CD4+/CD8+ mucosa-associated invariant T cells foster the development of endometriosis: a pilot study
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Immune dysregulation is one of the mechanisms to promote endometriosis (EMS). Various T cell subpopulations have been reported to play different roles in the development of EMS. The mucosa-associated invariant T cell (MAIT) is an important T cell subset in the pathogenesis of various autoimmune diseases. Evidence has indicated that there are three functionally distinct MAIT subsets: CD4+, CD8+ and CD4/CD8−/− (double negative, DN) MAIT cells. Till now, the associations between endometriosis and MAIT have not been studied. Our research investigates different MAIT subpopulations in peripheral blood (PB) and peritoneal fluid (PF) from EMS patients.
Thirty-two EMS patients and eighteen controls were included. PB and PF were collected. Tests of cytokines in plasma and PF were performed by ELISA kit. Characterisations of MAIT were done by flow cytometry. MAIT cells have been defined as CD3 + CD161 + Vα7.2+ cells. Based on CD4 and CD8 expression, they were divided into CD8+MAIT, CD4+MAIT and DN MAIT.
Enrichments of MAIT cells, especially CD4 and CD8 MAIT subsets were found. Moreover, CD8 MAIT cells had a high activation in the EMS group. EMS patients produced higher level of IL-8/12/17 as compared to these from controls. On the contrary, control patients exhibited an impressive upregulation of DN MAIT cells, however, these DN MAIT cells from controls showed a higher expression of PD-1. Lastly, we performed the relevance analysis, and discovered that the accumulation of PB MAIT cells positively correlated with an elevated level of serum CA125 production in EMS group.
These results suggest that different MAIT subsets play distinct roles in the progression of endometriosis.
KeywordsEndometriosis Mucosa-associated invariant T (MAIT) cells IL-17 Peritoneal fluid
- MAIT cells
Mucosa-associated invariant T cells
Myeloid derived suppressor cells
Major histocompatibility complex class
MHC I-related protein
Natural killer cell
Peripheral blood mononuclear cells
Programmed cell death protein
revised American Society for Reproductive Medicine
T cell receptor
Regulatory T cells
Endometriosis (EMS) is a chronic disease which is characterised by the presence of endometrial cells outside the uterus [1, 2]. It affects up to 10% of women of reproductive age, who suffer symptoms, such as dysmenorrhea, chronic pelvic pain, pelvic inflammatory reactions as well as infertility [3, 4]. The pathogenesis of endometriosis has been studied for decades, however, a clear answer is still missing.
Many reports have indicated that the process of endometriosis is related to a dysregulation of the host immune and some researchers even consider EMS to be an autoimmune disorder [5, 6, 7, 8, 9, 10, 11, 12]. On one hand, some studies have been focused on downregulation of anti-endometrial implants cells, such as NK cells, CD4+/CD8+ T cells, B cells and so on [5, 6, 7, 8], on the other hand, some studies demonstrated that increased immunosuppressive cells could promote the progression of endometriosis, such as Tregs (regulatory T cells), TH2 (T helper) cells and even MDSCs (myeloid derived suppressor cells), which have been suggested recently to promote the implantation of endometrial tissue [13, 14]. All these evidences pointed to the fact that the impaired immune response exists in endometriosis.
