Systemic Inflammation Induced by microRNAs: Endometriosis-Derived Alterations in Circulating microRNA 125b-5p and Let-7b-5p Regulate Macrophage Cytokine Production

Systemic Inflammation Induced by microRNAs: Endometriosis-Derived Alterations in Circulating... Abstract Context Endometriosis is characterized by aberrant inflammation. We previously reported increased levels of microRNA (miRNA) 125b-5p and decreased levels of miRNA Let-7b-5p in serum of patients with endometriosis. Objective Determine the regulatory function of miRNAs 125b-5p and Let-7b-5p on production of proinflammatory cytokines in endometriosis. Design Case-control study. Setting University hospital. Patients Women with (20) and without (26) endometriosis; human U937 macrophage cell line. Intervention Sera were collected from surgically diagnosed patients and differentiated U937 cells that were transfected with miRNAs 125b-5p and Let-7b-5p mimics and inhibitor. Main Outcome Measures Enzyme-linked immunosorbent assay for tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8, and IL-1β levels and quantitative real-time polymerase chain reaction for expression of miRNAs 125b-5p and Let-7b-5p in sera of patients with and without endometriosis. Transfected macrophages were evaluated for expression of inflammatory cytokines, intracellular production, and secretion of these cytokines. Results We noted substantial elevation of TNF-α, IL-1β, and IL-6, marked upregulation of miRNA 125b, and considerable downregulation of Let-7b in sera of patients with endometriosis vs control. There was a positive correlation between miRNA 125b levels and TNF-α, IL-1β, and IL-6 and a negative correlation between miRNA Let-7b levels and TNF-α in sera of patients with endometriosis. Transfection experiments showed a noteworthy upregulation of TNF-α, IL-1β, IL-6, and IL-8 in macrophages transfected with miRNA 125b mimic or Let-7b inhibitor. The secreted cytokine protein levels and intracellular imaging studies closely correlate with the messenger RNA changes. Conclusions Endometriosis-derived miRNAs regulate macrophage cytokine production that contributes to inflammation associated with this condition. Endometriosis is a common, inflammatory disease characterized by the growth of ectopic endometrial tissue outside the uterus (1, 2). It affects 5% to 15% of reproductive-age women and is present in as many as 30% to 50% of patients with infertility and/or pain (1, 3–5). Additionally, endometriosis has considerable social and psychological effects, with 63% of women reporting serious negative impact on their quality of life (6, 7). Societal costs for the treatment of endometriosis exceed $22 billion in the United States (8). Owing to the complexity of its development and symptoms, this disease has been widely studied; however, its systemic effects are still poorly understood (9, 10). An area of great significance in endometriosis is the increase in inflammatory activity and its implications in the pathogenesis of this condition (2, 11). The role of immune system and its response to endometriosis has been studied extensively. The endometriosis-associated inflammatory reaction has been described in previous studies, demonstrating the relative increase in the number and sensitivity to activation of macrophages and concurrent increase in inflammatory cytokines (12–15). Cytokines are distinct proteins that perform a fundamental role in controlling different cellular functions such as proliferation, chemotaxis, adhesion, morphological changes, and angiogenesis (13, 16). They play a crucial role in cell-to-cell communication within the immune system (9). Alterations in this immunological process is both local and systemic, and the disruption in this dynamic interaction contributes to the progression of the condition (10, 17, 18). Several cytokines have been associated with the evolution of endometriosis (19). Tumor necrosis factor-α (TNF-α) has potent inflammatory, cytotoxic, and angiogenic potential (20). Interleukin (IL)-6 promotes endometrial cell growth and is a potent stimulator of the vascular endothelial growth factor gene (21). IL-8 stimulates cell proliferation and has a role in endometriosis as an autocrine regulator of endometrial cell growth (22). It has likewise been implicated as a prominent cytokine involved in angiogenesis (23). IL-1β is a major proinflammatory cytokine secreted by macrophages that plays a vital role in endometriosis pathogenesis by orchestrating interaction between the microenvironment of the peritoneal cavity (24). Several studies have confirmed the increased concentrations of IL-1β, IL-6, and TNF-α in women with endometriosis compared with controls (14, 25, 26). Consistent with that finding, peritoneal macrophages secrete more IL-8, IL-10, and TNF-α in patients with endometriosis compared with healthy controls (25, 27). The mechanism by which endometriosis leads to systemic inflammation and macrophage alteration is still poorly characterized. MicroRNAs (miRNAs) are short, 18- to 22-nucleotide–size, noncoding RNAs that act as posttranscriptional modulators of gene expression (28–31). Circulating miRNAs are of interest because they are stable allowing for potential use as reproducible noninvasive biomarkers for various pathologies (32–34). miRNAs are involved in the pathogenesis of endometriosis (33–35), and several reports, including our own, have demonstrated the differential expression of miRNAs in the serum of patients with endometriosis (32, 36, 37). Of particular interest are two miRNAs: 125b-5p, which was found to be highly upregulated (36), and Let-7b-5p, which was significantly downregulated (32, 35) in patients with severe endometriosis. Changes in circulating miRNAs can regulate gene expression in cells remote from the location and cell type in which they are produced. This form of extensive cell-to-cell communication may serve as a mechanism by which endometriosis alters cytokine production in macrophages systemically. To investigate the potential role of these two miRNAs in the immunological response, particularly endometriosis-associated inflammation, we aimed to determine the levels of proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8 in macrophages with enforced overexpression or repression of these miRNAs. We expect that overexpression of miRNA 125b and inhibition of Let-7b will induce expression and subsequent production and secretion of these inflammatory cytokines. In addition, this study investigates the serum proinflammatory cytokine profile in patients with endometriosis compared with healthy controls. This allowed us to correlate the effects on macrophages with the cytokine milieu in patients with the disease. Materials and Methods Cell culture The human myelomonocytic cell line U937 was a generous gift from Robert Mean (Department of Pathology, Yale School of Medicine). Cells were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum and antibiotics (100 IU/mL penicillin and 50 μg/mL streptomycin) and maintained at 37°C in 5% CO2 atmosphere; media was changed every 48 to 72 hours. Differentiation to mature macrophages was carried out by treating with phorbol 12-myristate 13-acetate at 100 ng/mL of the cell suspension (1 × 106 cells/mL). After 48 hours, adherent cells (macrophages) were washed twice with ice-cold phosphate-buffered saline (PBS) solution and allowed to grow for 48 hours in fresh growth medium. In vitro experiments with U937 cell line were carried out using subculture passage 3. Transfection of miRNAs Transfection was carried out in 48-hour-old macrophages in a six-well plate (2 × 105 cells per well) without antibiotics using Lipofectamine™ RNAiMAX (Invitrogen) according to the manufacturer’s protocol. Briefly, the transfection medium contains 50 nmol miRNA and 3.5 μL Lipofectamine™ RNAiMAX in a volume of 500 μL of Opti-MEM® (Gibco) separately, without serum, and was allowed to sit for 5 minutes at room temperature, then combined, mixed gently, and incubated at room temperature for 20 minutes before adding to the well with cells and cultured overnight. Transfection medium was then replaced with fresh growth medium containing 10% fetal bovine serum and antibiotics. Each transfection condition was carried out under sterile conditions with respective controls in duplicate wells. The transfected macrophages were processed for experiments after 48 hours posttransfection. Quantitative real-time polymerase chain reaction Total RNA was extracted from 48-hour posttransfected macrophages using TRIzol reagent (Invitrogen) as described in the manual. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR Green (Bio-Rad) and optimized in the MyiQ Single Color Real-Time PCR Detection System (Bio-Rad). The specificity of the amplified transcript and absence of primer-dimers was confirmed by a melting curve analysis. All products yielded the predicted melting temperature. Gene expression was normalized to the expression of human β-actin for each sample. Relative messenger RNA (mRNA) expression for each gene was calculated using the comparative cycle threshold method, also known as the 2−ΔΔC(T) method (38). All experiments were carried out in triplicate, and nuclease-free water was used as a negative control replacing the cDNA template. Primers were obtained from the Keck Oligonucleotide Synthesis Facility (Yale University). Primer sequences for all the genes are: TNF-α forward, 5′-CACCATGAGCACTGAAAGCA-3′, and reverse, 5′-GCTCTTGATGGCAGAGAGGAG-3′; IL-1β forward, 5′-TTCGAGGCACAAGGCACAAC-3′, and reverse, 5′-CTGGAAGGAGCACTTCATCTGT-3′; IL-6 forward, 5′-ACCCCCAGGAGAAGATTCCA-3′, and reverse, 5′-GTCTTCCCCCACACCAAGTT-3′; IL-8 forward, 5′-TCTGTGTGAAGGTGCAGTTTTG-3′, and reverse, 5′-GGGGTGGAAAGGTTTGGAGT-3′; and β-actin forward, 5′-GAAGATCAAGATCATTGCTC-3′, and reverse, 5′-AACGCAACTAAGTCATAGTC-3′. Macrophage stimulation by lipopolysaccharide and cytokine protein measurements by enzyme-linked immunosorbent assay Transfected macrophages were stimulated with lipopolysaccharide (LPS) 48 hours posttransfection (Sigma-Aldrich) at different concentrations and durations (10, 100, and 1000 ng/mL for 6, 12, and 24 hours), and conditioned media was collected and stored in aliquots at −80°C until use for the determination of secreted cytokine proteins by enzyme-linked immunosorbent assay (ELISA) kit (Affymetrix) according to the manufacturer’s instructions. Optical density was read in a microplate reader at 450 nm, and a standard curve was subsequently generated to extrapolate cytokine concentration in the samples. Kit sensitivity was ∼4 pg/mL for TNF-α and 2 pg/mL for IL-1β, IL-6, and IL-8 with standard curve ranges of 4 to 500, 2 to 150, 2 to 200, and 2 to 250 pg/mL, respectively. Immunocytochemistry Transfected macrophages in culture chambers were fixed by incubation in 100% chilled methanol at room temperature for 5 minutes and then washed three times with ice-cold PBS. Permeabilization was carried out by incubation in PBS containing 0.25% Triton X-100 and subsequently washed with PBS three times for 5 minutes. Blocking was done with the use of 1% bovine serum albumin (BSA) in PBS plus 0.1% Tween 20 for 30 minutes. Immunostaining was done by incubation in the diluted primary antibody in 1% BSA in PBS plus 0.1% Tween 20 in a humidified chamber overnight at 4°C. Primary antibody (TNF-α, IL-1β, IL-6, and IL-8; Santa Cruz Biotechnology, Inc) use and dilution for all cytokines were based on the antibody data sheet recommendations. Slides were washed and incubated with the secondary antibody in 1% BSA in the dark at room temperature for 1 hour. The secondary antibody solution was decanted, and slides were washed three times with PBS for 5 minutes in the dark. Counterstaining for 1 minute with 4′,6-diamidino-2-phenylindole was done, and slides were mounted with a coverslip and a drop of mounting media. Study population Institutional Review Board approval was obtained from Yale School of Medicine (New Haven, CT) for using human samples. Written informed consent was obtained from subjects admitted to the hospital and undergoing laparoscopy or laparotomy for suspected benign indications such as pelvic masses, pelvic pain, infertility, and endometriosis. Inclusion criteria included women who were aged 19 to 49 years. Exclusion criteria included postmenopausal patients, hyperplasia or polyps, malignancy, autoimmune disease, and cardiovascular disease. All stages of endometriosis as well as untreated and treated subjects were included to provide a full spectrum of disease resulting in varied miRNA levels. The total number of women from whom serum was collected was 49. Among 49 women, 3 were excluded due to an unexpected comorbidity, whereas 46 were included in the study and divided into two groups as follows: the endometriosis group has 20 women with surgically diagnosed and histologically verified endometriosis, and the control group has 26 women, who were visually verified to be free from endometriosis during the surgery. The phase of the menstrual cycle was determined based on the patient’s menstrual history and last menstrual period. Cytokine measurements in human serum from patients with endometriosis by ELISA Whole-blood samples were collected in a sterile condition, kept at room temperature for 30 minutes, and the serum was obtained by centrifuging the blood clot at 2000g for 15 minutes at 4°C. The serum was immediately aliquoted for storage at −80°C until needed for ELISA, avoiding freeze-thaw cycles. Quantification of cytokine levels in individual samples was done twice each in duplicate well by ELISA (Affymetrix) following the manufacturer’s protocol. Quantitative real-time polymerase chain reaction (qRT-PCR) for miRNAs in sera Total miRNA was extracted from 200 μL of serum sample using the miRNeasy Mini Kit (Qiagen) and reverse transcribed using the TaqMan Advanced miRNA cDNA synthesis Kit (Thermo Fisher Scientific) according to the manufacturers’ specifications. miRNA levels were quantified with qRT-PCR using SYBR Green (Bio-Rad) and optimized in the MyiQ Single Color Real-Time PCR Detection System (Bio-Rad). Gene expression was normalized to the expression of human U6 small nuclear RNA to determine relative miRNA expression for each sample. Relative expression was calculated using the comparative cycle threshold method, also known as the 2−ΔΔC(T) method (38). All determinations were done twice, each using triplicate wells. Primers for miRNAs and the U6 genes were obtained from the Keck Oligonucleotide Synthesis Facility (Yale University). Primer sequences are as follows: miRNA-125b-5p forward, 5′-UCCCUGAGACCCUAACUUGUGA-3′; miRNA-Let7b-5p forward, 5′-TGAGGTAGTAGGTTGTGTGGTT-3′; and U6 forward, 5′-CTCGCTTCGGCAGCACA-3′. Statistical analysis Analysis was performed using Prism software version 7.0 (GraphPad Software, Inc.) and the Mann-Whitney U test. For correlation statistics between the relative expression of miRNAs and proinflammatory cytokine levels in serum, we made use of the Pearson correlation coefficient. Differences were considered as statistically significant for P values <0.05. Results Increased serum levels of proinflammatory cytokines in women with endometriosis We first examined the proinflammatory cytokine levels in women with histologically confirmed endometriosis and a comparison of serum cytokine concentrations between women with endometriosis and disease-free controls. The mean age [± standard deviation (SD)] of the women with endometriosis was 35.35 ± 8.66 years, which was not significantly different from the mean age of patients in the control group, 33.05 ± 7.5 years (P > 0.05). The mean body mass index (± SD) did not differ between groups (endometriosis group: 29.55 ± 8.76; control group: 26.66 ± 5.59). Supplemental Table 1 shows a summary of patient characteristics, including phase of the menstrual cycle and stage of endometriosis. As shown in Fig. 1, serum TNF-α (P < 0.001), IL-1β (P < 0.001), and IL-6 (P < 0.001) levels were significantly higher in the endometriosis group than in the control group, and IL-8 levels showed a trend toward increased secretion. Supplemental Table 2 shows the mean concentration of cytokines based on the phase of the menstrual cycle. There was no noteworthy difference in cytokine levels between phases in each subject group (i.e., control proliferative phase vs secretory phase or endometriosis proliferative phase vs secretory phase). The same considerable difference in cytokine concentration of TNF-α, IL-1β, and IL-6 between control and endometriosis groups matched by phase of menstrual cycle was observed (i.e., control proliferative phase vs endometriosis proliferative phase). We subsequently measured the levels of endometriosis-derived miRNA in the serum samples. miRNA 125b was significantly elevated in serum of patients with endometriosis compared with controls (P = 0.002), whereas miRNA Let-7b showed a considerable downregulation (P < 0.001), as shown in Fig. 2. Figure 1. View largeDownload slide Quantification of cytokines by ELISA in serum from women with and without endometriosis. The control group was comprised of 26 subjects, whereas the endometriosis group had 20 subjects. Determination of cytokine concentration for every serum sample was carried out twice in duplicate. Data points represent individual mean concentration and group mean ± SD. Distribution of cytokine serum levels showed a statistically significant increase in the level