Respiratory syncytial virus G protein CX3C motif impairs human airway epithelial and immune cell responses

Abstract

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infection in infants and young children and causes disease in the elderly and persons with compromised cardiac, pulmonary, or immune systems. Despite the high morbidity rates of RSV infection, no highly effective treatment or vaccine is yet available. The RSV G protein is an important contributor to the disease process. A conserved CX3C chemokine-like motif in G likely contributes to the pathogenesis of disease. Through this motif, G protein binds to CX3CR1 present on various immune cells and affects immune responses to RSV, as has been shown in the mouse model of RSV infection. However, very little is known of the role of RSV CX3C-CX3CR1 interactions in human disease. In this study, we use an in vitro model of human RSV infection comprised of human peripheral blood mononuclear cells (PBMCs) separated by a permeable membrane from human airway epithelial cells (A549) infected with RSV with either an intact CX3C motif (CX3C) or a mutated motif (CX4C). We show that the CX4C virus induces higher levels of type I/III interferon (IFN) in A549 cells, increased IFN-α and tumor necrosis factor alpha (TNF-α) production by human plasmacytoid dendritic cells (pDCs) and monocytes, and increased IFN-γ production in effector/memory T cell subpopulations. Treatment of CX3C virus-infected cells with the F(ab')2 form of an anti-G monoclonal antibody (MAb) that blocks binding to CX3CR1 gave results similar to those with the CX4C virus. Our data suggest that the RSV G protein CX3C motif impairs innate and adaptive human immune responses and may be important to vaccine and antiviral drug development.

Fig 1

In vitro model of human RSV infection. (A) Human airway epithelial cells (A549) were cultured in 24-well plates and infected with RSV strains or mock infected and cultured for 3 days, with culture medium replacement on day 2 p.i. At day 3 p.i., PBMCs from random human adult donors were added in permeable-membrane Transwell inserts and coincubated for an additional 24 h. (B to D) Responses of PBMCs after exposure to purified CX3C RSV (direct stimulation) or RSV-infected A549 cells (in vitro model). Shown are the percentages of CD69⁺ IFN-γ-producing CD4 and CD8 effector memory T cells (B), mean fluorescence intensity (MFI) of CD86 expression (C), and percentages of IFN-α-producing pDCs, mDCs, and monocytes (D). SEB or CpG was used as a positive control for T cell or innate cell responses. The data are represented as means ± SEM of independent experiments with PBMCs from 3 random donors. ∗, P < 0.05 versus mock infection in direct stimulation; △, P < 0.05 versus mock infection in the in vitro model; †, P < 0.05 versus direct stimulation (determined by a paired t test).

Fig 2

Replication of wild-type RSV (CX3C) and mutant RSV with an altered CX3C motif of the G protein (CX4C) in the in vitro model of human RSV infection. (A) Virus replication in A549 cells, determined by RSV-specific ELISA. A549 cells were infected and incubated for 3 days, with culture medium replacement on day 2 p.i. At day 3 p.i., PBMCs from random human adult donors were added in permeable-membrane inserts and coincubated for an additional 24 h. After coincubation and removal of the inserts and supernatants, A549 cells were washed and fixed, and an RSV-specific ELISA was performed. Absorbance represents the amount of surface and internal RSV antigens captured by ELISA. The data are represented as means ± SEM of independent experiments with PBMCs from 5 random donors. ∗, P < 0.05 versus mock infection (determined by the Mann-Whitney test). (B) Relative amount of viral RNA in basolateral supernatants of infected A549 cells determined by RT-PCR, represented as inverse cycle threshold (C_T) values. C_T levels reflect the number of cycles required to exceed the background level; inverse C_T levels (1/C_T) are proportional to the amount of target nucleic acid in the sample. RT-PCR underwent 40 cycles of amplification. The data are represented as means ± SEM of independent experiments with PBMCs from 5 random donors. (C) Viral titers in the bottom and top chambers after 6 or 24 h of coincubation of PBMCs with infected A549 cells, determined by a microtiter tissue culture infectivity assay. The data are represented as means ± SEM of 3 repeated independent experiments with PBMCs from one random donor. Mononuclear cells were coincubated with RSV-infected A549 cells. ∗, P < 0.05 versus the bottom chamber (determined by the Mann-Whitney test). (D) RSV-specific ELISA of A549 cells infected with CX3C and CX4C viruses with or without the presence of F(ab′)₂ fragments of monoclonal anti-G protein 131-2G antibody. Absorbance represents the amount of surface and internal RSV antigens detected by ELISA. The data are represented as means ± SEM of independent experiments with PBMCs from 3 random donors. ∗, P < 0.05 versus mock infection (determined by the Mann-Whitney test).

Fig 3

Antiviral responses of A549 cells infected with wild-type RSV (CX3C) and mutant RSV with an altered CX3C motif of the G protein (CX4C). A549 cells were mock infected or infected with the CX3C or CX4C RSV strain and incubated for 4 days, with culture medium changed on day 2 postinfection. Supernatants from the bottom chamber were collected on days 3 and 4 postinfection (6 h and 24 h after addition of inserts, respectively). The Luminex assay was performed with collected supernatants to measure production of IFN-α2, IFN-λ1, and IFN-λ2. The data are represented as means ± SEM of 3 repeated independent experiments. ∗, P < 0.05 versus mock infection; △, P < 0.05 versus CX3C (determined by the Mann-Whitney test).

