CpG oligodeoxynucleotides do not require TH1 cytokines to prevent eosinophilic airway inflammation in a murine model of asthma

doi:10.1016/S0091-6749(99)70022-9

Journal of Allergy and Clinical Immunology

Volume 104, Issue 6, December 1999, Pages 1258-1264

https://doi.org/10.1016/S0091-6749(99)70022-9 Get rights and content

Abstract

Background: Oligodeoxynucleotides (ODNs) containing the dinucleotide CpG in a specific sequence context (CpG-ODNs) have the ability to prevent the development of eosinophilic airway inflammation and bronchial hyperreactivity in a murine model of asthma. We have previously demonstrated that CpG-ODNs stimulate expression of the T_H1-inducing cytokines IFN-γ and IL-12 in a murine model of asthma and that this stimulation is associated with the protection against asthmatic inflammation. Objective: The purpose of this study was to examine whether the protection conferred by CpG-ODNs in a schistosome egg-egg antigen murine model of asthma is dependent on the induction of IFN-γ, IL-12, or both. Methods: C57BL/6 mice were sensitized to schistosome eggs in the presence or absence of CpG-ODNs or control ODNs and then stimulated with soluble egg antigen in the airway. The protection offered by CpG-ODNs in these mice was compared with the protection induced by CpG-ODNs in IL-12 and IFN-γ knockout mice and in mice treated with anticytokine blocking antibodies. Double-knockout mice (IL-12/IFN-γ) were also generated and used in these studies. Determinations included airway eosinophilic inflammation and bronchial hyperreactivity to inhaled methacholine. Results: We found that CpG-ODNs confer protection against both airway eosinophilia and bronchial hyperreactivity in the absence of IFN-γ or IL-12 or in the presence of both cytokines together. However, in the absence of either IL-12 or IFN-γ, mice require 10 times as much CpG-ODNs to be protected against the induction of airway eosinophilia. The T_H2 cytokines IL-4 and IL-5 were reduced in all of the CpG-treated mice, although less in the absence of IL-12 and IFN-γ. Conclusion: These data indicate that CpG-ODNs prevent the generation of T_H2-like immune responses by multiple mechanisms, which involve, but do not require, IL-12 and IFN-γ. A direct suppressive effect of CpG-ODNs on T_H2 responses is suggested by their reduction in IFN-γ and IL-12 knockout mice. (J Allergy Clin Immunol 1999;1258-64.)

Section snippets

Animals and murine model of asthma

Six-week-old female C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, Me). All animal care and housing requirements of the National Institutes of Health Committee on Care and Use of Laboratory Animals were followed. For some studies, mice on a C57BL/6 genetic background with disrupted genes for IFN-γ (C57BL/6-Ifng ^tm1Ts, Jackson Laboratory)¹⁰ or IL-12 (C57BL/6-Il12 ^atm1Jm, Jackson Laboratory)¹¹ were used. The IFN-γ knockout mice produce no IFN-γ; the IL-12 knockout mice produce

ELISA

Murine IL-4 and IL-5 were measured by using a sandwich ELISA (R&D) according to the manufacturer’s instructions.

Effect of T_H1 cytokines on airway eosinophilia and bronchial hyperreactivity

To ascertain whether T_H1 cytokines were capable of preventing the development of T_H2-mediated airway eosinophilia in the schistosome egg-SEA murine asthma model, we first examined the effects of administering IFN-γ and IL-12 at various time points relative to sensitization and airway challenge with schistosome eggs and SEA. We found that treatment with IL-12 (1 μg administered intraperitoneally on days 0, 1, 2, and 3), as well as with IFN-γ (10,000 U administered intraperitoneally on days 0, 1,

DISCUSSION

Immunostimulatory CpG-ODNs have a wide range of effects on inflammation and immune responses.8, 16, 17, 18, 19, 20 Recently, we have shown that CpG-ODNs are also effective in prevention of eosinophilic airway inflammation in a murine model of asthma.⁹ Most of these actions of CpG-ODNs have been associated with, or attributed to, the induction of a T_H1-like immune response. This response is characterized by production of IFN-γ and IL-12. To evaluate whether the association between the T_H1

