Model Of Human Airway Epithelium Research Articles

Fungal melanin suppresses airway epithelial chemokine secretion through blockade of calcium fluxing

Respiratory infections caused by the human fungal pathogen Aspergillus fumigatus are a major cause of mortality for immunocompromised patients. Exposure to these pathogens occurs through inhalation, although the role of the respiratory epithelium in disease pathogenesis has not been fully defined. Employing a primary human airway epithelial model, we demonstrate that fungal melanins potently block the post-translational secretion of the chemokines CXCL1 and CXCL8 independent of transcription or the requirement of melanin to be phagocytosed, leading to a significant reduction in neutrophil recruitment to the apical airway both in vitro and in vivo. Aspergillus-derived melanin, a major constituent of the fungal cell wall, dampened airway epithelial chemokine secretion in response to fungi, bacteria, and exogenous cytokines. Furthermore, melanin muted pathogen-mediated calcium fluxing and hindered actin filamentation. Taken together, our results reveal a critical role for melanin interaction with airway epithelium in shaping the host response to fungal and bacterial pathogens.

An original device to assess the respiratory impact of indoor air VOCs mixture using an in vitro approach

Exposure to indoor air pollution, particularly volatile organic compounds (VOCs), has been recognized as a risk factor in the development of respiratory and allergic diseases. VOCs are mainly emitted continuously at low concentrations from construction furniture and decoration products. Measurement campaigns carried out in new dwellings in France have shown that aldehydes predominate with a tendency to decrease formaldehyde concentrations and to increase those in hexanal. As the main route of VOCs exposure is inhalation, this project assessed the impact of a mixture of 17 VOCs representative of indoor air (in quality and quantity) on respiratory health using an in vitro approach. This original work was based on the set-up of an experimental device, combining a gas generation and dilution bench and exposure to the air-liquid interface (ALI) adapted to the reconstructed human airway epithelium model. The VOC mixture was enriched with formaldehyde or hexanal in different proportions (from 20 to 240µg.m-3) to study the biological impact of these aldehydes after repeated exposures of airway epithelium. After examination of the stability of the VOC concentrations in generated mixtures and the found of the optimal operating conditions for the dynamic gas generating system, the gaseous mixtures were distributed to the epithelium using the ALI exposure system providing direct contact between the epithelium and the tested mixtures. Our device lead to reproduce real conditions of human exposure. The results showed that the inflammatory response, assessed by the production of four cytokines, varied according to the nature of the aldehyde present in the VOC mixture (formaldehyde or hexanal), its concentration, and the duration and number of exposures applied. The most original and innovative results concern those obtained with hexanal, pollutant under-researched. Our results showed that this aldehyde could pose risks to respiratory health.

Inhibition of SARS-CoV-2 infection in human airway epithelium with a xeno-nucleic acid aptamer

BackgroundSARS-CoV-2, the agent responsible for the COVID-19 pandemic, enters cells through viral spike glycoprotein binding to the cellular receptor, angiotensin-converting enzyme 2 (ACE2). Given the lack of effective antivirals targeting SARS-CoV-2, we previously utilized systematic evolution of ligands by exponential enrichment (SELEX) and selected fluoro-arabino nucleic acid (FANA) aptamer R8-9 that was able to block the interaction between the viral receptor-binding domain and ACE2.MethodsHere, we further assessed FANA-R8-9 as an entry inhibitor in contexts that recapitulate infection in vivo.ResultsWe demonstrate that FANA-R8-9 inhibits spike-bearing pseudovirus particle uptake in cell lines. Then, using an in-vitro model of human airway epithelium (HAE) and SARS-CoV-2 virus, we show that FANA-R8-9 significantly reduces viral infection when added either at the time of inoculation, or several hours later. These results were specific to the R8-9 sequence, not the xeno-nucleic acid utilized to make the aptamer. Importantly, we also show that FANA-R8-9 is stable in HAE culture secretions and has no overt cytotoxic effects.ConclusionsTogether, these results suggest that FANA-R8-9 effectively prevents infection by specific SARS-CoV-2 variants and indicate that aptamer technology could be utilized to target other clinically-relevant viruses in the respiratory mucosa.

