Primary Bronchial Epithelial Cells Research Articles

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in cystic fibrosis.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lungs, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC (murine homolog of IL-8) secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection.NEW & NOTEWORTHY The experiments in this report identify a novel mechanism, whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet halves in OMVs secreted by P. aeruginosa, which reduced the OMV-LPS-induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF.

Selective Replacement of Cholesterol with Cationic Amphiphilic Drugs Enables the Design of Lipid Nanoparticles with Improved RNA Delivery.

The delivery of RNA across biological barriers can be achieved by encapsulation in lipid nanoparticles (LNPs). Cationic amphiphilic drugs (CADs) are pharmacologically diverse compounds with ionizable lipid-like features. In this work, we applied CADs as a fifth component of state-of-the-art LNPs via microfluidic mixing. Improved cytosolic delivery of both siRNA and mRNA was achieved by partly replacing the cholesterol fraction of LNPs with CADs. The LNPs could cross the mucus layer in a mucus-producing air-liquid interface model of human primary bronchial epithelial cells following nebulization. Moreover, CAD-LNPs demonstrated improved epithelial and endothelial targeting following intranasal administration in mice, without a marked pro-inflammatory signature. Importantly, quantification of the CAD-LNP molar composition, as demonstrated for nortriptyline, revealed a gradual leakage of the CAD from the formulation during LNP dialysis. Altogether, these data suggest that the addition of a CAD prior to the rapid mixing process might have an impact on the composition, structure, and performance of LNPs.

Alterations in the molecular control of mitochondrial turnover in COPD lung and airway epithelial cells

Abnormal mitochondria have been observed in bronchial- and alveolar epithelial cells of patients with chronic obstructive pulmonary disease (COPD). However, it is unknown if alterations in the molecular pathways regulating mitochondrial turnover (mitochondrial biogenesis vs mitophagy) are involved. Therefore, in this study, the abundance of key molecules controlling mitochondrial turnover were assessed in peripheral lung tissue from non-COPD patients (n = 6) and COPD patients (n = 11; GOLDII n = 4/11; GOLDIV n = 7/11) and in both undifferentiated and differentiated human primary bronchial epithelial cells (PBEC) from non-COPD patients and COPD patients (n = 4–7 patients/group). We observed significantly decreased transcript levels of key molecules controlling mitochondrial biogenesis (PPARGC1B, PPRC1, PPARD) in peripheral lung tissue from severe COPD patients. Interestingly, mRNA levels of the transcription factor TFAM (mitochondrial biogenesis) and BNIP3L (mitophagy) were increased in these patients. In general, these alterations were not recapitulated in undifferentiated and differentiated PBECs with the exception of decreased PPARGC1B expression in both PBEC models. Although these findings provide valuable insight in these pathways in bronchial epithelial cells and peripheral lung tissue of COPD patients, whether or not these alterations contribute to COPD pathogenesis, underlie changes in mitochondrial function or may represent compensatory mechanisms remains to be established.

Combination treatment to improve mucociliary transport of Pseudomonas aeruginosa biofilms.

People with muco-obstructive pulmonary diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) often have acute or chronic respiratory infections that are difficult to treat due in part to the accumulation of hyperconcentrated mucus within the airway. Mucus accumulation and obstruction promote chronic inflammation and infection and reduce therapeutic efficacy. Bacterial aggregates in the form of biofilms exhibit increased resistance to mechanical stressors from the immune response (e.g., phagocytosis) and chemical treatments including antibiotics. Herein, combination treatments designed to disrupt the mechanical properties of biofilms and potentiate antibiotic efficacy are investigated against mucus-grown Pseudomonas aeruginosa biofilms and optimized to 1) alter biofilm viscoelastic properties, 2) increase mucociliary transport rates, and 3) reduce bacterial viability. A disulfide bond reducing agent (tris(2-carboxyethyl)phosphine, TCEP), a surfactant (NP40), a biopolymer (hyaluronic acid, HA), a DNA degradation enzyme (DNase), and an antibiotic (tobramycin) are tested in various combinations to maximize biofilm disruption. The viscoelastic properties of biofilms are quantified with particle tracking microrheology and transport rates are quantified in a mucociliary transport device comprised of fully differentiated primary human bronchial epithelial cells. The combination of the NP40 with hyaluronic acid and tobramycin was the most effective at increasing mucociliary transport rates, decreasing the viscoelastic properties of mucus, and reducing bacterial viability. Multimechanistic targeting of biofilm infections may ultimately result in improved clinical outcomes, and the results of this study may be translated into future in vivo infection models.

