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

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.