Echinococcus granulosus antigen B regulates T‐cell function through inhibition of signal transducer and activator of transcription 3 in experimental immune thrombocytopenia

Summary

Dysregulated T‐cell homeostasis is central to the development of immune thrombocytopenia (ITP), characterized by reduced platelet counts. Antigen B (AgB), a key protein in Echinococcus granulosus cyst fluid, modulates T‐cell differentiation and reduces inflammation. Here, we explored the role of AgB in ITP and found that it enhances the generation and function of regulatory T cells (Tregs), boosting their immunosuppressive activity. In our passive ITP murine model, AgB treatment alleviated thrombocytopenia and restored the Treg–helper T‐cell (Th) balance. However, the therapeutic effects of AgB on CD4+ T cells were abolished by Treg depletion, highlighting the essential role of Tregs in AgB's mechanism of action. Moreover, AgB reduced proinflammatory cytokine production and inhibited signal transducer and activator of transcription 3 (STAT3) activation in ITP mice, with STAT3 inhibition negating the effects of AgB in Tregs. AgB promoted STAT3 degradation via tumour necrosis factor receptor‐associated factor 6 (TRAF6)‐mediated ubiquitination. In conclusion, by facilitating TRAF6‐mediated STAT3 ubiquitination, AgB restores T‐cell homeostasis and strengthens Treg immunosuppression, affording a potential therapeutic strategy for ITP.

During the pathological process of immune thrombocytopenia, signal transducer and activator of transcription 3 (STAT3) is activated by JAKs, leading to the production of a large number of inflammatory cytokines and promoting the activation of Th1, Th2 and Th17 cells. AgB enhances the binding of TRAF6 to STAT3, thereby increasing the degradation of STAT3, reducing the production of inflammatory cytokines and promoting the activation of Treg cells.

INTRODUCTION

Immune thrombocytopenia (ITP), an autoimmune disorder acquired through a breakdown in immune tolerance, manifests as accelerated destruction and diminished production of platelets. ¹ This loss of tolerance is central to the aetiology of ITP. ² Regulatory T cells (Tregs) are crucial for preserving self‐tolerance, and in patients with ITP, reduced numbers and compromised function of Tregs contribute to uncontrolled proliferation and activation of autoreactive effector T cells (Teffs). ³ In addition to unrestrained cytotoxic T‐lymphocyte activation and antiplatelet autoantibody generation, dysregulation of CD4+ helper T (Th) cells is a hallmark of ITP pathogenesis. ⁴ In patients with ITP, immune tolerance restoration, occurring through the rectification of Treg dysfunction and reduction in Th1 and Th17 cell proliferation, is a key mechanism underlying the effectiveness of current ITP‐specific therapeutic strategies. ⁵

Signal transducer and activator of transcription 3 (STAT3), a major transcription factor family member, is a critical regulator often overactivated in autoimmune diseases, including ITP. ⁶ , ⁷ Patients with ITP demonstrate notable increases in plasma STAT3 levels. Moreover, in T cells, STAT3 knockdown hinders Th17 differentiation, whereas STAT3 overexpression promotes it. ⁶ STAT3 inhibition also restores Treg–Th17 balance in ITP. ⁵ These findings highlight the critical role of STAT3 in regulating Treg and Th17 cell differentiation in the context of ITP.

Tumour necrosis factor (TNF) receptor‐associated factor 6 (TRAF6) is a crucial signalling intermediate for TNF receptor superfamily members, which regulates various signalling pathways. ⁸ The RING finger domain of TRAF6 functions as a ubiquitin ligase, allowing it to directly interact with and ubiquitinate multiple factors, including STAT3. ⁶ However, the specific role of TRAF6‐mediated ubiquitination in STAT3 activation, particularly in the context of T‐cell differentiation in ITP, warrants elucidation.

The hygiene hypothesis, introduced in 1989, postulates a correlation between the cleanliness that reduces helminth infections and a rise in autoimmune and allergic disease prevalence. ⁹ Research has revealed that helminths secrete various proteins that modulate host immune responses; this modulation is a survival mechanism mitigating a host's immune attack on parasites and attenuating the host's hypersensitivity during autoimmune or inflammatory conditions. ¹⁰ The immunomodulatory effects of helminth‐derived proteins encompass the regulation of antigen‐presenting cell function, the activation of Tregs to enhance the production of anti‐inflammatory cytokines such as transforming growth factor (TGF) β and interleukin (IL) 10, and the promotion of M2 polarization of macrophages. ¹¹ , ¹²

The larvae of Echinococcus granulosus, a parasitic tapeworm prevalent in both human and animal hosts, reside within human tissues, such as the liver or lung tissues, in the form of hydatid cysts over extended periods, potentially up to 53 years. ¹³ This longevity may be attributable to the release of immunomodulatory proteins mitigating host immune responses and suppressing allergic reactions, thus protecting a host from excessive inflammation. ¹⁴ Animal studies have substantiated the therapeutic potential of E. granulosus infection in intestinal inflammation and asthma alleviation. ¹⁵ , ¹⁶ Nevertheless, the use of live helminth infection is associated with serious health risks, making it clinically inapplicable. ¹⁷ Nevertheless, helminth‐derived proteins are risk‐free alternatives that can elicit similar anti‐inflammatory effects. ¹⁸ For instance, antigen B (AgB), a principal protein secreted by E. granulosus, can reduce the incidence of dextran sulphate sodium‐induced colitis by modulating M1/M2 macrophage polarization. ¹⁹ Furthermore, AgB can modulate host immune responses by inhibiting Th17 cell differentiation and promoting Treg development, thus mitigating airway inflammation. ²⁰ However, the role of AgB in immune system modulation for ITP treatment remains unexplored.