House dust mite allergens induce Ca2+ signalling and alarmin responses in asthma airway epithelial cells

Type 2 inflammation in asthma develops with exposure to stimuli to include inhaled allergens from house dust mites (HDM). Features include mucus hypersecretion and the formation of pro-secretory ion transport characterised by elevated basal Cl− current. Studies using human sinonasal epithelial cells treated with HDM extract report a higher protease activated receptor-2 (PAR-2) agonist-induced calcium mobilisation that may be related to airway sensitisation by allergen-associated proteases. Herein, this study aimed to investigate the effect of HDM on Ca2+ signalling and inflammatory responses in asthmatic airway epithelial cells. Primary bronchial epithelial cells (hPBECs) from asthma donors cultured at air-liquid interface were used to assess electrophysiological, Ca2+ signalling and inflammatory responses. Differences were observed regarding Ca2+ signalling in response to PAR-2 agonist 2-Furoyl-LIGRLO-amide (2-FLI), and equivalent short-circuit current (Ieq) in response to trypsin and 2-FLI, in ALI-asthma and healthy hPBECs. HDM treatment led to increased levels of intracellular cations (Ca2+, Na+) and significantly reduced the 2-FLI-induced change of Ieq in asthma cells. Apical HDM-induced Ca2+ mobilisation was found to mainly involve the activation of PAR-2 and PAR-4-associated store-operated Ca2+ influx and TRPV1. In contrast, PAR-2, PAR-4 antagonists and TRPV1 antagonist only showed slight impact on basolateral HDM-induced Ca2+ mobilisation. HDM trypsin-like serine proteases were the main components leading to non-amiloride sensitive Ieq and also increased interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP) from asthma hPBECs. These studies add further insight into the complex mechanisms associated with HDM-induced alterations in cell signalling and their relevance to pathological changes within asthma.

Alcohol and its metabolites dysregulate cellular bioenergetics and induce oxidative and endoplasmic reticulum stress in primary human bronchial epithelial cells.

Chronic alcohol consumption/misuse is a significant risk factor for pneumonia and lung infection leading to the development of chronic pulmonary disorders such as chronic obstructive pulmonary disease (COPD) and lung fibrosis. In this study, we sought to delineate the mechanism of alcohol-associated lung disease. We did so by measuring invitro mitochondrial, endoplasmic reticulum (ER) oxidative stress in human bronchial epithelial cells (hBECs) treated with ethanol and its oxidative (acetaldehyde) and nonoxidative (fatty acid ethyl esters or FAEEs) metabolites. Primary hBECs from a normal subject were treated with relevant concentrations of ethanol and its metabolites and incubated at 37°C for 24 h. Viability and cytotoxicity were determined using cell viability and lactate dehydrogenase (LDH) assay kits, respectively. Oxidized glutathione (GSSG) and reduced glutathione (GSH) were measured by colorimetric reaction, and 4-hydroxynenonal (4HNE) by immunohistochemistry. Endoplasmic reticulum stress and dysregulated cellular bioenergetics were determined by western blot analysis. Mitochondrial stress and real-time ATP production rates were determined using a Seahorse Extracellular Flux analyzer. Amelioration of ethanol-induced oxidative/ER stress and mitochondrial energetics was determined using an AMPKα agonist. Human bronchial epithelial cells treated with ethanol, acetaldehyde, and FAEEs showed a concentration-dependent increase in the secretion of LDH, oxidative/ER stress, deactivation of AMPKα phosphorylation and mitochondrial stress (decreased spare respiratory capacity) with concomitant decreases in mitochondrial and glycolytic ATP production rates. FAEEs caused greater cytotoxicity, ER stress, and dysregulated cellular bioenergetics than those ethanol and its oxidative metabolite. AMPKα agonist-pretreated cells significantly ameliorated ethanol-induced oxidative/ER stress, deactivation of AMPKα, and dysregulated cellular bioenergetics. Findings of this study suggest that ethanol and its metabolites contribute to cytotoxicity, oxidative/ER stress, and dysregulation of cellular bioenergetics in hBECs. The attenuation of ethanol-induced ER/oxidative stress and mitochondrial respiration by an AMPKα agonist may reflect a potential for it to be developed as a therapeutic agent for chronic alcohol-associated lung disease.

