Impaired CD8+ Mucosal-Associated Invariant T Cells and Myeloid-Derived Suppressor Cells Promote The Development of Polycystic Ovary Syndrome

Background: Immune dysfunction is one of the mechanisms to promote polycystic ovary syndrome (PCOS). Various immune cells have been reported to be involved in the development of PCOS. Meanwhile, the disturbance of metabolism is closely related to PCOS. This study is to explore the effect of mucosal-associated invariant T cells and myeloid-derived suppressor cells on the pathogenesis and the metabolic dysfunction of PCOS patients. Methods: 68 PCOS patients and 20 controls were recruited in this study and we collected the peripheral blood of participants’ during their follicular phase. The frequencies of MAIT cells and MDSCs were determined by ow cytometry after being stained with different monoclonal antibodies. And the concentrations of cytokines were determined by ELISA. Results: Compared to control group, the frequency of MDSCs, CD8 + MAIT cells and CD38 + CD8 + MAIT cells were signicantly decreased of PCOS patients with normal metabolism, however, proportion of CD4 + MAIT cells exhibited a noticeable increase. PCOS patients with abnormal weight showed a lower level and activation of CD8 + MAIT cells. On the contrary, they displayed an enrichment of CD4 + MAIT cells. PCOS patients with glucose metabolic disorder displayed a remarkable dysregulation of MDSCs and Mo-MDSCs. MDSCs were positively correlated with MAIT cells. Negative correlations between the frequency of CD8 + MAIT cells, CD38 + CD8 + MAIT cells and body mass index were revealed. CD4 + MAIT cell was positively correlated with BMI. Mo-MDSCs were found to be negatively related to the levels of 2hour plasma glucose and HOMA-IR index. Conclusion: The MAIT cells subpopulations and MDSCs played distinct roles in the etiology and metabolic disorders of PCOS. suppressor monocytic suppressor PMN-MDSC, suppressor


Introduction
Polycystic ovary syndrome (PCOS), which is one of the most prevalent endocrine diseases during reproductive age in female [1]. Approximately 8-13% women are affected by PCOS worldwide [2].
Patients with PCOS usually show typical clinical symptoms, including menstrual irregularity (oligomenorrhea or amenorrhea), hirsutism, acne and infertility. Nowadays, the metabolic dysfunction in PCOS have been payed close attention by physicians and researchers, because PCOS patients frequently diagnosed with diabetes/insulin resistance, obesity and other long-term health problem [3][4][5]. The causes of PCOS have been studied for decades, however, the pathogenesis of PCOS still remains unclear.
MAIT (Mucosal-associated invariant T) cells, one kind of unconventional T lymphocytes, which are characterized by a semi-invariant T cell receptor (TCR). A restricted α chain (Vα7.2-Jα33 in humans and Vα19-Jα33 in mice) and one of the several β chains make up the TCR, which has been revolutionaries conserved [12][13][14]. In human, MAIT cells have been de ned as CD3 + CD161 + Vα7.  [20]. Magalhaes and his colleagues reported abnormalities of circulating MAIT cells from T2D and obese patients [21,22]. These results revealed a close association between MAIT cells and disorders of metabolism. PCOS is considered to be related to metabolic irregularity, we hypothesize that MAIT cells are related to PCOS.
MDSCs (Myeloid-derived suppressor cells) have also been considered to have a vital role in the immunopathogenesis in many types of diseases [23,24]. MDSCs represent an intrinsic part of the

Sample collection
We collected peripheral blood from each participant in the morning during their menstrual day 2-3. According to the Biocoll (Biochrom, Berlin, Germany) protocol, we obtained the peripheral blood mononuclear cells with density gradient centrifugation. And we also collected plasma. The peripheral blood mononuclear cells and plasma cryopreserved in the cryopreservation medium at-80°C.

Flow cytometry analysis
Cells were treated with FcR Blocking Reagent (Biolegend, Germany) before stained with human antibodies. MAIT cells and MDSCs stained with conjugated antibodies. The conjugated antibodies and cell staining procedure were same with our previous study [27]. According to manufacturer's instructions, compensation control and cell acquisition was performed by FACSVerse and FACSuite software. Flow cytometry results were analyzed by FlowJo software (Tree Star, Ashland, OR).

Statistical analysis
We analyzed statistics by using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Statistical analyses of the differences between means in two groups were performed using unpaired, twotailed t-test. We performed Spearman analysis to nd the correlation. Correlation coe cients were presented as r. P-value < 0.05 was considered signi cantly different. Figure 1 showed the gating strategy of MAIT cells (Fig. 1A) and MDSCs (Fig. 1B) in peripheral blood.

