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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.

The innate immune system provides a rapid protective response against a wide range of cellular insults sensed by the host as danger signals, including those associated with viral infections. The NLRP3 inflammasome is a multiprotein complex that assembles in response to danger signals, including those generated by viruses, triggering self-cleavage of procaspase-1 and subsequent processing of proinflammatory cytokines. Precisely how viruses and inflammatory cells interact to control this process remains incompletely understood. The 2-prime, 5-prime oligoadenylate synthetase (OAS)/RNase L system is a component of the interferon-induced antiviral response that senses double-stranded RNA and activates endoribonuclease RNase L to cleave viral and cellular RNAs. We will report involvement of the mammalian OAS/RNase L system in viral activation of the NLRP3 inflammasome in mouse bone marrow-derived dendritic cells and in human THP-1-derived macrophages. Furthermore, RNase L deficiency reduced IL-1 β production in influenza A virus-infected mice. The signaling pathway includes RNA cleavage products with 2-prime, 3-prime-cyclic phosphorylated termini, DExD/H-box helicase, DHX33, and the mitochondrial adapter protein, MAVS. Data suggest that RNA cleavage events catalyzed by RNase L are required for optimal inflammasome activation during viral infections.

Inflammasomes are a key component of cytosolic surveillance. They are multi-protein complexes that regulate maturation of the potent pro-inflammatory cytokines interleukin (IL)-1β and IL-18, and drive cell death via pyroptosis. As well as responding to exogenous, pathogen-associated molecules, the inflammasomes also respond to endogenous signals, making these sensors prime mediators of inflammation. Since their groundbreaking description a decade ago, there has been an intense effort to discover the mechanisms that regulate the activity of inflammasomes, to enable therapeutic control of inflammation. Towards this, it has recently been reported that the antiviral protein kinase R (PKR) promotes inflammasome activity, endorsing the use of pharmacological inhibition of PKR to treat inflammation. However, this conclusion is contradicted by our findings that PKR has variable effects on the inflammasomes, even repressing their activity in specific instances. In these instances, inhibiting the activity of PKR could be counterproductive and even detrimental. Here we present data contradicting a pro-inflammatory function for PKR and identifying the mechanism by the kinase represses activity of the NLRP3 inflammasome.

Introduction Inflammatory bowel diseases (IBD) are characterized by chronic intestinal inflammation that potentiates the risk for developing colorectal cancer. Indeed, single nucleotide polymorphisms in NOD-like receptor (NLR) family pyrin domain containing-3 (NLRP3) gene has been linked to increased susceptibility to IBD in humans. However, the regulatory mechanisms of NLRP3 signaling pathways leading to the intestinal inflammation in IBD are not well defined. Previous studies have shown that Protein Kinase R (PKR), a kinase mediating stress response via phosphorylation of the eukaryotic initiation factor 2α (eIF2α), enhances Toll-like receptor signaling which is required to prime NLR inflammasomes. Here we tested the role of PKR in the NLR signaling pathway and its consequence to intestinal inflammation. Methods Wild-type (WT) or PKR-ablated mice were treated with dextran sodium sulphate in their drinking water and their condition monitored daily. After 9 days intestinal tissue sections were taken from both mouse strains and compared. Additionally, immortalized spleen and primary peritoneal macrophages from WT and PKR-ablated mice were primed with the Toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) for 4 h, and treated with NLR3 ligands for 1–2 h. Then, cell culture supernatants were analyzed for production of inflammatory cytokines. Results While WT mice demonstrated no conspicuous symptoms, PKR-ablated mice exhibited clinical symptoms of pathology and lost 20% of their weight by day 9. Correspondingly, the PKR-ablated mice had enhanced intestinal tissue damage when compared to WT mice. We showed that although similar levels of IL1β are induced in WT and PKR-ablated macrophages, higher levels of total IL-1β was released from PKR-ablated macrophages upon stimulations with nigercin or calcium pyrophosphate dehydrate crystals. In keeping with this, we show increased cleavage of pro-IL-1β to the active form in the absence of PKR. Moreover, activation of caspase-1 (CASP1), the protease that processes IL-1β, was increased in PKR-ablated compared to WT cells. Using kinase-dead PKR, we show that this repression of the inflammasome in part requires substrate phosphorylation. Conclusion We revealed that PKR is protective in a murine model of colitis and show this is due to repression of the activity of the NLRP3 inflammasome. These findings may provide insight into the development of therapeutic strategies against IBD.