Upregulated microRNA-125b-5p in patients with asthma-COPD overlap mediates oxidative stress and late apoptosis via targeting IL6R/TRIAP1 signaling

BackgroundAmong patients with chronic obstructive pulmonary disease (COPD), some have features of both asthma and COPD—a condition categorized as asthma-COPD overlap (ACO). Our aim was to determine whether asthma- or COPD-related microRNAs (miRNAs) play a role in the pathogenesis of ACO.MethodsA total of 22 healthy subjects and 27 patients with ACO were enrolled. We selected 6 miRNAs that were found to correlate with COPD and asthma. The expression of miRNAs and target genes was analyzed using quantitative reverse-transcriptase polymerase chain reaction. Cell apoptosis and intracellular reactive oxygen species production were evaluated using flow cytometry. In vitro human monocytic THP-1 cells and primary normal human bronchial epithelial (NHBE) cells under stimuli with cigarette smoke extract (CSE) or ovalbumin (OVA) allergen or both were used to verify the clinical findings.ResultsWe identified the upregulation of miR-125b-5p in patients with ACO and in THP-1 cells stimulated with CSE plus OVA allergen. We selected 16 genes related to the miR-125b-5p pathway and found that IL6R and TRIAP1 were both downregulated in patients with ACO and in THP-1 cells stimulated with CSE plus OVA. The percentage of late apoptotic cells increased in the THP-1 cell culture model when stimulated with CSE plus OVA, and the effect was reversed by transfection with miR-125b-5p small interfering RNA (siRNA). The percentage of reactive oxygen species-producing cells increased in the NHBE cell culture model when stimulated with CSE plus OVA, and the effect was reversed by transfection with miR-125b-5p siRNA. In NHBE cells, siRNA transfection reversed the upregulation of STAT3 under CSE+OVA stimulation.ConclusionsOur study revealed that upregulation of miR-125b-5p in patients with ACO mediated late apoptosis in THP-1 cells and oxidative stress in NHBE cells via targeting IL6R and TRIAP1. STAT3 expression was also regulated by miR-125b-5p.

COPD basal cells are primed towards secretory to multiciliated cell imbalance driving increased resilience to environmental stressors

IntroductionEnvironmental pollutants injure the mucociliary elevator, thereby provoking disease progression in chronic obstructive pulmonary disease (COPD). Epithelial resilience mechanisms to environmental nanoparticles in health and disease are poorly characterised.MethodsWe delineated...

GLUT1 mediates the release of HMGB1 from airway epithelial cells in mixed granulocytic asthma

Asthma is quite heterogenous and can be categorized as eosinophilic, mixed granulocytic (presence of both eosinophils and neutrophils in the airways) and neutrophilic. Clinically, mixed granulocytic asthma (MGA) often tends to be severe and requires large doses of corticosteroids. High mobility group box 1 (HMGB1) is one of the epithelium-derived alarmins that contributes to type 2 inflammation and asthma. This study was aimed to investigate the role of glucose transporter 1 (GLUT1) in modulation of airway epithelial HMGB1 production in MGA. Induced sputum and bronchial biopsy specimens were obtained from healthy subjects and asthma patients. BALB/c mice, the airway epithelial cell line BEAS-2B, or primary human bronchial epithelial cells (HBECs) were immunized with allergens. Intracellular and extracellular HMGB1 were both detected. The role of GLUT1 was assessed by using a pharmacological antagonist BAY876. MGA patients have a significant higher sputum HMGB1 level than the health and subjects with other inflammatory phenotypes. Nuclear-to-cytoplasmic translocation of HMGB1 was also observed in the bronchial epithelia. Allergen exposure markedly induced GLUT1 expression in murine lungs and cultured epithelial cells. Pharmacological antagonism of GLUT1 with BAY876 dramatically decreased airway hyperresponsiveness, neutrophil and eosinophil accumulation, as well as type 2 inflammation in murine models of MGA. Besides, the allergen-induced up-regulation of HMGB1 was also partly recovered by BAY876, accompanied by inhibited secretion into the airway lumen. In vitro, treatment with BAY876 relieved the allergen-induced over-expression and secretion of HMGB1 in airway epithelia. Taken together, our data indicated that GLUT1 mediates bronchial epithelial HMGB1 release in MGA.

Liquid application dosing alters the physiology of air-liquid interface (ALI) primary human bronchial epithelial cell/lung fibroblast co-cultures and in vitro testing relevant endpoints.

