Interleukin‐6 Downregulates T Helper 17 Cytokines via Indoleamine 2,3‐Dioxygenase 1 at the Maternal‐Fetal Interface

ABSTRACT

Background

The cytokines interleukin (IL)‐6 and transforming growth factor‐beta (TGF‐β) are established inducers of pathogenic T helper 17 (Th17) cells from naïve T cells. In the maternal‐fetal compartment, IL‐6 signaling elevates levels of indoleamine 2,3‐dioxygenase 1 (IDO1). This study investigated whether IL‐6 regulates Th17 cytokine production through IDO1.

Methods

Human decidua and chorionic villi (HDCV) were collected from normal early pregnant women undergoing elective termination. For HDCV culture, explants were treated with increasing doses of IL‐6 (0–50 ng/mL) or co‐treated with IL‐6 and the IDO1 inhibitor 1‐methyltryptophan (1‐MT) for 24 h. HDCV were also treated with IL‐6 (5 ng/mL) alone or in combination with TGF‐β (0.01–0.1 ng/mL) for 24 h. Recurrent pregnancy loss (RPL) mice were intraperitoneally injected with IL‐6 (2, 10, or 50 ng/mL), alone or combined with 1‐MT and/or Cosentyx, from gestational day (GD) 0.5 to 8. Protein expression levels of IL‐6, IDO1, IL‐17, and IL‐22 were assessed by Western blotting and immunohistochemistry. Embryo absorption rate (EAR) was calculated on GD 12.5. Cytokine concentrations in supernatants or serum were measured using enzyme‐linked immunosorbent assay.

Results

In HDCV tissues, IL‐6 positively correlated with IDO1 and negatively correlated with IL‐17/IL‐22. Exogenous IL‐6 dose‐dependently upregulated IDO1 and suppressed IL‐17/IL‐22 in cultured explants, effects reversed by 1‐MT. Physiological TGF‐β1 (0.01–0.1 ng/mL) enhanced IL‐6‐induced IDO1 upregulation and Th17 cytokine suppression. In RPL mice, IL‐6 reduced EAR and placental IL‐17/IL‐22 expression while increasing IDO1; these effects were blocked by 1‐MT and restored by Cosentyx.

Conclusions

During normal pregnancy, IL‐6 alone or the combination of IL‐6 and TGF‐β at the maternal‐fetal interface may inhibit Th17 cytokine production by upregulating IDO1 expression, thereby maintaining pregnancy stability.

1. Introduction

Recurrent pregnancy loss (RPL) is defined as two or more clinical pregnancy losses before 20 weeks of gestation, and affects 13% of reproductive‐aged couples [1]. The etiology of RPL encompasses anatomical, endocrinological, genetic, immunological, infectious, and thrombophilic factors; however, approximately 50% of cases are classified as unexplained recurrent pregnancy loss (URPL) [2]. Immune dysregulation has emerged as a significant contributor to URPL, with maternal‐fetal immune dysfunction identified as a key pathophysiological feature [1, 3, 4]. Despite extensive research, the exact mechanisms and pathogenesis underlying URPL remain unclear.

Embryo implantation in mammals establishes an intimate physical connection between the conceptus and the maternal uterus. This proximity presents a unique immunological paradox; the maternal immune system must actively tolerate the semi‐allogeneic fetus, which expresses paternal antigens. Genetically, the fetus resembles a semi‐allograft that shares half of its genetic material with its father. In the absence of regulatory immune mechanisms, these allografts are rejected. However, throughout gestation, the maternal immune system does not attack but rather establishes a state of tolerance, a critical mechanism for fetal protection [5]. This immunological privilege is maintained within the dynamic maternal‐fetal compartment, where the interaction between syncytiotrophoblast cells and the decidua occurs. Despite being immunologically inert, this interface is a hub of active immune processes that balance tolerance to the fetus with defense against pathogens, preventing rejection [6, 7, 8, 9].

A successful pregnancy relies on the precise regulation of the maternal immune system to recognize and tolerate the semi‐allogeneic fetus while simultaneously preventing infection [10]. A complex, multi‐layered network of immune cells and mediators operates at the maternal‐fetal interface to establish and maintain this tolerance [11]. Key cellular components include regulatory T cells (Tregs), which create a tolerogenic microenvironment and suppress fetal‐antigen‐specific effector responses [12]; decidual macrophages, which constitute approximately 20% of decidual leukocytes and contribute to immune regulation and tissue remodeling [13]; uterine natural killer (uNK) cells, the most abundant decidual leukocyte population (70%), which promote trophoblast invasion and vascular remodeling while exhibiting tolerogenic phenotypes [11, 13]; and dendritic cells involved in antigen presentation. At the molecular level, soluble factors such as interleukin (IL)‐35, indoleamine 2,3‐dioxygenase 1 (IDO1), and a predominant Th2‐type cytokine profile (e.g., IL‐6, IL‐10), is essential for sustaining maternal‐fetal tolerance [5, 14, 15]. In recent years, PD‐1, CTLA‐4, and Tim‐3 have emerged as critical regulators of immune responses during pregnancy [10]. Together, these cellular and molecular mechanisms act to ensure fetal survival while maintaining maternal immune competence.

Recent evidence highlights the pivotal role of Th2‐type cytokines within the maternal‐fetal compartment in promoting and maintaining fetomaternal immune tolerance. Cytokines such as IL‐6 and IL‐10 [16] are central to this process. These cytokines drive B‐cell expansion, maturation, and functional activity, thereby enhancing antibody production and humoral immunity, which collectively support pregnancy success. Among these cytokines, IL‐6 has garnered particular attention. Studies indicate that elevated IL‐6 levels in trophoblasts correlate with reduced embryo resorption and improved pregnancy outcomes in mice, a process mediated through IDO1 induction [17, 18, 19, 20].

