Th17 immunity in children with allergic asthma and rhinitis: a pharmacological approach

Abstract

Th17 cells and IL-17A play a role in the development and progression of allergic diseases. We analyzed the IL-17A levels in sputum supernatants (Ss), nasal wash (NW) and plasma (P) from Healthy Controls (HC) and children with Asthma/Rhinitis. We tested the expression of IL-17A, RORγ(t) and FOXP3 in peripheral blood T-lymphocytes from intermittent and mild-moderate asthma. The effect of Budesonide and Formoterol was tested "in vitro" on IL-17A, RORγ(t) and FOXP3 expression in cultured T-lymphocytes from mild-moderate asthma/persistent rhinitis patients, and on nasal and bronchial epithelial cells stimulated with NW and Ss from mild-moderate asthma/persistent rhinitis. Further, the effect of 12 weeks of treatment with Budesonide and Formoterol was tested "in vivo" in T-lymphocytes from mild-moderate asthma/persistent rhinitis patients. IL-17A was increased in Ss, NW and P from children with mild-moderate asthma compared with intermittent and HC. In cultured T-lymphocytes IL-17A and RORγ(t) expression were higher in mild-moderate asthma/persistent rhinitis than in mild-moderate asthma/intermittent rhinitis, while FOXP3 was reduced. Budesonide with Formoterol reduced IL-17A and RORγ(t), while increased FOXP3 in cultured T-lymphocytes from mild-moderate asthma/persistent rhinitis, and reduced the IL-8 release mediated by IL-17A present in NW and Ss from mild-moderate asthma/persistent rhinitis in nasal and bronchial epithelial cells. Finally, Budesonide with Formoterol reduced IL-17A levels in P and Ss, CD4(+)IL-17A(+)T-cells, in naïve children with mild-moderate asthma/persistent rhinitis after 12 weeks of treatment. Th17 mediated immunity may be involved in the airway disease of children with allergic asthma and allergic rhinitis. Budesonide with Formoterol might be a useful tool for its therapeutic control.

Figure 1. Levels of IL-17A in Control subjects (HC) and patients with asthma and rhinitis.

A, B, C) IL-17A levels in Ss, NW and plasma from HC, IA, and MA groups. Results are expressed as pg/g sputum for Ss, and as pg/ml for NW and plasma. Data are shown as individual values. Statistical analysis was performed by Kruskal Wallis and Mann-Whitney tests. D, E, F) IL-17A levels in Ss, NW and plasma from MA/IR in comparison with children with MA/PR. Bars show mean ± S.D. Statistical analysis was performed by Mann-Whitney test. Significance was accepted at p<0.05.

Figure 2. T-lymphocytes expressing intracellular IL-17A, RORγ(t) and Foxp3 in allergic children.

A) Expression of CD3⁺IL-17A⁺, CD3⁺RORγ(t)⁺ and CD3⁺Foxp3⁺ in T-cells from children with IA (n = 15) and MA (n = 19); B) a representative flow cytometry of the data; C) Expression of CD3⁺IL-17A⁺, CD3⁺RORγ(t)⁺ and CD3⁺Foxp3⁺ in T-cells from children with MA/IR (n = 9) in comparison with MA/PR (n = 10); D) a representative flow cytometry of the data. T-cells (1×10⁶ cells/ml) were cultured for 72 hours in 24-well cell culture plates in complete medium in presence of PMA (50 ng/ml) and ionomycin calcium salt (250 ng/ml). The analysis was performed by flow cytometry. Gates were set on CD3⁺cells. The bars represent mean+SD of the % of CD3⁺ expressing cells. Statistical analysis was performed by Mann-Whitney test. Significance was at p <0.05.

Figure 3. Effects of budesonide and formoterol on T-lymphocytes from children with MA/PR (n = 10).

