The role of selected cytokines from the interleukin-1 family in the peritoneal fluid of women with endometriosis

Endometriosis is an estrogen-dependent inflammatory gynecological disease characterized by the presence of endometrial tissue outside the uterine cavity. The most common location of endometrial lesions is the peritoneum of the sinus of Douglas and the vesico-uterine fold, the ovaries and fallopian tubes, the sacro-uterine ligaments and the outer membrane of the uterus and the rectovaginal septum [1]. More distant and unusual locations are also worth mentioning, such as the pericardium, lungs, pleura, diaphragm, brain, lower limbs, navel, as well as the caesarean section scar [2–4]. About 5–15% of women of reproductive age suffer from endometriosis, however, due to the non-specific symptoms and difficult diagnosis, the actual number of patients is unknown. The main clinical symptom of the disease is pain located in the pelvic area accompanied by prolonged and heavy menstruation. An important problem is infertility accompanying endometriosis, because 40–60% of affected women have problems with getting pregnant [5–7]. The classification created by the American Society for Reproductive Medicine (ASRM) determines the advancement of the disease, taking into account the extent and depth of endometriosis foci and the involvement of the peritoneum, fallopian tubes and ovary, as well as the presence and type of adhesions. ASRM has distinguished four stages of the disease, which are marked from I to IV [8, 9]. Despite numerous studies conducted over the years, the pathogenesis of endometriosis is still not fully understood. Even though there are many theories trying to explain the possibility of developing the disease, none of them fully explains the mechanisms involved in the formation and development of endometriosis. One of the oldest is Sampson’s theory from 1927 [10], which assumes that the source of cells may be endometrial fragments that move with the retrograde flow of menstrual blood through the fallopian tubes to the ovaries and peritoneal cavity, where they implant and then proliferate and vascularize. Many researchers are paying attention to the fact that an important element in the pathogenesis of endometriosis are disorders in the functioning of the immune system. In the early 1980s, Professor Dmowski [11] formulated the hypothesis that this disease occurs in women who have disorders in the functioning of the immune system. According to this researcher, a properly functioning system eliminates the ectopic endometrium, while because of its dysfunction, the environment of the peritoneal cavity favors the implantation of endometrial cells in ectopic sites. Endometriosis is similar to several autoimmune diseases, in the course of which there is an increased concentration of cytokines, disorders of apoptosis and angiogenesis, or increased activity of tissue metalloproteinases. Immunological factors and angiogenesis play a key role in the pathogenesis of endometriosis, which may affect a woman’s susceptibility to implantation of exfoliated endometrial cells [12, 13]. There are four stages in the development of endometriosis (Fig. 1). An important role in the pathogenesis of endometriosis is attributed to the interleukin-1 family. Interleukins belonging to the IL-1 family play an important role in the regulation of the immune and inflammatory response, affecting almost all types of cells [17]. The interleukin-1 family consists of 11 proteins, including 7 pro-inflammatory agonists (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ) and 4 anti-inflammatory antagonists (IL-1Ra, IL-36Ra, IL-37 and IL-38) [18, 19]. The characteristics of the interleukin-1 family are listed in Table 1 [16]. IL-1 family cytokines are released at the earliest stage of the immune response and act as a signal for the next cascade of pro-inflammatory cytokines [20]. The main cells secreting IL-1 are considered monocytes and macrophages, as well as T and B lymphocytes. It can also be secreted by organ-specific cells, such as keratinocytes, chondrocytes, dendritic