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Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, TaiwanDepartment of Microbiology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, TaiwanCenter for Resources, Research and Development, Kaohsiung Medical University, Kaohsiung, Taiwan
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, TaiwanCenter for Resources, Research and Development, Kaohsiung Medical University, Kaohsiung, TaiwanDepartment of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
Immune dysregulation may be involved in the development of endometriosis. The anti-inflammatory cytokine IL-10 plays an important role in eliminating unwanted cells and cellular debris in a silent way. We investigated the modulatory role of IL-10 in the development of endometriosis. We observed that the serum level of IL-10 in patients with endometriosis was significantly higher than that in healthy subjects or in control subjects with other gynecological disease. Monocyte-derived dendritic cells acquired from male donors and subsequently conditioned with serum from women with endometriosis exhibited a tolerogenic phenotype, including increased IL-10 production, lower IL-12 secretion, and down-regulation of CD86 and HLA-DR molecules. Depletion of IL-10 activity in a C57BL/6 mouse model of surgically induced endometriosis significantly decreased the size of endometrial lesions. In contrast, IL-10 administration promoted the growth of endometrial lesions in this model. In addition, infiltrated plasmacytoid dendritic cells were the primary IL-10–secreting immune cells in endometrial lesions. Our findings suggest that IL-10 may suppress immunity against endometrial implants, contributing to development of endometriosis.
Endometriosis is a common, benign, and estrogen-dependent gynecological disease characterized by the growth of endometrial cells outside the uterine cavity. The prevalence of endometriosis in the female reproductive years is estimated to be approximately 6% to 10%.
However, retrograde menstruation alone cannot completely explain the occurrence of endometriosis, because most women (76% to 90%) experience some retrograde menstruation, but far fewer women (6% to 10%) develop endometriosis.
Accumulating evidence suggests that defective immune surveillance permitting the survival of endometrial tissue after retrograde menstruation may play a crucial role in the genesis and development of endometriosis.
Endometrial tissue in the peritoneal cavity may, if host immunity cannot eliminate it, elicit aberrant inflammation. The anti-inflammatory cytokine IL-10 acts on many types of cells to limit and terminate local inflammation. The removal of apoptotic cells is mediated by dendritic cells (DCs) and macrophages. IL-10–producing macrophages are primarily involved in the silent clearance of early apoptotic cells in which the release of proinflammatory cytokines is prevented.
IL-10 can induce differentiation of DCs that leads to a tolerogenic phenotype, which includes decreased T-cell stimulatory activity, increased IL-10 production, and enhanced differentiation of adaptive regulatory T cells.
In contrast, late apoptotic cells or necrotic cells may produce danger signals and present self-antigens to DCs, thus activating autoimmune responses and finally leading to the development of autoimmunity, such as occurs in systemic lupus erythematosus.
Endometriosis is a chronic inflammatory disease that is associated with a complex cytokine pattern in the peritoneal fluid; these cytokines include proinflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α), T-cell–related cytokines (IL-4, IL-12, IL-17, and IFN-γ), and the anti-inflammatory mediator IL-10.
Thus, dysfunction of natural immunity may lead to inefficient recognition of endometrial antigens, with subsequent inadequate clearance of cellular debris, which then accumulates in the peritoneal cavity after cyclic intraperitoneal microhemorrhages. In the present study, we tested the hypothesis that dysregulation of IL-10 expression contributes to the development of endometriosis.
Materials and Methods
Subjects were enrolled after approval of the protocol by the Kaohsiung Medical University Hospital Institutional Review Board. Informed consent was obtained from all participants in accordance with the Declaration of Helsinki. The severity of endometriosis was determined according to the revised American Society of Reproductive Medicine (rASRM) classification in 1996. The endometriosis group consisted of 41 women (mean age, 34.0 ± 7.1 years) who underwent surgery for the treatment of advanced endometriosis (rASRM stages III–IV); none of these women had received any medical treatment for at least 3 months before surgery. The disease control group consisted of 26 women (mean age, 36.6 ± 7.9 years) who underwent surgery for benign gynecological disease and without evidence of endometriosis as diagnosed during surgery, including absence of endometrial polyps, teratoma, ovarian cysts, and myoma. The healthy control group consisted of 11 women (mean age, 33.1 ± 3.4 years) with no gynecological disease. All women in the two control groups had regular menstrual cycles. Clinicodemographic information for the three groups is summarized in Table 1.
Table 1Clinicodemographic Data for Control Subjects and Patients with Endometriosis Enrolled in This Study
All study participants had visited the outpatient Department of Obstetrics and Gynecology of the Kaohsiung Medical University Hospital. Exclusion criteria were the presence of any autoimmune disease, allergic disease, malignancy, or hepatitis B virus or hepatitis C virus infection, or any medical treatment or surgery within 3 months before the study-related surgery.
