Abstract
As the mean age of first-time mothers increases in the industrialized world, inquiries into causes of human reproductive senescence have followed. Rates of ovulatory dysfunction and oocyte aneuploidy parallel chronological age, but poor reproductive outcomes in women older than 35 years are also attributed to endometrial senescence. The current studies, using primary human endometrial stromal cell (ESC) cultures as an in vitro model for endometrial aging, characterize the proinflammatory cytokine, IL-1β-mediated and passage number-dependent effects on ESC phenotype. ESC senescence was accelerated by incubation with IL-1β, which was monitored by RNA sequencing, ELISA, immunocytochemistry and Western blotting. Senescence associated secreted phenotype (SASP) proteins, IL-1β, IL-6, IL-8, TNF-α, MMP3, CCL2, CCL5, and other senescence-associated biomarkers of DNA damage (p16, p21, HMGB1, phospho-γ-histone 2 A.X) were noted to increase directly in response to 0.1 nM IL-1β stimulation. Production of the corresponding SASP proteins increased further following extended cell passage. Using enzyme inhibitors and siRNA interference, these effects of IL-1β were found to be mediated via the c-Jun N-terminal kinase (JNK) signaling pathway. Hormone-induced ESC decidualization, classical morphological and biochemical endocrine responses to estradiol, progesterone and cAMP stimulation (prolactin, IGFBP-1, IL-11 and VEGF), were attenuated pari passu with prolonged ESC passaging. The kinetics of differentiation responses varied in a biomarker-specific manner, with IGFBP-1 and VEGF secretion showing the largest and smallest reductions, with respect to cell passage number. ESC hormone responsiveness was most robust when limited to the first six cell passages. Hence, investigation of ESC cultures as a decidualization model should respect this limitation of cell aging. The results support the hypotheses that “inflammaging” contributes to endometrial senescence, disruption of decidualization and impairment of fecundity. IL-1β and the JNK signaling pathway are pathogenetic targets amenable to pharmacological correction or mitigation with the potential to reduce endometrial stromal senescence and enhance uterine receptivity.
Similar content being viewed by others
Introduction
Advanced maternal age and reproductive senescence reduce pregnancy success and quality of life in affected women, their families and society. Postponing reproduction raises the likelihood of unwanted childlessness and, even when successful, poses a higher risk for clinical pregnancy complications, including fetal aneuploidy and preeclampsia [1]. While most research on reproductive aging in women has focused on epigenetic oocyte damage [2], endometrial senescence remains largely understudied [3, 4]. Assessment of serum glycodelin-A (PP14), an endometrial glycoprotein [5], failed to detect substantive changes in this biomarker among women 20–30 years vs. those 40–50 years old [6]. However, comprehensive microarray analyses of endometrial stromal cell (ESC) mRNAs derived from perimenopausal women exhibited a significantly altered transcriptome compared to ESC from premenopausal women [7].
In hemochorial placental species (viz, women, non-human old-world primates, bats, and mice), blastocyst nidation occurs within a narrow window of uterine receptivity synchronized to embryo development. In women, the “open” endometrial interval is regulated by ovarian steroid hormones, locally expressed growth factors and cytokines [8]. The implantation window is vulnerable to perturbation. A critical manifestation of receptive human endometrium is the decidual response, differentiation of endometrial mesenchyme into a secretory stroma supportive of placentation [9]. Biomarker proteins associated with receptive endometrium include prolactin [10]; IGFBP1 [11], IL-11 [12] and VEGF [13], among others. Another phenotypic change associated with decidualized ESCs is mesenchymal-to-epithelial transformation (MET), a cytological program shared with angiogenesis and tumorigenesis [14, 15].
Replicative senescence is an irreversible response to excessive cellular stress that limits cell longevity. It is characterized by resistance to mitogenesis and permanent cell cycle arrest. Established biomarkers of senescence associated secreted phenotype (SASP: IL-1β, IL-6, IL-8, TNF-α, MMP3, CCL2, CCL5) and nuclear protein indicators of DNA damage (p16, p21, HMGB1, phospho-γ-histone 2 A.X). Impaired decidualization in ESC derived from women with endometriosis [16, 17] and polycystic ovary disease (PCOS) [18, 19] have been reported. IL-1β can induce intrauterine inflammation and impair ESC decidualization and function [20,21,22,23]. Indeed, inverse correlations between in vitro fertilization (IVF) implantation rates and free serum [24] or intrauterine fluid IL-1β concentrations [25] are observed. In the current study we investigated whether IL-1β-treatment of primary human ESC cultures could recapitulate ESC senescence and whether its signature, so-called “inflammaging” biomarkers [26], were associated with decidual dysfunction.
Results
Critical role of inflammation as a modifier of ESC senescence
IL-1β effects on gene expression were agnostically evaluated in ESC derived from different subjects (n = 2, S1 and S2), treated without or with 0.1 nM IL-1β for 24 h. RNAseq identified several IL-1β-induced mRNA transcripts; the 10 that were most strongly upregulated are shown in the heat map (Fig. 1A): IL-1β itself, IL-6, IL-8, HMGB1, CCL2, MMP3, CXCL10, MAP2K1, CCL5 and TNF-α. The heatmap on the left (Fig. 1A) was generated using normalized gene expression values for each gene/sample. These values were standardized to center around zero. Genes (rows) were clustered based on their Euclidean distance using the complete linkage agglomeration method. The figure was created in R using the heatmap package. As shown in the volcano plot (Fig. 1B), IL-1β treatment upregulated six SASP mRNA transcripts (IL-1β, IL-6, IL-8, CCL2, CCL5 and TNF-α) and, to a lesser extent, MMP3. The volcano plot in Fig. 1B utilized adjusted p-values and fold-changes calculated by DESeq2. The adjusted p-value cut-off was set at 0.05, and the log2 (fold-change) cut-off was set at −1 and +1. The figure was generated in R using the Enhanced Volcano package.
