Chemokine CCL21 promotes the pathological progression of endometriosis by regulating inflammatory cytokine expression and activating the NF-κB signaling pathway

Endometriosis is characterized as a typical chronic inflammatory disease, and inflammation is pivotal in the pathogenesis of endometriosis, even in the process of endometriosis-related carcinogenesis [19, 20]. Inflammatory mediators (IL-1β, IL-6, and TNF-α) up-regulate the production of haptoglobin in endometrium of endometriosis patients, which binds to macrophages and subsequently reduces their phagocytic function, thereby promoting the establishment of endometriosis [21]. The enhanced inflammation in endometriosis is associated with the inhibition of endothelial function [21]. A. Catarina Neto et al. used metformin to reverse infertility associated with endometriosis, possibly due to its anti-inflammatory properties during pregnancy[22]. Despite this knowledge, the pathogenicity and molecular mechanisms of inflammation in endometriosis remain to be clarified. Therefore, early identification of IRGs that contribute to the pathogenesis of endometriosis is essential to provide better treatment strategies. In this study, we screened for potential hub IRGs through comprehensive bioinformatics analysis and established a predictive model based on three hub genes (CCL21, CFD, and ACKR1), which showed good predictive ability. These findings might strongly support further comprehensive research into precision treatment of endometriosis. To investigate the potential functions of hub genes further, we collected ectopic tissues from endometriosis patients and established endometriosis mouse model. Among hub genes, CCL21 was increased in both endometriosis patients and mouse model. Knockdown of CCL21 significantly inhibited the proliferation, migration, and invasion of endometriotic cells and promoted apoptosis. Further analysis revealed that reduced CCL21 could inhibit endometriosis development via suppressing NF-κB signaling pathway. Endometriosis is a complex chronic inflammatory gynecological disorder associated with infertility and endometriosis-related pain [23]. A study on patient generated data found that fatigue, headache, mental confusion, gastrointestinal problems, and pain are common in endometriosis phenotypes, all of which are characteristics of low-grade inflammation[24]. Although its mechanisms are yet to be fully elucidated, current research suggests that inflammatory changes in endometriotic lesions are related to the advancement of endometriosis. Inflammation is considered a significant contributor to pain [25]. An elevated level of IL-1β has been detected in the peritoneal fluid and lesion tissues of women with endometriosis and pelvic pain [26, 27]. The increased IL-1β directly enhances the production of nerve growth factors, correlating with localized neurogenesis around endometriotic lesions and deep dyspareunia [26]. The number of activated macrophages in the peritoneal fluid of women with endometriosis increases, secreting more cytokines, including IL-6 [28, 29]. The elevated IL-6 then inhibits the cytolytic activity of NK cells [30]. Higher concentrations of TNF-α in peritoneal fluid and serum of patients with endometriosis play a pivotal role in activating systemic and local inflammation associated with the progression of endometriosis [31]. In the present study, we also validated the expression of IL-6, TNF-α, and IL-1β in lesion tissues of endometriosis patients and mouse model. The mouse model of endometriosis constructed by transplanting cornu uteri into mesentery is characterized by proliferation and inflammation, but without epithelial mesenchymal transformation and fibrosis. This model simulates some but not all features of human endometriosis[32]. Considering the complex mechanisms of endometriosis and the sensitivity of patients to different drugs, the search for new diagnostic markers and therapeutic targets is critical. We identified 207 commonly DEGs between the endometriotic lesions and normal endometrium of endometriosis patients through GSE7305 and GSE51981 datasets. By using WGCNA analysis, we screened out the “lightcyan” module, which is most significantly positively correlated with endometriosis. After intersecting IRGs, DEGs, and “lightcyan” module genes from WGCNA, three hub genes (CCL21, CFD, and ACKR1) were identified. Among these, CCL21 was increased in the lesion tissues of endometriosis across multiple GEO datasets, indicating that upregulated CCL21 might promote the occurrence and progression of endometriosis. Yanzhen Zhou et al. reported the identification of three potential key genes (MET, IL4R, and BST2) associated with endometriosis[33]. This result is inconsistent with our research findings and may be due to differences in databases, but it cannot be ruled out that the sample may be different. Anyway, our experiment confirms the importance of CCL21. CCL21 is a key chemokine of the CC subfamily, a small molecular weight protein that exhibits chemotactic properties for cell migration [34]. It mediates a range of physiological and disease-related processes, encompassing stress responses, infection, wound healing, and cell differentiation. The abnormal expression of CCL21 is involved in inflammation and immune regulation in various autoimmune diseases [35, 36]. Researchers have found that CCL21 is significantly increased in the ectopic tissue of endometriosis patients through laser capture microdissection analysis [37]. However, the pathological role of CCL21 in endometriosis remains unknown. In our study, we found that the expression of CCL21 was elevated not only in the lesion tissues of endometriosis across multiple datasets but also in the lesion tissues of endometriosis patients and mouse model, suggesting that CCL21 might also be involved in the development of endometriosis. While endometriosis is a benign disease, it exhibits biological