Clinical significance of the CXCL6-CXCR1/CXCR2 chemokine signaling pathway in the peritoneal fluid of patients with endometriosis

Endometriosis is an estrogen-dependent, chronic inflammatory disease in which endometrial tissue is present outside the uterine cavity. It affects 10 to 15% of women of reproductive age but may also occur in postmenopausal patients [1]. It is a significant clinical problem, as it causes infertility in 35 to 50% of cases and causes intense pain [2, 3]. It is worth noting that despite many years of research on this disease, its pathogenesis and etiology have still not been fully explained. This disease may occur in the form of superficial endometrial foci on the peritoneum, endometrial cysts of the ovary or as deep endometriosis (DE). Over the years, numerous theories have been developed to explain the occurrence of ectopic endometrial foci, but few of them fully explain the development of all types of endometrioses [4]. Many researchers point out that an important element in the pathogenesis of endometriosis is the dysfunction of the immune system [5, 6]. Disturbances in the immune system within the peritoneal cavity led to an impaired immune response to endometrial cells, and the effect of these changes is the survival of endometrial cells, their implantation and development into foci of endometriosis [5, 7]. Moreover, it has been shown that there are interactions between immune cells and endometrial cells in the peritoneal cavity (Fig. 1), in which the primary role is played by immune system mediators — cytokines [8]. Chemokines are responsible for the influx of cells into the peritoneal cavity during endometriosis, the primary role of which is cell chemotaxis [9]. Studies conducted so far indicate that chemokines may play an important role in the pathogenesis of endometriosis and are considered as potential therapeutic targets. Among the chemokine/receptor signaling axes, researchers also take into consideration the important role of the CXC/CXCR axis [10]. Among those better known, the CXCL8/CXCR-1/CXCR-2 axis stands out. This signaling axis participates in the influx of neutrophils, monocytes and T lymphocytes into the peritoneal cavity, intensifying the ongoing inflammatory process [10]. Moreover, studies indicate that CXCL8 increases the proliferation of endometrial cells and inhibits the apoptosis process in an autocrine way [11]. An important role in the development of the disease is attributed to the CXCL5/CXCR-2 axis. It has been shown that it increases the influx of neutrophils into the peritoneal cavity, promotes the process of angiogenesis and participates in the remodeling of connective tissue [12]. Potential clinical significance is also indicated in the case of the CXCL12/CXCR-4 axis [10]. So far, the role of the CXCL6/CXCR-1/CXCR-2 axis in the pathogenesis of endometriosis is not fully known. The chemokine CXCL6 (GCP-2, granulocyte chemotactic protein 2) is a chemoattractant for neutrophils. Moreover, the presence of the glutamate–leucine–arginine (ELR) motif indicates its participation in the process of angiogenesis [13]. Its receptors are CXCR-1 and CXCR-2 (Fig. 2). Despite the demonstrated role of the chemokine CXCL6 in inflammatory processes, the participation of the CXCL6/CXCR-1/CXCR-2 signaling axis in the pathogenesis of endometriosis is not fully understood. Therefore, the aim of the present study was to evaluate the CXCL6/CXCR-1/CXCR-2 signaling axis in the peritoneal fluid of patients with endometriosis.