Senescence transcripts identified by RNAseq were confirmed at the protein level by ELISA in ESC conditioned media. MMP3, IL-6, IL-8, CCL5 and TNF-α were all stimulated in response to IL-1β (Fig. 1C). The analysis, conducted 72 hours post-treatment, compared IL-1β-treated samples with untreated controls using ANOVA, revealing a significant effect (p < 0.001). Subsequent post-hoc Scheffé tests identified significant differences between MMP3 and other biomarkers like IL-8, IL-6, RANTES, and TNFα (p < 0.001). However, no significant difference was noted between IL-8 and IL-6 (p > 0.05), while both CCL5 and TNFα showed significant disparities (p < 0.001). Comparatively, the differences for IL-6 versus RANTES and TNFα were significant (p < 0.001), but not between RANTES and TNFα (p > 0.05). These findings highlight the distinct regulation of SASP biomarkers in response to IL-1β stimulation in P3 ESCs.
Data in Fig. 1D are graphed with an expanded ordinate scale to resolve the lower absolute concentrations of CCL5 and TNF-α in ESC supernatants ( 800 pg/ml). Appreciable increases in biomarker protein secretion were not observed until after 80 min exposure to IL-1β. IFC experiments revealed increased p21 (green staining, Fig. 2A) accumulation in cells exposed to recombinant IL-1β for 24 h relative to controls. IFC with β-actin antibody (red staining, Fig. 2A, middle panels) showed that IL-1β induced more diffuse signal, consistent with its effect on ESC fibroblastic morphology [17]. Merged channel images show antibody overlap (Fig. 2A, right panels). β-Galactosidase staining at pH 6.0 was conducted on untreated ESC (left) and senescent ESC treated with IL-1β (right) for 24 h (Fig. 2B). The results revealed notably intense dark blue signals (Fig. 2B, right panels) indicative of cellular senescence following IL-1β treatment in ESC.
Western blotting of cell lysates confirmed that 24 h exposure to IL-1β upregulated senescence proteins IL-1β, IL-6, TNF-α, MMP3, p16, p21, HMGB1, CCL2, CCL5 along with phospho-histone H2A.X. β-Actin levels were not changed (Fig. 3A). Co-incubation with the JNK inhibitor SP suppressed upregulated SASP markers to near baseline levels over a 24-72 h time course (Fig. 3B, C). Notably, SP treatment alone (lane 8, Fig. 3B) reduced the levels of p21, HMGB1, CCL2 and phospho-histone H2A.X, SP alone (lane 8) reduced levels below those of untreated controls (lane 1, Fig. 3B). The latter phenomenon is in keeping with endogenous IL-1β production by ESC [17]. Additionally, analysis of three key senescence-associated biomarkers revealed that the baseline levels of IL-1β and IL-6 were notably low and remained undetectable by Western blot at all three time points examined, even under reasonable exposure times. Similarly, in the untreated control groups, MMP3 expression levels showed no significant changes across these time points (Fig. 3C).
ELISA of conditioned media confirmed findings in the Western blots. CCL5 secretion was upregulated by IL-1β in ESC derived from four subjects (S1, S2, S3, S4) and these effects were blocked by SP co-incubation (Fig. 4A). ANOVA showed significant upregulation of CCL5 from baseline after IL-1β-exposure in ESC from all four study subjects (p < 0.001), ANOVA with post-hoc Scheffé’s tests revealing significant inter-group differences (p < 0.001) between control vs. IL-1β-exposed ESC and IL-1β-exposed vs. IL-1β plus SP-treated cells). In experiments where IL-1β was co-incubated with a 100-fold excess of IL-1ra, complete abrogation of the IL-1β-induced upregulation of IL-8 and IL-6 was observed (Fig. 4B), suggesting that the effects were mediated via IL-1 receptor type 1 signaling. Two-factor ANOVA analysis revealed a significant difference in IL-6 and IL-8 (p < 0.001 for each). IL-1β time-dependently increased CCL5 and TNF-α, and SP suppressed CCL5 and TNF-α secretion after 24–72 h of IL-1β exposure (Fig. 4C, D, respectively). One-factor ANOVA analysis indicated that IL-1β stimulation induced IL-6 and IL-8 secretion, and this effect was completely blocked upon co-incubation with SP (p < 0.001 in each). The timing of addition of SP did not appear consequential. One h preincubation with the kinase inhibitor (PSP), had the same inhibitory effect as simultaneous co-incubation of IL-1β plus SP (SSP) on IL-1β, IL-6, TNF-α and MMP3 reduction (Fig. 4E). β-Actin levels were not affected. AS also suppressed IL-1β-induced CCL5 and TNF-α secretion, but its potency in ESC was only 43% that of SP (data not shown).
An alternative strategy to identify the responsible IL-1β signaling pathway was afforded using JNK1 and JNK2 siRNA interference. These constructs reduced IL-1β-induced IL-1β, IL-6, MMP3 and HMGB1 in ESC as determined by Western blotting (Fig. 4F) but had no effects on β-actin. Basal and IL-1β-stimulated p21 were inhibited with JNK2 siRNA but not as effectively by JNK1 siRNA (Fig. 4F). Pilot experiments using scrambled control siRNA showed no effects on IL-1β signaling (data not shown).
The effects of cell passage number on IL-1β as an inflammaging marker were assessed in Fig. 5A, B by IFC and IPC. P21 ESC showed high basal and IL-1β-induced IL-1β accumulation (Fig. 5B) compared to P3 cultures (Fig. 5A). Western blots of ESC lysates from P3, P12 and P21 cells after IL-1β stimulation showed increased protein expression in higher passage number cells, except for β-actin (Fig. 5C). ELISA confirmed that IL-6, IL-8, CCL5, TNF-α and MMP3 were all progressively more responsive to IL-1β in P12 and P21 than in P3 cells (Fig. 6A–E). Two-factor ANOVA analysis indicated a significant upregulation of five SASP components following after IL-1β stimulation: IL-8 (p < 0.01), IL-6 (p < 0.001), CCL5 (p < 0.05), MMP3 (p < 0.01) and TNF-α (p < 0.001). Scheffé Test was utilized to compare between passages (P3 vs. P21) following IL-1β treatment: IL-6 (p < 0.01), IL-8 (p < 0.001), CCL5 (p < 0.05), TNFα (p < 0.01), and MMP3 (p < 0.001); Additionally, under control conditions, subtle yet similar trends were observed with prolonged ESC passage.
