| BackgroundOvarian cancers are the common malignancy among women and there are almost 204,000 new cases and 125,000 deaths per year worldwide.The most common ovarian cancer(about 90%-95%)is epithelial ovarian cancer(EOC),which arises from the ovarian epithelium.Almost 70%of patients are diagnosed at an advanced stage of tumor dissemination because of the lack of sensitive and specific biomarkers and the fact that the disease tends to develop and spread rapidly.The control of EOC at advanced-stage remains difficult.Therefore.superior prognostic iarkers and novel therapeutic strategies are needed to improve the prognosis of EOC patients.EOC is naturally immune reactive.which is regulated by various immune cells.Immunologic reaction of EOC plays a significant role on disease control,and immunotherapy has emerged as a novel treatment method for EOCCD4+ T cells has an essential role in tumor immunology.Recently,it is demonstrated that T-helper(Th)cells(such as ThKTh2.and Thl7)and regulatory T(Treg)cells participate in the pathogenesis and progression of different solid tumors with opposite antitumor or protumor effectsTh22 cell is a newly discovered T cell subset and is identified by the production of Interleukin(IL)-22,but not interferon-γ(IFN-γ)and IL-17.Compare to other T cells(including Th1,Th2,Th17),Th22 subset showed distinct differences in the profile of altered genes.The naive CD4+ cells can differentiate into Th22 subset in the presence of IL-6 and tumor necrosis factor-α(TNF-α)with the aid of plasmacytoid dendritic cells and aryl hydrocarbon receptorin human body.The function of Th22 cells are mostly mediated by the cytokines of IL-22 and TNF-αIL-22 was first described as IL-10-related-T-cell-derived inducible factor(IL-TIF)IL-22 belongs to IL-10 family,which is a lineage-specific effector cytokine of Th22 cells.IL-22 maintains its function via a transmembrane receptor complex composed of IL-22R1 chain and IL-10R2 chain,and plays a dual role in inflammatory and autoimmune diseases.Notably,prosurvival and proliferative effects of IL-22 are confirmed by many cancers,such as lung and liver cancers.TNF-a is another main cytokine secreted by Th22 cells,which has been proved to be overproduced in several malignancies.Moreover,its tumor-promoting effect has been clearly proved in mouse cancermodelsHowever,the role of Th22 cells is not clear in human cancer.Recently,it has been demonstrated that Th22 cells participate in various tumors,including colon cancer,gastric cancer,hepatocellular carcinoma,and small-and large-cell lung cancer.These studies indicate the crucial role of Th22 cells in tumorigenesisTo the best of our knowledge,the roles of Th22 cells,IL-22 and their association with Th17 or Thl in EOC have not been reportedObjectivewe investigated the prevalence of peripheral Th22 cells,Thl 7 cells,Thl cells,and Treg cells in EOC,benign ovarian epithelial neoplasm(BOEN)patients and healthy control(HC);To examine and compare the plasma concentrations of IL-22,IL-17,IFN-γ,and TNF-α in EOC,benign ovarian epithelial neoplasm(BOEN)patients and healthy control(HC);The expressions of IL-22,TNF-α,IFN-γ,IL-17,IL-22R1 and IL-10R2 in the tissues of patients with EOC,benign epithelial ovarian neoplasms and healthy controls were detected by qPCR and Western Blot;The relationship among them and their associations with tumor stage were also evaluatedMaterials and Methods(1)A total of 34 untreated EOC patients diagnosed pathologically with a mean age of 55.6±10.1 years(range:32-79 ys)were enrolled in this study.The characteristics of the EOC patients are given in Table 1.Totally 15 BOEN patients and 15 healthy women were enrolled as control with a mean age of 54.1 ± 10.2 years and 53.7±10.6 years(range:36-73 and 33-71 ys).respectively(2)Briefly.200 L heparinized PB with an equal volume of Roswell Park Memorial Institute 1640 medium was incubated for 4hours at 37OC in the presence of 5%CO2.25ng/mL of phorbolmyristate acetate,1 g/mL of ionomycin and 1.7 g/mL ofMonensin(Alexis Biochemicals,San Diego,CA).After incubation,the cells were stained.Stained cells were analyzed by flow cytometric analysis using a FACS cytometer equipped with Cell Quest software.The expressions of Th22,Th17,Treg and Th1 cells in peripheral blood were detected.