Evaluation of in vitro rat and human airway epithelial models for acute inhalation toxicity testing.

In vivo models (mostly rodents) are currently accepted by regulatory authorities for assessing acute inhalation toxicity. Considerable efforts have been made in recent years to evaluate in vitro human airway epithelial models (HAEM) as replacements for in vivo testing. In the current work, an organotypic in vitro rat airway epithelial model (RAEM), rat EpiAirway, was developed and characterized to allow a direct comparison with the available HAEM, human EpiAirway, in order to address potential interspecies variability in responses to harmful agents. The rat and human models were evaluated in 2 independent laboratories with 14 reference chemicals, selected to cover a broad range of chemical structures and reactive groups, as well as known acute animal and human toxicity responses, in 3 replicate rounds of experiments. Toxicity endpoints included changes in tissue viability (MTT assay), epithelial barrier integrity (TEER, transepithelial electrical resistance), and tissue morphology (histopathology). The newly developed rat EpiAirway model produced reproducible results across all replicate experiments in both testing laboratories. Furthermore, a high level of concordance was observed between the RAEM and HAEM toxicity responses (determined by IC25) in both laboratories, with R2=0.78 and 0.88 when analyzed by TEER; and R2=0.92 for both when analyzed by MTT. These results indicate that rat and human airway epithelial tissues respond similarly to acute exposures to chemicals. The new in vitro RAEM will help extrapolate to in vivo rat toxicity responses and support screening as part of a 3Rs program.

123 Finding Optimal Methods for SbD Hazard Testing of Nanomaterials: The Effect of Cell Model and Exposure Method on Cytokine Response

Abstract The concept of safe-by-design (SbD) can help toxicologists and risk assessors to keep pace with the rapidly expanding field of nanotechnology. As part of SbD, the hazard potential of a new nanomaterial (NM) is identified at the first stages of product development. For this, simple yet predictive toxicity assays are crucial. We investigated the suitability of several in vitro models and exposure methods to predict the human (pulmonary) inflammation potential of NMs. Four silica NMs were selected of which in vivo pulmonary toxicity ranking had previously been established: DQ12 (quartz) > NM-203 (fumed silica) > Colloidal silica > Silanized colloidal silica. Several cell types including a 3D human airway epithelial model, lung epithelial cell lines, and immune cell lines, either in mono- or co-culture were exposed to the four materials either in a submerged setting or at the air-liquid interface (ALI). After 24 hours of exposure, cytokine response was assessed using either 13-plex LegendPlex, ELISA, or qPCR, and toxicity rankings were compared. The results indicate differences in sensitivity between cell models and exposure methods. Submerged exposure of a cell-line seems a suitable first tier for SbD hazard testing in case of NMs that can easily be dispersed. For NMs that are not compatible with submerged testing, ALI exposure might be needed. Higher tier testing might be used to confirm the ranking.

A Tracheal Aspirate-derived Airway Basal Cell Model Reveals a Proinflammatory Epithelial Defect in Congenital Diaphragmatic Hernia.

Rationale: Congenital diaphragmatic hernia (CDH) is characterized by incomplete closure of the diaphragm and lung hypoplasia. The pathophysiology of lung defects in CDH is poorly understood. Objectives: To establish a translational model of human airway epithelium in CDH for pathogenic investigation and therapeutic testing. Methods: We developed a robust methodology of epithelial progenitor derivation from tracheal aspirates of newborns. Basal stem cells (BSCs) from patients with CDH and preterm and term non-CDH control subjects were derived and analyzed by bulk RNA sequencing, assay for transposase accessible chromatin with sequencing, and air-liquid interface differentiation. Lung sections from fetal human CDH samples and the nitrofen rat model of CDH were subjected to histological assessment of epithelial defects. Therapeutics to restore epithelial differentiation were evaluated in human epithelial cell culture and the nitrofen rat model of CDH. Measurements and Main Results: Transcriptomic and epigenetic profiling of CDH and control BSCs reveals a proinflammatory signature that is manifested by hyperactive nuclear factor kappa B and independent of severity and hernia size. In addition, CDH BSCs exhibit defective epithelial differentiation invitro that recapitulates epithelial phenotypes found in fetal human CDH lung samples and fetal tracheas of the nitrofen rat model of CDH. Furthermore, blockade of nuclear factor kappa B hyperactivity normalizes epithelial differentiation phenotypes of human CDH BSCs invitro and in nitrofen rat tracheas invivo. Conclusions: Our findings have identified an underlying proinflammatory signature and BSC differentiation defects as a potential therapeutic target for airway epithelial defects in CDH.