cells, glial cells, mesangial cells, and endothelial cells [21]. Figure 2 show selected IL-1 family of cytokines. | Table 1. The characteristics of the interleukin-1 family [14] | ||| | Interleukin-1 family | Interleukin-1 family receptor | Interleukin-1 family coreceptor | Characteristics of the interleukin-1 family | | IL-1α | IL-1R1 | IL-1RacP(L-1R3) | Pro-inflammatory | | IL-1β | IL-1R1 | IL-1RacP(L-1R3) | Pro-inflammatory | | IL-1Ra | IL-1R1 | – | Antagonist for IL-1α, IL-1β | | IL-18 | IL-18Rα(IL-1R5) | IL-18R(βIL١R٧) | Pro-inflammatory | | IL-33 | IL-1R4ST2(IL-1R4 IL-1RL1, IL-33R) | IL-RacP | Pro-inflammatory | | IL-36Ra | IL-1Rrp2(IL-1R6) | IL-1RacP | Antagonist for IL-36α, IL-36β, IL-36γ | | IL-36α | IL-1Rrp2(IL-1R6, IL-36R) | IL-1RacP | Pro-inflammatory | | IL-36β | IL-1Rrp2(IL-1R6, IL-36R) | IL-1RacP | Pro-inflammatory | | IL-36γ | IL-1Rrp2(IL-1R6, IL-36R) | IL-1RacP | Pro-inflammatory | | IL-37 | IL-18Rα | IL-1R8(SIGIRR) | Anti-inflammatory | | IL-38 | IL-1R1 IL-1Rrp2 | IL1RAPL1(TIGIRR-2)IL1RAPL2(TIGIRR-1) | Partial antagonist for IL-1α and β and IL-36α, β and γ | Therefore, the aim of the conducted research was to understand the role of selected cytokines belonging to the IL-1 family, including IL-36α, IL-36β, IL-36γ, IL-36R, IL-37 and IL-38, in the onset and development of endometriosis by analyzing the concentration of the tested molecules and to determine whether their concentration depends on the stage of the disease.

The study group included 60 women aged between 21 and 49 years (mean age 32.6 ± 7.4 years) who had pelvic endometriosis diagnosed during laparoscopy and subsequently confirmed by histopathology. The research was conducted on women hospitalized at the Clinical Department of Gynecology and Obstetrics of the University Clinical Center in Katowice. Of the 60 women studied, 38 women were in the minimal/mild stage (stage I and II endometriosis) and 22 were in the moderate/severe stage (stage III and IV endometriosis). The reference group consisted of 20 women aged between 21 and 41 years (mean age 28.2 ± 5.6 years) who had no endometriosis or other pelvic lesions during laparoscopy. Inclusion criteria for both groups were at least 2 years primary infertility, stage from I to IV ovarian endometriosis, and regular cycles. Endometriosis was diagnosed by laparoscopy and histologically confirmed and classified according to the American Society of Reproductive Medicine classification. Peritoneal fluid collected during laparoscopy from women in the proliferative phase of the menstrual cycle was used for the study. Immediately after collection, the peritoneal fluid was centrifuged at an acceleration of 2500 rpm for 10 minutes. The resulting fluid was divided into aliquots and stored at –80°C until assays were performed. The concentration of cytokine in peritoneal fluid was determined by sandwich ELISA. The Enzyme-linked Immunosorbent Assay Kit for Interleukin 1 Epsilon (IL1e) was used to determine the concentration of the interleukin under study, Interleukin-36α. For IL-36β Enzyme-linked Immunosorbent Assay Kit for Interleukin 1 Eta (IL1h). IL-36γ Enzyme-linked Immunosorbent Assay Kit for Interleukin 1 Family, Member 9 (IL1F9). Determination of Interleukin-36 receptor (IL-36R) levels was performed using the Enzyme-linked Immunosorbent Assay Kit for Interleukin 1 Delta (FIL1d). The Enzyme-linked Immunosorbent Assay Kit for Interleukin 1 Zeta (IL1z) was used to determine the concentration of IL-37 under study. The Enzyme-linked Immunosorbent Assay Kit for Interleukin 1 Theta (IL1q) was used to determine the concentration of test IL-38 (Tab. 2). All assays were from Cloud-Clone Corp. All results are presented as amean ± standard deviation or median and interquartile range and were examined for normality of distribution by the Shapiro-Wilk test. Parametric data were analysed using Student’s t-test. For nonparametric data, Fisher’s exact test (analysis of variance) was applied to indicate statistical significance because it analyses the variance relationship both within and among the groups. The correlations were tested by Spearman’s rank