Role of inflammation as an inhibitor of ESC differentiation
Inflammation, particularly that mediated by IL-1β, can impair decidual differentiation in human ESC [23]. We postulated that prolonged passage number also may be associated with reduced ESC decidualization. ESC differentiation based upon cell shape was assessed by phase-contrast microscopy. Under control culture conditions (without added hormones) fibroblastic ESC morphology was indistinguishable among P3, P9 or P12 cells (Fig. 7A, left panels). Low passage cells ( ≤ P9) treated for seven days with a standardized decidualizing regimen (10 nM estradiol, 100 nM progesterone and 0.5 mM dibutyryl cAMP, H + 7) demonstrated cell “rounding” typical of MET [15]. The extent of MET in H + 7-treated ESC, reported as cell shape indices from 0.0 to 1.0, was inversely related to passage number. No significant differences in cell shape index were noted under control conditions (P3 = 0.18 ± 0.02, P9 = 0.40 ± 0.31, P12 = 0.29 ± 0.17, P = 0.19, 1-factor ANOVA). But following H + 7 hormone stimulation, statistically significant differences were observed among different ESC passage numbers (P3 = 0.95 ± 0.35, P9 = 0.64 ± 0.27, P12 = 0.43 ± 0.11, p < 0.01, 1-factor ANOVA). Post-hoc Scheffe’s tests revealed significant inter-group differences (p < 0.05) between hormone treated P3 vs. P9 cells and P3 vs. P12 cells. Two-factor ANOVA showed a significant interaction between the independent variables (passage number and days of hormone treatment) with F-values ranging from 12.6 to 99.9 (p < 0.001). Although attenuated at P12, H + 7 hormones still induced an increase in cell shape index over P3 controls without hormones (0.43 ± 0.11 vs. 0.18 ± 0.02, p < 0.01, Student’s t-test).
Cytological features of decidualization were associated with classical (prolactin, IGFBP-1) and emerging (IL-11, VEGF) biochemical correlates of ESC differentiation. ELISA quantified secreted biomarkers as continuous, dependent variables in conditioned media of P3, P9 and P12 ESC, under control (C) conditions and following three (H + 3), five (H + 5) or seven (H + 7) days of hormone exposure (Fig. 7B–E). Two-factor ANOVA showed a significant interaction between the independent variables (passage number and days of hormone treatment) with F-values ranging from 20.5 to 281.4 (p < 0.001) in all four decidualization biomarkers (Fig. 7B–E). The Scheffé test was utilized to compare control (C) versus seven days of hormone treatment (H + 7), C vs. H + 7, and within-group comparisons in PRL (Fig. 7B) revealed significant differences at P3 ( < 0.001), P9 (p < 0.001), and with no significant differences at P12 (P > 0.05). Further analyses using the Scheffe’s Test (H + 7) demonstrated significant inter-group differences at day seven post-hormone treatment (H + 7): P3 vs. P9 (P < 0.001), P3 vs. P12 (P < 0.001), and P9 vs. P12 (P < 0.01). Similarly, comparisons were conducted for IGFBP1 (Fig. 7C) using the Scheffé Test (H + 7), indicating significant inter-group differences in each comparison: P3 vs. P9, P3 vs. P12, and P9 vs. P12 (P < 0.001 for each). For IL-11 (Fig. 7D), significant differences were observed between P3 vs. P9 and P3 vs. P12 (P < 0.001), with no significant differences in P9 vs. P12 (P > 0.05). Finally, for VEGF (Fig. 7E), no significant differences were found in any of the comparisons: P3 vs. P9, P3 vs. P12, and P9 vs. P12 (P > 0.05 for each).
The results suggest notable variability in the response to hormone treatment across various passages of ESC, underscoring the heightened responsiveness of earlier passages (P3 and P9) compared to later passages (P12). Among the secreted biomarker proteins, all were most vigorously upregulated by hormones in P3 cells and their responsiveness was positively correlated with days of hormone exposure. The relative kinetics and magnitude of each biomarker were unique, with IGFBP-1 showing the largest differential response with respect to passage number (Fig. 7B) and VEGF having the smallest dynamic range (Fig. 7E). The results support the overall concept that primary ESC cultures lose hormone responsivity and differentiated function with extended passaging.
Discussion
The effects of inflammation on markers of ESC aging were assessed using an established IL-1β treatment protocol [17] that mimics aspects of ESC senescence and endometriosis. In RNAseq experiments, IL-1β upregulated a number of bona fide SASP transcripts, indicated in Fig. 1A, B. Corresponding proteins paralleled their mRNAs, as shown by ELISA (MMP3, CCL5 and TNF-α, Fig. 1C, D) and IFC (p21, Fig. 2). Western blots (Fig. 3B, C) in cells treated with JNK inhibitors SP, AS or JNK1 and JNK2 siRNA implicated that pathway as key to IL-1β signaling (Figs. 3B, C and 4A–E). The EC50 of IL-1β actions in these cells [17, 27], is consistent with its affinity for the IL-1 receptor 1/IL-1 receptor accessory protein complex [28]. SASP gene products identified by RNAseq include many inflammatory biomarkers that have been historically analyzed in endometriosis, including IL-1β, IL-6 [29], IL-8 [30], CCL5 [31], TNF-α [32], CCL2 [33], MMP3 [34], HMGB1 [35], p16, and p21 [36, 37]. The findings support the senescence hypothesis proposed by Malvezzi et al. [37] for this condition.
Recent studies addressing senescence-induced pregnancy loss in mice reveal the involvement of similar cytokines and inflammatory biomarkers. Conditional knock-out of the uterine Enhancer of zeste homolog 2 (Ezh2) gene, a histone methyltransferase that epigenetically regulates decidual gene transcription, resulted in smaller embryo implantation sites and midgestational pregnancy failure. Decidual IL-1β and MMP3 transcripts were noted to be upregulated [38] and p16 and p21 proteins were elevated in this model [39].
