Porcine Airway Epithelial Cells Research Articles

Validation of airway porcine epithelial cells as an alternative to human in vitro preclinical studies

Animal models are currently used in several fields of biomedical research as useful alternatives to human-based studies. However, the obtained results do not always effectively translate into clinical applications, due to interspecies anatomical and physiological differences. Detailed comparability studies are therefore required to verify whether the selected animal species could be a representative model for the disease or for cellular process under investigation. This has proven to be fundamental to obtaining reliable data from preclinical studies. Among the different species, swine is deemed an excellent animal model in many fields of biological research, and has been largely used in respiratory medicine, considering the high homology between human and swine airways. In the context of in vitro studies, the validation of porcine airway epithelial cells as an alternative to human epithelial cells is crucial. In this paper, porcine and human tracheal and bronchial epithelial cells are compared in terms of in vivo tissue architecture and in vitro cell behaviour under standard and airlifted conditions, analyzing the regenerative, proliferative and differentiative potentials of these cells. We report multiple analogies between the two species, validating the employment of porcine airway epithelial cells for most in vitro preclinical studies, although with some limitations due to species-related divergences.

Analysis of Nipah Virus Replication and Host Proteome Response Patterns in Differentiated Porcine Airway Epithelial Cells Cultured at the Air-Liquid Interface.

Respiratory tract epithelium infection plays a primary role in Nipah virus (NiV) pathogenesis and transmission. Knowledge about infection dynamics and host responses to NiV infection in respiratory tract epithelia is scarce. Studies in non-differentiated primary respiratory tract cells or cell lines indicate insufficient interferon (IFN) responses. However, studies are lacking in the determination of complex host response patterns in differentiated respiratory tract epithelia for the understanding of NiV replication and spread in swine. Here we characterized infection and spread of NiV in differentiated primary porcine bronchial epithelial cells (PBEC) cultivated at the air-liquid interface (ALI). After the initial infection of only a few apical cells, lateral spread for 12 days with epithelium disruption was observed without releasing substantial amounts of infectious virus from the apical or basal sides. Deep time course proteomics revealed pronounced upregulation of genes related to type I/II IFN, immunoproteasomal subunits, transporter associated with antigen processing (TAP)-mediated peptide transport, and major histocompatibility complex (MHC) I antigen presentation. Spliceosomal factors were downregulated. We propose a model in which NiV replication in PBEC is slowed by a potent and broad type I/II IFN host response with conversion from 26S proteasomes to immunoproteasomal antigen processing and improved MHC I presentation for adaptive immunity priming. NiV induced cytopathic effects could reflect the focal release of cell-associated NiV, which may contribute to efficient airborne viral spread between pigs.

Time-dependent viral interference between influenza virus and coronavirus in the infection of differentiated porcine airway epithelial cells

ABSTRACT Coronaviruses and influenza viruses are circulating in humans and animals all over the world. Co-infection with these two viruses may aggravate clinical signs. However, the molecular mechanisms of co-infections by these two viruses are incompletely understood. In this study, we applied air-liquid interface (ALI) cultures of well-differentiated porcine tracheal epithelial cells (PTECs) to analyze the co-infection by a swine influenza virus (SIV, H3N2 subtype) and porcine respiratory coronavirus (PRCoV) at different time intervals. Our results revealed that in short-term intervals, prior infection by influenza virus caused complete inhibition of coronavirus infection, while in long-term intervals, some coronavirus replication was detectable. The influenza virus infection resulted in (i) an upregulation of porcine aminopeptidase N, the cellular receptor for PRCoV and (ii) in the induction of an innate immune response which was responsible for the inhibition of PRCoV replication. By contrast, prior infection by coronavirus only caused a slight inhibition of influenza virus replication. Taken together, the timing and the order of virus infection are important determinants in co-infections. This study is the first to show the impact of SIV and PRCoV co- and super-infection on the cellular level. Our results have implications also for human viruses, including potential co-infections by SARS-CoV-2 and seasonal influenza viruses.