correlation test and are presented as a correlation coefficient (r). P < 0.05 was considered statistically significant. | Table 2. A summary of the tests used in the study together with Sensitivity, Interlaboratory reproducibility, Intralaboratory reproducibility [Instruction ELISA CLOUD-CLONE] | |||| | Parameter | Test | Sensitivity | Intra-assay | Inter-assay | | Interleukin-36α | Interleukin 1 Epsilon (IL1e) | < 6.3 pg/mL | CV < 10% | CV < 12% | | Interleukin-36 β | Interleukin 1 Eta (IL1h) | < 2.9 pg/mL | CV < 10% | CV < 12% | | Interleukin IL -36γ | Member 9 (IL1F9) | < 6.5 pg/ml | CV < 10% | CV < 12% | | Interleukin-36 receptor (IL-36R) | Interleukin 1 Delta (FIL1d) | < 2.9 pg/mL | CV < 10% | CV < 12% | | Interleukin-37 | Assay Kit for Interleukin 1 Zeta (IL1z) | < 6.1 pg/mL | CV < 10% | CV < 12% | | Interleukin-38 | Interleukin 1 Theta (IL1q) | < 2.8 pg/mL | CV < 10% | CV < 12% |

The study analysed the concentrations of the following cytokines IL-36α, IL-36β, IL-36γ, IL-36R, IL -37 and IL-38 in the peritoneal fluid of women with endometriosis and women from the reference group. The results were presented in Table 3 and Figure 3–8. | Table 3. Peritoneal level of studied chemokines in women with endometriosis and women from the reference group | ||| | Cytokines | Patients | p value | | | Endometriosis (n = 60) | Control (n = 20) | || | Interleukin-36α | 256. 81 (153.48–358.25) | 91.57 (73.08–124.54) | p < 0.001 | | Interleukin-36β | 46.69 (52.62–81.56) | 23.90 (18.18 – 30.20) | p < 0.05 | | Interleukin-36γ | 88.82 (36.07–132.66) | 11.49 (6.05–14.52) | p < 0.001 | | Receptor IL-36 | 47.39 (42.50–61.16) | 32.35 (29.83–35.27) | p < 0.001 | | Interleukin-37 | 493.05 (314.44–595.84) | 137.82 (105.38– 178.80) | p < 0.001 | | Interleukin-38 | 147.97 (102.55–238.86) | 54.37 (40.90–74.87) | p < 0.01 | Interleukin-36α, 36β, 36γ and receptor IL-36 The analysis showed a statistically significant increase in the concentration of IL-36α, 36β, 36γ and IL-36 receptor in the peritoneal fluid of women with endometriosis compared to the concentration of this parameter in the reference group. In the next step, we evaluated how the concentrations of these parameters in the peritoneal fluid developed in relation to the stage of endometriosis. For each parameter, we observed a statistically significant increase in the concentration in the group of women with moderate/severe endometriosis compared to the group of women with minimal/mild endometriosis (p < 0.001). The results are shown in Figure 3–6. Interleukin -37 The analysis of the results showed a statistically significant increase in the concentration of IL-37 in the peritoneal fluid of women with endometriosis compared to the concentration of this parameter in the reference group (p < 0.001). A statistically significant increase in the concentration of the studied parameter was observed in endometriosis with moderate/severe stage compared to the group of women with minimal/mild stage of the disease (p < 0.001). The results obtained are shown in Figure 7. Interleukin -38 The analysis showed a statistically significant increase in IL-38 levels in the group of women with endometriosis compared to the levels of this cytokine in the fluid of women in the reference group (p < 0.01). When comparing women with moderate/severe endometriosis with those with minimal/mild endometriosis, a statistically significant increase in concentration was also observed (p < 0.001). The results are shown in Figure 8. In addition, we analysed whether there was a correlation between the concentrations of IL-36α, IL-36β, IL-36γ and IL — IL-38 showing pro-inflammatory effects and IL-37 with anti-inflammatory effects in the peritoneal fluid of women with endometriosis. The analysis showed a positive, statistically significant correlation between the concentrations of IL-36α (r = 0.8; p p < 0.0001), IL-36β, (r = 0.5; p < 0.0001) IL-36γ (r = 0.7; p < 0.0001), IL-38 (r = 0.8; p < 0.0001) which show pro-inflammatory effects and IL-37 with anti-inflammatory effects. Linear regression curves showing the above relationships are illustrated in Figure 9.

