The Study On IL-36 Promoting The Expression Of IL-9 In CD4~+T Cells And Its Role In Tumor Immune Response

Part ? The preliminary study on IL-36 promoting the expression of IL-9 in CD4+ T Cells and Its mechanismObjective: The IL-1 family member IL-36 includes IL-36 ?,? and ?,which have a common receptor IL-36 R.The secretion of IL-9 is the main marker of Th9 cells,which is an important component of anti-tumor immune response mediated by Th9 subsets.Here we aime to analyze whether IL-36 can promote the expression of IL-9 of CD4+ T lymphocyte subsets and further explore its mechanism by using IL-36?stimulate IL-36/IL-36 R signal.Methods:Take mouse spleen to prepare single cell suspension,and use immunomagnetic beads to purify CD4+ T lymphocytes.Under the polarization condition of Th0,Th1,Th2,Th9,Th17 and Treg(CD4+Foxp3+),with or without addition of IL-36?,cells are collected and lysed after stimulation.After RNA extraction and reverse transcription,real-time fluorescence quantitative PCR(q RT-PCR)was used to detect the expression of IL-9 m RNA in different subpopulations.In addition,under the stimulation of different combinations of cytokines IL-4,TGF-?,IL-36?,collect cells and culture supernatant,the expression of IL-9 protein in each culture condition was detected by ELISA,the expression levels of IL-9 and Fox P3 in each culture condition were detected by Flow Cytometry.In addition,in the presence of STAT6 and IKK inhibitors,with or without addition of IL-36?,the expression of IL-9 under Th9 polarization were detected by ELISA.Results: The results showed that the expression of IL-9 m RNA was significantly up-regulated in CD4+ T cells stimulated by IL-36? for 24,48,and 72 hours,respectively.The m RNA expression levels of IL-9 and its transcription factor PU.1were significantly up-regulated after the addition of IL-36? to CD4+ T cells under Th9 polarization conditions.IL-36? dose-dependently upregulates IL-9 expression.In presence of TGF-? or TGF-? in combination with IL-4 group,the expression of IL-9 in protein level was significantly enhanced by IL-36? ELISA and Flow detection.In addition,the results showed that IL-36? inhibited the expression of Foxp3 while up-regulating IL-9 expression by determination with flow cytometry.Further,STAT6 and IKK inhibitors were found to have a significant inhibitory effect on the up-regulation of IL-9 under IL-36? mediated Th9 polarization.Conclusion: IL-36/IL-36 R signal can significantly promote the expression of IL-9 under polarized conditions of CD4+ T cells and up-regulate the expression of IL-9transcription factor PU.1.The role of IL-36 in the polarization of Th9 can replace or even exceed the function of IL-4 in the classical pathway;IL-36 has a regulatory effect on the balance between Th9/CD4+Foxp3+ regulatory T cells;the promotion of IL-36 on IL-9 expression depends on the STAT6 and IKK pathways.Part ? Preliminary analysis of the role of IL-9 in IL-36 mediated anti-tumor immune responseObjective : Previous studies have shown that IL-36 can significantly promote tumor immune response,inhibit tumor growth and prolong the survival of tumor-bearing mice,but the role of IL-9 as wells as Th9 cells in such outcome is not yet clear.Here we aime to investigate whether the inhibitory effect of IL-36 on tumors is partially dependent on the presence of IL-9,and whether the absence of IL-9 affects IL-36 mediated anti-tumor immune responses by using tumor cells that overexpress IL-36? to construct a tumor-bearing mouse model.Methods: B16-IL-36? as well as B16-vec tumor cells was respectively inoculated subcutaneously into C57BL/6J WT mice and IL-9 knockout(IL-9 KO)mice toconstruct a tumor-bearing mouse model,The size of tumor and the survival of mice were measured every 2 days,and the growth curve and survival curve of mice were drawn.The tumors from mice in each group were investigated at day 16.The immune cell subpopulations and cytokines in tumor microenvironment of tumor-bearing mice in each group were determined by using immunofluorescence antibody labeling and Flow Cytometry.Results: The results showed that IL-36? significantly inhibited tumor growth and prolonged survival of mice,while deletion of IL-9 partially inhibited IL-36?-mediated anti-tumor effects.After IL-9 depletion,infiltrated CD45+ cell population(CD45+ TILs)in B16-IL-36? tumor-bearing mice showed a decreasing trend without a statistical difference.The proportion of CD4+ and CD8+ T cells,CD19+ B cells and NK cells in CD45+ TILs also showed no statistically significant changes.However,the analysis of T cell activation showed that CD44,an activation marker on CD4+ TILs and CD8+TILs cells,was significantly down-regulated in the absence of IL-9.In addition,after IL-9 depletion,the expression of IFN-? in B16-IL-36? tumor-infiltrating CD8+ TILs cells showed a decreasing trend.Conclusion: The inhibitory effect of IL-36 on tumors partially depends on the presence of IL-9.Although the absence of IL-9 has no significant effect on the proportion and composition of tumor-infiltrating lymphocytes in the presence of IL-36 overexpression,the activation of CD4+ TILs and CD8+ TILs is suppressed due to IL-9deficiency.Thereby,IL-9 as well as Th9 subsets may be one of the important mechanisms by which it participates in the anti-tumor effects mediated by IL-36 and then affects IL-36 anti-tumor after deletion.