T helper 17 (Th17) cells represent a distinct lineage of CD4 + T helper cells defined by secretion of IL‐17 (IL‐17) family cytokines, including IL‐17A, IL‐17F, and IL‐22 [21]. Th17 cells arise from naïve CD4 + T cells, which retain multipotency and differentiate into various T helper subsets depending on cues from the local microenvironment [22]. This differentiation is initiated by activation of professional antigen‐presenting cells (APCs) and driven by cytokines, particularly IL‐6, IL‐21, and transforming growth factor‐beta (TGF‐β) [23, 24, 25]. In pregnancy, IL‐17 plays a functional role, as evidenced by studies showing that secukinumab (Cosentyx), a monoclonal antibody against IL‐17, suppresses IL‐17‐mediated inflammation and reduces miscarriage rates in experimental mouse models [26].

Th17 cells exert pivotal functions across diverse physiological and pathological contexts, including transplant rejection, antimicrobial defense, and antitumor immunity. Functionally, IL‐17, in synergy with IL‐1 and TNF‐α, orchestrates immune activation and cellular recruitment [27]. During gestation, a tightly regulated Th17 response supports host defense against extracellular pathogens through neutrophil‐mediated immunity [22]. However, excessive Th17 accumulation disrupts neutrophil recruitment at the maternal‐fetal interface [16], fostering a pro‐inflammatory milieu detrimental to pregnancy. For instance, IL‐17 has been implicated in inflammatory preeclampsia [28] and is strongly associated with intrahepatic cholestasis of pregnancy (ICP) [29]. Experimental evidence demonstrates that IL‐17 activates NK cells, impairs uterine artery function, and induces embryo resorption in rats [30]. Furthermore, fetal trophoblast invasion and subsequent exposure to paternal antigens may elicit a maternal inflammatory response that promotes Th17 differentiation, activates effector T cells, and undermines immune tolerance at the maternal‐fetal interface.

IDO, a heme‐containing enzyme catabolizing L‐tryptophan at the maternal‐fetal interface [31], is widely recognized as a central immunoregulatory factor across diverse physiological and pathological contexts, including pregnancy, infection, and autoimmunity [31, 32]. In human pregnancy, IDO exhibits high activity and broad expression in syncytiotrophoblasts, cytotrophoblasts, villous stroma, and fetal membrane macrophages [33]. Munn et al. demonstrated that fetuses are protected by local maternal immune tolerance mediated by the activity of IDO, and that 1‐methyltryptophan (1‐MT), a competitive IDO1 inhibitor, effectively abrogates IDO enzymatic activity in pregnant mice [31]. The activity of the IDO1 enzyme can be evaluated by the ratio of kynurenine (Kyn) to tryptophan (Try), with a higher ratio indicating higher activity of the IDO1 enzyme.

One of the mechanisms by which IDO1 induces maternal‐fetal immune tolerance is the downregulation of Th17‐type cytokines. IDO downregulates Th17‐type cytokine expression across multiple cell types and tissues [34]. Consistent with these findings, our previous work demonstrated that elevated IL‐6 expression in trophoblasts within the maternal‐fetal compartment promotes immune tolerance, potentially through the upregulation of IDO1 [19]. These results suggest a potential immunological pathway in which IL‐6 inhibits the Th17 response by inducing IDO1. This hypothesis appears paradoxical, since IL‐6, together with TGF‐β, is a well‐established driver of Th17 differentiation from naïve T cells, a mechanism central to numerous autoimmune diseases [35]. To resolve this apparent contradiction and elucidate the immunomodulatory role of IL‐6 within the maternal‐fetal interface, this study aimed to investigate whether IDO1 downregulates Th17‐type cytokine levels and whether IL‐6 modulates Th17 cytokine production through an IDO1‐dependent mechanism to maintain maternal‐fetal tolerance.

2. Materials and Methods

2.1. Materials

2.1.1. Human Decidual and Villous Tissues

Human decidua and chorionic villi (HDCV) tissues were collected from 37 females with normal pregnancies who underwent elective pregnancy termination at the Affiliated Hospital of Guizhou Medical University (Guiyang, China) between January and April 2025. Written informed consent was obtained from all participants before tissue collection. Inclusion criteria were gestational age between 6–8 weeks, normal embryonic development confirmed by ultrasound, and no use of mifepristone, misoprostol, or other pre‐abortion medications. Exclusion criteria included maternal age ≥ 35 years, thrombophilia, uterine malformations, endometrial disorders (e.g., adenomyosis), and endocrine diseases (e.g., diabetes, or hypothyroidism), and immune disorders (e.g., antiphospholipid syndrome) and a history of spontaneous pregnancy loss [36].

2.1.2. Experimental Animals

Ninety‐nine CBA/J female mice and 45 DBA/2 male mice were used in this study. All mice were 8 weeks old, weighed 20 ± 2 g, and belonged to inbred strains maintained under specific pathogen‐free (SPF) conditions. The animals were purchased from Beijing Huafukang Biotechnology Co., Ltd. (license no. SYXK(Gui)2023–0002). Upon arrival, the mice were housed in the SPF‐grade animal facility at the Clinical Research Center at Guizhou Medical University. They were maintained under strict specific pathogen‐free (SPF) conditions and provided with standard pellet feed from the Experimental Animal Center of Guizhou Medical University.

2.1.3. Reagents

Anti‐IL‐17A polyclonal antibody, rabbit anti‐IL‐22 antibody, IL‐6 polyclonal antibody, and IDO monoclonal antibody were provided by the Bio‐Byrd Company (China). HRP‐conjugated anti‐rabbit IgG was supplied by Proteintech (China), recombinant human IL‐6 by Abbkine (China), anti‐GAPDH polyclonal antibody by Baijia (China), and the Bicinchoninic Acid Assay (BCA) protein quantification kit and hematoxylin staining solution were from Servicebio (China). A rabbit two‐step detection kit (Rabbit Enhanced Polymer Method Detection System) was provided by the ZSGB‐BIO Company (China). Enzyme‐linked immunosorbent assay kits were from Elabscience (China). Recombinant human TGF‐β1 was from CoSinCayon (China). Recombinant Mouse IL‐17A protein was purchased from Proteintech (China). Mouse IL‐17/IL‐17A antibody and rat IgG2A isotype control were sourced from R&D Systems (USA). NIH‐3T3 cells were provided by Solarbio (China).

