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CXCL10/CXCR3-mediated responses promote immunity to respiratory syncytial virus infection by augmenting dendritic cell and CD8(+) T cell efficacy - PubMed

CXCL10/CXCR3-mediated responses promote immunity to respiratory syncytial virus infection by augmenting dendritic cell and CD8(+) T cell efficacy

Dennis M Lindell et al. Eur J Immunol. 2008 Aug.

Abstract

The induction of inflammatory cytokines during respiratory viral infections contributes to both disease pathogenesis and resolution. The present studies investigated the role of the chemokine CXCL10 and its specific receptor, CXCR3, in the host response to pulmonary respiratory syncytial virus (RSV) infection. Antibody-mediated neutralization of CXCL10 resulted in a significant increase in disease pathogenesis, including airway hyperresponsiveness (AHR), mucus gene expression, and impaired viral clearance. When the pulmonary cytokine levels were examined, only type I IFN and IL-12p70 were significantly reduced. These latter observations were reflected in reduced dendritic cell (DC) numbers and DC maturation in the lungs of RSV-infected mice treated with anti-CXCL10. Neutralization of the only known receptor for CXCL10, CXCR3, resulted in similar increases in pathogenic responses. When bone marrow-derived DC were incubated with CXCL10 and RSV, an up-regulation of type I IFN was observed. In addition, T lymphocytes were also examined and a significant decrease in the number of RSV M2 peptide-specific CD8(+) T cells was identified. These findings highlight a previously unappreciated role for the CXCL10:CXCR3 signaling axis in RSV-infected animals by recruiting virus-specific T cells into the lung and promoting viral clearance.

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Figures

Figure 1
Figure 1

Effect of CxCL10 neutralization on RSV-induced pathophysiology. (A) Airway hyperreactivity (AHR) was assessed in uninfected, Ig-treated/RSV-infected (Ctrl RSV) and antiCxCL10-treated/RSV-infected (aCxCL10) mice by plethysmography on day 8 post-infection. Change in resistance represents the increase over baseline in response to methacholine challenge. Bars represent the mean of 10 mice per group ± SEM . *p< 0.05 vs. uninfected, #p<0.05 vs. control RSV infected. (B) PAS staining in lungs of control Ig-treated/RSV-infected (Ctrl RSV) and antiCxCL10-treated/RSV-infected (aCxCL10) mice at day 8 post RSV-infection. Histologic sections were stained with periodic acid Schiff (PAS). (C) The expression of the mucus-associated genes Muc5ac and Gob5 was determined by real-time PCR of whole lung RNA (day 8 post-infection).

Figure 2
Figure 2

Role of CxCL10 neutralization on the impaired clearance of RSV from the lungs. (A) The number of infectious RSV particles (plaque forming units, PFU) in the lungs was assessed by plaque assay at day 3 post-infection. PFU were below the limit of detection at day 8 post-infection. (B) As a complementary measure of viral load, expression of the RSV G protein transcript was assessed at day 3 and day 8 post-infection. The relative increase in RSV G protein expression was compared to lungs of control Ig-treated/RSV-infected (Ctrl) mice. Bars represent the mean of 5 mice per group ± SEM. *p< 0.05 vs. ctrl. Similar results were obtained in two independent experiments.

Figure 3
Figure 3

Effect of CxCL10 neutralization on the production of cytokines in the lungs of RSV infected mice at day 8 post-infection. Mice were treated with anti-CxCL10 or control antibodies and infected with RSV. In (A), cytokine levels were assessed by ELISAs of homogenized lung samples. In (B), the expression of Ifnα4 was assessed by quantitative RT-PCR of lung RNA. Bars represent the mean of 5 mice per group ± SEM. *p< 0.05. Similar results were obtained in two independent experiments.

