205: A role for protein kinase R in regulating NLRP3 inflammasome assembly

Aim NLR family pyrin domain containing-3 (NLRP3) has been associated with inflammatory disorders. In response to exogenous and endogenous molecules, NLRP3 assemble an inflammasome containing the apoptosis-associated speck like protein containing a CARD (ASC) for activating caspase-1, inducing pyroptosis and processing interleukin (IL)-1β and IL-18. A recent model proposes that the assembly of the inflammasome is dependent on the cytoskeleton. As the protein kinase R (PKR) has been shown to regulate actin dynamics, we examined whether PKR regulates inflammasome activity. Methods Primary macrophages were isolated from the peritoneum, then primed with lipopolysaccharide and treated with NLRP3 inducers. Whole cells, lysates and culture supernatant were analyzed for the expression and spatial positions of constituents of the inflammasome and the production of cytokines. To investigate inflammation in vivo, mice were treated with dextran sodium sulphate (DSS) in their drinking water. After 9 days, intestinal tissue sections were isolated from different mouse strains and compared. Results We showed an increased interaction between NLRP3-ASC and related to this ASC speck formation, activation of caspase-1 and consequent processing of pro-IL-1β in PKR-ablated compared to wild-type macrophages. We demonstrated that this PKR-dependent repression of the inflammasome requires kinase activity by using cells from a kinase-dead PKR transgenic mouse. Consistently, we showed higher caspase-1 activity and increased pathology in PKR knockout compared to wild-type mice subjected to DSS. Mechanistically, we found that the increased proximity of NLRP3 and ASC in the absence of PKR reflected the juxtaposition of the endoplasmic reticulum and mitochondria. The spatial arrangement of these cellular organelles is mediated by microtubule-dependent affects. Consistent with this, we demonstrate that PKR interacts with microtubules in a kinase dependent fashion. Conclusion We demonstrated that PKR suppresses the NLRP3 inflammasome activation by modulating the cytoskeleton. These findings provide critical information to correctly develop therapeutic strategies to combat inflammatory disorders.