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. 2012 Nov;130(5):1175-1186.e9.
doi: 10.1016/j.jaci.2012.08.033. Epub 2012 Oct 1.

Responsiveness to Respiratory Syncytial Virus in Neonates Is Mediated Through Thymic Stromal Lymphopoietin and OX40 Ligand

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Free PMC article

Responsiveness to Respiratory Syncytial Virus in Neonates Is Mediated Through Thymic Stromal Lymphopoietin and OX40 Ligand

Junyan Han et al. J Allergy Clin Immunol. .
Free PMC article

Abstract

Background: Recent studies revealed a critical role for thymic stromal lymphopoietin (TSLP) released from epithelial cells and OX40 ligand (OX40L) expressed on dendritic cells (DCs) in T(H)2 priming and polarization.

Objectives: We sought to determine the importance of the TSLP-OX40L axis in neonatal respiratory syncytial virus (RSV) infection.

Methods: Mice were initially infected with RSV as neonates or adults and reinfected 5 weeks later. Anti-OX40L or anti-TSLP were administered during primary or secondary infection. Outcomes included assessment of airway function and inflammation and expression of OX40L, TSLP, and IL-12.

Results: OX40L was expressed mainly on CD11c(+)MHC class II (MHCII)(+)CD11b(+) DCs but not CD103(+) DCs. Treatment of neonates with OX40L antibody during primary RSV infection prevented the subsequent enhancement of airway hyperresponsiveness and the development of airway eosinophilia and mucus hyperproduction on reinfection. Administration of anti-TSLP before neonatal RSV infection reduced the accumulation of lung DCs, decreased OX40L expression on lung DCs, and attenuated the enhancement of airway responses after reinfection.

Conclusions: In mice initially infected as neonates, TSLP expression induced by RSV infection is an important upstream event that controls OX40L expression, lung DC migration, and T(H)2 polarization, accounting for the enhanced response on reinfection.

