After being stimulated by different cytokines, CD4 + T cells differentiate into different types of effector T cells including Th1, Th2, Th17 and Treg. The initial differentiation of Th17 and Treg cells share a common signaling pathway mediated by TGF-β. However, terminally differentiated cells perform the opposite function. Th17 cells can lead to autoimmune response and inflammation, while Treg cells inhibit these inflammatory phenomena and maintain immune homeostasis. Th17 and Treg cells also maintain the balance of maternal-fetal interface immunity and play an important role in recurrent pregnancy loss and preeclampsia. In addition to genetic and environmental factors, the important role of immune system in PCOS has received widespread attention in recent years.
In our study, the results of flow cytometry showed that compared with control group, the proportion of Treg cells in PCOS patients decreased, while the proportion of Th17 cells increased, and the proportion of Treg / Th17 cells in peripheral blood of PCOS patients decreased significantly. A series of previous studies have shown that compared with healthy women, the number of activated T cells in ovarian follicular fluid of PCOS patients increased, while the number of Treg cells in peripheral blood of PCOS patients decreased. The imbalance of Th1 / Th2 and Th17 / Treg was also observed in PCOS patients. The percentage of CD4 + CD25 + Foxp3 + T cells in PCOS patients decreased, and the proportion of Th17 subsets increased, although the difference was not statistically significant.These results were consistent with ours. It has been reported that a single injection of testosterone into female rodents can lead to the decrease of androgen in newborns and increase the relative and absolute number of CD4 + CD25 + Foxp3 + Treg cells in peripheral blood . Androgen can preserve the number of male Treg cells directly or indirectly through its metabolites. Our results showed that the androgen levels of PCOS patients were higher, but the ratio of Treg cells was lower than that of the control group. Spearman correlation analysis demonstrated that there was no correlation between androgen levels and Treg, Th17 cells. However, a significant positive correlation was observed between the ratio of Treg cells and luteinizing hormone levels. The reason may be that the serum hormones in PCOS patients, except LH, other hormones such as FSH, estradiol and androgen, will not affect the ratio of Treg cells. Congenital disability of peripheral Tregs may exist in PCOS women. According to previous study, the decrease of Tregs in peripheral blood of PCOS patients is due to the inherent low responsiveness of the body to IL-2, which lead to the abnormal activation of STAT5B and the reduction of Foxp3 expression.
In our previous study, elevated levels of inflammatory molecules in peripheral blood of PCOS rats were observed, including C-reactive protein, IL-6 and tumornecrosis factor-α. In this study, we detected the levels of Treg and Th17 cell-related cytokines including TGF-β, IL-10, IL-6, IL-17 and IL-23 in the serum of PCOS patients. As expected, we found that Treg cell-related cytokines TGF-β and IL-10 decreased in PCOS patients, while the levels of Th17 cell-related cytokines IL-6, IL-17 and IL-23 increased. This result further confirms the view that chronic low-grade inflammation is involved in the pathogenesis of PCOS. Besides, we found that the levels of insulin and glucose were negatively correlated with the proportion of Treg cells, positively correlated with the proportion of Th17 cells, and negatively correlated with the proportion of Treg/Th17 cells. Insulin resistance (IR) may be one of the main factors in the development of PCOS. IR and hyperinsulinemia play an important role in pathophysiology of PCOS. They aggravate the disorder of the reproductive endocrine and metabolism of glucose and lipid in PCOS patients. IR is one of the key factors affecting the efficacy of PCOS treatment. In recent years, more and more evidence showed that inflammation plays an important role in insulin resistance. Insulin resistance is related to immune factors such as adipocytokines and leptin. Chronic subclinical inflammation may be the initial cause of IR. Relevant studies have shown that insulin resistance can lead to Inflammatory response and Th17/Treg imbalance, which can be rescued by IL-6.
As chaperone proteins, heat shock proteins participate in the assembly of proteins in cells and contribute to protein homeostasis. Heat shock protein 70 has attracted more and more attention due to its important function in gametogenesis or pregnancy regulation. In this study, ELISA results showed that the serum HSP70 levels of PCOS patients were increased, which were consistent with the previous research. The increase of serum HSP70 levels were related to insulin resistance, oxidative stress and low-grade chronic inflammation in PCOS individuals. Elevated serum HSP70 levels were considered to indicate the ovarian damage of transgenic mice under oxidative/ischemic stress. Spearman correlation analysis showed that HSP70 and testosterone were significantly positively correlated. It has been reported that testosterone is necessary for the enhancement of HSP70 expression. Physiological testosterone can enhance the expression of HSP70 induced by ischemic pretreatment, which may be due to the fact that testosterone can stimulate phosphorylation and upregulate the mRNA expression of HSP70.
In addition to the chaperone function, HSP70 can also stimulate and inhibit inflammation. The correlation between HSP70 and Treg/Th17 cells ratio was studied for the first time in our work. Spearman rank correlation analysis demonstrated that the levels of HSP70 in human serum was significantly negatively correlated with Treg/Th17 ratio. The ability of HSP70 to induce autoimmune response may be part of natural autoimmunity, or it may be related to pathological autoimmune diseases. Anti-HSP70 antibody has been confirmed in cord, and may play a role in normal immune system.
