The Role Of T_H1/T_regImbalance In The Pathogenesis Of Premature Ovarian Insufficency

Chapter I The TH1/Treg cell imbalance in in human premature ovarian insufficiencyBackground:Premature ovarian insufficiency(POI)is defined as loss of ovarian function before 40 years of age,characterized by amenorrhea,hypergonadotrophin levels and reduced estrogen.POI is clinically complicated and etiologically heterogeneous.The majority of POI remain to be elucidated and defined as idiopathic.An autoimmune origin has long been considered to explain 5-30%of POI cases,which was also known as autoimmune primary ovarian insufficiency.Currently no reliable and specific predictive and diagnostic markers for autoimmune POI exist.The mechanisms of autoimmune disturbance underlying the ovarian senescence are largely unknown.Previously the histopathological analysis of autoimmune oophoritis revealed massive infiltration of lymphocytes,macrophages and dendritic cells,predominated by CD3+T cells,especially CD4+T cells,in the ovary.The abnormal amount of T cells and proinflammatory cytokines was also found in the peripheral blood of patients with POI.The activated T cells could release a series of proinflammatory cytokines and induce specific immune responses to destroy ovarian follicles and impair ovarian function.Regulatory T cell(Treg)is a subset of T lymphocyte cells with negative immune regulation,which play a key role in peripheral immune tolerance.The functional dysregulation of immune homeostasis in Treg cells underlies the augmented autoreactive response and tissue damage in multiple autoimmune disease.However,the few studies on cellular immunity of POI all had small sample sizes and the results remain controversial.There is no comprehensive description about subsets of immune cells and cytokine profiles in POI patients.Whether altered pathogenic T subsets and inflammatory cytokines,if any,are implicated in the pathogenesis of human POI remains poorly definedObjective:To investigate the involvement of aberrant cellular immunity in the pathogenesis of POI,T lymphocytes subpopulation and cytokines signatures were determined in periphery blood and local ovarian environment in patients with POI,and the correlation between inflammatory indicators and ovarian reserve markers were analyzedMethods:Cytokine profiles in serum in patients with POI and healthy women were determined by respective ELISAs.Flow cytometry was used to measure intracellular cytokines in T lymphocyte subsets and the number and phenotype of CD4+CD25hiFoxp3+ Treg cells from peripheral blood mononuclear cells(PBMC)ELISAs and quantitative real-time PCR were used to determine the inflammatory cytokine profiles in the ovarian microenvironment by measuring cytokines in follicular fluid and GCs in patients with biochemical POI(bPOI).The association of inflammatory indicators with the disease severity was analyzed by Spearman correlationResults:(1)Increased IFN-? and TNF-? in blood and ovary in patients with POITo investigate whether dysregulated cellular immunity occurs in POI,we firstly determined the cytokine profiles in serum in patients with POI and healthy women.POI patients showed significantly increased levels of type 1 proinflammatory cytokines IFN-?(P<0.0001)and TNF-?(P=0.0006),but reduced amounts of regulatory cytokine TGF-?(P<0.0001).No differences were detected for other cytokines,such as IL-4(TH2),IL-17(Th17)and IL-10.To determine whether the dysregulated cytokine profile was resulted from T lymphocytes,we analyzed intracellular cytokines in T cells from PBMC using flow cytometry.Compared to healthy women,patients with POI had increased frequency of CD3+TNF-?+T cells(P=0.0196),CD3+TNF-?+IFN-?+ T cells(P=0.0462)and CD3+IFN-?+T cells(P=0.0164).No differences were observed for IL-17+and IL-10+ CD3+ T cells between two groups(P>0.05).The percentages of CD4+and CD8+T cells were comparable between POI patients and control subjects We found that women with bPOI already had significantly higher levels of TNF-?