2.2. Methods

2.2.1. Tissue Processing and Culture

HDCV tissues were obtained from individuals with normal pregnancies following elective termination. Within 1 h of collection, tissues were washed with sterile phosphate‐buffered saline (PBS) to remove red blood cells and capillaries and were then dissected into small fragments (wet weight ≤ 1 mg). The tissues were cultured as follows. The prepared fragments were washed twice with F‐12/DMEM and then centrifuged at 500 × g for 5 min. The culture medium consisted of RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% Prime supplement, and 1% penicillin‐chloramphenicol mixture (100×). Cultures were maintained at 37°C in a humidified incubator with 5% CO₂. For IL‐6 treatment, tissues (0.5 g/well) were incubated for 24 h in a medium containing 0, 2, 10, or 50 ng/mL recombinant human IL‐6, corresponding to the control, 2, 10, or 50 ng/mL groups, respectively. For IDO inhibition studies, tissues were cultured for 24 h in medium containing 0, 50, or 50 ng/mL IL‐6, followed by the addition of 1‐methyltryptophan (1‐MT, 1 mg/mL, Sigma‐Aldrich) [37] and incubation for an additional 24 h. These groups were designated as the 0, 50, and 50 ng/Ml + 1‐MT groups. Supernatants were collected at 0, 24, and 48 h for enzyme‐linked immunosorbent assay (ELISA) detection of Try and Kyn, and at 48 h, the HDCV was collected for Western blot. For the combined study of IL‐6 and TGF‐β on Th17‐type cytokine levels, the tissues were cultured for 24 h with the following treatments: Group 1 received no cytokines; Group 2 received IL‐6 alone (5 ng/mL); Group 3 received IL‐6 (5 ng/mL) together with TGF‐β at concentrations of 0.01, 0.05, or 0.1 ng/mL [25, 38]. These groups were respectively named NC, IL‐6, and IL‐6 + 0.01, IL‐6 + 0.05, or IL‐6 + 0.1 groups. And at 24 h, the HDCV was collected for Western Blot.

2.2.2. Animal Modeling and Drug Administration

The RPL mouse model was established by co‐housing 8‐week‐old female CBA/J mice with DBA/2 males at a 2:1 ratio [39]. The presence of a vaginal plug was defined as gestational day (GD) 0.5, then pregnant CBA/J mice were randomized into six groups. Drug treatments were administered as follows (Figure 1): The control group (n = 21) received 0.2 mL reconstitution buffer intraperitoneally (i.p.) on GD 0.5, 5.5, 6, 7, and 8. The IL‐6 treatment groups received a single i.p. injection of 0.2 mL reconstitution buffer containing 2 ng/mL (n = 12), 10 ng/mL (n = 12), or 50 ng/mL (n = 21) IL‐6 (equivalent to 0.02, 0.1, or 0.5 µg/kg, respectively) on GD 0.5, followed by 0.2 mL buffer on GD 5.5, 6, 7, and 8. The 50 ng/mL IL‐6 + 1‐MT group (n = 21) received 50 ng/mL IL‐6 on GD 0.5, followed by 0.2 mL 1‐MT solution (10 mg/mL, 0.1 g/kg) [40] on GD 5.5, and buffer on GD 6, 7, and 8. The 50 ng/mL IL‐6 + 1‐MT + Cosentyx group (n = 12) received 50 ng/mL IL‐6 on GD 0.5, 1‐MT on GD 5.5, and 0.2 mL Cosentyx solution (20 mg/mL, 0.2 g/kg, Novartis Pharma Stein AG) on GD 6, 7, and 8 [26]. Serum was collected from three mice in each of the three treatment groups (0 ng/mL, 50 ng/mL, and 50 ng/mL + 1‐MT) at three time points: before pregnancy, 4 h after IL‐6 injection on GD 0.5, and 4 h after 1‐MT injection on GD 5.5. Serum Try and Kyn levels were measured by ELISA. The remaining 12 mice in each of these three groups, as well as all mice in the other three groups, were euthanized on GD 12.5. Embryonic resorption rates were determined, and placental tissues were collected for Western blotting and immunohistochemical staining.

FIGURE 1.

Schematic diagram of the animal model and drug administration protocol. GD, gestational day.

2.2.3. In Vitro Neutralization Assay of Cosentyx on Mouse IL‐17A

Cell culture. NIH‐3T3 cells were seeded in 96‐well plates at 1.5 × 10⁴ cells/well (100 µL) in RPMI 1640 supplemented with 10% FBS, 1% Prime supplement, and 1% penicillin‐streptomycin, and cultured overnight at 37°C in 5% CO₂. Antibody and IL‐17A stimulation. Recombinant mouse IL‐17A (10 ng/mL [41]) was pre‐incubated with increasing concentrations of Cosentyx (1, 10, 50, 100, 500, 1000, 2000 µg/mL) in equal volumes (50 µL + 50 µL) for 30 min at 37°C. Controls included unstimulated cells (medium only), IL‐17A alone (no antibody), positive control (IL‐17A + IL‐17A antibody, 1 µg/mL [41]), and negative control (IL‐17A + IgG isotype, 2000 µg/mL). After pre‐incubation, the mixtures were added to the cells. IL‐6 measurement. After 24 h, supernatants were collected and centrifuged at 1000×g for 5 min. IL‐6 concentration was measured [42] by ELISA (absorbance at 450 nm) following the manufacturer's instructions. All experiments were performed in triplicate, and data are presented as mean ± SD.

