Allergy Asthma Immunol Res.  2017 May;9(3):237-246. 10.4168/aair.2017.9.3.237.

Anti-Interleukin-9 Antibody Increases the Effect of Allergen-Specific Immunotherapy in Murine Allergic Rhinitis

• Shin JH
• Kim DH
• Kim BY
• Kim SW
• Hwang SH
• Lee J
• Kim SW

• KMID: 2371724
• DOI: http://doi.org/10.4168/aair.2017.9.3.237

Abstract

PURPOSE
Interleukin (IL)-9 induces allergic responses; however, the roles of anti-IL-9 antibody in the induction of tolerance remain unclear. This study investigated the effects of anti-IL-9 antibody on oral tolerance (OT) in a mouse model of allergic rhinitis (AR).
METHODS
BALB/c mice were divided into 4 groups: the control, AR, OT, and OT with anti-IL-9 antibody (OT+IL9AB) groups. Ovalbumin (OVA) was used for sensitization and challenge. Mice in the OT and OT+IL9AB groups were fed OVA for immunotherapy. During immunotherapy, OT+IL9AB mice were injected with anti-IL-9 antibody. Allergic symptoms, tissue eosinophil counts, and serum OVA-specific immunoglobulin E (IgE) were measured. The mRNA expressions of cytokines and transcription factors of T cells of nasal mucosa were determined by real-time polymerase chain reaction (PCR). The protein levels of GATA3, ROR-γt, and Foxp3 in nasal mucosa were determined by Western blot. CD4⁺CD25⁺Foxp3⁺ T cells in the spleen were analyzed by flow cytometry.
RESULTS
Administration of anti-IL-9 antibody decreased allergic symptoms, OVA-specific IgE levels, and eosinophil counts. In addition, it inhibited T-helper (Th) 2 responses, but had no effect on Th1 responses. Protein levels of ROR-γt and mRNA levels of PU.1 and ROR-γt were reduced by anti-IL-9 antibody. Anti-IL-9 antibody increased Foxp3 and IL-10 mRNA expression, Foxp3 protein, and induction of CD4⁺CD25⁺Foxp3⁺ T cells.
CONCLUSIONS
Anti-IL-9 antibody decreased allergic inflammation through suppression of Th2 and Th17 cells. Anti-IL-9 antibody enhanced the tolerogenic effects of regulatory T cells. These results suggest that anti-IL-9 antibody might represent a potential therapeutic agent for allergen immunotherapy in patients with uncontrolled allergic airway disease.

Keyword

Allergic rhinitis; interleukin-9; mouse; oral tolerance; regulatory T cells

MeSH Terms

Animals
Blotting, Western
Cytokines
Desensitization, Immunologic
Eosinophils
Flow Cytometry
Humans
Immunoglobulin E
Immunoglobulins
Immunotherapy*
Inflammation
Interleukin-10
Interleukin-9
Interleukins
Mice
Nasal Mucosa
Ovalbumin
Ovum
Real-Time Polymerase Chain Reaction
Rhinitis, Allergic*
RNA, Messenger
Spleen
T-Lymphocytes
T-Lymphocytes, Regulatory
Th17 Cells
Transcription Factors
Cytokines
Immunoglobulin E
Immunoglobulins
Interleukin-10
Interleukin-9
Interleukins
Ovalbumin
RNA, Messenger
Transcription Factors

Figure

• 

  Fig. 1 Schematic representation of the experimental AR and treatment protocol. AR, allergic rhinitis; OVA, ovalbumin; Alum, aluminum hydroxide; i.p., intraperitoneal administration; i.n., intranasal administration.

• 

  Fig. 2 Nasal symptom scores. The number of sneezes (A) and rubbing motions (B). Error bars represent standard deviations (SD). *P<0.05 vs the control group; †P<0.05 vs the Der f group.

• 

  Fig. 3 Decreased OVA-specific IgE synthesis and eosinophil infiltration in nasal mucosa by the administration of anti-IL-9 antibody during induction of OT. (A) OVA-specific IgE in serum. (B) Eosinophil counts in the nasal mucosa of each study group. (C) Infiltration of eosinophils (arrows) in the nasal mucosa of BALB/c mice. (a) Control, (b) AR, (c) OT, and (d) OT+IL9AB groups. H&E stain; original magnification, ×400 (scale bar=10 µm). Error bars represent standard deviations. *P<0.05. OVA, ovalbumin; IgE, immunoglobulin E; IL, interleukin; OT, oral tolerance; H&E, hematoxylin and eosin.

• 

  Fig. 4 Administration of anti-IL-9 antibody inhibits Th2 responses rather than Th1 responses. Quantitative analysis of IFN-γ (A), T-bet (B), IL-4 (C), and GATA3 (D) mRNA expression in nasal mucosa by real-time PCR. Error bars represent SD. *P<0.05. Th, T-helper; IFN, interferon; PCR, polymerase chain reaction; SD, standard deviation; other abbreviation as Fig. 3.

• 

  Fig. 5 Representative blot for determining GATA3, ROR-γt, and Foxp3 protein levels.

• 

  Fig. 6 Inhibition of mRNA expression of PU.1 and ROR-γt by the administration of anti-IL-9 antibody. Quantitative analysis of PU.1 (A) ROR-γt (B), and IL-17 (C) mRNA expression in nasal mucosa by real-time PCR. Error bars represent SD. Abbreviations as in Fig. 4. *P<0.05.

• 

  Fig. 7 Increased Foxp3, IL-10, and TGF-β mRNA expression and Foxp3 protein level by the administration of anti-IL-9 antibody. Quantitative analysis of Foxp3 (A), IL-10 (B), and TGF-β (C) mRNA expression in nasal mucosa by real-time PCR. Error bars represent SD. *P<0.05. TGF, transforming growth factor; other abbreviation as Fig. 4.

• 

  Fig. 8 Increased induction of CD4+CD25+Foxp3+ T cells by the administration of anti-IL-9 antibody. Flow cytometric analysis of CD4+CD25+Foxp3+ T cell subsets. (A) Representative fluorescence-activated cell sorting analysis of each group. The upper right quadrant represents CD4+CD25+Foxp3+ T cells. (B) The percentages of CD4+CD25+Foxp3+ T cells as a proportion of total splenic mononuclear cells. Error bars represent SD. Abbreviations as in Fig. 4. *P<0.05.

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