The Immune Cell And Cytokine Profile In Eosinophilic Asthma And The Regulation Of Fgfr1 On Airway Inflammation And Remodeling Of Asthma

BackgroundAsthma is a heterogeneous chronic airway inflammatory disease in which a variety of immune and inflammatory cells are involved.Eosinophilic asthma is the most common asthmatic phenotype mainly regulated by type 2 cytokines.Type 2helper T lymphocytes（Th2）,Type 2 cytotoxic T lymphocytes（Tc2）and Group 2innate lymphoid cells（ILC2）are all involved in the secretion of type 2 inflammatory cytokines in eosinophilic asthma.In previous studies investigating immune status in peripheral blood of eosinophilic asthma,enrolled patients used different levels of corticosteroids or/and other anti-inflammatory drugs,which could not accurately reflect true immune status of patients.Therefore,the inclusion of initially diagnosed and untreated patients with eosinophilic asthma could eliminate the interference of corticosteroids and other anti-inflammatory drugs on results,and more clearly and accurately reflect inherent characteristics of immune status of patients.Airway remodeling is also an important pathological change of asthma.Blood vessel regeneration and remodeling caused by hyperproliferation of vascular endothelium and neovascularization are important features of airway remodeling.Fibroblast growth factor 2（Fgf2）and Fibroblast growth factor receptor 1（Fgfr1）are both expressed in vascular endothelium,and have the effects of promoting endothelial cell proliferation and angiogenesis,implying their involvement in the process of airway remodeling in asthma.Up to date,no publications about the relationship between vascular endothelial Fgfr1 and airway remodeling in asthma were found.Elucidate the roles and mechanisms of Fgfr1 in airway inflammation and airway remodeling of asthma will help to explore a novel treatment target for asthma.Objective:1.To study the expression patterns of T lymphocytes,innate lymphoid cells and related cytokines in peripheral blood of initially diagnosed and untreated patients with eosinophilic asthma;2.To study the regulatory effects of vascular endothelial Fgfr1 on airway remodeling and inflammation of asthma;3.To study the effects of Fgfr1 inhibitor YTH17 on airway remodeling and the secretion of inflammatory cytokines by innate lymphoid cells of asthma.Materials and Methods:1.Clinical study:Initially diagnosed and untreated asthmatic patients who attended West China Hospital from June 2019 to December 2020 were enrolled.According to the absolute counts of eosinophils in peripheral blood,they were divided into eosinophilic asthma and non-eosinophilic asthma.Meanwhile,healthy volunteers were served as control group.In order to study the profile of T lymphocytes,innate lymphoid cells and related cytokines,baseline data of the included subjects were collected,cytokines in peripheral blood were detected by Luminex and the ratios of Th2,Tc2 and innate lymphoid cells in peripheral blood were detected by flow cytometry.2.In vivo mouse experiment:Chronic asthmatic mouse models were induced by repeated ovalbumin sensitization-nebulization,and the characteristics of OVA-induced chronic asthmatic mouse models and the effects of vascular endothelial Fgfr1and Fgfr1 inhibitor YTH17 on OVA-induced chronic asthmatic mouse models were studied.3.In vitro cell experiment:Effects of Fgfr1 inhibitor YTH17 on the secretion of inflammatory cytokines by group 2 and 3 innate lymphoid cells were observed.Results:1.The ratios of Th2 and Tc2 in peripheral blood of patients with eosinophilic asthma（n=27）were significantly higher than those of non-eosinophilic asthma（n=10）（median Th2 ratio:0.88[0.71-1.25]vs.0.23[0.17-0.72],P=0.010;median Tc2 ratio:0.53[0.31-0.92]vs.0.15[0.08-0.41],P=0.011）and healthy volunteers（n=11）（median Th2 ratio:0.88[0.71-1.25]vs.0.16[0.11-0.31],P=0.001;median Tc2 ratio:0.53[0.31-0.92]vs.0.08[0.07-0.23],P<0.001）;There were no significant differences in the ratios of Th2 and Tc2 in peripheral blood between non-eosinophilic asthma and healthy volunteers（P>0.05）.2.The ratio of ILC1 in peripheral blood of eosinophilic asthma was significantly lower than those of non-eosinophilic asthma（median:0.70[0.62-0.77]vs.0.82[0.73-0.92],P=0.014）and healthy volunteers（median:0.70[0.62-0.77]vs.0.87[0.85-0.88],P=0.001）;The ratio of ILC2 in peripheral blood of eosinophilic asthma was significantly higher than those of non-eosinophilic asthma（median:0.19[0.15-0.24]vs.0.12[0.05-0.15],P=0.009）and healthy volunteers（median:0.19[0.15-0.24]vs.0.04[0.03-0.05],P<0.001）;The ratio of ILC2 in peripheral blood of non-eosinophilic asthma were also significantly higher than that of healthy volunteers（median:0.12[0.05-0.15]vs.0.04[0.03-0.05],P=0.008）.There were no significant differences in the ratio of ILC3 in peripheral blood among eosinophilic asthma,non-eosinophilic asthma and healthy volunteers（all P>0.05）.3.The ACT score of initially diagnosed asthmatic patients was negatively