Venous blood (3 to 5 mL) was drawn from each study participant into a 5-mL sterile syringe and immediately transferred into a sterile clot tube. After the clotting, samples were centrifuged to obtain serum. The serum obtained from each sample was aliquoted and frozen at −20°C until cell culture or cytokine detection.
Generation of Monocyte-Derived DCs
Peripheral blood mononuclear cells were isolated from male volunteers (n = 5; mean age, 25 ± 3 years) after informed consent. Circulating CD14+ monocytes were purified using a human CD14 selection kit (STEMCELL Technologies, Vancouver, BC, Canada). The purity of isolated monocytes was ≥96%. CD14+ monocytes were differentiated into DCs in RPMI 1640 medium supplemented with 800 U/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), 500 U/mL recombinant human IL-4 (R&D Systems, Minneapolis, MN), 10% serum from each study subject, 2 mmol/L l-glutamine, 0.5 mmol/L sodium pyruvate, 50 μmol/L nonessential amino acids, 50 μmol/L 2-mercaptoethanol, 10 mmol/L HEPES (Gibco; Life Technologies, Carlsbad, CA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen; Life Technologies). After 6 days of culture, the monocyte-derived DCs (moDCs) were washed and either left unstimulated or stimulated with 1 μg/mL lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO) for another 24 hours. Cells were harvested for phenotypic analysis using flow cytometry; culture supernatants were used for cytokine pattern determination with ELISA (R&D Systems).
The moDCs were stained with the following fluorochrome-conjugated monoclonal antibodies (mAbs), or their corresponding isotype controls, purchased from BD Biosciences (San Jose, CA): anti–CD40-FITC (5C3), anti–CD86-PE (2331), anti–CD209-PerCP-Cy5.5 (DCN46), anti–CD11c-APC (B-lym6), anti–CD80-FITC (L307.4), anti–DEC205-PerCP (MG38), anti–HLA-DR (L243), and anti–CD83-APC (HB15e). All antibodies were used at optimal concentrations. Cells were analyzed with an LSR II flow cytometer (BD Biosciences) and FlowJo software version 10.0.5 (Tree Star, Ashland, OR).
Mice and the Surgical Endometriosis Model
All animal studies were conducted in accordance with the Kaohsiung Medical University guidelines for animal use and care (IACUC no. 99123). IL-10GFP mice on the C57BL/6 background were obtained from the Jackson Laboratory (Bar Harbor, ME) and were maintained at the Animal Center of Kaohsiung Medical University in a pathogen-free facility. Female control or IL-10GFP mice at the age of 6 to 10 weeks were used for surgery.
We established the endometriosis model as described previously,
with some modifications. In brief, the uterine horns from syngeneic female mice were opened longitudinally and removed using a dermal biopsy punch. Intraperitoneal endometrial lesions were surgically induced by suturing four identically sized uterine tissue samples to each side of the abdominal wall. To examine the role of IL-10, we injected each mouse with an anti–IL-10 blocking mAb (20 μg/mL, 20 μL/lesion) (BD Biosciences) or recombinant murine IL-10 (10 or 100 U/mL, 20 μL/lesion) (PeproTech, Rocky Hill, NJ) into the peritoneum under each transplanted tissue on the right side and corresponding controls on the left side. At days 3, 21, and 28 after surgery, the lesions were collected, and the lesion area was measured using ImageJ version 1.46r (NIH, Bethesda, MD) image analysis software. For histological analysis, formalin-fixed sections were stained with H&E. Images were acquired and analyzed with a TissueFAXS cell analysis system (TissueGnostics, Tarzana, CA).
Analysis of IL-10–Secreting Cells in Endometrial Lesions
To separate single cells from implanted tissues, the endometrial fragments from each mouse were pooled and incubated for 40 minutes in 0.05% trypsin, 0.53 mmol/L EDTA, 0.1% collagenase D, and 150 μg/mL DNase I and were mechanically disrupted with a gentleMACS dissociator (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. Each single-cell suspension was stained with anti–CD45-PE-Cy7 (30-F11), anti–CD11c-APC (N418), anti–PDCA-1-PE (ebio927), anti–CD3-APC (145-2C11), or anti–B220-PE (RA3-6B2) and analyzed with an LSR II flow cytometer (BD Biosciences).
Differences among the clinical information of study participants were analyzed using analysis of variance followed by a post hoc Scheffé’s multiple comparison test. Differences between any two groups in cytokine levels, surface markers, and lesion sizes were analyzed using the nonparametric U-test. Differences were considered significant at P < 0.05.
