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IkappaB kinase is a critical regulator of chemokine expression and lung inflammation in respiratory syncytial virus infection - PubMed

IkappaB kinase is a critical regulator of chemokine expression and lung inflammation in respiratory syncytial virus infection

Helene A Haeberle et al. J Virol. 2004 Mar.

Abstract

Respiratory syncytial virus (RSV) is the major etiologic agent of severe epidemic lower respiratory tract infections in infancy. Airway mucosal inflammation plays a critical role in the pathogenesis of RSV disease in both natural and experimental infections. RSV is among the most potent biological stimuli that induce the expression of inflammatory genes, including those encoding chemokines, but the mechanism(s) that controls virus-mediated airway inflammation in vivo has not been fully elucidated. Herein we show that the inoculation of BALB/c mice with RSV results in rapid activation of the multisubunit IkappaB kinase (IKK) in lung tissue. IKK transduces upstream activating signals into the rate-limiting phosphorylation (and proteolytic degradation) of IkappaBalpha, the inhibitory subunit that under normal conditions binds to the nuclear factor (NF)-kappaB complex and keeps it in an inactive cytoplasmic form. Mice treated intranasally with interleukin-10 or with a specific cell-permeable peptide that blocks the association of the catalytic subunit IKKbeta with the regulatory protein NEMO showed a striking reduction of lung NF-kappaB DNA binding activity, chemokine gene expression, and airway inflammation in response to RSV infection. These findings suggest that IKKbeta may be a potential target for the treatment of acute or chronic inflammatory diseases of the lung.

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Figures

FIG. 1.
FIG. 1.

Time course of lung NF-κB activation in RSV-infected mice. Nuclear proteins were extracted from the lungs of sham- or RSV-infected mice (at 1.5, 3, 6, 12, and 24 h postinoculation) and used for EMSA (with the RANTES NF-κB-1 oligonucleotide probe) as described in Materials and Methods. Each data point is for one animal that is representative of three animals tested for each time point.

FIG. 2.
FIG. 2.

Exogenous IL-10 blocks RSV-induced NF-κB activation by partially preventing phosphorylation and degradation of IκBα. The effect of IL-10 on IκBα phosphorylation (A) and total IκBα expression (B) was assessed by Western blot analysis and NF-κB activation by EMSA (C). BALB/c mice were infected with RSV alone (lanes 2, 4, 6, and 8) or together with i.n. administration of rIL-10 (10 μg) (lanes 3, 5, 7, and 9) or were sham infected (lane 1). At 0.5, 1.5, 6, and 24 h postinfection, lungs were removed and nuclear proteins or WCE was isolated as described in Materials and Methods. For Western blots, 50 μg of WCE was fractionated by SDS-PAGE (4 to 20%), transferred onto a PVDF membrane, and probed with an antibody against phosphorylated IκBα (A) or total IκBα (B). For determination of NF-κB activation, EMSAs were performed in parallel, using nuclear proteins from mouse lungs as described for Fig. 1 (C). Each data point and/or treatment condition is from a single animal and is representative of data for three animals. Results are representative of three independent experiments.

FIG. 3.
FIG. 3.

RSV induces IKK activity: inhibitory effect of IL-10. WCE from A549 cells (250 μg) or mouse lung tissue (500 μg) were immunoprecipitated with an antibody against IKKβ, and the immunoprecipitates were subjected to a kinase assay using GST-IκBα wild type (WT) or a mutated form (Mut) as substrates. Samples were subjected to SDS-PAGE and transferred to PVDF membranes. Membranes were dried and exposed to an XAR film. (A) RSV infection (6 h) induces IKKβ activity in A549 cells, as shown by specific phosphorylation of wild-type GST-IκBα but not a mutated substrate. (B) Time course of IKKβ activity in lung tissues of sham-infected mice, RSV-infected mice, and RSV-infected mice treated i.n. with IL-10. RSV infection induces the activation of IKKβ activity (lanes 2, 5, and 8), which is decreased by IL-10 treatment (lanes 3, 6, and 9). Equal sample loading was determined by Western blotting for IKKβ. (C) Densitometry analysis of phosphorylated GST-IκBα complexes expressed as percentages of activation relative to those of RSV-infected mice.

FIG. 4.
FIG. 4.