2.2.4. Assessment of the Embryonic Absorption Rate

On GD 12.5, all pregnant mice were euthanized, and the uteri were examined to evaluate embryonic development. Embryo absorption was identified by the fulfillment of any of the following criteria: marked reduction in embryo volume, visible hemorrhage, or signs of necrosis. The embryo absorption rate (EAR) of each group was calculated as follows:

$$\begin{array}{ccc}& & \text{Embryo absorption rate}(\%)\hfill \\ & & =\frac{\text{Embryo loss number}}{\text{Embryo loss number}\mathrm{+}\text{Live embryo number}}\times 100\%\hfill \end{array}$$

2.2.5. Western Blotting

Frozen tissues (HDCV tissues and mouse placenta) were minced and homogenized by sonication in RIPA buffer supplemented with PMSF (1:100, Solarbio) for 30 min at 4°C. The homogenate was then subjected to centrifugation (12,000 × g, 30 min, 4°C), and the protein concentration in the supernatant was measured using the Bicinchoninic Acid (BCA) method. The protein content was combined with 5× loading buffer at a 4:1 vol/vol ratio and denatured by heating at 95°C for 10 min. Equal protein quantities were separated by SDS‐PAGE and subjected to electroblotting onto PVDF membranes. Membranes were blocked for 20 min with a rapid blocking solution at ambient temperature, and subsequently incubated with primary antibodies for 16 h at 4°C. The following primary antibodies were used: anti‐IL‐17A (1:1000; BS90701), anti‐IL‐22 (1:1000; BS65609), anti‐IL‐6 (1:500; BZ16464), anti‐IDO1 (1:500; BS6639), and anti‐GAPDH (1:5000; IDS0103). After washing with TBS containing 0.1% Tween 20, the membranes were incubated with horseradish peroxidase‐conjugated anti‐rabbit IgG (1:10,000, SA00001–2) for 1 h at room temperature. Protein bands were visualized using an ECL kit and imaged by autoradiography. Band intensities were quantified using ImageJ software, and target protein expression levels were normalized to GAPDH as an internal control.

2.2.6. Immunohistochemistry

Fresh human decidua (HDCV) and mouse placental tissues were fixed in neutral formalin, paraffin‐embedded, and sectioned (4 µm). Sections were baked at 60°C for 30 min, deparaffinized in xylene, rehydrated through graded ethanol, and washed in distilled water. Antigen retrieval was performed in EDTA buffer (pH 9.0) by microwave heating (8 min). After blocking endogenous peroxidase with 3% H₂O₂ (10 min), sections were blocked with 5% normal goat serum (2 h). Primary antibodies against IL‐6, IDO1, IL‐17, and IL‐22 (all 1:200) were applied overnight at 4°C. Sections were then incubated with a reaction enhancer (37°C, 20 min) and HRP‐conjugated secondary antibody (37°C, 30 min). DAB was used for color development, followed by counterstaining with Mayer's hematoxylin. After dehydration and xylene clearance, sections were mounted with neutral balsam. Images were captured by light microscopy and quantified using ImageJ, with results expressed as average optical density (AOD) after background subtraction.

2.2.7. Enzyme‐linked Immunosorbent Assay

Detection of Try and Kyn in culture supernatants or mouse serum samples: (1) Sample preparation. Culture supernatants or serum samples were centrifuged at 12,000 × g for 15 min at 4°C, and the resulting supernatants were kept on ice. (2) Standard and sample loading. For standard wells, 20 µL of eight different standard solutions (0, 40, 80, 100, 120, 140, 160, and 200 µmol/L) were added in triplicate. For sample wells, 20 µL of each prepared sample was added in triplicate. (3) Reagent addition. Then, 20 µL of Reagent 4, 100 µL of Reagent 2, and 100 µL of Reagent 3 working solutions were added to all wells. (4) Incubation and measurement. The plate was shaken for 5 sec and incubated for 30 min at 37°C in the dark. Absorbance was measured at 535 nm (Try) and 450 nm (Kyn). (5) Calculation. A standard curve was generated using the linear equation y = kx + b, where y is absorbance and x is concentration. Sample concentrations were calculated by interpolation.

2.3. Statistical Analysis

All statistical analyses were performed using GraphPad Prism (v9.0.0). Continuous variables are expressed as the mean ± standard deviation (x ± s), and categorical variables are summarized as percentages (%). For comparisons between two groups, the independent‐samples t‐test was applied. For comparisons across multiple groups, two‐way analysis of variance (ANOVA) was employed. Group differences in abortion rates were assessed using the chi‐square test for a 2 × 2 contingency table. A p < 0.05 was set as the threshold for statistical significance.

2.4. Ethical Approval

The research was performed in compliance with the Helsinki Declaration and was approved by the relevant ethics boards, including the Ethics Committee of the Affiliated Hospital of Guizhou Medical University (No. 289, 2025) and the Laboratory Animal Ethics Committee of Guizhou Medical University (No. 2402746).

3. Results

3.1. IL‐6 and IDO1 Correlation With Th17‐Type Cytokines in HDCV Tissues from Early Pregnancy

To investigate the expression and relationship between IL‐6, IDO1, and Th17‐type cytokines (IL‐17/IL‐22) within the maternal‐fetal compartment, immunohistochemical and Western blotting analyses were performed on HDCV tissues.

Immunohistochemistry detected IL‐6, IDO1, and the Th17‐related cytokines IL‐17 and IL‐22 in both decidual (Figure 2A) and villous tissues (Figure 2B), with these proteins localized to the cytoplasm of syncytiotrophoblast cells. Positive signals were observed as yellow to yellowish‐brown granules.

FIGURE 2.

(A) Immunohistochemical analysis of human decidua tissues revealed expression of IL‐6, IDO1, IL‐17, and IL‐22. (B) Immunohistochemical analysis of human villous tissues revealed expression of IL‐6, IDO1, IL‐17, and IL‐22. Scale bar: 25 µm. Representative images (400× magnification) are from a subset of samples obtained from thirty‐seven healthy pregnant women.

Protein expression in decidual and villous tissues from normal early pregnancies was quantified via Western blotting. To assess the correlation patterns, IL‐6 expression was first analyzed in eight representative samples showing varying expression levels, numbered 1 to 8 in ascending order of intensity (Figures 3A, E). Western blotting analysis of all eight samples for IL‐6, IDO1, IL‐17, and IL‐22 (Figures 3B, F) revealed a positive correlation between IL‐6 and IDO1 expression (Figures 3C, G), whereas a negative correlation was observed between IL‐6 and both IL‐17 and IL‐22 (Figures 3C, G). Similarly, IDO1 expression negatively correlated with IL‐17 and IL‐22 (Figures 3D, H).

FIGURE 3.