In the present study, we assessed the impact of AgB, a major proportion of hydatid fluid, on the generation and suppressive function of Tregs from patients with ITP, with a focus on the STAT3 pathway as a key mediator. We also explored mechanisms through which AgB regulates STAT3 activation via TRAF6‐mediated ubiquitination. Finally, we established active ITP murine models to assess the in vivo effects of AgB on Tregs, providing insights into the potential therapeutic applications of AgB in ITP.

METHODS

Patient and control inclusion

Human peripheral blood samples (5 mL) were collected at the First Affiliated Hospital of Xinjiang Medical University (Table 1) from patients newly diagnosed as having E. granulosus sensu stricto (s.s.) infection [n = 6; 3 men and 3 women] or as ITP according to the 2011 American Society of Hematology guidelines ²¹ [n = 6; 3 men and 3 women; median platelet count = 16 × 10⁹/L (range: 1 × 10⁹/L to 28 × 10⁹/L)]. We also collected blood samples from healthy donors (HDs) at the hospital [controls; n = 6; 3 men and 3 women; median platelet count = 262 × 10⁹/L (range: 149 × 10⁹/L to 348 × 10⁹/L)]. Patients with severe heart, kidney, lung or liver dysfunction, or with primary immunodeficiency were excluded. Pregnant or lactating patients were also excluded.

TABLE 1.

Clinical characteristics of Egs, patients with ITP and HDs.

Patient no.	Sex	Age (years)
Eg 1	M	41
Eg 2	M	29
Eg 3	M	36
Eg 4	F	39
Eg 5	F	42
Eg 6	F	37
ITP1	M	27
ITP2	M	24
ITP3	M	31
ITP4	F	29
ITP5	F	33
ITP6	F	28
HD 1	M	32
HD 2	M	35
HD3	M	38
HD 4	F	35
HD 5	F	31
HD 6	F	36

Abbreviations: Egs, E. granulosus s.s.; HDs, healthy donors; ITP, immune thrombocytopenia.

This study was conducted in accordance with the ethical standards of the Helsinki Declaration, and the study protocol was approved by the Institutional Review Board of the First Affiliated Hospital of Xinjiang Medical University (IRB No.: 20210226–11). Before inclusion, all participants or their legally authorized representatives provided written informed consent, as appropriate.

Passive ITP mice model establishment

A passive ITP murine model was established using a widely recognized, validated protocol. ²² We used male and female C57BL/6 mice (age: 6–8 weeks) sourced from Xinjiang Medical University (Urumqi, China) and housed them in specific pathogen‐free environments provided by the study university's facilities. All animal experiments in this study were approved by the Animal Ethics Committee of Xinjiang Medical University (No.: 20210301–11).

In the experimental workflow, age‐ and sex‐matched mice were intraperitoneally administered anti‐mouse CD41 monoclonal antibodies (mAbs) (BD Biosciences, USA). The initial dosage was set at 68 μg/kg body weight, administered over the first 48 h. This dosage was increased by 34 μg/kg each subsequent day over 4 days. The animals were humanely euthanized by carbon dioxide (CO₂) on study day 6 (i.e. exactly 24 h after the final mAb injection). Throughout the study, ITP development was closely monitored based on daily assessments of platelet counts. A minimally invasive method was employed to quantify platelet counts, where whole blood samples (5 × 10^–6 L) were carefully collected from the tail vein of each mouse. These samples were then gently mixed with 45 × 10^–6 L of 2 mg/mL heparin solution to prevent coagulation and analysed on a Mindray BC‐5300 Auto Haematology Analyser.

AgB preparation

AgB was isolated from E. granulosus s.s. cyst fluid (EgCF) by using a well‐established protocol. ²³ EgCF was carefully extracted from ovines with echinococcal cysts from Xinjiang Uyghur Autonomous Region (China). We purified EgCF by first centrifuging it at 10 000× g at 4°C for 20 min and then filtering the supernatant through membranes with 0.45‐μm pores (Millipore).

To isolate AgB, the EgCF was precipitated with the addition of sodium acetate to a final concentration of 5 mM, with the pH adjusted to 5.0, followed by centrifugation at 5000× g at 4°C for 15 min. The pellet was resuspended in phosphate‐buffered saline (PBS) and heated in boiling water for 5 min, followed by centrifugation at 10 000× g at 4°C for 15 min. The supernatant was then collected and filtered through a 0.22‐μm filter to ensure sterility. Endotoxin contamination was rigorously eliminated using an endotoxin removal resin (ToxinEraser Endotoxin Removal Kit; GenScript Biotech, China), and the supernatant was verified to be lipopolysaccharide‐free by using gel clot assays (ToxinSensor Gel Clot Endotoxin Assay Kit; GenScript Biotech).

The concentration of purified AgB was determined using a Qubit quantitation fluorometer in combination with Quant‐iT reagents (Life Technologies). In in vivo studies, AgB‐treated mouse groups were administered a daily intraperitoneal injection of 100 μg of purified native AgB (1 mg/mL) for four consecutive days.

Peripheral blood mononuclear cell and CD4+ T‐cell isolation

Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood through Ficoll–Hypaque (Beyotime, Shanghai, China) density gradient centrifugation, as described previously. ⁵ Subsequently, CD4+ T cells were sorted using Dynabeads CD4 Positive Isolation Kits (Invitrogen, Carlsbad, CA), ensuring a high degree of purity. Flow cytometry analysis confirmed that the isolated cells were ≥90% pure.

Both PBMCs and the sorted CD4+ T cells were cultured in RPMI‐1640 medium (Beyotime) supplemented with 10% fetal bovine serum (Beyotime) and 1% penicillin and streptomycin (Beyotime). The cells were then stimulated with a cocktail of recombinant human IL‐2 (5 ng/mL; R&D Systems), antihuman CD3 antibodies (1 ng/mL; R&D Systems) and antihuman CD28 antibodies (1 ng/mL; R&D Systems). Finally, the cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 72 h.