Numerous studies indicate that cytokines have a significant impact on the development, course and effects of endometriosis. The effect of cytokines is noticeable already at the stage of implantation of ectopic endometrial foci. An abnormal increase in the number of activated macrophages within endometriotic lesions has been indicated, affecting the extracellular environment. Abnormal cytokine production by macrophages leads to stimulation of both epithelial and stromal cells significantly influencing the severity of the inflammatory response. Consequently, the increased interaction of differentiated cellular components in the microenvironment can lead to a continuous stimulation of inflammation, promoting the growth of endometrial lesions through increased proliferation or reduced apoptosis. Many pieces of data confirm that cytokines contained in the peritoneal fluid of women with endometriosis stimulate the proliferation of eutopic and ectopic endometriotic lesions [23]. Studies have shown elevated expression of IL-36 in the female reproductive system during the menstrual cycle, as well as an association with immune mediator production and cell recruitment to the genital tract. The involvement of IL-36 cytokine in inflammation and the observations to date on their potential role in the development of healthy or complicated pregnancies require further research [24]. The balance and correlation between Th1 and Th2 type responses and their regulatory mechanisms, as well as their mediators, require further research to understand the functioning of the immune response during pregnancy. Information regarding the involvement of IL-36 in reproductive processes is still poorly understood, but further studies may in the future indicate how cytokines may participate in the regulation of pregnancy [24]. In the studies presented in this paper, the concentration of individual interleukins from the IL-1 family and IL-37, IL-38 in the peritoneal fluid of women with endometriosis, which is the environment for developing changes, was checked. The results of our study are comparable to those obtained by other authors. The tested material was peritoneal fluid, as was the case in previous studies. In each of the compared studies, the ELISA method was used, and the samples of the test material were stored in an analogous manner, and moreover, the size of the study groups in each analysis was approximate. We used the division of endometriosis into early-grade I and II and late-grade III and IV based on the rAFS system, which is an older ASRM classification system, which in turn was included in our studies [25, 26]. As a result of the tests carried out in the peritoneal fluid of women with endometriosis, changes in the concentrations of the tested interleukins: IL-36α, IL-36β, IL-36γ and IL-36R were found in comparison to the values observed in women from the reference group. Moreover, an increase in the concentration of these proteins was observed with the subsequent stages of endometriosis. The highest concentrations of IL-36α, IL-36β, IL-36γ and IL-36R were observed in women with stage IV endometriosis and the lowest in women with stage I endometriosis. IL-36 represents 3 cytokines (IL-36α, IL-36β, IL-36γ), each of which has, pro-inflammatory effects and binds to the IL-36 receptor (IL-36R). Abnormal levels of their expression have an impact on the development of inflammation, autoimmunity, allergies and cancer [27]. Studies indicate the involvement of interleukins in inflammation. Signalling which covers IL-36, induces pro-inflammatory responses in fibroblasts, endothelium, epithelium, M2 macrophages, T cells, natural killer cells and dendritic cells (DC) [28]. Disorders associated with the IL-36 subfamily are associated with the pathogenesis of many diseases including: rheumatoid arthritis, inflammatory lung diseases, obesity, biliary obstruction and chronic glomerulonephritis [29, 30]. Studies conducted so far have shown an increase in the concentration of this cytokine in the presented diseases, which underlines the importance of this protein in chronic inflammation [31–33]. In addition, IL-36R signalling is involved in diseases such as psoriasis and colitis, mediated by pro-inflammatory cytokines and chemokines. The level of IL-36 in the peritoneal fluid of women with