(P=0.048)and reduced amounts of IL-10(P=0.0002)compared to the healthy control in follicular fluid.There was also significantly higher frequency of detectable IFN-y in bPOI patients than that in controls(P<0.0001).In addition,ovarian GCs isolated from women with bPOI showed significantly increased expression of inflammatory cytokines IFNG and TNF,and decreased TGFB expression compared with control groups(P<0.05).But no significant differences were observed for IL17,IL4 and IL10 mRNA expression(2)Treg cells deficiency in patients with POIWe firstly examined the number and phenotype of CD4+CD25hiFoxp3+Treg cells in PBMC of patients with POI and found the frequency and the absolute number of Treg cells was significantly decreased in women with POI compared with control women(P=0.0089;P=0.0371).To understand the underlying mechanisms for the decrease in Treg cells,we measured the proliferative rate of Treg cells ex vivo with Ki-67 staining,and observed that the fraction of Ki-67+Treg cells was decreased in patients with POI(P=0.028).In addition,patients with POI had a significantly higher proportion of apoptosis in Treg cells compared to control women(P=0.0345).In combination with the analysis of the expression of Foxp3,CTLA-4 and GITR that are indicators for Treg cell function.We found that Treg cells in women with POI exhibited significantly lower levels of Foxp3 expression determined by mean fluorescence intensity(MFI)(P 0.0318),and reduced CTLA-4 positive cells(P<0.0001)compared to healthy women The GITR expression was however comparable between the two groups(P=0.6660)(3)Increased ratio of TH1 cytokines to Treg cells correlates with severity of ovarian insufficiency in patientsTo confirm the dysregulated ratio of TH1:Treg cells is responsible for the severity of ovarian insufficiency,we conducted correlation analyses between inflammatory indicators and ovarian reserve markers in patients with POI We found that the amounts of proinflammatory cytokines IFN-y and TNF-? in the sera had strong positive correlations with FSH(IFN-?:FSH,R=0.36,P<0.001;TNF-?:FSH,R=0.43,P=0.002),but negative correlations with E2(IFN-?:E2,R=-0.29,P<0.001;TNF-?:E2,R=-0.47,P=0.001).The level of serum TGF-? negatively correlated with FSH and positively with E2,respectively(TGF-?:FSH,R=-0.37,P<0.001;TGF-?:E2,R=0.29,P<0.001).Significantly,Treg cells exhibited strong negative correlation with FSH and positive with E2 and T(Treg:FSH,R=-0.25,P=0.047;Treg:E2,R=0.27,P=0.04;Treg:T,R=0.27,P=0.04),suggesting their role in maintaining ovarian reserve and function.Similar correlations were also seen between the ratios of Treg:CD3+TNF-a+cells or Treg:CD3+TNF-?+IFN-?+cells and the levels of FSH,E2 and T(Treg:CD3+TNF-?+:FSH,R=-0.29,P=0.02;Treg:CD3+TNF-?+:E2,R=0.29,P=0.03;Treg:CD3+TNF-?+:T,R=0.31,P=0.02?Treg:CD3+TNF-?+IFN-?+:FSH,R=-0.33,P=0.01;Treg:CD3+TNF-?+IFN-?+:E2,R=0.31,P=0.02).Moreover,the negative correlation between FSH and Foxp3 intensity or CTLA-4 expression percentage further reinforced these associations(Foxp3:FSH,R=-0.26,P=0.04;CTLA-4:FSH,R=-0.38,P=0.01).Conclusions:POI patients exhibited systemically augmented TH1-like response in both periphery and ovarian microenvironment,the decreased number and impaired suppressive function of Treg cells might account for the increased proinflammatory cytokines IFN-y and TNF-? in the patients with POI.The correlation analyses between TH1:Treg and the severity of ovarian insufficiency highlight a potential causative role of abnormal cellular immunity in impairment of ovarian function and in the pathogenesis of POI?Chapter II The TH1/Treg cell imbalance in experimental premature ovarian insufficiency models in miceBackground:In chapter I,we have demonstrated that patients with POI showed augmented TH1 autoimmunity and decreased number and impaired suppressive function of Treg cells both in periphery and ovarian microenvironment.The decreased ratio of Treg to TH1 cells strongly correlated with progress of POI disease.It suggested that Treg cells might play a pivotal role in maintaining ovarian function,and TH1/Treg imbalance may be involved in the pathogenesis of human POI.Nevertheless,it remains unknown whether TH1/Treg imbalance play the pathogenic role in POI and the mechanism has not been uncovered yet.Given the unavailability of ovarian samples obtained from patients,experimental POI models in mice were warranted to determine the causative role of TH1/Treg imbalance-mediated TH1 inflammation in the pathogenesis of ovarian insufficiency.The experimental autoimmune oophoritis in mice can be induced by translational animal models,such as Aire gene knockout mouse,thymectomy on day 3,immunization with ovarian self-antigen or peptide of zona pellucida or MATER.The pathological phenotype of these mouse models is similar to that of ovaries in patients with POI.Previous studies mainly focused on the infiltration of inflammatory cells in the ovary and the mechanism that how inflammation induce ovary damage was not fully studied.The underlying mechanism by which Treg cells involved in the pathogenesis of POI remains largely unknown.Therefore,we utilized a new experimental model in mice mimicking ovarian insufficiency caused by TH1/Treg imbalance,which should have