| Endometriosis(EM)is a common and chronic gynecological disorder that heavily affects women of reproductive age.It has become one of rare persist ailment with invasion and dissemination.Recent years studies have showed that immune cell disfunction and disregulation are involved in the pathogenesis or progression of EM.Meyliod derived immune cells(MDSCs)could be recruited and activated in the pathologic microenvironment,participanting in kinds of diseases(bladder cancer、sepsis)by negtively regulating the immune response.In this study,we explored the existence of CD11 b and ARG-1 in normal endometrium as well as eutopic and ectopic endometrium from EM patients,the percentage of MDSCs in the peritoneal fluid(PF)and peripheral blood mononuclear cells(PBMCs)derived from EM patients and healthy subjects or women with other reproductive diseases(uterine myoma or ovary teratoma).The result showed that more MDSCs accumulated in PF and peripheral blood from EM patients than from women with other reproductive diseases(uterine myoma or ovary teratoma).The major MDSCs phenotype in EM patients was M-MDSC.M-MDSCs were recruited into the PF of EM patients by CCR9/CCL25 and CCR5/CCL5,and they coexpressed CCR1 and CCR9,this expression did not affected by granulocyte-macrophage colony-stimulating factor(GM-CSF).M-MDSCs from EM patients inhibited autologous T cell proliferation via arginase-1 and i NOS and macrophage phagocytosis.rh CCL25 promoted IL-10 and GM-CSF secretion from M-MDSCs.GM-CSF could induce the expansion of M-MDSCs.PF derived from EM patients promoted the expansion of M-MDSCs.rh CCL5 and rh CCL25 could increase the ARG1 enzymatic activity of M-MDSCs.And the ARG1 enzymatic activity of PBMCs or PF derived from EM patients were higher than that of women with other reproductive diseases(uterine myoma or ovary teratoma).So,we made the conclusion that CD14+CD33+HLADR-M-MDSCs recruited and activated by CCR9/CCL25 was crucial for pathogenic progression of endometriosis.This research can contribute to revealing the immunological pathogenesis of EM,then provide new evidence for treatment.Part Ⅰ.M-MDSCs were abnormally accumulated in EM patientsObjective:To explore the existence of M-MDSCs in EM patients.Methods:Immunohistochemistry and Real-Time PCR were performed on normal endometrium as well as eutopic and ectopic endometrium from women with other reproductive diseases(uterine myoma or ovary teratoma)and EM patients to explore the presence of CD11 b and ARG-1.Flow cytometry was performed on peritoneal fluid(PF)and peripheral blood mononuclear cells(PBMCs)derived from EM patients and healthy subjects or women with other reproductive diseases(uterine myoma or ovary teratoma)to explore the percentage of MDSCs or M-MDSCs.PBMCs and PF were also stimulated by granulocyte-macrophage colony-stimulating factor(GM-CSF)for 48 hours,then flow cytometry was performed to compare the percentage of M-MDSCs.We used microbeads to separate M-MDSCs from PBMCs,then stimulated them for 48 hours with GM-CSF,and finally analysed the purity of M-MDSCs before and after GM-CSF stimulation by flow cytometry.Results:(1)The immunohistochemistry and Real-Time PCR results showed that compared with the women with other reproductive diseases(uterine myoma or ovary teratoma),the expression of CD11 b and ARG-1 were significantly higher in EM patients,and they almostly existed in the cytoplasm.(2)The flow cytometry results showed that the percentage of MDSCs in the PBMCs derived from EM patients was higher than that from healthy subjects or women with other reproductive diseases(uterine myoma or ovary teratoma).The percentage of MDSCs in the peritoneal fluid derived from EM patients was also higher than that from women with other reproductive diseases(uterine myoma or ovary teratoma).And the major phenotype of MDSCs was CD11b+CD33+HLRDR-M-MDSCs.