NLRP3 inflammasome proteolysis in endothelial cell infected with Porphyromonas gingivalis

The infection of atheromatous plaque by Porphyromonas gingivalis (Pg) is considered a major component of periodontitis influence on atherosclerosis. Our study aims to analyse the effect of Pg infection and Pg lipopolysaccharide (LPS) stimulation on NLRP3 inflammasome regulation and IL-1β production in endothelial cells (EC).

Method: NLPR3 expression was induced in Endothelial cells (HUVEC) by a pre-treatment with ATP 5mM. Normal and pre-treated HUVEC were infected with Pg for 6 hours and stimulated by its purified LPS for 24 hours. RNA expression and protein concentration of NLRP3, IL-1β and caspase-1 were evaluated in cell lysates using RT-qPCR and Western blotting.

Result: Pg infection and Pg-LPS stimulation significantly increase NLRP3 mRNA especially in pre-treated cells. In Pg infected cells, an important proteolysis of NLPR3 proteins is observed resulting in a net decrease of the native protein level while in Pg-LPS stimulated cells an increase of NLPR3 protein level is observed without proteolysis. This proteolysis is not observed with heat-denatured Pg (HPg). Furthermore, the inhibition of protein synthesis by cycloheximide does not abolish NLRP3 protein degradation. Finally, a significant increase of secreted IL-1β is measured after ATP pre-treatment or Pg-LPS stimulation but not after Pg infection.

 Conclusion: Our results indicate that Pg and Pg-LPS differentially control the NLRP3 inflammasome pathway in ECs and that Pg proteases could be directly involved in NLRP3 proteolysis. Furthermore, these data highlight a novel potential mechanism used by Pg to block IL-1β secretion and to evade EC defense responses.