IL-37 Ameliorates Chronic Endometritis by Attenuating Epithelial-Mesenchymal Transition and Promoting M2 Macrophage Polarization

1. Introduction

Chronic endometritis (CE), a persistent inflammatory condition of the endometrium characterized by stromal plasma cell infiltration, edema, and aberrant glandular architecture, significantly contributes to infertility, recurrent implantation failure, and miscarriage [1,2]. Endometrial epithelial and stromal cells recognize pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) and activate the Toll-like receptor signaling pathway, thereby initiating the NF-κB and MAPK cascade reactions. This activation promotes a sustained release of inflammatory cytokines and chemokines, perpetuating immune cell infiltration [3,4]. The resulting chronic inflammatory milieu disrupts glandular function and endometrial receptivity, leading to progressive tissue fibrosis characterized by excessive collagen deposition, endometrial sclerosis, and loss of plasticity [5,6]. Current management strategies, predominantly antibiotic-based, face significant challenges including microbial resistance, disease recurrence, and the inability to reverse established fibrosis, thereby underscoring the urgent need for novel therapeutic strategies that simultaneously target inflammation and fibrotic remodeling [7,8,9].

Interleukin-37 (IL-37) is a multifunctional cytokine with potent immunosuppressive activity within the IL-1 family. Intracellular IL-37 binds to Smad3 and translocates to the nucleus, leading to upregulation of PTPN. This upregulation inhibits the activation of key pro-inflammatory signaling pathways, including ERK, p38, JNK, PI3K, and NF-κB [10,11,12,13,14]. Extracellularly, IL-37 binds the IL-18Rα/IL-1R8 complex, which activates inhibitory signaling axes—such as STAT3/6 and PTEN/FOXO/AMPK—and in turn blocks fibrosis-related pathways including MAPK, NF-κB, and mTOR [10,15,16,17,18]. Existing studies have confirmed the definitive anti-inflammatory and anti-fibrotic potential of IL-37 across multiple organ systems. In pulmonary fibrosis models, IL-37 significantly alleviates bleomycin-induced pulmonary inflammatory infiltration and collagen deposition [19]. In models of diabetic nephropathy, IL-37 mitigates fatty acid oxidation dysfunction and renal fibrosis by upregulating CPT1A [20]. Furthermore, in diabetic cardiomyopathy, IL-37 improves mitochondrial damage and inhibits myocardial fibrosis by maintaining the function of the SIRT1–AMPK–PGC1α axis [21].

IL-37 not only alleviates inflammation and reverses fibrosis but also regulates macrophage polarization. Specifically, it is produced by TLR-activated macrophages and can feed back to regulate their function. Moreover, studies show that in diseases such as hepatitis, liver fibrosis, and periodontitis, IL-37 inhibits M1 polarization and promotes M2 polarization, thereby helping to modulate the immune balance [16,22,23,24]. In addition, in atherosclerosis, IL-37 suppresses macrophage ferroptosis through the NRF2 pathway [25]. Finally, for tumor immunity, it enhances macrophage phagocytic capacity via the IL-18Rα/SIGIRR-STAT3 axis [26]. Collectively, these effects provide a critical foundation for IL-37's role in mitigating tissue damage.

The evidence presented indicates that IL-37 plays a pivotal role in regulating immunity and inhibiting fibrosis; however, its specific functions and therapeutic potential in CE remain unclear. In this study, we demonstrate that IL-37 exerts a protective effect on endometrial repair by inhibiting EMT and reprogramming macrophages towards M2 polarization, potentially co-activating the STAT6 and Smad3 signaling pathways. These findings may provide novel experimental evidence and strategies for the treatment of CE.