Differentiated primary human bronchial epithelial cell (dpHBEC) cultures grown under air-liquid interface (ALI) conditions exhibit key features of the human respiratory tract and are thus critical for respiratory research as well as efficacy and toxicity testing of inhaled substances (e.g., consumer products, industrial chemicals, and pharmaceuticals). Many inhalable substances (e.g., particles, aerosols, hydrophobic substances, reactive substances) have physiochemical properties that challenge their evaluation under ALI conditions in vitro. Evaluation of the effects of these methodologically challenging chemicals (MCCs) in vitro is typically conducted by "liquid application," involving the direct application of a solution containing the test substance to the apical, air-exposed surface of dpHBEC-ALI cultures. We report that the application of liquid to the apical surface of a dpHBEC-ALI co-culture model results in significant reprogramming of the dpHBEC transcriptome and biological pathway activity, alternative regulation of cellular signaling pathways, increased secretion of pro-inflammatory cytokines and growth factors, and decreased epithelial barrier integrity. Given the prevalence of liquid application in the delivery of test substances to ALI systems, understanding its effects provides critical infrastructure for the use of in vitro systems in respiratory research as well as in the safety and efficacy testing of inhalable substances.

The combination of propylene glycol and vegetable glycerin e-cigarette aerosols induces airway inflammation and mucus hyperconcentration

Despite concerns over their safety, e-cigarettes (e-cigs) remain a popular tobacco product. Although nicotine and flavors found in e-cig liquids (e-liquids) can cause harm in the airways, whether the delivery vehicles propylene glycol (PG) and vegetable glycerin (VG) are innocuous when inhaled remains unclear. Here, we investigated the effects of e-cig aerosols generated from e-liquid containing only PG/VG on airway inflammation and mucociliary function in primary human bronchial epithelial cells (HBEC) and sheep. Primary HBEC were cultured at the air–liquid interface (ALI) and exposed to e-cig aerosols of 50%/50% v/v PG/VG. Ion channel conductance, ciliary beat frequency, and the expression of inflammatory markers, cell type-specific markers, and the major mucins MUC5AC and MUC5B were evaluated after seven days of exposure. Sheep were exposed to e-cig aerosols of PG/VG for five days and mucus concentration and matrix metalloproteinase-9 (MMP-9) activity were measured from airway secretions. Seven-day exposure of HBEC to e-cig aerosols of PG/VG caused a significant reduction in the activities of apical ion channels important for mucus hydration, including the cystic fibrosis transmembrane conductance regulator (CFTR) and large conductance, Ca2+-activated, and voltage-dependent K+ (BK) channels. PG/VG aerosols significantly increased the mRNA expression of the inflammatory markers interleukin-6 (IL6), IL8, and MMP9, as well as MUC5AC. The increase in MUC5AC mRNA expression correlated with increased immunostaining of MUC5AC protein in PG/VG-exposed HBEC. On the other hand, PG/VG aerosols reduced MUC5B expression leading overall to higher MUC5AC/MUC5B ratios in exposed HBEC. Other cell type-specific markers, including forkhead box protein J1 (FOXJ1), keratin 5 (KRT5), and secretoglobin family 1A member 1 (SCGB1A1) mRNAs, as well as overall ciliation, were significantly reduced by PG/VG exposure. Finally, PG/VG aerosols increased MMP-9 activity and caused mucus hyperconcentration in sheep in vivo. E-cig aerosols of PG/VG induce airway inflammation, increase MUC5AC expression, and cause dysfunction of ion channels important for mucus hydration in HBEC in vitro. Furthermore, PG/VG aerosols increase MMP-9 activity and mucus concentration in sheep in vivo. Collectively, these data show that e-cig aerosols containing PG/VG are likely to be harmful in the airways.

