Role of CCR3 in respiratory syncytial virus infection of airway epithelial cells

Abstract

Respiratory syncytial virus (RSV) infection is the principal cause of severe lower respiratory tract disease and accounts for a significant risk for developing asthma later in life. Clinical studies have shown an increase in airway responsiveness and a concomitant Th₂ response in the lungs of RSV-infected patients. These indications suggest that RSV may modulate aspects of the immune response to promote virus replication. Here, we show that CCR3 facilitates RSV infection of airway epithelial cells, an effect that was inhibited by eotaxin-1/CCL11 or upon CCR3 gene silencing. Mechanistically, cellular entry of RSV is mediated by binding of the viral G protein to CCR3 and selective chemotaxis of Th₂ cells and eosinophils. In vivo, mice lacking CCR3 display a significant reduction in RSV infection, airway inflammation, and mucus production. Overall, RSV G protein-CCR3 interaction may participate in pulmonary infection and inflammation by enhancing eosinophils' recruitment and less potent antiviral Th₂ cells.

Keywords: Cell biology; Immunology; Virology.

Graphical abstract

Figure 1

CCR3-dependent infection by RSV and inhibition by eotaxin-1/CLL11 (A) RSV plaque reduction by chemokines in Hep-2 cells inoculated with RSV. Three-days postinfection, the plaques were counted and the percent inhibition of virus infectivity of treated cells was determined versus untreated control wells. (B) RSV plaque-forming units per well detected 3-days postinfection with or without chemokines (MCP-1/CCL3, eotaxin/CLL11, MIP-1β/CCL4, SDF-1/CXCL12, and Frk/CX3CL1) in Ghost cells expressing CCR2, CCR3, CCR5, CXCR4, or CX3CR-1. The number of parental cells that were infected with RSV was always less than in CCR3 transfected cells (p < 0.005). Eotaxin-1/CLL11 exhibited a dose-dependent inhibition of infection of G.CCR3⁺ cells as compared to PBS-treated cells (p < 0.05).

Figure 2

CCR3 associates with RSV G protein (A) Western blot of purified RSV sG using rabbit anti-sG glycoprotein Abs. Fractions were collected, separated by electrophoresis, and sG protein was identified through their binding to specific anti-sG antibodies. (B) A549 cells and G.CCR3⁺ lysates were incubated with sG protein (50 μg/mL) and the complexes formed by RSV G-protein and CCR3 were immunoprecipitated with anti-RSV G-protein mAb (5 μg/mL) and then analyzed with western blotting using goat anti-CCR3 polyclonal antibodies. mAb isotype controls were included as negative controls in these experiments. In B, eotaxin-1/CLL11 (2 μg/mL) and anti-Eotaxin-1/CLL11 mAb (5 μg/mL) were used as a positive control to immunoprecipitate CCR3. (C and D) Flow cytometry results showing Eotaxin-1/CLL11-FITC binding to G.CCR3⁺ or A549 (D) and inhibition of binding by anti-eotaxin-1/CLL11 NAbs. (E and F) Eotaxin-1/CLL11-FITC binding to G.CCR3⁺ cells and A549 and inhibition of binding by sG and RSV-F proteins. (G and H) sG, and RSV-F proteins had no effect on the binding of MIP-1β/CCL4 to G.CCR5⁺ and A549 cells.

Figure 3

CCR3 and CX3CR-1 protein and mRNA expression in epithelial cells and eosinophils (A) Detection of CCR3 and CX3CR1 immunoreactivity on airway epithelium. Airway sections cells from an asthmatic donor showed positive immunoreactivity for CCR3 and CX3CR1. Lung sections were stained with goat anti-CCR3 polyclonal antibodies and rabbit anti-CX3CR1 polyclonal antibodies. No staining was detected with the negative control antibodies (data not shown). Similar results were obtained in 5 other sections. (B) RNA purified from epithelial cell lines (A549 and Hep-2) and eosinophils was examined for the presence of CCR3 and CX3CR1 transcripts by RT-PCR. For positive controls, RNA from Ghost cells transfected with CCR3 or CX3CR1 were used. Results are representative of three independent experiments performed under the same conditions. (C) Surface expression of CCR3 and CX3CR-1 on A549 and eosinophils using flow cytometry and mouse anti-human CCR3 and anti-CX3CR1 mAbs or IgG isotype control mAbs (R&D Systems). Results are representative of three independent experiments performed under the same conditions. Scale bars indicate 125 μm. See also Figure S1