The study group included 54 women aged between 21 and 49 years (mean age 32.6 ± 7.4 years) who were diagnosed with pelvic endometriosis during laparoscopy and later confirmed by histopathology. The study was conducted on women hospitalized at the department of Gynecology and Obstetrics, Faculty of Medical Sciences in Katowice, Medical University of Silesia in Katowice. Out of 54 women studied, 15 women were in the I stage, 15 women in the II stage, 15 women in the III stage and 9 in the IV stage of endometriosis. The inclusion criteria for both groups were at least 2 years of primary infertility, stage of ovarian endometriosis from I to IV, and regular cycles. Endometriosis was diagnosed by laparoscopy and histologically confirmed and classified according to the American Society of Reproductive Medicine classification. The control group consisted of 16 women aged between 21 and 42 years (mean age 26.8 ± 5.5 years) who were not diagnosed with endometriosis or other lesions during laparoscopy. Peritoneal fluid collected during laparoscopy from women in the proliferative phase of the menstrual cycle was used for the study. The collected peritoneal fluid was then centrifuged at an acceleration of 2500 rpm for 10 minutes. The resulting fluid was divided into aliquots and stored at −80°C until the assays were performed. The concentration of the cytokine in peritoneal fluid was determined by sandwich ELISA. For this purpose, the Enzyme-linked Immunosorbent Assay Kit for Granulocyte Chemotactic Protein 2 (GCP2)® from Cloud-Clone Corp. was used to determine CXCL6 concentrations. The following assays were used to determine CXCR-1 and CXCR-2 receptor concentrations: Enzyme-linked Immunosorbent Assay Kit for Interleukin 8 Receptor Alpha (IL8Ra) ® from Cloud-Clone Corp. (CXCR-1) and Enzyme-linked Immunosorbent Assay Kit for Interleukin 8 Receptor Beta (IL8Rb)® from Cloud-Clone Corp. (CXCR-2). The results obtained were statistically analysed using Statistica version 13.3. In the first step, the normality of the distribution was assessed using the Shapiro–Wilk test. The Mann–Whitney U test was used to compare the concentrations of CXCL6, CXCR-1 and CXCR-2 between the study and control groups, and the Kruskal–Wallis ANOVA with multiple comparisons was used to assess the concentrations of the analysed parameters in terms of disease severity. Pearson’s correlation coefficient was used to determine the correlation between the chemokine and its receptors. The values of p < 0.05 were taken as the level of statistical significance.

The study analysed the concentrations of the following chemokines: CXCL6 and its receptors CXCR-1 i CXCR-2 in the peritoneal fluid of women with endometriosis and women from the reference control group. The results are presented in Table 1 and Figure 3. | Table 1. Peritoneal level of studied chemokines in women with endometriosis and women from the control group | ||| | Chemokines | Patients | p value | | | Endometriosis (n = 54) | Control (n = 16) | || | CXCL6 [pg/mL] | 141.94 (39.58–291.14) | 17.88 (13.54–23.10) | < 0.001 | | CXCR-1 [ng/mL] | 3.61 (1.49–6.52) | 1.92 (1.07–2.29) | < 0.05 | | CXCR-2 [ng/mL] | 2.30 (0.68–6.35) | 0.65 (0.23–1.15) | < 0.01 | Next, we assessed how the concentration of the chemokine CXCL6 and its receptors in the peritoneal fluid of the study group evolved in successive stages of endometriosis (Fig. 4). The analysis showed significant differences in CXCL6 levels between stage I and stages II (p < 0.05), III (p < 0.001) and IV (p < 0.001). Statistically significant differences were also found between grades II and IV (p < 0.01). No significant differences in CXCL6 levels were observed between grades II and III or grades III and IV. Significant differences in CXCR-1 concentrations were found between stage I and stages II (p < 0.05), III (p < 0.01) and IV (p < 0.01). The analysis also showed significant differences in concentration between grades II and IV (p < 0.05). No statistically significant differences in CXCR-1 concentration were observed between stage III and stage IV. The analysis showed statistically significant differences in CXCR-2 concentration between stage I and stage III (p < 0.05) and stage IV (p < 0.01). In addition, significant differences in concentration were observed between stage II, stage III (p < 0.05) and stage IV (p < 0.001). There were no significant differences in CXCR-2 concentration between stage I and stage III or between stage III and IV. In addition, we analysed whether there was a correlation between the concentrations of CXCL6 and the CXCR-1 I CXCR-2 receptors. Linear regression curves showing the above relationships are illustrated in Figure 5. The analysis showed a statistically significant positive correlation between CXCL6 levels and CXCR-1 receptor levels (p < 0.001; r = 0.6659) and between CXCL6 and CXCR-2 receptor levels (p < 0.001; r = 0.7525). ROC curves In the next step of the analysis, receiver operating characteristic (ROC) curves were plotted for all three parameters (Fig. 6). The following concentration values were taken as the dilution points for CXCL6: 0,042 pg/mL; for CXCR-1: 0.061 ng/mL; and for CXCR-2: 0.058 ng/mL. The area under the ROC curve (AUC) value amounts to for the chemokine CXCL6: 0.868; for CXCR-1: 0.771 and for CXCR-2: 0.759.

