| Background and objective:Bronchial asthma is a common chronic airway inflammatory disease.In the past,eosinophilic infiltration in the airway was considered to be the main inflammatory type of asthma.Otherwise,neutrophilic inflammation was found in some asthma phenotypies and sepecial cases such as acute attacks and severe asthma.Studies have found that neutrophilic asthmatics usually present with moderate to severe asthma and need to use large doses of inhaled glucocorticoids(ICS)or even oral steroids,which implied that neutrophilic asthma is closely related to severe asthma.Although the proportion of patients with severe asthma is relatively low,these patients respond poorly to existing treatments and consume most of the asthma-related medical resources.Considering the association of neutrophilic inflammation with severe asthma,further study of neutrophilic asthma is of important clinical significance.Innate lymphoid cells(ILCs)are a group of cells that have the apparent characteristics of lymphocytes,but do not express T or B cell specific antigen recognition receptors.According to the transcription factors needed and cytokines profiles secreted,ILCs can be divided into three categories,eg.ILC1 s,ILC2s and ILC3 s.Nowadays,it is found that ILC2 s are mainly involved in the pathogenesis of eosinophilic asthma by secreting type 2 inflammatory factors such as IL-5 and IL-13,while ILC3 s are activated by IL-23 and IL-1β to release IL-17 A and IL-22,and may be involved in the pathological process of neutrophilic asthma.However,the role and mechanism of ILC3 s in the pathogenesis of neutrophilic asthma are still unclear.Bronchial epithelial cells are not only the injured target but also initiative cells in the immunopathological mechanism of asthma.After being stimulated by inflammatory mediators or allergens,bronchial epithelial cells can secrete a large number of cytokines and chemokines,such as IL-6,CXCL1 and CXCL8.Then,these mediators can recruit or activate other inflammatory cells including eosinophils,neutrophils and macrophages.Fibroblast growth factor 2(FGF2)is a competent mitogenic factor,which is suggested to be involved in airway remodeling of asthma.It can promote airway structural cells to secrete a variety of inflammatory factors.However,there is no research on the relationship between ILC3 s,FGF2 and bronchial epithelial cells.Glucocorticoids are the first choice for the treatment of asthma,but they are poorly responded in neutrophilic asthma.The specific mechanism is not yet clear and needs further exploration.Based on existing evidences,our study attempt to elucidate the following questions: 1.What are the characteristics of airway inflammation,airway mucus secretion,airway reactivity in neutrophilic asthma and eosinophilic asthma mouse models? And more importantly,are there differences between the proportions of ILC2 s or ILC3 s to lymphocytes in lung tissue of neutrophilic asthma and eosinophilic asthma mouse models? 2.What are the effects of dexamethasone(DM)on airway inflammation,airway mucus secretion,airway hyperresponsiveness,and the proportions of ILC2 s and ILC3 s to lymphocytes in lung tissue of neutrophilic asthma and eosinophilic asthma mouse models? 3.Can ILC3 directly or by secreting other inflammatory factors promote bronchial epithelial cells to secrete neutrophil chemokines CXCL1 and CXCL8 to participate in neutrophilic asthma? 4.What is the specific mechanism of FGF2 to promote the secretion of IL-6,CXCL1 and CXCL8 by bronchial epithelial cells? 5.Will DM affect FGF2 activated bronchial epithelial cells to secrete IL-6,CXCL1 and CXCL8? The results of this study will provide a possibility to explore the pathogenesis of neutrophilic asthma,and provide a novel perspective for exploring the menchanism and treatment of asthma.Methods:1.In vivo animal experiments: 30 C57BL/6J mice were randomly divided into five groups: blank control group(Control group),eosinophilic asthma group(OVA/Alum group),neutrophilic asthma group(OVA/LPS group),eosinophilic asthma dexamethasone intervention group(OVA/Alum+DM group)and neutrophilic asthma dexamethasone intervention group(OVA/LPS+DM group).Eosinophilic and neutrophilic asthma models were established by OVA/Alum and OVA/LPS sensitization respectively and OVA challenge.DM was used as suppressive intervention.At the end of atomization chanllenge,the airway