Interleukin-12p40 Deficiency Attenuates Myocardial Ferroptosis in Doxorubicin-Induced Chronic Cardiomyopathy by Inhibiting Th17 Differentiation and Interleukin-17A Production

Rheumatology and Immunology Cardiovascular System Life Sciences Cell Biology Immunology Medicine
Interleukin-12p40 Doxorubicin-induced cardiotoxicity Ferroptosis Inflammation Adaptive immunity T lymphocyte differentiation

Academic Background

Doxorubicin (Dox) is an anthracycline drug widely used in the treatment of tumors, but it exhibits dose-dependent cardiotoxicity, potentially leading to cardiomyopathy and heart failure. Despite its significant anticancer effects, the cardiotoxicity of Dox limits its clinical application. Currently, Dexrazoxane is the only FDA-approved drug for treating Dox-related cardiomyopathy, but its use in pediatric patients is restricted due to the potential risk of increasing secondary malignancies in children with Hodgkin lymphoma. Therefore, identifying new therapeutic targets and strategies to prevent and treat Dox-induced cardiomyopathy has become an urgent clinical need.

In recent years, research has revealed that ferroptosis, a form of programmed cell death dependent on iron accumulation and lipid peroxidation, plays a crucial role in Dox-induced chronic cardiomyopathy (DICCM). However, the regulatory mechanisms of ferroptosis remain unclear. Additionally, the role of immune responses, particularly T cell-mediated adaptive immunity, in DICCM has garnered significant attention. IL-12p40, a common subunit of IL-12 and IL-23, possesses independent biological functions, but its role and mechanisms in DICCM have not been thoroughly investigated. Therefore, this study aims to explore the role of IL-12p40 in Dox-induced chronic cardiomyopathy and its regulatory mechanisms, providing new therapeutic targets for clinical treatment.

Source of the Paper

This paper was co-authored by Jishou Zhang, Wen Ding, Zheng Yin, and others from the Cardiovascular Research Institute of Renmin Hospital of Wuhan University. The research team also included scientists from the First Affiliated Hospital of Zhejiang University School of Medicine, the Center for Healthy Aging at Wuhan University, and other institutions. The paper was published online on September 19, 2024, in the journal Cardiovascular Research, titled “Interleukin-12p40 deficiency attenuates myocardial ferroptosis in doxorubicin-induced chronic cardiomyopathy by inhibiting Th17 differentiation and interleukin-17a production.”

Research Process and Results

1. Animal Model Establishment and Myocardial Ferroptosis Assessment

The study first induced a chronic cardiomyopathy model in mice using a cumulative dose of 12 mg/kg Dox (administered in three injections over two weeks). Echocardiography revealed a significant decline in left ventricular systolic function in the Dox-treated group, along with increased expression of apoptosis and ferroptosis markers (e.g., Bax, ACSL4, PTGS2) and elevated levels of the lipid peroxidation product 4-HNE. Furthermore, the study found that as the Dox dose increased, myocardial apoptosis and ferroptosis became more pronounced, indicating dose-dependent myocardial injury induced by Dox.

2. Expression and Function of IL-12p40 in DICCM

Through RNA sequencing and flow cytometry analysis, the study found that IL-12p40 expression was significantly upregulated in the hearts of Dox-treated mice, primarily in CD4+ T cells. In IL-12p40 knockout (IL-12p40 KO) mice, cardiac function significantly improved, and myocardial fibrosis and ferroptosis were alleviated following Dox treatment. Similar results were observed in Rag1−/− mice (lacking functional B and T cells), indicating that IL-12p40 exerts its pathological effects mainly through CD4+ T cells.

3. IL-12p40 Regulates DICCM via IL-23

To further elucidate the regulatory mechanisms of IL-12p40, the study used recombinant IL-12 (rIL-12), recombinant IL-23 (rIL-23), recombinant IL-12p40 monomer (rIL-12p40), and recombinant IL-12p80 (rIL-12p80). Results showed that only rIL-23 significantly reversed the cardioprotective effects of IL-12p40 KO mice, indicating that the protective effects of IL-12p40 deficiency are primarily mediated through the inhibition of IL-23. Additionally, IL-23p19 knockout mice exhibited significant improvements in cardiac function and reduced myocardial fibrosis and ferroptosis following Dox treatment, further confirming the critical role of IL-23 in DICCM.

4. IL-12p40 Attenuates Myocardial Ferroptosis by Inhibiting Th17 Differentiation and IL-17a Production

Through RNA sequencing and real-time quantitative PCR analysis, the study found that the expression of Th17 cell differentiation markers RORγt and IL-17a was significantly reduced in the hearts of IL-12p40 KO mice. Flow cytometry further confirmed a significant decrease in the proportion of CD4+IL-17a+ Th17 cells in the hearts of IL-12p40 KO mice. Moreover, treatment with an IL-17a neutralizing antibody significantly ameliorated Dox-induced myocardial injury, indicating that IL-12p40 attenuates myocardial ferroptosis by inhibiting Th17 differentiation and IL-17a production.

5. The IL-12p40/Th17/IL-17a Axis Regulates Myocardial Ferroptosis via the TRAF6/MAPK/p53 Signaling Pathway

Western blot analysis revealed that the expression of TRAF6 and p-JNK was significantly reduced in the hearts of IL-12p40 KO mice, and IL-17a neutralizing antibody treatment also significantly decreased TRAF6 and p-JNK expression. Additionally, the study found that the phosphorylation level of p53 was significantly reduced in the hearts of IL-12p40 KO mice, indicating that the IL-12p40/Th17/IL-17a axis regulates myocardial ferroptosis through the TRAF6/MAPK/p53 signaling pathway. In vitro experiments showed that silencing TRAF6 with siRNA or treating with a JNK inhibitor significantly reversed the pro-ferroptotic effects of IL-17a, further confirming this mechanism.

Conclusions and Significance

This study is the first to reveal the critical role of IL-12p40 in Dox-induced chronic cardiomyopathy and its regulatory mechanisms. The research demonstrates that IL-12p40 deficiency attenuates myocardial ferroptosis and apoptosis by inhibiting Th17 differentiation and IL-17a production, thereby improving cardiac function. This protective effect is primarily mediated through an IL-23-dependent mechanism and involves the TRAF6/MAPK/p53 signaling pathway. The study provides new therapeutic targets for Dox-induced cardiomyopathy, offering significant scientific value and clinical application prospects.

Research Highlights

  1. Innovative Discovery: First to reveal the critical role of IL-12p40 in Dox-induced chronic cardiomyopathy and elucidate its regulatory mechanisms through the IL-23/Th17/IL-17a axis.
  2. Multi-level Validation: Comprehensive validation of IL-12p40’s regulatory mechanisms through knockout mice, recombinant protein treatments, and in vitro experiments.
  3. Clinical Application Potential: The study provides new therapeutic targets for Dox-induced cardiomyopathy, offering significant clinical application value.

Other Valuable Information

The study also found that IL-12p40 expression is primarily localized to CD4+ T cells, indicating that T cell-mediated adaptive immunity plays a crucial role in Dox-induced cardiomyopathy. Additionally, the study revealed that the IL-12p40/Th17/IL-17a axis regulates myocardial ferroptosis through the TRAF6/MAPK/p53 signaling pathway, providing new insights into the regulatory mechanisms of myocardial ferroptosis.