The Role And Mechanism Of IL-35 In The Formation Of Immune Tolerance Microenvironment And Tumor Angiogenesis

BackgroundInterleukin-35?IL-35?is a new member of the IL-12 family which consists of two subunits: EBI3?Epstein Barr virus induced gene 3?and p35.It was found that IL-35 can act as immunosuppressive cytokines and participate in the formation of immune tolerance environment.In this study,the typical pathological?breast cancer?and physiological?pregnancy?immune environments were selected as the research objects to observe the induction of IL-35 on cells' 5 transformation in the microcirculation and the maintenance of immune tolerance in different states.Tumor microenvironment?TME?is a special environment for tumor cells,which is composed of tumor stromal cells?including tumor infiltrating lymphocytes,fibroblasts,vascular endothelial cells,etc.?,extracellular matrix and soluble molecules.TME plays an indispensable role in the local progression and distant metastasis of tumor,and participates in the regulation of tumor occurrence,development and drug response.Breast cancer cells and regulatory T cells?Treg?in TME can express and secrete immunosuppressive cytokine IL-35.The high level of IL-35 in breast cancer is considered as an independent factor of poor prognosis.However,it is not clear whether IL-35 can regulate tumor progression by inducing stromal cells?such as tumor infiltrating lymphocytes and vascular endothelial cells?in breast TME.As an important medium of cell communication,exosomes are extracellular vesicles secreted by cells,which have the structure of lipid bimolecular membrane,and participate in the transport of specific gene information to target cells and the regulation of their phenotype and biological process.New research shows that tumor cells can affect infiltrating lymphocytes and vascular endothelial cells in TME by secreting cytokines and exosomes,and then regulate the metabolism,immune escape and distant metastasis of tumor cells.Therefore,the exploration of breast cancer TME provides a new clue for the search of tumor molecular markers and therapeutic targets.At the same time,there are a lot of regulatory B cells?Breg?and Treg cells in breast TME.Breg and Treg can inhibit inflammation,maintain immune tolerance,and regulate immune response in inflammation,autoimmune diseases and transplantation rejection.Previous studies have confirmed that IL-35 inhibits the proliferation of B cells and T cells and induces them into special Breg cell subsets?IL-35 + Breg?and Treg cell subsets?iTR35?that play an immunomodulatory role by seCTeting IL-35.Therefore,in order to clarify the induction effect of IL-35 in breast TME,this study explore whether IL-35 can change the mRNA expression profile of tumor cell-derived exosomes,promote the angiogenesis in breast TME;or induce the transformation of T and B lymphocytes in breast TME,maintain immune tolerance microenvironment,help tumor cells escape immune surveillance and attack,accelerate the tumor progression and lung metastasis by simulating the tumor microenvironment witii high IL-35.Similar to tumor immune tolerance,there are large numbers of immune tolerant lymphocytes and suppressive cytokines in early pregnancy,which form a special"immune tolerant55 state.As an important part of peripheral immune tolerance in early pregnancy,Breg cells can promote Treg cell expansion,inhibit Thl differentiation and dendritic cells maturation while reducing the production of pro-inflammatory factors,such as TNF-a and IL-6.Treg cells inhibit effector immunity and support maternal vascular adaptations,thereby facilitating trophoblast invasion and placental access to the maternal blood supply.Therefore,Treg and Breg cells are considered to be highly related to pregnancy immune tolerance.Previous studies have found that the level of IL-35 in pregnant women's peripheral blood is increased,and IL-35 is expressed by human trophoblasts in early pregnancy.IL-35 can induce B and T cell transformation into IL-35+Breg and iTR35.We supposed that IL-35 may play an immune-modulatory role by inducing B and T lymphocytes conversion during pregnancy.In this study,we explored the role of BIO and IL-35+Breg,in maintaining maternal-fetal and peripheral immune tolerance,and further investigated the effect of pregnancy related hormones?hCG and E2?and IL-35 on BIO and IL-35+Breg cells.In the present study,we selected breast cancer microenvironment and pregnancy immunity,two typical immune tolerance environments,as research objects to explore the regulation of immunosuppressive cytokine IL-35 on angiogenesis and the induction of B cells in pathological or physiological microenvironment.Our study provides a new clue to further exploring the interaction between tumor cells and stromal cells in breast cancer TME,and is of