Transport properties in CFTR\u2212/\u2212 knockout piglets suggest normal airway surface liquid pH and enhanced amiloride-sensitive Na+ absorption

Previous analysis of CFTR-knockout (CFTR−/−) in piglets has provided important insights into the pathology of cystic fibrosis. However, controversies exist as to the true contribution of CFTR to the pH balance in airways and intestine. We therefore compared ion transport properties in newborn wild-type (CFTR+/+) and CFTR-knockout (CFTR−/− piglets). Tracheas of CFTR−/− piglets demonstrated typical cartilage malformations and muscle cell bundles. CFTR−/− airway epithelial cells showed enhanced lipid peroxidation, suggesting inflammation early in life. CFTR was mainly expressed in airway submucosal glands and was absent in lungs of CFTR−/− piglets, while expression of TMEM16A was uncompromised. mRNA levels for TMEM16A, TMEM16F, and αβγENaC were unchanged in CFTR−/− airways, while mRNA for SLC26A9 appeared reduced. CFTR was undetectable in epithelial cells of CFTR−/− airways and intestine. Small intestinal epithelium of CFTR−/− piglets showed mucus accumulation. Secretion of both electrolytes and mucus was activated by stimulation with prostaglandin E2 and ATP in the intestine of CFTR+/+, but not of CFTR−/− animals. pH was measured inside small bronchi using a pH microelectrode and revealed no difference between CFTR+/+ and CFTR−/− piglets. Intracellular pH in porcine airway epithelial cells revealed only a small contribution of CFTR to bicarbonate secretion, which was absent in cells from CFTR−/− piglets. In contrast to earlier reports, our data suggest a minor impact of CFTR on ASL pH. In contrast, enhanced amiloride-sensitive Na+ absorption may contribute to lung pathology in CFTR−/− piglets, along with a compromised CFTR- and TMEM16A-dependent Cl− transport.

Rock Inhibitor Y-27632 Enables Feeder-Free, Unlimited Expansion of Sus scrofa domesticus Swine Airway Stem Cells to Facilitate Respiratory Research.

Current limitations in the efficacy of treatments for chronic respiratory disorders position them as prospective regenerative medicine therapeutic targets. A substantial barrier to these ambitions is that research requires large numbers of cells whose acquisition is hindered by the limited availability of human tissue samples leading to an overreliance on physiologically dissimilar rodents. The development of cell culture strategies for airway cells from large mammals will more effectively support the transition from basic research to clinical therapy. Using readily available porcine lungs, we isolated conducting airway tissue and subsequently a large number of porcine airway epithelial cells (pAECs) using a digestion and mechanical scraping technique. Cells were cultured in a variety of culture media formulations, both foetal bovine serum-containing and serum-free media, in air (21%) and physiological (2%) oxygen tension and in the presence and absence of Rho kinase inhibitor Y-27362 (RI). Cell number at isolation and subsequent population doublings were determined; cells were characterised during culture and following differentiation by immunofluorescence, histology, and IL-8 ELISA. Cells were positive for epithelial markers (pan-cytokeratin and E-cadherin) and negative for fibroblastic markers (vimentin and smooth muscle actin). Supplementation of cultures with Y-27632 allowed for unlimited expansion whilst sustaining an epithelial phenotype. Early passage pAECs readily produced differentiated air-liquid interface (ALI) cultures with a capacity for mucociliary differentiation retained after substantial expansion, strongly modulated by the culture condition applied. Primary pAECs will be a useful tool to further respiratory-oriented research whilst RI-expanded pAECs are a promising tool, particularly with further optimisation of culture conditions.

Major histocompatibility complex I of swine respiratory cells presents conserved regions of influenza proteins.

Influenza A virus in swine (IAV-S) is a prevalent respiratory pathogen in pigs that has deleterious consequences to animal and human health. Pigs represent an important reservoir for influenza and potential mixing vessel for novel gene reassortments. Despite the central role of pigs in recent influenza outbreaks, much remains unknown about the impact of swine immunity on IAV-S transmission, pathogenesis, and evolution. An incomplete understanding of interactions between the porcine immune system and IAV-S has hindered development of new diagnostic tools and vaccines. In order to address this gap in knowledge, we identified swine leukocyte antigen (SLA) restricted IAV-S peptides presented by porcine airway epithelial cells using an immunoproteomics approach. The majority of MHC-associated peptides belonged to matrix 1, nucleoprotein and nonstructural 1 proteins. Future investigation of the potential cross-reactive nature of these peptides is needed to confirm antigen recognition by cytotoxic T lymphocytes and their utility as vaccine candidates.

