Autocrine Production of Interleukin-34 Promotes the Development of Endometriosis through CSF1R/JAK3/STAT6 signaling

Interleukin (IL)-34 plays a critical role in cell proliferation, differentiation, apoptosis, angiogenesis, inflammation and immunoregulation. Numerous diseases can be attributed to the dysregulation of IL-34 signaling. This study was performed to investigate the function of IL-34 in the pathogenesis of endometriosis. Firstly, by enzyme linked immunoabsorbent assay, we found that IL-34, VEGF, MMP-2 and MMP-9 were increased in the sera of patients with endometriosis. Secondly, exposure to IL-34 promoted the proliferation, migration and invasion of eutopic endometrial stromal cells (ESCs). Additionally, stimulation with IL-34 up-regulated colony-stimulating factor 1 receptor (CSF1R), p-JAK3, p-STAT6, VEGF, MMP-2 and MMP-9 in these eutopic ESCs. Treatment with AS1517499, an inhibitor of STAT6, remarkably abrogated the alterations induced by IL-34. A Chromatin immunoprecipitation (ChIP) assay demonstrated binding of STAT6 to the IL-34 promoter, further implicating STAT6 in IL-34 signaling. Notably, reverse results were obtained in ectopic ESCs with the application of an IL-34 neutralizing antibody. In vivo, AS1517499 suppressed the maintenance of endometriosis lesions in rats. In summary, autocrine production of IL-34, mediated by STAT6, promoted the development of endometriosis in vitro and in vivo through the CSF1R/JAK3/STAT6 pathway. Our research reveals the function of IL-34 in endometriosis, which may provide insight into novel therapeutic strategies for endometriosis.


IL-34 is increased in endometriosis.
Considering the crucial role of IL-34 in numerous disease states, we investigated the differential expression of IL-34 in endometrial samples with or without endometriosis using the microarray data in the Gene Expression Omnibus (GEO) database (GSE51981) 20 . As shown in Fig. 1A, IL-34 expression is increased in endometriosis tissues at all stages (Minimal/Mild, Moderate/Severe), implicating the role of IL-34 in the pathogenesis of endometriosis. We further collected the sera of endometriosis patients to confirm the changes in IL-34 expression levels. Results demonstrated that IL-34 secretion was elevated in endometriosis patients (Fig. 1B). Moreover, we also detected the serum levels of proteins associated with cell proliferation, angiogenesis, and epithelial-mesenchymal transition processes which are closely related to the pathogenesis of endometriosis, such as VEGF, MMP-2 and MMP-9, 21-23 , by ELISA. The results showed that VEGF, MMP-2 and MMP-9 were elevated in the sera of the patients suffering from endometriosis ( Fig. 1C-E). These results are consistent with previous findings 21-23 . IL-34 potentiates the proliferation, migration and invasion potential of eutopic ESCs. Next, eutopic ESCs were isolated and immunohistochemical (IHC) staining for CD10, a specific surface marker for endometrial stromal cells 24 , and CK19, a specific marker for epithelial cells, was performed to identify ESCs. A strong, positive expression of CD10 was detected in the isolated cells, whereas CK19 was almost undetected (Fig. S1). These data suggest a successful isolation of ESCs. Then, to detect the function of IL-34 in vitro, eutopic ESCs were treated with increasing doses of recombinant human IL-34 (0-200 ng/mL). We found that IL-34 potentiated eutopic ESC growth in a dose-dependent manner at doses of 25 to 100 ng/mL ( Fig. 2A). The proliferation rate of ESCs was similar at 100 ng/mL and 200 ng/mL doses of IL-34, thus doses of 50 ng/mL and 100 ng/mL were used in the following transwell assay. As indicated in Fig. 2B,C, IL-34 also enhanced migration and invasion in a dose-dependent manner. Taken together, elevated IL-34 expression may promote eutopic ESC proliferation, migration and invasion to facilitate the development of endometriosis. CSF1R/JAK3/STAT6 activation and IL-34 autocrine production are responsible for the increased proliferation, migration and invasion of eutopic ESCs. An early study demonstrated that STAT3 was involved in mediating the effect of IL-34 20 . We performed a screening for the potential downstream mediators of IL-34 by western blot analysis with antibodies for the JAK/STAT pathway. Recombinant IL-34 facilitated the phosphorylation and activation of Janus kinase 3 (JAK3) and STAT6 in a dose-dependent manner ( Fig. 3A) but had no effect on the expression of the other JAK and STAT family members (Fig. S2). Moreover, AS1517499, a specific STAT6 inhibitor, antagonized the pro-proliferative effect of IL-34 in eutopic ESCs (Fig. 3B). In addition, IL-34-induced migration and invasion were also abrogated upon inactivation of STAT6 (Fig. 3C,D). Collectively, these data demonstrate that the biological activity of IL-34 in eutopic ESCs is mediated by activation JAK3/STAT6 signaling.
