Transforming growth factor-beta promotes rhinovirus replication in bronchial epithelial cells by suppressing the innate immune response

Rhinovirus (RV) infection is a major cause of asthma exacerbations which may be due to a deficient innate immune response in the bronchial epithelium. We hypothesized that the pleiotropic cytokine, TGF-?, influences interferon (IFN) production by primary bronchial epithelial cells (PBECs) following RV infection. Exogenous TGF-?(2) increased RV replication and decreased IFN protein secretion in response to RV or double-stranded RNA (dsRNA). Conversely, neutralizing TGF-? antibodies decreased RV replication and increased IFN expression in response to RV or dsRNA. Endogenous TGF-?(2) levels were higher in conditioned media of PBECs from asthmatic donors and the suppressive effect of anti-TGF-? on RV replication was significantly greater in these cells. Basal SMAD-2 activation was reduced when asthmatic PBECs were treated with anti-TGF-? and this was accompanied by suppression of SOCS-1 and SOCS-3 expression. Our results suggest that endogenous TGF-? contributes to a suppressed IFN response to RV infection possibly via SOCS-1 and SOCS-3

The molecular mechanisms involved in rhinovirus-induced asthma exacerbation and its potential therapy

Rhinovirus (RV) infection is a major cause of asthma exacerbation in children and adults. Previous studies have shown that primary bronchial epithelial cells (PBECs) obtained from asthmatic subjects have a deficient interferon (IFN) response against RV infection, the molecular mechanism of which is unknown (Wark et al 2005; Contoli et al 2006).Initially it was hypothesised that this deficiency is inherent to other cell types in the airway, such as bronchial fibroblasts. In a rhinovirus infection model, it was shown that fibroblasts respond with a vigorous pro-inflammatory response. However this response was accompanied by no significant IFN response. Fibroblasts produced IFN when we treated with a synthetic double-stranded RNA. However, no differences were observed between normal and asthmatic cells, indicating that the deficient innate immune response in asthmatic epithelium is not inherent to other cell types. We suggest that in vivo, bronchial fibroblasts may contribute to the inflammatory state in asthma when infected with RV. This might occur when an epithelial cell barrier with disrupted tight junctions might allow penetration and infection of the underlying mesenchymal cell layer.To investigate the innate immune response of asthmatic PBECs we hypothesised that the anti-inflammatory cytokine transforming growth factor beta (TGF-β) dampened the innate immune response against rhinovirus infection. It has been shown previously that TGF-β is elevated in asthmatics. It was found that PBEC cultures from asthmatic subjects produce significantly more endogenous TGF-β2 compared to healthy controls. When PBECs from healthy donors were treated with exogenous TGF-β2 it promoted RV replication, which was coupled with a decreased IFN response. Conversely, treatment of PBECs from asthmatic subjects with a neutralizing antibody against TGF-β decreased RV replication. These observations provide an interesting link between an anti-inflammatory environment in the asthmatic airways contributing to a defective innate immune response in asthma.To understand the TGF-β-mediated effect on RV replication, the importance of src kinases as one of the upstream signaling molecules in TGF-β-dependent alterations of cellular physiology was investigated. We found that inhibitors of src, in particular the SU6656 compound, were very potent in inhibiting RV replication. Inhibition by SU6656 was coupled with a significant increase in IFN response. These findings may pave the way towards designing compounds of similar structure, which are able to augment the IFN response and therefore provide a new form of therapy against asthma exacerbation

Contribution of bronchial fibroblasts to the antiviral response in asthma.

Human rhinoviruses (HRV) are a major cause of asthma exacerbations and hospitalization. Studies using primary cultures suggest that this may be due to impaired production of type I and type III IFNs by asthmatic bronchial epithelial cells. Although epithelial cells are the main target for HRV infection, HRV can be detected in the subepithelial layer of bronchial mucosa from infected subjects by in situ hybridization. Therefore, we postulated that submucosal fibroblasts are also involved in the innate antiviral response to HRV infection in asthma. We found that regardless of subject group, bronchial fibroblasts were highly susceptible to RV1b infection. IL-8 and IL-6 were rapidly induced by either HRV or UV-irradiated virus, suggesting that these responses did not require viral replication. In contrast, RANTES expression was dependent on viral replication. Regardless of disease status, fibroblasts did not respond to HRV infection with significant induction of IFN-beta, even though both groups responded to synthetic dsRNA with similar levels of IFN-beta expression. Exogenous IFN-beta was highly protective against viral replication. Our data suggest that fibroblasts respond to HRV with a vigorous proinflammatory response but minimal IFN-beta expression. Their susceptibility to infection may cause them to be a reservoir for HRV replication in the lower airways, especially in asthmatic subjects where there is reduced protection offered by epithelial-derived IFNs. Their ability to support viral replication coupled with their vigorous proinflammatory response following infection may contribute to asthma exacerbations

Identification of host miRNAs that may limit human rhinovirus replication

Taken together, our results suggest that pathological changes in microRNA expression, as already reported for asthma or chronic obstructive pulmonary disease have the potential to affect Rhinovirus replication and therefore may play a role in virus-induced exacerbation

A microRNA network dysregulated in asthma controls IL-6 production in bronchial epithelial cells

