In vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo.

Respiratory syncytial virus (RSV) is the major viral cause of severe pulmonary disease in young infants worldwide. However, the mechanisms by which RSV causes disease in humans remain poorly understood. To help bridge this gap, we developed an ex vivo/in vitro model of RSV infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs), the primary targets of RSV infection in vivo. Our RSV/WD-PBEC model demonstrated remarkable similarities to hallmarks of RSV infection in infant lungs. These hallmarks included restriction of infection to noncontiguous or small clumps of apical ciliated and occasional nonciliated epithelial cells, apoptosis and sloughing of apical epithelial cells, occasional syncytium formation, goblet cell hyperplasia/metaplasia, and mucus hypersecretion. RSV was shed exclusively from the apical surface at titers consistent with those in airway aspirates from hospitalized infants. Furthermore, secretion of proinflammatory chemokines such as CXCL10, CCL5, IL-6, and CXCL8 reflected those chemokines present in airway aspirates. Interestingly, a recent RSV clinical isolate induced more cytopathogenesis than the prototypic A2 strain. Our findings indicate that this RSV/WD-PBEC model provides an authentic surrogate for RSV infection of airway epithelium in vivo. As such, this model may provide insights into RSV pathogenesis in humans that ultimately lead to successful RSV vaccines or therapeutics.

Glucocorticoid receptor ß and histone deacetylase 1 and 2 expression in the airways of severe asthma.

RATIONALE: Upregulation of glucocorticoid receptor ß (GRß) has been implicated in steroid resistance in severe asthma, although previous studies are conflicting. GRß has been proposed as a dominant negative isoform of glucocorticoid receptor α (GRα) but it has also been suggested that GRß can cause steroid resistance via reduced expression of histone deacetylase 2 (HDAC2), a key regulator of steroid responsiveness in the airway. OBJECTIVES: To examine GRß, GRα, HDAC1 and HDAC2 expression at transcript and protein levels in bronchial biopsies from a large series of patients with severe asthma, and to compare the findings with those of patients with mild to moderate asthma and healthy volunteers. METHODS: Bronchoscopic study in two UK centres with real-time PCR and immunohistochemistry performed on biopsies, western blotting of bronchial epithelial cells and immunoprecipitation with anti-GRß antibody. MEASUREMENTS AND MAIN RESULTS: Protein and mRNA expression for GRα and HDAC2 did not differ between groups. GRß mRNA was detected in only 13 of 73 samples (seven patients with severe asthma), however immunohistochemistry showed widespread epithelial staining in all groups. Western blotting of bronchial epithelial cells with GRß antibody detected an additional 'cross-reacting' protein, identified as clathrin. HDAC1 expression was increased in patients with severe asthma compared with healthy volunteers. CONCLUSIONS: GRß mRNA is expressed at low levels in a minority of patients with severe asthma. HDAC1 and HDAC2 expression was not downregulated in severe asthma. These data do not support upregulated GRß and resultant reduced HDAC expression as the principal mechanism of steroid resistance in severe asthma. Conflicting GRß literature may be explained in part by clathrin cross-reactivity with commercial antibodies.

Cytopathogenesis of Sendai virus in well-differentiated primary pediatric bronchial epithelial cells.

Sendai virus (SeV) is a murine respiratory virus of considerable interest as a gene therapy or vaccine vector, as it is considered nonpathogenic in humans. However, little is known about its interaction with the human respiratory tract. To address this, we developed a model of respiratory virus infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs). These physiologically authentic cultures are comprised of polarized pseudostratified multilayered epithelium containing ciliated, goblet, and basal cells and intact tight junctions. To facilitate our studies, we rescued a replication-competent recombinant SeV expressing enhanced green fluorescent protein (rSeV/eGFP). rSeV/eGFP infected WD-PBECs efficiently and progressively and was restricted to ciliated and nonciliated cells, not goblet cells, on the apical surface. Considerable cytopathology was evident in the rSeV/eGFP-infected cultures postinfection. This manifested itself by ciliostasis, cell sloughing, apoptosis, and extensive degeneration of WD-PBEC cultures. Syncytia were also evident, along with significant basolateral secretion of proinflammatory chemokines, including IP-10, RANTES, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), interleukin 6 (IL-6), and IL-8. Such deleterious responses are difficult to reconcile with a lack of pathogenesis in humans and suggest that caution may be required in exploiting replication-competent SeV as a vaccine vector. Alternatively, such robust responses might constitute appropriate normal host responses to viral infection and be a prerequisite for the induction of efficient immune responses.