The current experiments also demonstrate that hormone-induced ESC differentiation is compromised in a cell passage-dependent manner, manifested by temporal attenuation of MET morphology (Fig. 7A) and reduced secreted decidual biomarkers (prolactin, IGFBP-1, IL-11, and VEGF), pari passu as ESC passage number increased (Fig. 7B–E). The reliable utility of primary human ESC cultures as a model of decidual differentiation appears to be limited to the first six cell passages. A similar finding was published by Nayyar et al. [40], showing progressive reduction in IGFBP-1 secretion from menstrual effluent-derived ESC over weeks in culture. Also consistent with our observations, protein and mRNA biomarkers in ESC derived from clinically non-receptive patients in a Japanese IVF practice, exhibited significantly more senescence than ESC derived from subjects experiencing successful IVF pregnancies [41]. Based on these results we postulate that poor reproductive success (including failed implantation, miscarriage, and disorders associated with pathological placentation) are clinical manifestations of inflammaging.
Over the past decade, there has been significant advancement in understanding the influence of inflammation on infertility related to implantation failure. Traditionally, IL-1β, a key inflammatory mediator, was thought to be primarily produced by macrophages infiltrating the endometrium. Recent findings, however, suggest an alternative possibility, which is that IL-1β is notably auto-upregulated in ESC and not due to macrophage infiltration. We found that IL-1β levels in endometrial stromal cells (ESC) are low, often below the limits of detection by conventional methods like Western blotting. Remarkably, the introduction of human recombinant IL-1β markedly boosted the production of endogenous IL-1β by ESC themself, resulting in its exponential amplification in cultured ESC. In culture, exposure to IL-1β not only enhanced the release of IL-1β within ESC but also increased its reception by additional ESC. This led to a self-reinforcing feedback loop of IL-1β auto-upregulation, perpetuating a chronic inflammatory state. This pivotal discovery calls for a re-evaluation of the origins of IL-1β-associated inflammation and highlights the crucial role of ESC as participants in endometrial inflammation and receptivity. Further, our results suggest that inflammation may predispose to implantational delay or failure. The concept of “inflammaging,” which merges inflammation and aging, highlights this critical intersection, underscoring its significance for understanding the pathobiology of implantational failure and conditions that arise from poor placentation.
Brighton et al discovered striking similarities between cellular senescence and differentiation of ESC into decidual cells [42]. In our findings, we observed small increases in both IL-6 and CCL5 levels, each less than tenfold, after seven days of hormone treatment compared to controls (data not shown). In contrast, there was a greater than hundreds fold increase in IL-6 and CCL5 levels following 24 h of IL-1β treatment (Fig. 1C) [43, 44]. This significant divergence from hormone treatment highlights that acute decidual senescence plays a vital and necessary role in implantation, facilitating physiological repair and remodeling. In contrast, senescence induced by IL-1β may initiate persistent pathological changes that impair endometrial plasticity, reduce endometrial receptivity, and lead to poor implantation outcomes.
In this study, we made a novel observation, which is that IL-1β treatment led to a progressive rise in endogenous IL-1β expression levels in ESC throughout extended cell passages. To the best of our knowledge, this is the first description of this pattern. This intriguing pattern raises the question of whether there’s a correlation with heightened sensitivity to IL-1β stimulation, possibly linked to age-related alterations in cell generations, warranting further investigation.
Models, such as the one described here, and more complex 3-dimensional spheroids and organoids incorporating multiple cell types [45, 46], should inform further understanding of endometrial aging. We are hopeful that senescent changes associated with advanced maternal age might ultimately be reversed with properly adjudicated medicinal interventions, based on the IL-1β and JNK signaling pathways identified in this report.
Materials/subjects and methods
Sources of human tissues
Four participants undergoing elective laparoscopic surgery provided written informed consent. The study received ethics approval and consent to participate from the institutional review boards at Wake Forest School of Medicine, Winston-Salem, North Carolina (IRB #00019887). Patients were excluded if they did not read English, were unable to provide written informed consent, or were incarcerated, ensuring that informed consent was obtained from all subjects who fully understood the study. Fertile, parous subjects with regular menstrual cycles, ranging in age from 27 to 37 years, who had not received hormonal therapy for at least three months before surgery were recruited. Endometrial biopsies were collected in the proliferative phase by Pipelle aspiration under sterile conditions immediately prior to laparoscopy. Among the participants, no visible evidence of endometriosis, active infection or pelvic mass suspicious for malignancy was noted. The primary operative findings in one were intramural and subserosal fibroids, one had filmy periovarian adhesions, and two desired tubal sterilization without evidence of pelvic pathology.
Endometrial stromal cell (ESC) preparation, culture conditions and in vitro decidualization
Endometrial biopsies were digested with collagenase and glands and debris were separated by filtration [47]. ESC, in the flow-through fraction, were sub-cultured twice to eliminate macrophages and other leukocytes by plating directly onto 10-cm polystyrene dishes at a density of 25,000 cells per cm2 in DMEM/Ham’s F-12 medium, supplemented with 10% fetal calf serum and antibiotics (penicillin and streptomycin). P3 ESC were studied unless noted otherwise. Further passages at 1:2 splits were performed as noted below, up to passage P21. In some experiments, cells were plated on Nunc Lab-Tek chamber slides (Thermo-Fisher Scientific, Waltham, MA) for immunofluorescence (IFC)- and immunoperoxidase-cytochemistry (IPC). ESC prepared in this manner are >93% pure and retain functional estrogen and progesterone receptors, as well as other phenotypic endometrial markers, for up to six passages (P6) in vitro, but tend to dedifferentiate with more protracted passaging [48].