No significant differences in age, weight, or height were found among the healthy control subjects, disease control subjects, and patients with endometriosis; as expected, gravidity and parity were much lower in the endometriosis group than in the control group. Low gravidity and low parity are associated with risk of endometriosis.
To examine whether IL-10 overexpression is associated with development of endometriosis, we measured the levels of IL-10, as well as IL-12 and IL-6, in serum of study participants. In contrast to the anti-inflammatory activity of IL-10, IL-12 promotes the differentiation of Th1 cells, which guide immune effectors to clear unwanted cells and debris in an antigen-specific inflammatory process.
Increased IL-10 levels in serum were observed in patients with endometriosis, but not in disease control subjects or healthy control subjects (Figure 1). Similar levels of IL-6 and undetectable levels of IL-12 were found in all three study groups (data not shown).
Patient Serum-Conditioned moDCs Exhibit a Tolerogenic Phenotype
To examine whether serum IL-10 exerts suppressive activity, we used DCs cultured in endometriosis patient serum–containing medium, because IL-10 modulates DCs to differentiate into cells with a tolerogenic phenotype.
we used moDCs from healthy male donors, to avoid the possibility that the DCs from female subjects might be in a different state of maturity. We analyzed cytokine levels in supernatants obtained after culturing male monocytes with 10% serum from study participants during differentiation induced by GM-CSF plus IL-4. The moDCs cultured with serum from patients with endometriosis spontaneously secreted more IL-10 and IL-6 (Figure 2A). The moDCs stimulated with LPS and serum from patients with endometriosis expressed less IL-12 than when serum from healthy control subjects was used (Figure 2B). In contrast, the level of IL-6 expressed by moDCs in the presence of LPS was similar across the three groups. These findings indicate that patient serum–conditioned DCs produced higher levels of the anti-inflammatory cytokine IL-10 but lower levels of the inflammatory cytokine IL-12.
Next, we analyzed the phenotypic difference in moDCs conditioned with serum from endometriosis patients or control subjects. The moDCs conditioned with serum from healthy control subjects expressed high levels of CD86, HLA-DR (MHC class II molecule), and CD80 in response to LPS stimulation, representing the mature phenotype of moDCs (Figure 3). In contrast, moDCs conditioned with serum from patients with endometriosis exhibited reduced levels of CD86 and HLA-DR and decreased percentages of CD86high and HLA-DRhigh moDCs. The levels of other DC-associated molecules, including CD40, CD80, CD83, CD205, and CD209, were similar across the three study groups (data not shown). These findings indicate that patient serum–conditioned moDCs were resistant to LPS-induced maturation, which suggests a tolerogenic property of patient serum–conditioned DCs.
Local IL-10 Activity Enhances Development of Endometrial Lesions
Next, we examined whether IL-10 is involved in the development of endometriosis, using a mouse model of surgically induced endometriosis. Anti–IL-10 blocking mAb or its corresponding isotype control was locally injected into the sutured explants in mice that had undergone surgically induced endometriosis. Anti–IL-10 treatment at the beginning of lesion establishment significantly inhibited the growth of endometrial lesions at different time points, from day 3 to day 28 (Figure 4, A and B). In contrast, recombinant IL-10 administration in conjunction with lesion establishment significantly increased the development of endometrial lesions (Figure 4C). These findings suggested that low IL-10 activity may enhance the inflammatory response against the ectopic endometrium, whereas high IL-10 expression may suppress immunity and lead to the development of endometriosis.
Plasmacytoid Dendritic Cells in Endometrial Lesions Comprise the Major IL-10–Producing CD45+ Cell Subset
Next, we clarified the type of cell that secretes IL-10 in endometrial lesions. Many different types of immune cells can secrete IL-10 in the context of inflammation, including CD11c+ DCs, Th2 cells, type 1 regulatory T cells, Foxp3+ regulatory T cells, B cells, and macrophages.
Indeed, we observed that a substantial number of mononuclear cells had infiltrated into endometrial lesions (Figure 5). To easily track and identify IL-10–secreting cells in vivo, we used IL-10GFP mice to establish the endometriosis model. At 20 or 30 days after surgery, we used multiparameter flow cytometry to analyze the composition of immune cells in single-cell suspension that had infiltrated into the endometrial lesions. Approximately 20% and 40% of cells in the lesions secreted IL-10 at 20 and 30 days after surgery, respectively (Figure 6, A–D). Among the IL-10–secreting cells, approximately half were CD45+ immune cells; the remainder were CD45− nonimmune cells. In addition, CD45+ immune cells contained approximately 10% conventional DCs (cDCs) (CD11c+PDCA-1−) and 40% plasmacytoid DCs (pDCs) (CD11c+PDCA-1+) in the lesions (Figure 6, E–G). Interestingly, among the IL-10–secreting CD45+ immune cells, approximately 75% were pDCs at both 20 and 30 days after surgery (Figure 6, H and I). These findings suggested that local IL-10–secreting pDCs, at least in part, play an important role in the development of endometriosis.