endometriosis was analyzed by Heeyon Kim et al. [32] who showed that the concentration of this protein was significantly increased in the group of women with severe endometriosis. There are no unequivocal reports indicating the involvement of IL-36α, IL-36β, IL-36γ and IL-36R in this disease, however, studies covering other disease entities are available. Studies by Wang et al. [34] indicate the participation of IL-36γ, (an IL-36R agonist) in the proliferation and progression of lung cancer [34]. Similar conclusions were drawn by Yang et al. [35] indicating a strong association between IL-36γ and IL-36Ra with the development of intestinal inflammation and the development of intestinal cancer [35]. This is consistent with our study, which showed an increase in IL-36γ and IL-36R in women with endometriosis accompanied by a chronic inflammation. Moreover, these concentrations correlated with the progression of the disease, the highest concentration was observed in women with stage IV endometriosis and the lowest in women with stage I endometriosis. The concentration of IL-36α was higher in women with late endometriosis than in women with early endometriosis, which may reflect the increased production of this cytokine as the disease progresses [31, 33]. Li et al. [36] indicate a new mechanism of action of pro-inflammatory IL-36α with suppressed IL-36RA and IL-38 antagonists, thanks to which hyperosmolarity induces inflammation in a culture model and dry eye disease [36]. IL-36β is produced by the innate immune system cells and lymphocytes, inducing the production of pro-inflammatory cytokines, chemokines and co-stimulatory molecules, thus promoting the polarization of Th1 and Th17 cells. There is evidence that IL-36β is involved in innate immunity and inflammation of the skin and lungs, where epithelial cells may be the source of this cytokine. IL-36β is involved in pathological states of min. psoriasis and A. fumigatus infection [31]. In our study, we have shown an increase in the level of IL-36β in the peritoneal fluid of women with endometriosis, which correlated with an increase in patients’ inflammation. IL-37 is a new member of the IL-1 family that can reduce, inhibit inflammation and also affect immunity. In addition, IL-37 can inhibit macrophage proliferation, apoptosis, migration and expression of pro-inflammatory cytokines such as IL-1β, IL-6, and IL-10, and inhibit the occurrence and development of endometriosis by targeting multiple signalling pathways. Recent studies indicated that IL-37 expression in ectopic and eutopic endometrium and serum levels in patients with endometriosis were higher than in patients without endometriosis. IL-37 expression correlates with disease severity, but discrepancies still exist regarding the concentration of this parameter in peritoneal fluid [37, 38]. The analysis showed a statistically significant change in the concentration of IL-37 in the peritoneal fluid in women with endometriosis compared to women from the reference group.However, the mechanism of action of IL-37 is not fully known.Despite the ethnic differences in the women included in the study, the results of studies by Fan et al. [39] and Jiang et al. [40] also showed an increase in the concentration of IL-37 in the peritoneal fluid in women with endometriosis, which indicates a significant participation of this interleukin in the inflammatory processes associated with endometriosis. Interleukin 37 has been identified as a natural suppressor of the innate inflammatory response. IL-1β can increase IL-37 expression, the most effective stimulant identified so far is transforming growth factor β (TGFβ ), which is produced by peritoneal mesothelial cells and increased in areas close to endometriosis lesions. All studies conducted so far have shown higher concentrations of IL-37 in women with late endometriosis (stage III and IV) than in women with early endometriosis, suggesting increased secretion of IL-37 in more advanced stages of the disease. Considering that IL-37 is an anti-inflammatory cytokine, its increased expression in women with endometriosis is surprising. However, it is probably related to the inhibition of early pathogenic clearance of foreign tissue and facilitates lesion formation. According to the researchers, the obtained results indicate the