implications for the pathogenesis and therapeutic intervenes of the patients with POI.Objective:The Rag2-/-knockout mice adoptively transferred with Naive T cells were used to construct the experimental autoimmune POI models.The hormone levels,ovarian size,morphology,follicles numbers in different stages and function of granulosa cells were evaluated.To validate the causative role of abnormal TH1 cell-mediated inflammation in the pathogenesis of ovarian insufficiency,the infiltration of different subtypes of T lymphocytes and their related cytokines in inflamed ovary and peripheral lymphoid organs were measured.Mice receiving co-transferred Treg cells were utilized to elucidate the preventive and rescue role of Treg cells in ovarian damage and POI.Methods:The CD4+CD25-CD45RBhi T cells(Naive T cells)and CD4+CD25hiYFP+(Treg cells)from spleens and peripheral lymph nodes of Foxp3YFP-Cre mice were sorted by flow cytometry.Rag2-/-recipients were divided into three groups and intraperitoneally injected with PBS,CD4+CD25-CD45RBhi T cells in the presence or absence of CD4+CD25hi YFP+Treg cells.The ovary,spleen and ovarian draining lymph nodes were isolated to measure the distribution of inflammatory cells by FACS analysis.Ovarian tissues were harvested for histopathological and immunological analyses.Quantitative real-time PCR was used to measure the expressions of cytokines,chemokines and markers related to steroidogenesis in ovary.Serum was collected and isolated for estradiol and progesterone measurements by chemiluminescence immunoassay.Results:Compared with the untreated group,Rag2-/-mice transferred with CD4+CD25-CD45RBhi T cells(POI group)exhibited the phenotype of ovarian insufficiency with smaller ovarian size and decreased number of follicles in total and at different stages.The levels of estradiol and progesterone were also remarkably decreased.We found that the proportion of cleaved-PARP positive cells per follicle was much higher in POI group,and the apoptotic signals specifically distributed in GCs of growing antral follicles.Importantly,increased gene expression of proinflammatory cytokines(Ifng,Tnf and Il1b)and chemokines(Ccrl,Ccr2,Cxcl10 and Cxcl17),and decreased expression of genes relating to ovarian function(Cyp17al Cypl9al,Cypl1a1,Star and Lhr)were observed in the ovaries of POI group.Flow cytometry analysis of single cells in ovaries revealed massive infiltration of lymphocytes predominated by CD4+IFN-?+TNF-?+T cells,whereas Foxp3+Treg cells were virtually absent.In contrast,mice receiving co-transferred Treg cells(POI+Treg)exhibited little orno infiltration of lymphocytes in ovaries.The number of IFN-?-and TNF-?-producingCD4+T cells was also reduced in the ovary as well as in the spleen and draining lymphnodes.Co-transfer of Treg cells effectively prevented ovarian weight loss,improved ovarian function,reduced the amounts of proinflammatory cytokines in ovary and decreased the apoptosis of GCs.Consistently,the mRNA expression of the genes that reflected ovarian function,including Cyp17a1,Cyp19a1,Cyp11a1,Lhr were also augmented,which was accompanied by the reduction of mRNA expression of cytokines and chemokines.The data demonstrated a dramatically amelioration of ovarian insufficiency and resumption of ovarian function following Treg cell co-transfer.Conclusions:The massive infiltration of TH1 cells and its inflammatory cytokines in ovaries can promote granulosa cells apoptosis and suppress steroidogenesis,resulting in ovarian dysfunction and thus contributing to ovarian insufficiency.Treg cells plays a key role in suppressing the pathogenic function of TH1 inflammation in the ovary.Chapter ? Function and mechanism study of TH1 cytokines in granulosa cellsBackground:A cascade of cytokines recruited from periphery circulation or secreted by immune/non-immune cells in ovary coexist in the ovarian microenvironment under physiological conditions.They are involved in follicular growth,ovulation and luteinization,and luteolysis.The proliferation and differentiation of granulosa cells is critical for folliculogenesis.The dysfunction of granulosa cells is one of the main causes contributing to the acceleration of follicular atresia.Previous studies have demonstrated that cytokines had impact on granulosa cells growth and steroidogenesis,however the underlying mechanisms remain largely unknown.Our findings in previous two chapters provided compelling evidence that patients with POI showed increased TH1 dominant inflammation.And the causative effects were validated in experimental POI in mice evidenced by increased apoptosis of granulosa cells and ovarian dysfunction.However,the mechanism of TH1 inflammatory cytokines underlying the granulosa cells dysfunction is still poorly understoodObjective:In vitro study was conducted to determine the involvement of TH1 cytokines IFN-? and TNF-? in the granulosa cells function.The signaling pathway and its downstream target gene was studied to clarify the relevant mechanisms.Methods:KGN cells were cultured in the presence of either rhIFN-y(50.0 ng/ml)and rhTNF-?