2. Materials and Methods

2.1. Expression and Purification of Recombinant IL-37 Protein

The pET22b-TAT-IL37d plasmid was kindly provided by Shandong University. Bacteria carrying the plasmid were cultured in LB medium with ampicillin. After reaching log phase, IPTG-induced expression at 16°C. Cells were lysed by ultrasonication, and the supernatant was purified by Ni-NTA affinity chromatography. After dialysis, endotoxin removal, and testing, the samples were confirmed as qualified. Concentration was measured by BCA, and purity by SDS-PAGE. Samples were sterilized, filtered, aliquoted, and stored at -80°C.

2.2. Cell Culture

NRK-52e, HEK-293T, and RAW 264.7 cells were all purchased from ATCC. All cells were cultured in DMEM high-glucose medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco), and maintained at 37°C in an incubator with 5% CO₂.

2.3. Establishment and Management of Animal Models

All animal protocols were approved by the Biomedical Research Ethics Committee of Hunan Normal University (permit number: 2025674). Female SD rats (8 weeks old) were housed in an SPF-grade facility and randomly divided into four groups: control, LPS, IL-37 1 μg, and IL-37 2 μg. Chronic endometritis was induced by uterine curettage combined with LPS suture embedding. The control group underwent laparotomy and uterine cornua exposure only, without curettage or embedding. After suture removal, treatment groups received intraperitoneal IL-37 injections every 3 days. Other groups were given the same volume of saline. Rats were euthanized at the third estrous cycle post-model establishment. Uterine tissues were then collected for examination.

2.4. Hematoxylin and Eosin (HE) Staining

After deparaffinization and hydration, the paraffin sections were stained with hematoxylin for 5 to 8 minutes and rinsed in running water. They were then counterstained with eosin for 1 to 3 minutes, dehydrated in a graded ethanol series, cleared in xylene, and mounted with neutral balsam. Observations were performed under a microscope, and images were analyzed using ImageJ software.

2.5.Masson Staining

After dewaxing and hydration, paraffin sections were stained with Weigert's iron hematoxylin for 5-10 minutes, differentiated with acid ethanol, and blued. They were then immersed in Lichun red and acid eosin for 5-10 minutes, followed by phosphomolybdic acid treatment for 5 minutes. Without water washing, sections were counterstained with aniline blue for 5 minutes and rinsed with a weak acid solution. The sections were dehydrated through graded ethanol, cleared with xylene, and mounted with neutral gum. Samples were examined microscopically, photographed, and analyzed using ImageJ software.

2.6. Immunofluorescence Staining

After embedding in OCT, frozen tissue sections were prepared, fixed, permeabilized, and blocked. Sections were then incubated overnight at 4 °C with primary antibodies: IL-37 (Abcam), E-Cadherin (CST), Vimentin (ABclonal), CD86 (Proteintech), CD206 (Proteintech), Smad3 (CST), and Stat6 (Proteintech). The following day, sections were incubated with fluorescent secondary antibodies at room temperature in the dark for 1 hour, then stained with DAPI. Microscopy was used for imaging, and ImageJ was used for analysis.

2.7.Transwell Assay

Transwell chambers were placed in a 24-well plate. A cell suspension in serum-free medium was added to the upper chamber, and complete medium with 10% fetal bovine serum was added to the lower chamber as a chemotactic agent. Plates were incubated at 37°C with 5% CO₂ for 24 hours. After incubation, the medium was removed from the upper chamber, and non-migrated cells were wiped from the upper surface with a cotton swab. Migrated cells on the lower surface were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and analyzed using ImageJ.

2.8. Western Blot

Total protein was extracted using RIPA lysis buffer. Equal amounts of protein were separated by 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% non-fat milk, membranes were incubated overnight at 4°C with primary antibodies: E-Cadherin (CST), Vimentin (ABclonal), STAT6 (Proteintech), p-STAT6 (CST), Smad3 (CST), p-Smad3 (CST), Arg 1 (CST), and β-actin (CST). The next day, membranes were incubated with the secondary antibody for 1 hour at room temperature. Chemiluminescence was detected and images captured using the Tanon system, followed by grayscale analysis in ImageJ.