great significance for elucidating the unique mechanisms of tumor cell immune escape and distant metastasis.Part I EL-35 promotes tumor angiogenesis by regulating the mRNA expression profile of exosomes derived from breast cancer cellsObjective:In the process of tumor development and metastasis,angiogenesis is essential for the escape of tumor cells into blood stream and distant metastasis establishment.It has been proved that tumor cells can promote tumor angiogenesis by secreting a variety of bioactive mediators.Exosomes is an extracellular vesicle with bilayer phospholipid membrane,which can be secreted by most cells and internalized by recipient cells.The study of breast TME shows that exosomes from tumor cells can communicate with the surrounding stromal cells to transmit specific information to the recipient cells,so as to improve the growth environment of tumor cells and promote tumor growth.Our previous studies showed that both breast cancer cells and tumor infiltrating lymphocytes can express suppressor cytokine IL-35.In the breast tumor microenvironment,IL-35 secreted by tumor cells and infiltrating lymphocytes mediates the transformation of tumor local T lymphocytes into suppressive iTr35 cells,further promoting and expanding the positive feedback regulation of tumor local immunosuppression.In addition to the regulation of lymphocyte transformation,whether the high concentration of IL-35 has an effect on the activity and function of breast cancer cells? Can exosomes secreted by IL-35-stimulated breast tumor cells regulate vascular endothelial cells in TME? In order to answer the above questions,this part of the study will focus on the angiogenesis of exosomes secreted by breast cancer cells in TME and the effect of IL-35 on this process.In order to answer the above questions,this part of the study will focus on the angiogenesis of exosomes secreted by breast cancer cells in TME and the effect of IL-35 on this process.Methods:1.The exosomes of breast cancer cells were isolated by differential centrifugation;the morphology and diameter of exosomes were identified by transmission electron microscopy and nanoparticle tracking analysis;the expression of CD9 and TSG101 were detected by Western blotting.2.Exosomes were dyed with PKH67,human umbilical vein endothelial cells?HUVECs?were labeled by phalloidin.Internalization of exosomes by HUVECs was observed by laser confocal microscopy.3.HUVECs were cultured with exosomes from breast cancer cells?con-exo?or exosomes stimulated by IL-35?IL-35-exo?.The proliferation activity and cell cycle of HUVECs were detected by CCK8 and flow cytometry.4.HUVECs were induced by con-35-exo and IL-35-exo respectively.Tube form abilities of HUVECs were analyzed by tube formation test and chick chorioallantoic membrane model.5.Gene chip technology was used to analyze the mRNA expression profiles in Con-exo and IL-35-exo,and the significant different genes were screened out.6.By using bioinformatics to cluster and enrich the differentially expressed genes,the related genes and signal pathways involved in angiogenesis were obtained.By constructing protein protein interaction?PPI?network,key central genes were screened out_Results:1.The exosomes were typical double-layer vesicles with diameter of about 100 nm and can express CD9 and TSG101.2.Exosomes derived from breast tumor cells can be internalized by HUVECs and mainly enriched in cytoplasm.3.The results of CCK-8 showed that the exosomes from breast cancer cells significantly promoted the proliferation of HUVECs;the results of flow cytometry showed that the exosomes from breast cancer cells significantly increased the number of HUVECs cells in S and G2 / M stages.Compared with the negative control group,IL-35-exo has a more significant effect on cell proliferation.4.Tube formation results showed that exosomes from breast cancer cells significantly increased the total branch length of HUVECs network.Both con-exo and IL-35-exo can promote the formation of chorioallantoic branching vessels.Compared with con-exo,IL-35-exo has more obvious effect on angiogenesis.5.By comparing the mRNA expression profiles of Con-exo and IL-35-exo,3508 significantly different expressed genes were identified.Cluster analysis and functional enrichment showed that these genes were significantly enriched in RAS and PPAR signaling pathways.PP1 network analysis further identified hub genes such as R-Ras,INSR and KDR.Conclusion:Our study demonstrates that exosomes derived from breast cancer cells can be internalized by HUVECs and promote angiogenesis in tumor microenvironment.IL-35 can enhance the angiogenesis by changing the mRNA of exosomes.This study provides new clues for better understanding the role of tumor secreting exosomes and IL-35 in tumor microenvironment and breast tumor