of TNF-α, IL-1β, and IL-6 in patients with endometriosis compared with control. *Denotes statistical significance (P < 0.05) vs control group. Figure 1. View largeDownload slide Quantification of cytokines by ELISA in serum from women with and without endometriosis. The control group was comprised of 26 subjects, whereas the endometriosis group had 20 subjects. Determination of cytokine concentration for every serum sample was carried out twice in duplicate. Data points represent individual mean concentration and group mean ± SD. Distribution of cytokine serum levels showed a statistically significant increase in the level of TNF-α, IL-1β, and IL-6 in patients with endometriosis compared with control. *Denotes statistical significance (P < 0.05) vs control group. Figure 2. View largeDownload slide Expression levels of miRNA in serum of women with and without endometriosis by qRT-PCR. The control group was comprised of 26 subjects, whereas the endometriosis group had 20 subjects. Experiments were carried out twice each time in triplicate. Data represent mean ± SD. There is a statistically significant upregulation of miRNA-125b and a significant downregulation in patients with endometriosis compared with controls. *Denotes statistical significance (P < 0.05) vs control group. Figure 2. View largeDownload slide Expression levels of miRNA in serum of women with and without endometriosis by qRT-PCR. The control group was comprised of 26 subjects, whereas the endometriosis group had 20 subjects. Experiments were carried out twice each time in triplicate. Data represent mean ± SD. There is a statistically significant upregulation of miRNA-125b and a significant downregulation in patients with endometriosis compared with controls. *Denotes statistical significance (P < 0.05) vs control group. Correlation between expression levels of miRNAs and serum levels of cytokines in patients with endometriosis Although we have established the increase in amount of proinflammatory cytokines as well as determined the relative expression of endometriosis-derived miRNAs 125b and Let-7b in serum of patients with endometriosis compared with control, we next evaluated the correlation between the miRNA levels and cytokine concentration. Figure 3(a) shows the Pearson correlation scatter plot illustrating a positive correlation between expression levels of miRNA 125b-5p and concentration of TNF-α (P < 0.001; r = 0.759), IL-1β (P < 0.001; r = 0.761), and IL-6 (P < 0.001; r = 0.961) in serum of patients with endometriosis. IL-6 showed the highest positive correlation. In contrast, Fig. 3(b) shows the negative correlation between expression levels of miRNA Let-7b and concentration of TNF-α (P = 0.017; r = −0.53) in serum of patients with endometriosis. Figure 3. View largeDownload slide Pearson correlation scatter plots. (a) Positive correlation between expression levels of miRNA 125b-5p and TNF-α (P < 0.001), IL-1β (P < 0.001), and IL-6 (P < 0.001). IL-6 showed the highest positive correlation (r = 0.961). (b) Negative correlation between expression levels of miRNA Let-7b-5p and TNF-α (P = 0.017). IL-1β and IL-6 showed a trend toward a negative correlation. Pearson correlation coefficient (r) as shown for serum of subjects (n = 20) with endometriosis. Figure 3. View largeDownload slide Pearson correlation scatter plots. (a) Positive correlation between expression levels of miRNA 125b-5p and TNF-α (P < 0.001), IL-1β (P < 0.001), and IL-6 (P < 0.001). IL-6 showed the highest positive correlation (r = 0.961). (b) Negative correlation between expression levels of miRNA Let-7b-5p and TNF-α (P = 0.017). IL-1β and IL-6 showed a trend toward a negative correlation. Pearson correlation coefficient (r) as shown for serum of subjects (n = 20) with endometriosis. Increased expression of proinflammatory cytokines in macrophages transfected with miRNA-125b mimic and Let-7b inhibitors We have previously demonstrated a notable increase in circulating miRNA 125b and decrease in miRNA Let-7b in women with endometriosis (35). In this study, we investigated changes in the relative expression of cytokines resulting from overexpression of miRNAs 125b and Let-7b inhibitor in macrophages. As shown in Fig. 4, qRT-PCR was used to demonstrate that there was a statistically marked increase in mRNA expression of cytokines TNF-α (2.66-fold; P < 0.001), IL-1β (1.86-fold; P = 0.0019), IL-6 (1.51-fold; P ≤ 0.001), and IL-8 (1.70-fold; P = 0.0078) in macrophages transfected with miRNA 125b-5p mimic. Transfection with miRNA Let-7b mimic caused noteworthy decrease in expression of TNF-α (0.58-fold difference; P < 0.001), IL-8 (0.66-fold difference; P = 0.0019), and a trend toward downregulation for IL-1β (0.88-fold difference), and IL-6 (0.74-fold difference). Although miRNA 125 is increased, Let-7b levels are decreased in women with endometriosis; we therefore specifically examined the effect of inhibiting this miRNA. The miRNA Let-7b inhibitor transfection caused a considerable increase in expression of TNF-α (1.5-fold, P = 0.002), IL-1β (2.94-fold; P = 0.002), IL-6 (1.59-fold; P = 0.006), and IL-8 (6.19-fold; P = 0.008). Figure 4. View largeDownload slide Transfection of miRNAs in macrophages altered cytokine expression. miRNA-125b is increased in the circulation of women with endometriosis, whereas Let-7b is decreased. Cytokine mRNA expression in macrophages transfected with miRNA-125b and Let-7b mimic and inhibitor were assessed by qRT-PCR and presented as fold change relative to the control (scramble). Increased relative expression of TNF-α (2.66-fold; P < 0.001), IL-1β (1.86-fold; P = 0.0019), IL-6 (1.51-fold; P < 0.001), and IL-8 (1.70-fold; P = 0.0078) in macrophages transfected with miRNA 125b-5p; decreased expression of TNF-α (0.58-fold; P < 0.001), and IL-8 (0.66-fold; P = 0.0019) in group transfected with miRNA Let-7b-5p; and increased expression of TNF-α (1.5-fold; P = 0.002), IL-1β (2.94-fold; P = 0.002), IL-6 (1.59-fold; P = 0.006), and IL-8 (6.19-fold; P = 0.008) in macrophages transfected with Let-7b-5p inhibitor shown. Bars represent the mean ± standard error (SE) of three individual experiments, each performed in triplicate. *Denotes statistical significance (P < 0.05) vs control. Figure 4. View largeDownload slide Transfection of miRNAs in macrophages altered cytokine expression. miRNA-125b is increased in the circulation of women with endometriosis, whereas Let-7b is decreased. Cytokine mRNA expression in macrophages transfected with miRNA-125b and Let-7b mimic and inhibitor were assessed by qRT-PCR and presented as fold change relative to the control (scramble). Increased relative expression of TNF-α (2.66-fold; P < 0.001), IL-1β (1.86-fold; P = 0.0019), IL-6 (1.51-fold; P < 0.001), and IL-8 (1.70-fold; P = 0.0078) in macrophages transfected with miRNA 125b-5p; decreased expression of TNF-α (0.58-fold; P < 0.001), and IL-8 (0.66-fold; P = 0.0019) in group transfected with miRNA Let-7b-5p; and increased expression of TNF-α (1.5-fold; P = 0.002), IL-1β (2.94-fold; P = 0.002), IL-6 (1.59-fold; P = 0.006), and IL-8 (6.19-fold; P = 0.008) in macrophages transfected with Let-7b-5p inhibitor shown. Bars represent the mean ± standard error (SE) of three individual experiments, each performed in triplicate. *Denotes statistical significance (P < 0.05) vs control. To confirm the intracellular changes in cytokine protein production, we then proceeded with immunostaining of the transfected macrophages. Fixed cells were visualized under a fluorescence microscope. Figure 5 gives the representative merged confocal images of the different cytokines in the three miRNA experimental groups and their corresponding scramble (control) sets. Macrophages transfected with miRNA 125b and Let-7b inhibitor showed significantly greater proportion of highly fluorescent cells for all four cytokines—TNF-α, IL-1β, IL-6, and IL-8. In contrast, miRNA Let-7b–transfected macrophages did not display any considerable difference from the controls. Figure 5. View largeDownload slide Confocal analysis of cytokine protein expression. Merged confocal images comparing different miRNA mimic or inhibitor transfection are shown. In each image, blue represents cell nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI); for each row, red indicates TNF-α, purple indicates IL-1β, yellow indicates IL-6, and green indicates IL-8. Images are representative of three random fields in each slide, with n = 4 in each group, done in two independent experiments. Scale bar, 100 μm. Macrophages transfected with miRNA 125b (elevated in endometriosis) and Let-7b (decreased in endometriosis) inhibitor showed a higher percentage of fluorescent cells for all four cytokines compared with their