Rhinovirus increases Moraxella catarrhalis adhesion to the respiratory epithelium.

Rhinovirus causes many types of respiratory illnesses, ranging from minor colds to exacerbations of asthma. Moraxella catarrhalis is an opportunistic pathogen that is increased in abundance during rhinovirus illnesses and asthma exacerbations and is associated with increased severity of illness through mechanisms that are ill-defined. We used a co-infection model of human airway epithelium differentiated at the air-liquid interface to test the hypothesis that rhinovirus infection promotes M. catarrhalis adhesion and survival on the respiratory epithelium. Initial experiments showed that infection with M. catarrhalis alone did not damage the epithelium or induce cytokine production, but increased trans-epithelial electrical resistance, indicative of increased barrier function. In a co-infection model, infection with the more virulent rhinovirus-A and rhinovirus-C, but not the less virulent rhinovirus-B types, increased cell-associated M. catarrhalis. Immunofluorescent staining demonstrated that M. catarrhalis adhered to rhinovirus-infected ciliated epithelial cells and infected cells being extruded from the epithelium. Rhinovirus induced pronounced changes in gene expression and secretion of inflammatory cytokines. In contrast, M. catarrhalis caused minimal effects and did not enhance RV-induced responses. Our results indicate that rhinovirus-A or C infection increases M. catarrhalis survival and cell association while M. catarrhalis infection alone does not cause cytopathology or epithelial inflammation. Our findings suggest that rhinovirus and M. catarrhalis co-infection could promote epithelial damage and more severe illness by amplifying leukocyte inflammatory responses at the epithelial surface.

Pre-clinical evaluation of antiviral activity of nitazoxanide against SARS-CoV-2.

SummaryBackgroundTo address the emergence of SARS-CoV-2, multiple clinical trials in humans were rapidly started, including those involving an oral treatment by nitazoxanide, despite no or limited pre-clinical evidence of antiviral efficacy.MethodsIn this work, we present a complete pre-clinical evaluation of the antiviral activity of nitazoxanide against SARS-CoV-2.FindingsFirst, we confirmed the in vitro efficacy of nitazoxanide and tizoxanide (its active metabolite) against SARS-CoV-2. Then, we demonstrated nitazoxanide activity in a reconstructed bronchial human airway epithelium model. In a SARS-CoV-2 virus challenge model in hamsters, oral and intranasal treatment with nitazoxanide failed to impair viral replication in commonly affected organs. We hypothesized that this could be due to insufficient diffusion of the drug into organs of interest. Indeed, our pharmacokinetic study confirmed that concentrations of tizoxanide in organs of interest were always below the in vitro EC50.InterpretationThese preclinical results suggest, if directly applicable to humans, that the standard formulation and dosage of nitazoxanide is not effective in providing antiviral therapy for Covid-19.FundingThis work was supported by the Fondation de France “call FLASH COVID-19”, project TAMAC, by “Institut national de la santé et de la recherche médicale” through the REACTing (REsearch and ACTion targeting emerging infectious diseases), by REACTING/ANRS MIE under the agreement No. 21180 (‘Activité des molécules antivirales dans le modèle hamster’), by European Virus Archive Global (EVA 213 GLOBAL) funded by the European Union's Horizon 2020 research and innovation program under grant agreement No. 871029 and DNDi under support by the Wellcome Trust Grant ref: 222489/Z/21/Z through the COVID-19 Therapeutics Accelerator”.

Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium.

