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

Potentially Pathogenic Airway Bacteria and Neutrophilic Inflammation in Treatment Resistant Severe Asthma

BACKGROUND:Molecular microbiological analysis of airway samples in asthma has demonstrated an altered microbiome in comparison to healthy controls. Such changes may have relevance to treatment-resistant severe asthma, particularly those with neutrophilic airway inflammation, as bacteria might be anticipated to activate the innate immune response, a process that is poorly steroid responsive. An understanding of the relationship between airway bacterial presence and dominance in severe asthma may help direct alternative treatment approaches. OBJECTIVE:We aimed to use a culture independent analysis strategy to describe the presence, dominance and abundance of bacterial taxa in induced sputum from treatment resistant severe asthmatics and correlate findings with clinical characteristics and airway inflammatory markers. METHODS:Induced sputum was obtained from 28 stable treatment-resistant severe asthmatics. The samples were divided for supernatant IL-8 measurement, cytospin preparation for differential cell count and Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiling for bacterial community analysis. RESULTS:In 17/28 patients, the dominant species within the airway bacterial community was Moraxella catarrhalis or a member of the Haemophilus or Streptococcus genera. Colonisation with these species was associated with longer asthma disease duration (mean (SD) 31.8 years (16.7) vs 15.6 years (8.0), p = 0.008), worse post-bronchodilator percent predicted FEV1 (68.0% (24.0) vs 85.5% (19.7), p = 0.025) and higher sputum neutrophil differential cell counts (median (IQR) 80% (67-83) vs 43% (29-67), p = 0.001). Total abundance of these organisms significantly and positively correlated with sputum IL-8 concentration and neutrophil count. CONCLUSIONS:Airway colonisation with potentially pathogenic micro-organisms in asthma is associated with more severe airways obstruction and neutrophilic airway inflammation. This altered colonisation may have a role in the development of an asthma phenotype that responds less well to current asthma therapies

Exogenous IFN-? has antiviral and anti-inflammatory properties in primary bronchial epithelial cells from asthmatic subjects exposed to rhinovirus

Background: Rhinoviruses are the major cause of asthma exacerbations. Previous studies suggest that primary bronchial epithelial cells (PBECs) from asthmatic subjects are more susceptible to rhinovirus infection because of deficient IFN-? production. Although augmenting the innate immune response might provide a novel approach for treatment of virus-induced asthma exacerbations, the potential of IFN-? to modulate antiviral and proinflammatory responses in asthmatic epithelium is poorly characterized.Objectives: We sought to compare responses of PBECs from nonasthmatic and asthmatic subjects to exogenous IFN-? and test the inflammatory effects of IFN-? in response to rhinovirus infection.Methods: PBECs were treated with IFN-? and infected with a low inoculum of human rhinovirus serotype 1B to simulate a natural viral infection. Expression of interferon-responsive genes and inflammatory responses were analyzed by using reverse transcription–quantitative real-time PCR, cytometric bead arrays, or both; viral titers were assessed by using the 50% tissue culture infection dose.Results: Expression of IFN-?–stimulated antiviral genes was comparable in PBECs from nonasthmatic or asthmatic donors. Exogenous IFN-? significantly protected PBECs from asthmatic donors against rhinovirus infection by suppressing viral replication. Interferon-inducible protein 10 (IP-10), RANTES, and IL-6 release in response to rhinovirus infection was triggered only in PBECs from asthmatic donors. Although exogenous IFN-? alone stimulated some release of IP-10 (but not IL-6 or RANTES), it significantly reduced rhinovirus-induced IP-10, RANTES, and IL-6 expression when tested in combination with rhinovirus.Conclusions: PBECs from asthmatic donors have a normal antiviral response to exogenous IFN-?. The ability of IFN-? to suppress viral replication suggests that it might limit virus-induced exacerbations by shortening the duration of the inflammatory response