(3)Plasma was isolated from PB by centrifugation.The plasma levels of IL-22,IL-17,IFN-γ and TNF-α were determined with a quantitative sandwich enzyme immunoassay technique according to the manufacturer’s instructions.(4)The tumor stage(stage Ⅰ,Ⅱ and stage Ⅲ,Ⅳ)and histological grade(G1,well-differentiated tumor;G2,moderate-differentiated tumor;G3,poor-differentiated tumor)were assessed and classified according to FIGO and World Health Organization classifications.The expressions of Th cells and related cytokines in different groups were analyzed.(5)Tissues were taken and the expression levels of IL-22、TNF-α、INF-γ、L-17、IL-22R1 and IL-10R2 were detected by qPCR and Western Blot.Results(1)To determine the percentage of Th cells and Treg cells in PB,flow cytometry was performed.Representative flow cytometry results were shown.①significantly elevated frequencies of Th22(CD4+IFN-F-IL17+IL22+T cells)were found in EOC(2.57±0.88%)compared with BOEN patients(0.87±0.21%,P<0.001)and HC(0.82±0.12%,P<0.001).However,no significant difference was found in Th22 cells between BOEN patients and HC(P=0.838).②However,no significant difference was found in Treg(CD4+CD25+Foxp3+T cells)cells and Thl cells(CD4+IFN-γ+T cells)among EOC patients,BOEN patients,and HC.But,the percentage of Thl in EOC patients(27.04±12.21%)was higher as compared with that in BOEN patients(20.14±1.49%)and HC(20.36±1.45%).③ The percentage of Thl 7(CD4+IL17+IFN-γ-T cells)of EOC patients(Thl7:3.15±1.35%)significantly increased compared with BOEN patients(Th17:1.06±0.26%,P<0.001)and HC(Th17:1.04±0.17%,P<0.001).However,no significant difference was found between BOEN patients and HC(Th17:P=0.961).These results demonstrate that frequencies of Th22 and Th17 cells in EOC patients are higher than those in BOEN patients and HC.(2)IL-22,IL-17,IFN-γ,and TNF-α in sera of EOC patients were detected with ELISA.CDsignificantly higher levels of IL-22 were found in EOC patients(34.19±4.58pg/mL)than those in BOEN patients(23.1±1.78pg/mL,P<0.001)and HC(22,1±1.71pg/mL,P<0.001)②Remarkable differences of TNF-α were revealed in EOC patients(median,114;range,5.50-595pg/mL)than those in BOEN patients(median,7.15;range,4.31-9.31pg/mL,P<0.001)and HC(median,6.32;range,3.76-9.31pg/mL,P<0.001)③However,there was no significant difference in the IL-17 and IFN-γ levels among EOC,BOEN patients and HC(P>0.05).These results indicate that the levels of IL-22 and TNF-α are significantly higher in peripheral blood of EOC patients than those in BOEN patients and HC.(3)The correlations among Th22、Thl7,Treg.and Thl Cells in EOC patients were analyzed.① A positive correlation was seen between Th22 cells and Thl7 cells in EOC patients(r=0.534,P＝0.001)②.Similarly,a positive correlation was detected between Th22 and Thl cells(r=0.464.P=0.006)or Thl7 and Thl cells(r=0.541.p＝0.001)③ However,Treg cells did not show significant correlation with Th22(P=0.760),Thl7(P=0.966),and Thl(P=0.237)cells in patients with EOC.These results show that positive correlations are found between Th22 cells and Thl7 cells,Th22 and Thl cells,and Thl 7 and Thl cells in EOC patients.(4)Then,we further analyzed the correlations of Thl cells and Th22 cells with their cytokines.