The clinical impact of rhinovirus C (RV-C) is well-documented; yet, the viral life cycle remains poorly defined. Thus, we characterized RV-C15 replication at the single-cell level and its impact on the human airway epithelium (HAE) using a physiologically-relevant in vitro model. RV-C15 replication was restricted to ciliated cells where viral RNA levels peaked at 12 hours post-infection (hpi), correlating with elevated titers in the apical compartment at 24hpi. Notably, infection was associated with a loss of polarized expression of the RV-C receptor, cadherin-related family member 3. Visualization of double-stranded RNA (dsRNA) during RV-C15 replication revealed two distinct replication complex arrangements within the cell, likely corresponding to different time points in infection. To further define RV-C15 replication sites, we analyzed the expression and colocalization of giantin, phosphatidylinositol-4-phosphate, and calnexin with dsRNA. Despite observing Golgi fragmentation by immunofluorescence during RV-C15 infection as previously reported for other RVs, a high ratio of calnexin-dsRNA colocalization implicated the endoplasmic reticulum as the primary site for RV-C15 replication in HAE. RV-C15 infection was also associated with elevated stimulator of interferon genes (STING) expression and the induction of incomplete autophagy, a mechanism used by other RVs to facilitate non-lytic release of progeny virions. Notably, genetic depletion of STING in HAE attenuated RV-C15 and -A16 (but not -B14) replication, corroborating a previously proposed proviral role for STING in some RV infections. Finally, RV-C15 infection resulted in a temporary loss in epithelial barrier integrity and the translocation of tight junction proteins while a reduction in mucociliary clearance indicated cytopathic effects on epithelial function. Together, our findings identify both shared and unique features of RV-C replication compared to related rhinoviruses and define the impact of RV-C on both epithelial cell organization and tissue functionality–aspects of infection that may contribute to pathogenesis in vivo.

Avian Cell Line DuckCelt®-T17 Is an Efficient Production System for Live-Attenuated Human Metapneumovirus Vaccine Candidate Metavac®.

The development of a live-attenuated vaccine (LAV) for the prevention of human metapneumovirus (HMPV) infection is often hampered by the lack of highly efficient and scalable cell-based production systems that support eventual global vaccine production. Avian cell lines cultivated in suspension compete with traditional cell platforms used for viral vaccine manufacture. We investigated whether the DuckCelt®-T17 avian cell line (Vaxxel), previously described as an efficient production system for several influenza strains, could also be used to produce a new HMPV LAV candidate (Metavac®, SH gene-deleted A1/C-85473 HMPV). To that end, we characterized the operational parameters of MOI, cell density, and trypsin addition to achieve the optimal production of Metavac®, and demonstrated that the DuckCelt®-T17 cell line is permissive and well-adapted to the production of the wild-type A1/C-85473 HMPV and the Metavac® vaccine candidate. Moreover, our results confirmed that the LAV candidate produced in DuckCelt®-T17 cells conserves its advantageous replication properties in LLC-MK2 and 3D-reconstituted human airway epithelium models, and its capacity to induce efficient neutralizing antibodies in a BALB/c mouse model. Our results suggest that the DuckCelt®-T17 avian cell line is a very promising platform for the scalable in-suspension serum-free production of the HMPV-based LAV candidate Metavac®.

Early nasal type I IFN immunity against SARS-CoV-2 is compromised in patients with autoantibodies against type I IFNs.

IFN-I and IFN-III immunity in the nasal mucosa is poorly characterized during SARS-CoV-2 infection. We analyze the nasal IFN-I/III signature, namely the expression of ISGF-3-dependent IFN-stimulated genes, in mildly symptomatic COVID-19 patients and show its correlation with serum IFN-α2 levels, which peak at symptom onset and return to baseline from day 10 onward. Moreover, the nasal IFN-I/III signature correlates with the nasopharyngeal viral load and is associated with the presence of infectious viruses. By contrast, we observe low nasal IFN-I/III scores despite high nasal viral loads in a subset of critically ill COVID-19 patients, which correlates with the presence of autoantibodies (auto-Abs) against IFN-I in both blood and nasopharyngeal mucosa. In addition, functional assays in a reconstituted human airway epithelium model of SARS-CoV-2 infection confirm the role of such auto-Abs in abrogating the antiviral effects of IFN-I, but not those of IFN-III. Thus, IFN-I auto-Abs may compromise not only systemic but also local antiviral IFN-I immunity at the early stages of SARS-CoV-2 infection.

