Effect Of Sema4A On HDM Stimulated Human Treg Cell Function And Its Mechanism

Allergic asthma is a disease characterized by airway hyperreaction and chronic airway inflammation involving a variety of cells,such as eosinophilic T lymphocytes and airway epithelial cells.Excessive inflammatory response caused by immune dysfunction of helper T（Th）cells plays a key role in the occurrence and development of asthma.Regulatory T cells（Tregs）are a subset of CD4⁺T cells that specifically express Foxp3 and play their roles in suppressing immune responses to prevent autoimmunity and maintain self-tolerance.The number of Treg in asthmatic patients is reduced,and the appearance of inflammatory Th2-like and Th17-like Treg cells is more obvious.To explore the mechanism of impaired phenotype and function of Treg in asthmatic state will contribute to the in-depth study of the pathogenesis of asthma and provide new ideas and methods for clinical diagnosis and treatment.Previous studies in our laboratory showed that the number of human Treg cells in spleen cells of humanized HDM asthmatic mice was reduced,and the expression of Foxp3 and other functional genes was down-regulated.Transcriptome sequencing showed that Sema family molecules were down-regulated in human CD4⁺CD25^-effector T cells of HDM asthmatic mice,including Sema4A（Semaphorins 4A）,a signaling factor associated with T cell development.Whether the down-regulation of Sema4A is related to the dysfunction of the reduction of the number of human Treg cells in anthropogenic asthmatic mice,and what is the specific molecular mechanism,needs further research.Objective:To explore the effects of HDM stimulation on the expression of Sema4A in CD4⁺CD25^-T cells in vitro,as well as the effects of Sema4A on the phenotype and function of human Treg cells and its specific molecular mechanism.Methods:After human CD4⁺CD25⁺Treg cells and CD4⁺CD25^-T cells were isolated from the spleen of HDM induced mouse humanized asthma model,transcriptome sequencing was performed to screen differential genes and gene set enrichment analysis was performed.Human Peripheral blood mononuclear cells（h PBMC）were stimulated by HDM in vitro,and the proliferation rate of CD4⁺CD25^-effector T cells in PBMC was determined by flow cytometry.The expression of Sema4A in CD4⁺CD25^-effector T cells and the expression of Sema4A receptor Plexin B1 and NRP-1 on CD4⁺CD25⁺CD127^dim/-Treg cells were isolated from human PBMC.Sema4A was added and co-cultured with Treg cells for a week,and the expression level of Foxp3、IL-10、Helios in Treg cells was detected by flow cytometry.Methylation Specific PCR（MSP）was used to determine the demethylation levels of Foxp3 and Ikzf2 in Sema4A cocultured Treg cells.Results:Transcriptome sequencing results showed that the function of human Treg cells in HDM asthmatic mice was impaired,and the expressions of effector molecules Tgfb1,Il-10ra and Il-10rb were down-regulated,and the expressions of inflammatory molecules were up-regulated.The expression of Ifng and Il-21 genes in effector T cells was up-regulated,while the expression of Sema family molecules was down-regulated.Flow cytometry results showed that the proliferation of CD4⁺CD25^-T cells in PBMC of offspring stimulated by HDM for 7 days is 22.98±1.85%（P=0.0004）,and Foxp3（P=0.0074）、IL-10（P=0.0098）、CTLA-4（P=0.0338）in Treg cells.The expression of effector molecules such as Helios（P=0.0064）was down-regulated.Meanwhile,Sema4A expression was decreased in CD4⁺CD25^-T cells（P=0.0414）and Plexin B1（P=0.0096）、NRP-1（P=0.0062）in CD4⁺CD25⁺Treg cells in PBMC.Flow cytometry results showed that after Sema4A was added,the proliferation of CD4⁺CD25^-T cells in HDM stimulated PBMC was significantly inhibited（14.85±2.07%）（P=0.0007）,and the expression of Foxp3（P=0.0405）、IL-10（P=0.0348）.CTLA-4（P=0.0032）、Helios（P=0.0237）and Sema4A receptor Plexin B1（P=0.0008）、NRP-1（P=0.0463）in Tregs increased.Flow cytometry results showed that the expressions of Treg effector Foxp3（P=0.0061）、Helios（P=0.0057）and Plexin B1（P=0.0009）were significantly up-regulated after co-culture with Sema4A.MLR results showed that Treg cells inhibited the proliferation of CD4⁺CD25^-T cells after co-culture with Sema4A（12.42±1.05%）（P=0.0014）.MSP results showed that the degree of demethylation of Foxp3 gene was significantly increased（P=0.0366）and Ikzf2 gene was slightly increased（P=0.0478）after Sema4A co-culture in Treg cells.Conclusion:1.The expressions of human Treg effector molecules Tgfb1,Il-10ra and Il-10rb were down-regulated and the expressions of inflammatory molecules were up-regulated in humanized HDM asthmatic mice,The expression of Ifng and IL-21 pro-inflammatory molecules in effector T cells increased,while the expression of Sema family molecules decreased,2.HDM stimulation can down-regulate the expression of Sema4A in CD4⁺CD25^-T cells in PBMC and Plexin B1 in Treg cells,3.Sema4A is involved in stabilizing the expression of effector molecules such as Foxp3、Helios and immunosuppressive function of Treg cells,and its molecular mechanism may be related to the promotion of demethylation of Foxp3 and Ikzf2 gene by Sema4A combined with Plexin B1.