Differential cytopathogenesis of respiratory syncytial virus prototypic and clinical isolates in primary pediatric bronchial epithelial cells.

BACKGROUND: Human respiratory syncytial virus (RSV) causes severe respiratory disease in infants. Airway epithelial cells are the principle targets of RSV infection. However, the mechanisms by which it causes disease are poorly understood. Most RSV pathogenesis data are derived using laboratory-adapted prototypic strains. We hypothesized that such strains may be poorly representative of recent clinical isolates in terms of virus/host interactions in primary human bronchial epithelial cells (PBECs). METHODS: To address this hypothesis, we isolated three RSV strains from infants hospitalized with bronchiolitis and compared them with the prototypic RSV A2 in terms of cytopathology, virus growth kinetics and chemokine secretion in infected PBEC monolayers. RESULTS: RSV A2 rapidly obliterated the PBECs, whereas the clinical isolates caused much less cytopathology. Concomitantly, RSV A2 also grew faster and to higher titers in PBECs. Furthermore, dramatically increased secretion of IP-10 and RANTES was evident following A2 infection compared with the clinical isolates. CONCLUSIONS: The prototypic RSV strain A2 is poorly representative of recent clinical isolates in terms of cytopathogenicity, viral growth kinetics and pro-inflammatory responses induced following infection of PBEC monolayers. Thus, the choice of RSV strain may have important implications for future RSV pathogenesis studies.

Effects of IL-13 on mucociliary differentiation of pediatric asthmatic bronchial epithelial cells.

Goblet cell hyperplasia (GCH) and decreased ciliated cells are characteristic of asthma. We examined the effects of IL-13 (2 and 20 ng/mL) on in vitro mucociliary differentiation in pediatric bronchial epithelial cells (PBECs) of normal PBEC [PBEC(N)] and asthmatic PBEC [PBEC(A)] children. Markers of differentiation, real-time PCR for MUC5AC, MUC5AC ELISA, and transepithelial electrical resistance (TEER) were assessed. Stimulation with 20 ng/mL IL-13 in PBEC(N) resulted in GCH [20 ng/mL IL-13: mean, 33.8% (SD, 7.2) versus unstimulated: mean, 18.9% (SD, 5.0); p < 0.0001] and decreased ciliated cell number [20 ng/mL IL-13: mean, 8% (SD, 5.6) versus unstimulated: mean, 22.7% (SD,7.6); p < 0.01]. PBEC(N) stimulated with 20 ng/mL IL-13 resulted in >5-fold (SD, 3.2) increase in MUC5AC mRNA expression, p < 0.001, compared with unstimulated PBEC(N). In PBEC(A), GCH was also seen [20 ng/mL IL-13: mean, 44.7% (SD, 16.4) versus unstimulated: mean, 30.4% (SD, 13.9); p < 0.05] with a decreased ciliated cell number [20 ng/mL IL-13: mean, 8.8% (SD, 7.5) versus unstimulated: mean, 16.3% (SD, 4.2); p < 0.001]. We also observed an increase in MUC5AC mRNA expression with 20 ng/mL IL-13 in PBEC(A), p < 0.05. IL-13 drives PBEC(N) toward an asthmatic phenotype and worsens the phenotype in PBEC(A) with reduced ciliated cell numbers and increased goblet cells.

Wild-type measles virus infection of primary epithelial cells occurs via the basolateral surface without syncytium formation or release of infectious virus.

The lymphotropic and myelotropic nature of wild-type measles virus (wt-MV) is well recognized, with dendritic cells and lymphocytes expressing the MV receptor CD150 mediating systemic spread of the virus. Infection of respiratory epithelial cells has long been considered crucial for entry of MV into the body. However, the lack of detectable CD150 on these cells raises the issue of their importance in the pathogenesis of measles. This study utilized a combination of in vitro, ex vivo and in vivo model systems to characterize the susceptibility of epithelial cells to wt-MV of proven pathogenicity. Low numbers of MV-infected epithelial cells in close proximity to underlying infected lymphocytes or myeloid cells suggested infection via the basolateral side of the epithelium in the macaque model. In primary cultures of human bronchial epithelial cells, foci of MV-infected cells were only observed following infection via the basolateral cell surface. The extent of infection in primary cells was enhanced both in vitro and in ex vivo cornea rim tissue by disrupting the integrity of the cells prior to the application of virus. This demonstrated that, whilst epithelial cells may not be the primary target cells for wt-MV, areas of epithelium in which tight junctions are disrupted can become infected using high m.o.i. The low numbers of MV-infected epithelial cells observed in vivo in conjunction with the absence of infectious virus release from infected primary cell cultures suggest that epithelial cells have a peripheral role in MV transmission.

