Villainous role of estrogen in macrophage-nerve interaction in endometriosis

Different estrogen receptors have abnormal distribution in the ectopic endometrium of women with endometriosis [18]. A higher ERβ and a lower ERα expression profile in endometriotic lesions have been proposed as a major background of estrogen action in endometriosis [19]. Several studies have shown significantly elevated ERβ levels and decreased ERα levels in endometriotic stromal cells and tissues compared to the eutopic endometrium [20]. ERβ-induced the formation of ASK-1/STRAP complex can prevent the activation of tumor necrosis factor α (TNFα)-mediated apoptosis in endometriotic tissues [21]. Increased expression of Ras-like and estrogen-regulated growth inhibitor (RERG) has also been identified after the activation of ERβ [22]. ERβ binds to the promoter regulatory region of RERG, inducing gene expression of RERG, and then enhancing the proliferation of endometriotic cells. Moreover, multiple molecules in endometriosis are able to stimulate the expression of ERβ, such as insulin-like growth factors 1 (IGF1) [23], activated platelets [24], serum and glucocorticoid-regulated kinase (SGK1) [25]. However, cross-talk between ERα and interleukin (IL) 6 pathways is recently shown to promote the early initiation of endometriosis [26]. IL-6 mediates the recruitment of monocytes which can differentiate into macrophages expressing ERα. Estrogen-ERα binding in turn regulates the IL-6 promoter through activation of NF-κB and CEBPβ. Further study characterizes the heterogeneous expression of ERs in different types of endometriosis. Ovarian lesions show the lowest expression of ERα and the highest expression of ERβ, whereas the fallopian tube lesions show a high expression of both receptors [27]. The largest ERβ to ERα ratio is observed in ovarian lesions compared with peritoneal, fallopian tube, and extra pelvic lesions. Moreover, aberrant expression of ERα and ERβ are both correlated to the production of proinflammatory cytokines in endometriosis [28]. The combinational interaction of ERβ with caspase 1 and NLR family pyrin domain-containing 3 (NALP3) activates caspase 1 and subsequently elevates the level of IL-1β in ectopic lesions, enhancing the inflammation in the endometriotic microenvironment [21]. In addition, the expression of GPER is also significantly increased in ectopic endometrium compared to eutopic endometrium [29], which specifically promotes the proliferation of endometrial fragment. Although abnormal expression of different ERs has been identified in endometriotic lesion, their functions involved in the pathogenesis of endometriosis are far from clarified.

As discussed above, activation of the estrogen receptors can induce inflammatory events. In the endometriotic microenvironment, multiple immune cells, such as macrophages, mast cells, neutrophils, and mature dendritic cells are mobilized by the retrograded endometrial cells. Among them, macrophages are the primary contributor of pro-inflammatory cytokines and chemokines [30]. Both ERα and ERβ are significantly overexpressed in macrophages of endometriosis [28]. Treatment with 17β-estradiol in human macrophages in vitro induces an increase of ERα expression in macrophages. Furthermore, the estrogen-ER interaction can up-regulate the production of IL-6 and TNFα from lipopolysaccharide (LPS)-activated macrophages [31]. Specifically, macrophages with overexpression of ERβ in endometriotic lesions have been shown to exhibit enhanced phagocytic activity, and secrete inflammatory cytokines including IL1β, TNFα and IL6 [21, 32]. Deficiency of ERα, but not ERβ, increased TNFα production by mouse peritoneal macrophages in response to bacterial stimuli, indicating a prominent role of ERα in mediating the anti-inflammatory effect of estrogen. However, an opposite finding shows the ability of selective ERβ activation to inhibit the expression of inflammatory mediators. Some researchers even argue that ERα is only detected in a subset of macrophages in both peritoneal cavity and endometriotic biopsy specimens, which is different from previous studies [13]. Activation of ERα by estrogen is able to inhibit NF-κB-dependent inflammation by promoting synthesis of the negative regulator IκBα [33]. And this discrepancy about the ERs on macrophages is due to the inherent differences between peritoneal fluid macrophages and tissue macrophages. Moreover, the expression of GPER on macrophage in endometriosis is also significantly increased compared with that of healthy tissue, suggesting an alternative way that the immune response induced by macrophages is modulated by estrogen [34]. Treatment with the selective GPER agonist G-1 is able to inhibit LPS-induced TNFα production in human macrophages. Activation of GPER down-regulates NF-κB promoter activation through increase phosphorylation of JNK, resulting in the inhibition of LPS-induced IL-6 secretion [35]. GPER can also affects function macrophages by decreasing the expression of TLR4. These data indicate that the pro- or anti-inflammatory role of estrogen on macrophages is closely related to different types of estrogen receptors. Activation of ERβ has the potential to arouse pro-inflammatory response, while both ERα and GPER play significant roles in anti-inflammation.

