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

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 anti-TGF-β antibodies on release of IFN-β and IFNλ1/IL-29 protein in response to poly IC.

<p>PBECs from 6 asthmatic donors were treated with 0.1–10 µg/ml poly IC in the presence of neutralizing anti-TGF-β antibodies (black bars) or an IgG isotype control (open bars) and incubated for 24 h. Supernatants were removed and IFN-β (A) or IFN-λ1/IL-29 protein levels (B) were measured by ELISA. Graphs show (mean±SEM) IFN produced in pg/ml the presence of the control or anti TGF-β antibodies.</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

The effect of TGF-β neutralization on basal SMAD2 activation.

<p>PBECs from asthmatic donors were treated with RV and anti TGF-β antibody, as indicated, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044580#pone-0044580-g005" target="_blank">Figure 5</a>. Cell lysates were harvested at 1, 4, and 6 hours post-virus infection and Smad-2 phosphorylation was analysed by Western blotting. A representative Western blot is shown in (A) and densitometric quantification of the experiment repeated using PBECs from 3 different asthmatic subjects is shown in (B).</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-λ1/IL-29 release from virally infected (A) (n = 10) or poly IC (n = 4) exposed (B) PBEC cultures from non-asthmatic donors.

<p>IFNλ1/IL-29 protein levels were measured by ELISA from RV-infected or poly IC exposed PBECs treated with TGF-β₂ as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044580#pone-0044580-g002" target="_blank">Figure 2</a>. Median (IQR) IFN-λ₁ release  = 3896 (2766) and 4932 (4941) pg/ml for cells treated with 1 or 10 µg/ml Poly IC, respectively.</p

Neutralizing endogenous TGF-β suppresses RV1B mediated SOCS-1 and SOCS-3 gene expression in asthmatic PBECs.

<p>Samples were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044580#pone-0044580-g005" target="_blank">Figure 5</a>. SOCS-1 (A) and SOCS-3 (B) gene expression were measured in 7 asthmatic subjects at 48 h p.i. by RT-qPCR and normalized to housekeeping genes. Results were plotted as relative fold-induction using the ΔΔCt method. The Wilcoxon rank sum test was used to analyse statistical significance.</p