Fig 4

Activation/maturation of human dendritic cells and monocytes during coincubation with RSV CX3C- or CX4C-infected A549 cells. Histograms show expression of HLA-DR and the costimulatory molecule CD86 on the surface of single-donor PBMCs coincubated for 24 h with A549 cells that were mock infected (gray area) or infected with CX3C (solid lines) and CX4C (dotted lines) RSV strains. Graphs show the geometric mean fluorescence intensity (MFI) of CD86 and HLA-DR expressed on myeloid (mDC) and plasmacytoid (pDC) dendritic cells and monocytes. The data are represented as means ± SEM of independent experiments with PBMCs from 5 random donors. ∗, P < 0.05 versus mock infection (determined by the Wilcoxon matched-pairs test).

Fig 5

Innate immune responses of human PBMCs coincubated with A549 cells infected with CX3C or CX4C virus. A549 cells were mock infected or infected with the CX3C or CX4C RSV strain and cultured for 3 days, with culture medium replacement on day 2 p.i. and addition of PBMCs in permeable-membrane inserts on day 3 p.i. After coincubation for an additional 24 h, PBMCs were harvested and analyzed. The counterplot shows the distribution of plasmacytoid dendritic cells expressing CD86 and intracellular IFN-α (data for PBMCs from a single donor). CpG (ODN 2336), a human TLR9 ligand, was used as a positive control for IFN-α production. Graphs show the percentages of myeloid (mDC) and plasmacytoid (pDC) dendritic cells and monocytes producing IFN-α, TNF-α, and MCP-1. The data are represented as means ± SEM of independent experiments with PBMCs from 5 random donors and individual paired results for each donor. †, P < 0.05 versus mock infection; △, P < 0.05 versus CX3C (determined by the Wilcoxon matched-pairs test).

Fig 6

Innate immune responses of human PBMCs coincubated with A549 cells infected with CX3C or CX4C virus in the presence of F(ab′)₂ fragments of anti-G protein antibody. A549 cells were mock infected or infected with the CX3C or CX4C RSV strain and cultured for 3 days, with culture medium replaced on day 2 p.i. and PBMCs added to the permeable-membrane inserts on day 3 p.i. For the F(ab′)₂-treated wells, A549 cells were pretreated with F(ab′)₂ 131-2G MAb for 2 h before infection, and MAbs were included in medium changes and medium for the PBMCs. The PBMCs were harvested 24 h after exposure to A549 cells and studied by flow cytometry. Graphs show the percentages of myeloid (mDC) and plasmacytoid (pDC) dendritic cells and monocytes producing IFN-α and TNF-α. The data are represented as means ± SEM of independent experiments with PBMCs from 3 random donors. ∗, P < 0.05 (determined by the Wilcoxon matched-pairs test).

Fig 7

Memory T cell responses of human PBMCs coincubated with A549 cells infected with CX3C or CX4C virus. A549 cells were mock infected or infected with the CX3C or CX4C RSV strain and cultured for 3 days, with culture medium replacement on day 2 p.i. and addition of PBMCs in permeable-membrane inserts on day 3 p.i. After coincubation for an additional 24 h, PBMCs were harvested and analyzed. Counterplots show the distribution of CD8 effector memory T cells expressing CD69 and intracellular IFN-γ and of CD4 central memory T cells expressing CD69/IL-4. SEB was used as a positive control for IFN-γ/IL-4 production. Graphs show the percentages of CD69⁺ central memory (T_CM), effector memory (T_EM), and CD45RA⁺ effector memory (T_EMRA) T cells producing IFN-γ and IL-4. The data are represented as means ± SEM of independent experiments with PBMCs from 5 random donors and individual paired results for each donor. †, P < 0.05 versus mock infection; △, P < 0.05 versus CX3C (determined by the Wilcoxon matched-pairs test).

Fig 8

Memory T cell responses of human PBMCs coincubated with A549 cells infected with CX3C or CX4C virus in the presence of F(ab′)₂ fragments of anti-G protein antibody. A549 cells were mock infected or infected with the CX3C or CX4C RSV strain and cultured for 3 days, with culture medium replaced on day 2 p.i. and PBMCs added to the permeable-membrane inserts on day 3 p.i. For the F(ab′)₂-treated wells, A549 cells were pretreated with F(ab′)₂ 131-2G MAb for 2 h before infection, and MAbs were included in medium changes and medium for the PBMCs. The PBMCs were harvested 24 h after exposure to A549 cells and studied by flow cytometry. The graph shows the percentages of CD69⁺ central memory (T_CM), effector memory (T_EM), and CD45RA⁺ effector memory (T_EMRA) T cells producing IFN-γ. The data are represented as means ± SEM of independent experiments with PBMCs from 3 random donors. ∗, P < 0.05 (determined by the Wilcoxon matched-pairs test).