In addition to macrophage, ERs are also expressed on the nervous system. Whether in the myometrium of women with or without adenomyosis, the expression of ERβ in sympathetic nerves is much more than sensory nerves [36, 37]. Activation of ERβ in sympathetic nerve could promote the down-regulation of cyclin E, IGF1, or cytokines [38], leading to a prominent reduction of the sympathetic innervation. ERα and ERβ are also expressed in the uterine-related dorsal root ganglion (DRG) neurons [39, 40], suggesting an important plasticity regulation and developmental function in uterine sensory nerves. Although ERα and ERβ are detected in DRG, the impacts of ER agonists on neuron in endometriosis have been clarified to depend on the subtypes of ERs. Activation of ERβ in DRG can down-regulate the expression of nerve repellent factor, SLIT3. Conversely, activation of ERα upregulates SLIT3 expression in DRGs, highlighting the potentially opposite impacts of different ERs in neuron [41]. Acute activation of membrane-associated ERα in peripheral sensory neurons by estrogen is confirmed to enhance allodynia elicited by the inflammatory mediator bradykinin [42]. Moreover, GPER is also identified in peripheral sensory neurons and the expression of GPER is increased after peripheral nerve injury [43]. However, the exact mechanism of GPER in neurogenesis and peripheral sensitization remains unclear.

There have been multiple studies demonstrating a significantly high level of macrophages in endometriotic milieu [44, 45]. Among different mechanisms involved in the recruitment of macrophages, nerve fibers exhibit an extraordinary ability to recruit macrophages to the site of lesion. Many factors have been confirmed to mediate the recruitment of macrophages toward the nerve fibers, including leukemia inhibitory factor (LIF) [46], IL1α, IL1β [47] and pancreatitis- associated protein 3 (PAP3) [48]. The role of estrogen during the process of nerve fiber-induced macrophage recruitment has been recently clarified [13]. Following peripheral axon injury, the neuron-derived exosomal miR-21-5p is phagocytosed by macrophages, which can mediate the macrophages infiltrations towards peripheral nerves [49]. Analysis has proven that exosomal miR-21-5p release is mediated by the estrogen signaling pathway [50]. Estrogen can also stimulate the secretion of colony-stimulating factor 1 (CSF1) and C-C motif ligand 2 (CCL2) from peripheral nerves, which enhances macrophages migration towards the endometriotic lesion [13].

Macrophages are special immune cells with high plasticity and different functional profiles. These immune cells can be divided into two subtypes as classically activated (M1) and alternatively activated (M2) macrophages based on the distinct pathways of activation [51]. The function of M1 macrophages is to produce pro-inflammatory cytokines which is induced by Th1 cytokines, such as Interferon γ (IFNγ) and TNFα. M2 macrophages, which are anti-inflammatory, are polarized by Th2 cytokines, such as IL-4 and IL-13 via activation of STAT6 through IL-4 receptor alpha [52]. Interestingly, the macrophages in endometriosis are predominantly of M2 phenotype [53]. Further evidence confirms that abnormal endometriotic milieu can induce the polarization of M2 macrophages [54]. Estrogen-dependent regulation of CCL2 in peritoneal cavity is confirmed to polarize macrophages from M1 toward M2 phenotype via CCL2/CCR pathway [55]. 17β-estradiol and TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin) also have a synergistic effect on inducing M2 macrophages polarization through activation of the STAT3 and P38-MAPK pathway [56]. Moreover, ERα signaling is recently clarified to preferentially polarize macrophages from a range of sources to an alternative phenotype [33]. Estrogen is able to inhibit NFκB-dependent inflammation by promoting synthesis of the negative regulator IκBα [57]. However, an opposed result shows that estrogen inhibits alternative activation polarization in a tumor-associated macrophage via the inhibition of JAK1-STAT6 pathway [58]. Although the role of estrogen in polarization of macrophages remains controversial, studies discussed above indicate the capacity of estrogen in mediating the recruitment of macrophages toward nerve fibers accompanied with M2 polarization of macrophages (Fig. 1).