Novel GP64 envelope variants for improved delivery to human airway epithelial cells.

Lentiviral vectors pseudotyped with the baculovirus envelope protein GP64 transduce primary cultures of human airway epithelia (HAE) at their apical surface. Our goal in this study was to harness a directed evolution approach to develop a novel envelope glycoprotein with increased transduction properties for HAE. Using error-prone PCR, a library of GP64 mutants was generated and used to prepare a diverse pool of lentiviral virions pseudotyped with GP64 variants. The library was serially passaged on HAE and three GP64 mutations were recovered. Single-, double- and the triple-combination mutant envelope glycoproteins were compared with wild-type GP64 for their ability to transduce HAE. Our results suggest that lentiviral vectors pseudotyped with evolved GP64 transduced HAE with greater efficiency than wild-type GP64. This effect was not observed in primary cultures of porcine airway epithelial cells, suggesting that the directed evolution protocol was species specific. In summary, our studies indicate that serial passage of a GP64 mutant library yielded specific variants with improved HAE cell tropism, yielding tools with the potential to improve the success of gene therapy for airway diseases.

Characterization of miRNAs involved in response to poly(I:C) in porcine airway epithelial cells.

MicroRNAs (miRNA) have been implicated in a variety of pathological conditions including infectious diseases. Knowledge of the miRNAs affected by poly(I:C), a synthetic analog of viral double-stranded RNA, in porcine airway epithelial cells (PAECs) contributes to understanding the mechanisms of swine viral respiratory diseases, which bring enormous economic loss worldwide every year. In this study, we used high throughput sequencing to profile miRNA expression in PAECs treated with poly(I:C) as compared to the untreated control. This approach revealed 23 differentially expressed miRNAs (DEMs), five of which have not been implicated in viral infection before. Nineteen of the 23 miRNAs were down-regulated including members of the miR-17-92 cluster, a well-known polycistronic oncomir and extensively involved in viral infection in humans. Target genes of DEMs, predicted using bioinformatic methods and validated by luciferase reporter analysis on two representative DEMs, were significantly enriched in several pathways including transforming growth factor-β signaling. A large quantity of sequence variations (isomiRs) were found including a substitution at position 5, which was verified to redirect miRNAs to a new spectrum of targets by luciferase reporter assay together with bioinformatics analysis. Twelve novel porcine miRNAs conserved in other species were identified by homology analysis together with cloning verification. Furthermore, the expression analysis revealed the potential importance of three novel miRNAs in porcine immune response to viruses. Overall, our data contribute to clarifying the mechanisms underlying the host immune response against respiratory viruses in pigs, and enriches the repertoire of porcine miRNAs.

Species-specific and individual differences in Nipah virus replication in porcine and human airway epithelial cells.

Highly pathogenic Nipah virus (NiV) causes symptomatic infections in pigs and humans. The severity of respiratory symptoms is much more pronounced in pigs than in humans, suggesting species-specific differences of NiV replication in porcine and human airways. Here, we present a comparative study on productive NiV replication in primary airway epithelial cell cultures of the two species. We reveal that NiV growth substantially differs in primary cells between pigs and humans, with a more rapid spread of infection in human airway epithelia. Increased replication, correlated with higher endogenous expression levels of the main NiV entry receptor ephrin-B2, not only significantly differed between airway cells of the two species but also varied between cells from different human donors. To our knowledge, our study provides the first experimental evidence of species-specific and individual differences in NiV receptor expression and replication kinetics in primary airway epithelial cells. It remains to be determined whether and how these differences contribute to the viral host range and pathogenicity.

Differential Induction of Type I and Type III Interferons by Swine and Human Origin H1N1 Influenza A Viruses in Porcine Airway Epithelial Cells.