use of IL-37 as a potential diagnostic tool in endometriosis [39, 40]. In addition, Fan et al. [39] suggested that IL-37 should also be considered in future studies using different estrogen concentrations, given that endometriosis is a hormone-dependent disease. Therefore, it seems important to direct research to clarify the mechanism by which IL-37 affects the eutopic and ectopic endometrium [39]. Another evaluated cytokine parameter was IL-38. Its concentration in the peritoneal fluid of women with endometriosis was significantly increased compared to the concentration of this cytokine in the fluid of women from the reference group. An increase in concentration was also observed in stage III–IV of the disease compared to stages I–II. IL-38 is highly expressed in immune cells and plays a key role in autoimmune diseases, but the exact signalling and functional pathway is still poorly known. In the available literature, there are few studies on the occurrence of this cytokine in women with endometriosis. Interleukin-38 (IL-38) shares structural features with the IL-1 receptor antagonist (IL-1Ra) and IL-36Ra. IL-36R is a specific IL-38 receptor, a partial antagonist of the IL-36 receptor. IL-38 inhibits the production of T cell cytokines IL-17 and IL-22. IL-38 also inhibits the production of IL-8 induced by IL-36γ, thereby suppressing inflammatory responses. IL-38-related cytokines, including IL-1Ra and IL-36Ra, are involved in the regulation of inflammation and the immune response. Study of cytokines related to IL-36 and IL-38 may provide new information to help develop anti-inflammatory therapies [41]. In addition, the study showed a positive correlation between the levels of the pro-inflammatory interleukins: IL-36α, IL-36β, IL-36γ and IL-38, and the anti-inflammatory interleukin IL-37. The demonstrated correlation between the concentrations of these parameters in a group of patients with endometriosis indicates a disturbance of the immune system taking place with the involvement of the IL-1 family of cytokine systems studied and may suggest that these proteins may directly or indirectly participate in increasing inflammation in the formation and development of the disease. The interactions taking place in the microenvironment of the peritoneal fluid may influence the direction of biological action, which is related to the severity of internalisation abnormalities in the pro/anti-inflammatory cytokine system. Understanding these mechanisms may allow us to find potential biomarkers useful for the diagnosis of endometriosis in the future, but this requires further research. Furthermore, it is also noteworthy that changes in the concentrations of the analysed cytokines promote chronic inflammation, lesion proliferation, local hormonal imbalance, and may result in poorer oocyte quality, poor sperm motility, embryotoxicity and reduced endometrial receptivity, suggesting adverse effects on the reproductive capacity of patients. In this aspect, only increased levels of IL-1β in the peritoneal fluid have so far been indicated in women with endometriosis and associated infertility. It also seems interesting to know the role of other cytokines belonging to the IL-1 family in these patients.

Disturbances of the immune system involving the network of cytokines belonging to the IL-1 family occurring in the peritoneal fluid environment testify to the involvement of these molecules in the development of the disease and are one of many factors involved in the pathogenesis of endometriosis. The use of some of them in the treatment of endometriosis may be a hope for effective causal treatment of this disease, but this requires further, more advanced research. Article information and declarations Author contributions Conceptualization — AMP, MSK; AW — methodology; DW, MSK — writin, original draft preparation; AMP, MSK, AW — writing, review andediting; AMP, MSK — visualization, MSK, DW, AMP — supervision;. All authors have read and agreed to the published version ofthe manuscript. Funding This research was funded by Medical University of Silesia in Katowice, Poland, grant number BNW-1-025/K/3/O. Acknowledgments Bioethics Committee of the Medical University of Silesia in Katowice (decision no. KNW/0022/KB/35/15). Conflict of interest The authors declare no conflict of interest.