Assessment of Treg immunosuppression in patients with ITP

CD4+CD25− T cells (i.e. Teffs) and CD4+CD25+Foxp3+ T cells (i.e. Tregs) were meticulously isolated from PBMCs by using a Dynabeads Regulatory CD4+/CD25+ T‐cell kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. CD4+CD25− T cells were fluorescently labelled with 5,6‐carboxyfluorescein diacetate succinimidyl ester (CFSE; 2.5 μM; MedChemExpress) and plated at 2 × 10⁵ cells/well in a 96‐well plate. Cocultures were established with or without Tregs at a Teff–Treg ratio of 4:1. The cells were stimulated with a combination of recombinant human IL‐2 (5 ng/mL; R&D Systems), antihuman CD3 antibodies (1 ng/mL; R&D Systems) and antihuman CD28 antibodies (1 ng/mL; R&D Systems). To evaluate their immunosuppressive function, cells were incubated with 100 ng/mL AgB or an equivalent volume of PBS for 6 days. Finally, the cells were harvested, and their proliferation was analysed using FlowJo. The results afforded a quantitative assessment of Treg‐mediated suppression.

In vitro suppression assay of murine Tregs

CD4+CD25+Foxp3+ T cells and CD4+CD25− T cells were isolated from splenocyte single‐cell suspensions by using an EasySep Mouse CD4+CD25+ Regulatory T Cell Isolation Kit II (Stemcell Technologies, Vancouver, Canada), according to the manufacturers' protocols.

Teffs were labelled with CFSE (2.5 μM; MedChemExpress) and then seeded at 1 × 10⁵ cells per well in round‐bottom 96‐well plates precoated with 5 μg/mL anti‐CD3 antibodies (R&D Systems). These plates also contained 2 μg/mL soluble anti‐CD28 antibodies (R&D Systems) for costimulation. The Teffs were cocultured with Tregs at a Treg–Teff ratio of 1:4. After 6 days of culture, Teff proliferation was analysed, as described previously. ²⁴ Suppression (%) was calculated as 100 − [(proliferation in Treg coculture/proliferation in Teff‐only culture) × 100].

Depletion of Tregs in ITP mice

To induce in vivo depletion of CD25+ T cells, we administered ITP mice with 400 μg of monoclonal antimouse CD25 antibodies (R&D Systems) intraperitoneally at 2‐day intervals. The control group was administered an equivalent amount of purified isotype IgG (R&D Systems). The CD25+ T‐cell depletion efficacy was assessed through flow cytometry analysis on splenocytes collected on day 6. In general, we observed the CD25+ T‐cell depletion efficiency to be >90%, indicating that our depletion strategy was robust and effective.

Flow cytometry

Cells were detected by flow cytometry, as described previously. ⁵ In brief, cells were prepared at 1 × 10⁶ cells/mL. To quantify the Th17 cell population, these cells were incubated with human anti‐CD3, anti‐CD4 and anti‐IL‐17A antibodies (all from Abcam, Cambridge, UK) for 1 h in the dark. The Treg percentage was determined by incubating the cells with antihuman CD25, CD4 and Foxp3 antibodies (all from Abcam) for 1 h in the dark. Flow cytometry data were acquired on an FACS Canto II flow cytometer (BD, USA).

In total, 1 × 10⁶ cultured human PBMCs or CD4+ T cells were collected from 24‐well plates after coculture with AgB at 100 ng/mL. For ITP murine models, single‐cell suspensions were prepared at 1 × 10⁶ cells/mL through mechanical disaggregation of murine spleens. All cells were then stained with antihuman or antimouse conjugated antibodies according to the manufacturer's instructions.

Apoptosis and regulatory T‐cell analysis

To assess apoptosis, PBMCs were stained with fluorescein isothiocyanate‐conjugated annexin V and propidium iodide (PI) using a Cell Apoptosis Kit (Beyotime). Tregs (i.e. CD4+CD25+Foxp3+ cells) were stained with a regulatory t‐cell staining kit (Invitrogen) and enumerated. Teffs (i.e. CD4+ cells) were stained with a T‐cell subset staining kit (eBioscience) and enumerated. Th1, Th17 and Th22 cells were characterized as CD4+IFN‐γ+, CD4+IFN‐γ−IL17+ and CD4+IFN‐γ−IL22+ populations respectively. Flow cytometric analysis was conducted on an FACS Canto II flow cytometer (BD, USA) to evaluate cellular immune responses comprehensively.

Enzyme‐linked immunosorbent assay

Inflammatory cytokine levels were quantified using appropriate enzyme‐linked immunosorbent assay kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturers' protocols. ⁵

Serum biochemical analysis

Next, we performed a comprehensive biochemical assessment of serum isolated through centrifugation at 2000× g at 4°C for 10 min. The assessment included measurement of creatine kinase (CK), creatine kinase isoenzyme (CK‐MB), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate transaminase (AST), creatinine and serum urea nitrogen (BUN) levels by using commercial diagnostic kits from Nanjing Jiancheng Biological Company (Nanjing, China), according to the manufacturers' protocols.

Western blotting

Western blot analysis was performed, as described previously. ²⁵ Samples were collected and homogenized to obtain whole‐cell lysates. The protein content in the supernatants of these lysates was then determined using the bicinchoninic acid (BCA) reagent (Beyotime), according to the manufacturer's instructions. Aliquots of the samples, each containing 30 μg of protein, were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis to separate the proteins based on their molecular weights. Subsequently, the resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. These membranes were incubated with primary antibodies targeting the proteins of interest (Table 2). The immunoreactive bands on the membranes were visualized using horseradish peroxidase‐conjugated secondary antibodies that produced a colorimetric reaction after protein binding. The intensity of these bands was quantified using a Kodak Scientific Imaging System (Kodak, Rockville, MD, USA) and further analysed using ImageJ to obtain numerical data for comparative assessments.

