However, studies focusing on the airway epithelium are limited and many questions remain unanswered; including whether abnormalities are a direct result of interrupted development, a consequence of exposure to inflammatory stimuli in the perinatal period or a combination of the two. In addition, the difficulty of accessing airway tissue has resulted in the majority of evidence being collected in the pre-surfactant era which may not reflect contemporary preterm birth. This review examines the consequences of preterm birth on the airway epithelium and explores the clinical relevance of currently available models whilst highlighting the need to develop a clinically relevant in vitro model to help further our understanding of the airway epithelium in preterm birth.