(A, E) Representative Western blot images showing IL‐6 protein expression in eight individual samples of decidual and chorionic tissues from healthy pregnant women. Samples are arranged in lanes 1–8 in order of increasing IL‐6 expression. (B, F) Corresponding Western blot images showing the expression levels of IL‐6, IDO1, IL‐17, and IL‐22 in the same set of samples. (C, G) Correlation analysis of IL‐6 with IDO1, IL‐17, and IL‐22 expression in decidua and chorionic villi. (D, H) Correlation analysis of IDO1 with both IL‐17 and IL‐22 in decidua and chorionic villi. R², the coefficients of determination. Grey scale ratio represents the band intensity of each target protein normalized to GAPDH.

3.2. IL‐6 Upregulated IDO1 and Suppressed Th17‐Type Cytokine Expression in Cultured HDCV Tissues

To evaluate the regulatory effects of IL‐6 on IDO1 and Th17‐type cytokines, HDCV explants were exposed to increasing concentrations of IL‐6 (0, 2, 10, and 50 ng/mL) for 24 h, followed by western blotting. In Figures 4A, B, E, and F, exogenous IL‐6 treatment led to a dose‐dependent increase in the production of both IL‐6 and IDO1 while simultaneously reducing the levels of Th17‐type cytokines in both tissue types. Correlation analysis further revealed a positive correlation between IL‐6 and IDO1 and a negative correlation between IL‐6 and Th17‐type cytokines in IL‐6‐stimulated decidua (Figure 4C) and chorionic villi (Figure 4G). Additionally, a negative correlation was observed between IDO1 and Th17‐type cytokines in decidual (Figure 4D) and villous (Figure 4H) cultures.

FIGURE 4.

(A, E) Representative blots of IL‐6, IDO1, IL‐17, and IL‐22 from decidual and chorionic villi explants treated with IL‐6 for 24 h. (B, F) Regression analysis between IL‐6 concentration and protein expression in decidual and chorionic villi explants treated with IL‐6 for 24 h. (C, G) Correlation analysis of IL‐6 with IDO1, IL‐17, and IL‐22 expression in decidual and chorionic villi explants treated with IL‐6 for 24 h. (D, H) Correlation analysis of IDO1 with IL‐17 and IL‐22 in decidual and chorionic villi explants treated with IL‐6 for 24 h. b, regression coefficients. R2, the coefficients of determination. Grey scale ratio represents the band intensity of each target protein normalized to GAPDH.

3.3. 1‐MT Reversed IL‐6‐Mediated Suppression of Th17‐Type Cytokine Expression

To determine whether IL‐6 suppresses Th17‐type cytokine expression via an IDO1‐dependent mechanism, human decidual and chorionic villus explants were cultured for 24 h in medium containing 0, 50, or 50 ng/mL IL‐6. In the third group (50 ng/mL IL‐6), 1‐MT (1 mg/mL) was then added, and the culture continued for an additional 24 h. Western blotting showed that IL‐6 treatment significantly reduced the levels of Th17‐type cytokines in both decidual (Figure 5A, middle column; 5B) and chorionic villi (Figure 5C, middle column; 5D). Conversely, cotreatment with 1‐MT restored cytokine expression in IL‐6‐stimulated tissues (Figure 5A, right column, 5B; Figure 5C, right column, 5D). The supernatants from each group were collected at 0 h, 24 h, and 48 h after culture, respectively, for ELISA detection of Try and Kyn concentrations. The ELISA results are as follows: For the decidual and chorionic supernatant at 48 h of culture, the ratio of Kyn to Try was significantly lower in the 50 ng/mL + 1‐MT group compared to the 50 ng/mL group (Figure 5E; Figure 5F). These results indicate that 1‐MT effectively reverses the IL‐6‐induced downregulation of IL‐17 and IL‐22, providing evidence supporting the involvement of IDO1 in this regulatory pathway, since the higher the ratio of Kyn to Try, the higher the activity of IDO1.

FIGURE 5.

Tissues were cultured for 24 h in medium containing 0, 50, or 50 ng/mL IL‐6, followed by the addition of 1‐MT (1 mg/mL) and incubated for an additional 24 h. (A, C) Western blot analysis of IL‐17 and IL‐22 expression in decidua and chorionic villi cultured. (B, D) Quantitative analysis of IL‐17 and IL‐22 protein levels. Data are presented as mean ± SE. Šidák's multiple comparisons test was used as the post‐hoc method following two‐way repeated measures ANOVA. (E, F) The ratio of Kyn to Try in the supernatant of decidua and villi cultured for different durations in each group. Data are presented as mean ± SE. Tukey's multiple comparisons test was used as the post‐hoc method following two‐way repeated measures ANOVA. Grey scale ratio represents the band intensity of each target protein normalized to GAPDH. Statistical significance was set at p < 0.05. * indicates a p‐value less than 0.05; ** indicates a p‐value less than 0.01; *** indicates a p‐value less than 0.001; ns indicates a p‐value greater than 0.05.

3.4. Physiological Concentration TGF‐β1 Enhances IL‐6‐Induced Upregulation of IDO1 and Suppression of IL‐17/IL‐22 in a Concentration‐Dependent Manner in HDCV Tissues

To compare the regulatory effects of IL‐6 combined with TGF‐β1 on IDO1 or Th17‐related cytokine expression, tissue explants were treated with 0 or 5 ng/mL IL‐6 in the presence or absence of increasing concentrations of TGF‐β1 (0.01, 0.05, or 0.1 ng/mL) for 24 h.

Protein expression levels of IDO1, IL‐17, and IL‐22 in decidual and villous tissues were determined by western blotting (Figures 6A, C). In decidual tissue (Figure 6A), compared with the NC control (IL‐6 0 ng/mL + TGF‐β1 0 ng/mL) group, the use of exogenous IL‐6 alone or in combination with physiological concentrations of TGF‐β1 (0.01, 0.05, or 0.1 ng/mL) significantly increased the expression of IDO1, and this increase (Figure 6B) was accompanied by an increase in the concentration and a decrease in the expression of IL‐17 and IL‐22 in a TGF‐β1 dose‐dependent manner. Similar trends were observed in villous tissue (Figures 6C, D).

FIGURE 6.