Surfactant protein D prevents mucin overproduction in airway goblet cells via SIRPα

Mucin overproduction is a common feature of chronic airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), and exacerbates their underlying respiratory condition. Surfactant protein D (SP-D) protects against airway diseases through modulation of immune reactions, but whether it also exerts direct effects on airway epithelial cells has remained unclear. Therefore, we sought to investigate the inhibitory role of SP-D on mucin production in airway epithelial cells. We prepared air–liquid interface (ALI) cultures of human primary bronchial epithelial cells (HBECs), which recapitulated a well-differentiated human airway epithelium. Benzo(a)pyrene (BaP), a key toxicant in cigarette smoke, induced mucin 5AC (MUC5AC) production in ALI-cultured HBECs, airway secretory cell lines, and airway epithelia of mice. Then, the protective effects of SP-D against the BaP-induced mucin overproduction were examined. BaP increased MUC5AC production in ALI cultures of HBECs, and this effect was attenuated by SP-D. SP-D also suppressed the BaP-induced phosphorylation of extracellular signal-regulated kinase (ERK) and MUC5AC expression in NCI-H292 goblet-like cells, but not in NCI-H441 club-like cells. Signal regulatory protein α (SIRPα) was found to be expressed in HBECs and NCI-H292 cells but absent in NCI-H441 cells. In NCI-H292 cells, SP-D activated SH2 domain-containing tyrosine phosphatase-1 (SHP-1), downstream of SIRPα, and knockdown of SIRPα abolished the suppressive effects of SP-D on BaP-induced ERK phosphorylation and MUC5AC production. Consistent with these in vitro findings, intratracheal instillation of SP-D prevented the BaP-induced phosphorylation of ERK and Muc5ac expression in airway epithelial cells in a mouse model. SP-D acts directly on airway epithelial cells to inhibit mucin secretion through ligation of SIRPα and SHP-1-mediated dephosphorylation of ERK. Targeting of SIRPα is therefore a potential new therapeutic approach to suppression of mucin hypersecretion in chronic airway diseases such as COPD and asthma.

Assessment of wood smoke induced pulmonary toxicity in normal- and chronic bronchitis-like bronchial and alveolar lung mucosa models at air–liquid interface

BackgroundChronic obstructive pulmonary disease (COPD) has the highest increased risk due to household air pollution arising from biomass fuel burning. However, knowledge on COPD patho-mechanisms is mainly limited to tobacco smoke exposure. In this study, a repeated direct wood smoke (WS) exposure was performed using normal- (bro-ALI) and chronic bronchitis-like bronchial (bro-ALI-CB), and alveolar (alv-ALI) lung mucosa models at air–liquid interface (ALI) to assess broad toxicological end points.MethodsThe bro-ALI and bro-ALI-CB models were developed using human primary bronchial epithelial cells and the alv-ALI model was developed using a representative type-II pneumocyte cell line. The lung models were exposed to WS (10 min/exposure; 5-exposures over 3-days; n = 6–7 independent experiments). Sham exposed samples served as control. WS composition was analyzed following passive sampling. Cytotoxicity, total cellular reactive oxygen species (ROS) and stress responsive NFkB were assessed by flow cytometry. WS exposure induced changes in gene expression were evaluated by RNA-seq (p ≤ 0.01) followed by pathway enrichment analysis. Secreted levels of proinflammatory cytokines were assessed in the basal media. Non-parametric statistical analysis was performed.Results147 unique compounds were annotated in WS of which 42 compounds have inhalation toxicity (9 very high). WS exposure resulted in significantly increased ROS in bro-ALI (11.2%) and bro-ALI-CB (25.7%) along with correspondingly increased NFkB levels (bro-ALI: 35.6%; bro-ALI-CB: 18.1%). A total of 1262 (817-up and 445-down), 329 (141-up and 188-down), and 102 (33-up and 69-down) genes were differentially regulated in the WS-exposed bro-ALI, bro-ALI-CB, and alv-ALI models respectively. The enriched pathways included the terms acute phase response, mitochondrial dysfunction, inflammation, oxidative stress, NFkB, ROS, xenobiotic metabolism of AHR, and chronic respiratory disorder. The enrichment of the ‘cilium’ related genes was predominant in the WS-exposed bro-ALI (180-up and 7-down). The pathways primary ciliary dyskinesia, ciliopathy, and ciliary movement were enriched in both WS-exposed bro-ALI and bro-ALI-CB. Interleukin-6 and tumor necrosis factor-α were reduced (p < 0.05) in WS-exposed bro-ALI and bro-ALI-CB.ConclusionFindings of this study indicate differential response to WS-exposure in different lung regions and in chronic bronchitis, a condition commonly associated with COPD. Further, the data suggests ciliopathy as a candidate pathway in relation to WS-exposure.