Decidualization was induced in vitro by exposing the cultures to 10 nM 17β-estradiol + 100 nM progesterone + 0.5 mM dibutyryl cAMP in 0.1% EtOH (“hormones, H”) in phenol red-free medium (DMEM/Ham’s F-12 cat# 10-092 cv, CellGro, Manassas, VA, USA) supplemented with 5% charcoal-stripped (hormone-depleted) fetal calf serum to mimic the endocrine milieu of early pregnancy, as described [49]. Passage numbers of the cell preparations are indicated as P3, P9, P12 or P21. Control conditions were achieved by incubation in the same phenol red-free medium for seven days, but solvent alone (0.1% EtOH) was substituted for hormones. Morphological changes consistent with mesenchymal-epithelial transition (MET) were determined after phase contrast microscopy using cell shape index values ranging from 0.0 to 1.0 in 10 randomly selected ESC per photomicrographs as described [15, 50].
IL-1β-treatment to recapitulate cellular inflammation and induce senescence
To mimic inflammatory aging, termed “inflammaging,” [26], ESCs were incubated in the presence of 0.1 nM human recombinant IL-1β (Sigma-Aldrich, St. Louis MO, cat# I9401), which we showed was the EC50 for several biomarker endpoints in ESC [17]. Cellular lysates for mRNA sequencing (RNAseq) and Western blotting and culture supernatants for ELISA were prepared after 24-72 h IL-1β exposure, as described below, and compared to untreated ESC cultures. IL-1ra (R&D, cat # 280-RA-010), a natural inhibitor that binds avidly to IL-1 type 1 receptors [51] was added at 10 nM (a 100-fold molar excess) to block IL-1 receptor signaling [17]. Pathway analyses were assessed using selective pharmacological c-Jun-N terminal kinase [JNK] inhibitors. Effects of exposure of ESC to IL-1β or IL-1β + SP600125 (“SP,” NSC 75890, Sigma, cat #S5567 [43]) or AS602801 (“AS,” bentamapimod, Sigma, cat# SML-3358), were compared after 24 to 72 h. A concentration of 30 μM was used for SP and AS, based on prior optimization in ESC [17]. As an alternative approach to block JNK action in ESC, JNK1 and JNK2 double-stranded siRNA constructs or a scrambled duplex control purchased from Cell Signaling Technologies (cat# 6232, 6233 and 6568), were introduced into ESC via transient transfection at a concentration of 100 nmol per well using Lipofectamine RNAiMAX (Thermo-Fisher) as described [17]. Successful inhibition of JNK1 and JNK2 protein expression was verified by Western blotting.
RNAseq, transcriptome and statistical analyses
Total RNA was isolated from cell lysates using the TRIzol® Plus RNA Purification Kit (Life Technologies, Grand Island, NY). RNA concentrations and quality were measured by the Nanodrop 2000® method. cDNA was synthesized from mRNA samples and subsequently used as template for bulk RNAseq as described [52]. Per-cycle basecall (BCL) files generated by the Illumina NextSeq 500 were converted to per-read FASTQ files using bcl2fastq version 2.20.0.422 with standard default parameters. Sequencing quality was reviewed using FastQC version 0.11.9, detection of potential contamination was done using FastQ Screen version 0.14.1. FastQC and FastQ Screen quality reports were summarized using MultiQC version 1.9.0. RNA sequence accession codes are GSE261130 in NCBI tracking system.
No appreciable adapter sequences were detected so no trimming was performed. Genomic alignments were performed using HISAT2 version 2.2.1 with default parameters. NCBI reference GRCh38 was used for the reference genome and gene annotation set. Sequence alignments were compressed and sorted into binary alignment map (BAM) files using Samtools version 1.15.1. Counting of mapped reads for genomic features was performed using Subread featureCounts version 1.6.2 with the parameters “-s 2 –g gene_name –t exon –Q 60 -B -C”. The annotation file specified with—a was the NCBI GRCh38 reference provided by Illumina iGenomes. Alignment statistics and feature assignment statistics were again summarized using MultiQC.
Differentially expressed genes were detected using the Bioconductor package DESeq2 version 1.34.0. Genes with one count or less were filtered, and alpha was set to 0.05. DESeq2 tests used a negative binomial generalized linear model, dispersion estimates, and logarithmic fold changes. DESeq2 calculates log2 fold changes and Wald test P values, performs independent filtering and adjusts for multiple testing using the Benjamini-Hochberg procedure to limit false discovery rate (FDR) to p < 0.05. Volcano plots confirmed the upregulation of SASP gene transcripts, and they were corroborated at the protein level by immunocytochemistry, ELISA and Western blots.
Immunofluorescence- (IFC) and Immunoperoxidase-cytochemistry (IPC)
To verify the cellular localization of biomarker proteins, ESC were plated on Lab-Tek® chamber sides and grown to 80% confluence. Some cultures were stimulated with 0.1 nM recombinant IL-1β for 24 h, followed by extensive washing. ESC were fixed in 4% paraformaldehyde and permeabilized with ice cold acetone. The cells were stained with β-actin to label cytoskeleton microfilaments (cat# 3700, Cell Signaling, 1:600 antibody dilution). SASP and DNA damage biomarkers included IL-1β, p21. The suppliers, conditions and concentrations of antibodies used for IFC and IPC are described in Antibody Table 1. In IPC experiments nuclei were counterstained with hematoxylin.
Senescence-associated-β-galactosidase (SAβG) staining
SAβG staining was conducted on confluent endometrial stromal cells (ESC) in 6-well plates using the Senescence β-Galactosidase Staining Kit (#9860; Cell Signaling Technology, MA, USA), in strict accordance with the manufacturer’s instructions. ESC were seeded onto 6-well plates and cultured until they reached 50–60% confluence. Selected cultures were then stimulated with 0.1 nM recombinant IL-1β for 24 h. Following this, cells were fixed in 1× fixative solution for 10–15 min at room temperature and stained overnight with 2 ml of β-galactosidase staining solution at 37 °C in a dry incubator (no CO2). To prevent evaporation, plates were sealed. The next day, the staining solutions were removed, and the cells were overlaid with 70% (v/v) glycerol in water and stored at 4 °C. The staining results were visualized using light microscopy, and images were captured.
