| Background and ObjectivesPremature Ovarian Failure?POF?,also known as primary ovarian insufficiency?POI?,is a highly heterogeneous disease affecting 0.5-3.0%of childbearing age women.The risks of osteoporosis,cardiovascular disease,cognitive decline and Parkinson's disease are multiplied in untreated patients with POF,which in turn seriously impair reproductive health and quality of life of patients[1].Therefore,the study on the prevention and treatment of POF has attracted more and more scholars'attention.All forms of premature ovarian senescence?POS?will successively experienced concealed phase,biochemical stage and end stage.The early stage of POF is asymptomatic.Therefor,most POF are discoved at a late stage when the ovarian function is completely lost,and there is almost no effective treatment method for these patients with POF[2].The etiology and pathogenesis of POF are not fully understood.The known causes of POF include genetics,infection,environment,immunity,chemotherapy,radiotherapy and surgery.9%-40%of patients with POF have other autoimmune diseases such as primary adrenal insufficiency?Addison's disease?and autoimmune thyroid disease?Grave's or Hashimoto disease?,which in turn promote the research of autoimmune cause of POF.In recent years,many researchers have suggested that autoimmune ovarian disease?AOD?may lead to POF[3-5].Previous study have demonstrated that Regulatory T cells?Treg?from normal mice can effectively prevent autoimmune ovarian disease,which suggested that Treg cell abnormalities/depletion play a key role in the development of AOD[6,7].The study also observed a large number of T cells and B cells in the ovarian tissue of thymectomize on day3?D3tx?mice before rather than after adoptive transfer of Treg cells,which suggested that dysfunction of Treg cells are closely related to the subsequent effective changes of humoral immunity/B cells[8].It's unclear whether Treg cells maintain their own tolerance and immune homeostasis by directly inhibiting and regulating effector T cell function or directly inhibit and regulate B cell function.To answer the question,our study examined the temporal changes of Treg cells and B cells in a mouse model of AOD,and preliminarily explored the mechanism of the regulation of B cell function by Treg cells.The ultimate aim of this study is providing new clues for the early immunological changes of POF,and developing a new method for POF/POI treatment.MethodsAOD model was set up by removing the thymus of BALB/c mice on day 3?D3tx group/experimental group?,and peripheral blood,ovary,spleen and pelvic lymph node tissues were collected.The levels of bothserum sex hormones and helper T cell-associated inflammatory factors were detected by ELISA.The morphological changes of mouse ovarian tissue were examined by HE staining.Flow cytometry was used to detect CD4+T cells,Treg cells and B cell subsets in spleen and pelvic lymph nodes.In vitro co-culture combined Flow cytometry?CSFE?was used to detect the effects of two different types of Treg cells?nTreg,iTreg?on B cell proliferation,and the effects of iTreg cells on B cell proliferation after treated with transwell chambers,TGF-?or IL-10 inhibitor.In vitro co-culture combined Flow cytometry?Annexin-V-FITC/PI staning?was used to detectthe effects of iTreg cells on B cell apoptosis.Magnetic beads were used to separate CD4+CD25+Treg?nTreg?and NaiveCD4+T cells from spleen of normal mice.iTreg cells were obtained by stimulating NaiveCD4+T cells with TGF-?in vitro.The effects of nTreg or iTreg cells on AOD were further evaluated by adoptive transfer of nTreg or iTreg cells into d3tx mice in the 1st and 8th week after surgery,and pathological changes in ovarian tissue were evaluated again at the before and after the transfer.The main results1.The levels of serumsex hormones?E2,FSH,and LH?in peripheral blood of mice were examined from the 1st to the 10th week after operation.The results showed that the average level of E2 in the experimental group was significantly lower than that of the control group,while the average level of FSH and LH in the experimental group was significantly higher than that of the control group from the 5th to the 10th week after operation.2.The pathological changes of ovarian tissue were observed from the 1st to the 10th week after operation.The results