Mucosal-associated invariant T (MAIT) cells are non-classical T lymphocytes characterised by a semi-invariant T cell receptor (TCR) which has been evolutionarily conserved [15, 16, 17, 18]. This TCR consists of a restricted α chain (Vα7.2-Jα33 in humans and Vα19-Jα33 in mice) and one of the several β chains [16, 17]. It has been known that MAIT cells take part in both innate and adaptive immune response and can be found in peripheral blood and other tissues [18, 19]. However, till now there is no study about the relationship between MAIT cells and EMS, even though other kinds of T cells have been fully studied, such as TH17, TH1, TH2 and so on [10, 12, 20, 21]. In humans, MAIT cells have been defined as CD3+CD161+Vα7.2+ cells. Based on CD4 and CD8 expression, MAIT cells can be divided into CD8+MAIT cells and CD4/CD8−/− (double negative, DN) MAIT cells which are the major population of MAIT cells, and CD4+MAIT cells which are only up to 2–11% of MAIT cells in human blood [18, 19]. MAIT cells have the ability to recognize microbial-derived vitamin B metabolites presented by the major histocompatibility complex class I (MHC I) - related protein 1 (MR1) and allow them to detect various strains of bacteria and yeasts in vitro and in vivo [22, 23]. Recently, multiple evidences showed that human MAIT cells with high expression of IL-18α could be activated by the pro-inflammatory cytokines IL-12 and IL-18 in a TCR independent manner [24, 25, 26]. Once activated, MAIT cells show cytotoxic properties, and secrete pro-inflammatory cytokines such as IL-17 and IFN-γ [23, 27]. It has been reported that both blood and peritoneal fluid from endometriosis patients are rich in IL12 and IL18 . Therefore, we have the hypothesis that MAIT cells can be activated in endometriosis patients.
Next to the role of MAIT cells in mediating anti-microbial defenses, they have been implicated in the development of autoimmune and immunological diseases (multiple sclerosis, inflammatory bowel disease and inflammatory arthritis) [26, 29, 30, 31]. This suggests that MAIT cells may play a role in the manifestation of inflammatory responses in the absence of infection. Due to the functions of MAIT cells, many studies about different types of cancer suggested that MAIT cells have a great ability to destroy cancer cells [32, 33, 34]. However, some recent researches revealed that MAIT cells showed a potential to promote the progression of tumor by enhancing an immunosuppressive microenvironment which is induced by the cytokines produced by MAIT cells [35, 36]. For instance, it has been indicated that MAIT cells have the ability to facilitate the recruitment of MDSCs . Since endometriosis has been reported to be a chronic inflammatory disease with malignant activities, what kind of role will MAIT cells play in endometriosis? Will they promote or prevent the development of endometriosis? Our study aims to assess the immune disorder of endometriosis by analyzing MAIT cell subpopulations and cytokine levels in the PF and PB from patients with endometriosis and controls. The present study reveals the relation between MAIT cells and endometriosis, thus finds out dysregulation of MAIT cells might be related to the immune disorder of endometriosis.
Characteristics of EMS and control patients
EMS stage, n (%)
Ovarian benign cysts
32.6 ± 1.10
33.8 ± 1.53
31.3 ± 1.58
33.3 ± 1.23
27.6 ± 0.63
27.4 ± 0.88
27.9 ± 0.92
27.8 ± 0.76
83.0 ± 10.2
72.4 ± 14.9
95.1 ± 13.7
9.48 ± 0.86
a) Age at the sample collecting; b) CA125 before surgery; NS Not statistically significant
Heparinised PB samples from all patients were taken in a sterile condition and centrifugated with the density gradient centrifugation by Biocoll (Biochrom, Berlin, Germany). The procedure was conducted according to the manufacturer’s instructions. Peripheral blood mononuclear cells (PBMCs) and plasma were collected. Clear PF samples were centrifuged with a speed of 2000 rpm for 10 min. PF supernatant and plasma was stored at − 80 °C. The PBMC and cell pellets from PF were counted using a Neubauer counting chamber and adjusted to 107 cells. All cell samples were cryopreserved in the medium containing X-VIVO supplemented with 30% human serum and 10% DMSO at − 80 °C. All samples were stored until needed for analysis avoiding freeze-thaw cycles.