HA N193D substitution in the HPAI H5N1 virus alters receptor binding affinity and enhances virulence in mammalian hosts

ABSTRACT During the 2021/2022 winter season, we isolated highly pathogenic avian influenza (HPAI) H5N1 viruses harbouring an amino acid substitution from Asparagine(N) to Aspartic acid (D) at residue 193 of the hemagglutinin (HA) receptor binding domain (RBD) from migratory birds in South Korea. Herein, we investigated the characteristics of the N193D HA-RBD substitution in the A/CommonTeal/Korea/W811/2021[CT/W811] virus by using recombinant viruses engineered via reverse genetics (RG). A receptor affinity assay revealed that the N193D HA-RBD substitution in CT/W811 increases α2,6 sialic acid receptor binding affinity. The rCT/W811-HA193N virus caused rapid lethality with high virus titres in chickens compared with the rCT/W811-HA193D virus, while the rCT/W811-HA193D virus exhibited enhanced virulence in mammalian hosts with multiple tissue tropism. Surprisingly, a ferret-to-ferret transmission assay revealed that rCT/W811-HA193D virus replicates well in the respiratory tract, at a rate about 10 times higher than that of rCT/W811-HA193N, and all rCT/W811-HA193D direct contact ferrets were seroconverted at 10 days post-contact. Further, competition transmission assay of the two viruses revealed that rCT/W811-HA193D has enhanced growth kinetics compared with the rCT/W811-HA193N, eventually becoming the dominant strain in nasal turbinates. Further, rCT/W811-HA193D exhibits high infectivity in primary human bronchial epithelial (HBE) cells, suggesting the potential for human infection. Taken together, the HA-193D containing HPAI H5N1 virus from migratory birds showed enhanced virulence in mammalian hosts, but not in avian hosts, with multi-organ replication and ferret-to-ferret transmission. Thus, this suggests that HA-193D change increases the probability of HPAI H5N1 infection and transmission in humans.

Membrane lipid composition of bronchial epithelial cells influences antiviral responses during rhinovirus infection

ABSTRACT Lipids and their mediators have important regulatory functions in many cellular processes, including the innate antiviral response. The aim of this study was to compare the lipid membrane composition of in vitro differentiated primary bronchial epithelial cells (PBECs) with ex vivo bronchial brushings and to establish whether any changes in the lipid membrane composition affect antiviral defense of cells from donors without and with severe asthma. Using mass spectrometry, we showed that the lipid membrane of in vitro differentiated PBECs was deprived of polyunsaturated fatty acids (PUFAs) compared to ex vivo bronchial brushings. Supplementation of the culture medium with arachidonic acid (AA) increased the PUFA-content to more closely match the ex vivo membrane profile. Rhinovirus (RV16) infection of AA-supplemented cultures from healthy donors resulted in significantly reduced viral replication while release of inflammatory mediators and prostaglandin E2 (PGE2) was significantly increased. Indomethacin, an inhibitor of prostaglandin-endoperoxide synthases, suppressed RV16-induced PGE2 release and significantly reduced CXCL-8/IL-8 release from AA-supplemented cultures indicating a link between PGE2 and CXCL8/IL-8 release. In contrast, in AA-supplemented cultures from severe asthmatic donors, viral replication was enhanced whereas PTGS2 expression and PGE2 release were unchanged and CXCL8/IL-8 was significantly reduced in response to RV16 infection. While the PTGS2/COX-2 pathway is initially pro-inflammatory, its downstream products can promote symptom resolution. Thus, reduced PGE2 release during an RV-induced severe asthma exacerbation may lead to prolonged symptoms and slower recovery. Our data highlight the importance of reflecting the in vivo lipid profile in in vitro cell cultures for mechanistic studies.

Modelling bronchial epithelial-fibroblast cross-talk in idiopathic pulmonary fibrosis (IPF) using a human-derived in vitro air liquid interface (ALI) culture