Due to its functions, the chemokine CXCL6 is considered involved in both physiological and pathological processes. So far, there have been few studies on the evaluation of the chemokine CXCL6 and its receptors in gynecological disorders. One of them was the analysis conducted by Mittal et al. [14], which showed that CXCL6 is a physiological element in the composition of amniotic fluid. In addition, the potential participation of CXCL6 in the development of intrauterine inflammation was demonstrated. The study also indicated an important role of chemokines during normal and abnormal labor. It is believed that GCP-2 is also involved in other clinical syndromes associated with inflammation [15]. Despite the proven role of the chemokine CXCL6 in inflammatory processes, the role of the CXCL6/CXCR-1/CXCR-2 signaling axis in the pathogenesis of endometriosis is not fully understood. The aim of this study was to assess the CXCL6/CXCR-1/CXCR-2 signaling axis in the peritoneal fluid of patients with endometriosis. The conducted studies assessed the concentration of the chemokine CXCL6 and its receptors in the peritoneal fluid of women with endometriosis and women from the control group. In addition, the concentrations of the tested parameters were determined in terms of the stage of disease progression determined by the American Society for Reproductive Medicine (ASRM) classification. The statistical analysis showed that the concentration of GCP-2 in the peritoneal fluid of women with endometriosis was significantly increased compared to the concentration in women from the control group (p < 0.001). Moreover, it was shown that the concentration of CXCL6 increased in the subsequent stages of the disease. Statistically significant differences were observed between stage I and stage II, III and IV (p < 0.01; p < 0.001; p < 0.001, respectively), indicating the increased secretion of the chemokine CXCL6 with diseases progression which probably occurs due to the increased influx of inflammatory cells into the peritoneal cavity. Moreover, in the advanced stages of the disease, there is impaired control over the activity of the chemokine CXCL6, which may lead to the intensification of the inflammation in the peritoneal cavity. Moreover, the increased secretion of CXCL6 in stages III and IV may indicate the participation of the chemokine in the process of growth of endometriosis foci including the induction of the angiogenesis process. Similar studies were conducted by Suzumori et al. [16], who assessed the concentration of GCP-2 in the peritoneal fluid of women with endometriosis. The concentration of CXCL6 was compared between the group of women with endometriosis and the control group. The analysis also considered the phase of the menstrual cycle and the stage of disease progression according to the ASRM classification. It was shown that in the peritoneal fluid of women with endometriosis, in the proliferative phase of the menstrual cycle, the concentration of GCP-2 was significantly increased compared to the concentration in the control group. It was shown that the level of CXCL6 in this group positively correlated with the stage of disease severity. Mine et al. [17] assessed the ability of endometrial cells to secrete GCP-2 under the influence of and without stimulation by various inflammatory mediators. The cells were cultured in the presence of interleukin-1 alpha (IL-1α) and beta (IL-1β), tumor necrosis factor-alpha (TNF-α), tumor necrosis factor-beta (TNF-β), interferon gamma (IFN-γ) and lipopolysaccharide (LPS). The assessment of CXCL6 secretion was performed by determining its concentration in the supernatant using ELISA. Studies showed that endometrial stromal cells had a small ability to secrete the chemokine CXCL6. Moreover, it was observed that IL-1α, IL-1β, TNF-α, TNF-β and LPS increased the secretion of CXCL6 by endometrial cells. Studies indicate that the ability of the endometrium to produce the chemokine CXCL6 may regulate the migration of neutrophils within the endometrium, which has a significant impact on both normal and pathological processes occurring within the uterine cavity. The ability of ectopic endometrial cells to express the chemokine CXCL6 was also analyzed by Zhu et