Preclinical evaluation of Imatinib does not support its use as an antiviral drug against SARS-CoV-2

Following the emergence of SARS-CoV-2, the search for an effective and rapidly available treatment was initiated worldwide based on repurposing of available drugs. Previous reports described the antiviral activity of certain tyrosine kinase inhibitors (TKIs) targeting the Abelson kinase 2 against pathogenic coronaviruses. Imatinib, one of them, has more than twenty years of safe utilization for the treatment of hematological malignancies. In this context, Imatinib was rapidly evaluated in clinical trials against Covid-19. Here, we present the pre-clinical evaluation of imatinib in multiple models. Our results indicated that imatinib and another TKI, the masitinib, exhibit an antiviral activity in VeroE6 cells. However, imatinib was inactive in a reconstructed bronchial human airway epithelium model. In vivo, imatinib therapy failed to impair SARS-CoV-2 replication in a golden Syrian hamster model despite high concentrations in plasma and in the lung. Overall, these results do not support the use of imatinib and similar TKIs as antivirals in the treatment of Covid-19.

A Complete In Vitro Toxicological Assessment of the Biological Effects of Cerium Oxide Nanoparticles: From Acute Toxicity to Multi-Dose Subchronic Cytotoxicity Study.

Engineered nanomaterials (ENMs) are of significant relevance due to their unique properties, which have been exploited for widespread applications. Cerium oxide nanoparticles (CeO2-NPs) are one of most exploited ENM in the industry due to their excellent catalytic and multi-enzyme mimetic properties. Thus, the toxicological effects of these ENMs should be further studied. In this study, the acute and subchronic toxicity of CeO2-NPs were assessed. First, an in vitro multi-dose short-term (24 h) toxicological assessment was performed in three different cell lines: A549 and Calu3 were used to represented lung tissue and 3T3 was used as an interstitial tissue model. After that, a sub-chronic toxicity assessment (90 days) of these NPs was carried out on a realistic and well-established reconstituted primary human airway epithelial model (MucilAir™), cultured at the Air–Liquid Interface (ALI), to study the long-term effects of these particles. Results showed minor toxicity of CeO2-NPs in acute exposures. However, in subchronic exposures, cytotoxic and inflammatory responses were observed in the human airway epithelial model after 60 days of exposure to CeO2-NPs. These results suggest that acute toxicity approaches may underestimate the toxicological effect of some ENMs, highlighting the need for subchronic toxicological studies in order to accurately assess the toxicity of ENM and their cumulative effects in organisms.

In vitro characterisation of SARS-CoV-2 and susceptibility of domestic ferrets (Mustela putorius furo).

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an emerging virus that has caused significant human morbidity and mortality since its detection in late 2019. With the rapid emergence has come an unprecedented programme of vaccine development with at least 300 candidates under development. Ferrets have proven to be an appropriate animal model for testing safety and efficacy of SARS-CoV-2 vaccines due to quantifiable virus shedding in nasal washes and oral swabs. Here, we outline our efforts early in the SARS-CoV-2 outbreak to propagate and characterize an Australian isolate of the virus in vitro and in an ex vivo model of human airway epithelium, as well as to demonstrate the susceptibility of domestic ferrets (Mustela putorius furo) to SARS-CoV-2 infection following intranasal challenge.