Idiopathic Pulmonary Fibrosis (IPF) is a devastating form of respiratory disease with a life expectancy of 3–4 years. Inflammation, epithelial injury and myofibroblast proliferation have been implicated in disease initiation and, recently, epithelial-fibroblastic crosstalk has been identified as a central driver. However, the ability to interrogate this crosstalk is limited due to the absence of in vitro models that mimic physiological conditions. To investigate IPF dysregulated cross-talk, primary normal human bronchial epithelial (NHBE) cells and primary normal human lung fibroblasts (NHLF) or diseased human lung fibroblasts (DHLF) from IPF patients, were co-cultured in direct contact at the air–liquid interface (ALI). Intercellular crosstalk was assessed by comparing cellular phenotypes of co-cultures to respective monocultures, through optical, biomolecular and electrical methods. A co-culture-dependent decrease in epithelium thickness, basal cell mRNA (P63, KRT5) and an increase in transepithelial electrical resistance (TEER) was observed. This effect was significantly enhanced in DHLF co-cultures and lead to the induction of epithelial to mesenchymal transition (EMT) and increased mRNA expression of TGFβ-2, ZO-1 and DN12. When stimulated with exogenous TGFβ, NHBE and NHLF monocultures showed a significant upregulation of EMT (COL1A1, FN1, VIM, ASMA) and senescence (P21) markers, respectively. In contrast, direct NHLF/NHBE co-culture indicated a protective role of epithelial-fibroblastic cross-talk against TGFβ-induced EMT, fibroblast-to-myofibroblast transition (FMT) and inflammatory cytokine release (IL-6, IL-8, IL-13, IL-1β, TNF-α). DHLF co-cultures showed no significant phenotypic transition upon stimulation, likely due to the constitutively high expression of TGFβ isoforms prior to any exogenous stimulation. The model developed provides an alternative method to generate IPF-related bronchial epithelial phenotypes in vitro, through the direct co-culture of human lung fibroblasts with NHBEs. These findings highlight the importance of fibroblast TGFβ signaling in EMT but that monocultures give rise to differential responses compared to co-cultures, when exposed to this pro-inflammatory stimulus. This holds implications for any translation conclusions drawn from monoculture studies and is an important step in development of more biomimetic models of IPF. In summary, we believe this in vitro system to study fibroblast-epithelial crosstalk, within the context of IPF, provides a platform which will aid in the identification and validation of novel targets.

SARS-CoV-2-infected human airway epithelial cell cultures uniquely lack interferon and immediate early gene responses caused by other coronaviruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a member of a class of highly pathogenic coronaviruses. The large family of coronaviruses, however, also includes members that cause only mild symptoms, like human coronavirus-229E (HCoV-229E) or OC43 (HCoV-OC43). Unravelling how molecular (and cellular) pathophysiology differs between highly and low pathogenic coronaviruses is important for the development of therapeutic strategies. Here, we analysed the transcriptome of primary human bronchial epithelial cells (PBEC), differentiated at the air-liquid interface (ALI) after infection with SARS-CoV-2, SARS-CoV, Middle East Respiratory Syndrome (MERS)-CoV and HCoV-229E using bulk RNA sequencing. ALI-PBEC were efficiently infected by all viruses, and SARS-CoV, MERS-CoV and HCoV-229E infection resulted in a largely similar transcriptional response. The response to SARS-CoV-2 infection differed markedly as it uniquely lacked the increase in expression of immediate early genes, including FOS, FOSB and NR4A1 that was observed with all other coronaviruses. This finding was further confirmed in publicly available experimental and clinical datasets. Interfering with NR4A1 signalling in Calu-3 lung epithelial cells resulted in a 100-fold reduction in extracellular RNA copies of SARS-CoV-2 and MERS-CoV, suggesting an involvement in virus replication. Furthermore, a lack in induction of interferon-related gene expression characterised the main difference between the highly pathogenic coronaviruses and low pathogenic viruses HCoV-229E and HCoV-OC43. Our results demonstrate a previously unknown suppression of a host response gene set by SARS-CoV-2 and confirm a difference in interferon-related gene expression between highly pathogenic and low pathogenic coronaviruses.

The Asthma Risk Gene, GSDMB, Promotes Mitochondrial DNA-induced ISGs Expression.

Released mitochondrial DNA (mtDNA) in cells activates cGAS-STING pathway, which induces expression of interferon-stimulated genes (ISGs) and thereby promotes inflammation, as frequently seen in asthmatic airways. However, whether the genetic determinant, Gasdermin B (GSDMB), the most replicated asthma risk gene, regulates this pathway remains unknown. We set out to determine whether and how GSDMB regulates mtDNA-activated cGAS-STING pathway and subsequent ISGs induction in human airway epithelial cells. Using qPCR, ELISA, native polyacrylamide gel electrophoresis, co-immunoprecipitation and immunofluorescence assays, we evaluated the regulation of GSDMB on cGAS-STING pathway in both BEAS-2B cells and primary normal human bronchial epithelial cells (nHBEs). mtDNA was extracted in plasma samples from human asthmatics and the correlation between mtDNA levels and eosinophil counts was analyzed. GSDMB is significantly associated with RANTES expression in asthmatic nasal epithelial brushing samples from the Genes-environments and Admixture in Latino Americans (GALA) II study. Over-expression of GSDMB promotes DNA-induced IFN and ISGs expression in bronchial epithelial BEAS-2B cells and nHBEs. Conversely, knockout of GSDMB led to weakened induction of interferon (IFNs) and ISGs in BEAS-2B cells. Mechanistically, GSDMB interacts with the C-terminus of STING, promoting the translocation of STING to Golgi, leading to the phosphorylation of IRF3 and induction of IFNs and ISGs. mtDNA copy number in serum from asthmatics was significantly correlated with blood eosinophil counts especially in male subjects. GSDMB promotes the activation of mtDNA and poly (dA:dT)-induced activation of cGAS-STING pathway in airway epithelial cells, leading to enhanced induction of ISGs.