Next, we further dissected the precise molecular mechanism by which IL-34 promoted proliferation, migration and invasion. As IL-34 is an alternative ligand of CSF1R 14,15 , CSF1R expression increased with the IL-34 stimulation (Fig. 3E). In addition, the IL-34-induced phosphorylation of JAK3/STAT6 was suppressed by AS1517499 (Fig. 3E). Moreover, IL-34 promoted the transcription and translation of VEGF, MMP-2 and MMP-9, and this alteration was abrogated by treatment with AS1517499 (Fig. 3E,F). Interestingly, we also observed an increased transcription and translation of IL-34 after IL-34 treatment, which was suppressed by AS1517499 (Fig. 3E,F). Thus, we hypothesized that JAK3/STAT6 signaling might regulate IL-34 expression. To test whether JAK3/STAT6 signaling regulates IL-34 expression, STAT6 knockdown and overexpression plasmids were constructed to modulate the expression of STAT6 (Fig. S3). Silencing of STAT6 also showed the same effect as AS1517499 on the mRNA levels of related genes (Fig. 3G). A ChIP assay revealed the presence of an IL-34 promoter sequence in the precipitated complexes (Fig. 3H). Overall, the results suggest that activated STAT6 binds to the promoter of IL-34 to regulate its expression in ESCs. In summary, STAT6 mediated autocrine production of IL-34 triggers the CSF1R/JAK3/STAT6 signaling cascade, which, in turn, up-regulates VEGF, MMP-2 and MMP-9 to promote the pathological progression of ESCs.
Anti-IL-34 suppresses the proliferation, migration and invasion of ectopic ESCs. Ectopic ESCs were isolated and pretreated with IL-34 neutralizing antibody to validate the effects of IL-34 in endometriosis.  www.nature.com/scientificreports www.nature.com/scientificreports/ A STAT6 specific inhibitor suppresses endometriosis in vivo. An endometriosis rat model was established, as previously described 25 , to validate the essential role of the IL-34/CSF1R/JAK3/STAT6 signaling pathway in vivo. Endometriotic lesions harvested from the Endometriosis + AS1517499 group appeared smaller than those from Endometriosis group (Fig. S5). Unsurprisingly, IL-34, VEGF, MMP-2 and MMP-9 were increased in the sera of the experimental endometriosis rats, which could be abrogated by STAT6 signaling blockage with AS1517499 (Fig. 5A). Eutopic endometrial tissues and ectopic implants were also collected for qRT-PCR and western blot analysis. The transcription and translation of IL-34, CSF1R, VEGF, MMP-2 and MMP-9 were elevated (Fig. 5B,C). Moreover, results revealed that p-JAK3 and p-STAT6 proteins increased in the ectopic implants (Fig. 5C). Inhibition of STAT6 signaling with AS1517499 suppressed the expression of the above proteins in the endometriosis rats (Fig. 5B,C). In general, targeting the IL-34/CSF1R/JAK3/STAT6 pathway with a STAT6 inhibitor was an effective means of treating endometriosis in rats.

Discussion
In our present study, through reanalysis of the expression profile of IL-34 in the microarray data of the Gene Expression Omnibus (GEO) database (GSE51981), we found that IL-34 was increased in clinical samples with endometriosis. IL-34 plays a critical role in cell proliferation, differentiation, apoptosis, angiogenesis, inflammation and immunoregulation 18,26 . Numerous diseases are attributed to the dysregulation of IL-34 signaling [27][28][29] . Thus, we hypothesized that IL-34 may play an important role as a multifunctional inflammatory cytokine in the pathogenesis and persistence of endometriotic lesions.