corresponding controls. Ratio of positive cells did not differ from the control in macrophages transfected with miRNA Let-7b. Figure 5. View largeDownload slide Confocal analysis of cytokine protein expression. Merged confocal images comparing different miRNA mimic or inhibitor transfection are shown. In each image, blue represents cell nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI); for each row, red indicates TNF-α, purple indicates IL-1β, yellow indicates IL-6, and green indicates IL-8. Images are representative of three random fields in each slide, with n = 4 in each group, done in two independent experiments. Scale bar, 100 μm. Macrophages transfected with miRNA 125b (elevated in endometriosis) and Let-7b (decreased in endometriosis) inhibitor showed a higher percentage of fluorescent cells for all four cytokines compared with their corresponding controls. Ratio of positive cells did not differ from the control in macrophages transfected with miRNA Let-7b. LPS increased the cytokine secretion in macrophages transfected with miRNA 125b mimic and Let-7b inhibitor Multiple cytokines are secreted from activated macrophages (39). Therefore, we stimulated the 48-hour posttransfected macrophages with LPS in varying doses and time intervals to know if translation of cytokines and subsequent secretion reflect mRNA changes. LPS dose and timing of maximal cytokine secretion of macrophages vary across literature (25, 40–43), but an optimal time point was set at 24 hours following a dose of 100 ng/mL for this study. Figure 6 shows ELISA results from conditioned media of transfected macrophages treated with 100 ng/mL LPS for 24 hours. A significant increase in TNF-α (P = 0.004), IL-1β (P = 0.016), IL-6 (P = 0.029), and IL-8 (P = 0.016) protein secretion in macrophages transfected with miRNA 125b. In contrast, the macrophages transfected with miRNA Let-7b showed a noted decrease in protein secretion of TNF-α (P = 0.0095), IL-1β (P = 0.016), and IL-6 (P = 0.029). Figure 6 also shows a substantial increase in secretion of all these cytokines in macrophages transfected with miRNA Let-7b inhibitor TNF-α (P = 0.016), IL-1β (P = 0.029), IL-6 (P = 0.016), and IL-8 (P = 0.004). Figure 6. View largeDownload slide LPS stimulation induces cytokine secretion after miRNA alterations associated with endometriosis. LPS stimulation (100 ng/mL) of transfected macrophages showing protein levels determined by ELISA. TNF-α, IL-1β, IL-6, and IL-8 were significantly higher in conditioned media of cells transfected with miRNA 125b and Let-7b inhibitor compared with the controls. Bars represent the mean ± standard error (SE) of three individual experiments, each performed in triplicate. Differences were considered as statistically significant for P values <0.05. *Denotes statistical significance between control mimic vs miRNA mimic; #denotes statistical significance between control inhibitor vs miRNA inhibitor. Figure 6. View largeDownload slide LPS stimulation induces cytokine secretion after miRNA alterations associated with endometriosis. LPS stimulation (100 ng/mL) of transfected macrophages showing protein levels determined by ELISA. TNF-α, IL-1β, IL-6, and IL-8 were significantly higher in conditioned media of cells transfected with miRNA 125b and Let-7b inhibitor compared with the controls. Bars represent the mean ± standard error (SE) of three individual experiments, each performed in triplicate. Differences were considered as statistically significant for P values <0.05. *Denotes statistical significance between control mimic vs miRNA mimic; #denotes statistical significance between control inhibitor vs miRNA inhibitor. Discussion Endometriosis has been associated with inflammation and increased inflammatory cytokine production. In this study, we confirm an elevation of several inflammatory cytokines in the circulation of women with endometriosis (14, 44). Endometriosis-associated inflammation is thought to be mediated in part by macrophages; increased macrophage activity is reflected by a rise in number and activation potential, resulting in an increase in secretion of cytokines and chemokines (45). However, it is not clear how endometriosis leads to the increased systemic inflammation. We demonstrate in this study that alterations in circulating miRNAs are one mechanism by which endometriosis causes immune dysfunction and inflammation. As these miRNAs are altered in the circulation of women with the disease, they can influence the cytokine expression of macrophages systemically, including areas remote from endometriosis. Alterations of circulating miRNA in endometriosis furthers the concept that endometriosis is a systemic disease. We have previously demonstrated systemic effects of endometriosis remote from the site of lesions. Although peritoneal endometriosis affects the uterus and endometrial receptivity, we have demonstrated that endometriosis placed under the skin of mice far from the uterus still affects the eutopic endometrium (46). Similarly, endometriosis can affect liver metabolism, leading to weight loss (47). Further endometriosis alters stem cell trafficking, leading to stem cell deficiency in the uterus and infertility (48, 49). All of these examples demonstrate that endometriosis is not a disease in which the effects are localized to the pelvis. The ability to systemically alter macrophage function and induce an inflammatory state adds to the list of widespread, whole-body effects of endometriosis. Endometriosis should be considered a disease with multiorgan involvement. Alterations in circulating miRNAs clearly induce inflammation and may well contribute substantially to the systemic manifestations of the disease. Let-7 family also targets the KRAS gene, and its loss may permit initiation of the progression of ectopic lesions and represent a link between inflammation and the tumor-like growth in endometriosis (32, 50–52). KRAS signaling leads to enhanced proliferation and invasion of cells, including many tumors (51). We have previously demonstrated a role for Let-7b regulation of KRAS in endometriosis (52). Similarly, Let-7 family also targets aromatase (53). Let-7 family may target multiple pathways that influence endometriosis and other processes in addition to the role described in this study. The exact role of Let-7b in the regulation of macrophages is still an ongoing area of research. A known target of importance is C/EBP-δ, which is a transcription factor required for sustained responsiveness to Toll-like receptor signaling (54). Our data clearly demonstrate that overexpression of miRNA Let-7b caused a marked decrease in the expression of the proinflammatory genes, whereas blocking of Let-7b caused a reciprocal increase in cytokine expression. These results are further confirmed by the data obtained for consistent protein pattern in intracellular protein production as well as secreted cytokine levels post–LPS stimulation in the respective transfection experiments. It is important to recognize that it is the collective effects of these miRNAs that determine the disease outcome. miRNAs each have multiple targets, and it is likely that the inflammatory cytokines identified in this study represent important, but not exclusive, targets of these miRNAs. The inhibition or stimulation of proinflammatory cytokines by these miRNAs identifies prospective novel targets for interventions in endometriosis. Restoration of normal peripheral miRNA signaling between cells and organs may well be a means to treat the systemic manifestations of this disease. miRNA mimics and inhibitors are exciting new therapeutics for endometriosis. In summary, circulating miRNA 125b-5p and Let-7b-5p function as regulators of the inflammatory response in endometriosis. They induce macrophage inflammatory cytokine production, defining a mechanism responsible for the systemic effects of this disease. The miRNAs with altered levels in endometriosis identified in this study have potential as both diagnostic tools and therapeutic targets. Abbreviations: BSA bovine serum albumin ELISA enzyme-linked immunosorbent assay IL interleukin LPS lipopolysaccharide miRNA microRNA mRNA messenger RNA qRT-PCR quantitative real-time polymerase chain reaction SD standard deviation TNF-α tumor necrosis factor-α. Acknowledgments Disclosure Summary: The authors have nothing to disclose. References 1. Giudice LC. Clinical practice. 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Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Clinical Endocrinology and Metabolism Oxford University Press

Systemic Inflammation Induced by microRNAs: Endometriosis-Derived Alterations in Circulating microRNA 125b-5p and Let-7b-5p Regulate Macrophage Cytokine Production