Interferons (IFNs) have been shown to inhibit influenza A virus (IAV) replication and play an essential role in controlling viral infection. Here we studied the kinetics and magnitude of induction of type I and type III IFN transcripts by primary porcine airway epithelial cells (pAECs) in response to swine and human origin IAV. We observed that swine influenza viruses (SIV) replicate more efficiently than the human pandemic influenza A/California/2009 (pH1N1 CA/09) in pAECs. Interestingly, we also found significant difference in kinetics of IFN-β, IFN-λ1 and IFN-λ3 gene expression by these viruses. While there was delay of up to 12 hours post infection (h p.i.) in induction of IFN genes in pAECs infected with swine IAV A/Sw/Illinois/2008 (H1N1 IL/08), human pH1N1 CA/09 rapidly induced IFN-β, IFN-λ1 and IFN-λ3 gene expression as early as 4 h p.i. However, the magnitude of IFN-β and IFN-λ3 induction at 24 h p.i. was not significantly different between the viral strains tested. Additionally, we found that swine H1N1 IL/08 was less sensitive to dsRNA induced antiviral response compared to human pH1N1 CA/09. Our data suggest that the human and swine IAVs differ in their ability to induce and respond to type I and type III interferons in swine cells. Swine origin IAV may have adapted to the pig host by subverting innate antiviral responses to viral infection.

Immune responses of porcine airway epithelial cells to poly(I:C), a synthetic analogue of viral double-stranded RNA

Yang, X.-q., Wang, L., Li, H.-t. and Liu, D. 2015. Immune responses of porcine airway epithelial cells to poly(I:C), a synthetic analogue of viral double-stranded RNA. Can. J. Anim. Sci. 95: 13–20. Swine respiratory disease (SRD) is one of the most economically important diseases affecting the pig industry. The main infectious agents that cause SRD are viruses, but the molecular pathogenesis of viral SRD has not been extensively studied. Here, using digital gene expression tag profiling, the global transcriptional responses to poly(I:C), a synthetic analogue of viral double-stranded RNA, was analyzed in porcine airway epithelial cells (PAECs). The profiling analysis revealed numerous differentially expressed genes (DEGs), including unknown sequences in the porcine nucleotide databases. Gene ontology enrichment analysis showed that DEGs were mainly enriched in response to stress (GO: 0006950), of which, defense response is one sub-process. Poly(I:C) challenge induced a general inflammation response as indicated by marked upregulation of a variety of pathogen recognition receptors, interferon-stimulated genes, proinflammatory cytokines, and chemokines, together with the significant downregulation of anti-inflammatory molecules. Furthermore, the antiapoptotic pathway was triggered, as demonstrated by the significant suppression of molecules involved in the induction of apoptosis, together with the significant stimulation of putative inhibitor of apoptosis. The results indicate that PAECs initiated defense against poly(I:C) challenge through the inflammation responses, whereas poly(I:C) can utilize antiapoptotic pathway to evade host defense.

Infection of Differentiated Porcine Airway Epithelial Cells by Influenza Virus: Differential Susceptibility to Infection by Porcine and Avian Viruses

BackgroundSwine are important hosts for influenza A viruses playing a crucial role in the epidemiology and interspecies transmission of these viruses. Respiratory epithelial cells are the primary target cells for influenza viruses.Methodology/Principal FindingsTo analyze the infection of porcine airway epithelial cells by influenza viruses, we established precision-cut lung slices as a culture system for differentiated respiratory epithelial cells. Both ciliated and mucus-producing cells were found to be susceptible to infection by swine influenza A virus (H3N2 subtype) with high titers of infectious virus released into the supernatant already one day after infection. By comparison, growth of two avian influenza viruses (subtypes H9N2 and H7N7) was delayed by about 24 h. The two avian viruses differed both in the spectrum of susceptible cells and in the efficiency of replication. As the H9N2 virus grew to titers that were only tenfold lower than that of a porcine H3N2 virus this avian virus is an interesting candidate for interspecies transmission. Lectin staining indicated the presence of both α-2,3- and α-2,6-linked sialic acids on airway epithelial cells. However, their distribution did not correlate with pattern of virus infection indicating that staining by plant lectins is not a reliable indicator for the presence of cellular receptors for influenza viruses.Conclusions/SignificanceDifferentiated respiratory epithelial cells significantly differ in their susceptibility to infection by avian influenza viruses. We expect that the newly described precision-cut lung slices from the swine lung are an interesting culture system to analyze the infection of differentiated respiratory epithelial cells by different pathogens (viral, bacterial and parasitic ones) of swine.