ELISA for detection of SASP biomarkers and in vitro decidualization
Sensitive and specific sandwich ELISA kits for senescence biomarkers, IL-6, IL-8, TNFα, MMP3 and CCL5 (cat# D6050, D8000C, TDA00D, DMP300 and DRN00B, R&D Biosystems, Minneapolis, MN), were measured to define the effects of IL-1β on ESC cultures. ELISA also were used to quantify secreted biomarkers of ESC decidualization: prolactin (cat# 0300, Alpha Diagnostic International, San Antonio, TX); IGFBP-1, VEGF, and IL-11 (cat# DGB100, DVE00, and D1100, R&D Biosystems, Minneapolis, MN). We and others previously established these assays to be precise and linear in ESC and they are increasingly recognized as reliable biochemical surrogates for ESC decidualization [11, 12, 49].
Western blot analysis
To quantify intracellular protein expression, Western blots were performed on whole-cell lysates collected in extraction buffer (cat# FNN0011, Life Technologies, Grand Island, NY), followed by protein quantification using the Thermo Scientific-Pierce BCA™ Protein Assay method (cat# PI-23227, Thermo-Fisher Scientific, Waltham, MA) [15]. 30 μg protein per lane were loaded onto NuPAGE® Novex® 4-12% Bis-Tris protein gels (Thermo-Fisher), electrophoresed, and transferred to PVDF membranes. After blocking with 5% skim milk in PBS, primary antibodies to senescence biomarkers (IL-1β, IL-6, IL-8, TNF-α, MMP3, p16, p21, HMGB1, CCL2, CCL5 and phospho-γ-histone 2 A.X (listed in Antibody Table 1) were incubated overnight and the blots were then exposed to secondary goat anti-rabbit or anti-mouse antibodies (1:300 ,000; cat# 31460 and 31430, Pierce Biotechnology Inc., Rockford, IL) linked to horseradish peroxidase and visualized by SuperSignal™ West Pico PLUS (cat# PI34577, Thermo Scientific). Blots were washed, re-probed with mouse monoclonal anti-human β-actin antibodies (1:1000 dilution, cat# 3700, Cell Signaling) to confirm evenness of loading. SeeBlue Plus2® pre-stained molecular weight standards (cat# LC5925, Life Technologies, Grand Island, NY), were used to calibrate migration of proteins. The molecular mass of each band is indicated at the right of each Figure as kDa. Full-length copies of the original Western gels are provided as a single Supplemental Fig. 1.
Data presentation and statistical analyses
Results and statistical evaluation of RNAseq and transcriptome findings were assessed as described above. Data from biomarker ELISA and cell shape indices were all found to be normally distributed (Kolmogorov-Smirnov tests) and are presented as means ± SD when performed in triplicate. Each experiment was repeated a minimum of three independent times with representative findings shown. SASP biomarkers in our model of ESC inflammation were compared by 1-factor ANOVA in control ESC vs. those stimulated with IL-1β in the absence or presence of SP (NSC 75890), AS (bentamapimod) or JNK1 or JNK2 siRNA. Post hoc Scheffé’s tests were used to compare inter-group differences among the SP-, AS- or siRNA-treated samples. Comparisons among cell shape indices and ELISA data for decidual markers were made using 2-factor ANOVA, with passage number and hormone treatment as the categorical independent variables. Cell shape indices compared among different passage numbers with or without hormone treatment were analyzed by 2- and 1-factor ANOVA. 10 randomly selected ESC photomicrograph images were used for cell shape measurements under each experimental condition. Statistical significance was accepted when two-tailed tests yielded p < 0.05.
Availability of data and materials
All datasets will be made available to readers promptly upon request.
References
Cooke CM, Davidge ST. Advanced maternal age and the impact on maternal and offspring cardiovascular health. Am J Physiol Heart Circ Physiol 2019;317:H387–h94.
Chamani IJ, Keefe DL. Epigenetics and female reproductive aging. Front Endocrinol (Lausanne). 2019;10:473.
Garg D, Johnstone EB, Lomo L, Fair DB, Rosen MP, Taylor R, et al. Looking beyond the ovary for oncofertility care in women: uterine injury as a potential target for fertility-preserving treatments. J Assist Reprod Genet. 2020;37:1467–76.
Pathare ADS, Loid M, Saare M, Gidlöf SB, Zamani Esteki M, Acharya G, et al. Endometrial receptivity in women of advanced age: an underrated factor in infertility. Hum Reprod Update. 2023;29:773–93.
Seppala M, Taylor RN, Koistinen H, Koistinen R, Milgrom E. Glycodelin: a major lipocalin protein of the reproductive axis with diverse actions in cell recognition and differentiation. Endocr Rev. 2002;23:401–30.
Batista MC, Cartledge TP, Zellmer AW, Merino MJ, Axiotis C, Bremner WJ, et al. Effects of aging on menstrual cycle hormones and endometrial maturation. Fertil Steril. 1995;64:492–9.
Erikson DW, Barragan F, Piltonen TT, Chen JC, Balayan S, Irwin JC, et al. Stromal fibroblasts from perimenopausal endometrium exhibit a different transcriptome than those from the premenopausal endometrium. Biol Reprod. 2017;97:387–99.
Yu J, Berga SL, Zou W, Rajakumar A, Man M, Sidell N, et al. Human endometrial stromal cell differentiation is stimulated by PPARβ/δ activation: New targets for infertility? J Clin Endocrinol Metab. 2020;105:2983–95.
Bell SC. Secretory endometrial and decidual proteins: Studies and clinical significance of a maternally derived group of pregnancy-associated serum proteins. Hum Reprod. 1986;1:129–43.
Maslar IA, Kaplan BM, Luciano AA, Riddick DH. Prolactin production by the endometrium of early human pregnancy. J Clin Endocrinol Metab. 1980;51:78–83.
Irwin JC, de las Fuentes L, Giudice LC. Growth factors and decidualization in vitro. Ann N. Y Acad Sci. 1994;734:7–18. 7-18.
Dimitriadis E, Salamonsen LA, Robb L. Expression of interleukin-11 during the human menstrual cycle: coincidence with stromal cell decidualization and relationship to leukaemia inhibitory factor and prolactin. Mol Hum Reprod. 2000;6:907–14.