reactivity of mice was assessed by whole body plethysmograph.Mice lung tissue and bronchoalveolar lavage fluid(BALF)were obtain from mice after they were sacrificed under anesthesia.The number of various types of white blood cells in BALF were counted by Wright-Giemsa staining,the degree of airway inflammation and lung structure damage were assessed by HE staining,the degree of airway epithelial goblet metaplasia was assessed by PAS staining,the proportions of ILC2 s and ILC3 s to lymphocytes in lung tissue were detected by flow cytometry,the m RNA expression levels of IL-17 A,IL-22 and CXCL1 in mice lung tissue were assayed by real-time fluorescent quantitative polymerase chain reaction(RT-PCR).2.In vitro cell experiments:(1)Firstly,the effects of ILC3 s on 16 HBE cells and neutrophil chemotaxis were studied.Peripheral blood neutrophils in healthy subjects were isolated by neutrophilic isolation kit and peripheral blood ILC2 s and ILC3 s were sorted by flow cytometry.ILC2 s,ILC3s and neutrophils purity were detected by flow cytometry.IL-23 and IL-1β were used to interfere with ILC3 s.ILC3s culture supernatant,IL-6,IL-22,FGF2 and FGFR1 inhibitors(YTH17)were used separately or in combination to interfere with 16 HBE cells.FGF2 and YTH17 were used separately or in combination to intervene 16 HBE cells and co-cultured with neutrophils.The m RNA expression levels of IL-17 A,IL-22,IL-6 and FGF2 in ILC3 s in each group,the m RNA expression level of FGF2 in ILC2 s with no intervention,and the m RNA expression levels of CXCL1 and CXCL8 in each group of 16 HBE cells were detected by RT-PCR.The protein expression levels of IL-17 A,IL-22,IL-6 and FGF2 in supernatant of ILC3 s in each group,the protein expression level of FGF2 in supernatant of ILC2 s with no intervention and the protein expression levels of CXCL1 and CXCL8 in supernatant of 16 HBE cells in each group were measured by enzyme linked immunosorbent assay(ELISA),the level of neutrophil migration was assessed by crystal violet staining and CCK8.(2)Secondly,the effects of FGF2 on the JAKSTAT signal pathway of 16 HBE were analyzed,while the expression of inflammatory factors in 16 HBE cells and the effects of DM intervention were studied.FGF2,YTH17,DM,JAK2 inhibitor(AZD1480),STAT3 inhibitor(Stattic),JAK2/STAT3 inhibitor(WP1066)separately or in combination were used to interfere with 16 HBE cells.The m RNA expression levels of IL-6,CXCL1 and CXCL8 in each group of cells were detected by RT-PCR.The protein expression levels of JAK2,STAT3,phosphorylated JAK2(p-JAK2)and phosphorylated STAT3(p-STAT3)in each group of cells were detected by western blotting(WB).The protein expression levels of IL-6,CXCL1 and CXCL8 in each group of the cell culture supernatant were detected by ELISA.Results:1.In vivo animal experiments(1)The total number of white blood cells in BALF(P<0.01,P<0.05),airway inflammation score and airway reactivity in OVA/LPS and OVA/Alum group mice were significantly higher than those in control group(all P<0.01).The metaplasia score of airway epithelial cells in OVA/Alum group was significantly higher than that in control group(P<0.01).The number of neutrophils in the BALF and airway reactivity in OVA/LPS group were significantly higher than those in OVA/Alum group(P<0.05,P<0.01),while the number of eosinophils in the BALF in OVA/LPS group was significantly lower than that in OVA/Alum group(P<0.01).The proportions of ILC3 s to lymphocytes in lung tissue in OVA/LPS group was significantly higher than that in control group and OVA/Alum group(both P<0.01).The proportions of ILC2 s to lymphocytes in lung tissue in OVA/Alum group was significantly higher than that in OVA/LPS group and control group(both P<0.01).(2)After DM intervention,the number of eosinophils in BALF,airway inflammation score,metaplasia score of airway epithelial cells and the proportions of ILC2 s to lymphocytes in lung tissue in OVA/Alum group were all significantly decreased(all P<0.05).After DM intervention,the metaplasia score of airway epithelial cells decreased significantly in OVA/LPS group(P<0.05),but the number of neutrophils in BALF,airway inflammation score,airway reactivity and the proportions of ILC3 s to lymphocytes in lung tissue were not significantly changed in OVA/LPS group(all P>0.05).