Aberrant of innervation in endometriotic lesions is also demonstrated to be involved in the pathogenesis of endometriosis-associated pain [59]. Pain sensation of endometriosis is correlated with neural hypertrophy and increased neural density in the endometriotic lesion [60, 61]. Further studies discovered that there exists an imbalanced distribution of sensory, sympathetic and parasympathetic nerve fibers in endometriosis [7]. As a result, abnormal neurogenesis is important in endometriosis. Since the coexistence of macrophages and nerve fibers as well as their close correlation, macrophages can affect the neurogenesis in endometriosis. In addition, the secretion of multiple NGF from macrophages can be induced by estrogen, indicating a pivotal role of estrogen in mediating the above process [62].

Peripheral nerve sensitization is a process involving reduced threshold of ion channels, increased membrane excitability, and increased nociceptors expression. Inflammatory cytokines in endometriotic milieu can influence these aspects by binding to their corresponding nociceptors on peripheral nerves. Macrophages are thought to be the major source of inflammatory mediators, which can subsequently sensitize peripheral nerve fibers. The mRNA concentration of transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential cation channel, subfamily A, member 1 (TRPA1) are significantly higher in women with endometriosis [83]. TNFα, IL1β and monocyte chemotactic protein 1 (MCP1) from macrophages can influence the membrane potential of sensory nerve via TRPV-1-dependent mechanism [83‐85]. The NGF from macrophages can stimulate the activation of TRPV1 receptors through the activation of NGF receptors on neurons. The voltage-gated sodium channels, encoded by SCNA genes, are also stimulated by MCP1 through a CCR/Gβγ-dependent mechanism [86]. P2X (purinergic X) receptors, are another increased nociceptor in endometriosis which can be activated by extracellular ATP [87].As mentioned above, aberrant expression of ERα and ERβ in macrophages of endometriosis is correlated with the function of inflammatory cytokines, indicating the active role of estrogen in the secretion of inflammatory cytokines from macrophages [31]. The expression of those nociceptors discussed above is also up-regulated by estrogen in endometriotic cell-derived sensory neurons. These alternations by estrogen may be related to the presence of specificity protein 1 (SP1) transcription factor binding sites in the promoter regions of nociceptive genes [87]. In short, estrogen not only mediates the secretion of inflammatory cytokines from macrophages to activate nociceptors, but also increases the expression of nociceptors on peripheral nerves, which eventually exacerbates the local inflammation to induce peripheral hyper-sensitization.

Sustained peripheral sensitization by the inflammatory mediators from macrophages elicits the release of neuropeptides with inflammatory and nociceptive function, which induces a vicious circle to deteriorate the progression of endometriosis-associated pain. TRPV1-positive nerves can induce a neurogenic inflammation through the release of substance P (SP) and calcitonin gene-related peptide (CGRP) [88]. Further research demonstrates that estrogen can modulate corneal sensitivity by up-regulation of SP [89].The release of CGRP is also regulated by 17β-estradiol in the visceral pain sensitivity, implicating the role of estrogen in regulating of inflammatory neuropeptides [90]. The function of SP has been reported to stimulate the secretion of chemokine (C-X-C motif) ligand 8 (CXCL8) and IL-1β from macrophages, which exhibits a classically pro-inflammatory profile [91]. Furthermore, activation of neurokinin 1 receptor (NK1R), the receptor of SP, also depends upon estrogen in uterine [92]. However, activation of NK1R on macrophage has a special effect on M2 polarization through activation of P13K/Akt/mTOR signaling pathway, which may be beneficial for the repair of nerve injury [93]. The function of CGRP also exhibit compromised feature to promote M2 polarization of murine peritoneal macrophages via calmodulin, PKC and PKA pathway, leading to an attenuation of inflammation and tissue damage [94]. Although neuropeptides released from peripheral nerves generate a positive feedback to amplify the inflammation between nerves and macrophages, their function on macrophages polarization suggest that more evidence is necessary to clarify the effect of estrogen on these neuropeptides in endometriosis (Fig. 3).