Laws MJ, Taylor RN, Sidell N, DeMayo FJ, Lydon JP, Gutstein DE, et al. Gap junction communication between uterine stromal cells plays a critical role in pregnancy-associated neovascularization and embryo survival. Development. 2008;135:2659–68.
Zhang XH, Liang X, Liang XH, Wang TS, Qi QR, Deng WB, et al. The mesenchymal-epithelial transition during in vitro decidualization. Reprod Sci. 2013;20:354–60.
Yu J, Berga SL, Johnston-MacAnanny EB, Sidell N, Bagchi IC, Bagchi MK, et al. Endometrial stromal decidualization responds reversibly to hormone stimulation and withdrawal. Endocrinology. 2016;157:2432–46.
Yu J, Boicea A, Barrett KL, James CO, Bagchi IC, Bagchi MK, et al. Reduced connexin 43 in eutopic endometrium and cultured endometrial stromal cells from subjects with endometriosis. Mol Hum Reprod. 2014;20:260–70.
Yu J, Berga SL, Zou W, Yook DG, Pan JC, Andrade AA, et al. IL-1beta inhibits connexin 43 and disrupts decidualization of human endometrial stromal cells through ERK1/2 and p38 MAP kinase. Endocrinology. 2017;158:4270–85.
Piltonen TT, Chen JC, Khatun M, Kangasniemi M, Liakka A, Spitzer T, et al. Endometrial stromal fibroblasts from women with polycystic ovary syndrome have impaired progesterone-mediated decidualization, aberrant cytokine profiles and promote enhanced immune cell migration in vitro. Hum Reprod. 2015;30:1203–15.
Popovic M, Sartorius G, Christ-Crain M. Chronic low-grade inflammation in polycystic ovary syndrome: is there a (patho)-physiological role for interleukin-1? Semin Immunopathol. 2019;41:447–59.
Kariya M, Kanzaki H, Takakura K, Imai K, Okamoto N, Emi N, et al. Interleukin-1 inhibits in vitro decidualization of human endometrial stromal cells. J Clin Endocrinol Metab. 1991;73:1170–4.
Frank GR, Brar AK, Jikihara H, Cedars MI, Handwerger S. Interleukin-1 beta and the endometrium: An inhibitor of stromal cell differentiation and possible autoregulator of decidualization in humans. Biol Reprod. 1995;52:184–91.
Vicovac LM, Starkey PM, Aplin JD. Comment: Effect of cytokines on prolactin production by human decidual stromal cells in culture: studies using cells freed of bone marrow-derived contaminants. J Clin Endocrinol Metab. 1994;79:1877–82.
Yu J, Berga SL, Zou W, Taylor RN. Interleukin-1beta inhibits estrogen receptor-alpha, progesterone receptors A and B and biomarkers of human endometrial stromal cell differentiation: implications for endometriosis. Mol Hum Reprod. 2019;25:625–37.
Spandorfer SD, Neuer A, Liu HC, Rosenwaks Z, Witkin SS. Involvement of interleukin-1 and the interleukin-1 receptor antagonist in in vitro embryo development among women undergoing in vitro fertilization-embryo transfer. J Assist Reprod Genet. 2003;20:502–5.
Boomsma CM, Kavelaars A, Eijkemans MJ, Lentjes EG, Fauser BC, Heijnen CJ, et al. Endometrial secretion analysis identifies a cytokine profile predictive of pregnancy in IVF. Hum Reprod. 2009;24:1427–35.
Franceschi C, Bonafè M, Valensin S, Olivieri F, De Luca M, Ottaviani E, et al. Inflamm-aging. An evolutionary perspective on immunosenescence. Ann N. Y Acad Sci. 2000;908:244–54.
Yu J, Berga SL, Meng Q, Xia M, Kohout TA, van Duin M, et al. Cabergoline stimulates human endometrial stromal cell decidualization and reverses effects of interleukin-1β in vitro. J Clin Endocrinol Metab. 2021;106:3591–604.
Greenfeder SA, Nunes P, Kwee L, Labow M, Chizzonite RA, Ju G. Molecular cloning and characterization of a second subunit of the interleukin 1 receptor complex. J Biol Chem. 1995;270:13757–65.
Tseng JF, Ryan IP, Milam TD, Murai JT, Schriock ED, Landers DV, et al. Interleukin-6 secretion in vitro is up-regulated in ectopic and eutopic endometrial stromal cells from women with endometriosis. J Clin Endocrinol Metab. 1996;81:1118–22.
Ryan IP, Tseng JF, Schriock ED, Khorram O, Landers DV, Taylor RN. Interleukin-8 concentrations are elevated in peritoneal fluid of women with endometriosis. Fertil Steril. 1995;63:929–32.
Khorram O, Taylor RN, Ryan IP, Schall TJ, Landers DV. Peritoneal fluid concentrations of the cytokine RANTES correlate with the severity of endometriosis. Am J Obstet Gynecol. 1993;169:1545–9.
Hornung D, Klingel K, Dohrn K, Kandolf R, Wallwiener D, Taylor RN. Regulated on activation, normal T-cell-expressed and -secreted mRNA expression in normal endometrium and endometriotic implants: assessment of autocrine/paracrine regulation by in situ hybridization. Am J Pathol. 2001;158:1949–54.
Akoum A, Lemay A, Brunet C, Hebert J. Secretion of monocyte chemotactic protein-1 by cytokine-stimulated endometrial cells of women with endometriosis. Le groupe d’investigation en gynecologie. Fertil Steril. 1995;63:322–8.
Sharpe-Timms KL, Cox KE. Paracrine regulation of matrix metalloproteinase expression in endometriosis. Ann N. Y Acad Sci. 2002;955:147–56.
Cao Y, Liu X, Guo SW. Plasma High Mobility Group Box 1 (HMGB1), Osteopontin (OPN), and hyaluronic acid (HA) as admissible biomarkers for endometriosis. Sci Rep. 2019;9:9272.