MicroRNAs are short non-coding single stranded RNAs that regulate gene expression. While much is known about the effects of individual microRNAs, there is now growing evidence that they can work in co-operative networks. MicroRNAs are known to be dysregulated in many diseases and affect pathways involved in the pathology. We investigated dysregulation of microRNA networks using asthma as the disease model. Asthma is a chronic inflammatory disease of the airways characterized by bronchial hyperresponsiveness and airway remodelling. The airway epithelium is a major contributor to asthma pathology and has been shown to produce an excess of inflammatory and pro-remodelling cytokines such as TGF-?, IL-6 and IL-8 as well as deficient amounts of anti-viral interferons. After performing microRNA arrays, we found that microRNAs -18a, -27a, -128 and -155 are down-regulated in asthmatic bronchial epithelial cells, compared to cells from healthy donors. Interestingly, these microRNAs are predicted in silico to target several components of the TGF-?, IL-6, IL-8 and interferons pathways. Manipulation of the levels of individual microRNAs in bronchial epithelial cells did not have an effect on any of these pathways. Importantly, knock-down of the network of microRNAs miR-18a, -27a, -128 and -155 led to a significant increase of IL-8 and IL-6 expression. Interestingly, despite strong in silico predictions, down-regulation of the pool of microRNAs did not have an effect on the TGF-? and Interferon pathways. In conclusion, using both bioinformatics and experimental tools we found a highly relevant potential role for microRNA dysregulation in the control of IL-6 and IL-8 expression in asthma. Our results suggest that microRNAs may have different roles depending on the presence of other microRNAs. Thus, interpretation of in silico analysis of microRNA function should be confirmed experimentally in the relevant cellular context taking into account interactions with other microRNAs when studying diseas

The innate antiviral response upregulates IL-13 receptor ?2 in bronchial fibroblasts

IFN-? induces IL-13R?2 expression, leading to a consequential suppression of responsiveness to IL-13. These data suggest cross-talk between TH1 and TH2 pathways and point to an immunomodulatory role for IL-13R?2 in human bronchial fibroblasts during viral infectio

Double-stranded RNA induces disproportionate expression of thymic stromal lymphopoietin versus interferon-beta in bronchial epithelial cells from donors with asthma

Background Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that strongly activates dendritic cells and can initiate allergic inflammation. Since exposure to rhinovirus or double-stranded (ds) RNA (a surrogate of viral infection) induces TSLP expression in bronchial epithelial cells (BECs), this cytokine may link innate antiviral responses and the type 2 adaptive immune response.Objective As BECs from donors with asthma have a deficient interferon (IFN) response to rhinovirus infection, a study was undertaken to test the hypothesis that their antiviral response shows a bias towards TSLP production.Methods Primary BECs were grown from subjects with asthma and healthy volunteers. After exposure to dsRNA, interleukin (IL)-8, IFN? and TSLP mRNA and protein expression were measured by RT-qPCR and ELISA, respectively.Results dsRNA dose-dependently increased IL-8 expression in BECs with no significant difference between the groups. However, BECs from subjects with asthma expressed less IFN? and more TSLP mRNA and protein in response to dsRNA than BECs from those without asthma (median (IQR) 57 (38–82) pg/ml vs 106 (57–214) pg/ml for IFN? (p&lt;0.05) and 114 (86–143) pg/ml vs 65 (32–119) pg/ml for TSLP (p&lt;0.05) in response to 10 ?g/ml dsRNA for 24 h). Induction of TSLP mRNA by dsRNA was blocked by Toll-like receptor 3 or protein kinase inhibitors or by preventing de novo protein synthesis, but not by neutralisation of type I IFN receptors.Conclusion BECs from subjects with asthma are biased towards higher TSLP and lower IFN? production in response to dsRNA, suggesting that viral infection in asthma may lead to an altered mediator profile that biases towards a Th2 immune response.<br/

Inhibition of Pim1 kinase, new therapeutic approach in virus-induced asthma exacerbations

Therapeutic options to treat virus-induced asthma exacerbations are limited and urgently needed. Therefore, we tested Pim1 kinase as potential therapeutic target in human rhinovirus (HRV) infections. We hypothesised that inhibition of Pim1 kinase reduces HRV replication by augmenting the interferon-induced anti-viral response due to increased activity of the janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Air-liquid interface (ALI) cultures of primary bronchial epithelial cells (PBECs) from healthy individuals and moderate-to-severe asthmatic volunteers were infected with HRV-16 with or without a specific Pim1 inhibitor; viral replication and induction of anti-viral responses were measured using RT-qPCR. Viral titres were measured by 50% tissue culture infective dose and release of interferon-gamma-induced protein 10 (IP-10) and RANTES protein assessed by ELISA. Phosphorylation of STAT-1 was determined using western blotting. Viral replication was reduced in ALI cultures of healthy and asthmatic PBECs treated with the Pim1 inhibitor. Using cultures from healthy donors, enhanced STAT-1 phosphorylation upon inhibition of Pim1 kinase activity resulted in increased mRNA expression of interferon-beta, interleukin-29, IP-10 and RANTES 12 h after infection and increased protein levels of IP-10 and RANTES 24 h after infection. We have identified Pim1 kinase as novel target to reduce viral replication in ALI cultures of PBECs. This may open new avenues for therapeutic interventions in virus-induced asthma exacerbations