Expression And Immune Property Of IL-37 To The Human Peripheral Blood CD4~+ Effector T Cells

OBJECTIVE1. To prove that interleukin-37 is expressed in human peripheral blood CD4~+effector T cells.2. To reaserch the relation between activative degree of CD4~+effector T cells and expression level of IL-37.3. To prove that the expression of IL-37 has the immunosuppressive effect on CD4~+ effector T cells.METHODSPart I:We isolated CD4~+T cells from health volunteers’peripheral blood mononuclear cells (PBMCs) with Human CD4~+T Cell Isolation kit. Analyze the purity with CD4-FITC by flow cytometry.1. Proliferation activity detection:Set stimulant group (lipopolysaccharide-LPS: lug/ml), control normal group (without LPS), and test CD4~+T cell proliferation activity of the two groups, obtain the time-effect relationship (24 h,48 h,72 h) with CCK-8..2. IL-37 gene expression:Make sure the effector T cell can express IL-37-mRNA in gene aspect with PCR technology.3.IL-37 protein expression:To investigate the IL-37 protein expression level in CD4~+T cells under the stimulation with LPS at different times(24h、48h、72h) through Western blot and flow cytometry methods.4. IL-37 express position detection:To detect the IL-37 expression and location in effector T cells with immunofluorescence technique and laser confocal detection.Part II:Separate CD4~+effector T cells from healthy volunteers peripheral blood mononuclear cells (PBMCs) with the Human CD4~+T Cell Isolation kit. Use siRNA to silence the IL-37 expression. Set groups as siRNA-IL-37-、siRNA-Scramble and Normal control.1.IL-37 silence effect test:To investigate the IL-37 protein expression level in siRNA、siRNA-scramble and Normal CD4~+T cells groups under the stimulation with LPS for 72 hours through Flow cytometry methods.2. Proliferation activity test:Use CCK-8 to test different groups of CD4~+T cells proliferation activity after the LPS stimulation for 72 hours.3. To investigate the surface molecule (CD 152) expression level in siRNA、 siRNA-scramble and Normal CD4~+T cells groups under the stimulation with LPS for 72 hours through Flow cytometry methods.4. Using Elisa technology to test the supernatant fluid IL-2、IL-4、IL-17 and IFN-γ expression level of CD4~+effector T cells in siRNA-IL-37 and normal control groups.Results:1. After the separation of CD4~+T cells with Human CD4~+T Cell Isolation kit, the activity of the cells were above 98% detected in Trypan blue; the purity were higher than 99% detected in flow cytometry.2. Stimulated the cells with LPS, Use CCK-8 to detect the activity in different stimulated times (24h、48h、72h), the result shows at 72 hour, the cells reach the top activity.3. The PCR technology analysis the IL-37-mRNA expression in CD4~+T cells, with the extension of the stimulation, the IL-37-mRNA expression level become higher.4. The Western blot and Flow cytometry show the IL-37 protein expression in CD4~+ T cells, and the expression level enhanced with the extension of LPS stimulation (P<0.05).5. With laser scanning confocal microscope technology, expression of IL-37 was obvious in the cytoplasm and it showed high density around the nucleus by laser scanning confocal microscope.6. With the Flow cytometry, the CD 152 expression level in siRNA-IL-37 group CD4~+T cells was obviously lower than siRNA-scramble and the Normal groups (P<0.05).7. In testing the proliferation activity in CCK-8,siRNA-IL-37 cells have a higher value at OD450 compared with siRNA-scramble and normal groups(P<0.05). Indicate that siRNA-IL-37 silenced cells are more actively. However siRNA-scramble group and the normal group have no significant difference between each other(P>0.05).8. With Elisa technology, we tested IL-2、IL-4、IFN-y and IL-17 expression level in the cell culture fluid of siRNA-IL-37 silenced CD4~+T cells and the contral group after the 72 hours-LPS stimulation. The resultssuggest that the four cytokines in siRNA group are obviously higher than those in the contrastgroup(P<0.05). Investigate IL-37 may have a inhibitory effect to the cytokines production of CD4~+effector T cells.9. Analysis the IFN-γ/IL-4 involved in cell culture of the normal and the siRNA-IL-37 group, no significant difference obtained compared between each other.CONCLUSIONS1. IL-37 can positively expressed in CD4~+effector T cells in healthy people peripheral blood.2. With the stimulation of inflammatory factors such as LPS, the IL-37 expression level enhanced with the proliferation activity level of the effector T cells. Under this experiment environment, IL-37 reach the top activity at 72 hours. The IL-37-mRNA and protein express in the same trend.3. IL-37 could express both in and out the cell,and most abundant around the nucleus.4. IL-37 can affect the expression level of CD4~+effector T cells. Interfere the expression of IL-37 could decrease the inflammation effect degree of CD4~+T cells.