Figures

FIG 1
FIG 1
RSV infection induces OX40L expression on lung DCs. Mice were infected as neonates or at 5 weeks of age. Age-matched control mice were inoculated with PBS. One day after RSV infection, lung single-cell suspensions were prepared, and lung DC subsets were identified following the gating strategy described in Fig E1. The percentage of OX40L+ cells among CD11c+CD11b+CD103 DCs (A) and the percentage of IL-12+ cells among CD11c+ cells (B) was analyzed by using flow cytometry, as described in the Methods section. Results are from 3 independent experiments (n = 12). **P < .01.
FIG 2
FIG 2
Effect of anti-OX40L on airway responsiveness to primary RSV infection in adult mice. Mice were infected on day 0 at 5 weeks of age. Anti-OX40L (RM134L) or control antibody was administered intraperitoneally at 15 mg/kg on days −1, +1, and +2. Airway responsiveness to inhaled MCh (A), BAL cellularity (B), and BAL fluid IFN-γ levels (C) were assessed on day 7 after infection. Results are from 3 independent experiments (n = 12). Data are expressed as means ± SEMs. *P < .05 and **P < .01. Ab, Antibody; Eos, eosinophil; Lym, lymphocyte; Mac, macrophage; Neu, neutrophil; RL, lung resistance.
FIG 3
FIG 3
Effect of anti-OX40L administered during primary infection on secondary RSV infection in adult mice. Mice were initially infected with RSV at 5 weeks of age and reinfected with RSV 5 weeks later. Anti-OX40L or control antibody was administered during primary RSV infection. Airway responsiveness to inhaled MCh (A), BAL cellularity (B), and BAL fluid IFN-γ levels (C) were assessed on day 7 after reinfection. Results are from 3 independent experiments (n = 12). Data are expressed as means ± SEMs. *P < .05 and **P < .01. Ab, Antibody; Eos, eosinophil; Lym, lymphocyte; Mac, macrophage; Neu, neutrophil; RL, lung resistance.
FIG 4
FIG 4
Effect of anti-OX40L administered during neonatal primary infection on the response to RSV reinfection. Newborn mice were infected with RSV on day 0. Anti-OX40L or control antibody was administered on days −1, +1, and +2. Five weeks later, mice were reinfected with RSV. Airway responsiveness to inhaled MCh (A), BAL cellularity (B), and BAL fluid cytokine levels (C) were assessed on day 7 after secondary RSV infection. Results are from 3 independent experiments (n = 12). Data are expressed as means ± SEMs. *P < .05 and **P < .01. Ab, Antibody; Eos, eosinophil; Lym, lymphocyte; Mac, macrophage; Neu, neutrophil; RL, lung resistance.
FIG 5
FIG 5
Administration of anti-OX40L during neonatal infection affects cytokine responses in PBLNs. Newborn mice were infected with RSV and reinfected with RSV 5 weeks later. Anti-OX40L or control antibody was administered either during primary RSV (A) or secondary RSV (B) infection. Seven days after reinfection, PBLN cells were collected and seeded in 96-well plates and stimulated with UV-RSV. IL-4, IL-5, IL-6, IL-13, and IFN-γ levels in the supernatants were measured 3 days after culture. Results are from 3 independent experiments (n = 12). Data are expressed as means ± SEMs. *P < .05 and **P < .01. Ab, Antibody.
FIG 6
FIG 6
RSV induces TSLP expression in the lung and neutralization of TSLP before primary infection affects the responses to RSV reinfection. A, Lung mRNA was isolated 1 day after primary infection of neonates or adult mice and TSLP mRNA levels were measured by quantitative RT-PCR. Increases in expression levels were compared with those in adult noninfected control animals. B-D, Newborn mice were infected with RSV and reinfected with RSV 5 weeks later. Anti-TSLP or control antibody was administered 1 day before neonatal infection. Seven days after reinfection, airway responsiveness to inhaled MCh (Fig 6, B), BAL cellularity (Fig 6, C), and BAL fluid cytokine levels (Fig 6, D) were assessed. E-G, Mice were infected with RSV as neonates. Anti-TSLP or control antibody was administered 1 day before infection. Lung single-cell suspensions were prepared 1 day after infection, and OX40L expression was analyzed by flow cytometry. Fig 6, E, Percentage of OX40L+ cells among lung CD11c+CD11b+ DCs. Fig 6, F, Total number of OX40L+CD11c+ cells in the lung. Fig 6, G, OX40L MFI expression on CD11c+CD11b+ DCs. Representative histogram and combined MFI depicting expression of OX40L on CD11c+CD11b+ DCs; the isotype control is shown as a shaded line. Results are from 2 to 4 independent experiments (n = 6-12). Data are expressed as means ± SEMs. **P < .01. Ab, Antibody; Eos, eosinophil; Lym, lymphocyte; Mac, macrophage; Neu, neutrophil; RL, lung resistance.
FIG 6
FIG 6
RSV induces TSLP expression in the lung and neutralization of TSLP before primary infection affects the responses to RSV reinfection. A, Lung mRNA was isolated 1 day after primary infection of neonates or adult mice and TSLP mRNA levels were measured by quantitative RT-PCR. Increases in expression levels were compared with those in adult noninfected control animals. B-D, Newborn mice were infected with RSV and reinfected with RSV 5 weeks later. Anti-TSLP or control antibody was administered 1 day before neonatal infection. Seven days after reinfection, airway responsiveness to inhaled MCh (Fig 6, B), BAL cellularity (Fig 6, C), and BAL fluid cytokine levels (Fig 6, D) were assessed. E-G, Mice were infected with RSV as neonates. Anti-TSLP or control antibody was administered 1 day before infection. Lung single-cell suspensions were prepared 1 day after infection, and OX40L expression was analyzed by flow cytometry. Fig 6, E, Percentage of OX40L+ cells among lung CD11c+CD11b+ DCs. Fig 6, F, Total number of OX40L+CD11c+ cells in the lung. Fig 6, G, OX40L MFI expression on CD11c+CD11b+ DCs. Representative histogram and combined MFI depicting expression of OX40L on CD11c+CD11b+ DCs; the isotype control is shown as a shaded line. Results are from 2 to 4 independent experiments (n = 6-12). Data are expressed as means ± SEMs. **P < .01. Ab, Antibody; Eos, eosinophil; Lym, lymphocyte; Mac, macrophage; Neu, neutrophil; RL, lung resistance.
FIG 6
FIG 6
RSV induces TSLP expression in the lung and neutralization of TSLP before primary infection affects the responses to RSV reinfection. A, Lung mRNA was isolated 1 day after primary infection of neonates or adult mice and TSLP mRNA levels were measured by quantitative RT-PCR. Increases in expression levels were compared with those in adult noninfected control animals. B-D, Newborn mice were infected with RSV and reinfected with RSV 5 weeks later. Anti-TSLP or control antibody was administered 1 day before neonatal infection. Seven days after reinfection, airway responsiveness to inhaled MCh (Fig 6, B), BAL cellularity (Fig 6, C), and BAL fluid cytokine levels (Fig 6, D) were assessed. E-G, Mice were infected with RSV as neonates. Anti-TSLP or control antibody was administered 1 day before infection. Lung single-cell suspensions were prepared 1 day after infection, and OX40L expression was analyzed by flow cytometry. Fig 6, E, Percentage of OX40L+ cells among lung CD11c+CD11b+ DCs. Fig 6, F, Total number of OX40L+CD11c+ cells in the lung. Fig 6, G, OX40L MFI expression on CD11c+CD11b+ DCs. Representative histogram and combined MFI depicting expression of OX40L on CD11c+CD11b+ DCs; the isotype control is shown as a shaded line. Results are from 2 to 4 independent experiments (n = 6-12). Data are expressed as means ± SEMs. **P < .01. Ab, Antibody; Eos, eosinophil; Lym, lymphocyte; Mac, macrophage; Neu, neutrophil; RL, lung resistance.

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