Autophagy, TGF-β, and SMAD-2/3 Signaling Regulates Interferon-β Response in Respiratory Syncytial Virus Infected Macrophages

Abstract

Human respiratory syncytial virus (RSV) is a lung tropic virus causing severe airway diseases including bronchiolitis and pneumonia among infants, children, and immuno-compromised individuals. RSV triggers transforming growth factor-β (TGF-β) production from lung epithelial cells and TGF-β facilitates RSV infection of these cells. However, it is still unknown whether RSV infected myeloid cells like macrophages produce TGF-β and the role of TGF-β if any during RSV infection of these cells. Our study revealed that RSV infected macrophages produce TGF-β and as a consequence these cells activate TGF-β dependent SMAD-2/3 signaling pathway. Further mechanistic studies illustrated a role of autophagy in triggering TGF-β production from RSV infected macrophages. In an effort to elucidate the role of TGF-β and SMAD-2/3 signaling during RSV infection, we surprisingly unfolded the requirement of TGF-β-SMAD2/3 signaling in conferring optimal innate immune antiviral response during RSV infection of macrophages. Type-I interferon (e.g., interferon-β or IFN-β) is a critical host factor regulating innate immune antiviral response during RSV infection. Our study revealed that loss of TGF-β-SMAD2/3 signaling pathway in RSV infected macrophages led to diminished expression and production of IFN-β. Inhibiting autophagy in RSV infected macrophages also resulted in reduced production of IFN-β. Thus, our studies have unfolded the requirement of autophagy-TGF-β-SMAD2/3 signaling network for optimal innate immune antiviral response during RSV infection of macrophages.

Keywords: SMAD; TGF-β; autophagy; interferon-β; macrophages; respiratory syncytial virus.

Figure 1

RSV triggers TGF-β production and activates SMAD-2/3 signaling in macrophages. (A) Primary bone marrow derived macrophages (BMDMs) and (B) mouse macrophage cell line RAW 264.7 were infected with RSV (1MOI) for different time points. Following infection, the medium supernatant was collected to assess active TGF-β secretion by ELISA analysis. (C) BMDMs and (D) RAW 264.7 cells were infected with RSV (1MOI) for 2 and 4 h. Cell lysates were subjected to western blot analysis with phospho-SMAD2 (p-SMAD2), SMAD2, and β-actin antibodies. The ELISA values (A,B) represent the mean ± standard deviation. ^*p, ^**p, and ^***p ≤ 0.05 using a Student's t-test.

Figure 2

Autophagy induction during RSV infection promotes TGF-β production and SMAD-2/3 signaling pathway activation. (A) BMDMs were infected with RSV (1MOI) for 1, 2, and 4 h. Cell lysates from mock and RSV infected cells were subjected to western blotting with LC3 antibody. (B) Band intensity of LC3-I and LC3-II (Figure 2A) was quantified and the ratio of LC3-II/LC3-I was plotted to denote autophagy induction during RSV infection. (C) BMDMs were pre-treated with autophagy inhibitor (3 MA; 5 mM) for 2 h and infected with RSV (1MOI) in presence of DMSO (vehicle control) or 3 MA. Following infection, TGF-β levels in the medium supernatant was measured by ELISA. (D) BMDMs were pre-treated with 3 MA (5 mM) for 2 h and infected with RSV (1MOI) in presence of DMSO or 3 MA. Cell lysates were subjected to western blot analysis with phospho-SMAD2 (p-SMAD2), SMAD2, and β-actin antibodies. The ELISA value (C) represents the mean ± standard deviation. ^*p ≤ 0.05 using a Student's t-test.

Figure 3

Autophagy induction during RSV infection promotes SMAD-2/3 signaling pathway activation. BMDMs (A) and RAW 264.7 cells (B) were transfected with either control siRNA or beclin-1 siRNA (60 pmol). Cell lysates collected from these cells were subjected to western blot analysis with beclin-1 and β-actin antibodies. RAW 264.7 cells (C) and BMDMs (D) cells silenced for beclin-1 expression were infected with RSV. Cell lysate collected from these cells were subjected to western blot analysis with phospho-SMAD2 (p-SMAD2), SMAD2, and β-actin antibodies. NT; non-transfected cells.

