The Regulation Of IL-17D On Recruiments Of MDSCs In Tumor Microenvironment

Backgroud The occurrence and development of tumors depend not only on the malignant proliferation and invasion characteristics of the tumor cells themselves,but also depend on the multiple factors in tumor microenvironment.The role of tumor microenvironment in promoting proliferation of tumor cells,inactivating immune attack,generating immune tolerance,and evading immune surveillance cannot be underestimated.The tumor microenvironment is closely related to the process of tumorigenesis,development and metastasis.MDSCs is an important cell component in the tumor microenvironment and a unique group of cells that negatively regulates the body’s anti-tumor immune response during tumor development.MDSCs are abundantly produced in various infections,tumors,acute and chronic inflammation,and other diseases.Accumulation in tissues and organs such as bone marrow,blood,spleen,lymph nodes,and disease sites,which has a wide impact on the overall immune system of the body’s immune system.MDSCs can inhibit the body’s acquired immunity and natural anti-tumor immunity through various ways,especially to inhibit the tumor immunity of T cell subsets,such as significantly inhibiting the anti-tumor effect of Th1/Tc1,Promote Treg-induced tumor immune escape and tumor progression in further.IL-17D(Interleukin-17D)is one of the IL-17 family members.At present,the specific role of IL-17 D is still unknown.Our previous studies have confirmed that as the tumor progresses,the expression of IL-17 D gradually declines,and by the middle of the tumor,the expression of IL-17 D is almost absent.Although studies have demonstrated that IL-17 D can influence the local chemotaxis and migration of NK cells,we found that tumor growth was slowed in the subcutaneous tumor bearing model of IL-17 D knockout mice,with a concomitant decrease of MDSCs.Based on this phenomenon and related research,this study aims to further explore the correlation between the major cellular sources of IL-17 D and the absence expression of the factor in the tumor microenvironment and changes in the immune microenvironment of the tumor;To clarify the regulatory mechanism of IL-17 D on MDSCs in tissue microenvironment and its effect on tumor microenvironment;to determine the main cell source of IL-17 D expression in skin tissue and its effect on the recruitment and function of MDSCs.Pueposes 1.To explore the correlation between the major cellular source of IL-17 D and the absence expression of the factor in tumor microenvironment and the change of tumor immune microenvironment.2.To clarify the specific regulatory mechanism of IL-17 D on MDSC in tissue microenvironment and its impact on tumor microenvironment.Methods and Materials 1.To establish a wild-type mouse B16 metastatic lung tumor,skin tumor model and EG7 skin tumor model,and determine the expression level of IL-17 D in different tumor models.2.RT-PCR or Real-time PCR was used to detect the expression lever of IL-17 D and m RNA of chemokine CXCL1,CXCL2,CCL2 and CCL7 in tumor-bearing mice(B16/EG7).3.Using flow cytometry to detect the secreting lever of IL-17 D in the local of B16 tumor-burdened miceand anlyze the main cell source of IL-17 D in IL-17D+CD45-culster and detect the MDSC cell proportiontumor in tumer infiltrating lymphocytes;In addition,the simultaneous using flow cytometry analysis the expression lever and cell source of IL-17 D in normal mice lung,spleen and skin tissue,analyzing the changes of IL-17 D content in epithelial cells and fibroblasin from skin.4.The primary mouse skin fibroblasts were isolated and cultured using tissue block adherence method,purified by differential adherence method,and identified by flow cytometry detection of primary fibroblast surface markers.5.Using cell transfection technology,lentivirus as vector,NIH/3T3 cells were transfected with a lentivirus containing a highly expressed IL-17 D or IL-17 D Si RNA gene fragment to observe the effect of changes in IL-17 D expression on the biological characteristics of the cells.6.Using Transwell technology,the NIH/3T3 cells transfected with lentivirus were co-cultured with spleen lymphocytes or tumor-infiltrating lymphocytes of tumor mice.7.Flow cytometry was used to detect the effect of changes in IL-17 D expression in NIH/3T3 cells on the chemotaxis of MDSCs.Results 1.Tumor growth inhibition the expression of IL-17 D in local tissue.1.1 Tumor growth inhibits the transcription level of IL-17 D in local tissue.The mouse lung metastatic tumor(B16)model and subcutaneous tumor(B16/EG7)model were established.Using RT-PCR and Real-time PCR method to detect the local transcription lever of IL-17 D gene in tumors.The results showed that compared with the normal control tissues,the transcription level of IL-17 D in the tumor-bearing mice was