TABLE 2.

Mouse antibodies used in this study.

Antibody	Source	Identifier
β‐actin	Proteintech	20 536‐1‐AP
Histone H3	zenbio	R24572
Na, K‐ATPase	abcam	ab76020
PI3K	zenbio	R381065
p‐PI3K	zenbio	310 164
Akt	zenbio	342 529
p‐Akt	zenbio	310 021
p‐STAT3	zenbio	R381552
STAT3	abcam	ab32500
Ubiquitin	abcam	ab134953
TRAF6	abcam	ab137452

Immunoprecipitation

Immunoprecipitation was performed, as described previously. ²⁵ Cells were lysed using RIPA buffer (Beyotime) supplemented with a protease inhibitor cocktail, followed by incubation on ice for 30 min to ensure complete lysis. After centrifugation to clarify the lysates, equal amounts of protein from each sample were incubated with anti‐STAT3 primary antibody at 4°C overnight under gentle rotation. Immune complexes were precipitated by the addition of protein A sepharose (GE Healthcare), and the resulting pellets were washed thoroughly to remove complexes with nonspecific binding. The immune complexes were then resolved through 5% sodium dodecyl sulphate polyacrylamide gel electrophoresis. Next, the resolved proteins were transferred onto PVDF membranes. Nonspecific binding sites on the membrane were blocked through incubation with 3% bovine serum albumin diluted in tris‐buffered saline containing Tween 20. The membrane was subsequently probed with antiubiquitin antibodies to detect ubiquitinated proteins and then washed to remove unbound antibodies (Table 2). The blots were incubated with a horseradish peroxidase‐conjugated antirabbit IgG secondary antibody for immune complex detection. The intensity of the bands was quantified using the Kodak Scientific Imaging System.

Assessment of STAT3 and AKT inhibition in vitro and in vivo

In our in vitro experiments, cryptotanshinone (a selective STAT3 inhibitor; Targetmol, Boston, MA, USA) and ARQ 092 (an AKT inhibitor; Targetmol) were reconstituted in dimethyl sulphoxide (DMSO) and introduced into the culture media of PBMCs at final concentrations of 10 μM and 500 nM respectively. After 72 h of incubation, the PBMCs were collected for subsequent flow cytometry analysis to evaluate the effects of the inhibitors on cellular signalling pathways.

In our in vivo experiments, cryptotanshinone and ARQ 092 were dissolved in sterile 0.5% carboxymethylcellulose sodium and administered to our mice at 20 and 30 mg/kg once every 2 days via gavage respectively.

Statistical analysis

The experimental outcomes are expressed as means ± standard errors of the means (SEMs). Statistical significance was assessed using Student's t test or using analysis of variance followed by Tukey's post hoc test for multiple comparisons, when appropriate. In instances where the assumption of homogeneity of variances was violated, group comparisons were made using the Mann–Whitney U‐test and Kruskal–Wallis test, followed by Dunn's multiple comparison test. The Kaplan–Meier test was used to compare survival data between groups.

All statistical analyses were performed on GraphPad Prism (version 8.0). A threshold of p < 0.05 was used to define statistical significance.

RESULTS

E. granulosus s.s. infection enhances the immunosuppressive capacity of human Tregs

E. granulosus infection can modulate Treg differentiation. ²⁶ To assess the impact of E. granulosus s.s. infection (Egs) on the functional capabilities of human Tregs, we isolated PBMCs from patients diagnosed with Egs and HDs. An initial comparison revealed no significant differences in the baseline proportions of Tregs in the CD4+ T‐cell population between the Eg and HD groups (Figure 1A).

FIGURE 1.

E. granulosus s.s. infection augments Treg inhibitory function. (A) Flow cytometry to quantify Tregs among the CD4+ T‐cell population in peripheral blood mononuclear cells (PBMCs) from Egs (n = 6) and healthy donors (HDs) (n = 6). (B) In vitro suppression assays for the suppressive capacity of Tregs isolated from Egs and HDs. CFSE‐labelled Teffs were stimulated and co‐cultured with Tregs. Representative histograms depict the proliferative response of Teffs in the presence or absence of Tregs. Data are presented as mean ± SEMs from two to three independent experiments. Statistical comparisons were made using Student's t test. **p < 0.01.

Subsequently, we sorted CD4+CD25+CD127− Tregs from both the Egs and HDs and cocultured them with CFSE‐labelled Teffs cells at a ratio of 1:4 to determine the suppressive activity of Tregs. Notably, cocultured with Tregs considerably reduced the division index of Teffs in the Eg group compared with the HD group (Figure 1B). Thus, Tregs from the Egs demonstrated enhanced immunosuppressive function.

AgB enhances Treg differentiation and immunosuppressive function in vitro

We isolated and cultured PBMCs from patients with ITP and HDs and then exposed them to varying concentrations of AgB for 72 h. Next, these PBMCs were stained with annexin V and PI to assess their apoptotic levels. The findings demonstrated that AgB did not affect apoptosis of PBMCs from either patients with ITP or HDs, indicating that AgB was not cytotoxic to PBMCs (Figure 2A–C). Notably, 1–100 ng/mL AgB significantly and dose‐dependently increased Treg proportions in the CD4+ T‐cell population; however, no further increase was observed at higher AgB concentrations (Figure 2D–F). Moreover, AgB slightly elevated CD4+ T‐cell percentage in PBMCs (Figure 2G,H). Consequently, 100 ng/mL AgB was selected for further analysis. To exclude the influence of other cell types, CD4+ T cells were sorted and cocultured with AgB, and AgB was noted to considerably increase the Treg count (Figure 2I,J).

