Effect Of PKC-θ Gene Knockout On Genome-wide Methylation During The Differentiation Of CD4~+T Cells In Asthmatic Mice

OBJECTIVE:Analyze the effect of PKC-θknockout on the whole-genome methylation of splenic CD4⁺T cells derived from asthma model mice,and provide an experimental basis for the treatment of allergic asthma.METHODS:Methods:Twenty C57BL/6 mice were knocked out of PKC-θgene,half of which were male and female.Ten mice were sensitized by intraperitoneal injection of 200μL PBS buffer[containing 100μg ovalbumin（OVA）and 1 mg Al（OH）₃]on day 0,day 7,and 14 respectively.On the 15th day,the mice were placed in a self-made closed container,and the asthma was induced by ultrasonic nebulization and inhalation with PBS buffer containing 5%OVA and 2%Al（OH）₃once a day for 30 minutes for 7 consecutive days.They were sacrificed on the 21st day（within 24h of the last inhalation）（named KO＿21d group）.In the control group,PBS was used instead of OVA,and the other treatments were the same（KO＿0d group）.The contents of the test are as follows:（1）Take lung tissues from all mice for H&E and PAS staining to observe pathological changes in mouse lung tissue;（2）Collect alveolar lavage fluid（BALF）,and detect IL-4,IL-17,IFNγby ELISA;（3）Separate the spleen,prepare a single cell suspension,and detect Th1（CD4⁺IFN-γ）,Th2（CD4⁺IL-4）,Th17（CD4⁺IL）in mouse spleen cells by flow cytometry-17),the proportion of CD4⁺CD25⁺Treg cells;（4）CD4⁺T cells in spleen cells were sorted by flow cytometry,and after the genome was extracted,PKC was detected by whole-genome bisulfite sequencing（WGBS）-Theta knockout CD4⁺T cell whole genome methylation,using R software and its program package to analyze the differentially methylated region（differentially methylated region,DMR）,as well as the GO classification of DMR-related genes and their promoters,and KEGG signaling pathway Enriched.RESULT:1.The results of H&E and PAS staining of lung tissue showed that PKC-θcan reduce airway inflammatory cell infiltration and airway mucus secretion.2.ELISA results showed that the levels of cytokine IFN-γin BAFL of mice in KO-0d group and KO-21d group were（117.8±16.8）and（85.3±24.2）pg/ml,respectively.Compared with the KO-0d group,the IFN-γcontent in the BALF of the KO-21d group was significantly reduced（P<0.05）.The levels of cytokine IL-4 in BALF in KO-0d group and KO-21d group were（7.1±2.3）and（14.5±3.6）pg/ml,respectively.Compared with the KO-0d group,the IL-4 content of the KO-21d group was significantly higher（P<0.01）.The levels of cytokine IL-17 in the KO-0d group and KO-21d group were（4.3±1.1）and（9.2±4.3）pg/ml,respectively.Compared with the KO-0d group,the IL-17 content in the alveolar lavage fluid of the KO-21d group was increased（P<0.05）.3.The results of flow cytometry showed that the number of cells in BALF of mice in KO-0d group and KO-21d group was（1.41±0.32）and（2.53±0.62）10-4/m L,respectively.The number of cells in BALF in the KO-21d group was significantly higher than that in the KO-0d group（P<0.01）.The proportion of Th1 cells in the spleen cells of the KO-0d group and KO-21d group were（9.45±1.12）and（6.28±0.94）%,respectively.The proportion of Th1 cells in the KO-21d group was significantly lower than that in the KO-0d group（P<0.01）.The proportion of Th2cells in the spleen cells of the KO-0d group and KO-21d group were（6.62±1.33）%and（7.62±0.89）%,respectively.There was no significant change in the proportion of Th2 cells between the KO-21d group and the KO-0d group（P>0.05）.The proportion of Th17 cells in the spleen cells of the KO-0d group and KO-21d group were（2.12±0.49）%and（4.85±0.66）%,respectively.Compared with the KO-0d group,the proportion of Th17 cells in the KO-21d group was significantly higher（P<0.01）.The proportion of Treg cells in the spleen cells of the KO-0d group and KO-21d group were（6.48±1.62）%and（3.00±0.99）%,respectively.Compared with the KO-0d group,the proportion of Treg cells in the KO-21d group was lower（P<0.01）.4.After data analysis of WGBS,a total of 14,000 significant DMRs were obtained.Further GO classification results show that these DMRs have extensive effects on biological processes,cellular components,and molecular functions.The KEGG analysis results show that these DMR-related genes are mainly enriched in the biologically related signal pathways closely related to allergic asthma such as cell proliferation,hypoxia,immunity,etc.,such as Rap1 signaling pathway,MAPK signaling pathway,and Calcium signaling.pathway,PI3K-Akt signaling pathway,T cell receptor signaling pathway,HIF-1 signaling pathway,HIF-1 signaling pathway,etc.CONCLUSION:1.OVA-sensitized PKC-θknockout mice showed reduced airway inflammatory cell infiltration and airway mucus secretion;2.The secretion of inflammatory factors IL-4 and IL-17 in BALF of allergic asthma PKC-θknockout mice is reduced;3.The proportion of Th1（CD4⁺IFN-γ）,Th2（CD4⁺IL-4）and Th17（CD4⁺IL-17）in the spleen CD4⁺T cells of allergic asthma PKC-θknockout mice decreased;4.The genes affected by methylation in CD4⁺T cells from spleen of allergic asthmatic PKC-θknockout mice are mainly related to signal pathways related to cell proliferation,hypoxia,and immunity.