showed that compared with the control group,a little CD19+cells were observered in the ovarian stroma at the 3rd week,a large number of CD3+and CD19+lymphocytes were observed in the ovarian stroma at the 5th week,a large number of fibrous tissue hyperplasia was observed,the number of growing follicles was significantly reduced and the number of atresia follicles was significantly increased in the experimental group.The AOD performance reached 2 to 3 grades at the 5th week after surgery,and reached 4 grades by the 8th week.3.Helper T cell-associated inflammatory factors detection results showed that compared with the control group,the levels of TGF-?and IL-4 were significantly decreased and the levels of IFN-r and IL-21 were significantly increased in the experiment group from the 5th to 10th week after operation.4.Cells dynamic monitoring results showed that compared with the control group,CD4+T cells were slightly elevated from the 1st to the 10th week in experimental group after operation,Treg cells increased slightly from the 1st to the 3rd week,and gradually decreased after 3 weeks,and continued to be lower than the control group from the 5th to the 10th week.Activated B cells?CD138-IgD+?gradually increased from the 1st to the 10th week.Plasma cells?CD138+CD19-IgD-?did not change significantly from the 1st to the 4th week,and gradually increased from the 5th to the 10th week.5.The results of correlation analysis showed that there was no correlation between Treg and plasma cells?CD138+CD19-IgD-??r=-0.1457,P=0.05640?,while Treg was negatively correlated with activated B cells?CD138-IgD+??r=-0.6622,P=0.0028?.6.The purities of nTreg cells,na?ve CD4+T cells,iTreg cells and B cells were 96.1%,97.5%,83.2%and 96.8%,respectively.7.B cell proliferation assay showed that the levels of B cell proliferation in two co-culture groups?nTreg+B cells/iTreg+B cells?were both significantly lower than that in the B cell alone group.There was no significant difference in two co-culture groups.8.Both transwell assay and inhibitor assay showed that compared with the control group,the levels of B cells proliferation significantly increased in the group using Transwell chamber and the two groups treated with TGF-?inhibitor or IL-10 inhibitor.9.B cell apoptosis analysis showed that there was no significant difference in apoptosis between B cells alone group and B+iTreg cells group.10.Treg cells adoptive transfer assay showed that adoptive transfer of iTreg or nTreg cells at the 1st week rather than the 8th week can effectively prevent the development of POF.ConclusionsThymic-extracted D3tx mice developed typical features similar to human POF,such as the infiltration of inflammatory cells including lymphocytes,plasma cells,and mononuclear Macrophages cells in the ovarian follicles and stroma,which resulted in structure disturbance of ovarian and the loss and atresia of follicle.Mouse AOD originates from the damage of cellular immune function?the abnormal number and dysfunction of T cells?.In the later stage,the ovarian appears plasma cells and activated B cells infiltration in the ovarian stroma and follicles.So,D3tx mouse is a good animal model of human POF.The number of Treg cells in local lymphoid organs and tissues of D3tx mice was depleted after thymus removal,and then experienced a short-term protective and compensatory elevation.Pathological changes of ovary were found in the 5th week after surgery.Treg cells were negatively correlated with activated B cells.iTreg cells directly inhibit B cell proliferation through cell-cell contact and secretion of TGF-?,IL-10cytokines.It is speculated that B cell dysfunction may be a follow-up effect of Treg cell dysfunction and related to local ovarian tissue damage.This immunosuppressive function of iTreg cells may be caused by directly inhibiting B cell proliferation,antibody production and inflammatory infiltration.Before the pathological damage of ovarian tissue occurred,the adoptive transfer of nTreg and iTreg cells could effectively inhibit the occurrence of AOD.In comparison,the adoptive transfer of Treg cells can not effectively improved AOD ending after the pathological damage occurred.It is suggested that the treatment of clinical immune POF should also have a certain window period,and early diagnosis is significantly needed. |
PDF Full Text Request