Extracellular vesicles secreted by primary human bronchial epithelial cells reduce Pseudomonas aeruginosa burden and inflammation in cystic fibrosis mouse lung.

Cystic fibrosis (CF) results in a reduction in the volume of airway surface liquid, increased accumulation of viscous mucus, persistent antibiotic-resistant lung infections that cause chronic inflammation, and a decline in lung function. More than 50% of adults with CF are chronically colonized by Pseudomonas aeruginosa (P. aeruginosa), the primary reason for morbidity and mortality in people with CF (pwCF). Although highly effective modulator therapy (HEMT) is an important part of disease management in CF, HEMT does not eliminate P. aeruginosa or lung inflammation. Thus, new treatments are required to reduce lung infection and inflammation in CF. In a previous in vitro study, we demonstrated that primary human bronchial epithelial cells (HBECs) secrete extracellular vesicles (EVs) that block the ability of P. aeruginosa to form biofilms by reducing the abundance of several proteins necessary for biofilm formation as well as enhancing the sensitivity of P. aeruginosa to β-lactam antibiotics. In this study, using a CF mouse model of P. aeruginosa infection, we demonstrate that intratracheal administration of EVs secreted by HBEC reduced P. aeruginosa lung burden and several proinflammatory cytokines including IFN-γ, TNF-α, and MIP-1β in bronchoalveolar lavage fluid (BALF), even in the absence of antibiotics. Moreover, EVs decreased neutrophils in BALF. Thus, EVs secreted by HBEC reduce the lung burden of P. aeruginosa, decrease inflammation, and reduce neutrophils in a CF mouse model. These results suggest that HBEC via the secretion of EVs may play an important role in the immune response to P. aeruginosa lung infection.NEW & NOTEWORTHY Our findings show that extracellular vesicles secreted by primary human bronchial epithelial cells significantly reduce Pseudomonas aeruginosa burden, inflammation, and weight loss in a cystic fibrosis mouse model of infection.

Inhibition of SARS-CoV-2 infection and replication by Petasites hybridus CO2-extract (Ze 339)

BackgroundThe intensified search for low-threshold herbal anti-viral drugs would be of great advantage in prevention of early stages of infection. Since the SARS-CoV-2 Omicron variant has prevailed in western countries, the course has only been mild, but there are still no widely available drugs that can alleviate or shorten disease progression and counteract the development of Long-COVID. This study aimed to investigate the antiviral effects of a CO2-extract from Petasites hybridus (Ze 339). MethodsWe analyzed the infection and replication rate of SARS-CoV-2 in primary normal human bronchial epithelial cells (NHBEs) using a GFP-expressing version of the wild-type SARS-CoV-2 virus and live cell imaging. Upon infection with a clinical isolate of the Omicron variant, viral RNA content was quantified, and plaque assays were performed. In addition, the human transcriptome was analyzed after 4- and 24-hours post infection using whole genome microarrays. ResultsZe 339 had a protective effect on primary airway epithelial cells during SARS-CoV-2 infection and impeded SARS-CoV-2 infection and replication in NHBE. Notably, Ze 339 inhibited the expression of infection-induced IFNA10 by 8.6-fold (p < 0.05) and additionally reduced a wide range of other interferons (IFNA6, IFNA7, IFNA8, IFNA21, IFNE, IFNW1). ConclusionThereby, Ze 339 attenuated epithelial infection by SARS-CoV-2 and modeled the IFN response. In conclusion, this study highlights Ze 339 as a potential treatment option for COVID-19 that limits infection-associated cell intrinsic immune responses.