(3)The flow cytometry results showed that granulocyte-macrophage colony-stimulating factor could promote the expansion of M-MDSCs from PF and PBMCs,increase the purity of M-MDSCs separated by macrobeads.Conclusions:MDSCs were abnormally accumulated in EM patients,and their major phenotype was CD11b+CD33+HLRDR-M-MDSCs.Part Ⅱ.The recruitment and function of CD11b+CD33+HLRDRM-MDSCsObjective : to explore the recruitment and function of CD14+CD33+HLADRM-MDSCs on CD8+ T cells and macrophagesMethods:A phagocytosis assay was performed by flow cytometry.Enzyme-linked immunosorbent assay was performed to explore the concentration of CCL5 or CCL25 in the PF of EM patients.Flow cytometry was performed to explore the expression of CCR1 or CCR9 on the surface of M-MDSCs before and after GM-CSF stimulation.Chemotactic effect of M-MDSCs to different chemotactic factor(CCL5、CCL25)and their neutralizing antibodies were explored by Transwell assay.The number of migratory M-MDSCs were counted under a microscope.We separated M-MDSCs and CD8+ T cells with microbeads,then stimulated M-MDSCs with GM-CSF for 48 hours to get high purity.Then the CD8+ T cells were activated with CD3/CD28 antibody,these two kinds of cells were cocultured directly with different ratios(1:1,1:5,1:10)with or without i NOS or arginase inhibitors for 72 hours later.3H-Td R incorporation was measured by a cell harvester and liquid scintillation counter to explore the proliferation of CD8+ T cells.The macropahges cell line was induced by PMA,then cocultured directly with M-MDSCs with the ratio of 1:1 for 6 hours.Results:(1)Enzyme-linked immunosorbent assay results showed that there were high concentration of CCL5 or CCL25 in the PF of EM patients.(2)M-MDSCs coexpressed CCR1 and CCR9,with CCR9 a higher expression than that of CCR1,and GM-CSF did not effect the level of these two moleculars on the surface of M-MDSCs.(3)M-MDSCs were mainly recruited by CCR9/CCL25 and CCL5/CCR1.(4)3H-Td R incorporation results showed that M-MDSCs could inhibit the proliferation of CD8+T cells via i NOS or ARG.(5)The flow cytometry results showed that M-MDSCs inhibited the phagocytosis of macrophages.Conclusions: M-MDSCs were recruited by CCR9/CCL25 and CCL5/CCR1,and they could inhibit the proliferation of CD8+ T cells and the phagocytosis of macrophages.Part Ⅲ.The regulation of EM microenvironment on M-MDSCsObjective:To explore the effect of EM microenvironment on the M-MDSCsMethods : Enzyme-linked immunosorbent assay was performed to asasy the concentration of IL-10 in the PF of EM patients.High purity of M-MDSCs were cultured in the presence of rh CCL25 or CCR9 neutralizing antibody or CCL25 neutralizing antibody for 48 hours.Enzyme-linked immunosorbent assay was performed to asasy the concentration of IL-10 and GM-CSF in the supertanant.M-MDSCs were labeled with CFSE,then cultured in the presence of GM-CSF or PF from EM patients for 48 hours.Flow cytometry was performed to explore the peoliferation of M-MDSCs.Arginase activity assay was was performed to explore arginase activity of PBMCs or PF from EM patients and PF from women with other reproductive diseases(uterine myoma or ovary teratoma).We cultured M-MDSCs in the presence of rh CCL5 or rh CCL25 or CCL25 plus CCR9 antibody for 48 hours,then performed the arginase activity assay again.Results:(1)Enzyme-linked immunosorbent assay results showed that there was high concentration of IL-10 in the PF of EM patients.rh CCL25 could promote the secretion of IL-10 and GM-CSF of M-MDSCs by binding to the CCR9 on the surface of M-MDSCs.(2)The flow cytometry results showed that peritoneal fluid derived from EM patients could promote proliferation of M-MDSCs.(3)Arginase activity assay showed that the arginase activity of EM PF was higher than that of EM PBMCs and PF from women with other reproductive diseases(uterine myoma or ovary teratoma).rh CCL5 and rh CCL25 both could increase the arginase activity of M-MDSCs.Conclusions:EM microenvironment could positively promote the proliferation,arginase activity and cytokine secretion of M-MDSCs. |
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