Severe asthma disease duration and inflammation related to abundance of <i>Haemophilus., Streptococcus</i>, and <i>Moraxella sp.</i>

<p>The relationship in treatment-resistant severe asthma between total abundance of <i>Haemophilus sp., Streptococcus sp.</i>, and <i>Moraxella catarrhalis</i> in induced sputum samples and [A] neutrophil differential cell count (%), [B] asthma duration (years), [C] interleukin (IL)-8 concentration in induced sputum and [D] the relationship between <i>M. catarrhalis</i> abundance and induced sputum IL-8 concentration.</p

The effect of neutralizing endogenous TGF-β on RV replication.

<p>PBECs from 8 asthmatic donors or 6 non-asthmatic control subjects were pretreated for 24 h in the presence of a neutralizing anti pan TGF-β antibody or isotype control antibody before infection with RV1B (1000 units/10⁵ cells) for 48 h. The fold-decrease in viral replication by the neutralizing antibody was plotted as a ratio of the TCID₅₀/ml of antibody-treated versus isotype controls. The figure shows median and interquartile range, with individual data points superimposed. Data were analysed using a Mann Whitney U test.</p

Interleukin-8 sputum supernatant concentrations in severe asthma related to sputum neutrophil count and lung function.

<p>The relationship in treatment-resistant severe asthma between IL-8 concentrations in induced sputum and (A) neutrophil differential cell counts and (B) Post bronchodilator percent predicted FEV₁.</p

Suppression of viral replication in PBECs from asthmatic donors by neutralization of endogenous TGF-β.

<p>PBECs from asthmatic donors were pretreated for 24 h in the presence of a neutralizing anti pan TGF-β antibody or isotype control antibody before infection with RV1B (1000 units/10⁵ cells) for 48 h. In A, viral titre was determined as TCID₅₀/ml using culture supernatants obtained 48 h p.i. In B, IFN-β protein was measured at 48 h and was expressed as a ratio of the viral load measured as TCID₅₀ units. The data were analyzed using Wilcoxon’s rank sum test.</p

Exogenous TGF-β₂ suppresses IFN-β release from virally infected (A) or poly IC exposed (B) PBEC cultures from non-asthmatic donors.

<p>PBEC cultures were infected with RV1B (5000 TCID₅₀ units/10⁵ cells) (n = 10) or treated with poly IC (n = 5) in the presence or absence of TGF-β₂ which was used at 1 (black bars in B) or 10 ng/ml (panel A and grey bars in B). Culture supernatants were harvested 48 hours p.i (A) or 8 h post stimulation (B) and IFN-β protein levels were measured by ELISA. In B, the data are expressed as a % of control cultures treated with poly IC in the absence of TGF-β₂ (median (IQR) IFN-β release  = 346 (1135) and 369 (1390) pg/ml for cells treated with 1 or 10 µg/ml Poly IC, respectively. The data were analyzed using Wilcoxon’s rank sum test (A) or using a paired t-test for normally distributed data (B).</p

The effect of exogenous TGF-β₂ on RV replication.

<p>PBECs from 3 non-asthmatic volunteers were pre-incubated with 0, 1, 10, and 25 ng/ml of TGF- β₂ for 24 h, followed by infection with RV1B at 5000 TCID₅₀ units/10⁵ cells. Cells were then further incubated for 48 h in the presence or absence of TGF-β₂, as indicated. Viral replication at 24 h was measured as vRNA by RT-qPCR (A) and at 48 h by release of infectious virions into culture supernatants by TCID₅₀ assays (B). The graph (C) shows data for infectious virus release from PBECs from 10 non-asthmatic donors treated without or with 10 ng/ml TGF-β₂, followed by infection with RV1B at 5000 TCID₅₀ units/10⁵ cells for 48 hours. Statistical comparison was made using a Wilcoxon rank sum test. The # mark in C indicates where 2 data points overlap (1.8e⁶→3.1e⁶ TCID₅₀ units/ml).</p