First, we showed that excessive secretion of IL-34 was detected in the sera of endometriosis patients and in rat models of endometriosis. Second, CSF1R, p-JAK3, p-STAT6, VEGF, MMP-2 and MMP-9 were up-regulated www.nature.com/scientificreports www.nature.com/scientificreports/ in response to recombinant IL-34 in vitro. VEGF, MMP-2 and MMP-9 are closely related to the pathogenesis of endometriosis [21][22][23] . According to our study, recombinant IL-34 promoted the expression of these proteins in eutopic ESCs, which indicates that IL-34 may participate in several important cellular processes that are dysregulated in ESCs, such as proliferation, migration and invasion. Third, a chromatin immunoprecipitation (ChIP) assay confirmed the binding of STAT6 to the IL-34 promoter. AS1517499, an inhibitor of STAT6, remarkably abrogated the cellular alterations induced by IL-34. As a transcription factor of the STAT family, STAT6 is implicated in the pathology of a wide variety of diseases [30][31][32] . A genetic polymorphism analysis demonstrated the association between STAT6 and endometriosis 33 . The present study suggests that IL-34 is also an activator of STAT6 signaling by binding to CSF1R in ESCs. Further, an IL-34 neutralizing antibody could attenuate CSF1R/JAK3/ STAT6 activation and down-regulate VEGF, MMP-2 and MMP-9, eventually leading to suppression of the development of endometriosis in vitro. These results were further validated in vivo through endometriosis lesions in a rat model. Taken together, these results indicate that IL-34 facilitates endometriosis progression by promoting ESC proliferation, migration and invasion.
Based on previous reports 34-36 , we chose IL-34 concentrations of 0-200 ng/mL for the functional assays. We found that IL-34 concentrations of 25 to 100 ng/mL promoted eutopic ESC growth in a dose-dependent manner. Here, the doses of IL-34 for in vitro experiments were much higher than the level of IL-34 in serum samples from endometriosis patients, which were typically less than 400 pg/mL. The possible reason was that the IL-34 concentration surrounding ESCs could be higher than the serum concentration of Il-34.
Because CSF1R is not an exclusive receptor for IL-34, IL-34 may exert pathophysiological effects through other receptor molecules. Receptor-type protein-tyrosine phosphatase ζ (PTPRZ1) was recently identified as an additional IL-34 receptor 37 . IL-34 binds to the extracellular domain of PTPRZ1 to stimulate the phosphorylation of paxillin and focal adhesion kinase, which influences the growth and migration of glioblastoma cells. Whether CSF1R is necessary and sufficient for the activation of JAK3/STAT6 signals induced by IL-34 needs further investigation. In addition, CSF-1 is structurally and functionally analogous to IL-34 and its effects are mediated exclusively through CSF1R 15 . Mounting evidence suggests that CSF-1 is increased in endometriosis and is conducive to the formation of endometriotic lesions 38,39 . Whether CSF-1 is involved in the activation of CSF1R/JAK3/STAT6 signaling is still unclear. Moreover, the recruitment of immune cells in the endometriotic microenvironment 40 may also influence the production of IL-34 and deserves further investigation.
In conclusion, our research reveals the functional role of IL-34 in endometriosis as well as the mechanism by which IL-34 mediates its effects. With this study, we hope to provide insight for the development of novel therapeutic strategies in endometriosis.

Reanalysis of the Gene Expression Omnibus (GEO) dataset.
The endometriosis dataset, which contains microarray data of endometrial specimens from women with Non-Endometriosis and No Uterine P/pelvic pathology, women with Non-Endometriosis but with Uterine P/pelvic pathology, women with Minimal/Mild endometriosis and women with Moderate/Severe endometriosis, was downloaded from the GEO database using accession number GSE51981 20 , and the expression of IL-34 was compared using an one-way ANOVA analysis followed by Tukey's test.
Clinical samples. A total of 90 endometriosis patients (age range 25-43, M ± SD. 35.2 ± 5.7) and 90 non-endometriosis patients (age range 26-48, M ± SD. 36.6 ± 4.8) admitted at Women's Hospital, Zhejiang University School of Medicine were enrolled in this study after written informed consent was obtained. The endometriosis patients were at stage III-IV (according to revised AFS classification) (23cases of stage III/67 cases of stage IV), and 75.6% (68 of 90) of the patients had chronic pelvic pain and 41.1% (37 of 90) had infertility. The non-endometriosis patients included 36 (40.0%) cases of mature teratoma and 54 (60.0%) cases of tubal infertility. There was no difference in body mass index (BMI) between the two groups. The samples were all obtained during the proliferative phase of the menstrual cycle. The menstrual cycle phase was determined by preoperative history and histological examination. The inclusion criteria were as follows: regular menstruation with a period of 28 to 32 days; no other endocrine, immune and metabolic diseases, no hormones or other medications were received within three months before surgery; non-lactating; and no history of serious drug allergies. The sera were collected from all the participants and stored at −80 °C until ELISA analysis. The eutopic endometrial samples and the non-fibrotic red ectopic endometria were collected during laparoscopic excision of endometrioma and used for the isolation of eutopic and ectopic endometrial stromal cells (ESCs), respectively.