Alteration of MDSCs, MAIT cells in PCOS patients with normal metabolism
MAIT cells were divided into DN, CD8 + and CD4 + three subpopulations. Finally, CD38 + cells were isolated. 3.3. Attenuation of MDSCs from PCOS patients with glucose metabolic disorder 32 patients with normal weight were divided into two groups, normal glucose (NG) subgroup and abnormal glucose (AG) subgroup. PCOS patients with a fasting glucose level less than 6.1mmol/L and a 2hour glucose level less than 7.8mmol/L were assigned to the NG group. The remaining patients were divided into the AG group. There were 23patients in the NG group and 9 patients in the AG group. Compared with NG group, AG group have decreased levels of MDSC (Fig. 4A, P < 0.05). And AG group have lower levels of Mo-MDSC, as compared to that from NG group (Fig. 4B, P < 0.05). Negative correlation between Mo-MDSC and the level of 2hour plasma glucose (Fig. 4C, r =-0.2453, P = 0.0438) as well as HOMA-IR index (Fig. 4D, r =-0.2427, P = 0.0461) were also found. For the frequencies of MAIT cells and PMN-MDSCs population, no signi cant differences between subgroups were found.

Discussion
In our study, the association between cytokines, MAIT cells and MDSCs were explored in PCOS patients.
In the last decades, abundant evidences have indicated that immune responses play an important role in PCOS, including the immunological activities of different T-cells, macrophages and associated in ammatory factors [6][7][8][9][10][11]. However, till now there is no study that showed investigation of MAIT cells and MDSCs in PCOS patients. In this study, we revealed that the disorders of circulatory MAIT cells as well as MDSCs might be an immunological pathogenesis for PCOS and was associated with metabolic dysfunction.
It has been reported that the abnormality of Th17 cells percentage was associated with PCOS [29]. Due to the same secretion of IL-17, MAIT cells have been reported to have a similar function of Th17 cells [30]. In this study, a decreased frequency of CD8 + MAIT cells in PCOS patients was revealed. Moreover, CD38 + CD8 + MAIT cells, known as activated MAIT cells, were also reduced. MAIT cells are divided into three subgroups, CD8 + MAIT cells, CD4 + MAIT cells, and CD8 − CD4 − MAIT cells [15]. Our results indicated that the impaired frequency and function of CD8 + MAIT cells might have a close link with PCOS. Early studies have suggested that CD8 + MAIT cells played a protective role in some diseases [31,32].
Consistent with previous studies, our study also revealed the protective role of CD8 + MAIT cells in PCOS.
Nonetheless, CD4 + MAIT cells displayed an expansion in PCOS. It has been known that the functional roles and frequencies of MAIT cells differ between different subsets and different diseases [33,34]. In this study, our results also support the distinct roles of CD8 + MAIT and CD4 + MAIT cells. Recently, PCOS was also recognized as a chronic in ammatory disease [35,36]. Therefore, our ndings would suggest that the protective role of CD8 + MAIT cells in PCOS are weakened, which is similar to the results from other chronic in ammatory diseases.
Two of the major problems from patients with PCOS are overweight and obesity. In our study, we found that with the increase of BMI, the frequencies of CD8 + MAIT cells and CD38 + CD8 + MAIT cells decreased.
Whereas, the proportion of CD4 + MAIT cells displayed increased levels. Previous researches have depicted that obese adults displayed a depletion of circulating MAIT cells [20,21]. Touch [11,39]. Based on the immunosuppressive properties of MDSCs and Tregs, the two populations are expected to share a similar role in different diseases [40,41]. Reduced immunosuppression is a key factor in the overreaction in in ammatory response. As mentioned previously, patients with PCOS have a strong chronic in ammatory background. Therefore, we suggested that an impaired immunosuppressive microenvironment with reduced MDSCs might be a cause of long-term chronic in ammation in PCOS.
Furthermore, our ndings also revealed that PCOS patients with disorders of glucose metabolism had a strong reduction of MDSCs and Mo-MDSCs. What's more, with the progression of glucose metabolic dysfunction, the frequencies of MDSCs and Mo-MDSCs decreased. The roles of MDSCs in metabolic diseases have been widely studied. Yin et al. have demonstrated that transferred MDSCs could downregulate autoimmune responses and prevent diabetes onset in mice model [42]. Researchers have previously clari ed that MDSCs could improve metabolic dysfunctions and act as a protector in obesity [43]. Combined with these results, our study indicated that a decreased frequency of MDSCs and Mo-MDSCs might induce the occurrence of metabolic dysfunction in PCOS patients.

Conclusion
The outcomes of our research gave a clue that the combined reduction of CD8 + MAIT cells and MDSCs, and increased population of CD4 + MAIT cells might contribute to the pathogenesis of PCOS and its metabolic disorders. We propose that the prominently reduced amount of CD8 + MAIT cells and MDSCs, shown in PCOS patients' PB, probably promote the chronic in ammation in PCOS immune microenvironment, therefore might promote the onset of PCOS and increase the severity of PCOS. The results suggested that inducing CD8 + MAIT cells and MDSCs might be a potential therapeutic target for PCOS patients. However, the limitation of this study is that we only analyzed the PB from PCOS patients, which might not present the immune status in ovary microenvironment. In-depth researches about the mechanisms of MAIT cells and MDSCs could lead to new therapeutic ideas.

Consent for publication
All participants volunteered to enroll in this study and signed a written consent inform.

Availability of data and materials
The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.