Intranasal administration of IL-35 inhibits allergic responses and symptoms in mice with allergic rhinitis

Abstract

Background: IL-35 was recently identified as an anti-inflammatory cytokine. We previously reported that recombinant fusion protein of murine IL-35 and human IgG1 Fc fragment (rIL-35) reduced Th2 cytokines (IL-4 and IL-5) in vitro. However, it is unclear whether IL-35 can attenuate nasal allergic responses and symptoms of allergic rhinitis in vivo.

Methods: To investigate the in vivo effect of IL-35 on allergic rhinitis in mice, mice were sensitized with ovalbumin (OVA). Intranasal administration of rIL-35 and intranasal challenge of OVA were then performed. Nasal symptoms were estimated after the last nasal challenge. Nasal tissue and cervical lymph nodes (CLN) were collected. OVA-specific IgE in sera, OVA-specific T cell response, and the production of cytokines (IL-4, IL-5, and IL-10) stimulated by the OVA antigen were measured. The transcription level of Foxp3 and the frequency of CD4⁺CD25⁺ regulatory T cells were also measured.

Results: rIL-35 significantly inhibited the number of sneezes and nasal rubbing movements. It also reduced the number of eosinophils in the nasal mucosa and significantly decreased the level of OVA-specific IgE, the OVA-specific T cell proliferation, and the production of IL-4 and IL-5. Furthermore, rIL-35 significantly increased the production of IL-10, the transcription level of Foxp3, and the frequency of CD4⁺CD25⁺ regulatory T cells.

Conclusions: This study showed for the first time that rIL-35 inhibits nasal allergic responses and symptoms in mice, and that rIL-35 increases IL-10, Foxp3, and CD4⁺CD25⁺ regulatory T cells in CLN. This study also suggests that intranasal administration of IL-35 can attenuate allergic rhinitis.

Keywords: Allergic rhinitis; Cervical lymph node; IL-10; IL-35; Regulatory T cell.

Graphical abstract

Fig. 1

Schematic protocol of experiment. Intranasal administration of PBS alone (PBS), recombinant human IgG1 Fc fragment alone (rFc), or recombinant fusion protein of murine IL-35 and human IgG1 Fc fragment (rIL-35) was performed one hour before OVA intranasal challenge.

Fig. 2

Intranasal administration of rIL-35 inhibited nasal sneezes, nasal rubbing movements, and nasal eosinophilia. Mice received PBS alone (PBS); recombinant human IgG1 Fc fragment alone (rFc); or recombinant fusion protein of murine IL-35 and human IgG1 Fc fragment (rIL-35). The number of nasal sneezes (A) and nasal rubbing movements (B) was counted for 20 min immediately after the last nasal challenge. The number of eosinophils infiltrated into the nasal mucosa of the nasal septum was also measured microscopically in a field of view at 400× magnification (C). Data are indicated as mean ± SD of five mice per group. Data are representative of two independent experiments with similar results. **P < 0.01 by ANOVA.

Fig. 3

The effects of rIL-35 on OVA-specific T cell response. Mice received PBS alone (PBS); recombinant human IgG1 Fc fragment alone (rFc); or recombinant fusion protein of murine IL-35 and human IgG1 Fc fragment (rIL-35). Lymphoid cells were collected from CLN, and OVA-specific T cell response was measured. Data are indicated as mean ± SD of five mice per group. Data are representative of two independent experiments with similar results. **P < 0.01 versus counterpart group by ANOVA.

Fig. 4

Intranasal administration of rIL-35 inhibited OVA-specific IgE and IgG1. Intranasal administration of PBS alone (PBS); recombinant human IgG1 Fc fragment alone (rFc); or recombinant fusion protein of murine IL-35 and human IgG1 Fc fragment (rIL-35) was performed. Sera were collected, and OVA-specific IgE (A) and IgG1 (B) in sera were measured using ELISA. Data are indicated as mean ± SD of five mice per group. Data are representative of two independent experiments with similar results. **P < 0.01 versus counterpart group by ANOVA.

Fig. 5

The effect of intranasal administration of rIL-35 on production of IL-4, IL-5, and IL-10 in CLN. Intranasal administration of PBS alone (PBS); recombinant human IgG1 Fc fragment alone (rFc); or recombinant fusion protein of murine IL-35 and human IgG1 Fc fragment (rIL-35) was performed. Lymphoid cells were collected from CLN, and cells were cultured with OVA antigen. IL-4 (A), IL-5 (B), and IL-10 (C) in the supernatant were measured by ELISA. Data are indicated as mean ± SD of five mice per group. Data are representative of two independent experiments with similar results. **P < 0.01 versus counterpart group by ANOVA.

Fig. 6

The effect of rIL-35 on Foxp3 gene expression and the frequency of CD4⁺CD25⁺ T cells in CLN. Mice received PBS alone (PBS); recombinant human IgG1 Fc fragment alone (rFc); or recombinant fusion protein of murine IL-35 and human IgG1 Fc fragment (rIL-35). Real time PCR analysis of Foxp3 (A) and flow cytometric analysis of CD4⁺CD25⁺ T cells (B) are shown. Data are indicated as mean ± SD of five mice per group. Data are representative of two independent experiments with similar results. **P < 0.01 versus counterpart group by ANOVA.