FIGURE 2.

AgB enhances counts and inhibitory function of Tregs in peripheral blood mononuclear cells (PBMCs). (A) Representative dot plots of flow cytometry analyses depicting apoptosis in PBMCs from patients with immune thrombocytopenia (ITP) after treatment with AgB at varying concentrations. The apoptosis rate is indicated by the percentage of Annexin V‐positive and PI‐negative cells. (B, C) Apoptosis levels in PBMCs compared between healthy donors (HDs) and patients with ITP. (D) Representative dot plots of flow cytometry analyses of Tregs among the CD4+ T‐cell population in PBMCs from ITP patients treated with specified AgB dosages. (E, F) AgB dose‐dependently increases Treg proportions among the CD4+ T‐cell population in PBMCs from HDs and patients with ITP. (G, H) AgB administration does not significantly alter CD4+ T‐cell percentages in PBMCs in HDs (G) and patients with ITP (H). (I) Representative dot plots of flow cytometry analysis showing Tregs among the CD4+ T‐cell population from ITP patients treated or not treated with AgB. (J) AgB (100 ng/mL) significantly elevates Treg percentages among the CD4+ T‐cell population from both HDs and patients with ITP. (K) Representative histograms of flow cytometry analysis displaying CFSE‐labelled CD4+ Teffs after 6 days of culture. The right panel shows the quantification of Teff proliferation under the indicated conditions. AgB augmented Tregs' inhibitory function in patients with ITP but not in HDs. Data are presented as mean ± SEMs from two to three independent experiments (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001; ^## p < 0.01.

We further isolated Tregs from patients with ITP and HDs and cocultured them with CFSE‐labelled Teffs to evaluate the suppressive activity of Tregs in the presence of AgB. The division index of ITP patient Teffs was significantly lower after coculture with Tregs and AgB than coculture with Tregs alone (Figure 2K), indicating that AgB‐treated Tregs demonstrate enhanced immunosuppressive function. However, AgB alone did not influence Teff proliferation significantly (Figure 2K). Taken together, these results suggested that AgB significantly improves the immunosuppressive function of Tregs in ITP.

AgB alleviates thrombocytopenia in ITP mice by increasing the splenic Treg population

To extend our in vitro findings to an in vivo context, we established a passive immune ITP murine model (Figure 3A). After AgB administration, all mice survived without any apparent toxic effects (Figure S1). AgB led to a considerable increase in platelet counts in ITP mice (Figure 3B). In flow cytometry analysis, AgB‐treated ITP mouse spleens demonstrated a significant elevation in the proportion of Tregs in the CD4+ T‐cell population (Figure 3C,D). In contrast, AgB treatment reduced the percentages of Th1, Th17 and Th22 cells among the CD4+ T‐cell population (Figure 3E–G). Moreover, Tregs from AgB‐treated ITP mice demonstrated an enhanced suppressive function (Figure 3H–J).

FIGURE 3.

AgB alleviates thrombocytopenia in our passive immune thrombocytopenia (ITP) murine model through Tregs. (A) A schematic overview of passive ITP murine model development. (B) AgB treatment significantly increases platelet counts in ITP mice. Platelet counts were monitored daily. (C) Representative dot plots of flow cytometry analyses of the proportion of Tregs in the CD4+ T‐cell population in mouse spleens. (D–G) Flow cytometry assays assessing the percentages of Treg (D), Th1 (E), Th17 (F) and Th22 (G) cells in the CD4+ T‐cell population in mouse spleens. (H–J) In vitro suppression assays to evaluate the suppressive activity of Tregs from the spleens of ITP mice treated with or not treated with AgB. CFSE‐labelled CD4+CD25− Teffs stimulated and co‐cultured with Tregs, and representative histograms show CD4+ Teff proliferation in the presence or absence of Tregs. (K) Mice treated with anti‐CD25 antibody exhibit a significant depletion of Tregs in the spleen. (L) Depletion of Tregs by anti‐CD25 antibodies partially reverses the effects of AgB on increases in platelet count in ITP mice. (M–O) After Treg depletion with anti‐CD25 antibodies, AgB does not significantly influence splenic Th1, Th17 and Th22 cell counts in ITP mice. Data are presented as means ± SEMs from two to three independent experiments (n = 6). *p < 0.05; **p < 0.01; ^# p < 0.05; ^## p < 0.01; ^### p < 0.001; ^$$ p < 0.01.

To determine whether the protective effect of AgB on platelets is mediated by Tregs, we used an ITP mouse model with anti‐CD25‐induced Treg depletion. These mice demonstrated a significant reduction in splenic Treg counts (Figure 3K), which partially reversed the AgB‐induced increase in platelet counts (Figure 3L). Furthermore, after Treg depletion, AgB no longer significantly influenced splenic Th1, Th17 or Th22 cell counts (Figure 3M–O). Thus, the therapeutic effect of AgB on platelet counts at least partially involves Treg modulation.

Regulatory effects of AgB on cytokines are associated with T‐cell differentiation in ITP mice

AgB treatment significantly reduced the serum levels of proinflammatory cytokines TNF‐α, IL‐17A and IFN‐γ, which are all essential in T‐cell differentiation. Moreover, AgB reduced the levels of various inflammatory cytokines, including IL‐1β, IL‐2, IL‐4, IL‐6, IL‐8 and IL‐12, as well as chemokines, further modulating the immune environment. In contrast, a significant increase in the levels of the anti‐inflammatory cytokine TGF‐β was observed. However, serum IL‐10 and IL‐13 levels did not change significantly (Figure 4). These findings underscore the potential of AgB to reshape the cytokine landscape, influencing T‐cell differentiation and function in the context of ITP.