(50.0 ng/ml)alone,or in combination for 48 hours.The cell apoptosis,Edu assays and estradiol synthesis were measured to evaluate apoptosis,proliferation and steroidogenesis.The proliferation,apoptosis and steroidogenesis markers were also detected by Real-time PCR and Western Blot.Several functional signature genes in granulosa cell were examined by quantitative real-time PCR and Western Blot.Given that CTGF exhibited consistent downregulation on both transcription and protein level,CTGF was identified as the downstream target of the effects of IFN-y and TNF-? on granulosa cells.By employing shRNA transfection to knock down the endogenous CTGF gene in KGN cells,combing with supplement of exogenous rhCTGF into the KGN cells,the granulosa cell growth and steroidogenesis was detected to reveal the effect of CTGF on granulosa cell.The involvement of JAK-STAT1 and NF-?B pathways in the regulation of cytokines on granulosa cell function was studied with the addition of inhibitors for JAK-STAT1 or NF-?B pathway.The data was further validated by murine primary granulosa cells in cultures.Results:(1)TH1 cytokine impaired cell growth and steroidogenesis of human granulosa cells in culturesBoth IFN-? and TNF-? could induce profound increase in cell death and decrease in cell proliferation of human KGN cells.Consistently,both cytokines substantially increased cleaved-PARP but decreased PCNA expression,further indicating IFN-y and TNF-? could impair cell growth by promoting apoptosis and decreasing proliferation.We found that estradiol production by human KGN cells was significantly impaired upon IFN-? or TNF-? treatment.Meanwhile,IFN-? or TNF-? treatment significantly decreased the expression of CYP19A1 and E2 synthesis.Importantly,synergistic reduction in GCs growth and steroidogenesis was observed in IFN-y plus TNF-?combination compared to either cytokine alone(2)TH1 cytokine promoted granulosa cells apoptosis by down-regulating CTGF through JAK-STAT1 and NF-?B activationA number of functional signature genes of GCs including CTGF,INHBA,WT1,FOXO1,and GATA6 were firstly examined by RT-qPCR.We found only CTGF exhibited consistent downregulation on both transcription and protein level.To further determine whether effects of IFN-? and TNF-? on GCs was mediated by CTGF,we down-regulated endogenous CTGF expression by employing shRNA transfection in KGN cells and found CTGF reduction had no effect on CYP19A1 expression and estradiol synthesis in response to IFN-? and TNF-? treatment.However,downregulation of CTGF significantly enhanced the apoptosis and suppressed proliferation of KGN cells which was evidenced further by increased cleaved-PARP expression and decreased PCNA expression.Conversely,addition of exogenous rhCTGF into the KGN cells effectively rescued the cell death induced by both cytokines These data suggest that CTGF deficiency is critical for TH1 cytokine-induced growth impairment in granulosa cell.We next explored how IFN-y and TNF-? regulated CTGF expression.The janus kinase(JAK)/signal transducer,activator of transcription 1(STAT1)and nuclear factor kappa-B(NF-?B)signaling was activated by IFN-y and TNF-?,evidenced by increased phosphorylation of STAT1,IKBa and p65 in human KGN cells.Addition of inhibitors for JAK or IKB? phosphorylation attenuated IFN-y and TNF-?-induced inhibitory effect on CTGF expression in KGN cells.However,the suppression of E2 synthesis by IFN-y and TNF-? could not be reversed by both JAK/STAT1 and NF-?B inhibitors.These data reveal that IFN-y and TNF-? influence granulosa cells growth,at least partially by down-regulating CTGF through of JAK-STAT1 and NF-?B pathways,respectively.Finally,we cultured murine primary GCs with cytokines and similar results were obtained.The data indicate that IFN-y and TNF-a downregulate CTGF in granulosa cells via JAK-STAT1 and NF-?B activationConclusions:The increased TH1 inflammatory cytokines IFN-? and TNF-? could impair steroidogenesis by targeting CYP19A1,and promote apoptosis of GCs partially by down-regulation of CTGF via JAK-STAT1 and NF-?B pathway activation,hence contribute to follicle atresia,ovarian dysfunction and premature insufficiency.