characteristics similar to cancer, such as excessive proliferation, metastasis, and invasion [38]. To further investigate the impact of CCL21 on endometriosis, we used small interfering RNA methods to reduce CCL21 expression in 12z cells. Compared to the control group, knockdown of CCL21 suppressed the proliferation, migration, and invasion capabilities of 12z cells and promoted apoptosis. These results suggest that CCL21 could alleviate the pathology of endometriosis. To further investigate how CCL21 regulates the development of endometriosis, we divided patients with endometriosis into high- and low-CCL21 groups according to median CCL21 value. GSEA results showed that the high-CCL21 group was enriched in NF-κB and chemokine signaling pathway, while the low-CCL21 group was primarily enriched in oocyte meiosis. These results suggest that the inflammation in lesion tissues of patients in high-CCL21 group was increased, and the maturation of follicles was inhibited, which also indicated that patients in the high-CCL21 group might experience infertility. NF-κB regulates multiple genes in response to various stimuli, such as pro-inflammatory cytokines, thereby participating in the inflammatory response [39]. The activation of NF-κB leads to its translocation from cytoplasm to nucleus, where it activates the transcription of target genes, primarily encoding factors involved in inflammatory response. The abnormal level of NF-κB is associated with the pathogenesis of autoimmune diseases and autoinflammatory conditions [40, 41]. Our results indicated that NF-κB was elevated in lesion tissues of patients and mouse model with endometriosis, which also suggested that endometriosis patients were in a state of activated inflammation. Knocking down CCL21 in 12z cells significantly inhibited NF-κB and its downstream inflammatory factors (IL-6, TNF-α, and IL-1β). Overall, reducing CCL21 alleviated the progression of endometriosis by suppressing the activation of NF-κB signaling pathway and inhibiting the expression of IL-6, TNF-α, and IL-1β. There are still limitations in evaluating the severity and location of endometriosis, as well as diagnostic strategies[42]. More and more evidences suggest that endometriosis is a systemic disease, and there are complex interactions between angiogenesis, hormones, and immune changes caused by chronic inflammation in endometriosis. Developing new and safer anti-inflammatory and immunomodulatory drugs may become a long-term treatment option for endometriosis patients [43]. In summary, we have confirmed the important role of CCL21 in endometriosis, providing reference information for the diagnosis and treatment of endometriosis. Although there are still some issues that need to be addressed, such as the relationship between CCL21 and the severity of endometriosis, and how to develop targeted CCL21 drugs to avoid adverse reactions to other tissues, the results of this study may still help clarify the pathogenesis of endometriosis development and promote the establishment of treatment methods. Endometriosis is characterized as a typical chronic inflammatory disease, and inflammation is pivotal in the pathogenesis of endometriosis, even in the process of endometriosis-related carcinogenesis [19, 20]. Inflammatory mediators (IL-1β, IL-6, and TNF-α) up-regulate the production of haptoglobin in endometrium of endometriosis patients, which binds to macrophages and subsequently reduces their phagocytic function, thereby promoting the establishment of endometriosis [21]. The enhanced inflammation in endometriosis is associated with the inhibition of endothelial function [21]. A. Catarina Neto et al. used metformin to reverse infertility associated with endometriosis, possibly due to its anti-inflammatory properties during pregnancy[22]. Despite this knowledge, the pathogenicity and molecular mechanisms of inflammation in endometriosis remain to be clarified. Therefore, early identification of IRGs that contribute to the pathogenesis of endometriosis is essential to provide better treatment strategies. In this study, we screened for potential hub IRGs through comprehensive bioinformatics analysis and established a predictive model based on three hub genes (CCL21, CFD, and ACKR1), which showed good predictive ability. These findings might strongly support further comprehensive research into precision treatment of endometriosis. To investigate the potential functions of hub genes further, we collected ectopic tissues from endometriosis patients and established endometriosis mouse model. Among hub genes, CCL21 was increased in both endometriosis patients and mouse model. Knockdown of CCL21 significantly inhibited the proliferation, migration, and invasion of endometriotic cells and promoted apoptosis. Further analysis revealed that reduced CCL21 could inhibit endometriosis development via suppressing NF-κB signaling pathway. Endometriosis is a complex chronic inflammatory gynecological disorder associated with infertility and endometriosis-related pain [23]. A study on patient generated data found that fatigue, headache, mental confusion, gastrointestinal problems, and pain are common in endometriosis phenotypes, all of which are characteristics of low-grade inflammation[24]. Although its mechanisms are yet to be fully elucidated, current research suggests that inflammatory changes in endometriotic lesions are related to the advancement of endometriosis. Inflammation is considered a significant contributor to pain [25]. An elevated level of IL-1β has been detected in the peritoneal fluid and lesion tissues of women with endometriosis and pelvic pain [26, 27]. The increased IL-1β directly enhances the production of nerve growth factors, correlating with localized neurogenesis around endometriotic lesions and deep dyspareunia [26]. The number of activated macrophages in the peritoneal fluid of women with endometriosis increases, secreting more