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Endocrine Society
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Copyright © 2018 Endocrine Society
ISSN
0021-972X
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1945-7197
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10.1210/jc.2017-01199
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Abstract

Abstract Context Endometriosis is characterized by aberrant inflammation. We previously reported increased levels of microRNA (miRNA) 125b-5p and decreased levels of miRNA Let-7b-5p in serum of patients with endometriosis. Objective Determine the regulatory function of miRNAs 125b-5p and Let-7b-5p on production of proinflammatory cytokines in endometriosis. Design Case-control study. Setting University hospital. Patients Women with (20) and without (26) endometriosis; human U937 macrophage cell line. Intervention Sera were collected from surgically diagnosed patients and differentiated U937 cells that were transfected with miRNAs 125b-5p and Let-7b-5p mimics and inhibitor. Main Outcome Measures Enzyme-linked immunosorbent assay for tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8, and IL-1β levels and quantitative real-time polymerase chain reaction for expression of miRNAs 125b-5p and Let-7b-5p in sera of patients with and without endometriosis. Transfected macrophages were evaluated for expression of inflammatory cytokines, intracellular production, and secretion of these cytokines. Results We noted substantial elevation of TNF-α, IL-1β, and IL-6, marked upregulation of miRNA 125b, and considerable downregulation of Let-7b in sera of patients with endometriosis vs control. There was a positive correlation between miRNA 125b levels and TNF-α, IL-1β, and IL-6 and a negative correlation between miRNA Let-7b levels and TNF-α in sera of patients with endometriosis. Transfection experiments showed a noteworthy upregulation of TNF-α, IL-1β, IL-6, and IL-8 in macrophages transfected with miRNA 125b mimic or Let-7b inhibitor. The secreted cytokine protein levels and intracellular imaging studies closely correlate with the messenger RNA changes. Conclusions Endometriosis-derived miRNAs regulate macrophage cytokine production that contributes to inflammation associated with this condition. Endometriosis is a common, inflammatory disease characterized by the growth of ectopic endometrial tissue outside the uterus (1, 2). It affects 5% to 15% of reproductive-age women and is present in as many as 30% to 50% of patients with infertility and/or pain (1, 3–5). Additionally, endometriosis has considerable social and psychological effects, with 63% of women reporting serious negative impact on their quality of life (6, 7). Societal costs for the treatment of endometriosis exceed $22 billion in the United States (8). Owing to the complexity of its development and symptoms, this disease has been widely studied; however, its systemic effects are still poorly understood (9, 10). An area of great significance in endometriosis is the increase in inflammatory activity and its implications in the pathogenesis of this condition (2, 11). The role of immune system and its response to endometriosis has been studied extensively. The endometriosis-associated inflammatory reaction has been described in previous studies, demonstrating the relative increase in the number and sensitivity to activation of macrophages and concurrent increase in inflammatory cytokines (12–15). Cytokines are distinct proteins that perform a fundamental role in controlling different cellular functions such as proliferation, chemotaxis, adhesion, morphological changes, and angiogenesis (13, 16). They play a crucial role in cell-to-cell communication within the immune system (9). Alterations in this immunological process is both local and systemic, and the disruption in this dynamic interaction contributes to the progression of the condition (10, 17, 18). Several cytokines have been associated with the evolution of endometriosis (19). Tumor necrosis factor-α (TNF-α) has potent inflammatory, cytotoxic, and angiogenic potential (20). Interleukin (IL)-6 promotes endometrial cell growth and is a potent stimulator of the vascular endothelial growth factor gene (21). IL-8 stimulates cell proliferation and has a role in endometriosis as an autocrine regulator of endometrial cell growth (22). It has likewise been implicated as a prominent cytokine involved in angiogenesis (23). IL-1β is a major proinflammatory cytokine secreted by macrophages that plays a vital role in endometriosis pathogenesis by orchestrating interaction between the microenvironment of the peritoneal cavity (24). Several studies have confirmed the increased concentrations of IL-1β, IL-6, and TNF-α in women with endometriosis compared with controls (14, 25, 26). Consistent with that finding, peritoneal macrophages secrete more IL-8, IL-10, and TNF-α in patients with endometriosis compared with healthy controls (25, 27). The mechanism by which endometriosis leads to systemic inflammation and macrophage alteration is still poorly characterized. MicroRNAs (miRNAs) are short, 18- to 22-nucleotide–size, noncoding RNAs that act as posttranscriptional modulators of gene expression (28–31). Circulating miRNAs are of interest because they are stable allowing for potential use as reproducible noninvasive biomarkers for various pathologies (32–34). miRNAs are involved in the pathogenesis of endometriosis (33–35), and several reports, including our own, have demonstrated the differential expression of miRNAs in the serum of patients with endometriosis (32, 36, 37). Of particular interest are two miRNAs: 125b-5p, which was found to be highly upregulated (36), and Let-7b-5p, which was significantly downregulated (32, 35) in patients with severe endometriosis. Changes in circulating miRNAs can regulate gene expression in cells remote from the location and cell type in which they are produced. This form of extensive cell-to-cell communication may serve as a mechanism by which endometriosis alters cytokine production in macrophages systemically. To investigate the potential role of these two miRNAs in the immunological response, particularly endometriosis-associated inflammation, we aimed to determine the levels of proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8 in macrophages with enforced overexpression or repression of these miRNAs. We expect that overexpression of miRNA 125b and inhibition of Let-7b will induce expression and subsequent production and secretion of these inflammatory cytokines. In addition, this study investigates the serum proinflammatory cytokine profile in patients with endometriosis compared with healthy controls. This allowed us to correlate the effects on macrophages with the cytokine milieu in patients with the disease. Materials and Methods Cell culture The human myelomonocytic cell line U937 was a generous gift from Robert Mean (Department of Pathology, Yale School of Medicine). Cells were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum and antibiotics (100 IU/mL penicillin and 50 μg/mL streptomycin) and maintained at 37°C in 5% CO2 atmosphere; media was changed every 48 to 72 hours. Differentiation to mature macrophages was carried out by treating with phorbol 12-myristate 13-acetate at 100 ng/mL of the cell suspension (1 × 106 cells/mL). After 48 hours, adherent cells (macrophages) were washed twice with ice-cold phosphate-buffered saline (PBS) solution and allowed to grow for 48 hours in fresh growth medium. In vitro experiments with U937 cell line were carried out using subculture passage 3. Transfection of miRNAs Transfection was carried out in 48-hour-old macrophages in a six-well plate (2 × 105 cells per well) without antibiotics using Lipofectamine™ RNAiMAX (Invitrogen) according to the manufacturer’s protocol. Briefly, the transfection medium contains 50 nmol miRNA and 3.5 μL Lipofectamine™ RNAiMAX in a volume of 500 μL of Opti-MEM® (Gibco) separately, without serum, and was allowed to sit for 5 minutes at room temperature, then combined, mixed gently, and incubated at room temperature for 20 minutes before adding to the well with cells and cultured overnight. Transfection medium was then replaced with fresh growth medium containing 10% fetal bovine serum and antibiotics. Each transfection condition was carried out under sterile conditions with respective controls in duplicate wells. The transfected macrophages were processed for experiments after 48 hours posttransfection. Quantitative real-time polymerase chain reaction Total RNA was extracted from 48-hour posttransfected macrophages using TRIzol reagent (Invitrogen) as described in the manual. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR Green (Bio-Rad) and optimized in the MyiQ Single Color Real-Time PCR Detection System (Bio-Rad). The specificity of the amplified transcript and absence of primer-dimers was confirmed by a melting curve analysis. All products yielded the predicted melting temperature. Gene expression was normalized to the expression of human β-actin for each sample. Relative messenger RNA (mRNA) expression for each gene was calculated using the comparative cycle threshold method, also known as the 2−ΔΔC(T) method (38). All experiments were carried out in triplicate, and nuclease-free water was used as a negative control replacing the cDNA template. Primers were obtained from the Keck Oligonucleotide Synthesis Facility (Yale University). Primer sequences for all the genes are: TNF-α forward, 5′-CACCATGAGCACTGAAAGCA-3′, and reverse, 5′-GCTCTTGATGGCAGAGAGGAG-3′; IL-1β forward, 5′-TTCGAGGCACAAGGCACAAC-3′, and reverse, 5′-CTGGAAGGAGCACTTCATCTGT-3′; IL-6 forward, 5′-ACCCCCAGGAGAAGATTCCA-3′, and reverse, 5′-GTCTTCCCCCACACCAAGTT-3′; IL-8 forward, 5′-TCTGTGTGAAGGTGCAGTTTTG-3′, and reverse, 5′-GGGGTGGAAAGGTTTGGAGT-3′; and β-actin forward, 5′-GAAGATCAAGATCATTGCTC-3′, and reverse, 5′-AACGCAACTAAGTCATAGTC-3′. Macrophage stimulation by lipopolysaccharide and cytokine protein measurements by enzyme-linked immunosorbent assay Transfected macrophages were stimulated with lipopolysaccharide (LPS) 48 hours posttransfection (Sigma-Aldrich) at different concentrations and durations (10, 100, and 1000 ng/mL for 6, 12, and 24 hours), and conditioned media was collected and stored in aliquots at −80°C until use for the determination of secreted cytokine proteins by enzyme-linked immunosorbent assay (ELISA) kit (Affymetrix) according to the manufacturer’s instructions. Optical density was read in a microplate reader at 450 nm, and a standard curve was subsequently generated to extrapolate cytokine concentration in the samples. Kit sensitivity was ∼4 pg/mL for TNF-α and 2 pg/mL for IL-1β, IL-6, and IL-8 with standard curve ranges of 4 to 500, 2 to 150, 2 to 200, and 2 to 250 pg/mL, respectively. Immunocytochemistry Transfected macrophages in culture chambers were fixed by incubation in 100% chilled methanol at room temperature for 5 minutes and then washed three times with ice-cold PBS. Permeabilization was carried out by incubation in PBS containing 0.25% Triton X-100 and subsequently washed with PBS three times for 5 minutes. Blocking was done with the use of 1% bovine serum albumin (BSA) in PBS plus 0.1% Tween 20 for 30 minutes. Immunostaining was done by incubation in the diluted primary antibody in 1% BSA in PBS plus 0.1% Tween 20 in a humidified chamber overnight at 4°C. Primary antibody (TNF-α, IL-1β, IL-6, and IL-8; Santa Cruz Biotechnology, Inc) use and dilution for all cytokines were based on the antibody data sheet recommendations. Slides were washed and incubated with the secondary antibody in 1% BSA in the dark at room temperature for 1 hour. The secondary antibody solution was decanted, and slides were washed three times with PBS for 5 minutes in the dark. Counterstaining for 1 minute with 4′,6-diamidino-2-phenylindole was done, and slides were mounted with a coverslip and a drop of mounting media. Study population Institutional Review Board approval was obtained from Yale School of Medicine (New Haven, CT) for using human samples. Written informed consent was obtained from subjects admitted to the hospital and undergoing laparoscopy or laparotomy for suspected benign indications such as pelvic masses, pelvic pain, infertility, and endometriosis. Inclusion criteria included women who were aged 19 to 49 years. Exclusion criteria included postmenopausal patients, hyperplasia or polyps, malignancy, autoimmune disease, and cardiovascular disease. All stages of endometriosis as well as untreated and treated subjects were included to provide a full spectrum of disease resulting in varied miRNA levels. The total number of women from whom serum was collected was 49. Among 49 women, 3 were excluded due to an unexpected comorbidity, whereas 46 were included in the study and divided into two groups as follows: the endometriosis group has 20 women with surgically diagnosed and histologically verified endometriosis, and the control group has 26 women, who were visually verified to be free from endometriosis during the surgery. The phase of the menstrual cycle was determined based on the patient’s menstrual history and last menstrual period. Cytokine measurements in human serum from patients with endometriosis by ELISA Whole-blood samples were collected in a sterile condition, kept at room temperature for 30 minutes, and the serum was obtained by centrifuging the blood clot at 2000g for 15 minutes at 4°C. The serum was immediately aliquoted for storage at −80°C until needed for ELISA, avoiding freeze-thaw cycles. Quantification of cytokine levels in individual samples was done twice each in duplicate well by ELISA (Affymetrix) following the manufacturer’s protocol. Quantitative real-time polymerase chain reaction (qRT-PCR) for miRNAs in sera Total miRNA was extracted from 200 μL of serum sample using the miRNeasy Mini Kit (Qiagen) and reverse transcribed using the TaqMan Advanced miRNA cDNA synthesis Kit (Thermo Fisher Scientific) according to the manufacturers’ specifications. miRNA levels were quantified with qRT-PCR using SYBR Green (Bio-Rad) and optimized in the MyiQ Single Color Real-Time PCR Detection System (Bio-Rad). Gene expression was normalized to the expression of human U6 small nuclear RNA to determine relative miRNA expression for each sample. Relative expression was calculated using the comparative cycle threshold method, also known as the 2−ΔΔC(T) method (38). All determinations were done twice, each using triplicate wells. Primers for miRNAs and the U6 genes were obtained from the Keck Oligonucleotide Synthesis Facility (Yale University). Primer sequences are as follows: miRNA-125b-5p forward, 5′-UCCCUGAGACCCUAACUUGUGA-3′; miRNA-Let7b-5p forward, 5′-TGAGGTAGTAGGTTGTGTGGTT-3′; and U6 forward, 5′-CTCGCTTCGGCAGCACA-3′. Statistical analysis Analysis was performed using Prism software version 7.0 (GraphPad Software, Inc.) and the Mann-Whitney U test. For correlation statistics between the relative expression of miRNAs and proinflammatory cytokine levels in serum, we made use of the Pearson correlation coefficient. Differences were considered as statistically significant for P values <0.05. Results Increased serum levels of proinflammatory cytokines in women with endometriosis We first examined the proinflammatory cytokine levels in women with histologically confirmed endometriosis and a comparison of serum cytokine concentrations between women with endometriosis and disease-free controls. The mean age [± standard deviation (SD)] of the women with endometriosis was 35.35 ± 8.66 years, which was not significantly different from the mean age of patients in the control group, 33.05 ± 7.5 years (P > 0.05). The mean body mass index (± SD) did not differ between groups (endometriosis group: 29.55 ± 8.76; control group: 26.66 ± 5.59). Supplemental Table 1 shows a summary of patient characteristics, including phase of the menstrual cycle and stage of endometriosis. As shown in Fig. 1, serum TNF-α (P < 0.001), IL-1β (P < 0.001), and IL-6 (P < 0.001) levels were significantly higher in the endometriosis group than in the control group, and IL-8 levels showed a trend toward increased secretion. Supplemental Table 2 shows the mean concentration of cytokines based on the phase of the menstrual cycle. There was no noteworthy difference in cytokine levels between phases in each subject group (i.e., control proliferative phase vs secretory phase or endometriosis proliferative phase vs secretory phase). The same considerable difference in cytokine concentration of TNF-α, IL-1β, and IL-6 between control and endometriosis groups matched by phase of menstrual cycle was observed (i.e., control proliferative phase vs endometriosis proliferative phase). We subsequently measured the levels of endometriosis-derived miRNA in the serum samples. miRNA 125b was significantly elevated in serum of patients with endometriosis compared with controls (P = 0.002), whereas miRNA Let-7b showed a considerable downregulation (P < 0.001), as shown in Fig. 2. Figure 1. View largeDownload slide Quantification of cytokines by ELISA in serum from women with and without endometriosis. The control group was comprised of 26 subjects, whereas the endometriosis group had 20 subjects. Determination of cytokine concentration for every serum sample was carried out twice in duplicate. Data points represent individual