FIGURE 4.

T‐cell differentiation cytokines detected in mouse serum through enzyme‐linked immunosorbent assay. Data are presented as mean ± SEMs from two to three tests (n = 6). *p < 0.05, **p < 0.01.

AgB suppresses STAT3 activation to modulate T‐cell homeostasis in ITP

In ITP, the Janus kinase‐STAT (Jak‐STAT) and phosphoinositide 3‐kinase (PI3K)–protein kinase B (AKT) pathways—integral to the rapid signalling module crucial for CD4+ T‐cell homeostasis—are hyperactivated, as indicated by elevated phosphorylated STAT3 (p‐STAT3) and phosphorylated AKT (p‐AKT) levels. Here, AgB treatment reduced STAT3 and p‐STAT3 expression in lysates derived from PBMCs of patients with ITP. Although AgB did not affect PI3K and AKT expression, it reduced phosphorylated PI3K and p‐AKT levels (Figure 5A).

FIGURE 5.

AgB ameliorates immune thrombocytopenia (ITP) in mice by inhibiting STAT3 activation. (A) Immunoblotting of the expression of STAT3, PI3K, AKT and their phosphorylated forms in peripheral blood mononuclear cells (PBMCs) from healthy donors (HDs) and patients with ITP. The right panel shows densitometry analysis results, quantifying the relative levels of phosphorylated proteins normalized to total protein or β‐Actin. (B, C) Treg proportions among the CD4+ T‐cell population in PBMCs from HDs (B) and patients with ITP (C) cultured under specified conditions for 72 h and analysed through flow cytometry. (D, E) Platelet counts in mice administered AgB, a STAT3 inhibitor or an AKT inhibitor. (F) Immunoblotting of the expression of STAT3, AKT, and their phosphorylated forms in PBMCs from Egs and HDs. The right panel presents densitometry analysis of the corresponding blots. (G) Immunoblotting for the subcellular distribution of STAT3 and p‐STAT3 in CD4+ T cells from patients with ITP. The right panel provides quantification of STAT3 and p‐STAT3 levels within these fractions. (H) Immunofluorescence staining for cellular localization of STAT3 and p‐STAT3 in CD4+ T cells from patients with ITP. Scale bar: 20 μm. Data are presented as means ± SEMs from two to three independent experiments (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001; ^## p < 0.01; ^### p < 0.001; ^$$ p < 0.01.

We then used STAT3 and AKT inhibitors in our in vitro and in vivo experiments. In vitro, the proportions of Tregs in the CD4+ T‐cell population from patients with ITP and HDs after treatment with a combination of STAT3 inhibitor and AgB were comparable to those after treatment with the STAT3 inhibitor alone (Figure 5B). Similarly, in vivo, platelet counts in mice treated with both the STAT3 inhibitor and AgB did not significantly differ from those treated with the STAT3 inhibitor alone (Figure 5D). In contrast, the AKT inhibitor combined with AgB significantly increased the percentages of Tregs from ITP patients in vitro (Figure 5C). Furthermore, in mice, the coadministration of the AKT inhibitor and AgB led to an increase in platelet counts compared with the AKT inhibitor alone (Figure 5E). Moreover, a dampening of STAT3 and AKT signalling was observed in PBMCs from Egs compared with those in HDs (Figure 5F). Taken together, these findings suggested that, in the context of ITP, AgB promotes Treg restoration via STAT3 inhibition but not AKT activation.

Subcellular fractionation analyses revealed that AgB mainly reduced STAT3 and p‐STAT3 recruitment to the membranes of CD4+ T cells from ITP patients (Figure 5G). Similarly, immunofluorescence imaging revealed that AgB reduced the STAT3 signal at the plasma membrane and endomembrane and reduced phosphorylation in CD4+ T cells (Figure 5H). These data further supported the notion that AgB inhibits STAT3 phosphorylation by limiting its membrane recruitment at the site where the JAK2 is typically localized, thereby influencing the CD4+ T‐cell activation status in ITP.

AgB enhances STAT3 degradation through TRAF6‐mediated ubiquitination

Next, we investigated the mechanism through which AgB modulates STAT3 signalling and found that AgB did not influence STAT3 mRNA expression in CD4+ T cells (Figure 6A). Thus, we hypothesized that AgB regulates STAT3 stability. As such, we treated CD4+ T cells with the protein synthesis inhibitor cycloheximide (CHX), followed by AgB, and subsequently assessed STAT3 expression. The results demonstrated that AgB significantly enhanced STAT3 degradation (Figure 6B), indicating that AgB‐mediated regulation of STAT3 occurs through its protein stability. Moreover, we treated CD4+ T cells with the proteasome inhibitor MG132. The results indicated that MG132 substantially reversed the decrease in STAT3 induced by AgB (Figure 6C). Thus, the degradation may, at least in part, be mediated by the proteasomal pathway. Moreover, AgB increased STAT3 ubiquitination (Figure 6D). Given that TRAF6 is a well‐characterized ubiquitin ligase that interacts with STAT3 and facilitates its ubiquitination, we examined STAT3–TRAF6 interactions. Our results demonstrated that AgB enhanced the association between STAT3 and TRAF6, as evidenced by increased coimmunoprecipitation of STAT3 with TRAF6 (Figure 6E). Thus, AgB may promote STAT3 degradation through augmentation of TRAF6‐mediated ubiquitination.

FIGURE 6.

