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

Abstract

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

Figure 1. The effect of exogenous TGF-β₂ on RV replication.

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).

Figure 2. Exogenous TGF-β₂ suppresses IFN-β release from virally infected (A) or poly IC exposed (B) PBEC cultures from non-asthmatic donors.

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).

Figure 3. 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.

IFNλ1/IL-29 protein levels were measured by ELISA from RV-infected or poly IC exposed PBECs treated with TGF-β₂ as described in Figure 2. Median (IQR) IFN-λ₁ release  = 3896 (2766) and 4932 (4941) pg/ml for cells treated with 1 or 10 µg/ml Poly IC, respectively.

Figure 4. The effect of neutralizing endogenous TGF-β on RV replication.

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.

Figure 5. Suppression of viral replication in PBECs from asthmatic donors by neutralization of endogenous TGF-β.

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.

Figure 6. The effect of anti-TGF-β antibodies on release of IFN-β and IFNλ1/IL-29 protein in response to poly IC.

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.

Figure 7. The effect of RV infection on TGF-β isoform expression.

TGF-β1, β2, β3 mRNA levels were measured in PBECs from 4 asthmatic and 4 non-asthmatic donors following infection with RV; TGF-β mRNA expression was measured relative to GAPDH/UBC using the ΔΔCt method (A). Total TGF-β₂ protein levels were measured in conditioned media from PBEC cultures of 15 non-asthmatic and 23 asthmatic donors which were harvested at 48 h. Latent TGF-β₂ was activated by acid-treatment and total TGF-β₂ measured by ELISA. Statistical significance was tested using Mann Whitney U test (B). PBEC culture supernatants of 2 non-asthmatic and 4 asthmatic donors were tested in a transformed mink lung cell luciferase bioassay in order to measure active TGF-β in the presence (black bars) or absence (grey bars) of rhinovirus at 5000 TCID₅₀ units/10⁵ cells. Superimposed is a standard curve obtained using 250 or 500 pg of active TGFβ (C). Data in panels A and B are given as box and whisker plots showing median, interquartile range and 95% confidence intervals; individual data points are superimposed.

Figure 8. The effect of TGF-β neutralization on basal SMAD2 activation.

PBECs from asthmatic donors were treated with RV and anti TGF-β antibody, as indicated, as described in Figure 5. 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).

Figure 9. Neutralizing endogenous TGF-β suppresses RV1B mediated SOCS-1 and SOCS-3 gene expression in asthmatic PBECs.

Samples were treated as described in Figure 5. 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.