A 3-D well-differentiated model of pediatric bronchial epithelium demonstrates unstimulated morphological differences between asthmatic and nonasthmatic cells.

There is a need for reproducible and effective models of pediatric bronchial epithelium to study disease states such as asthma. We aimed to develop, characterize, and differentiate an effective, an efficient, and a reliable three-dimensional model of pediatric bronchial epithelium to test the hypothesis that children with asthma differ in their epithelial morphologic phenotype when compared with nonasthmatic children. Primary cell cultures from both asthmatic and nonasthmatic children were grown and differentiated at the air-liquid interface for 28 d. Tight junction formation, MUC5AC secretion, IL-8, IL-6, prostaglandin E2 production, and the percentage of goblet and ciliated cells in culture were assessed. Well-differentiated, multilayered, columnar epithelium containing both ciliated and goblet cells from asthmatic and nonasthmatic subjects were generated. All cultures demonstrated tight junction formation at the apical surface and exhibited mucus production and secretion. Asthmatic and nonasthmatic cultures secreted similar quantities of IL-8, IL-6, and prostaglandin E2. Cultures developed from asthmatic children contained considerably more goblet cells and fewer ciliated cells compared with those from nonasthmatic children. A well-differentiated model of pediatric epithelium has been developed that will be useful for more in vivo like study of the mechanisms at play during asthma.

Staphylococcus aureus enterotoxins induce IL-8 secretion by human nasal epithelial cells.

BACKGROUND: Staphylococcus aureus produces a set of proteins which act both as superantigens and toxins. Although their mode of action as superantigens is well understood, little is known about their effects on airway epithelial cells. METHODS: To investigate this problem, primary nasal epithelial cells derived from normal and asthmatic subjects were stimulated with staphylococcal enterotoxin A and B (SEA and SEB) and secreted (supernatants) and cell-associated (cell lysates) IL-8, TNF-alpha, RANTES and eotaxin were determined by specific ELISAs. RESULTS: Non-toxic concentrations of SEA and SEB (0.01 microg/ml and 1.0 microg/ml) induced IL-8 secretion after 24 h of culture. Pre-treatment of the cells with IFN-gamma (50 IU/ml) resulted in a further increase of IL-8 secretion. In cells from healthy donors pretreated with IFN-gamma, SEA at 1.0 mug/ml induced release of 1009 pg/ml IL-8 (733.0-1216 pg/ml, median (range)) while in cells from asthmatic donors the same treatment induced significantly higher IL-8 secretion - 1550 pg/ml (1168.0-2000.0 pg/ml p = 0.04). Normal cells pre-treated with IFN-gamma and then cultured with SEB at 1.0 mug/ml released 904.6 pg/ml IL-8 (666.5-1169.0 pg/ml). Cells from asthmatics treated in the same way produced significantly higher amounts of IL-8--1665.0 pg/ml (1168.0-2000.0 pg/ml, p = 0.01). Blocking antibodies to MHC class II molecules added to cultures stimulated with SEA and SEB, reduced IL-8 secretion by about 40% in IFN-gamma unstimulated cultures and 75% in IFN-gamma stimulated cultures. No secretion of TNF-alpha, RANTES and eotaxin was noted. CONCLUSION: Staphylococcal enterotoxins may have a role in the pathogenesis of asthma.

Epidermal Growth Factor Removal or Tyrphostin AG1478 Treatment Reduces Goblet Cells & Mucus Secretion of Epithelial Cells from Asthmatic Children Using the Air-Liquid Interface Model.

RATIONALE: Epithelial remodelling in asthma is characterised by goblet cell hyperplasia and mucus hypersecretion for which no therapies exist. Differentiated bronchial air-liquid interface cultures from asthmatic children display high goblet cell numbers. Epidermal growth factor and its receptor have been implicated in goblet cell hyperplasia. OBJECTIVES: We hypothesised that EGF removal or tyrphostin AG1478 treatment of differentiating air-liquid interface cultures from asthmatic children would result in a reduction of epithelial goblet cells and mucus secretion. METHODS: In Aim 1 primary bronchial epithelial cells from non-asthmatic (n = 5) and asthmatic (n = 5) children were differentiated under EGF-positive (10 ng/ml EGF) and EGF-negative culture conditions for 28 days. In Aim 2, cultures from a further group of asthmatic children (n = 5) were grown under tyrphostin AG1478, a tyrosine kinase inhibitor, conditions. All cultures were analysed for epithelial resistance, markers of differentiation using immunocytochemistry, ELISA for MUC5AC mucin secretion and qPCR for MUC5AC mRNA. RESULTS: In cultures from asthmatic children the goblet cell number was reduced in the EGF negative group (p = 0.01). Tyrphostin AG1478 treatment of cultures from asthmatic children had significant reductions in goblet cells at 0.2 µg/ml (p = 0.03) and 2 µg/ml (p = 0.003) as well as mucus secretion at 2 µg/ml (p = 0.04). CONCLUSIONS: We have shown in this preliminary study that through EGF removal and tyrphostin AG1478 treatment the goblet cell number and mucus hypersecretion in differentiating air-liquid interface cultures from asthmatic children is significantly reduced. This further highlights the epidermal growth factor receptor as a potential therapeutic target to inhibit goblet cell hyperplasia and mucus hypersecretion in asthma.

