Neutrophil-derived PAD4 induces citrullination of CKMT1 exacerbates mucosal inflammation in inflammatory bowel disease

Infectious Diseases Rheumatology and Immunology Life Sciences Immunology Gastroenterology Medicine
neutrophil citrullination CKMT1 intestinal epithelial cells inflammatory bowel disease intestinal inflammation apoptosis

In recent years, increasing evidence from research on Inflammatory Bowel Disease (IBD) has shown that neutrophils play a crucial role in the occurrence and development of intestinal inflammation. IBD includes Crohn’s Disease (CD) and Ulcerative Colitis (UC), and while its pathogenesis is not yet fully understood, abnormal immune responses are clearly one of the main factors affecting the condition.

Research Background and Objectives

The aim of this study was to investigate the specific role of Peptidyl Arginine Deiminase 4 (PAD4) and its potential substrates in the pathological process of IBD. The research team pointed out that neutrophils can combat pathogenic microorganisms by forming a network structure called Neutrophil Extracellular Traps (NETs), but at the same time, this mechanism may also disrupt cellular connections and induce apoptosis of Intestinal Epithelial Cells (IECs). Therefore, understanding the role of PAD4 in this process is particularly important.

Researchers and Institutions

This paper was jointly completed by Shuling Wang, Yihang Song, Zhijie Wang, and others, with the authors mainly from the Naval Medical University, Changhai Hospital in Shanghai, China, and the University of Science and Technology of China. The paper was published in “Cellular & Molecular Immunology” on May 8, 2024.

Research Methods and Procedures

The research team systematically decoded the role of PAD4 in a mouse model of colitis induced by dextran sulfate sodium (DSS) using single-cell RNA sequencing (scRNA-seq) and substrate citrullination mapping techniques.

  1. Animal Experiments: Researchers first generated PAD4-deficient mice (PAD4−／−) through gene knockout techniques and found that this deficiency significantly improved DSS-induced colitis inflammation.
  2. Single-Cell RNA Sequencing: Through single-cell RNA sequencing of colon tissue, the research team identified 17 different cell clusters and found a significant reduction in the number of neutrophils in PAD4-deficient mice. In addition, they observed significant changes in epithelial cell subpopulations.
  3. Substrate Citrullination Mapping: Using liquid chromatography-mass spectrometry (LC-MS) techniques, they further validated the role of PAD4 in colitis inflammation and discovered that Mitochondrial Creatine Kinase 1 (CKMT1) is a key substrate regulated by PAD4.

Main Research Findings

  1. PAD4 Deficiency Reshapes the Intestinal Cellular Landscape:

    • The study found that PAD4 deficiency led to a significant reduction in the number of neutrophils in the colon tissue of DSS-treated mice, accompanied by a significant decrease in the expression of a series of inflammation-related genes.
    • In the analysis of IECs, a decrease in immature distal epithelial cells was found, but NOS2+CLCA3b+ epithelial cells increased significantly, all of which were closely related to oxidative phosphorylation and autophagy pathways.

  2. PAD4 Regulates Intestinal Barrier Function through EVs:

    • The study found that during the formation of NETs, PAD4 is released into IECs through EVs, affecting their cell survival and barrier function.
    • In vitro experiments using PAD4 overexpressing IEC cell lines further demonstrated the regulatory role of PAD4 in cellular citrullination processes, increasing apoptosis markers and decreasing cell junction markers.

  3. CKMT1 is a Key Substrate for PAD4 Citrullination:

    • In a series of mass spectrometry analyses, the research team found that the R243 site of CKMT1 is the main site of PAD4-mediated citrullination.
    • In PAD4-deficient mice, the protein stability of CKMT1 was significantly improved in the DSS-induced colitis mouse model, indicating that PAD4 regulated CKMT1 expression through the autophagy pathway.

Research Conclusions and Significance

Through this series of in-depth studies, the team demonstrated the important role of PAD4 in IBD, revealing the crucial mechanism by which PAD4 affects IEC homeostasis by citrullinating CKMT1, thereby exacerbating IBD conditions. These findings not only provide new insights into the pathological mechanisms of IBD but also point to PAD4 as a potential therapeutic target.

Research Highlights

  1. First discovery that PAD4 is transmitted to IECs through EVs during NETs formation and exerts its effects.
  2. Identification of CKMT1 as a key substrate for PAD4 citrullination, revealing its specific role in intestinal pathology.
  3. Systematic analysis of the molecular mechanisms of PAD4 in intestinal inflammation through single-cell RNA sequencing and mass spectrometry analysis.

Clinical Relevance

Through clinical analysis of IBD patients, the research team further validated the expression changes of PAD4 and CKMT1 in IBD. The data showed that PAD4 and citrullination levels were significantly elevated in IBD patients, while CKMT1 expression was markedly decreased, which negatively correlated with the degree of colon inflammation. This finding provides important clues for the clinical diagnosis and targeted treatment of IBD.

Outlook and Recommendations

This study provides strong theoretical support for the application prospects of PAD4 in IBD treatment. Future research can further explore the specific regulatory mechanisms of PAD4 and CKMT1 in IBD, develop new therapeutic targets and drugs, with the hope of bringing more effective treatment options for patients.