Discrepancy in the suppressive function of regulatory T cells in allergic asthmatic vs. allergic rhinitis subjects upon low-dose allergen challenges

Abstract

Background: Regulatory T cells (Tregs) contribute to the maintenance of immunological tolerance. There is evidence of impaired function of these cells in people with asthma and allergy. In this study, we evaluated and compared the function of Tregs in allergic asthmatic and allergic non-asthmatic patients, both before and after low-dose allergen challenges.

Methods: Three groups of subjects were recruited for a baseline evaluation: healthy controls without allergy or asthma, allergic asthmatic subjects, and allergic non-asthmatic subjects. All of them were subjected to expiratory flow measurements, sputum induction, and blood sampling. In addition, both groups of allergic subjects underwent low-dose allergen challenges. Tregs were isolated from whole blood using CD4⁺CD25^high and CD127^low staining. The suppression function was measured by flow cytometry. The levels of IL-10, IFN-γ, IgG4, IgA, and TGF-β were measured using ELISA, and sputum Foxp3 was evaluated using qRT-PCR.

Results: The suppressive function of Tregs in healthy controls was significantly higher than in allergic asthmatic or allergic non-asthmatic subjects. Repeated exposure to low doses of allergen increased the suppressor function of Tregs in allergic non-asthmatic subjects but decreased it in allergic asthmatic subjects. Foxp3 gene expression was increased in induced sputum in allergic non-asthmatic subjects, whereas it did not change in asthmatic subjects. Serum IL-10 level was decreased in allergic asthmatic subjects after allergen challenge but not in allergic non-asthmatic subjects. IFN-γ level increased upon allergen challenge in allergic non-asthmatic subjects. IgG4 level was higher in allergic non-asthmatic subjects than in allergic asthmatic subjects.

Conclusions: Low-dose allergen challenges stimulate the suppressor function of Tregs in non-asthmatic allergic subjects but not in allergic asthmatic subjects.

Keywords: Foxp3; IL-10; allergen exposure; allergy; bronchoalveolar lavage fluid (BALF), Tregs.

Figure 1

PBMC percentage of Tregs (CD4⁺CD25⁺FoxP3⁺CD127⁻) before and after allergen challenges. (A,B) Tregs were isolated from patients’ PBMC, gated, and quantified by flow cytometry using CD4⁺CD25⁺Foxp3⁺CD127⁻ discriminating markers. (C,D) Treg PBMC frequency before and after allergen challenges. Tregs from healthy controls (black square, n = 7), allergic non-asthmatics (white triangle, n = 7), and allergic asthmatics (black lozenge, n = 5) are expressed in terms of percentage among CD4⁺ T cells. Data are expressed using mean ± SD.

Figure 2

PBMC Foxp3 expression among Tregs before and after allergen challenges. (A–C) Foxp3 MFI among CD4⁺CD25⁺Foxp3⁺CD127⁻ Tregs were quantified by flow cytometry before and after allergen challenges in healthy controls (black square, n = 7), allergic non-asthmatics (white triangle, n = 7), and allergic asthmatics (black lozenge, n = 5). Data were expressed using mean ± SD.

Figure 3

Allergic non-asthmatic Tregs are more suppressive. (A) Isolated Tregs (CD25⁺ FoxP3⁺CD127⁻) co-cultured with CFSE-labeled CD4⁺CD25⁻ cells from PBMC before and after allergen exposure. (B) Suppressive function of Tregs from healthy controls (black square, n = 7), allergic asthmatic subjects (black lozenge, n = 5), and allergic non-asthmatic subjects (white triangle, n = 7) are presented as median and percentage of inhibition before and after allergen exposure. Data were expressed using mean ± SD.

Figure 4

IL-10 and IFN-γ increases in culture supernatants from suppression assay. (A,B) IL-10 and IFN-γ from healthy controls (black square, n = 7), allergic asthmatic subjects (black lozenge, n = 5), and allergic non-asthmatic subjects (white triangle, n = 7) before and after allergen exposure were measured in supernatants from suppression assay by ELISA. Data were expressed using mean ± SD.

Figure 5

Foxp3 gene expression in induced sputum. Cells were isolated from the mucus plug and were counted using a viability marker. Live cell mRNAs were extracted and Foxp3 gene expression was quantified by qRT-PCR before and after allergen exposure. Foxp3 gene expression in healthy controls (black square, n = 7), allergic non-asthmatic subjects (white triangle, n = 7), and allergic asthmatic subjects (black lozenge, n = 5) was normalized on the GAPDH housekeeping gene. Data were expressed using mean ± SD.

Figure 6

IL-10 level (A) decreases in allergic asthmatic patients, while total IgG4 (B) increases in allergic non-asthmatic patients' serum after allergen exposure. IL-10 and total IgG4 were measured in the serum of healthy controls (black square, n = 7), allergic asthmatics (black lozenge, n = 6–7) and allergic non-asthmatic subjects (white triangle, n = 7) by ELISA before and after allergen exposure. Data were expressed using mean ± SD.