The role of hepatocyte growth factor/c-met interactions in the immune system.

Hepatocyte growth factor (HGF) is a pleiotropic cytokine with mitogenic, motogenic and morphogenic activity for mainly epithelial and endothelial target cells. All the different effects of HGF are mediated through its specific receptor met, a heterodimeric transmembrane tyrosine kinase. The broad activity of HGF and its impact on many physiologic and pathologic processes are reflected by met expression in a variety of organs and cell types. This paper discusses expression of HGF and c-met within the immune system and their interactions in the control of immune cell functions.

Nasal epithelial cells can act as a physiological surrogate for paediatric asthma studies.

INTRODUCTION: Differentiated paediatric epithelial cells can be used to study the role of epithelial cells in asthma. Nasal epithelial cells are easier to obtain and may act as a surrogate for bronchial epithelium in asthma studies. We assessed the suitability of nasal epithelium from asthmatic children to be a surrogate for bronchial epithelium using air-liquid interface cultures. METHODS: Paired nasal and bronchial epithelial cells from asthmatic children (n = 9) were differentiated for 28 days under unstimulated and IL-13-stimulated conditions. Morphological and physiological markers were analysed using immunocytochemistry, transepithelial-electrical-resistance, Quantitative Real-time-PCR, ELISA and multiplex cytokine/chemokine analysis. RESULTS: Physiologically, nasal epithelial cells from asthmatic children exhibit similar cytokine responses to stimulation with IL-13 compared with paired bronchial epithelial cells. Morphologically however, nasal epithelial cells differed significantly from bronchial epithelial cells from asthmatic patients under unstimulated and IL-13-stimulated conditions. Nasal epithelial cells exhibited lower proliferation/differentiation rates and lower percentages of goblet and ciliated cells when unstimulated, while exhibiting a diminished and varied response to IL-13. CONCLUSIONS: We conclude that morphologically, nasal epithelial cells would not be a suitable surrogate due to a significantly lower rate of proliferation and differentiation of goblet and ciliated cells. Physiologically, nasal epithelial cells respond similarly to exogenous stimulation with IL-13 in cytokine production and could be used as a physiological surrogate in the event that bronchial epithelial cells are not available.

Chronic IL9 and IL-13 exposure leads to an altered differentiation of ciliated cells in a well-differentiated paediatric bronchial epithelial cell model.

Asthma is a chronic inflammatory disease characterised by airways remodelling. In mouse models IL-9 and IL-13 have been implicated in airways remodelling including mucus hypersecretion and goblet cell hyperplasia. Their role, especially that of IL-9, has been much less studied in authentic human ex vivo models of the bronchial epithelium from normal and asthmatic children. We assessed the effects of IL-9, IL-13 and an IL-9/IL-13 combination, during differentiation of bronchial epithelial cells from normal (n = 6) and asthmatic (n = 8) children. Cultures were analysed for morphological markers and factors associated with altered differentiation (MUC5AC, SPDEF and MMP-7). IL-9, IL-9/IL-13 combination and IL-13 stimulated bronchial epithelial cells from normal children had fewer ciliated cells [14.8% (SD 8.9), p = 0.048, 12.4 (SD 6.1), p = 0.016 and 7.3% (SD 6.6), p = 0.031] respectively compared with unstimulated [(21.4% (SD 9.6)]. IL-9 stimulation had no effect on goblet cell number in either group whereas IL-9/IL-13 combination and IL-13 significantly increased goblet cell number [24.8% (SD 8.8), p = 0.02), 32.9% (SD 8.6), p = 0.007] compared with unstimulated normal bronchial cells [(18.6% (SD 6.2)]. All stimulations increased MUC5AC mRNA in bronchial epithelial cells from normal children and increased MUC5AC mucin secretion. MMP-7 localisation was dysregulated in normal bronchial epithelium stimulated with Th2 cytokines which resembled the unstimulated bronchial epithelium of asthmatic children. All stimulations resulted in a significant reduction in transepithelial electrical resistance values over time suggesting a role in altered tight junction formation. We conclude that IL-9 does not increase goblet cell numbers in bronchial epithelial cell cultures from normal or asthmatic children. IL-9 and IL-13 alone and in combination, reduce ciliated cell numbers and transepithelial electrical resistance during differentiation of normal epithelium, which clinically could inhibit mucociliary clearance and drive an altered repair mechanism. This suggests an alternative role for IL-9 in airways remodelling and reaffirms IL-9 as a potential therapeutic target.

