Nortriptyline Induces Sterile Inflammation by Directly Targeting NLRP3 Inflammasome Activation

Background: Increasing evidence shows that tricyclic antidepressant s ( TCA s) could induce relative rare but severe idiosyncratic drug-induced liver injury (IDILI) in clinical applications, while the underlying mechanisms are still poorly understood. We sought to determine the molecular mechanism of TCAs-induced hepatotoxicity, and facilitated the potential approach to increase of the biosafety of TCAs. Methods: We examined the effect of TCAs on downstream effector cytokines.  MCC950 pretreatment and conditional knockouts of Nlrp3 were applicated to verify the specificity of TCAs for NLRP3 inflammasome and inductively coupled plasma mass spectrometry (ICP-MS), immunoblot, immunoprecipitation, Enzyme-linked immunosorbent assay (ELISA) and histology staining were used to investigate the related mechanisms. Findings: Nortriptyline, used as a common TCA agent, induced the altering of aminotransferase level, hepatic inflammation and hepatocyte death in a NLRP3-dependent manner. In parallel in vitro studies, Nortriptyline directly induced inflammasome activation as evidenced by the caspase-1 activation, IL-1β generation and gasdermin D (GSDMD)-mediated pyroptosis, while these changes were blocked by the selective NLRP3 inhibitor MCC950 treatment or Nlrp3 deficiency. Mechanistically, Nortriptyline treatment led to increased accumulation of mitochondrial reactive oxygen species (mtROS) resulting in the aberrant activation of NLRP3 inflammasome, and ROS scavenger pretreatment completely abrogated Nortriptyline-induced caspase-1 maturation and IL-1β release. Moreover, exposure to other TCAs that have been reported to induced liver injury, including Imipramine, Amitriptyline and Protriptyline, also led to NLRP3 inflammasome aberrant activation, as well as subsequent inflammatory cytokines release. Interpretation: NLRP3 inflammasome may acts as a crucial target for TCA agents and suggest that aberrant activation of NLRP3 inflammasome may contribute to pathogenesis of TCAs-induced liver injury. Funding: This work was granted by National Natural Science Foundation of China (81630100, 81874368), National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (2018ZX09101002- 001-002), Beijing Nova Program (Z181100006218001), Chinese Postdoctoral Science Foundation (grant number: 2020M673676), Youth Foundation of Chinese PLA General Hospital (QNF19065). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: This study was approved by the Animal Ethics Committee of the Fifth Medical Center, Chinese People's Liberation Army General Hospital (Beijing, China).