Figure 4

Inhibition of CCR3-dependent RSV-GFP infection by eotaxin-1/CLL11 using flow cytometry Human primary airway epithelial cells (PAEC) and G.CCR3⁺ cells were inoculated with RSV-GFP in the presence or absence of eotaxin-1/CLL11 and 3 days post-infection, cells were trypsinized and the percentage of infected cells was determined by assessing GFP fluorescence via flow cytometry. A representative flow cytometry plot of the mean percentage of GFP-positive PAEC and Ghost transfected cells for each of the conditions tested is shown in (A–F). (A) Human PAEC were infected with RSV-GFP in virus medium with 0 and 1 μg/mL of eotaxin-1/CLL11 after 3 days of infection (neg Ctl: no RSV and no eotaxin-1/CLL11). (B) Fluorescence measured by flow cytometry in parental Ghost cells inoculated or noninoculated with RSV-GFP. (C) GFP fluorescence in parental Ghost and G.CCR3⁺ cells inoculated with or without RSV-GFP during 3 days. (D) G.CCR3⁺ cells after 3 days infection with RSV-GFP in the absence or presence of eotaxin-1/CLL11 (1 μg/mL). (E and F) G.CCR4⁺ and G.CCR5⁺ cells 3-days postinfection with RSV-GFP, respectively.

Figure 5

Silencing CCR3 reduces RSV infection in Hep-2 cells (A) Chemokine receptors-targeting and control siRNA were transfected into Hep-2 cells and harvested 24h after transfection. Cultures were challenged with RSV and 3 days post-infection the percentage inhibition was determined by dividing the mean plaque-forming units (PFU) of siRNA-treated Hep-2 cells by the number of PBS-treated Hep-2 cells. Each CCR siRNA was compared to its siRNA control. Error bars represent the standard deviation of triplicates. (B) siRNA compounds were assessed to block RSV infection of Ghost transfected cells. The compounds were added to cells followed by infection with RSV. Three-days postinfection, the PFU were counted in siRNA-treated cells versus PBS-treated control wells. Error bars represent the standard deviation of three independent experiments with triplicates for each experimental condition. See also Figures S2 and S3

Figure 6

CCR3 deficiency prevented lung RSV-induced inflammation and mucus production (A) Effect of CCR3 deficiency on lung viral titer 5 days after infection. Bar graphs show quantitative plaque assays and ELISA of RSV in lung homogenates. p < 0.001, n = 7, and n = 8 per group for experiments in RSV plaque assay and ELISA, respectively. (B) Paraffin-embedded lung tissues obtained 5-days post-infection from RSV-infected wild type and CCR3^−/− mice were stained by periodic acid-Schiff (PAS) and hematoxylin and eosin (H&E). B, show reduced inflammation in CCR3 deficient mice and mucus production as evidenced by significantly fewer PAS-positive cells. The significance of difference between selected groups was determined by unpaired t test. ∗, p < 0.05. Scale bars indicate 200 μm.

Figure 7

Eosinophil migration induced by sG protein and eotaxin-1/CLL11 (Eot) in filter assays Eosinophils (Eos) were collected, purified, and incubated with or without anti-CCR3 or anti-CX3CR1 mAbs and a chemotaxis assay was performed in Boyden chambers as described in STAR Methods. (A) Transmigration through polycarbonate filters showing chemotactic activity of purified human eosinophils to sG protein and eotaxin-1/CLL11 in the absence or presence of anti-CCR3 Abs. (B) Eotaxin-1/CLL11 induced eosinophils chemotaxis and blockade of CCR3 with antibodies significantly inhibited eosinophil migration (p < 0.01 compared with chemotaxis to eotaxin-1/CLL11 alone). Incubation with anti-CCR3 mAb decreased the potency of sG protein to attract eosinophils markedly (p < 0.001 compared with sG protein alone). Whereas, preincubation of eosinophils with anti-CX3CR1 Ab or control antibodies had no effect on chemotaxis (not shown). Data are presented as the number of cells per field (magnification, X400). The results shown are representative experiments and are presented as the mean ± SD of 5 high power fields per well. Med, medium. Scale bars indicate 75 μm.

Figure 8

RSV sG protein attracts preferentially Th₂ cells Th₁ and Th₂ cells were generated as described in the STAR Methods. As shown at the single-cell level by measuring intracellular cytokine production (A) neonatal T cells primed under the Th₁ conditions differentiated into T cells producing IFNγ but little IL-4, (A, upper left), whereas naive T cells primed in the presence of IL-4 and anti-IL-12 resulted in a population of T cells producing mainly IL-4. (A, upper right). (B) Chemotactic response of Th₁ cells (B, lower left) and Th₂ cells (B, lower right) to sG protein (50 ng/mL), eotaxin-1/CLL11 (100 ng/mL) or MIP-1β/CCL4 (100 ng/mL). The chemotactic activity of sG protein, eotaxin-1/CLL11, and MIP-1β/ CCL4 were compared to medium alone with the use of modified Boyden chambers. The anti-CCR3, anti-CCR5 or anti-CX3CR1 Abs were added to the upper chamber to examine the antagonism of cell migration toward the chemoattractants (sG protein, eotaxin-1/CLL11, or MIP-1β/CCL4) in the lower chamber. Data are presented as the number of cells per field (magnification, ×400). Isotype control mAbs had no effect on the chemotaxis (not shown). The results shown are representative experiments and are presented as the mean ± SD of 5 high power fields per well. Med, medium.