In the decidual and villus samples, the cells were treated with 0 or 5 ng/mL of IL‐6 for 24 h, and different concentrations of TGF‐β1 were added respectively. (A, C) Representative Western blot bands of IDO1, IL‐17, and IL‐22 after the treatment. (B, D) Regression analysis between TGF‐β1 concentration and protein expression. b, regression coefficients. Grey scale ratio represents the band intensity of each target protein normalized to GAPDH.

3.5. IL‐6 Reduced EAR and Modulated IDO1 and Th17‐type Cytokine Expression in RPL Mouse Models

To assess the therapeutic potential of IL‐6 in RPL, mice were administered graded concentrations of IL‐6 (0, 2, 10, or 50 ng/mL) via intraperitoneal injection from GD 0.5 to GD 8. On GD 12.5, placental tissues were collected for analysis of protein expression, and the EAR was calculated.

The EAR was significantly lower in the 2, 10, and 50 ng/mL IL‐6 treatment groups compared to the control group (Table 1). This protective effect was diminished in the 50 ng/mL + 1‐MT group, where the EAR was notably higher than in the 50 ng/mL group. In contrast, the addition of Cosentyx (IL‐17 inhibitor) to the 50 ng/mL + 1‐MT regimen significantly reduced the EAR compared with both the 50 ng/mL IL‐6 + 1‐MT group and the control group (Table 1).

TABLE 1.

Comparison of embryo absorption rates in each group.

Group	Mice (n)	Surviving embryos (n)	Embryos lost (n)	Embryo absorption rate (%)	p
Control group	12	44	28	38.89
2 ng/mL group	12	75	24	24.24	* 0.0447
10 ng/mL group	12	78	14	15.22	** 0.0010
50 ng/mL group	12	91	16	14.95	*** 0.0004
50 ng/mL + 1‐MT group	12	77	41	34.75	**** 0.0007
50 ng/mL + 1‐MT + cosentyx group	12	110	14	12.73	**** < 0.0001
					**** < 0.0001

Compared with control group: ^* p = 0.0447 for 2 ng/mL group, ^** p = 0.0010 for 10 ng/mL group, ^*** p = 0.0004 for 50 ng/mL group, and ^**** p <0.0001 for 50 ng/mL + 1‐MT + cosentyx group. Compared with 50 ng/mL group: ^**** p = 0.0007 for 50 ng/mL + 1‐MT group. Compared with 50 ng/mL + 1‐MT group: ^**** p <0.0001 for 50 ng/mL + 1‐MT + cosentyx group.

In vitro neutralization assay confirmed the efficacy of Cosentyx against mouse IL‐17A. Cosentyx inhibited IL‐17A‐induced IL‐6 production in a dose‐dependent manner. A significant inhibitory effect (>80%) was observed starting from 500 µg/mL. At a concentration of 2000 µg/mL, Cosentyx achieved a neutralization rate of 94.95%, which was comparable to that of the positive control antibody (94.80%) (Table 2). Based on these in vitro data, this concentration was selected as a reference for in vivo administration.

TABLE 2.

Neutralization of IL17Ainduced proliferation in NIH‐3T3 cells.

Group	Concentration (µg/mL)	IL‐6 concentration (pg/mL) (Mean ± SD)	Neutralization rate (%)
untreated control	—	94.82 ± 3.78	—
IL17Astimulated control	—	1967.93 ± 38.03	—
IL17A Antibody	—	102.30 ± 11.57	94.80
IgG Isotype Control	—	1937.09 ± 147.96	1.57
Cosentyx	1	1766.32 ± 132.96	10.24
	10	1206.13 ± 15.97	38.71
	50	675.99 ± 43.43	65.65
	100	490.15 ± 4.32	75.09
	500	265.57 ± 5.93	86.51
	1000	158.48 ± 6.50	91.95
	2000	99.42 ± 3.19	94.95

Note: The concentration changes of IL‐6 were measured by ELISA. Neutralization rate = 1 − (Treatment group IL‐6 concentration / IL‐6 concentration in the IL‐17‐only group) × 100%.

Western blotting analysis revealed a significant increase in IL‐6 and IDO1 expression, whereas Th17‐type cytokines were suppressed in the placentas of mice treated with 2, 10, and 50 ng/mL IL‐6, compared with the 0 ng/mL IL‐6 control group (Figure 7A). Exogenous IL‐6 also induced dose‐dependent increases in IL‐6 and IDO1, alongside decreases in IL‐17 and IL‐22 levels in the placentas of mice (Figure 7B). Correlation analysis further revealed a positive correlation between IL‐6 and IDO1 and a negative correlation between IL‐6 and Th17‐type cytokines in the placentas of mice (Figure 7C). Immunohistochemistry confirmed elevated IL‐6 and IDO1 expression and reduced Th17‐type cytokine levels in the 50 ng/mL group compared with the 0 ng/mL group (Figures 7D, E).

FIGURE 7.

(A) Western blot analysis of IDO1, IL‐6, IL‐17, and IL‐22 expression in placental tissues from RPL mice treated in a concentration‐dependent manner of IL‐6. (B) Dose‐response relationship of IDO1, IL‐6, IL‐17, and IL‐22 expression in placental tissues from RPL mice treated with increasing concentrations of IL‐6. (C) Correlation of IL‐6 with IDO1, IL‐17, and IL‐22 in placenta of RPL mice treated with increasing IL‐6. (D) Immunohistochemical analysis of the different proteins in RPL mice placental sections (scale bar: 25 µm, 40× magnification). (E) Comparison of mean optical density (MOD) in the placental tissues of RPL mice injected intraperitoneally with IL‐6 (0 and 50 ng/mL). Data are presented as mean ± SE. Šidák's multiple comparisons test was used as the post‐hoc method following two‐way repeated measures ANOVA. (F) Western blot results from RPL mice treated with IL‐6 (50 ng/mL) alone or combined with 1‐MT. (G) Quantitative analysis of IL‐17 and IL‐22 protein levels in RPL mice treated with IL‐6 (50 ng/mL) alone or combined with 1‐MT. Data are presented as mean ± SE. Tukey's multiple comparisons test was used as the post‐hoc method following two‐way repeated measures ANOVA. (H) Analysis of the Kyn/Try ratio in the serum of RPL mice at non‐pregnant (before pregnancy), GD0.5 + 4 h (4 h after IL‐6 injection on GD 0.5), and GD5.5 + 4 h (4 h after 1‐MT injection on GD 5.5). Data are presented as mean ± SE. Tukey's multiple comparisons test was used as the post‐hoc method following two‐way repeated measures ANOVA. Grey scale ratio represents the band intensity of each target protein normalized to GAPDH. Statistical significance was set at p < 0.05. * indicates a p‐value less than 0.05; ** indicates a p‐value less than 0.01; *** indicates a p‐value less than 0.001; **** indicates a p‐value less than 0.0001; ns indicates a p‐value greater than 0.05.

