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Non-canonical NF-κB contributes to endothelial pyroptosis and atherogenesis dependent on IRF-1

  • Author Footnotes
    # Xing Fan, Qiannan Li, and Yiying Wang contributed equally to this study.
    Xing Fan
    Footnotes
    # Xing Fan, Qiannan Li, and Yiying Wang contributed equally to this study.
    Affiliations
    Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China

    Key laboratory of Human Functional Genomics of Jiangsu Province, Nanjing, China
    Search for articles by this author
  • Author Footnotes
    # Xing Fan, Qiannan Li, and Yiying Wang contributed equally to this study.
    Qiannan Li
    Footnotes
    # Xing Fan, Qiannan Li, and Yiying Wang contributed equally to this study.
    Affiliations
    Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China

    Key laboratory of Human Functional Genomics of Jiangsu Province, Nanjing, China
    Search for articles by this author
  • Author Footnotes
    # Xing Fan, Qiannan Li, and Yiying Wang contributed equally to this study.
    Yiying Wang
    Footnotes
    # Xing Fan, Qiannan Li, and Yiying Wang contributed equally to this study.
    Affiliations
    Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China

    Key laboratory of Human Functional Genomics of Jiangsu Province, Nanjing, China
    Search for articles by this author
  • Dai-Min Zhang
    Affiliations
    Department of Cardiology, Sir Run Run Hospital, Nanjing Medical University, Nanjing, China
    Search for articles by this author
  • Jingchao Zhou
    Affiliations
    Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China

    Key laboratory of Human Functional Genomics of Jiangsu Province, Nanjing, China
    Search for articles by this author
  • Qing Chen
    Affiliations
    Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China

    Key laboratory of Human Functional Genomics of Jiangsu Province, Nanjing, China
    Search for articles by this author
  • Liang Sheng
    Affiliations
    Department of Pharmacology, Nanjing Medical University, Nanjing, China
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  • Anthony G. Passerini
    Affiliations
    Department of Biomedical Engineering, University of California Davis, Davis, California
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  • ChongXiu Sun
    Correspondence
    Reprint requests: ChongXiu Sun, Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, 101 Longmian Avenue, Jiangning District, Nanjing 211166, China
    Affiliations
    Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China

    Key laboratory of Human Functional Genomics of Jiangsu Province, Nanjing, China
    Search for articles by this author
  • Author Footnotes
    # Xing Fan, Qiannan Li, and Yiying Wang contributed equally to this study.
Published:November 12, 2022DOI:https://doi.org/10.1016/j.trsl.2022.11.001

      Abstract

      Cell inflammation and death are closely linked processes contributing to endothelial dysfunction, which plays a critical role in atherogenesis. Activation of the NLRP3 inflammasome causes pyroptosis, the Gasdermin D (GSDMD)-mediated inflammatory cell death. The non-canonical NF-κB pathway has been implicated in inflammation; however, its role in NLRP3 inflammasome-mediated endothelial dysfunction has not been investigated. This study investigated a role for the non-canonical NF-κB pathway in regulating endothelial pyroptosis as it relates to atherogenesis. Immunohistochemistry indicated inflammasome activation in the endothelial cells (EC) of human atherosclerotic arteries. Flow cytometry and Western blot analysis revealed that oxidized low-density lipoprotein (oxLDL) activated the NLRP3 inflammasome, concomitant with the activation of non-canonical NF-κB in primary human aortic EC. Interference of NF-κB inducing kinase (NIK), the key regulator of the non-canonical pathway, significantly attenuated oxLDL- or LPS/ATP-induced NLRP3 inflammasome activation, pyroptosis, IL-1β, and IL-18 secretion. In contrast, overexpression of NIK exacerbated these responses. Chromatin immunoprecipitation revealed that activation of the non-canonical NF-κB pathway upregulated the transcription factor IRF-1 through RelB/p52 binding to its promoter region at -782/-770. In addition to the known target CASP1, RNA sequencing further identified GSDMD as a target gene of IRF-1. IRF-1 but not RelB/p52 interacted with the GSDMD promoter at -526/-515 and the CASP1 promoter at -11/10 to promote the expression and CASP1-mediated activation of GSDMD. Consistent with the observations in cultured endothelium, endothelial-specific deficiency of NIK or IRF-1 attenuated atherosclerosis in high-fat diet-fed Apoe-null mice. These data demonstrate that the non-canonical NF-κB pathway contributes to NLRP3 inflammasome-mediated endothelial pyroptosis and the development of atherosclerosis through GSDMD activation in a manner dependent on IRF-1. Further investigation may facilitate the identification of specific therapeutic targets for atherosclerotic heart diseases.

      Abbreviations:

      ChIP (chromatin immunoprecipitation assay), DAMP (danger-associated molecular patterns), ELISA (enzyme linked immunosorbent assay), GSDMD (gasdermin D), HAEC (human aortic endothelial cell), IRF-1 (interferon regulatory factor 1), MAEC (murine aortic endothelial cell), NF-κB (nuclear factor-kappa B), NIK (NF-kB inducing kinase), NLRP3 (NOD-like receptor P3), oxLDL (oxidized low-density lipoprotein), PAMP (pathogen-associated molecular patterns), RHD (Relhomology domain), TAD (transcription activation domain)
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