al. [18], who used the microarray method to assess the expression profile of selected chemokines from the CXC family. The studies were performed using a 3D culture of cells forming endometrial cysts and a culture of normal endometrial cells. It was shown that in the culture of endometrial cells the expression of the chemokine CXCL6 was increased compared to the culture of normal cells. According to the researchers, the ectopic expression of the chemokine CXCL6 indicates its participation in the initiation of the inflammatory process during endometriosis. Tan et al. [19] assessed how hypoxic conditions affect the expression of proangiogenic factors in cultures of endometrial stromal cells that are part of ectopic endometriosis foci. The proangiogenic factors studied were vascular endothelial growth factor (VEGF) and the chemokine CXCL6. The studies showed that hypoxic conditions increased the expression of CXCL6 by endometrial cells. Based on the results obtained, it was concluded that hypoxia affects the progression of endometriosis foci by inducing the expression of molecules that enhance angiogenesis by the cells building these foci. Our studies have shown that CXCR-1 and CXCR-2 levels in the group of women with endometriosis were significantly higher than in the control group (p < 0.05 for CXCR-1 receptor; p < 0.01 for CXCR-2 receptor). Moreover, as the stage of the disease progressed, the levels of the tested receptors increased. In the case of CXCR-1, significant differences in the levels were demonstrated between stage I and stages II, III and IV (p < 0.05; p < 0.001; p < 0.001, respectively). On the other hand, the level of the CXCR-2 receptor differed significantly between stage I and stages III and IV (p < 0.01; p < 0.001, respectively). The results indicate that CXCR-1/2 receptors may be involved in disease progression and may also activate pathways initiating the angiogenesis process. Li et al. [11] analyzed the expression profile of CXCR-1 and CXCR-2 receptors in eutopic endometrial tissue of women with endometriosis. Immunohistochemical method was used to assess expression. It was observed that the expression of CXCR-1 receptor in the examined tissues was significantly higher in women with endometriosis than in women in the control group. The level of CXCR-2 receptor expression did not show significant differences between both groups. The results suggest that a higher expression of CXCR-1 in eutopic endometrium may be important in disease progression. This may occur through the activation of cellular signaling pathways which induce processes that enable cell survival. Ulukus et al. [20] examined the ability of cells that make up ectopic endometrial tissue to express CXCR-1 and CXCR-2 receptors. Immunohistochemical methods were used in the study. It was shown that epithelial cells that make up ectopic endometrial tissue show an increased expression of CXCR-1 and CXCR-2 receptors compared to normal tissue. The researchers indicate that these receptors may play an important role in the pathogenesis of endometriosis.

- Changes in the concentrations of the chemokine CXCL6 and its receptors CXCR-1 and CXCR-2 in the peritoneal fluid of women with endometriosis indicate an impaired immune response in the peritoneal cavity during endometriosis related to the stage of the disease. - The CXCL6/CXCR-1/CXCR-2 system plays an important role in the development and progression of endometriosis, which may have significant clinical significance as a potential therapeutic target but requires further research. Article information and declarations Data availability statement Data is contained in the article. Author contributions Concept, methodology, article draft, corresponding author — MSK; methodology, analysis and article draft — KR; concept, acquisition of data, article draft — AW; concept, article draft, supervision — AMP. Ethics statement Bioethics Committee of the Medical University of Silesia in Katowice (decision no. KNW/0022/KB/35/15). Acknowledgments None. Funding This research was funded by Medical University of Silesia in Katowice, Poland; grant number BNW-1-012/K/4/O. Conflicts of interest The authors declare that there is no conflict of interest. Supplementary material None.