Cell isolation and cell culture. Primary cells were isolated as previously described 41 . Tissue samples were washed with PBS and cut into pieces. The tissue pieces were digested with 0.125% collagenase IV (Sigma, St. Louis, MO, USA) for 60 min and centrifuged at 500 rpm for 5 min. Then, the cell suspension was collected and centrifuged at 3000 rpm for 10 min. Cell pellets were resuspended in DMEM/F12 medium (Hyclone, SH30023.01B; Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, 16000-044; Carlsbad, CA, USA) and 1% penicillin-streptomycin (Solarbio, Beijing, China) and incubated at 37 °C with 5% CO 2 . After 2 h, ESCs attached to surface of the plate, and the culture medium was replaced to discard the immunocytes and epithelial glands. The isolated cells were identified by IHC staining with anti-CK19 and anti-CD10 antibodies (Supplemental Table 2). www.nature.com/scientificreports www.nature.com/scientificreports/ Enzyme linked immunoabsorbent assay (ELISA). The concentrations of IL-34, MMP-2, MMP-9 and VEGF in the sera were detected by ELISA according to the manufacturer's instructions (X-Y Biotechnology, Shanghai, China). Briefly, the prepared standard samples and serum samples were added to the wells coated with the specific antibody and incubated at 37 °C. Then the liquids were discarded and the wells were washed with wash buffer. Horseradish peroxidase-conjugate reagent was added to each well except the blank group. After incubation and another wash, tetramethylbenzidine (TMB) substrate solution was added and the plate was incubated in dark. Finally, the reaction was terminated by addition of a stop solution and the absorbance at 450 nm was measured.
Cell counting kit-8 (CCK-8) assay. CCK-8 assay was performed using a Cell Proliferation and Cytotoxicity Assay Kit (SAB, CP002; College Park, MD, USA). Briefly, 100 μL of cell suspension containing 2 × 10 3 ESCs was added to each well of the 96-well plates. After incubating overnight, the cells were divided into different groups and exposed to different treatments. Finally, 10 μL of CCK-8 solution was added to each well. Cell viability was evaluated by measuring the absorbance at 450 nm.
Western blot analysis. Protein levels were detected by western blot analysis. Briefly, the lysates were separated by gel electrophoresis. Proteins were then transferred onto polyvinylidene fluoride membranes and blocked with 5% nonfat milk. The membranes were then incubated with optimally diluted primary antibodies and second antibodies sequentially. Protein expression was assessed with a chemiluminescent imaging system (Tanon 5200, Shanghai, China). The antibodies used for Western blot are listed in Supplemental Table 2.
Chromatin immunoprecipitation assay (ChIP). After transfection with si-STAT6 or pcDNA3.1-STAT6, eutopic ESCs were cross-linked with 1% formaldehyde. Then the cells were lysed and chromatin was sheared to DNA fragments of 150-900 bp by sonication. Immunoprecipitation was performed using an anti-STAT6 antibody. Rabbit IgG was used as a negative control. The precipitated complexes were washed and reverse cross-linked. After purification, the extracted DNA was amplified by qRT-PCR to identify the presence of an IL-34 promoter sequence. The primers which were specific for the IL-34 promoter that contained STAT6 binding site are listed as follows. Forward: 5′-GGTTGAAGACTCCCTCCTAC-3′. Reverse: 5′-AAAGCAGGCCACTGCAGCTC-3′.
Endometriosis rat model and in vivo study. Eighteen female wistar rats weighting 210-240 g were obtained to establish the endometriosis model. The rats accepted preoperative diethylstilbestrol injection with a dose of 0.1 mg/kg. Rat surgeries were conducted as previously described 25 . Briefly, a vaginal secretion smear was applied to confirm the estrus cycle. After anesthetization with chloral hydrate (0.4 mL/100 g), a right abdominal incision was made to isolate the right uterine horn. Then the uterine horn was excised. The stripped endometrium was cut into fragments and planted in the abdominal cavity and subcutis. Penicillin was applied to prevent infection. The rats were divided into three groups (n = 6 per group) according to the treatment: Normal, rats underwent laparotomy surgery without the transplant of endometrial tissues; Endometriosis, experimental endometriosis rats; Endometriosis + AS1517499, experimental endometriosis rats intraperitoneally injected with AS1517499. The rats that underwent laparotomy surgery without the transplant of endometrial tissues were used as a control.. Then, exploratory laparotomy was performed to check the formation of ectopic lesions after two weeks, which indicated the successful establishment of an endometriosis rat model. The endometriosis rats were