| Asthma is a chronic inflammatory disorder of the airways in which many cells play a role, in particular, T lymphocytes. Th1/Th2 paradigm is regarded as a very important immunological mechanism in asthma. Th2 cells play a key role in asthma pathogenesis by producing of cytokines IL-4, IL-13, IL-5, which promote eosinophil differentiation, IgE production, and induce airway eosinophilic inflammation. Despite the research focus on Th2-driven asthma, our studies have shown that as many as 49.37% of asthmatics children are characterized by noneosinophilic inflammation, more than half of these patients are characterized by neutrophilic asthma,not all asthma can be explained by Th2 mechanisms.The mechanisms of neutrophilic asthma remain largely unknown.Th17 cells are a new subset of CD4+T helper cells.Expression of IL-17A is a hallmark of Th17 cells, and RORγt is the key transcription factor that orchestrates the differentiation of Th17 cells. IL-17A plays an important role in neutrophil recruitment.Recent studies have showed that, Th17 cells may be involved in the pathogenesis of asthma. We assume the immunological mechanisms of neutrophilic asthma should be different from that of eosinophilic asthma. Th2 cells may mediate eosinophilic airway inflammation; Th17 cells may mediate neutrophilic airway inflammation. But so far, related research is rare, moreover, traditional model of eosinophilic asthma are widely used in Th17 cells and asthma research.With the aim of finding the molecular mechanism of Th17 cells involving in regulation of airway inflammation in neutrophilic asthma, finding a new target for neutrophilic asthma therapy, in this study we established neutrophilic asthma model, with the eosinophilic asthma model as a control, discussed the role of Th17 cells in neutrophilic asthma pathogenesis and the mechanism,the asthmatic mice were treated with dexamethsone to discuss the role of glucocorticoids in the management of neutrophilic asthma and the mechanism. Our study was divided into four parts. Objective To establish a mouse model of neutrophilic asthma, providing a basis for further research.Methods Thirty-six BALB/c mice were randomly divided into three groups: normal control group (NC group), eosinophilic asthma control group (EA group), and neutrophilic asthma group (NA group). A mouse model of eosinophilic asthma was established by conventional methods. A mouse model of neutrophilic asthma was established: after being anesthetized, the mice were sensibilized by an i.p. injection of 50μg OVA and received intranasal applications of 10μg of lipopolysaccharide (LPS) on days 0, 7 and 14, on days21-34 mice were challenged with 5%OVA aerosol inhalation. The control group were received PBS instead of OVA and LPS, challenged with saline. Physiologic responses to allergen challenge of mice were observed .Whole lung lavage was performed; cell counts and differentials were measured. The change of air way histology was observed by HE stain.Results The mice of asthmatic group presented restlessness, fast breathing, bowing the back, urinary and fecal incontinence and so on. Such presentation did not occur in NC group.The total cell counts, the percentage of eosinophil, neutrophil in BALF of EA group were significantly higher than that of NC group (all P<0.01). The total cell counts, the percentage of eosinophil, neutrophil in BALF of NA group also were significantly higher than that of NC group (all P<0.01). The percentage of neutrophil in BALF of NA group was significantly higher than that of EA group (P<0.01).The total cell counts, the percentage of eosinophil in BALF of NA group were significantly lower than that of EA group (both P<0.01).Histology of lung tissue: in EA group, the airway wall thickening, goblet cell hyperplasia, numerous inflammatory cells, especially EOS, infiltrating around the bronchioles and blood vessels; in NA group, besides EOS, NEU and LYM also infiltrating around the bronchioles and blood vessels.Conclusion We successfully established a mouse model of neutrophilic asthma using LPS and OVA. Objective To discuss the immunological mechanism of neutrophilic asthma.Methods The mouse model of eosinophilic asthma and the mouse model of neutrophilic asthma were established as previously described. Concentrations of IL-17A, IL-4 and IFN-γin BALF were measured by ELISA. After preparation of spleen and lung single cell suspension, flow cytometry was used to quantify the percentage of IL-17A-producing CD4+CD8-T cells, IFN-gamma-producing CD4+CD8-T cells and IL-4-producing CD4+CD8-T cells.Results The level of IL-17A in BALF of NA group was significantly higher than that of NC group and EA group (both P<0.01). The level of IL-4 in BALF of EA group and NA group was significantly higher than that of NC group (both P<0.01), but there was no significant difference between them (P>0.05). The level of IFN-γin BALF of EA group and NA group was significantly lower than that of NC group (P<0.01), but there was no significant difference between them (P>0.05).The percentage of IL-17A–producing cells as well as the percentage of IL-4–producing cells in NA group were significantly higher than that in NC group (both P<0.01), the percentage of IFN-gamma–producing cells in NA group was significantly lower than that in NC group (P<0.01), both in the spleen and in the lung. The percentage of IL-17A–producing cells in NA group was significantly higher than that in EA group, both in the spleen and in the lung (both P<0.01). The percentage of IFN-gamma–producing cells in NA group was significantly lower than that in EA group in the spleen (P<0.01), but was significantly higher than that in EA group in the lung (P<0.01). The percentage of IL-4–producing cells in NA group was significantly lower than that in EA group in the lung (P<0.01), but there was no significant difference between them in the spleen (P>0.05).The percentage of IL-4–producing cells as well as IL-17A-producing cells in EA group was significantly higher than that in NC group (P<0.01, P<0.05, respectively); the percentage of IFN-gamma–producing cells in EA group was significantly lower than that in NC group, both in the spleen and in the lung (both P<0.01). The level of IL-17 in BALF, the percentage of IL-17A–producing cells in the lung had a positive correlation with the percentage of NEU in BALF.The level of IL-4 in BALF, the percentage of IL-4–producing cells in the lung had a positive correlation with the percentage of EOS in BALF.The level of IFN-γin BALF, the percentage of IFN-gamma–producing cells in the lung had a negative correlation with the percentage of EOS in BALF. The percentage of IL-4–producing cells in the lung had a positive correlation with the percentage of NEU in BALF.Conclusion Th17 cells may mediate neutrophilic airway inflammation via IL-17. Objective To discuss possible mechanisms of transcriptional regulation of Th17 cells-mediated neutrophilic asthma.Methods The mouse model of eosinophilic asthma and the mouse model of neutrophilic asthma were established as previously described. The protein expression of RORγt, GATA-3 and T-bet in the lungs was detected by immunohistochemical analysis and western blot analysis.Results The levels of RORγt as well as GATA-3 protein expression in NA group were significantly higher than those in NC group (both P<0.01); the levels of T-bet protein expression in NA group were significantly lower than those in NC group (P<0.01). The levels of RORγt as well as T-bet protein expression in NA group were significantly higher than those in EA group (both P<0.01);there was no significant difference of the levels of GATA-3 protein expression between EA group and NA group (P>0.05) .The levels of GATA-3 protein expression in EA group were significantly higher than those in NC group (P<0.01). The levels of T-bet protein expression in EA group were significantly lower than those in NC group (P<0.01). There was no significant difference of the levels of RORγt protein expression between EA group and NC group (P>0.05).The levels of RORγt protein expression in the lung had a positive correlation with the level of IL-17 in BALF, the percentage of NEU in BALF. The levels of GATA-3 protein expression in the lung had a positive correlation with the level of IL-4 in BALF, the percentage of EOS, NEU in BALF.The levels of T-bet protein expression in the lung had a positive correlation with levels of INF-γin BALF, but had a negative correlation with the level of IL-4 in BALF, the percentage of EOS in BALF.Conclusion RORγt and GATA-3 may be involved in the regulation of Th17 cells-mediated neutrophilic airway inflammation. Objective To discuss the role of glucocorticoids in the management of neutrophilic asthma and the mechanism.Methods Ninty BALB/c mice were randomly divided into five groups: NC group, EA group, NA group, dexamethasone intervention group (eosinophilic asthma model, EAD group), dexamethasone intervention group (neutrophilic asthma model, NAD group).The mouse model of eosinophilic asthma and the mouse model of neutrophilic asthma were established as previously described. Mice in dexamethasone intervention group were pre-treated with dexamethasone (1mg/kg, i.p) 30min before each challenge. Physiologic responses to allergen challenge of mice were observed.Whole lung lavage was performed; cell counts and cell differentials were measured.The change of air way histology was observed by HE stain.Concentrations of IL-17A, IL-4 and IFN-γin BALF were measured by ELISA. After preparation of lung single cell suspension, flow cytometry was used to quantify the percentage of IL-17A-producing cells,IFN-gamma-producing cells,IL-4-producing cells.The protein expression of RORγt,GATA-3 and T-bet in the lungs was detected by western blot analysis.Results The total cell counts, the percentages of neutrophil, the percentages of eosinophil in BALF of NAD group were significantly lower than those of NA group (all P<0.01), but were significantly higher than those of NC group (P<0.01, P<0.05, P<0.01, respectively).The total cell counts in BALF of NAD group were significantly higher than those of EAD group (P<0.05).The total cell counts, the percentages of neutrophil, the percentages of eosinophil in BALF of EAD group were significantly lower than those of EA group (all P<0.01),but were significantly higher than those of NC group(P<0.01, P<0.05, P<0.01, respectively). Compared with the asthmatic groups, no obvious bronchial wall thickening, and inflammatory cell infiltration around bronchioles significantly decreasing could be observed in dexamethasone intervention groups. Compared with NA group, the expressions of RORγt,the expressions of GATA-3,the expressions of T-bet,the percentage of IL-17–producing cells,the level of IL-17,the level of IFN-γin NAD group were significantly decreased(all P<0.01),there was no significant difference of the expressions of RORγt,the percentage of IL-17–producing cells,the level of IL-17 between NAD group and NC group (both P>0.05),the expressions of GATA-3 was significantly higher than that of NC group(P<0.01);the percentage of IL-4–producing cells was significantly elevated(P<0.01);there was no significant difference of the percentage of IFN-gamma–producing cells,the level of IL-4 between NA group and NAD group (both P>0.05).Compared with EA group, the expressions of GATA-3, the percentage of IL-4–producing cells, the level of IL-4 in EAD group were significantly decreased (all P<0.01), but were significantly higher than those of NC group (all P<0.01); the percentage of IFN-gamma–producing cells was elevated, and there was no significant difference between EAD group and NC group (P>0.05). There was no significant difference of the expressions of T-bet, the level of IFN-γbetween EA group and EAD group (both P>0.05).Conclusion Dexamethasone reduces Th17-driven neutrophilic airway inflammation of asthma, may be by directly inhibiting RORγt expression or by suppressing GATA-3 expression leading to down-regulation RORγt expression.
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