A) Expression of CD3⁺IL-17A⁺; C) Expression of CD3⁺RORγ(t)⁺; E) Expression of CD3⁺Foxp3⁺;B, D, F) representative flow cytometry of the data. The cells (1×10⁶ cells/ml) were cultured for 72 hours in 24-well cell culture plates in complete medium in presence of PMA (50 ng/ml) and ionomycin calcium salt (250 ng/ml), and then the effect of Budesonide 10⁻⁸ M and Formoterol 10⁻⁸ M alone or in combination was evaluated. The analysis was performed by flow cytometry. Gates were set on CD3⁺cells. The bars represent mean+SD of the % of CD3⁺ expressing cells. Statistical analysis was performed by ANOVA with Fisher test correction. Significance was at p <0.05.

Figure 4. Effect of budesonide and formoterol on T-lymphocytes from children with MA/PR (n = 10). CD4⁺IL-17A⁺ and CD4⁺CD25⁺Foxp3⁺ T-cells in children with MA/PR (n = 10).

A) Expression of CD4⁺IL-17A⁺; B) a representative flow cytometric detection of CD4⁺ T-cell cytokine expression in PBMC. C) Expression of CD4⁺CD25⁺Foxp3⁺; D) a representative flow cytometry analysis of CD4⁺CD25⁺ T-cells. PBMC (1×10⁶ cells/ml) were cultured for 72 hours in 24-well cell culture plates in complete medium in presence of PMA (50 ng/ml) and ionomycin calcium salt (250 ng/ml), in the presence or absence of Budesonide 10⁻⁸ M and Formoterol 10⁻⁸ M alone or in combination. The analysis was performed by flow cytometry. Gates were set on CD3⁺cells followed by CD4⁺cells and CD4⁺CD25⁺cells. Statistical analysis was performed by ANOVA with Fisher test correction. Significance was at p<0.05.

Figure 5. Ss and NW from children with MA/PR (n = 6) induced the IL-8 release from epithelial cells.

16-HBE and RPMI2650 cells were stimulated in the presence or absence of Budesonide 10⁻⁸ M and Formoterol 10⁻⁸ M alone or in combination A) with Ss (n = 6) or B) with NW from children with MA/PR; 16-HBE and RPMI2650 cells were stimulated in the presence or absence of anti IL-17R monoclonal antibody C) with Ss or D) with NW from children with MA/PR. Results are expressed as pg/ml. Data are shown as mean ± S.D. ANOVA with Fisher test correction was used to compare the different experimental conditions. **p<0.001 vs baseline; *p<0.001 vs IS; #p<0.05 vs IS. **p<0.001 vs baseline; ##p<0.01 vs NW; #p<0.05 vs NW.

Figure 6. rhIL-17A induced IL-8 release from epithelial cells.

16HBE cells were stimulated with A) rhIL-17A in the presence or absence of Budesonide 10⁻⁸ M and Formoterol 10⁻⁸ M alone or in combination and B) with rhIL-17A in the presence or absence of anti IL-17R monoclonal antibody. RPMI2650 cells were stimulated with C) rhIL-17A in the presence or absence of Budesonide 10⁻⁸ M and Formoterol 10⁻⁸ M alone or in combination, and D) with rhIL-17A in the presence or absence of anti IL-17R monoclonal antibody. Results are expressed as pg/ml. Data are shown as mean ± S.D. of six separate experiments. ANOVA with Fisher test correction was used to compare the different experimental conditions. °°p<0.001 vs baseline; °p<0.01 vs rhIL-17A. ###p<0.001 vs rhIL-17A.

Figure 7. Effect of 12 weeks treatment with inhaled Budesonide and Formoterol.

Pediatric patients with MA/PR were treated with Budesonide and Formoterol for 12 weeks and the effect was evaluated before and after the treatment on A) plasma IL-17A levels (pg/ml); B) CD4⁺ IL-17A⁺T-cells (%); C) Ss IL-17A levels (pg/g sputum). Results are expressed as individual data points and the lateral bars represent median (25–75 percentiles). Statistical analysis was performed by Wilcoxon U-test. Significance was at p<0.05.

Figure 8. Scheme of the role of Th17/Treg dysbalance in children with asthma and rhinitis.