Notably, the suppression of Th17‐type cytokines by IL‐6 was reversed by 1‐MT, as evidenced by higher cytokine levels in the 50 ng/mL + 1‐MT group than in the 50 ng/mL group (Figures 7F, G). Furthermore, in the 50 ng/mL + 1‐MT group, the ratio of Kyn to Try in the mouse serum decreased at GD 5.5 + 4 h (Figure 7H), indicating that 1‐MT successfully inhibited IDO1 activity in the mice.

4. Discussion

In this study, we initially identified the co‐expression of IL‐6, IDO1, and Th17‐type cytokines (IL‐17 and IL‐22) in HDCV within the maternal‐fetal compartment. Importantly, we observed a significant positive correlation between IL‐6 and IDO1 protein levels, whereas both molecules negatively correlated with Th17‐type cytokine expression in HDCV. These results suggest a potential regulatory axis in which IL‐6 enhances IDO1 production while simultaneously suppressing Th17‐type cytokines, and that IDO1 itself contributes to the downregulation of these Th17‐type cytokines. Subsequently, using in vitro cultures of HDCV, we experimentally confirmed that IL‐6 treatment upregulates IDO1 expression and concurrently downregulates Th17‐type cytokine production. Furthermore, in a RPL mouse model, IL‐6 administration significantly reduced the EAR, and this protective effect was reversed by treatment with the IDO1 inhibitor, 1‐MT. Collectively, these results suggest that IL‐6 improves pregnancy outcomes by suppressing Th17 cytokine production through IDO1 upregulation in the maternal‐fetal compartment.

It is reported that IL‐6 regulates IDO1 expression in many cells and tissues. For instance, the siRNA‐mediated knockdown of IL‐6 abrogated both the transcript and protein levels of IDO1 in SKOV‐3 and NCI‐H596 cells, consequently suppressing kynurenine production [19]. Similarly, IL‐6 enhances STAT3 phosphorylation in dendritic cells, leading to significant upregulation of IDO1 [43]. Our previous work further elucidated that at the maternal‐fetal interface during normal pregnancy, IL‐6 enhances IDO1 expression through a dual mechanism: upregulating SHP‐1/2 expression through STAT3 phosphorylation while concurrently suppressing SOCS3 [20]. This regulatory cascade has been shown to increase the pregnancy rate in a mouse model of RPL, which aligns with the results of the present study, demonstrating that IL‐6 upregulates IDO1 and reduces the embryo resorption rate in an RPL mouse model.

Previous studies have established that the synergistic action of IL‐6 and TGF‐β drives the switching of naïve T cells into pathogenic Th17 cells [23]. Further mechanistic research revealed that IL‐6 triggers IL‐21 expression, initiating an autocrine loop that amplifies both IL‐21 and IL‐23 receptor expression in naïve CD4 + T cells; IL‐21 and IL‐23, together with TGF‐β, subsequently promote IL‐17 production [44]. In contrast, our results demonstrate a divergent role of IL‐6, showing that IL‐6 downregulated the expression of IL‐17 and IL‐22 at the maternal‐fetal interface, where the expression of TGF‐β was abundant [37]. Our results indicate that the decreased expression of IL‐17 and IL‐22 was associated with the increased expression of IDO1 by IL‐6, suggesting a tissue‐specific immunomodulatory pathway wherein IL‐6 counteracts Th17 cytokine production through IDO1 activation.

Accumulating evidence indicates that the IDO‐mediated downregulation of Th17‐type cytokines represents a broad immunoregulatory mechanism observed in mouse kidney tissue [45], gastric mucosa of Helicobacter pylori‐infected humans [46], joint synovial fluid [34], mouse liver tissue [47], and murine and human dendritic cells [48]. As a pacemaker enzyme in tryptophan catabolism, IDO1 catalyzes the production of kynurenine metabolites, including L‐kynurenine, 3'‐hydroxykynurenine, and 3'‐hydroxyanthranilic acid. Desvignes et al. demonstrated that a mixture of these metabolites dose‐dependently inhibited IL‐17 production during Th17 cell differentiation, with 3'‐hydroxyanthranilic acid exhibiting the most potent inhibitory activity [49]. Mechanistically, kynurenine metabolites abrogate the enhancing effect of IL‐23, a key cytokine for Th17 cell maintenance, thereby suppressing Th17 differentiation and IL‐17 production. Our experimental data were consistent with this paradigm. We demonstrated that in cultured human decidual and chorionic villi explants and in the placenta of RPL mice, IL‐6 reduced IL‐17 expression. The inhibitory effect of IL‐6 on IL‐17 can be reversed by using 1‐MT to inhibit IDO1 and reduce the ratio of Kyn to Try, indicating that IL‐6 induces IDO1 expression, leading to the production of kynurenine metabolites that contribute to the downregulation of IL‐17 at the maternal‐fetal interface. Due to technical limitations, we did not perform flow cytometry to directly assess Th17 cell polarization. Nevertheless, the observed decrease in IL‐17A protein levels supports the conclusion that IDO1 plays a functional role in modulating the Th17 cell response. Future studies should employ naïve CD4 + T cells stimulated with IL‐6 and TGF‐β to induce Th17 differentiation, followed by culture with IL‐6 alone, combined with intracellular IL‐17A staining, to fully establish this mechanism.