AgB facilitates STAT3 degradation through promotion of TRAF6‐mediated ubiquitination. (A) STAT3 mRNA expression in CD4+ T cells from immune thrombocytopenia (ITP) patients treated with or not treated with AgB evaluated through quantitative reverse transcription polymerase chain reaction. (B, C) STAT3 protein expression and stability in CD4+ T cells from ITP patients treated with 10 μg/mL cycloheximide (CHX) or 10 μM MG132 through western blotting. (D) Immunoprecipitation assay for STAT3 ubiquitination, providing insights into posttranslational modifications affecting STAT3 protein stability. (E) Immunoprecipitation assay for STAT3–TRAF6 interactions. AgB could enhance the association between STAT3 and TRAF6. Data are presented as means ± SEMs from two to three independent experiments (n = 6). ***p < 0.001; ^### p < 0.01.

DISCUSSION

ITP is a heterogeneous autoimmune disorder characterized by a range of bleeding manifestations. Despite extensive research, the precise pathogenesis of ITP remains to be determined. ²⁷ , ²⁸ Here, we delineated the role and impact of AgB on the suppressive function of Tregs in the ITP context. Our findings suggested that AgB augments Treg quantity and functionality and suppresses STAT3 activation, eventually ameliorating thrombocytopenia.

T‐cell homeostasis dysregulation, particularly Treg depletion and unchecked Teff activation, is pivotal in ITP aetiology. In recent years, helminths and their derived molecules have emerged as promising therapeutic agents for autoimmune and inflammatory conditions. Their capacity to modulate host immune responses, encompassing Th2‐biased response induction, Treg activity enhancement and alternatively activated macrophage promotion, has been leveraged in various experimental and clinical applications. ²⁹ The immunomodulatory potential of helminths and their derived molecules may afford a novel therapeutic strategy for ITP and other immune‐mediated diseases.

E. granulosus infection involves modulation of host immune responses by prompting a shift towards regulatory phenotypes in various immune cells, reconfiguring the immune homeostasis within the host. ³⁰ Individuals with cystic echinococcosis exhibit a notable increase in the counts of Tregs and the levels of associated cytokines, such as IL‐10 and TGF‐β, alongside a reduction in the proinflammatory Th17 cells in their peripheral blood. ³¹ Considering its potent immunomodulatory capabilities, E. granulosus infection can be effectively harnessed in experimental settings to mitigate airway inflammation ¹⁶ and ameliorate colitis ³² in murine models. Furthermore, extracts from the hydatid‐laminated layer of E. granulosus have demonstrated therapeutic benefits in dextran sulphate sodium‐induced colitis in mice, characterized by a reduction in inflammatory responses. ³³

AgB, an immunodominant lipoprotein predominant among the excretory and secretory metacestode products of E. granulosus, is a key player in the immunological interplay of the helminth with its host during infection. AgB is a member of the family of hydrophobic ligand‐binding proteins characterized by their high abundance and immunogenicity and an oligomeric structure composed of 7–10‐kDa α‐helix‐rich subunits. ³⁴ AgB can suppress Th17 cell differentiation and promote Treg development, thereby mitigating airway inflammation associated with asthma. ²⁰ The present study further demonstrated that in vitro, AgB increases the counts of Tregs and bolsters their immunosuppressive function in patients with ITP. In addition to its effects on Tregs, AgB was noted to modulate the balance of CD4+ T‐cell subpopulations by curbing Th17 cell differentiation in active ITP murine models. This regulatory effect may underlie the therapeutic mechanism through which AgB increases platelet counts in ITP mice. Furthermore, consistent with our in vitro and in vivo findings, AgB significantly elevated the counts and inhibitory function of Tregs and reduced the counts of Th1, Th17 and Th22 cells. Treg depletion negated the restorative effects of AgB on T‐cell subpopulations, underscoring the pivotal role of Tregs in AgB‐mediated modulation of T‐cell homeostasis.

Alternative therapeutic strategies, including intravenous immunoglobulin (IVIg) and anti‐CD20 antibody therapies, can upregulate Foxp3 expression in CD4+ T cells and restore Treg populations in ITP murine models. ²⁷ , ³⁵ Corresponding outcomes have been documented in clinical settings in patients with ITP: Rituximab, thrombopoietin‐receptor agonists (TPO‐RAs), dexamethasone and IVIgs have demonstrated significant benefits in enhancing Treg function and modulating CD4+ T‐cell subpopulations in these patients. ²⁸ , ³⁶ , ³⁷ In the current study, AgB increased the counts of Tregs and strengthened their immunosuppressive capabilities in vitro, even in the absence of platelets, suggesting that the immunomodulatory effects of AgB are platelet independent.

Dysregulated cytokine profiles are strongly linked to immune tolerance disruption in ITP. ³⁸ , ³⁹ Treatment modalities such as high‐dose dexamethasone, rituximab and TPO‐RAs are often associated with normalizing cytokine imbalances. ²⁸ , ³⁹ , ⁴⁰ In line with the altered Treg–Th17 cell ratio, IFN‐γ, IL‐2 and IL‐17a levels are typically elevated, whereas IL‐4 and IL‐10 are reduced in patients with ITP. Moreover, cytokines such as IL‐6, TGF‐β and TNF‐α have been implicated in ITP pathogenesis. In the present study, AgB significantly increased TGF‐β levels, whereas it reduced TNF‐α, IFN‐γ, IL‐17a, IL‐12, IL‐8 and IL‐6 levels. These findings correspond to the reestablishment of a balanced Treg–Teff equilibrium in ITP, highlighting the potential of AgB as an immunotherapeutic agent.