significantly decreased(P<0.05),and it was more obvious in the late stage of tumor development.1.2 The proportion of IL-17D-secreting cells in tumor-bearing mice decreased significantly.The results of flow cytometry showed that the proportion of IL-17D-secreting cells in tumor tissue decreased significantly(P<0.05),and the CD45-cells decreased more significantly than CD45+ cells(P<0.05).2.IL-17 D deletion reduces local MDSCs content and inhibits the growth of subcutaneous tumors.2.1 IL-17 D deletion can inhibit the growth of B16,EG7 and other tumors.IL-17 D knockout mice were injected subcutaneously with B16 melanoma cells and EG7 lymphoma cells to construct two skin tumor models.With the growth of the tumor,compared with the wild-type tumor-bearing mice,the tumor growth of B16 melanoma and EG7 lymphoma in IL-17 D knockout mice was significantly inhibited,especially in the middle and late stage,and IL-17 DKO subcutaneous tumors in mice.The volume was significantly different from the control group(P<0.05).2.2 The absence of IL-17 D inhibits the content of MDSCs in tumor infiltrating lymphocytes. To further explore how to limit the rapid growth of tumors in the absence of IL-17 D,We detected changes in the composition of tumor-infiltrating lymphocytes by flow cytometry.Compared with the wild type control group,the proportion of MDSCs cells in the tumor infiltrating lymphocytes of IL-17 DKO tumor was significantly reduced(P<0.01).2.3 The effect of injecting supplementary IL-17 D on tumor growth in IL-17 D deficient tumor mice.To further confirm the correlation between IL-17 D and tumor volume changes,we injected IL-17 D into the IL-17 DKO tumor-bearing mice(250 ng/50 ml/mouse).The effect of replenishing IL-17 D on tumor growth was observed,the experimental results showed that the tumor size of IL-17 DKO tumor-bearing mice after topical IL-17 D preparation had no significant difference compared with the control group,and the tumor growth difference between IL-17 DKO tumor-bearing mice and wild-type tumor-bearing mice disappeared(P>0.01),the content of MDSCs in tumor-infiltrating lymphocytes was significantly higher than that before IL-17 D,and there was no significant difference in the content of MDSCs between tumor-infiltrating lymphocytes in wild-type mice(P>0.05).2.4 IL-17 D deletion down-regulates the expression Level of MDSCs-related Chemokines in Skin Tumor Tissues.Real-time PCR was used to detect the expression levels of IL-17 D and MDSCs-related chemokines in B16 tumor tissues.The results showed that compared with the wild-type tumor control group,the expression level of MDSCs-related chemokines CCL2 and CCL7 in IL-17 DKO tumor-bearing mice were significantly lower(P<0.05).Therefore,we believe that in skin tissue,IL-17 D can regulate the accumulation of MDSCs by affecting the expression of MDSCs-related chemokines.In the late stage of tumor,the absence of IL-17 D can limit the tumor’s excessive proliferation.3.Fibroblasts are important cell sources of IL-17 D in skin tissue.Flow cytometry results showed that CD45-stromal cells expressed IL-17 D abundantly in lung,spleen and skin tissues of mice.Among them,the amount of IL-17 D produced by skin fibroblasts can account for 60%-80% of the total,which becomes an important cell source for IL-17 D in local tissue.4.The effect of IL-17 D on the expression of chemokines in NIH/3T3 cell line. Real-time PCR was used to detect the expression levels of IL-17 D and MDSCs-related chemokines in NIH/3T3 cells after transfection.IL-17D-overexpressing in NIH/3T3 cell line(LV-IL-17D)was found to have significantly increased of the expression levels of CCL2 and CCL7 related to MDSCs(P<0.05);after interference with Si-IL-17 D,the NIH/3T3 cell line significantly down-regulated the transcription levels of CCL2 and CCL7 related to MDSCs(P<0.05).5.The expression lever Changes of IL-17 D in NIH/3T3 cells can affect the migration of MDSCs.Transwell results showed that IL-17 D overexpression in NIH/3T3 cells promoted the migration of MDSCs in splenic lymphocytes(P<0.05);after the interference with Si-IL-17 D,the migration of MDSCs was significantly decreased compared with the control group(LV-CTRL)(P<0.01).The results were the same with the tumor infiltrating lymphocyte repeat test.Conclusions 1.Tumor growth suppresses the expression of IL-17 D in local tissue.The absence of IL-17 D can down-regulate the expression of chemokines CCL2 and CCL7 which related to recruitment of MDSCs in tumor tissues,and down-regulate the content of MDSCs in local tissues of tumor,thereby inhibiting the overgrowth of subcutaneous tumors.2.Fibroblasts are important regulatory cells of the tumor microenvironment,which can limit the excessive chemotaxis and recruiments of MDSCs by down-regulating the expression of IL-17 D,and further more inhibit the excessive proliferation of tumors.