2.9. Real-Time Quantitative PCR (RT-qPCR)

Total RNA was extracted using Trizol (Invitrogen). Concentration and purity were measured, then cDNA was synthesized with a reverse transcription kit (TAKARA). qPCR was performed with a TAKARA kit on a Bio-Rad instrument using these conditions: 95°C for 30 seconds, then 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. β-actin or GAPDH served as internal controls, and relative gene expression was determined by the 2^−ΔΔCt method.

2.10. Flow Cytometry

RAW 264.7 cells from each group were collected and incubated with Fc blocking reagent (BD) on ice for 15 minutes to prevent non-specific binding. Then the cells were fixed, permeabilized and incubate with APC-conjugated CD206 antibody (Biolegend) in the dark at room temperature for 30 minutes. The cells were washed twice with pre-cooled PBS and detected using a BD flow cytometer.

2.11. Dual-Luciferase Reporter Gene Assay

HEK-293T cells were co-transfected with the pLenti-Smad-minP-Luc reporter plasmid and the pRL-TK internal reference plasmid using Lipo8000TM transfection reagent (Beyotime). After 24 hours, the cells were treated with IL-37 at 10, 100, or 1000 ng/mL for an additional 24 hours. Cells were then lysed, and luciferase activity was measured using a dual-luciferase reporter assay kit (Beyotime). Smad pathway transcriptional activity was expressed as the ratio of firefly to Renilla luciferase activity.

2.12. Statistical Analysis

All data were derived from at least three independent experiments and expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8.0 software. Unpaired t-tests were employed for comparisons between two groups, one-way ANOVA for multi-group comparisons, and Tukey's post-hoc test for pairwise comparisons between groups. A P-value <0.05 was considered statistically significant.

3. Results

3.1. Exogenous IL-37 Protein Ameliorates LPS-Induced Rat Chronic Endometritis

To evaluate the therapeutic potential of IL-37 for CE, we successfully expressed and purified recombinant IL-37 protein, which was administered via intraperitoneal injection to treat CE. Immunofluorescence analysis of uterine tissues revealed that, compared to the control group, the LPS-induced model group showed no significant change in IL-37 expression. In contrast, the IL-37 (1 μg and 2 μg) groups exhibited a marked increase in IL-37-specific fluorescence signals, primarily localized at the cell periphery (Figure 1A, B). These findings indicate that exogenous IL-37 protein can be effectively taken up by rat uterine cells.

Gross morphological observation demonstrated that, compared to the control group, the LPS group exhibited significant pathological changes in the uterus, including intrauterine fluid accumulation, tissue edema, and thinning of the uterine wall with increased transparency. However, following intervention with IL-37 (1 μg and 2 μg), these pathological manifestations were markedly alleviated, as evidenced by reduced intrauterine fluid and restoration of normal uterine morphology (Figure 1A).

H&E staining results (Figure 1A, C, D) illustrated that the LPS group exhibited damaged endometrial structure, accompanied by extensive inflammatory cell infiltration and stromal edema. IL-37 treatment significantly attenuated inflammatory infiltration and edema, promoting the repair of the endometrial structure, which was reflected in the restoration of the endometrial area ratio and glandular number. Furthermore, Masson staining (Figure 1A, E) demonstrated that the LPS group exhibited significantly increased collagen fiber deposition, while IL-37 intervention effectively reduced the collagen deposition area and reversed the fibrotic process.

Therefore, we concluded that exogenous IL-37 can be effectively delivered to injured uteri and exerts therapeutic effects including anti-inflammatory action, promotion of tissue repair, and anti-fibrotic activity.

3.2. IL-37 Inhibits the EMT Process In Vitro and In Vivo

Based on previous findings that IL-37 can reverse endometrial fibrosis, we hypothesized that its effects may be associated with the inhibition of the EMT process. To evaluate the occurrence of EMT and the intervention effect of IL-37 at the organizational level, we conducted immunofluorescence staining of rat uterine tissues. The results (Figure 2) indicated that, compared with the control group, the LPS group exhibited significantly downregulated expression of the epithelial marker E-cadherin and significantly upregulated expression of the mesenchymal marker vimentin in uterine tissues. Following IL-37 treatment, the fluorescence intensity of E-cadherin was effectively restored, whereas the expression of vimentin was significantly inhibited.