Figure 4
Figure 4

(A) Representative gating of dendritic cell populations from the lungs of RSV infected mice. Total lung leukocytes were isolated by enzymatic digest, and analysed without further purification by flow cytometry. Monocytes/macrophages (gates I and III) were defined as CD11b+CD11c lo and CD11bloCD11c+; mDC were defined by low auto fluorescence, CD45+, CD11bhi, CD11chi MHC-II+ (gate II); pDC were defined by low forward scatter, low side scatter, CD45R/B220+, CD11cint (right panel). The expression of MHC II by CD11chiCD11bhi mDC is shown in the histogram in the middle panel. (B) The frequency of CXCR3 expression by various leukocyte subsets from the lungs of uninfected and day 8 RSV infected mice. Macrophage/monocyte (gates I, III) and CD11chiCD11bhi mDC (gate II) were gated as shown in (A). Similar results were obtained in three independent experiments.

Figure 5
Figure 5

(A) Absolute numbers of myeloid dendritic cells (mDC), plasmacytoid dendritic cells (pDC), NK cells, and monocyte/macrophages in the lungs of RSV-infected, Ig-treated █ OK? █ control and RSV-infected, CxCL10 neutralized (aCxCL10) mice at day 8 post-infection. Frequencies were determined by flow cytometry, and absolute numbers were determined as described in the Materials and methods. Gating of myeloid cells was done as shown in Fig 4. Monocytes/macrophages represent gates I and III defined in Fig. 4. Bars represent the mean of 9–10 mice per group ± SEM. Dashed lines represent cell numbers in naive animals. *p < 0.05 (B) Representative histograms depict the expression of MHC II by mDC from the lungs of naïve mice, Ig-treated/RSV-infected controls and CxCL10-neturalized/RSV-infected mice at day 8 post-infection. (C) The median fluorescence intensity (MedFI) of MHC II staining is shown. Bars represent the mean of five mice ± SEM. *p<0.03. Similar results were obtained in 3 independent experiments.

Figure 6
Figure 6

Lung T cell numbers in anti-CxCL10/RSV-infected and control Ig-treated/RSV-infected mice. The absolute numbers of CD4, CD8, and RSV M82–90 tetramer+ T cells at day eight post-infection were determined by flow cytometric analysis of enzymatically digested lungs. *p < 0.05 versus control Ig treated mice. Similar results were obtained in two independent experiments.

Figure 7
Figure 7

Effect of CxCR3 neutralization on RSV-induced pathophysiology. (A) Airway hyperreactivity (AHR) was assessed in uninfected, Ig-treated/RSV-infected (Ctrl RSV) and antiCxCL10-treated/RSV-infected (aCxCL10) mice by plethysmography on day 8 post-infection. Change in resistance represents the increase over baseline in response to methacholine challenge. Bars represent the mean of 10 mice per group ± SEM . (B) The expression of the mucus-associated genes Muc5ac and Gob5 was determined by real-time PCR of whole lung RNA (day 8 post-infection). Bars represent the mean of 5 mice per group from one of two independent experiments. *p < 0.05 vs. uninfected, #p<0.05 vs. control Ig-treated/RSV infected.

Figure 8
Figure 8

Effect of CxCL10 on type I IFN production and viral replication/transcription in vitro. BMDC were prepared as described in the Methods. (A) BMDC were stained with either anti-CxCR3 (shaded) or isotype control (hollow line) antibodies and analyzed by flow cytometry. One representative histogram is shown. Similar results were obtained in 2 independent experiments. (B) Effect of recombinant CxCL10 on RSV-induced expression of Ifna4 mRNA and RSV G transcript. BMDC were infected with RSV (MOI =1) alone, or in the presence of 100ng/ml CxCL10, and the levels of Ifnα4 mRNA and RSV G transcript were assessed by real time PCR 24 hours after infection. Bars represent the Mean of 3 replicates +/− SEM. *p<0.05 versus control. Similar results were obtained in two independent experiments. (C) Effect of CxCL10 on RSV-induced IL-12 p70 and MHC II expression. BMDC were cultured as in (B), IL-12p70 was measured in the supernatant at 24 hours post-infection. The expression of MHC II was determined by flow cytometry.

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