Flow cytometry analysis
The staining procedure was the same as described before. In short, after thawing, samples were treated with FcR Blocking Reagent (Miltenyi Biotech, Germany) and stained accordingly with human anti-bodies (mAbs). They were anti-CD8-PerCP-Cy5.5, anti-CD3-APC-Cy7 (SP34–2), anti-CD4-FITC, anti-Vα 7.2-PE, anti-CD161-APC, anti-CD38-PE-Cy7, anti-CD279-PE-Cy7 and all of them were from BD Biosciences (Heidelberg, Germany). Then samples were washed. Acquisition was carried out by six-color flow cytometry using FACSVerse™ flow cytometry (BD Biosciences) with FACSuite software (BD Biosciences). Analyses of the data were made by FlowJo software (Tree Star, Ashland, OR, USA).
Cytokines measurements in plasma and PF from patients by ELISA
IL-8, 12, 18, 17, MMP-9, INF-γ from the peritoneal fluid and plasma were analysed by ELISA kit (Multisciences Biotech, Hangzhou, China). The procedure was performed as the manufacturer indicated. Briefly, a polystyrene microplate of 96 well pre-coated with monoclonal antibody specific for each cytokine was used for each test. After final staining and washing, the optical density was determined.
GraphPad Prism software (GraphPad Software, San Diego, USA) was used for statistical analysis. A one-way ANOVA test and an unpaired Student’s t test were performed respectively for multiple groups’ results or two groups’ results. Spearman analysis was performed for correlation test. Correlation coefficient is presented as r. A p value less than 0.05 was considered statistical difference.
Presence of MAIT cells in PB and PF from patients
Enrichment of the cytokines IL-8/12/17 in EMS patients
Enhancement of different MAIT cells in EMS patients and controls
Frequencies of MAIT subpopulations in PB and PF
2.45 ± 0.34
1.42 ± 0.19
1.03 ± 0.09
2.50 ± 0.33
1.70 ± 0.24
1.29 ± 0.13
2.77 ± 0.42
3.39 ± 0.56
0.30 ± 0.05
< 0.0001b, c
6.52 ± 1.05
1.73 ± 0.22
0.67 ± 0.09
< 0.0001a, b
Functional activation of MAIT cells from EMS patients and rapid consumption of MAIT cells in controls
The correlation between different MAIT cells and related cytokines and CA125 in EMS patients
Endometriosis has been extensively studied for decades, however the clear mechanisms for its pathogenesis are still poorly understood, due to the complexity of its initiation and process. One of the most known hypotheses is the retrograde menstruation theory of Sampson . His theory indicated that endometrial cells (epithelium and/or glands) flowed through the fallopian tubes to the pelvis, and then implanted on other organs in the pelvis or even outside pelvis . The haze is why only 10% of women have endometriosis, while retrograde menstrual flow happens to a high number of women [9, 10]. One explanation could be the microenvironment, especially the immunological and inflammatory factors.
An area of great interest in endometriosis is soaring in immunological activity and its function in development of this condition [6, 9, 10, 12]. The endometriosis-associated immunological reactions were well reported in previous studies indicating the abnormalities in the frequency and function of T cells and their associated cytokines [9, 10, 20]. Our study indicates that alterations in circulating and local MAIT cells may be one mechanism which causes immunological disorder in endometriosis.
MAIT cells are abundant in the host immune system [17, 41]. As the name implies, they reside in the mucosa, but they are also found in other organs or tissues such as the peripheral blood, lymphoid tissues, lung and liver [23, 30]. We are the first study to analyse them in the peritoneal fluid. Firstly, we found that MAIT cells also exist in the peritoneal fluid. They are CD8+CD4−MAIT cells, CD8−CD4−MAIT cells, and CD8−CD4+MAIT cells. In accordance with other studies, our results showed that for endometriosis patients in both PB and PF the CD8 MAIT cells were the major subpopulation and CD4 MAIT cells were the minority, with DN MAIT cells in between (Fig. 1 and Table 2). As IL-17 producing cells, MAIT cells have been reported to have a similar function of TH17 cells [23, 30]. Multiple studies have discovered that TH17 cells are enriched in EMS patients and play a critical role in the progression of EMS [10, 20, 42]. Therefore, we assumed that augment of MAIT cells could also contribute to the pathogenesis of EMS, and our results showed that EMS patients had increased frequencies of MAIT cells in PB and PF. Furthermore, we found that PF IL-17 was higher in EMS patients as compared to controls, which was identical to other reports .
