The Role And Mechanisms Of TGF-β In Eosinophil Phenotypic Transformation In Asthma

Introduction:Asthma is a disease characterized by airway hyperresponsiveness,chronic airway inflammation,and airway remodeling.And its inflammation is manifested by a large amount of eosinophil infiltration aroung small bronchial and blood vessels as well as hypersecretion of mucus.Eosinophil is one of the most important immune cells in the development of asthma whose number is positively correlated with the severity of asthma.Existing evidence suggests that eosinophils are not only the effector cells in asthma,but also have key regulatory effects on a variety of structure and immune cells in the asthma microenvironment.Recent studies have shown that there were different subtypes of eosinophils.In the asthma microenvironment,a large number of newly-emerged CD101⁺eosinophils appear in a mature and activated stage,with different functions comparing CD101^-eosinophils in steady stage.Through transcriptome analysis we found that the expression of transforming growth factor-β（TGF-β）receptors was different in these two types of eosinophils.This suggests that TGF-βsignaling may play a role in the phenotypic transformation of eosinophils.And TGF-βhas been proved to regulate the differentiation and function of various immune cells such as dendritic cells and alveolar macrophages.Therefore,based on these,this study will explore the effect of TGF-βsignaling pathway on eosinophils and its role and mechanisms in asthma.Objective:To clarify the effect of TGF-βon the phenotypic transformation of eosinophils in asthma and its specific mechanism.Methods:1.Animal model:6-8 weeks old gender-consistent mice wild type（WT）mice were used to construct an house dust mite（HDM）model by airway instillation,and then flow cytometry was used to analyze the number of Eos and CD101^-/⁺Eos ratio in the lung,peripheral blood and bone marrow and;Eos-specific Tgfbr2 gene knockout(Eocre-Tgfbr2^f/f)mice were were used to construct an HDM model to further study the effect of TGF-βon Eos and its effect on asthma inflammation by comparing the difference between this mouse and control mice influences.2.Transcriptome sequencing:Use flow cytometry to sort different subtypes of Eos,and screen differentially expressed genes by transcriptome sequencing.3.Cell experiments:NJ.1638（IL-5 overexpressing mice,hypereosinophil mouse）were used to isolate and purify Eos for in vitro intervention experiments.Then test the expression of effector genes in asthma,and explore the effects of TGF-βon Eos function and its specific signaling pathway mechanism.4.Clinical specimen collection and analysis:Anticoagulated peripheral blood was collected from non-asthmatic and asthmatic patients,and the Eos quantity and CD101 phenotype were analyzed by flow cytometry to confirm the conclusions in the mouse model.Results:This study found that CD101⁺eosinophils in the lung of a mouse HDM asthma model were increased and were mainly transformed from peripheral CD101^-eosinophils into the lung in the local microenvironment;omics analysis showed that CD101^-/⁺had differential expression of TGF-βsignaling pathway molecules.TGF-βtreatment of eosinophils isolated from NJ.1638 mice in vitro can induce the increase of CD101expression and promote expression of effectors related to asthma inflammation,while inhibition of TGF-βsignaling pathway and knockout of receptor gene can inhibit this phenomenon.In addition,we collected and analyzed peripheral blood of asthma patients,and found that CD101 expression also increased in asthmatic patients.The above results indicate that TGF-βcan promote the phenotypic transformation and function of eosinophils,but the use of eosinophil TGF-βreceptor-specific knockout(Eocre-Tgfbr2^f/f)mice to construct HDM asthma models does not suggest significant inflammation-protection phenomenon.So the role of this process in asthma remains to be refined and studied.Conclusion:CD101⁺Eos increased in the lungs and peripheral blood of asthma patients in mice;TGF-βinduced eosinophil CD101 expression and expressed asthma inflammatory effector molecules in vitro.