Goumenou AG, Matalliotakis IM, Tzardi M, Fragouli IG, Mahutte NG, Arici A. p16, retinoblastoma (pRb), and cyclin D1 protein expression in human endometriotic and adenomyotic lesions. Fertil Steril. 2006;85:1204–7.
Malvezzi H, Dobo C, Filippi RZ, Mendes do Nascimento H, Palmieri da Silva ESL, Meola J, et al. Altered p16(Ink4a), IL-1β, and lamin b1 protein expression suggest cellular senescence in deep endometriotic lesions. Int J Mol Sci. 2022;23:2476.
Osokine I, Siewiera J, Rideaux D, Ma S, Tsukui T, Erlebacher A. Gene silencing by EZH2 suppresses TGF-β activity within the decidua to avert pregnancy-adverse wound healing at the maternal-fetal interface. Cell Rep. 2022;38:110329.
Sirohi VK, Medrano TI, Kannan A, Bagchi IC, Cooke PS. Uterine-specific Ezh2 deletion enhances stromal cell senescence and impairs placentation, resulting in pregnancy loss. iScience. 2023;26:107028.
Nayyar A, Saleem MI, Yilmaz M, DeFranco M, Klein G, Elmaliki KM, et al. Menstrual effluent provides a novel diagnostic window on the pathogenesis of endometriosis. Front Reprod Health. 2020;2.
Tomari H, Kawamura T, Asanoma K, Egashira K, Kawamura K, Honjo K, et al. Contribution of senescence in human endometrial stromal cells during proliferative phase to embryo receptivity. Biol Reprod. 2020;103:104–13.
Brighton PJ, Maruyama Y, Fishwick K, Vrljicak P, Tewary S, Fujihara R, et al. Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium. Elife 2017;6:e31274.
Yu J, Francisco AMC, Patel BG, Cline JM, Zou E, Berga SL, et al. IL-1beta stimulates brain-derived neurotrophic factor production in eutopic endometriosis stromal cell cultures: A model for cytokine regulation of neuroangiogenesis. Am J Pathol. 2018;188:2281–92.
Yu J, Berga SL, Zou E, Schrepf AD, Clauw DJ, As-Saine S, et al. Neurotrophins and their receptors, novel therapeutic targets for pelvic pain in endometriosis, are coordinately regulated by IL-1β via the JNK signaling pathway. Am J Pathol. 2023;193:1046–58.
Rawlings TM, Tryfonos M, Makwana K, Taylor DM, Brosens JJ, Lucas ES. Endometrial assembloids to model human embryo implantation in vitro. Methods Mol Biol. 2023.
Gnecco JS, Brown A, Buttrey K, Ives C, Goods BA, Baugh L, et al. Organoid co-culture model of the human endometrium in a fully synthetic extracellular matrix enables the study of epithelial-stromal crosstalk. Med. 2023;4:554–79.e9.
Ryan IP, Schriock ED, Taylor RN. Isolation, characterization, and comparison of human endometrial and endometriosis cells in vitro. J Clin Endocrinol Metab. 1994;78:642–9.
Chowdhury I, Banerjee S, Driss A, Xu W, Mehrabi S, Nezhat C, et al. Curcumin attenuates proangiogenic and proinflammatory factors in human eutopic endometrial stromal cells through the NF-kappaB signaling pathway. J Cell Physiol. 2019;234:6298–312.
Yu J, Wu J, Bagchi IC, Bagchi MK, Sidell N, Taylor RN. Disruption of gap junctions reduces biomarkers of decidualization and angiogenesis and increases inflammatory mediators in human endometrial stromal cell cultures. Mol Cell Endocrinol. 2011;344:25–34.
Schutte SC, Taylor RN. A tissue-engineered human endometrial stroma that responds to cues for secretory differentiation, decidualization, and menstruation. Fertil Steril. 2012;97:997–1003.
Banda NK, Guthridge C, Sheppard D, Cairns KS, Muggli M, Bech-Otschir D, et al. Intracellular IL-1 receptor antagonist type 1 inhibits IL-1-induced cytokine production in keratinocytes through binding to the third component of the COP9 signalosome. J Immunol. 2005;174:3608–16.
Horeth E, Bard J, Che M, Wrynn T, Song EAC, Marzullo B, et al. High-resolution transcriptomic landscape of the human submandibular gland. J Dent Res. 2023:220345221147908.
Acknowledgements
We thank the Genomics and Bioinformatics Core, Pathololgy and Anatomical Sciences Research Service Center, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York 14203, for their assistance with the performance and interpretation of the RNAseq analyses and developing images and we are grateful to the participants who donated endometrial biopsies.
Author information
Authors and Affiliations
Contributions
All authors contributed substantively to the work. R.N.T., S.L.B., E.Z., J.W., S.S., B.J.M., I.C.B., M.K.B., and J.Y. designed the experiment, analyzed and interpreted the data, and prepared the manuscript, tables, and figures; S.L.B. recruited the participants and collected the endometrial samples; J.Y. and E.Z. performed Immunofluorescence- (IFC) and Immunoperoxidase-cytochemistry (IPC), cell culture isolation, ELISA, and Western blotting experiments; R.N.T. secured funding; and R.N.T. and J.Y. wrote the initial draft of the manuscript and is the guarantor of this work and, as such, has full access to all the data in the study and takes responsibility for the integrity and the accuracy of the data analysis. All authors approved the final version of the paper.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Ethics approval and consent to participate
The studies were carried out in full compliance with the Declaration of Helsinki. All participants undergoing elective laparoscopic surgery provided written informed consent. The study received ethics approval and consent from the institutional review boards at Wake Forest School of Medicine, Winston-Salem, North Carolina (IRB #00019887).
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Taylor, R.N., Berga, S.L., Zou, E. et al. Interleukin-1β induces and accelerates human endometrial stromal cell senescence and impairs decidualization via the c-Jun N-terminal kinase pathway. Cell Death Discov. 10, 288 (2024). https://doi.org/10.1038/s41420-024-02048-6
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41420-024-02048-6
- Springer Nature Limited