Moreover, we conducted in vitro validation using LPS-stimulated rat renal tubular epithelial cells. Transwell migration assays demonstrated that IL-37 treatment effectively inhibited the LPS-induced enhancement of cell migration capacity (Figure 3A, B). Western blot and RT-qPCR results confirmed that IL-37 intervention could reverse the dysregulated expression of key EMT markers induced by LPS. Specifically, IL-37 restored the expression of the epithelial marker E-cadherin and downregulated the elevated levels of several mesenchymal markers, including vimentin and N-cadherin(Figure 3C, D). .These finding provide a critical mechanistic basis for IL-37's role in ameliorating endometrial fibrosis.

3.3. IL-37 Inhibits M1 and Promote M2 Polarization

To elucidate the regulatory role of IL-37 in the immune microenvironment of CE, we systematically evaluated its effects on macrophage polarization..First, immunofluorescence staining of uterine tissue revealed that LPS induced a pro-inflammatory environment characterized by an expansion of CD86⁺ M1 macrophages and a contraction of CD206⁺ M2 macrophages. Administration of IL-37 dose-dependently recalibrated this ratio, suppressing CD86⁺ fluorescence while amplifying CD206⁺ signals, with maximal restoration achieved at 2 µg .(Figure 4).

Next, we assessed the effect of IL-37 on macrophage polarization in RAW264.7 cells. In LPS-driven M1 cultures, IL-37 curtailed the transcription of IL-6, iNOS, and IL-1β, alongside the inhibition of the master M1 transcription factor STAT3 (Figure 5A). Conversely, under IL-4-driven M2 conditions, IL-37 collaborated with IL-4 to enhance the expression of TGF-β, CD206, and Arg1, while elevating STAT6 mRNA (Figure 5B). Flow cytometric quantification confirmed that the combination of IL-37 and IL-4 synergistically increased the CD206⁺ subpopulation in a concentration-dependent manner (Figure 5C). Collectively, these data establish IL-37 suppresses the pro-inflammatory M1 phenotype while promoting the reparative M2 phenotype, thereby reprogramming the endometrial immune niche toward a reparative phenotype in CE.

3.4. IL-37 Promotes M2 Macrophage Polarization via Coordinated STAT6/Smad3 Activation

To determine whether IL-37 reprograms macrophage function by modulating STAT6 and Smad3 signaling pathways, we conducted a systematic investigation of its effects in IL-4-induced M2 polarization model. Western blot analysis revealed that IL-4 alone elevated phosphorylated STAT6 (p-STAT6) , IL-37, at concentrations ranging from 10 to 1000 ng/mL, dose-dependently amplified p-STAT6 and independently triggered robust phosphorylation of Smad3 (p-Smad3). Consequently, the ratios of p-STAT6 to total STAT6 and p-Smad3 to total Smad3 were both significantly increased, indicating a synergistic potentiation of STAT6 and de novo activation of Smad3(Figure 6A, B).

Moreover, the results from the smad-responsive luciferase reporter (pLenti-Smad-minP-Luc) demonstrated that IL-37 enhanced luciferase activity in a concentration-dependent manner, demonstrating amplified Smad-mediated transcription (Figure 6C). Additionally, immunofluorescence results (Figure 6D) visually illustrate the dynamic influence of IL-37 on this process. After 30 minutes of treatment, STAT6 and Smad3 signals were primarily localized to the cytoplasm; after 1 hour, they were significantly enhanced and distributed throughout the nucleus. This time-dependent nuclear accumulation clearly demonstrates that IL-37 effectively drives the nuclear translocation of these two key transcription factors.