Heat shock protein is a stress-induced protein with immunomodulatory properties. It contains peptide binding domain, which can bind proteins and non-protein molecules with exposed hydrophobic residues. The function of antigen presentation and the ability of inducing cytokines in vitro are the results of the binding between HSPs and molecules or molecular chaperone role of HSPs. In inflammatory disease models, T cells that respond to heat shock proteins inhibit the disease by producing anti-inflammatory cytokines. The anti-inflammatory activity of HSP-specific T cells depends on their recognition of endogenous HSP epitopes. It has been reported that these T cells can be induced by the conservative sequence of HSP of microorganisms. The upregulation of endogenous HSP expression induced by drugs can promote the production of anti-inflammatory T cells. HSP70 can induce protective, anti-inflammatory regulatory T-cell response.
In our study, serum HSP70 levels were significantly negatively correlated with the Treg/Th17 ratio. The possible reason may be that in addition to regulating protein homeostasis, HSPs also participate in the enhancement of immune response under a variety of stress conditions, including fever, oxidative stress and inflammatory cytokine signaling activation during viral infection. In response to these stress signals, HSPs mediate the constitutive and inducible danger signals that activate the immune response.HSP70 has been reported to activate the innate immune system. Studies have shown that the E3 ligase Stub1, which is expressed in response to danger signals during inflammation, is responsible for the ubiquitination of Foxp3 with the help of HSP70 chaperone protein, which leads to the degradation of major Treg cell transcription factors.The high level of heat shock protein 70 directly promotes the expression of Th17 gene after TCR stimulation. This effect stems from the direct intracellular interaction of HSP70 with a RISC complex. The activity of heat shock protein 70 in Th17 promotion depends on the regulation of a set of specific miRNA expression. Selective inhibition of these microRNAs or directly blocking the function of heat shock protein 70 will downregulate the expression of Th17 gene.
In our study, Spearman rank correlation analysis showed that serum HSP70 levels were significantly negatively correlated with IL-10 and TGF-β levels, while significantly positively correlated with IL-6, IL-17, IL-23 and other inflammatory cytokines. Our previous study has shown that the levels of serum heat shock protein 70 in PCOS rats were decreased, and they were strongly negative correlated to testosterone, luteinizing hormone and inflammatory factors such as C-reactive protein, IL-6, IL-18, and tumor necrosis factor-α。This is contrary to our results in PCOS patients. The possible reason is that the transient superphysiological testosterone characteristics caused by the construction of PCOS rat model down-regulated the expression of HSP70 in rat serum. HSP 72 is another member of the heat shock protein 70 family. According to the previous research, transient hyperphysiological testosterone can down-regulate the expression of heat shock protein 72. The regulation of HSP70 expression is mediated by heat shock transcription factor (HSF)-1. Testosterone can induce the inhibition of heat shock protein 72 expression or HSF1 activation, which may directly block the trimerization and phosphorylation of HSF1, or indirectly inhibit HSF1 activation, leading to the reduced expression of HSP70.
Insulin has the capacity to promote the occurrence and development of PCOS through PI3K and MAPK signaling pathways. Here, our current investigation found that there was a significant positive correlation between serum insulin levels and testosterone levels. 17α- Hydroxylase was found to be decreased due to the inhibition of PI3K in follicular cells of PCOS patients, indicating that insulin may promote steroidogenesis through PI3K pathway.The existence of specific high affinity insulin receptor on human follicular membrane indicates that insulin can directly mediate the physiological effects induced by follicular membrane cells. Insulin can directly increase the secretion of androstenedione in theca cells. Previous studies have shown that the interaction between insulin and luteinizing hormone upregulates the expression of StAR and CYP17A1 genes, and then increases androgen levels. In addition, increased insulin can reduce SHBG synthesis, thereby reducing its binding with testosterone, leading to hyperandrogenemia. Studies have shown that physiological concentration of testosterone can promote insulin secretion and Ca2 + uptake through membrane edema mechanism. Graham et al. proposed a mathematical model of menstrual cycle. The model showed that with the increase of insulin-mediated testosterone production, ovulation interruption increased, indicating that the role of insulin in ovulation dysfunction is usually related to the increase of ovarian androgen production.
Our research also proved that insulin levels and anti-Mullerian hormone (AMH) levels were significantly positively correlated. The relationship between insulin levels and AMH levels in PCOS patients is controversial. A series of studies have found that compared with PCOS patients without insulin resistance (IR), the concentration of AMH in PCOS patients with IR is significantly higher, The serum AMH is positively correlated with HOMA-IR, which is consistent with our results. However, other studies have not found a link between AMH and IR[51, 52]. It has also been reported that there is a negative correlation between AMH and HOMA-IR. These contradictory data may be partly caused by the heterogeneity of the study population.
Our study has several limitations. Due to its cross-sectional design, the causal relationship between HSP70 and Treg/Th17 is unclear. In addition, due to the limitation of sample collection, the small number of patients limits the statistical power and the generalizability of our findings. Besides, both insulin and glucose are not tested under fasting conditions, thus the judgment of insulin resistance is not clear. The relationship between HSP70 and Treg/Th17 ratio still needs further research. Although this study confirmed that the upregulation of HSP70 expression was negatively correlated with Th17/Treg ratio, it is still necessary to study the specific regulatory mechanism of molecular biology to provide new targets for disease diagnosis and treatment.