Activated protein C and PAR1-derived and PAR3-derived peptides are anti-inflammatory by suppressing macrophage NLRP3 inflammasomes.

BACKGROUND Activated protein C (APC) has been shown to restrict murine inflammasome activity. However, whether APC can exert anti-inflammatory activity in part through suppression of inflammasome activation in human systems is unknown. OBJECTIVES Studies were made to determine whether either APC or protease activated receptor (PAR)-derived peptides can reduce NLRP3 inflammasome activity in differentiated human THP-1 macrophage-like cells or in primary human monocytes stimulated to activate the inflammasome. METHODS Human THP-1 cells or primary human monocytes were differentiated, treated with APC or PAR-derived peptides, and then stimulated with LPS and ATP to induce caspase-1 activity, a product of inflammasome activation. RESULTS APC or non-canonical PAR1-derived or PAR3-derived peptides significantly reduced caspase-1 activity, detection of fluorescent NLRP3 and IL-1β release from THP-1 cells. At low concentrations where no effect was observed for each individual peptide, combinations of the PAR1-derived peptide and the PAR3-derived peptide resulted in a significant synergistic decrease in caspase-1 and IL-1β release. Caspase-1 activity was also reduced in primary human monocytes. Studies using blocking antibodies and small molecule PAR1 inhibitors suggest that EPCR, PAR1 and PAR3 each play roles in the observed anti-inflammatory effects. Several shortened versions of the PAR1-derived and PAR3-derived peptide reduced caspase-1 activity and exhibited synergistic anti-inflammatory effects. CONCLUSIONS The results indicate that both APC and certain PAR1-derived and PAR3-derived peptides which are biased agonists for PAR1 or PAR3 can reduce inflammasome activity in stimulated human monocytes as measured by caspase-1 activity and IL-1β release and that PAR-derived biased peptide agonist combinations are synergistically anti-inflammatory.