Collectively, IL-37 functions as a dual co-activator: it amplifies IL-4-triggered STAT6 signaling while independently activating the Smad3 cascade, thereby converging two canonical M2 pathways to enforce macrophage alternative activation.

4. Discussion

The endometrium is a cyclically reprogrammed mucosal surface whose capacity for scarless restoration underpins fertility. When this process is disrupted, CE and intrauterine adhesion (IUA) occur, transforming a transient inflammatory insult into a self-amplifying fibrotic circuit [27,28,29]. This study systematically elucidates, for the first time, the dual protective role of IL-37 in promoting endometrial injury repair: it directly inhibits EMT to preserve epithelial integrity and reprograms macrophage polarization to enhance the repair microenvironment through the coordinated activation of the STAT6/Smad3 axis. These two mechanistic pathways collectively provide robust experimental evidence supporting IL-37 as a potential novel therapeutic target for CE.

IL-37, a member of the interleukin-1 family, exhibits extensive anti-inflammatory and immunoregulatory properties[30]. These characteristics confer protective effects in various disease models, including cardiovascular diseases, autoimmune disorders, and tumors [31,32,33,34]. In recent years, the role of IL-37 in diseases of the female reproductive system has garnered increasing attention. For instance, in endometriosis (EMS), recombinant human IL-37 can inhibit the growth of ectopic lesions by suppressing dendritic cell STAT3 phosphorylation and modulating the Th1/Th2 balance[35]. In endometrial cancer, IL-37b targets the Rac1/NF-κB signaling pathway to reduce the expression of matrix metalloproteinase 2 (MMP2), effectively suppressing cancer cell migration and invasion [36]. Furthermore, endometrial regenerative cells that secrete IL-37 can significantly downregulate pro-inflammatory factors such as TNF-α, IL-1β, and IL-6, while upregulating IL-10, thereby exerting therapeutic effects in chronic transplant vasculopathy[37].Our study is the first to demonstrate that exogenous IL-37 alleviates endometritis by inhibiting EMT and promoting M2 polarization. These findings collectively reveal the crucial regulatory role of IL-37 in both inflammatory and tumor-related pathological processes within the female reproductive system.

EMT is a pivotal event in the fibrotic process of various organs, characterized by the downregulation of epithelial markers, such as E-cadherin, and the upregulation of stromal markers, including N-cadherin and Vimentin [38]. Maintaining epithelial cell homeostasis is crucial for preventing fibrosis [39]. Inhibiting EMT serves as a common mechanism underlying the protective effects of IL-37 in several fibrotic diseases. In hepatic fibrosis, the intracellular form of IL-37 directly interacts with Smad3, thereby suppressing TGF-β signaling and hepatic stellate cell activation [16]. In asthma models, IL-37 alleviates airway EMT by modulating the ERK1/2 and STAT3 pathways [40]. Furthermore, in diabetic cardiomyopathy, IL-37 exerts anti-fibrotic effects by inhibiting the JAK2-STAT3 axis [41].Our study confirms that IL-37 can reverse EMT in CE. In vivo, IL-37 restored the epithelial phenotype of the damaged endometrium. In vitro, it inhibited LPS-induced migration of renal tubular epithelial cells while simultaneously reversing the abnormal expression of EMT markers. These findings indicate that IL-37 can prevent the initiation of fibrosis at its source by directly stabilizing epithelial cells, thereby laying the foundation for tissue regeneration. In CE, microbial-associated molecular patterns, such as LPS, drive the release of pro-inflammatory factors, including TNF-α and IL-6, as well as pro-fibrotic factors like TGF-β, through the activation of the TLR4/NF-κB signaling axis [42,43,44]. IL-37 has been shown to effectively suppress the activation of the NF-κB pathway[30], which may inhibit this upstream inflammatory signaling and downregulate the regulatory network that drives EMT.

