Blockade of NLRP3 expression in primary hepatocytes by inhibitors of NF-κB signalling

The activation of NLRP3 inflammasome is performed in two steps. In a first step pro-inflammatory cytokines such as interleukin-1β (IL-1β) become activated by proteolytic cleavage of their proforms. As a consequence they stimulate the assembly of the NLRP3 inflammasome representing a large cytoplasmic multiprotein complex that process a large variety of danger and stress signals that are triggered by lipopolysaccharides (LPS), ATP or crystals that induce lysosomal destabilization [1]. In a previous study from our laboratory we showed that LPS stimulation of primary hepatocytes leads to a significant increase expression of NLRP3, IL-1β, and TNF-α [2]. In the present study we extended these investigations and analyzed the consequences of NLRP3 inflammasome activation in hepatocytes. We found that the LPS-induced expression of NLRP3, IL-1β, and TNF-α can be drastically reduced by the NF-κB activation inhibitor (QNZ), while other inflammasome branches (e.g. ASC) are not affected at all by this small component. Furthermore, cells in which the NF-κB pathway was blocked in the presence of LPS were unable to synthesise pro-IL-1β that is mandatory for NLRP3 inflammasome activation and function. The observed activation of NLRP3 and the elevated activity of IL-1β were also demonstrated in reporter gene assays. Using immunofluorescence staining in primary murine hepatocyte, we confirmed the gene expression data for NLRP3 and IL-1β at the protein level. Altogether, our findings confirm the existence of a tight regulatory network between IL-1β, NF-κB and activity of the cellular NLRP3 inflammasome branch.

References cited:

[1] Gross O, Thomas CJ, Guarda G, Tschopp J. The inflammasome: an integrated view. Immunol Rev 2011;243:136 – 51.

[2] Boaru SG, Borkham-Kamphorst E, Tihaa L, Haas U, Weiskirchen R. Expression analysis of inflammasome components in experimental models of inflammatory and fibrotic liver disease. J Inflamm, 2012, 9(1):48.