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Toll-like receptor (TLR) mediated signaling induces pro-inflammatory responses and can both suppress and exacerbate allergic responses in the airways. The aim of our study was to directly compare the efficacy of different TLR agonists in inhibiting or exacerbating the development of Th2-mediated responses in the airways and investigate if the suppressive effects were associated with increased pro-inflammatory responses. Mice were immunized on day 0, 14 and 21 by intraperitoneal injection of ovalbumin/alum and exposed to ovalbumin aerosol on day 26 and 27. TLR2, TLR3, TLR4, TLR7 and TLR9 agonists (0.001, 0.01, 0.1, or 1 mg/kg) were administered intratracheally 1 h before each allergen exposure. Both the TLR7 and TLR9 agonists dose dependently reduced airway eosinophilia, while the TLR3 agonist only reduced airway eosinophilia at a dose of 1.0 mg/kg. The TLR2 and TLR4 agonists potentiated eosinophilia. All TLR agonists enhanced neutrophil numbers at doses as low as 0.01 mg/kg, in particular TLR2 and TLR4 agonists. TLR7 and TLR9 agonists also significantly reduced IL-4 and IL-5 levels and all TLR agonists, with the exception of TLR7, enhanced the amount IL-1β, IL-6, and TNF-α detected in the whole lung lavage. Only application of TLR9 agonist induced detectable levels of IL-10 in the lung. Suppressive effects of the TLR agonists were not dependent upon IFN-γ and IL-10 or associated with increased numbers of Foxp3⁺CD4⁺ Tr cells in the lavage fluid. Airway resistance was reduced significantly only when TLR7 agonist was administered. When applied therapeutically 2 days after allergen exposure, all TLR agonists, except TLR2, similarly reduced airway eosinophilia and IL-4 levels. Taken together our results show that TLR7 agonists had the strongest anti-asthmatic effects with the lowest pro-inflammatory potential, suggesting that activating TLR7 may have the greatest potential to treat allergic disorders in humans.
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^☆: Supported by grants from the National Institutes of Health (HL59324, HL02950, and ES05605), the American Lung Association, the Department of Veteran’s Affairs (Career Award), the Center for Advanced Studies-Spellman Rockefeller, and CpG Immunopharmaceuticals.

^☆☆: Reprint requests: Joel N. Kline, MD, C33GH UIHC, Iowa City, IA 52242.

^★: 1/1/102873

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CpG oligodeoxynucleotides do not require TH1 cytokines to prevent eosinophilic airway inflammation in a murine model of asthma☆,☆☆,★

Abstract

Section snippets

Animals and murine model of asthma

ELISA

Effect of TH1 cytokines on airway eosinophilia and bronchial hyperreactivity

DISCUSSION

Immunity

Clin Immunol Immunopathol

Blood

Trends Microbiol

Immunol Lett

National Asthma Education and Prevention Program. Expert Panel Report 2: guidelines for the diagnosis and management of asthma

CD4 T-lymphocyte activation in asthma is accompanied by increased serum concentrations of interleukin 5. Effect of glucocorticoid therapy

Am Rev Respir Dis

Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma

N Engl J Med

Two types of murine helper T cell clones. I. Definition according to profiles of lymphokine activities and secreted proteins

J Immunol

Interleukin 12 inhibits antigen-induced airway hyperresponsiveness, inflammation, and Th2 cytokine expression in mice

J Exp Med

CpG motifs in bacterial DNA trigger direct B-cell activation

Nature

Bacterial DNA induces NK cells to produce IFN-gamma in vivo and increases the toxicity of lipopolysaccharides

J Immunol

Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA

J Immunol

Modulation of airway inflammation by CpG oligodeoxynucleotides in a murine model of asthma

J Immunol

CpG oligodeoxynucleotides do not require T_H1 cytokines to prevent eosinophilic airway inflammation in a murine model of asthma☆,☆☆,★

Effect of T_H1 cytokines on airway eosinophilia and bronchial hyperreactivity