MAIT cells and their ability of IL-17 production were previously studied in many autoimmune diseases and immunological disorder, such as multiple sclerosis, inflammatory arthritis, Type 1 diabetes, primary Sjogren’s syndrome and so on [26, 27, 30]. It was well studied that IL-17 was increased and took part in the pathological process in the above-mentioned diseases [30, 31]. Meanwhile, some researches also suggested that MAIT cells were equipped to launch TH17-skewed immune reactions . Taken together, MAIT, TH17 and IL-17 could initiate a pro-inflammatory condition and even induce an immunosuppressive microenvironment. Interestingly, Rudak and his colleagues proposed that MAIT cells may promote the tumor development in tumorigenesis due to their potency of inducing MDSCs . However, the relation between these cells needs further investigation. Moreover, our findings revealed that PF samples from EMS patients had elevated levels of IL-8 and IL-12 (Fig. 2). These results give further evidences that MAIT cells can be activated and function in endometriosis. Unfortunately, we could not find any association between measured cytokines and the frequencies of MAIT cells. Although, we discovered that in peripheral blood there was a positive relation between frequencies of MAIT cells and the level of serum CA125. Another study also found that TH17 cells positively correlated with serum CA125 . This result might give a further hint that increased levels of MAIT cells are associated with the severity of endometriosis.
As mentioned before, there are three subpopulations of MAIT cells, and they were reported to have different functions and participate in various diseases [43, 44, 45]. The present study showed an interesting outcome. In our study, we found increased PF CD4 and CD8 MAIT cells in EMS group and highly activated CD8 MAIT cells in EMS group and early EMS group. In contrast, there was an elevation of DN MAIT cells within control group as compared to EMS patients, both early and late stage groups. One recent study by Dias et al. indicated that DN MAIT cells might derive from CD8 MAIT subset, but with different functions. In their study, they pointed out that DN MAIT cells had less cytolytic effect and were more prone to apoptosis . Similar to their study, there was a positive relation between CD8 MAIT cells and DN MAIT cells. We also found that DN MAIT cells had a high expression of PD-1 in control group. Meanwhile, these two subsets of MAIT cells play opposite roles in endometriosis. One explanation would be that DN MAIT cells function as guardians in the immune system, and unlike the two other subsets, they exhaust quickly without producing overloaded pro-inflammatory cytokines. However, further researches about these mechanisms are needed.
Our study revealed the role of MAIT cells in endometriosis and different profiles of their three subpopulations (i.e. CD8 MAIT, CD4 MAIT and DN MAIT cells). The outcomes of our research have identified that the disorder of MAIT cells might contribute to the immune dysregulation of endometriosis patients. CD4 and CD8 MAIT cells could be drivers in the development of in endometriosis, whereas DN MAIT cells might be protectors for the host. Therefore, manipulation of these cells might open new therapeutic strategies in the future.
The authors acknowledge Nicky Werner, an English teacher in Anhui Medical University, for proof reading the article. We would like to thank the doctors and nurses who performed the surgeries for our patients.
HJ and YC participated in study design and manuscript drafting; CL and ZL collected the samples and performed the main experiments. KB, KW, PG and YX helped to collect the clinical data and flow cytometry experiments; YC, PZ and ZW helped with the sample preparations. All authors read and approved the final manuscript.
This work was supported by the National Natural Science funds of China for Young Scholar [grant number 81701421] and the Province Natural Science funds of Anhui [grant number 1808085QH273].
Ethics approval and consent to participate
This study was approved by the Ethics Review Board of the First Affiliated Hospital of Anhui Medical University, China (No. 20170026). Written informed consent was signed by all patients.
Consent for publication
The authors declare that they have no competing interests.
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