The Jak–STAT pathway, an evolutionarily conserved mechanism in eukaryotes, is essential for Treg–Teff homeostasis initiation and maintenance. ⁴¹ STAT5 and STAT3 compete for promoter binding sites, exerting opposing effects on T‐cell differentiation. ⁴² , ⁴³ , ⁴⁴ STAT3 activation by cytokines such as IL‐6 and IL‐21 is essential for promoting Th17 cell development and facilitating the conversion of natural Tregs into Teffs. ⁴⁵ In the current study, AgB inhibited STAT3 activation; this appears to be a central mechanism underlying the therapeutic efficacy of AgB. Moreover, under the conditions of AKT inhibition, AgB demonstrated considerable effects on Tregs. Thus, the therapeutic effects of AgB in ITP may not primarily depend on AKT signalling.

To assess the mechanisms underlying the reduction of STAT3 expression, we first examined STAT3 production and found that AgB did not influence STAT3 mRNA expression. Thus, posttranscriptional modifications may be responsible for STAT3 regulation. Various factors, including increased protein tyrosine phosphatase and protein inhibitor levels and ubiquitination, suppress STAT3 expression. STAT3 ubiquitination enhances STAT3 degradation and inhibits Th17 cell differentiation. An increase in STAT3 ubiquitination thus correlates with the functional inhibition of STAT3. ⁴⁶ Consequently, we performed assays to detect STAT3 ubiquitination and observed that AgB administration increased STAT3 ubiquitination. This ubiquitination may be partly mediated by TRAF6, an E3 ubiquitin ligase that targets STAT3. These results provide novel insights into the molecular mechanisms underlying AgB‐mediated modulation of STAT3 signalling.

AgB oligomers predominantly have a molecular mass of 150–230 kDa; nevertheless, higher molecular mass aggregates have also been detected. ⁴⁷ The oligomeric structure of AgB comprises five 8‐kDa subunits, designated AgB8/1 to AgB8/5 and encoded by a multigene family. ⁴⁸ These subunits exhibit differential expression across various lifecycle stages of E. granulosus, its metacestode tissues and different individuals. ⁴⁷ , ⁴⁹ , ⁵⁰ Heterogeneity of the AgB oligomeric structure may confer distinct immunomodulatory functions. Nevertheless, the specific AgB oligomeric structure essential for its immune modulation of Th17 cells and regulation of STAT3 warrants further investigation. The therapeutic potential of AgB, a helminth‐derived protein, is a complex, understudied area in the field of immunology. Considering that murine immune responses may not accurately reflect human immune conditions, we acknowledge that our in vivo findings may not directly apply to humans. Therefore, further exploration of variations in the effects of AgB between murine models and humans is required to bridge the existing knowledge gap.

The current results offer an initial glimpse into the potential therapeutic role of AgB in ITP. However, further research comprehensively assessing the long‐term safety and efficacy of AgB is imperative. Given that AgB influences the immune system through multiple signalling pathways, exploring the interplay between other cytokine signalling pathways and STAT3 is essential. The prospect of synergistic effects when AgB is combined with established treatments, such as TPO‐RAs or rituximab, is particularly compelling; however, this area was not explored in the current study. Although preliminary studies have suggested that AgB influences B‐cell differentiation, studies have yet to investigate this in the context of ITP. This represents a crucial avenue of research, as elucidating the impact on B cells could yield profound insights into the capacity of AgB to modulate immune responses in ITP.

The current therapeutic methodologies for primary ITP treatment include corticosteroids, TPO‐RAs, IVIgs, immunosuppression and splenectomy. ⁵¹ Although approximately 80% of patients may achieve substantial therapeutic benefits from these medical or surgical strategies, many require additional treatment options. However, the available treatment modalities cannot resolve ITP or halt its progression to a chronic state, and their side effects can be deleterious. ⁵¹ As such, the development of less toxic, more effective novel therapeutic options is urgently needed. In the current study, we observed that AgB has promising treatment outcomes in ITP, suggesting that the protein may be an alternative or complementary strategy to the current treatment modalities for ITP.

CONCLUSION

The mechanism underlying the therapeutic efficacy of AgB in ITP involves suppression of STAT3 activation, along with enhancement of its ubiquitination and subsequent degradation. This increases the production of Tregs and bolsters their immunosuppressive capabilities, thus ameliorating ITP symptoms. Therefore, its immunomodulatory properties make AgB a promising avenue for the development of innovative ITP treatment approaches.

AUTHOR CONTRIBUTIONS

YY conceived and designed the study; YC collected the data; HJ analysed and interpreted the data; YZ drafted the manuscript and provided critical revisions important to the intellectual content; and MY approved the final version of the manuscript.

FUNDING INFORMATION

This work was supported by the Key Research and Development Project of the Xinjiang Uygur Autonomous Region of China (grant number 2024B03038‐1), the National Natural Science Foundation of China (grant number 82160031), Graduate Innovation Project of Xinjiang Uygur Autonomous Region (grant number XJ2025G182), State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia Fund (SKL‐HIDCA‐2024‐BC7) and the Youth Voyage Fund Program of The First Affiliated Hospital of Xinjiang Medical University (2023YFY‐QKMS‐01).

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no competing interests.

ETHICS STATEMENT

The experimental protocols were approved by the Institutional Review Board of the First Affiliated Hospital of Xinjiang Medical University and the Institutional Animal Care and Usage Committee of Xinjiang Medical University. Written informed consent was obtained from all participants or their legally authorized representatives prior to their participation in the study.

Supporting information

Figure S1.

Yue Y, Zhang Y, Cheng Y, Jiao H, Yan M. Echinococcus granulosus antigen B regulates T‐cell function through inhibition of signal transducer and activator of transcription 3 in experimental immune thrombocytopenia. Br J Haematol. 2025;206(6):1627–1641. 10.1111/bjh.20064

DATA AVAILABILITY STATEMENT

The datasets analysed during this study are available from the corresponding author upon reasonable request.