TRAF!-Mediated Regulation of Inflammasome Complex via Linear Ubiquitin Complex

Inflammasomes are multimeric protein complexes that act as a critical component of the innate immune system that control the caspase-1 activation and release of a potent pro-inflammatory cytokine IL-1β in response to pathogens and cellular damage. The NLRP3 inflammasome is the most well-characterized inflammasome that consists of the NLRP3 sensor, ASC adaptor protein, and pro-caspase-1. Interestingly, excessive activation of NLRP3 inflammasome has been associated with autoinflammatory and autoimmune diseases such as rheumatoid arthritis (RA), gout, and lupus. Therefore, the NLRP3 inflammasome activation is tightly regulated, among others, by linear ubiquitination, a form of post-translational modification mediated by linear ubiquitin chain assembly (LUBAC). Moreover, tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1), a key signalling adaptor protein, has been shown to negatively regulate LUBAC and interfere with linear ubiquitination. Therefore, we explored whether TRAF1 can regulate inflammasome activation and the underlying mechanism. To this end, we knocked down the expression of TRAF1 by RNA interference (shRNA) in the human macrophage cell line (THP-1) and induced NLRP3 inflammasome activation using various stimuli including LPS, nigericin, ATP, and urate crystals (MSU). The activation of NLRP3 inflammasome, including secretion of IL-1β, caspase-1, and ASC oligomerization were assessed by immunoblotting, ELISA, bioluminescence, and immunoprecipitation. Our findings demonstrated that with reduced TRAF1 levels, there was an increased activation of NLRP3 inflammasome characterized by elevated caspase-1 processing and active IL-1b secretion. Mechanistically, we showed that TRAF1 knockdown cells exhibited increased linear ubiquitination and subsequent oligomerization of ASC. Thereby, TRAF1 limits NLRP3 inflammasome activation by interfering with linear ubiquitination of ASC, which results in reduced caspase-1 activation and IL-1β secretion. These findings provide insights that might lead to the development of novel therapies that target TRAF1 to improve outcomes of NLRP3 inflammasome-related diseases such as RA