mean concentration and group mean ± SD. Distribution of cytokine serum levels showed a statistically significant increase in the level of TNF-α, IL-1β, and IL-6 in patients with endometriosis compared with control. *Denotes statistical significance (P < 0.05) vs control group. Figure 1. View largeDownload slide Quantification of cytokines by ELISA in serum from women with and without endometriosis. The control group was comprised of 26 subjects, whereas the endometriosis group had 20 subjects. Determination of cytokine concentration for every serum sample was carried out twice in duplicate. Data points represent individual mean concentration and group mean ± SD. Distribution of cytokine serum levels showed a statistically significant increase in the level of TNF-α, IL-1β, and IL-6 in patients with endometriosis compared with control. *Denotes statistical significance (P < 0.05) vs control group. Figure 2. View largeDownload slide Expression levels of miRNA in serum of women with and without endometriosis by qRT-PCR. The control group was comprised of 26 subjects, whereas the endometriosis group had 20 subjects. Experiments were carried out twice each time in triplicate. Data represent mean ± SD. There is a statistically significant upregulation of miRNA-125b and a significant downregulation in patients with endometriosis compared with controls. *Denotes statistical significance (P < 0.05) vs control group. Figure 2. View largeDownload slide Expression levels of miRNA in serum of women with and without endometriosis by qRT-PCR. The control group was comprised of 26 subjects, whereas the endometriosis group had 20 subjects. Experiments were carried out twice each time in triplicate. Data represent mean ± SD. There is a statistically significant upregulation of miRNA-125b and a significant downregulation in patients with endometriosis compared with controls. *Denotes statistical significance (P < 0.05) vs control group. Correlation between expression levels of miRNAs and serum levels of cytokines in patients with endometriosis Although we have established the increase in amount of proinflammatory cytokines as well as determined the relative expression of endometriosis-derived miRNAs 125b and Let-7b in serum of patients with endometriosis compared with control, we next evaluated the correlation between the miRNA levels and cytokine concentration. Figure 3(a) shows the Pearson correlation scatter plot illustrating a positive correlation between expression levels of miRNA 125b-5p and concentration of TNF-α (P < 0.001; r = 0.759), IL-1β (P < 0.001; r = 0.761), and IL-6 (P < 0.001; r = 0.961) in serum of patients with endometriosis. IL-6 showed the highest positive correlation. In contrast, Fig. 3(b) shows the negative correlation between expression levels of miRNA Let-7b and concentration of TNF-α (P = 0.017; r = −0.53) in serum of patients with endometriosis. Figure 3. View largeDownload slide Pearson correlation scatter plots. (a) Positive correlation between expression levels of miRNA 125b-5p and TNF-α (P < 0.001), IL-1β (P < 0.001), and IL-6 (P < 0.001). IL-6 showed the highest positive correlation (r = 0.961). (b) Negative correlation between expression levels of miRNA Let-7b-5p and TNF-α (P = 0.017). IL-1β and IL-6 showed a trend toward a negative correlation. Pearson correlation coefficient (r) as shown for serum of subjects (n = 20) with endometriosis. Figure 3. View largeDownload slide Pearson correlation scatter plots. (a) Positive correlation between expression levels of miRNA 125b-5p and TNF-α (P < 0.001), IL-1β (P < 0.001), and IL-6 (P < 0.001). IL-6 showed the highest positive correlation (r = 0.961). (b) Negative correlation between expression levels of miRNA Let-7b-5p and TNF-α (P = 0.017). IL-1β and IL-6 showed a trend toward a negative correlation. Pearson correlation coefficient (r) as shown for serum of subjects (n = 20) with endometriosis. Increased expression of proinflammatory cytokines in macrophages transfected with miRNA-125b mimic and Let-7b inhibitors We have previously demonstrated a notable increase in circulating miRNA 125b and decrease in miRNA Let-7b in women with endometriosis (35). In this study, we investigated changes in the relative expression of cytokines resulting from overexpression of miRNAs 125b and Let-7b inhibitor in macrophages. As shown in Fig. 4, qRT-PCR was used to demonstrate that there was a statistically marked increase in mRNA expression of cytokines TNF-α (2.66-fold; P < 0.001), IL-1β (1.86-fold; P = 0.0019), IL-6 (1.51-fold; P ≤ 0.001), and IL-8 (1.70-fold; P = 0.0078) in macrophages transfected with miRNA 125b-5p mimic. Transfection with miRNA Let-7b mimic caused noteworthy decrease in expression of TNF-α (0.58-fold difference; P < 0.001), IL-8 (0.66-fold difference; P = 0.0019), and a trend toward downregulation for IL-1β (0.88-fold difference), and IL-6 (0.74-fold difference). Although miRNA 125 is increased, Let-7b levels are decreased in women with endometriosis; we therefore specifically examined the effect of inhibiting this miRNA. The miRNA Let-7b inhibitor transfection caused a considerable increase in expression of TNF-α (1.5-fold, P = 0.002), IL-1β (2.94-fold; P = 0.002), IL-6 (1.59-fold; P = 0.006), and IL-8 (6.19-fold; P = 0.008). Figure 4. View largeDownload slide Transfection of miRNAs in macrophages altered cytokine expression. miRNA-125b is increased in the circulation of women with endometriosis, whereas Let-7b is decreased. Cytokine mRNA expression in macrophages transfected with miRNA-125b and Let-7b mimic and inhibitor were assessed by qRT-PCR and presented as fold change relative to the control (scramble). Increased relative expression of TNF-α (2.66-fold; P < 0.001), IL-1β (1.86-fold; P = 0.0019), IL-6 (1.51-fold; P < 0.001), and IL-8 (1.70-fold; P = 0.0078) in macrophages transfected with miRNA 125b-5p; decreased expression of TNF-α (0.58-fold; P < 0.001), and IL-8 (0.66-fold; P = 0.0019) in group transfected with miRNA Let-7b-5p; and increased expression of TNF-α (1.5-fold; P = 0.002), IL-1β (2.94-fold; P = 0.002), IL-6 (1.59-fold; P = 0.006), and IL-8 (6.19-fold; P = 0.008) in macrophages transfected with Let-7b-5p inhibitor shown. Bars represent the mean ± standard error (SE) of three individual experiments, each performed in triplicate. *Denotes statistical significance (P < 0.05) vs control. Figure 4. View largeDownload slide Transfection of miRNAs in macrophages altered cytokine expression. miRNA-125b is increased in the circulation of women with endometriosis, whereas Let-7b is decreased. Cytokine mRNA expression in macrophages transfected with miRNA-125b and Let-7b mimic and inhibitor were assessed by qRT-PCR and presented as fold change relative to the control (scramble). Increased relative expression of TNF-α (2.66-fold; P < 0.001), IL-1β (1.86-fold; P = 0.0019), IL-6 (1.51-fold; P < 0.001), and IL-8 (1.70-fold; P = 0.0078) in macrophages transfected with miRNA 125b-5p; decreased expression of TNF-α (0.58-fold; P < 0.001), and IL-8 (0.66-fold; P = 0.0019) in group transfected with miRNA Let-7b-5p; and increased expression of TNF-α (1.5-fold; P = 0.002), IL-1β (2.94-fold; P = 0.002), IL-6 (1.59-fold; P = 0.006), and IL-8 (6.19-fold; P = 0.008) in macrophages transfected with Let-7b-5p inhibitor shown. Bars represent the mean ± standard error (SE) of three individual experiments, each performed in triplicate. *Denotes statistical significance (P < 0.05) vs control. To confirm the intracellular changes in cytokine protein production, we then proceeded with immunostaining of the transfected macrophages. Fixed cells were visualized under a fluorescence microscope. Figure 5 gives the representative merged confocal images of the different cytokines in the three miRNA experimental groups and their corresponding scramble (control) sets. Macrophages transfected with miRNA 125b and Let-7b inhibitor showed significantly greater proportion of highly fluorescent cells for all four cytokines—TNF-α, IL-1β, IL-6, and IL-8. In contrast, miRNA Let-7b–transfected macrophages did not display any considerable difference from the controls. Figure 5. View largeDownload slide Confocal analysis of cytokine protein expression. Merged confocal images comparing different miRNA mimic or inhibitor transfection are shown. In each image, blue represents cell nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI); for each row, red indicates TNF-α, purple indicates IL-1β, yellow indicates IL-6, and green indicates IL-8. Images are representative of three random fields in each slide, with n = 4 in each group, done in two independent experiments. Scale bar, 100 μm. Macrophages transfected with miRNA 125b (elevated in endometriosis) and