In the 1920s, John A. Sampson provided the first theory for the pathogenesis of endometriosis, in terms of retrograde menstruation.
were the first to demonstrate deficient cellular immunity in patients with endometriosis. Since then, functional changes have been observed in several immunological components of the peritoneal fluid, as well as in the serum, of women with endometriosis.
of women with endometriosis. In support of such observations, in the present study, increased IL-10 levels were found in serum from patients with endometriosis, and patient serum–conditioned moDCs exhibited a semimature phenotype in response to LPS stimulation, which suggests dysregulated IL-10 activity in endometriosis. In the mouse model of surgically induced endometriosis, blockage of IL-10 activity decreased the size of the endometrial lesions, whereas IL-10 administration enhanced the growth of endometriosis. To our knowledge, this is the first study to demonstrate an adverse effect of IL-10 overexpression on the development of endometriosis in a mouse model. This is also the first study to identify innate pDCs as IL-10–secreting cells in the microenvironment of endometriosis. These findings suggest that overexpression of IL-10 or high IL-10 activity may suppress cell-mediated immune responses and then promote ectopic implantation of endometrial cells.
Several studies have shown that IL10 gene promoter polymorphisms are associated with the risk of endometriosis. Some studies have shown that the frequency of the C allele at −819 or −592 in the IL10 promoter is significantly increased in patients with endometriosis, compared with control subjects.
Recently, a meta-analysis showed that IL10 −592 A/C polymorphisms are associated with a significantly increased risk of endometriosis in the Asian population in all genetic models and allele contrasts.
These conflicting results may be due to the different ethnic origins and different scales in the study populations. In addition, a functional study of promoter polymorphisms should be performed in a microenvironment in the context of endometriosis, because gene regulation occurs in a cell type–specific and tissue-specific manner.
Elucidation of the mechanism underlying how human IL10 promoter polymorphisms and IL-10 protein expression lead to endometriosis requires further detailed investigation.
In the present study, we found that an abundance of IL-10–secreting pDCs (IL-10GFPCD11c+PDCA-1+) infiltrate into the endometrial lesions. This observation is in accord with previous findings showing that infiltrated CD11c+ DCs exhibit an immature phenotype (MHC class IIlow). The infiltrated CD11c+ DCs express VEGFR2 and are associated with increased angiogenesis.
we speculate that infiltrating IL-10–secreting pDCs may participate in the development of endometriosis, at least in part, by promoting angiogenesis or suppressing anti-self responses in the lesions. In support of this idea, depletion of IL-10 activity inhibited lesion development, whereas enhancement of IL-10 levels increased the size of lesions (Figure 4). Elucidation of the detailed mechanism involved in the pDC–IL-10 axis in the pathogenesis of endometriosis requires further investigation.
IL-10 plays a critical role in regulating physiological responses, including dampening immunity to pathogens in the context of infection, maintenance of immune tolerance,
IL-10 is secreted by a wide variety of cell types, including both immune (CD45+) and nonimmune (CD45−). In accord, we observed that approximately 50% of IL-10–secreting cells were immune cells, with the remainder being nonimmune cells (Figure 6). To our knowledge, this is the first report of IL-10 production in local pDCs in the context of endometriosis. Because others have reported that pDCs do not secrete IL-10 in response to microbial products,
our novel observation suggests that pDCs control endometrial inflammation in response to endogenous danger signals, such as local apoptotic cells. The roles of pDCs in the anti-inflammatory response and the pathogenesis of endometriosis should be explored in greater depth.
We thank the Center for Resources, Research and Development of Kaohsiung Medical University for providing the LSR II and TissueFAXS system.
J.-L.S. and E.M.T. conceived and designed experiments; Y.C., P.L.C., T.H.H., E.H., Y.C.C., and Y.F.C. performed experiments and analyzed data; all authors were involved in writing the paper and had final approval of the submitted and published versions.
Supported by grants NSC-99-2628-B-037-009-MY3, NSC100-2314-B-037-043, NSC 102-2628-B-037-011-MY3 from the National Science Council (Taiwan), and by grants KMUH101-1R27, KMUH100-0R24, KMUH 99-9I04, and KMUH 99-9R30 from Kaohsiung Medical University Hospital.
J.-L.S. and Y.C. contributed equally to this work.