Inhibitory effect of IL-10 on chemokine mRNA expression in lung tissue of RSV-infected mice. The expression of nine murine chemokine mRNAs was determined by RPA. (A) BALB/c mice were sham infected (lane 1), infected with RSV (lane 2), or infected with RSV together with i.n. administration of rIL-10 (lane 3). At 24 h postinfection, RNA was isolated from lung tissue and hybridized with a 32P-labeled RiboQuant MultiProbe (Pharmingen) containing DNA templates for the mouse chemokines RANTES, eotaxin, MIP-1β, MIP-1α, MIP-2, MCP-1, and TCA-3 and the housekeeping genes L32 (rRNA) and GAPDH. After RNase treatment and purification, protected probes were run on a QuickPoint sequence gel, exposed to an XAR film, and developed. The identity of each protected fragment was established as described in Materials and Methods. (B) The quantity of each mRNA species in the original RNA sample was determined based on the signal intensity (by optical densitometry) given by the appropriately sized, protected probe fragment band. Sample loading was normalized with the housekeeping gene L32, which was included in each template set. The density of each chemokine mRNA is expressed relative to that of L32. The data shown are representative of four independent experiments. Data are expressed as means ± standard errors of the means (SEM) of four animals per group. *, P < 0.05, compared with RSV plus IL-10.

FIG. 5.
FIG. 5.

Inhibitory effect of IL-10 on RANTES, MCP-1, and MIP-1α protein production. The concentrations of RANTES, MCP-1, and MIP-1α were determined by enzyme-linked immunosorbent assay in BAL obtained from groups of mice that were sham infected, RSV infected, or RSV infected and treated with rIL-10 (at 24 h postinfection). Data are expressed as means ± SEM of five animals per group. #, P < 0.001 compared to sham-infected mice; *, P < 0.05 compared to mice infected with RSV plus IL-10.

FIG. 6.
FIG. 6.

Lung pathology scores in RSV-infected mice. Mice were sham infected, infected with RSV, or infected with RSV and treated with rIL-10. At 24 h postinfection, mice were sacrificed and lungs were removed, fixed in 10% buffered formalin, and embedded in paraffin. Multiple 4-μm-thick sections were stained with H&E and scored for cellular inflammation. The number of abnormal perivascular and peribronchial spaces divided by the total spaces counted is the percentage reported as the pathology score. Each data point represents the pathology score of an individual animal (group means are indicated by horizontal lines) in each independent experiment (n = 4 experiments). The pathology score for sham-infected mice is zero. P values were <0.01 for each experiment for RSV-infected mice compared to RSV-infected mice treated with IL-10.

FIG. 7.
FIG. 7.

Viral replication in lungs of BALB/c mice infected with pooled RSV and treated with IL-10. BALB/c mice were infected with RSV (final administered dose, 107 PFU) together with 10 μg of IL-10 or control vehicle (PBS) in a final volume of 50 μl. One, 5 and 21 days after infection, the lung tissue was removed and homogenized and the concentration of infectious virus was determined by a plaque assay. The bar graph shows means ± SEM of three mice for each treatment group.

FIG. 8.
FIG. 8.

NBD peptide blocks NF-κB activation and reduces lung chemokine expression and inflammation in RSV-infected mice. BALB/c mice were infected with RSV and concomitantly injected i.n. with NBD peptide (20 μg/20 μl) or a mutated control peptide. On day 5, 8 h before mice were sacrificed, a second dose of peptide or vehicle was inoculated i.n. (A) Nuclear proteins were extracted from the lungs and used for EMSAs as described for Fig. 1. Results for one sample of sham infection (lane 1) and two separate samples of RSV plus mutated NBD (mNBD; lanes 2 and 3) or RSV plus NBD (NBD; lanes 4 and 5) are shown. (B) Densitometry of lung chemokine mRNA expression by RPA (means ± SEM of three mice per treatment group). (C) Multiple H&E-stained lung sections were scored for cellular inflammation as described in Materials and Methods (means ± SEM of three mice per treatment group). *, P < 0.05 for RSV-infected mice treated with NBD compared to RSV-infected mice.

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References

    1. Aherne, W. T., T. Bird, S. D. B. Court, P. S. Gardner, and J. McQuillin. 1970. Pathological changes in virus infections of the lower respiratory tract in children. J. Clin. Pathol. 23:7-18. - PMC - PubMed
    1. Beg, A. A., and A. S. J. Baldwin. 1993. The I kappa B proteins: multifunctional regulators of Rel/NF-kappa B transcription factors. Genes Dev. 7:2064-2070. - PubMed
    1. Berg, D. J., R. Kuhn, K. Rajewsky, W. Muller, S. Menon, N. Davidson, G. Grunig, and D. Rennick. 1995. Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance. J. Clin. Investig. 96:2339-2347. - PMC - PubMed
    1. Bitko, V., A. Velazquez, L. Yank, Y.-C. Yang, and S. Barik. 1997. Transcriptional induction of multiple cytokines by human respiratory syncytial virus requires activation of NF-kB and is inhibited by sodium salicylate and aspirin. Virology 232:369-378. - PubMed
    1. Casola, A., R. P. Garofalo, H. Haeberle, T. F. Elliott, A. Lin, M. Jamaluddin, and A. R. Brasier. 2001. Multiple cis regulatory elements control RANTES promoter activity in alveolar epithelial cells infected with respiratory syncytial virus. J. Virol. 75:6428-6439. - PMC - PubMed

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