correlated with the ratio of ILC2 in peripheral blood.The ACQ-7 score was negatively correlated with the ratio of ILC1 in peripheral blood,and was significantly positively correlated with the ratio of ILC2 in peripheral blood（all P<0.05）.FEV₁value was negatively correlated with the ratios of Th2,Tc2 and ILC2 in peripheral blood,and was positively correlated with the ratio of ILC1.FEV₁/FVC%was negatively correlated with the ratio of ILC2 in peripheral blood（P<0.05）.Fe NO,eosinophil percentage（%）and IL-5 level in peripheral blood were positively correlated with the ratios of Th2,Tc2 and ILC2,and negatively correlated with the ratio of ILC1 in peripheral blood（all P<0.05）.Eosinophil percentage（%）in peripheral blood was positively correlated with IL-5 level and Fe NO（both P<0.05）.4.In initially diagnosed asthmatic patients,the ratio of Th2 cells in peripheral blood was positively correlated with the ratios of Tc2 and ILC2,and negatively correlated with the ratio of ILC1（all P<0.05）.The ratio of Tc2 was positively correlated with the ratio of ILC2,and negatively correlated with the ratio of ILC1（all P<0.05）;The ratio of ILC1 was negatively correlated with the ratios of ILC2 and ILC3（all P<0.05）.5.After OVA sensitization-challenge,mice showed characteristics of chronic asthmatic models such as airway hyperresponsiveness,airway inflammation,high mucus secretion,and airway subepithelial collagen deposition.Meanwhile,the expression of type 2 inflammatory cytokines（IL-33,IL-5 and IL-13）,and the ratios of ILC2 and ILC3 in lung tissue were increased（all P<0.05）.6.After OVA sensitization-challenge,lung vascular endothelial cells of mouse model highly expressed fibroblast growth factor receptor 1（Fgfr1）,and the expression of fibroblast growth factor 2（Fgf2）had a tendency to increase.After OVA sensitization-challenge,Fgfr1^iΔEC/iΔEC（vascular endothelial Fgfr1 gene knockout）mouse model showed significantly alleviated airway hyperresponsiveness,airway inflammation,airway subepithelial collagen deposition,reduced type 2 inflammatory factors（IL-5 and IL-13 level）and ratios of ILC2 and ILC3 in the lung tissue,compared with Fgfr1^iΔEC/+control group（all P<0.05）;Transcriptome sequencing found that the expression of Malat1 gene in lung endothelial cells of Fgfr1^iΔEC/iΔECmice was significantly lower than that of control group（P<0.05）.7.The percentage of positive area of phosphorylated Fgfr1 in lung tissue of asthmatic patients was significantly higher than that of control group（12.054±2.230vs.5.709±1.742,P=0.018）.The expressions of Fgf2 and Fgfr1 in lung tissue of OVA-induced chronic asthmatic mouse model also increased（both P<0.05）.8.Pathological results showed that Fgfr1 inhibitor YTH17 can significantly inhibit airway inflammation,airway mucus hypersecretion and airway subepithelial collagen deposition in OVA-induced chronic asthmatic mouse models.9.Both ILC2 and ILC3 cells expressed Fgfr1-4,and the expression of Fgfr1 was significantly higher than Fgfr2-4（all P<0.05）.10.YTH17 at 100μmmol/L can significantly inhibit the stimulatory effect of IL-33 at 100ng/ml on ILC2（IL-5 protein level:9755.96±2370.33 pg/ml vs.58996.69±1013.46 pg/ml vs.2656.91±1042.48pg/ml;IL-13 protein level:18666.32±2467.33pg/ml vs.34395.14±2437.57 pg/ml vs.1853.55±1373.39 pg/ml,all P<0.05）.11.YTH17 at 100μmmol/L can significantly inhibit the costimulatory effect of IL-1β+IL-23 at 50ng/ml on ILC3（IL-22 protein level:35.88±3.60 pg/ml vs.262.90±39.56 pg/ml vs.75.82±6.28 pg/ml,all P<0.001）.Conclusion:1.The ratios of Th2,Tc2,and ILC2 in peripheral blood of initially diagnosed and untreated eosinophilic asthma were significantly increased,and the ratio of ILC1significantly decreased,suggesting that not only the adaptive immunity but also the innate immunity participate in the regulation of type 2 inflammation in eosinophilic asthma.2.OVA-induced chronic asthmatic mouse model showed characteristics of airway hyperresponsiveness,airway inflammation,high mucus secretion,airway subepithelial collagen deposition,increased expression of type 2 inflammatory cytokines（IL-5 and IL-13）and increased ratios of ILC2 and ILC3 in lung tissue.Vascular endothelium Fgfr1 had positive regulatory effects on airway hyperresponsiveness,airway inflammation,airway subepithelial collagen deposition,expression of type 2 inflammatory cytokines（IL-5 and IL-13）and ratios of ILC2 and ILC3 in lung tissue of asthma.3.The expression of phosphorylated Fgfr1 in lung tissue of asthmatic patient was significantly increased.ILC2 and ILC3 expressed Fgfr1.After inhibiting Fgfr1,OVA-induced airway inflammation,airway mucus hypersecretion and airway subepithelial collagen deposition of asthma significantly reduced.Inhibition of Fgfr1 can significantly impaired the ability of IL-33 to stimulate ILC2 to secrete IL-5 and IL-13,and weakened the ability of IL-1β+IL-23 to costimulate ILC3 to secrete IL-22,suggesting that Fgfr1 may become a potential therapeutic target for asthma in the future.