β-catenin/Tcf signaling in squamous differentiation of porcine airway epithelial cells

For a preliminary study of the role of beta-catenin/Tcf signaling in squamous differentiation of airway (tracheobronchial) epithelial cells, a stable mutant of beta-catenin was transfected into primarily cultured porcine airway epithelial cells. Western blotting revealed that exogenous protein was observed in large quantity in cytoplasm and nucleus. When co-transfected with Tcf luciferase reporter plasmids, beta-catenin mutant increased the reporter's transcriptional activities. However, mRNA expression of a squamous differentiation marker, small proline-rich protein (SPRP), was not elevated, as shown by reverse transcription-polymerase chain reaction. These findings suggest that beta-catenin/Tcf signaling may not be directly involved in the squamous differentiation of porcine airway epithelial cells.

Effects of cigarette smoke extract on E-cadherin expression in cultured airway epithelial cells.

To investigate whether the change of E-cadherin (ECD) expression plays a role in the injury and repair of airway epithelial cells (AEC) caused by smoking, porcine AECs were cultured by using an enzyme-dispersed method. After exposure of the AECs to cigarette smoke extract (CSE), the ECD expression in the cells was detected by using immunocytochemistry and in situ hybridization. The results showed that ECD was distributed on the plasma membrane at the cell junctions of AECs. After exposure to 20% CSE, the membranous ECD expression was decreased, the cytoplasmic ECD expression was increased (P < 0.01) as the exposure time went on. But the content of ECD mRNA in the AECs did not chang. It suggests that the change of ECD expression is regulated at the posttranslational level and plays a role in the injury and repair of AEC caused by smoking.

Interaction of liposomal and polycationic transfection complexes with pulmonary surfactant.

The delivery of genes to the airways holds promise for the treatment of lung diseases such as cystic fibrosis and asthma. Current non-viral gene delivery systems lack sufficient transfection efficiency. Pulmonary surfactant has been reported to be a barrier to gene transfer into the airways. Here we analyze the interaction of liposomal and polycationic transfection complexes with pulmonary surfactant. The efficiency of non-viral transfection of cultured human airway epithelial cells (16HBE14o-), COS7 cells and porcine primary airway epithelial cells was studied in the presence of various surfactant preparations in order to model the conditions prevailing in the airways during transfection. The natural pulmonary surfactant, Alveofact, an extract from bovine lung lavage, was found to inhibit lipofection with lipofectAMINE for all cell lines investigated. Dendrimer meditated polyfection was unaffected for pulmonary cell lines and was weakly affected for COS7 cells. PEI-mediated polyfection was unaffected for all cell lines tested. The synthetic surfactant preparation Exosurf containing L-alpha-phosphatidylcholine-dipalmitoyl (DPPC) as the sole lipid ingredient had no statistically significant effect on polymer- and lipid-mediated transfection. The transfection efficiencies are related to structural changes in the DNA complexes as demonstrated by DNase-accessibility tests and fluorescence spectroscopy. In the presence of the phospholipid POPG, which is a constituent of Alveofact, DNA condensed in lipofectAMINE lipoplexes became accessible to DNaseI, while DNA condensed with PAMAM dendrimer or PEI was less accessible to DNase I as compared to lipoplexes. Consistently, the fluorescence of a DNA-intercalating dye increased after addition of Alveofact only in the case of lipoplexes. In contrast to lipofection, gene transfer with cationic polymers to airway epithelial cells is not inhibited by pulmonary surfactant in vitro. Depending on the surfactant concentration even an increase in polymermediated transfection can be seen. In conclusion, cationic polymers appear to be the more stable gene delivery systems for topical application into the airways.