(3)The m RNA expression levels of IL-17 A and CXCL1 in the lung tissues of mice in OVA/LPS group were significantly higher than those in control group(P<0.05,P<0.01).The m RNA expression levels of IL-17 A and CXCL1 in the lung tissues of mice after DM intervention were not decreased(all P>0.05).2.In vitro cell experiments(1)In control group,the m RNA and protein expression levels of FGF2 in ILC3 s were significantly higher than those in ILC2s(P<0.01).Compared with control group,the m RNA(both P<0.01)and protein(P<0.05,P<0.01)expression levels of IL-22 and IL-6 in ILC3 s stimulating by IL-23+IL-1β were significantly increased,while the m RNA expression level of IL-17 A in ILC3 s stimulating by IL-23+IL-1β was significantly increased but the protein of IL-17 A was unable to be detected.After IL-23+IL-1β stimulating,the m RNA expression level of FGF2 in ILC3 s did not change significantly.(2)Compared with control group,the m RNA expression levels of CXCL1 and CXCL8 in 16 HBE cells were significantly increased after FGF2 intervention(both P<0.05),but did not change significantly after IL-6 and IL-22 intervention(both P>0.05).The m RNA and protein expression levels of CXCL1 and CXCL8 in 16 HBE cells were significantly increased after ILC3 s culture supernatant intervention(both P<0.05),and those phenomenons can be all inhibited by YTH17(both P<0.01).(3)Compared with control group,the level of neutrophil migration was significantly increased in the transwell co-cultured with 16 HBE cells after FGF2intervention(P<0.01).Compared with the FGF2 intervention alone,pre-treated16 HBE cells with YTH17 resulted in significantly decreased the level of neutrophil migration in the transwell co-cultured with 16 HBE cells(P<0.01).(4)Compared with control group,the m RNA expression levels of FGFR1/FGFR4 not FGFR2/FGFR3 in 16 HBE cells were significantly increased after FGF2intervention(P<0.01 P<0.01).(5)Compared with control group,the protein phosphorylation levels of JAK2 and STAT3,the m RNA and protein expression levels of IL-6,CXCL1 and CXCL8 were all increased in 16 HBE cells after FGF2 intervention(all P<0.01).Compared with FGF2 intervention alone,pre-treated 16 HBE cells with YTH17 reduced the protein phosphorylation levels of JAK2 and STAT3,the m RNA and protein expression levels of IL-6,CXCL1 and CXCL8(all P<0.01).(6)Compared with FGF2 intervention alone,pre-treated 16 HBE cells with Stattic decreased the protein levels of CXCL1(P<0.01),but the m RNA and protein expression levels of IL-6 and CXCL8 in 16 HBE cells did not change significantly(both P>0.05).Pre-treated 16 HBE cells with AZD1480 decreased the m RNA and protein expression levels of IL-6,CXCL1 and CXCL8(all P<0.01).Pre-treated16 HBE cells with WP1066 decreased the protein expression levels of CXCL1 and CXCL8(both P<0.01),but the m RNA and protein expression levels of IL-6 did not change significantly.(7)Compared with FGF2 intervention alone,pre-treated 16 HBE cells with DM decreased the protein phosphorylation levels of JAK2(P<0.05),the m RNA and protein expression levels of IL-6,CXCL1 and CXCL8(P<0.01),but the protein phosphorylation levels of STAT3 in 16 HBE cells did not change significantly(P>0.05).Conclusion:1.The mouse model of neutrophilic asthma showed characteristics of neutrophilic airway inflammation,airway hyperresponsiveness,increased expression of cytokines IL-17 A,CXCL1 and increased proportions of ILC3 s to lymphocytes in lung tissue.The mouse model of eosinophilic asthma showed characteristics of eosinophilic airway inflammation,airway hyperresponsiveness,airway mucus hypersecretion and increased proportions of ILC2 s to lymphocytes in lung tissue.2.ILC3 s can produce FGF2,which can activate the FGFR1-JAK2-STAT3 signaling pathway in 16 HBE cells,and stimulate 16 HBE cells to secrete CXCL1 and CXCL8.FGF2 may become a potential therapeutic target for neutrophilic asthma in the future.3.The airway inflammation,airway mucus secretion and the proportions of ILC2 s to lymphocytes in lung tissue were all decreased in mice with eosinophilic asthma after DM intervention.DM has no significant impact on airway inflammation,airway reactivity,the proportions of ILC3 s to lymphocytes in lung tissue,the m RNA expression levels of IL-17 A and CXCL1 in mice with neutrophilic asthma.However,DM can inhibit FGF2-induced JAK2 protein phosphorylation in 16 HBE cells,and inhibit the CXCL1,CXCL8 and IL-6 secretion by 16 HBE cells. |
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