Multi-endpoint analysis of human 3D airway epithelium following repeated exposure to whole electronic vapor product aerosol or cigarette smoke

Smoking is a cause of serious diseases in smokers including chronic respiratory diseases. This study aimed to evaluate the tobacco harm reduction (THR) potential of an electronic vapor product (EVP, myblu™) compared to a Kentucky Reference Cigarette (3R4F), and assessed endpoints related to chronic respiratory diseases. Endpoints included: cytotoxicity, barrier integrity (TEER), cilia function, immunohistochemistry, and pro-inflammatory markers. In order to more closely represent the user exposure scenario, we have employed the in vitro 3D organotypic model of human airway epithelium (MucilAir™, Epithelix) for respiratory assessment. The model was repeatedly exposed to either whole aerosol of the EVP, or whole 3R4F smoke, at the air liquid interface (ALI), for 4 weeks to either 30, 60 or 90 puffs on 3-exposure-per-week basis. 3R4F smoke generation used the ISO 20778:2018 regime and EVP aerosol used the ISO 20768:2018 vaping regime.Exposure to undiluted whole EVP aerosol did not trigger any significant changes in the level of pro-inflammatory mediators, cilia beating function, barrier integrity and cytotoxicity when compared with air controls. In contrast, exposure to diluted (1:17) whole cigarette smoke caused significant changes to all the endpoints mentioned above. To our knowledge, this is the first study evaluating the effects of repeated whole cigarette smoke and whole EVP aerosol exposure to a 3D lung model at the ALI. Our results add to the growing body of scientific literature supporting the THR potential of EVPs relative to combustible cigarettes and the applicability of the 3D lung models in human-relevant product risk assessments.

Transcriptional Profiling of Immune and Inflammatory Responses in the Context of SARS-CoV-2 Fungal Superinfection in a Human Airway Epithelial Model.

An increasing amount of evidence indicates a relatively high prevalence of superinfections associated with coronavirus disease 2019 (COVID-19), including invasive aspergillosis, but the underlying mechanisms remain to be characterized. In the present study, to better understand the biological impact of superinfection, we determine and compare the host transcriptional response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) versus Aspergillus superinfection, using a model of reconstituted human airway epithelium. Our analyses reveal that both simple infection and superinfection induce strong deregulation of core components of innate immune and inflammatory responses, with a stronger response to superinfection in the bronchial epithelial model compared to its nasal counterpart. Our results also highlight unique transcriptional footprints of SARS-CoV-2 Aspergillus superinfection, such as an imbalanced type I/type III IFN, and an induction of several monocyte and neutrophil associated chemokines, that could be useful for the understanding of Aspergillus-associated COVID-19 and also the management of severe forms of aspergillosis in this specific context.

In Vitro Combinations of Baloxavir Acid and Other Inhibitors against Seasonal Influenza A Viruses.

Two antiviral classes, the neuraminidase inhibitors (NAIs) and polymerase inhibitors (baloxavir marboxil and favipiravir) can be used to prevent and treat influenza infections during seasonal epidemics and pandemics. However, prolonged treatment may lead to the emergence of drug resistance. Therapeutic combinations constitute an alternative to prevent resistance and reduce antiviral doses. Therefore, we evaluated in vitro combinations of baloxavir acid (BXA) and other approved drugs against influenza A(H1N1)pdm09 and A(H3N2) subtypes. The determination of an effective concentration inhibiting virus cytopathic effects by 50% (EC50) for each drug and combination indexes (CIs) were based on cell viability. CompuSyn software was used to determine synergism, additivity or antagonism between drugs. Combinations of BXA and NAIs or favipiravir had synergistic effects on cell viability against the two influenza A subtypes. Those effects were confirmed using a physiological and predictive ex vivo reconstructed human airway epithelium model. On the other hand, the combination of BXA and ribavirin showed mixed results. Overall, BXA stands as a good candidate for combination with several existing drugs, notably oseltamivir and favipiravir, to improve in vitro antiviral activity. These results should be considered for further animal and clinical evaluations.

Multiparametric investigation of non functionalized-AGuIX nanoparticles in 3D human airway epithelium models demonstrates preferential targeting of tumor cells

Liquid deposit mimicking surface aerosolization in the airway is a promising strategy for targeting bronchopulmonary tumors with reduced doses of nanoparticle (NPs). In mimicking and studying such delivery approaches, the use of human in vitro 3D culture models can bridge the gap between 2D cell culture and small animal investigations. Here, we exposed airway epithelia to liquid-apical gadolinium-based AGuIX® NPs in order to determine their safety profile. We used a multiparametric methodology to investigate the NP’s distribution over time in both healthy and tumor-bearing 3D models. AGuIX® NPs were able to target tumor cells in the absence of specific surface functionalization, without evidence of toxicity. Finally, we validated the therapeutic potential of this hybrid theranostic AGuIX® NPs upon radiation exposure in this model. In conclusion, 3D cell cultures can efficiently mimic the normal and tumor-bearing airway epitheliums, providing an ethical and accessible model for the investigation of nebulized NPs.

