IL-21 receptor signalling partially mediates Th2-mediated allergic airway responses

Abstract

Background: Interleukin-21 (IL-21) has been implicated in the development of Th2-mediated immune responses; however, the exact role it plays in allergic diseases is not well understood.

Objective: To elucidate the contribution of IL-21 receptor signalling to Th2-dependent immune responses in the lung.

Methods: We compared allergic airway responses in wild-type BALB/c and Il21r-deficient mice exposed to local airway challenge with house dust mite (HDM).

Results: We demonstrate that IL-21R-deficiency reduces HDM-driven airway hyperresponsiveness (AHR) with only partial effects on airway inflammation. Concomitant with the reduction in AHR in Il21r-deficient mice, significant suppression was observed in protein levels of the Th2 cytokines IL-4, and IL-13. In contrast, IL-21R-deficiency was associated with an increase in PBS- and allergen-driven IgE levels, while IgG1 and IgG2a levels were decreased. Moreover, our results suggest that IL-21 may contribute to AHR through its ability to both directly induce Th2 cell survival and to impair regulatory T-cell suppression of Th2 cytokine production. Importantly, we show that IL-21-positive cells are increased in the bronchial mucosa of asthmatics compared with non-asthmatics.

Conclusion: These results suggest that IL-21 plays an important role in the allergic diathesis by enhancing Th2 cytokine production through multiple mechanisms including the suppression of Treg inhibitory effects on Th2 cell cytokine production.

Keywords: IL-21; IL-21R; Tregs; airway hyperresponsiveness; asthma.

Figure 1. Il21r deficiency attenuates allergen-driven airway hyperresponsiveness

A/J mice were exposed to HDM intratracheally on days 0 and 14 and sacrificed on day 17. Il21 mRNA was measured in lungs and mediastinal lymph nodes by real-time PCR (A, B). Expression of Il21 was normalized to ribosomal protein S14 mRNA. Wildtype BALB/c and Il21r-deficient mice were sensitized and challenged with PBS or HDM as described in Materials and Methods. Airway responsiveness to i.v. acetylcholine was assessed prior to sacrifice, 72 hours after final HDM challenge (C). BALF was harvested at time of sacrifice and BAL macrophages, eosinophils, and neutrophils were enumerated (D). Lung cells were cultured ex vivo and restimulated for 72 hours with 30 µg/ml HDM extract, and IL-4, IL-5, and IL-13 levels in culture supernatants were measured by ELISA (E). Levels of Il4 and Il13 mRNA were measured by real-time PCR from total lung RNA (F). Data represents mean ± SEM. n = 5–8 mice per group (*p<0.05, **p<0.01, ***p<0.001). Data is representative of 2 independent experiments.

Figure 2. IL-21 regulates allergen-induced IgE synthesis

Serum levels of total IgE (A), HDM-specific IgE (B), HDM-specific IgG2a (C), and HDM-specific IgG1 (D) were measured by ELISA 72 hours after the final HDM challenge. Data shown are mean ± SEM. n = 8 mice per group (*p<0.05, **p<0.01, ***p<0.001). Data is representative of 2 independent experiments.

Figure 3. IL-21 expression is increased in human asthmatic airways

Immunohistochemical analysis for IL-21 was conducted on sections from bronchial biopsies obtained from non-asthmatic (A), moderate asthmatic (B), and severe asthmatic (C) individuals (20× magnification). A section from a severe patient was also stained with an isotype control (D). Quantification of the IL-21+ cells/field in the subepithelium is presented in panel E (*p < 0.05, **p < 0.01).

Figure 4. IL-21 overrides Treg-mediated suppression of T effector cytokine production and proliferation

CFSE-labeled effector T cells (CD4+CD25−) derived from lymph nodes were stimulated with anti-CD3 and anti-CD28 and treated with IL-21 in the presence of increasing numbers of Tregs (CD4+CD25^bright). After 5 days, (A, B) supernatants were collected for cytokine analysis, and (C) cells harvested for detection of proliferation by CFSE dilution. Proliferation is expressed as % divided T cells and expressed as the mean ± SEM of three wells. Effector T cells (CD4+CD25−) derived from lymph nodes were in were incubated with increasing numbers of IL-2 or IL-2+IL-21-pretreated Tregs (CD4+CD25^bright), after 5 days supernatants were collected for IL-2 analysis (D). Supernatants from CD4+CD25^bright cells (5.0×10⁴/well) were collected for cytokine analysis, from media or IL-21-treated Tregs, data is expressed as the mean ± SEM of three wells (C). The influx of IL-21R+ Tregs (CD4+CD25+Foxp3+) in the lungs is greater than IL-21R+ effector T cells after HDM challenge. Tregs also strongly upregulate the IL-21R on their surface (MFI) compared to CD4+ effector T cells following allergen exposure (F), data is expressed as the mean ± SEM of 4–8 mice (*p < 0.05, **p < 0.01). Data is representative of 2–5 independent experiments.