①Results showed that a positive correlation was found between Thl cells and IFN-γ(r=0.418,P=0.014)②Similarly,Th22 cells were positively correlated with IL-22(r=0.585,P<0.001)and TNF-α concentration(r=0.495,P=0.009).These results demonstrate that Th22 cells have positive correlation with IL-22 or TNF-α and Th1 cells have positive correlation with IFN-γ.(5)① EOC patients in stage Ⅲ-Ⅳ exhibited significantly higher circulating Th22 than those in stage Ⅰ-Ⅱ(stage Ⅰ-Ⅱ,1.91±0.23%;stage Ⅲ-Ⅳ,2.77±0.91%,P=0.012).②Furthermore,there was an increased trend of IL-22 and TNF-α in stage Ⅲ-Ⅳpatients(IL-22:35.6±3.98pg/mL;TNF-α:median 133.2,range5.50-595pg/mL)compared with patients in stage Ⅰ-Ⅱ(IL-22:29.8±3.61 pg/mL,P=0.002;TNF-α:median,51.6,range 21.6-109.5pg/mL,P=0.01).③However,no significant difference was found in Thl7 cells,Thl cells,IL-17,and IFN-γ between patients in stage Ⅰ-Ⅱ and stage Ⅲ-Ⅳ(data not shown).④Besides,no significant difference was found in Th22,Th17,Th1,IL-22,and TNF-α among 3 types of differentiation in EOC patients(data not shown).These results show that the levels of Th22,IL-22,and TNF-α are significantly higher in stage Ⅲ-Ⅳ patients than those in stage Ⅰ-ⅡI patients.(6)mRNA was detected by qPCR and protein level was detected by Western Blot.The expression of IL-22 in EOC patients was significantly higher than that in BOEN group(P<0.001)and HC group(P<0.001).There was no significant difference in the expression of IL-22 between BOEN and HC(P>0.05).The expression of TNF-α in EOC patients was significantly higher than that in BOEN group(P<0.001)and HC group(P<0.001).There was no significant difference in the expression of TNF-αbetween BOEN and HC(P>0.05).There was no significant difference in IFN-γexpression between EOC,BOEN and HC groups(P>0.05).There was no significant difference in IL-17 expression between EOC,BOEN and HC groups(P>0.05).The expression of IL-22 and TNF-α was significantly increased in EOC tissues.(7)mRNA PCR was performed to detect the expression levels of IL-22R1 and IL-10R2 in tissues of EOC patients,benign ovarian epithelial tumors patients and healthy controls.The expression of IL-22R1 in EOC patients was significantly higher than that in BOEN group(P<0.001)and HC group(P<0.001).IL-10R2 in EOC patients was significantly higher than that in BOEN group(P<0.001)and HC group(P<0.001).There was no significant difference in the expression of IL-22R1 and IL-10R2 between BOEN and HC(P>0.05).Western Blot analysis of protein levels revealed that the expression of IL-22R1 in EOC patients was significantly higher than that in BOEN group(P<0.001)and HC group(P<0.001).IL-10R2 in EOC patients was significantly higher than that in BOEN group(P<0.001)and HC group(P<0.001).There was no significant difference in the expression of il-22 between BOEN and HC(P>0.05).Conclusions(1)Our data showed that the expression of Th22 cells and Thl7 were significantly higher in the peripheral blood of EOC patients compared with BOEN patients and healthy controls.(2)Our data showed that the level of IL-22 and TNF-α was significantly higher in the plasma of EOC patients compared with BOEN patients and healthy controls.And,the percentage of Th22 cells in peripheral blood and level of IL-22 and TNF-α in plasma were elevated consistently.(3)Our data showed that Th22 cells,IL-22 and TNF-α were elevated in the an advanced stage of EOC dissemination.(4)The expressions of IL-22 and TNF-α were significantly increased in the EOC tissues.(5)The expression levels of IL-22R1 and IL-10R2 in EOC tissues were significantly increased.In general,the elevated frequencies of Th22 cells and related cytokines in the peripheral blood of EOC patients suggested the roles of Th22 cells,IL-22 and TNF-αin the pathogenesis of EOC. |
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