Strain-Dependent Impact of G and SH Deletions Provide New Insights for Live-Attenuated HMPV Vaccine Development.

Human metapneumovirus (HMPV) is a major pediatric respiratory pathogen with currently no specific treatment or licensed vaccine. Different strategies to prevent this infection have been evaluated, including live-attenuated vaccines (LAV) based on SH and/or G protein deletions. This approach showed promising outcomes but has not been evaluated further using different viral strains. In that regard, we previously showed that different HMPV strains harbor distinct in vitro fusogenic and in vivo pathogenic phenotypes, possibly influencing the selection of vaccine strains. In this study, we investigated the putative contribution of the low conserved SH or G accessory proteins in such strain-dependent phenotypes and generated recombinant wild type (WT) and SH- or G-deleted viruses derived from two different patient-derived HMPV strains, A1/C-85473 and B2/CAN98-75. The ΔSH and ΔG deletions led to different strain-specific phenotypes in both LLC-MK2 cell and reconstituted human airway epithelium models. More interestingly, the ΔG-85473 and especially ΔSH-C-85473 recombinant viruses conferred significant protection against HMPV challenge and induced immunogenicity against a heterologous strain. In conclusion, our results show that the viral genetic backbone should be considered in the design of live-attenuated HMPV vaccines, and that a SH-deleted virus based on the A1/C-85473 HMPV strain could be a promising LAV candidate as it is both attenuated and protective in mice while being efficiently produced in a cell-based system.

Airway epithelial cells exposed to wildfire smoke extract exhibit dysregulated autophagy and barrier dysfunction consistent with COPD

BackgroundIndividuals with respiratory disease are being increasingly exposed to wildfire smoke as populations encroach further into forested regions and climate change continues to bring higher temperatures with lower rainfall. Frequent exposures have significant potential to accelerate conditions such as chronic obstructive pulmonary disease (COPD) which is characterised by an exaggerated inflammatory response to environmental stimuli. Here we employ models of human airway epithelium exposed to wildfire smoke-extract (WFSE) to examine modulation in airway epithelial cell (AEC) survival, fragility and barrier function.MethodsSubmerged cultures of small airway epithelial cells (SAEC) and differentiated air-liquid interface (ALI) cultures of primary bronchial AEC (bAEC) were treated for 1–24 h with 1–10% WFSE generated from plant species found in the Australian bushland. Autophagy (LC3-II and Sequestosome), apoptosis (Poly-(ADP)-Ribose Polymerase (PARP) cleavage) and tight junction proteins were measured using western blot. Barrier function was assessed via permeability of fluorescein tracers and measuring trans-epithelial electrical resistance. The production of IL-6 was assessed using ELISA.ResultsPrimary epithelial models exposed to WFSE exhibited a significant blockade in autophagy as evidenced by an increase in LC3-II coupled with a concomitant elevation in Sequestosome abundance. These exposures also induced significant PARP cleavage indicative of apoptotic changes. ALI cultures of bAEC treated with 5% WFSE demonstrated barrier dysfunction with significant increases in paracellular molecular permeability and ionic conductance, and a reduction in the abundance of the tight junction proteins ZO-1 and Claudin-1. These cultures also exhibited increased IL-6 secretion consistent with the aberrant and pro-inflammatory repair response observed in the COPD airways. Further, blocks in autophagy and barrier disruption were significantly elevated in response to WFSE in comparison to similar exposures with cigarette smoke-extract.ConclusionWFSE inhibits autophagic flux and induces barrier dysfunction in the airway epithelium. As autophagy is a central regulator of cellular repair, viability, and inflammation, targeting the block in autophagic flux may ameliorate the consequences of wildfire smoke-exposure for individuals with pre-existing respiratory conditions.