Let-7b (decreased in endometriosis) inhibitor showed a higher percentage of fluorescent cells for all four cytokines compared with their corresponding controls. Ratio of positive cells did not differ from the control in macrophages transfected with miRNA Let-7b. Figure 5. View largeDownload slide Confocal analysis of cytokine protein expression. Merged confocal images comparing different miRNA mimic or inhibitor transfection are shown. In each image, blue represents cell nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI); for each row, red indicates TNF-α, purple indicates IL-1β, yellow indicates IL-6, and green indicates IL-8. Images are representative of three random fields in each slide, with n = 4 in each group, done in two independent experiments. Scale bar, 100 μm. Macrophages transfected with miRNA 125b (elevated in endometriosis) and Let-7b (decreased in endometriosis) inhibitor showed a higher percentage of fluorescent cells for all four cytokines compared with their corresponding controls. Ratio of positive cells did not differ from the control in macrophages transfected with miRNA Let-7b. LPS increased the cytokine secretion in macrophages transfected with miRNA 125b mimic and Let-7b inhibitor Multiple cytokines are secreted from activated macrophages (39). Therefore, we stimulated the 48-hour posttransfected macrophages with LPS in varying doses and time intervals to know if translation of cytokines and subsequent secretion reflect mRNA changes. LPS dose and timing of maximal cytokine secretion of macrophages vary across literature (25, 40–43), but an optimal time point was set at 24 hours following a dose of 100 ng/mL for this study. Figure 6 shows ELISA results from conditioned media of transfected macrophages treated with 100 ng/mL LPS for 24 hours. A significant increase in TNF-α (P = 0.004), IL-1β (P = 0.016), IL-6 (P = 0.029), and IL-8 (P = 0.016) protein secretion in macrophages transfected with miRNA 125b. In contrast, the macrophages transfected with miRNA Let-7b showed a noted decrease in protein secretion of TNF-α (P = 0.0095), IL-1β (P = 0.016), and IL-6 (P = 0.029). Figure 6 also shows a substantial increase in secretion of all these cytokines in macrophages transfected with miRNA Let-7b inhibitor TNF-α (P = 0.016), IL-1β (P = 0.029), IL-6 (P = 0.016), and IL-8 (P = 0.004). Figure 6. View largeDownload slide LPS stimulation induces cytokine secretion after miRNA alterations associated with endometriosis. LPS stimulation (100 ng/mL) of transfected macrophages showing protein levels determined by ELISA. TNF-α, IL-1β, IL-6, and IL-8 were significantly higher in conditioned media of cells transfected with miRNA 125b and Let-7b inhibitor compared with the controls. Bars represent the mean ± standard error (SE) of three individual experiments, each performed in triplicate. Differences were considered as statistically significant for P values <0.05. *Denotes statistical significance between control mimic vs miRNA mimic; #denotes statistical significance between control inhibitor vs miRNA inhibitor. Figure 6. View largeDownload slide LPS stimulation induces cytokine secretion after miRNA alterations associated with endometriosis. LPS stimulation (100 ng/mL) of transfected macrophages showing protein levels determined by ELISA. TNF-α, IL-1β, IL-6, and IL-8 were significantly higher in conditioned media of cells transfected with miRNA 125b and Let-7b inhibitor compared with the controls. Bars represent the mean ± standard error (SE) of three individual experiments, each performed in triplicate. Differences were considered as statistically significant for P values <0.05. *Denotes statistical significance between control mimic vs miRNA mimic; #denotes statistical significance between control inhibitor vs miRNA inhibitor. Discussion Endometriosis has been associated with inflammation and increased inflammatory cytokine production. In this study, we confirm an elevation of several inflammatory cytokines in the circulation of women with endometriosis (14, 44). Endometriosis-associated inflammation is thought to be mediated in part by macrophages; increased macrophage activity is reflected by a rise in number and activation potential, resulting in an increase in secretion of cytokines and chemokines (45). However, it is not clear how endometriosis leads to the increased systemic inflammation. We demonstrate in this study that alterations in circulating miRNAs are one mechanism by which endometriosis causes immune dysfunction and inflammation. As these miRNAs are altered in the circulation of women with the disease, they can influence the cytokine expression of macrophages systemically, including areas remote from endometriosis. Alterations of circulating miRNA in endometriosis furthers the concept that endometriosis is a systemic disease. We have previously demonstrated systemic effects of endometriosis remote from the site of lesions. Although peritoneal endometriosis affects the uterus and endometrial receptivity, we have demonstrated that endometriosis placed under the skin of mice far from the uterus still affects the eutopic endometrium (46). Similarly, endometriosis can affect liver metabolism, leading to weight loss (47). Further endometriosis alters stem cell trafficking, leading to stem cell deficiency in the uterus and infertility (48, 49). All of these examples demonstrate that endometriosis is not a disease in which the effects are localized to the pelvis. The ability to systemically alter macrophage function and induce an inflammatory state adds to the list of widespread, whole-body effects of endometriosis. Endometriosis should be considered a disease with multiorgan involvement. Alterations in circulating miRNAs clearly induce inflammation and may well contribute substantially to the systemic manifestations of the disease. Let-7 family also targets the KRAS gene, and its loss may permit initiation of the progression of ectopic lesions and represent a link between inflammation and the tumor-like growth in endometriosis (32, 50–52). KRAS signaling leads to enhanced proliferation and invasion of cells, including many tumors (51). We have previously demonstrated a role for Let-7b regulation of KRAS in endometriosis (52). Similarly, Let-7 family also targets aromatase (53). Let-7 family may target multiple pathways that influence endometriosis and other processes in addition to the role described in this study. The exact role of Let-7b in the regulation of macrophages is still an ongoing area of research. A known target of importance is C/EBP-δ, which is a transcription factor required for sustained responsiveness to Toll-like receptor signaling (54). Our data clearly demonstrate that overexpression of miRNA Let-7b caused a marked decrease in the expression of the proinflammatory genes, whereas blocking of Let-7b caused a reciprocal increase in cytokine expression. These results are further confirmed by the data obtained for consistent protein pattern in intracellular protein production as well as secreted cytokine levels post–LPS stimulation in the respective transfection experiments. It is important to recognize that it is the collective effects of these miRNAs that determine the disease outcome. miRNAs each have multiple targets, and it is likely that the inflammatory cytokines identified in this study represent important, but not exclusive, targets of these miRNAs. The inhibition or stimulation of proinflammatory cytokines by these miRNAs identifies prospective novel targets for interventions in endometriosis. Restoration of normal peripheral miRNA signaling between cells and organs may well be a means to treat the systemic manifestations of this disease. miRNA mimics and inhibitors are exciting new therapeutics for endometriosis. In summary, circulating miRNA 125b-5p and Let-7b-5p function as regulators of the inflammatory response in endometriosis. They induce macrophage inflammatory cytokine production, defining a mechanism responsible for the systemic effects of this disease. The miRNAs with altered levels in endometriosis identified in this study have potential as both diagnostic tools and therapeutic targets. Abbreviations: BSA bovine serum albumin ELISA enzyme-linked immunosorbent assay IL interleukin LPS lipopolysaccharide miRNA microRNA mRNA messenger RNA qRT-PCR quantitative real-time polymerase chain reaction SD standard deviation TNF-α tumor necrosis factor-α. Acknowledgments Disclosure Summary: The authors have nothing to disclose. References 1. Giudice LC. Clinical practice. 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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Jan 1, 2018

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