Studies have indicated that in normal pregnancy, Th1 immunity is dominant during the peri‐implantation period, which benefits embryo implantation. Subsequently, Th1 immunity switches to Th2 immunity, and the predominant Th2 anti‐inflammatory responses at the implantation site may induce maternal‐fetal tolerance and protect the fetus from the maternal immune system. Generally, successful pregnancies are associated with Th2 cytokine profiles, whereas elevated proinflammatory Th1 and Th17 responses are associated with pregnancy loss [50]. This is corroborated by clinical observations in patients with URSA who exhibit high expression of Th17 cells [51]. Thus, restoring immune balance is a promising therapeutic strategy for URSA. In support of this, Xu et al. demonstrated a significant upregulation of IL‐17 in miscarriage‐prone mice. Exogenous IL‐17 administration induced miscarriage in normal mice, whereas anti‐IL‐17 antibodies prevented pregnancy loss in abortion‐prone models [26]. This is consistent with our data showing that a decrease in IL‐17 by IL‐6 in the placenta of RPL mice reduced the EAR, and that the IL‐17‐blocking antibody cosentyx also reduces the EAR of RPL mice treated with the IDO1 inhibitor 1‐MT to increase the expression of IL‐17. To determine the in vivo dosage, we calculated the predicted blood concentration based on the injection volume (0.2 mL) and estimated mouse blood volume (1.4 mL). The selected intraperitoneal dose of 200 mg/kg (4 mg per 20 g mouse) was calculated to yield an initial blood concentration of approximately 2.5 mg/mL (2500 µg/mL) (4 mg / 1.6 mL total volume). This predicted in vivo concentration is slightly above the optimal in vitro neutralization concentration of 2000 µg/mL identified in our study (Table 2). This dose escalation was considered necessary for the following reasons. Secukinumab (Cosentyx) is a humanized anti‐human IL‐17A monoclonal antibody with limited cross‐reactivity to mouse IL‐17A [52]. Therefore, a higher dose was administered to achieve sufficient target engagement and to ensure sustained neutralization throughout the experimental period in mice. We acknowledge that using a higher dose than the optimal in vitro concentration carries a potential risk of non‐specific effects. However, this approach is common when using humanized antibodies in murine models due to species‐specific affinity differences. Nevertheless, to rigorously confirm the specificity of the observed effects, future studies should consider using mouse‐specific anti‐IL‐17A neutralizing antibodies or IL‐17A knockout mice. Collectively, these results indicate that IL‐6 suppresses placental IL‐17 production via IDO1, reduces EAR, and maintains pregnancy in RPL mice.

Previous studies have established that the synergistic action of IL‐6 and TGF‐β drives the differentiation of naïve T cells into pathogenic Th17 cells [23]. Further mechanistic research revealed that IL‐6 triggers IL‐21 expression, initiating an autocrine loop that amplifies both IL‐21 and IL‐23 receptor expression in naïve CD4 + T cells; IL‐21 and IL‐23, together with TGF‐β, subsequently promote IL‐17 production [44]. In contrast, our results demonstrate a divergent role of IL‐6 at the maternal‐fetal interface, where TGF‐β is abundantly expressed, showing that IL‐6 downregulated the expression of IL‐17 and IL‐22. It has been reported that the baseline levels of IL‐6 and TGF‐β in placental tissue are approximately 10–20 ng/g (15.4 ± 4.1 ng/g) and 1.19 (0.713–2.051) ng/g of tissue, respectively [17, 53]. In our experiment, 0.5 g explants of chorionic villi and decidual tissue were cultured in 1 mL of culture medium containing 5 ng/mL IL‐6 (equivalent to 10 ng/g tissue) and TGF‐β at concentrations of 0.01, 0.05, and 0.1 ng/mL, corresponding to tissue TGF‐β levels of 0.02, 0.1, and 0.2 ng/g, respectively. Our previous reported showed that physiological concentrations of TGF‐β (ranging from 0.01 to 0.1 ng/mL) increase IDO1 expression in chorionic villi and decidual tissues [38]. Our present results indicated that in the presence of baseline IL‐6 (10 ng/g or 5 ng/mL), physiological concentrations of TGF‐β (ranging from 0.01 to 0.1 ng/mL, that is, 0.02–0.2 ng/g) increased IDO1 expression and reduced the expression levels of IL‐17 and IL‐22 in chorionic villi and decidual tissue explants in a dose‐dependent manner. Although the mechanism by which physiological concentrations of TGF‐β increase IDO1 expression has been reported [19], future studies should investigate whether pathological concentrations of TGF‐β increase the expression of IDO1.

5. Conclusion

In individuals with normal pregnancy, IL‐6, IDO1, and Th17‐type cytokines are co‐expressed in the cytoplasm of human decidual and chorionic tissues. Our results indicate that Th17 cytokines are detrimental to pregnancy maintenance at the maternal‐fetal interface. We propose that one mechanism by which IL‐6 promotes pregnancy is the upregulation of IDO1, and the kynurenine metabolites of IDO1 subsequently suppress Th17 cytokine production. This is supported by our observations in an RPL mouse model in which exogenous IL‐6 administration reduced the EAR by downregulating placental Th17 cytokine expression. Moreover, in vitro experiments revealed that baseline levels of IL‐6 and TGF‐β exerted a certain effect on upregulating IDO1 and suppressing Th17 cytokine production. This finding stands in sharp contrast to the classical paradigm in which the combination of IL‐6 and TGF‐β promotes the production of Th17 cytokines.

Funding

This work was supported by the National Natural Science Foundation of China presided over by Guanyou Huang (http://www.nsfc.gov.cn/ Grants 82260312), Guizhou Provincial Health Commission Science and Technology Fund over by Guanyou Huang (Grant gzwkj2021‐319), and Guizhou Provincial Health Commission Science and Technology Fund presided over by Leilei Zhu (Grant gzwkj2025‐094).

Ethics Statement

The research was performed in compliance with the Helsinki Declaration and was approved by the relevant ethics boards, including the Ethics Committee of the Affiliated Hospital of Guizhou Medical University (No. 289,2025) and the Laboratory Animal Ethics Committee of Guizhou Medical University (No. 2402746).

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available upon request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.