Identification and function of cyclic nucleotide phosphodiesterase isoenzymes in airway epithelial cells.

Epithelial cells actively participate in inflammatory airway disease by liberating mediators such as arachidonate metabolites and cytokines. Inhibition of phosphodiesterases (PDEs) may be a useful anti-inflammatory approach. The PDE isoenzyme pattern and the effects of PDE inhibition on mediator generation were analyzed in primary cultures of human and porcine airway epithelial cells (AEC) and in the bronchial epithelial cell line BEAS-2B. PDE4 and PDE5 were detected in lysates of all cell types studied. In primary cultures of human AEC, the PDE4 variants PDE4A5, PDE4C1, PDE4D2, and PDE4D3 were identified by polymerase chain reaction analysis. Evidence of the recently described PDE7 was obtained by rolipram- insensitive cyclic adenosine monophosphate (cAMP) degradation, and its presence was verified by the demonstration of PDE7 messenger RNA. Primary cultures of human airway epithelium also expressed PDE1. Enhanced epithelial cAMP levels, induced by forskolin and PDE4 inhibition, increased formation of prostaglandin E2 (PGE2), but not of interleukin (IL)-8 or 15-hydroxyeicosatetraenoic acid (15-HETE) in airway epithelial cells. Increased cyclic guanosine monophosphate levels in these cells provoked by sodium nitroprusside and the PDE5 inhibitor zaprinast reduced the PGE2 synthesis, whereas 15-HETE and IL-8 formation were unchanged. The data suggest that PDE isoenzymes are important in airway inflammation and that PDE inhibitors exert anti-inflammatory effects by acting on AEC.

Endothelin-1 Stimulates Proliferation of Normal Airway Epithelial Cells

To investigate the influence of endothelins on airway epithelial cell growth, we measured [3H]thymidine incorporation and cell numbers of cultured porcine tracheal epithelial cells in the presence or absence of endothelin-1 or -3 with or without PD-145065 (a combined endothelin-A and -B receptor antagonist), BQ-123 (an antagonist specific for endothelin-A receptors) or phosphoramidon (an inhibitor, in part, of endothelin converting enzymes). We found that endothelin-1 stimulated the proliferation of airway epithelial, cells and this response was progressively inhibited by increasing concentrations of either PD-145065 or BQ-123. In contrast to endothelin-l, airway epithelial cells were not responsive to endothelin3. They also appeared to be producing endothelin-l endogenously. Phosphoramidon significantly decreased basal growth of cells incubated in the absence of exogenous endothelin, and was associated with a significant diminution in their endothelin production. We conclude that endothelin-l is mitogenic for porcine airway epithelial cells and may be involved in both autocrine and paracrine control of them.

Histochemical and morphologic changes of porcine airway epithelial cells in response to infection with Mycoplasma hyopneumoniae

SUMMARY Mycoplasma hyopneumoniae causes pneumonia in pigs. The effect of infection by this organism on histochemical characteristics of airway mucin within epithelial cells was studied. Seven- to 10-week-old pigs were inoculated intratracheally with M hyopneumoniae or culture broth, and lung tissues were collected from inoculated and control pigs at 2, 4, and 6 weeks after inoculation. Tissue sections were stained with periodic acid-Schiff/Alcian blue, pH 2.5 or high iron diamine/Alcian blue. Histologic features of randomly selected bronchi, bronchioles, and submucosal glands were compared in sections stained with periodic acid-Schiff/Alcian blue. Bronchial goblet cell sulfomucin and sialomucin were quantitated by image analysis of sections stained with high iron diamine/Alcian blue. Bronchi and bronchioles of infected pigs contained proportionately fewer goblet cells with mucin at all stages of infection than age-matched control pigs. Goblet cells in bronchi of infected pigs contained significantly less total mucin and sialomucin, and significantly more sulfomucin than goblet cells of control pigs. Increased sulfated mucin in bronchial goblet cells may reflect altered glycoprotein production or secretion in response to infection with M hyopneumoniae.