Reactive Oxygen Species Regulation by NCF1 Governs Ferroptosis Susceptibility of Kupffer Cells to MASH

Hepatobiliary System Life Sciences Immunology Medicine Cell Biology Biochemistry
Reactive Oxygen Species NCF1 Kupffer Cells Iron Homeostasis Ferroptosis MASH Lipid Peroxidation

NCF1 Regulates Reactive Oxygen Species Vulnerability to Ferroptosis in Kupffer Cells and Its Impact on MASH

Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), has a global prevalence of up to 25.2% and is a leading cause of chronic liver disease in adults and children. MASLD encompasses a spectrum of hepatic histopathological abnormalities, ranging from simple steatosis to metabolic dysfunction-associated steatohepatitis (MASH), which is an inflammatory disease that may further progress to cirrhosis, hepatocellular carcinoma (HCC), and end-stage liver disease. Currently, no approved pharmacotherapy exists for MASH. Therefore, gaining a deep understanding of the pathological mechanisms of MASH is crucial for developing effective treatment options.

Research Background and Objectives

Recent studies indicate that self-maintaining Kupffer cells (KCs) are impaired in MASH. The increased death of KCs gradually generates monocyte-derived macrophages (MoMcs) to replenish the KC reservoir. These new macrophages exhibit a transcriptional landscape significantly different from that of KCs, displaying more pronounced inflammatory characteristics. Research indicates that oxidative stress is a key factor in the progression from steatosis to MASH. Neutrophil cytosolic factor 1 (NCF1) is a critical regulator of oxidative stress, and its polymorphisms are associated with susceptibility to various chronic inflammatory autoimmune diseases. However, it remains unclear whether and how NCF1 regulates oxidative responses and inflammation in diet-induced MASH. Furthermore, elucidating which cell type mediates the pathogenic function of NCF1 in MASH is of significant clinical importance.

Research Institutions and Publication Information

This study was conducted by researchers from several institutes and hospitals affiliated with Xi’an Jiaotong University, including Jing Zhang, Yu Wang, Meiyang Fan, and Liesu Meng, and was published on August 6, 2024, in the journal “Cell Metabolism” (DOI: https://doi.org/10.1016/j.cmet.2024.05.008).

Research Methods and Process

Experimental Procedure

The study employed the following process:

  1. NCF1 Gene Expression Analysis

    • Flow cytometry was used to analyze liver cells from MASH mice to detect NCF1 expression levels.

  2. Phenotype Studies

    • Studies were conducted using wild-type (WT) mice, NCF1* mice, and transgenic mice specifically expressing NCF1 in CD68+ macrophages (NCF1*.NCF1tg-CD68), which were maintained under different dietary conditions (methionine/choline-deficient diet and high-fat diet) to assess liver function markers and histological changes.

  3. Measurement of Iron Levels and Reactive Oxygen Species (ROS)

    • Phen Green SK probes and Ferritin antibodies were used in flow cytometry to measure iron content and lipid peroxidation levels in liver KCs and MoMcs of the different mouse models.

  4. Ferroptosis Inhibitor Experiments

    • Ferroptosis inhibitor Ferrostatin-1 (Fer-1) was administered by injection to assess its effect on MASH symptoms and ferroptosis in mice.

  5. Cell Co-culture Experiments

    • A co-culture system of Kupffer cells and hepatocytes was established to investigate how conditioned medium (CM) from PA-stimulated macrophages regulates Hepcidin production in hepatocytes and affects iron deposition in KCs.

Data Analysis and Algorithms

RNA sequencing analysis combined with flow cytometry data was utilized. Various statistical methods were employed to analyze and compare gene expression, protein levels, and biological markers.

Research Results

  1. NCF1 Expression and Its Impact

    • NCF1 was predominantly expressed in Kupffer cells (KCs) and monocyte-derived macrophages (MoMcs), with significant upregulation observed in the livers of MASH mice.

  2. Phenotypic Changes

    • NCF1* mutant mice exhibited less severe steatosis, inflammatory responses, and fibrosis. However, these indices worsened with the restoration of NCF1 expression in CD68+ macrophages, demonstrating that macrophage-expressed NCF1 exacerbates MASH.

  3. Iron Levels and Lipid Peroxidation in KCs and MoMcs

    • The study found elevated iron levels in KCs of MASH mice, which were notably decreased in NCF1* mutant mice. Through detecting lipid peroxidation products, it was established that NCF1 promotes ferroptosis in KCs.

  4. Effectiveness of Ferroptosis Inhibitors

    • Ferrostatin-1 (Fer-1) significantly improved liver function in MASH mice, reduced hepatic inflammation and fibrosis, and decreased iron deposition and lipid peroxidation in KCs, confirming the critical role of ferroptosis in MASH.

  5. Relationship Between OxPLs and Hepcidin

    • The research demonstrated that NCF1 enhances ROS production in KCs, promoting the formation of oxidized phospholipids (OxPLs). These OxPLs act as messengers, activating Toll-like receptor 4 (TLR4) in hepatocytes, leading to increased Hepcidin production, resulting in iron deposition and ferroptosis in KCs.

Conclusion

This study elucidates the role of NCF1 in macrophages, revealing the mechanism of ferroptosis in Kupffer cells in the context of MASH. NCF1 increases the formation of oxidized phospholipids (OxPLs), activating the TLR4 pathway in hepatocytes, thus promoting Hepcidin production, ultimately leading to iron deposition and ferroptosis in Kupffer cells. The findings suggest that targeting NCF1 and the described signaling pathways could provide new therapeutic strategies for patients with MASH.

Research Highlights and Value

  1. Significant Discoveries

    • For the first time, the specific mechanism by which NCF1 promotes ferroptosis in Kupffer cells in MASH has been revealed, offering new perspectives for pathological research on MASH.

  2. Therapeutic Potential

    • The study indicates that NCF1 and its related signaling pathways could serve as potential therapeutic targets to improve the fate of Kupffer cells and limit the progression of MASH.

  3. Theoretical Innovation

    • The study proposes the “vicious cycle” theory of OxPLs activating the TLR4-Hepcidin axis to promote iron deposition and ferroptosis, providing an important theoretical basis for future MASH treatment.

  4. Methodological Innovation

    • Innovative use of multiple mouse models and cell co-culture systems was employed to elucidate the role of macrophage NCF1 in MASH and to verify the critical role of key signaling pathways.

These discoveries lay an important foundation for further understanding the pathogenesis of MASH and for the development of related treatment methods.