| BackgroundAs a chronic heterogeneous airway inflammatory disease,bronchial asthma has a variety of typical clinical symptoms including chest tightness,wheezing,coughing,etc.Although studies have identified several risk factors for the development of asthma,including environmental and genetic factors,the accurate cause of asthma is still unclear,which makes the treatment of asthma difficult.To address the difficulty,a series of asthma phenotype has been defined,including allergic asthma,non-allergic asthma,and so on.Among the many types of asthma,allergic asthma is undoubtedly the most important one.The phenotype of asthma has improved the effective control rate of asthma in clinical practice,but some asthma patients still suffer from the disease.Given that asthma is an inflammatory disease,researches on the intrinsic mechanisms of the inflammatory responses and the targeted interventions in asthma are of high sociological value and clinical significance.Macrophages,which originate from the bone marrow,are an important part of the immune system.Depending on the type of external stimuli,macrophages are usually classified into two main types:classically activated macrophages(M1 macrophages),which receive stimuli such as IFN-γ,and alternatively activated macrophages(M2 macrophages),which are stimulated by IL-4.As the most abundant immune cells in the lung,the association of macrophages with the development of asthma has been widely demonstrated.For example,Robbe P et al.found that M1 macrophages could promote the development of non-allergic pulmonary inflammatory response and Byers DE et al.found that M2 macrophages were closely related to Th2 type cytokines such as IL-4,which is a major factor in triggering allergic asthma.There are seven members of the activator protein 1(AP-1)transcription factor family,of which Fra2 is the most recently identified.As an emerging molecule,Fra2 has an important role in the growth and development of individuals.For example,a study by Eferl found that mice with Fra2 deficiency died within one week after birth.Fra2 is closely associated with the development of several diseases,such as pulmonary fibrosis and pulmonary hypertension.JAK-STAT is a classical signaling pathway that mediates inflammatory responses in a variety of diseases,such as asthma.Rani A et al.found that Fra2 is a downstream target of STAT5,while JAK3 is an upstream molecule of STAT5.The above clues suggest that macrophages and Fra2 are likely to have relevance in the pathogenesis of allergic asthma,while the intrinsic molecular mechanism of their interaction has not been reported.Thus,the research was designed to study the role of macrophages and Fra2 in allergic asthma.,with the aim of demonstrating the relationship between them and exploring the underlying molecular mechanisms,which could help improve the treatment of allergic asthma.ObjectivesTo investigate the association between macrophages and Fra2 in allergic asthma and the corresponding molecular mechanisms,and identifying new targets for the treatment of allergic asthma.Methods1.Expression of Fra2 in airway tissues of patients with asthma1.1 PatientsSeventeen patients with asthma who visited the respiratory department of Shandong Qianfo Mountain Hospital in 2021 were selected to form the asthma group,and 17 patients from the health care department formed the control group.1.2 H&E(hematoxylin&eosin)staining to investigate the pathological features of the airway tissue in both groups.1.3 Immunohistochemical staining was used to detect the expression of Fra2 in the airway tissues of the two groups.2.Expression of Fra2 in airway tissues of asthmatic mice2.1 Experimental animal modelingThe method of ovalbumin(OVA)sensitization plus excitation was used.2.2 Experimental animal groupingEqual numbers of mice were randomly divided into the asthma group and the control group.2.3 H&E staining to investigate the pathological changes in the airway tissues of the two groups of mice.2.4 Immunohistochemical staining and Immunofluorescence to test the expression of Fra2 in the airway tissues of the two groups of mice.3.Correlation of JAK3-STAT5 pathway with Fra2 in asthma3.1 Quantitative Real-time Polymerase Chain Reaction(qRT-PCR)to test the expression of JAK3,STAT5,and Fra2 in the airway tissues of patients with asthma.3.2 qRT-PCR and western blotting to examine the expression of JAK3,STAT5,and Fra2 in the airway tissues of asthmatic mice.4 Correlation of pulmonary macrophages with asthma4.1 Flow cytometry analysis of the ratio of M1 and M2 macrophages in the lungs of two groups of mice4.2 Flow cytometry sorting of lung M1 and M2 macrophages in asthmatic mice4.3 Western blotting for optimal culture cycle of in vitro mice lung macrophages4.4 qRT-PCR and western blotting to determine the expression of JAK3,STAT5,and Fra2 in M1 and M2 macrophages in the lungs of two groups of mice4.5 In vitro culture of M2 macrophages with inhibitors of JAK3 as well as STAT5 and small interfering RNA(small interfering RNA,si-RNA)of Fra2(si-Fra2),followed by detection of the expression of JAK3,STAT5,and Fra2 using western blotting.5.Correlation of Fra2 with airway inflammatory response in asthma5.1 qRT-PCR to determine the levels of Th2-type cytokines IL-4,IL-5,and IL-13 in M2 macrophages with or without the addition of si-Fra2.5.2 Enzyme linked immunosorbent assay(ELISA)to determine the levels of Th2-type cytokines IL-4,IL-5,and IL-13 in the culture medium of M2 macrophages with or without the addition of si-Fra2Results1.The airway tissues of the patients with asthma showed significant inflammatory response and increased Fra2 expression1.1 H&E staining results showed that there was significant inflammatory cell infiltration in the airway tissues of patients with asthma compared with the control group.1.2 Immunohistochemical staining results showed that the expression of Fra2 was significantly higher in the airway tissues of the patients with asthma compared with the control group.2.The airway tissues of the asthmatic mice showed significant inflammatory response and increased Fra2 expression2.1 The H&E staining results showed that there was a significant inflammatory cell infiltration in the airway tissues of asthmatic mice compared with the control group.2.2 Immunohistochemical staining and immunofluorescence results showed that the expression of Fra2 was significantly higher in the airway tissues of asthmatic mice compared with the control group.3.JAK3-STAT5-Fra2 is closely related to allergic asthma3.1 The qRT-PCR results showed that the expression of JAK3,STAT5,and Fra2 were significantly higher in the airway tissues of patients in the asthma group compared with the control group.3.2 The results of qRT-PCR and western blotting indicated that the expression of JAK3,STAT5,and Fra2 were significantly higher in the airway tissues of mice in the asthma group compared with the control group.4.M2 macrophages are closely related to allergic asthma4.1 The results of flow cytometry showed that the proportion and number of M2 macrophages in the lungs of asthmatic mice were significantly higher compared with the control group.However,this distinction was not seen in M1 macrophages4.2 The results of western blotting indicated that 24 hours of in vitro culture was the optimal culture cycle for macrophages.4.3 The results of qRT-PCR and western blotting indicated that the expression of p-JAK3,p-STAT5,and Fra2 in M2 macrophages of asthmatic mice was significantly higher than that in the control group,while the expression of p-JAK3,p-STAT5,and Fra2 in M1 macrophages of the two groups was not significantly different.4.4 The inhibitors of JAK3 and STAT5 and the use of si-Fra2 demonstrated the effectiveness of the JAK3-STAT5-Fra2 pathway.5.M2 macrophages could promote asthma airway inflammatory response via Fra2qRT-PCR and ELISA results showed that Fra2 could promote the production of Th2-type cytokines(IL-4,IL-5,and IL-13)in M2 macrophages of asthmatic mice,which could lead to the enhanced airway inflammatory response.ConclusionM2 macrophages could promote the airway inflammatory response in allergic asthma via JAK3-STAT5-Fra2. |
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