cytokines, including IL-6 [28, 29]. The elevated IL-6 then inhibits the cytolytic activity of NK cells [30]. Higher concentrations of TNF-α in peritoneal fluid and serum of patients with endometriosis play a pivotal role in activating systemic and local inflammation associated with the progression of endometriosis [31]. In the present study, we also validated the expression of IL-6, TNF-α, and IL-1β in lesion tissues of endometriosis patients and mouse model. The mouse model of endometriosis constructed by transplanting cornu uteri into mesentery is characterized by proliferation and inflammation, but without epithelial mesenchymal transformation and fibrosis. This model simulates some but not all features of human endometriosis[32]. Considering the complex mechanisms of endometriosis and the sensitivity of patients to different drugs, the search for new diagnostic markers and therapeutic targets is critical. We identified 207 commonly DEGs between the endometriotic lesions and normal endometrium of endometriosis patients through GSE7305 and GSE51981 datasets. By using WGCNA analysis, we screened out the “lightcyan” module, which is most significantly positively correlated with endometriosis. After intersecting IRGs, DEGs, and “lightcyan” module genes from WGCNA, three hub genes (CCL21, CFD, and ACKR1) were identified. Among these, CCL21 was increased in the lesion tissues of endometriosis across multiple GEO datasets, indicating that upregulated CCL21 might promote the occurrence and progression of endometriosis. Yanzhen Zhou et al. reported the identification of three potential key genes (MET, IL4R, and BST2) associated with endometriosis[33]. This result is inconsistent with our research findings and may be due to differences in databases, but it cannot be ruled out that the sample may be different. Anyway, our experiment confirms the importance of CCL21. CCL21 is a key chemokine of the CC subfamily, a small molecular weight protein that exhibits chemotactic properties for cell migration [34]. It mediates a range of physiological and disease-related processes, encompassing stress responses, infection, wound healing, and cell differentiation. The abnormal expression of CCL21 is involved in inflammation and immune regulation in various autoimmune diseases [35, 36]. Researchers have found that CCL21 is significantly increased in the ectopic tissue of endometriosis patients through laser capture microdissection analysis [37]. However, the pathological role of CCL21 in endometriosis remains unknown. In our study, we found that the expression of CCL21 was elevated not only in the lesion tissues of endometriosis across multiple datasets but also in the lesion tissues of endometriosis patients and mouse model, suggesting that CCL21 might also be involved in the development of endometriosis. While endometriosis is a benign disease, it exhibits biological characteristics similar to cancer, such as excessive proliferation, metastasis, and invasion [38]. To further investigate the impact of CCL21 on endometriosis, we used small interfering RNA methods to reduce CCL21 expression in 12z cells. Compared to the control group, knockdown of CCL21 suppressed the proliferation, migration, and invasion capabilities of 12z cells and promoted apoptosis. These results suggest that CCL21 could alleviate the pathology of endometriosis. To further investigate how CCL21 regulates the development of endometriosis, we divided patients with endometriosis into high- and low-CCL21 groups according to median CCL21 value. GSEA results showed that the high-CCL21 group was enriched in NF-κB and chemokine signaling pathway, while the low-CCL21 group was primarily enriched in oocyte meiosis. These results suggest that the inflammation in lesion tissues of patients in high-CCL21 group was increased, and the maturation of follicles was inhibited, which also indicated that patients in the high-CCL21 group might experience infertility. NF-κB regulates multiple genes in response to various stimuli, such as pro-inflammatory cytokines, thereby participating in the inflammatory response [39]. The activation of NF-κB leads to its translocation from cytoplasm to nucleus, where it activates the transcription of target genes, primarily encoding factors involved in inflammatory response. The abnormal level of NF-κB is associated with the pathogenesis of autoimmune diseases and autoinflammatory conditions [40, 41]. Our results indicated that NF-κB was elevated in lesion tissues of patients and mouse model with endometriosis, which also suggested that endometriosis patients were in a state of activated inflammation. Knocking down CCL21 in 12z cells significantly inhibited NF-κB and its downstream inflammatory factors (IL-6, TNF-α, and IL-1β). Overall, reducing CCL21 alleviated the progression of endometriosis by suppressing the activation of NF-κB signaling pathway and inhibiting the expression of IL-6, TNF-α, and IL-1β. There are still limitations in evaluating the severity and location of endometriosis, as well as diagnostic strategies[42]. More and more evidences suggest that endometriosis is a systemic disease, and there are complex interactions between angiogenesis, hormones, and immune changes caused by chronic inflammation in endometriosis. Developing new and safer anti-inflammatory and immunomodulatory drugs may become a long-term treatment option for endometriosis patients [43]. In summary, we have confirmed the important role of CCL21 in endometriosis, providing reference information for the diagnosis and treatment of endometriosis. Although there are still some issues that need to be addressed, such as the relationship between CCL21 and the severity of endometriosis, and how to develop targeted CCL21 drugs to avoid adverse reactions to other tissues, the results of this study may still help clarify the pathogenesis of endometriosis development and promote the establishment of treatment methods.