As pivotal regulatory cells within the immune microenvironment of CE, macrophages directly influence the processes of endometrial tissue damage and repair through their polarization states [45,46,47,48]. Previous studies have demonstrated that modulating macrophage polarization can serve as an effective therapeutic strategy for CE. For instance, glycyrrhizic acid can alleviate endometrial hyperplasia and inflammation in CE mice by inhibiting HMGB1-mediated macrophage pyroptosis [49]. Adipose-derived stem cell exosomes suppress M1 polarization by regulating the SIRT2/NLRP3 axis, thereby mitigating endometritis [50]. Additionally, recombinant humanized type III collagen (rhCol III) has been shown both in vitro and in rat CE models to promote macrophage polarization toward the M2 phenotype by inhibiting the NF-κB/YAP pathway, consequently improving inflammation and facilitating the restoration of the immune microenvironment [51].

Although IL-37 has been established as a crucial immunoregulatory factor in various inflammatory models, its specific molecular mechanism in regulating macrophage polarization in chronic inflammation remains unclear. This study is the first to demonstrate that IL-37 can induce 'functional reprogramming' of macrophages in chronic inflammation models. Experimental results indicate that under M1 polarization conditions, IL-37 significantly suppresses the expression of pro-inflammatory genes such as IL-6, iNOS, and IL-1β. Conversely, under M2 polarization conditions, IL-37 not only further upregulates the expression of classic M2 markers including TGF-β, Arg1, and CD206 but also increases the proportion of CD206+ M2 cells. In-depth mechanistic studies revealed that IL-37 treatment significantly enhanced the phosphorylation levels of STAT6 and Smad3, thereby activating the downstream Smad signaling pathway to promote the expression of the repair-related protein ARG1. Notably, classical M2 macrophages typically activate the Smad pathway through TGF-β secretion, promoting EMT and fibrosis [52,53]. However, this study found that under IL-37 intervention, although the STAT6/Smad3 signaling was synergistically activated, its downstream effects were not pro-fibrotic; instead, they were accompanied by EMT inhibition and upregulation of ARG1 expression. This indicates that IL-37 does not induce M2 polarization in the conventional sense but rather achieves precise 'reprogramming' of macrophage function by remodeling the activation pattern of the STAT6/Smad3 signaling network. This enables macrophages to enhance anti-inflammatory and repair functions while avoiding potential pro-fibrotic effects.

In summary, this study proposes an integrated mechanistic model regarding IL-37's role in promoting endometrial repair. Within the CE-damaged microenvironment, IL-37 operates through dual synergistic pathways. On one hand, it directly inhibits EMT to stabilize epithelial architecture; on the other hand, it reprograms macrophages by coordinately activating and remodeling the functional output of the STAT6/Smad3 axis, thereby shaping an efficient and safe pro-repair immune microenvironment. These two pathways mutually reinforce each other to collectively drive high-quality tissue regeneration. Although the precise mechanism by which IL-37 coordinately activates STAT6 and Smad3 remains to be elucidated further, this study provides a solid theoretical foundation and experimental basis for developing novel therapies targeting IL-37 or its downstream signaling nodes for the treatment of CE and related endometrial fibrotic diseases.

5. Conclusions

Our results demonstrated that IL-37 accumulated in injured uterine tissue, significantly ameliorating inflammatory infiltration, tissue destruction, and collagen deposition. Treatment with IL-37 attenuated epithelial cell migration and reversed abnormal EMT by upregulating E-cadherin and downregulating Vimentin. Furthermore, IL-37 inhibited M1 macrophage infiltration and promoted a shift toward the M2 phenotype. Mechanistic studies revealed that IL-37 co-activated the STAT6 and Smad3 signaling pathways, enhancing their phosphorylation and nuclear translocation, which subsequently boosted ARG1 expression. These findings indicate that IL-37 ameliorates CE by attenuating EMT and promoting M2 macrophage polarization through the coordinated activation of the STAT6/Smad3 axis, underscoring its potential as a therapeutic agent for CE.