progression.Part II IL-35 promotes tumor progression by inducing lymphocyte transformation in breast tumor microenvironmentObjective:Recurrence and metastasis of breast cancer are the important reasons for its poor prognosis.The main mechanisms include drug resistance of tumor cells,tumor immune tolerance microenvironment formation and tumor cells escaping immune surveillance.In tumor immunology,the balance between immune effector cells and immunosuppressive cells plays an important role in tumor development.Tumor cells can acclimate the immune cells in TME,induce the generation of immunosuppressive cell subsets,thus accelerate the immune escape and lead to tumor malignant progress.Recently,a series of immune cells have been identified,such as Treg and Breg,which are the key immune modulatory factors to inhibit anti-tumor immune response and promote tumor progression.Breast tumor cells and Treg in TME can express and secrete IL-35.The increased level of IL-35 in tumor tissue is considered to be a poor prognostic factor.Our in vitro study has confirmed that IL-35 secreted by breast cancer cells can induce the formation of local immune tolerance microenvironment by mediating the transformation of T cells into 1Tr35 cells.On the other hand,IL-35 can also inhibit the proliferation of B cells,and mediate the transformation of B cells into Breg cell subsets that can secrete IL-10 and IL-35,which is IL-35+Breg.Whether the high level of IL-35 in breast TME affects tumor progression by inducing lymphocyte differentiation in TME? The purpose of this study was to investigate the effect of IL-35 on the differentiation of infiltrating lymphocytes in breast TME by establishing tumor bearing mice model and simulating tumor microenvironment with high IL-35.At the same time,the effects of IL-35 on angiogenesis,local tumor progression and distant metastasis were studied by in vivo experiments.Methods:1.The breast cancer model of BALB/c mice was established by injecting the breast cancer 4T1 cell suspension into the subcutaneous chest of female BALB/c mice.2.After tumor formation,BALB/c mice were randomly divided into two groups.The tumor in the control group was injected with PBS,and the tumor in the experimental group was injected with IL-35.The growth curve was detected and recorded.3.CD19+B and CD4+ were separated by magnetic beads,and lymphocyte differentiation was detected by flow cytometry.4.Immunohistochemical method was used to detect the expression of Ki67 and CD31 in different groups of tumor cells,and the expression intensity and range of Ki67 and CD31 in tumor tissues were scored to evaluate the tumor proliferation and vascular density.At the same time,the mouse lung was separated and the metastasis was detected by immunohistochemistry.Results:1.Compared with the control group,the tumor growth rate of IL-35 treated group was faster and the tumor volume increased significantly.The results of immunohistochemistry showed that the proportion of Ki67 positive cells in tumor tissue of IL-35 treatment group increased significantly.2.Compared with the control group,the number of lung metastases in IL-35 treatment group was significantly increased3.The results of flow cytometry showed that IL-35 could induce the transformation of lymphocytes in breast tumor TME into JTr35 and IL-35+Breg.4.The results of immunohistochemistry showed that the proportion of CD31 positive cells in breast tumor from IL-35 treated group was significantly higher than that of the control group.Conclusion:This study confirmed that IL-35 not only promote the development of breast cancer by directly stimulating the proliferation of tumor cells,but also indirectly create favorable conditions for the growth and metastasis of tumor cells by increasing the micro-vessel density in tumor microenvironment and inducing immune tolerance.Therefore,IL-35 plays an important role in the development and prognosis of breast cancer and may become a new target of treatment.Part III IL-35 maintains immune tolerance during pregnancy by inducing lymphocyte transformationObjective:In the second part,we mainly discussed the role and mechanism of IL-35 in promoting local immune tolerance in pathological environment?tumor microenvironment?.Here we focused on the role of IL-35 in promoting immune tolerance in physiological environment?maternal fetal tolerance microenvironment?.There are a large number of immunotolerant lymphocytes and suppressive cytokines in early pregnancy,which are similar to the characteristics of tumor immunotolerance.The immune tolerance during pregnancy mainly includes maternal fetal interface and peripheral immune tolerance.There are a lot of T cells in the maternal fetal interface.Trophoblast cells can regulate Th] / Th2 balance,increase Th2 ce?? content and inhibit the immune effect of Thl7 