IL-31 does not induce normal human ciliated epithelial cells to differentiate into a phenotype consistent with the pathophysiology of asthma.

BACKGROUND: IL-31 is a novel cytokine that has been implicated in allergic diseases such as atopic dermatitis and more recently asthma. While IL-31 has been well studied in skin conditions such as atopic dermatitis, little is known about the role IL-31 plays in asthma and specifically the differentiation process of the bronchial epithelium, which is central to the pathogenesis of allergic asthma. METHODS: We examined the effects of IL-13 (20 ng/ml), IL-31 (20 ng/ml) and an IL-13/IL-31 combination stimulation (20 ng/ml each) on the in vitro mucociliary differentiation of paediatric bronchial epithelial cells (PBECs) from healthy patients (n=6). IL-31 receptor (IL-31-RA) expression, markers of differentiation (goblet and ciliated cells), transepithelial electrical resistance (TEER), quantification of goblet and ciliated cells, real time PCR for MUC5AC, ELISA for VEGF, EGF and MCP-1 (CCL-2) and ELISA for MUC5AC were assessed. RESULTS: We found that well-differentiated PBECs expressed IL-31-RA however it's expression did not increase upon stimulation with IL-31 or either of the other treatments. TEER indicated good formation of tight junctions which was found to be similar across all treatment groups (p=0.9). We found that IL-13 alone significantly reduced the number of ciliated cells compared with unstimulated (IL-13 stimuation: mean=4.8% (SD=2.5); unstimulated: mean=15.9%, (SD=7.4), p<0.01). IL-31 stimulation alone had no effect on ciliated cells whereas the IL-13/IL-31 combination stimulation significantly reduced the number of ciliated cells compared with control (IL-13/IL-31 combination: mean=5.1% (SD=4.6); unstimulated: mean=15.9%, (SD=7.4), p<0.01). We did not find that the combination of IL-13 and IL-31 had any additional effects to that of IL-13 alone. MUC5AC mRNA and secreted mucin was found in similar levels between unstimulated and all treatments, however IL-13 increased levels of MUC5AC mRNA by a factor of 2.84, albeit not significantly, compared with unstimulated cultures (IL-13 stimulation: mean=2.84 (SD=3.79); unstimulated: mean=1.0). CONCLUSIONS: IL-31RA receptor is present on well-differentiated paediatric bronchial epithelial cells. IL-31 does not exhibit any detrimental effects on mucociliary differentiation. IL-31 does not appear to have a synergistic effect when combined in culture with IL-13, in the differentiation process.

Bronchial epithelial cell growth regulation in fibroblast cocultures: the role of hepatocyte growth factor.

Proliferation of bronchial epithelial cells is an important biological process in physiological conditions and various lung diseases. The objective of this study was to determine how bronchial fibroblasts influence bronchial epithelial cell proliferation. The proliferative activity in cocultures was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and direct cells counts. Concentration of cytokines was measured in cell culture supernatants by means of ELISA. In primary cell cocultures, fibroblasts or fibroblast-conditioned medium enhanced 1.85-fold the proliferation of primary bronchial epithelial cells (P < 0.02) compared with bronchial epithelial cells cultured alone. The proliferative activity in cocultures and in fibroblast-conditioned medium was reduced by neutralizing antibody to hepatocyte growth factor (HGF) and HGF receptor c-met. Neutralizing antibodies to FGF-7 and IGF-1 had no effect. Treatment of fibroblast-epithelial cocultures with anti-IL-6 and anti-TNF-alpha neutralizing antibodies and with indomethacin decreased production of HGF. These results indicate that cytokines and PGE(2) may indirectly mediate epithelial cell proliferation via the regulation of HGF in bronchial stromal cells and that HGF plays a crucial role in proinflammatory cytokine-induced proliferation in the experimental system studied.