Th17 Immunity In Patients With Allergic Asthma

Allergic asthma is a complex disease characterized by chronic airway inflammatory, airway remodeling, mucous oversecretion and airway hyperreactivity. Many inflammatory cells and pro-inflammatory cytokines, such as lymphocyte, eosinophil, mast cell and so on, have been suggested to be associated with dysfunction of airway. A polarization bias of T lymphocytes is now recognized as the main mechanism that involved in pathogenesis of allergic asthma. T lymphocyte is both an important inflammatory cell and necessary immunocyte. T lymphocytes can be categorized into two subsets according to their different surface antigens:CD4+and CD8+T lymphocytes. CD4+T lymphocytes include Thl and Th2 cells, according to the different cytokines they released. The Thl/Th2 imbalance and their cytokines have been well documented and are essential in the pathogenesis of allergic asthma. Recently, the identification of Th17 leads to the revision of Thl/Th2 imbalance paradigm and provides us with the new perspectives on the basis of asthma. Th17 immunity has been found to play a critical role in the pathogenesis of allergic asthma, although these studies just reflected the abnormal Th17 immunity in local sites or in animal models. However, this abnormal immunity may not be the case in the systemically allergic reaction and immune responses in allergic asthmatic patients, whether Th17 immunity contributes to the systemically immune responses in allergic asthmatic patients is unclear.In our study, firstly, we investigated the numbers of Th17 cells and concentrations of related cytokines in the peripheral blood from both allergic asthmatic patients and healthy controls by flow cytometry, enzyme-linked immunosorbent assay (ELISA) and Luminex multi-analyte technology. We found that the percentages of Th17 cells and levels of Th-17 related cytokines, such as IL-17、IL-22 and IL-23, were all increased in the patients with allergic asthma and tended to increase with the severity of the disease, although some patients were treated with inhaled glucocorticoid. In allergic asthmatics, IL-17 concentrations both in plasma and from activated PBMCs were positively correlated with IL-23 concentrations, which indicated that IL-23 may promote the differentiation of Th-17. RORyt is the crucial transcription factor for the differentiation of Th-17. The levels of RORyt mRNA were increased in patients with allergic asthma as compared with those in healthy controls which analysised by RT-PCR. IL-25 also belongs to the IL-17 family. Interestingly, IL-25 and IL-17 appear to play different roles in the pathogenesis of allergic asthma. After separating patients based on the severity of allergic asthma, we found the levels of IL-17 in plasma were highest in the severe group, while the levels of IL-25 were only increased in the mild group.Next, we assessed the effects of IL-25 on the release of IFN-y, IL-4 and IL-17 from cultured PBMCs in both allergic asthmatics and healthy controls. We found IL-25 could induce the release of IL-4 from activated PBMCs in a dose dependent manner, and inhibit the release of IL-17.Above all, Th 17 cells play a pivotal role in the pathogenesis of allergic asthma and provide an important supplement to the classical mode of Thl/Th2. It may supply us with the new theoretical evidences to the pathogenesis and treatment of non-Th2 immunity type of asthma. Part 1. Th17 Immunity in Patients with Allergic AsthmaObjective:To investigate the Th17 immunity in the pathogenesis of allergic asthma.Methods:Peripheral blood mononuclear cells (PBMCs) were isolated from allergic asthmatics (n=29) and healthy controls (n=12). The frequencies of Th1, Th2 and Th17 cells were analyzed by flow cytometry. The related cytokines (IFN-y, IL-4, IL-17, IL-22, IL-23 and IL-25) concentrations in plasma and those produced from activated PBMCs were measured by enzyme-linked immunosorbent assay (ELISA) and Luminex multi-analyte technology. The level of retinoic acid-related orphan receptor yt (ROR yt), a key transcription factor controlling Th17 differentiation, was examined by real time quantitative polymerase chain reaction (RT-PCR).Results:1. The percentages of Thl cells were lower but the percentages of Th2 cells were higher in allergic asthmatics than those in healthy controls (p<0.05). The ratio of Thl/Th2 cytokine (IFN-y/IL-4) was lower in allergic asthmatics than that in healthy controls (p<0.05).2. The percentages of Th2 and Th17 cells, the concentrations of thier related cytokines (IL-4,IL-17, IL-22 and IL-23) were higher in allergic asthmatics than those in healthy controls (p<0.05), although some patients were treated with inhaled glucocorticoid.3. After separating patients based on the severity of allergic asthma, we found that the plasma concentrations of IL-17 and IL-22 tended to enhance with the severity of the disease, while IL-25 level was elevated only in mild patients.4. In allergic asthmatics, IL-17 concentrations both in plasma and from activated PBMCs were positively correlated with IL-23 concentration (r=0.526, p=0.003 and r=0.428, p=0.021).5. The ROR yt mRNA level was increased in allergic asthmatics as compared with that in healthy controls (p<0.05)Conclusion:Our results suggest that besides predominant Th2 immunity, abnormal Th17 immunity may be also involved in the pathogenesis of allergic asthma. Part 2. The Influences of IL-25 on Thl, Th2 and Th17 Cytokines Secretion from Activated PBMCs in Patients with Allergic AsthmaObjective:To investigate the influences of IL-25 on Thl, Th2 and Th17 cytokines secretion from activated PBMCs in both patients with allergic asthma and healthy controls.Methods:Peripheral blood mononuclear cells (PBMCs) were isolated from allergic asthmatics (n=29) and healthy controls (n=12). Then PBMCs from the two groups were devided into 4 groups:spontaneous culture without stimulation; costimulated with PMA and Ionomycin; stimulated with IL-25 alone; stimulated with PMA+Ionomycin costimulation and different doses of IL-25. The concentrations of Th1, Th2 and Th17 cytokines (IFN-y, IL-4 and IL-17) from cell culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA).Results:1. The levels of IFN-y, IL-4 and IL-17 could not be detected by ELISA in supernants from spontaneous cultured PBMCs and PBMCs stimulated with IL-25 alone in both groups, but when stimulated with PMA and Ionomycin costimulation the concentrations of IL-4 and IL-17 were higher in allergic asthmatics than those in heathy controls (p<0.05). When co-stimulated with IL-25, the release of IL-4 was increased in both allergic asthmatics and healthy controls, and the increasement was higher in the former. And IL-25 could suppress the production of IL-17 in allergic asthmatics (p< 0.05).2. When PBMCs co-stimulated with different doses of IL-25, significant elevation of IL-4 secretion could be enhanced by IL-25 in a dose-dependent fashion (1 ng/ml—50 ng/ml) in allergic asthmatic patients, but no dose-dependent influence on suppression of IL-17. Conclusion:IL-25 exhibited synergistic effect on the secretion of IL-4 and suppressive effects on the release of IL-17 in patients with allergic asthma, and had a dose-dependent relationship with the former.