cells at the maternal fetal interface by secreting IL-10,thymic stromal lymphopoietin and other cytokines.Our previous studies have shown that trophoblasts in early pregnancy can express and secrete immunosuppressive cytokine IL-35,and the level of IL-35 in peripheral blood of early pregnant women is significantly increased.Considering the immunosuppressive effect of IL-35 and its expression in trophoblast cells,we speculated that IL-35 might be involved in the maintenance of maternal fetal tolerance by inducing the differentiation of iTR35 cells.In addition,as an important part of inhibitory immune cells,Breg cells can play a regulatory role in autoimmune diseases and infection by regulating the peripheral immune response.As an important subgroup of Breg cells,IL-10+ Breg cells?also known as BIO?exert the immunosuppressive effects through secreting IL-10.BIO cells present CD]9+CD5+CDldh,phenotype in mouse spleen.In addition,IL-35 can inhibit the proliferation of B cells,and mediate the transformation of B cells into a new subgroup of Breg cells: IL-35+ Breg,which plays an immunosuppressive role by secreting IL-35.In the previous study,we confirmed that IL-35 can promote the tumor progression and the formation of local immune tolerance by promoting the expansion of tumor infiltrating Treg and Breg cells.This study will further explore the role of IL-35 in promoting the transformation of T and B lymphocytes and maintaining the immune tolerance during pregnancy.Because B lymphocytes rarely presente at the maternal fetal interface,we mainly studied the regulatory role of Breg cells in peripheral immune tolerance during pregnancy.In addition,during pregnancy,the expression levels of estradiol?E2?and human chorionic gonadotropin?hCG?in peripheral blood change dramatically.In autoimmune encephalomyelitis?EAE?,exogenous estradiol?E2?can increase the production of IL₁0 by spleen cells.Human chorionic gonadotropin?hCG?is also considered to be an important factor in the regulation of B cell phenotype and antibody production.Therefore,we also discussed the role and mechanism of these hormones in the transformation of Breg cellsMethod:1. CBA / J x DBA / 2J model of spontaneous abortion and CBA / J x BALB / C model of normal pregnancy were constructed.2.The expression level of IL-35 at the maternal fetal interface was detected by Western blotting.Flow cytometry was used to detect the proportion of BIO cells in the spleen and the iTR35 at the maternal fetal interface.3.Intraperitoneal injection of recombinant IL-35 cytokines and IL-35 neutralizing antibody into pregnant mice was carried out to observe the effect of IL-35 on abortion and the change of 1Tr35 proportion at the maternal fetal interface.4.The primary CD19+B cells in the spleen of non-pregnant mice were isolated and cultured with pregnancy related hormones?hCG,E2?and IL-35.CCK8 was used to detect the B cell proliferation.The ratio of BIO and IL-35+Breg was analyzed by flow cytometry.5.Western blotting was used to detect the signaling pathways that hCG or IL-35 induced BregResults:1.The embryo loss rate of spontaneous abortion mice was significantly higher than that of normal pregnant mice.2.The expression level of IL-35 and the proportion of iTR35 cells at the maternal fetal interface of spontaneous abortion mice were significantly lower than that of normal pregnant mice.3.IL-35 significantly increased the iTR35 content at the maternal fetal interface and inhibited abortion;IL-35 neutralizing antibody decreased the iTr35 propotion at the matemalfetalinterfaceandincreasedtherateofplacentaloss.4.The peripheral BIO cells in normal pregnant mice were significantly expanded,while the proportion of peripheral BIO cells in spontaneous abortion mice was significantly lower than that of normal pregnant mice.5.IL-35 and hCG inhibited the proliferation of B cells in mice spleen.IL-35 activated STAT1 and STAT3 proteins in B cells and mediated their transform into BIO and IL-35+Breg.hCG activated the phosphorylation of STAT3 in B cells and induced its transformation into BIO.E2 had no significant effect on the proliferation and transformation of spleen B cells.Conclusion:This study confirmed that decidual iTR35 cells and spleen BIO cells play an important role in maintaining maternal fetal interface and peripheral immune tolerance during pregnancy.IL-35 can maintain normal pregnancy by inducing the differentiation of iTr35 cells at the maternal fetal interface.In addition,hCG and IL*35 can inhibit the proliferation of B cells and mediate their transformation into BIO and IL-35+Breg cells.Our study provides a new basis for the application of IL-35 in the treatment of recurrent abortion.At the same time,it also provides a new way to study the mechanism of IL-35 maintaining immune tolerance in tumor microenvironment.