Figure 4

TGF-β and SMAD-2/3 signaling is required for optimal interferon-β (IFN-β) production during RSV infection. BMDMs (A) and RAW 264.7 cells (B) treated with either DMSO (vehicle control) or TGF-β inhibitor (SB-431542; 10 μM) were infected with RSV (1MOI; 16 h). Following infection, medium supernatant was assessed for IFN-β secretion by ELISA analysis. (C) RAW264.7 cells treated with either DMSO or SB-431542 (SB) were infected with RSV (1MOI). At 16 h post-infection, RT-PCR analysis was performed to assess IFN-β expression. (D) RAW 264.7 cells were transfected with either control siRNA or SMAD2 siRNA. Cell lysates collected from these cells were subjected to western blot analysis with SMAD2 and β-actin antibodies. (E) RAW 264.7 cells transfected with either control siRNA or SMAD2 siRNA were infected with RSV (1MOI). At 16 h post-infection, medium supernatant was collected to assess IFN-β level by ELISA. The ELISA values (A,B,E) represent the mean ± standard deviation. ^*p ≤ 0.05 using a Student's t-test. NT; non-transfected cells.

Figure 5

TGF-β regulates RSV replication in macrophages and IFN-β production kinetics following RSV infection. (A) RAW 264.7 cells transfected with either control siRNA or SMAD2 siRNA were infected with RSV (1MOI). At 16 h post-infection, RT-PCR analysis was performed to assess expression RSV nucleocapsid (N) protein mRNA. (B) Band intensity of RSV N and GAPDH in control siRNA and SMAD2 siRNA transfected cells (Figure 5A) was quantified and the ratio of N/GAPDH was plotted to denote enhanced expression of RSV N in SMAD2 siRNA transfected cells. (C) RAW 264.7 were infected with RSV (1MOI) for 0 h (mock), 2, 4, and 8 h. Following infection, the medium supernatant was collected to assess IFN-β production by ELISA analysis. The ELISA value represents the mean ± standard deviation. ^*p and ^**p ≤ 0.05 using a Student's t-test.

Figure 6

Autophagy regulates IFN-β production from RSV infected macrophages. BMDMs (A) and RAW 264.7 cells (B) treated with either DMSO (vehicle control) or autophagy inhibitor (3 MA; 5 mM) were infected with RSV (1MOI). At 16 h post-RSV infection, medium supernatant was collected to assess IFN-β production by ELISA. (C) RAW 264.7 cells treated with either DMSO or 3 MA were infected with RSV (1MOI). At 16 h post-infection, RT-PCR analysis was performed to analyze IFN-β expression. (D) RAW 264.7 cells transfected with either control siRNA or beclin-1 siRNA were infected with RSV (1MOI). At 16 h post-infection, RT-PCR analysis was performed to analyze IFN-β expression. (E) RAW 264.7 cells treated with either DMSO (vehicle control) or autophagy inhibitor (3 MA; 5 mM) were infected with RSV (1MOI). At 16 h post-RSV infection, cell lysate was subjected to Western blot analysis with RSV fusion (F) protein antibody. (F) Band intensity of RSV F and actin in DMSO and 3 MA treated cells (Figure 6E) was quantified and the ratio of F protein/actin was plotted to denote enhanced expression of RSV F protein in 3 MA treated cells. The ELISA values (A,B) represent the mean ± standard deviation. ^*p ≤ 0.05 using a Student's t-test.

Figure 7

A model depicting the role of autophagy—TGF-β—SMAD2/3 signaling network in positively regulating IFN-β production during RSV infection of macrophages. RSV infection triggers autophagy in macrophages and this event promotes TGF-β production. Cell surface TGF-β receptor (TGFR) is then activated by extracellular TGF-β via paracrine/autocrine action. Interaction of extracellular TGF-β with TGFR will result in activation of SMAD-2/3 signaling comprising of phosphorylation of SMAD-2/3. Activation (i.e., phosphorylation of SMAD-2/3) of SMAD-2/3 will result in IFN-β expression and production.