Site-specific phosphorylation and microtubule dynamics control Pyrin inflammasome activation.

Pyrin, encoded by the MEFV gene, is best known for its gain-of-function mutations causing familial Mediterranean fever (FMF), an autoinflammatory disease. Pyrin forms a caspase-1-activating inflammasome in response to inactivating modifications of Rho GTPases by various bacterial toxins or effectors. Pyrin-mediated innate immunity is unique in that it senses bacterial virulence rather than microbial molecules, but its mechanism of activation is unknown. Here we show that Pyrin was phosphorylated in bone marrow-derived macrophages and dendritic cells. We identified Ser-205 and Ser-241 in mouse Pyrin whose phosphorylation resulted in inhibitory binding by cellular 14-3-3 proteins. The two serines underwent dephosphorylation upon toxin stimulation or bacterial infection, triggering 14-3-3 dissociation, which correlated with Pyrin inflammasome activation. We developed antibodies specific for phosphorylated Ser-205 and Ser-241, which confirmed the stimuli-induced dephosphorylation of endogenous Pyrin. Mutational analyses indicated that both phosphorylation and signal-induced dephosphorylation of Ser-205/241 are important for Pyrin activation. Moreover, microtubule drugs, including colchicine, commonly used to treat FMF, effectively blocked activation of the Pyrin inflammasome. These drugs did not affect Pyrin dephosphorylation and 14-3-3 dissociation but inhibited Pyrin-mediated apoptosis-associated Speck-like protein containing CARD (ASC) aggregation. Our study reveals that site-specific (de)phosphorylation and microtubule dynamics critically control Pyrin inflammasome activation, illustrating a fine and complex mechanism in cytosolic immunity.

A Burkholderia Type VI Effector Deamidates Rho GTPases to Activate the Pyrin Inflammasome and Trigger Inflammation.

Burkholderia cenocepacia is an opportunistic pathogen of the cystic fibrosis lung that elicits a strong inflammatory response. B. cenocepacia employs a type VI secretion system (T6SS) to survive in macrophages by disarming Rho-type GTPases, causing actin cytoskeletal defects. Here, we identified TecA, a non-VgrG T6SS effector responsible for actin disruption. TecA and other bacterial homologs bear a cysteine protease-like catalytic triad, which inactivates Rho GTPases by deamidating a conserved asparagine in the GTPase switch-I region. RhoA deamidation induces caspase-1 inflammasome activation, which is mediated by the familial Mediterranean fever disease protein Pyrin. In mouse infection, the deamidase activity of TecA is necessary and sufficient for B. cenocepacia-triggered lung inflammation and also protects mice from lethal B. cenocepacia infection. Therefore, Burkholderia TecA is a T6SS effector that modifies a eukaryotic target through an asparagine deamidase activity, which in turn elicits host cell death and inflammation through activation of the Pyrin inflammasome.

Non-canonical activation of inflammatory caspases by cytosolic LPS in innate immunity.

Lipopolysaccharide (LPS) is the major component of Gram-negative bacteria cell wall. In innate immunity, extracellular LPS is recognized by Toll-like receptor 4 to stimulate cytokine transcription. Recent studies suggest a 'non-canonical inflammasome' that senses cytoplasmic LPS and activates caspase-11 in mouse macrophages. Unexpectedly, biochemical studies reveal that caspase-11 and its human orthologs caspase-4/caspase-5 are LPS receptors themselves. Direct LPS binding induces caspase-4/caspase-5/caspase-11 oligomerization and activation, triggering cell pyroptosis and anti-bacterial defenses. Caspase-4/caspase-5/caspase-11 recognition of intracellular LPS requires bacterial escape from the vacuole; this process is promoted by interferon-inducible GTPases-mediated lysis of the bacteria-containing vacuole. Non-canonical activation of these inflammatory caspases by LPS not only represents a new paradigm in innate immunity but also critically determines LPS-induced septic shock in mice.

Immunological function of familial Mediterranean fever disease protein Pyrin.

Pyrin, encoded by MEFV gene, is conserved in humans and mice. Mutations in the MEFV gene are associated with the human autoinflammatory disease familial Mediterranean fever (FMF). Pyrin can interact with the inflammasome adaptor ASC and induce inflammatory caspase-1 activation in monocytic cells, but the physiological function of Pyrin has been unknown for many years. Here we summarize previous studies of Pyrin function under the context of FMF and immunity, and discuss a recent study demonstrating that Pyrin forms an inflammasome complex for caspase-1 activation in innate immunity. Pyrin inflammasome detects inactivating modifications of host Rho GTPases by diverse bacterial toxins and infections, including Clostridium difficile glucosylating cytotoxin TcdB, FIC-domain adenylyltransferase effectors from Vibrio parahaemolyticus and Histophilus somni, ADP-ribosylating Clostridium botulinum C3 toxin as well as Burkholderia cenocepacia infection. The mode of Pyrin action, i.e., sensing pathogen virulence activity rather than directly recognizing a microbial molecule, represents a new paradigm in innate immunity.

Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome.

Cytosolic inflammasome complexes mediated by a pattern recognition receptor (PRR) defend against pathogen infection by activating caspase 1. Pyrin, a candidate PRR, can bind to the inflammasome adaptor ASC to form a caspase 1-activating complex. Mutations in the Pyrin-encoding gene, MEFV, cause a human autoinflammatory disease known as familial Mediterranean fever. Despite important roles in immunity and disease, the physiological function of Pyrin remains unknown. Here we show that Pyrin mediates caspase 1 inflammasome activation in response to Rho-glucosylation activity of cytotoxin TcdB, a major virulence factor of Clostridium difficile, which causes most cases of nosocomial diarrhoea. The glucosyltransferase-inactive TcdB mutant loses the inflammasome-stimulating activity. Other Rho-inactivating toxins, including FIC-domain adenylyltransferases (Vibrio parahaemolyticus VopS and Histophilus somni IbpA) and Clostridium botulinum ADP-ribosylating C3 toxin, can also biochemically activate the Pyrin inflammasome in their enzymatic activity-dependent manner. These toxins all target the Rho subfamily and modify a switch-I residue. We further demonstrate that Burkholderia cenocepacia inactivates RHOA by deamidating Asn 41, also in the switch-I region, and thereby triggers Pyrin inflammasome activation, both of which require the bacterial type VI secretion system (T6SS). Loss of the Pyrin inflammasome causes elevated intra-macrophage growth of B. cenocepacia and diminished lung inflammation in mice. Thus, Pyrin functions to sense pathogen modification and inactivation of Rho GTPases, representing a new paradigm in mammalian innate immunity.

Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation.

Inflammasome mediated by central nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) protein is critical for defense against bacterial infection. Here we show that type III secretion system (T3SS) needle proteins from several bacterial pathogens, including Salmonella typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, and Burkholderia spp., can induce robust inflammasome activation in both human monocyte-derived and mouse bone marrow macrophages. Needle protein activation of human NRL family CARD domain containing 4 (NLRC4) inflammasome requires the sole human neuronal apoptosis inhibitory protein (hNAIP). Among the seven mouse NAIPs, NAIP1 functions as the mouse counterpart of hNAIP. We found that NAIP1 recognition of T3SS needle proteins was more robust in mouse dendritic cells than in bone marrow macrophages. Needle proteins, as well as flagellin and rod proteins from five different bacteria, exhibited differential and cell type-dependent inflammasome-stimulating activity. Comprehensive profiling of the three types of NAIP ligands revealed that NAIP1 sensing of the needle protein dominated S. flexneri-induced inflammasome activation, particularly in dendritic cells. hNAIP/NAIP1 and NAIP2/5 formed a large oligomeric complex with NLRC4 in the presence of corresponding bacterial ligands, and could support reconstitution of the NLRC4 inflammasome in a ligand-specific manner.

The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus.

Inflammasomes are large cytoplasmic complexes that sense microbial infections/danger molecules and induce caspase-1 activation-dependent cytokine production and macrophage inflammatory death. The inflammasome assembled by the NOD-like receptor (NLR) protein NLRC4 responds to bacterial flagellin and a conserved type III secretion system (TTSS) rod component. How the NLRC4 inflammasome detects the two bacterial products and the molecular mechanism of NLRC4 inflammasome activation are not understood. Here we show that NAIP5, a BIR-domain NLR protein required for Legionella pneumophila replication in mouse macrophages, is a universal component of the flagellin-NLRC4 pathway. NAIP5 directly and specifically interacted with flagellin, which determined the inflammasome-stimulation activities of different bacterial flagellins. NAIP5 engagement by flagellin promoted a physical NAIP5-NLRC4 association, rendering full reconstitution of a flagellin-responsive NLRC4 inflammasome in non-macrophage cells. The related NAIP2 functioned analogously to NAIP5, serving as a specific inflammasome receptor for TTSS rod proteins such as Salmonella PrgJ and Burkholderia BsaK. Genetic analysis of Chromobacterium violaceum infection revealed that the TTSS needle protein CprI can stimulate NLRC4 inflammasome activation in human macrophages. Similarly, CprI is specifically recognized by human NAIP, the sole NAIP family member in human. The finding that NAIP proteins are inflammasome receptors for bacterial flagellin and TTSS apparatus components further predicts that the remaining NAIP family members may recognize other unidentified microbial products to activate NLRC4 inflammasome-mediated innate immunity.

Chemical probing reveals insights into the signaling mechanism of inflammasome activation.

Caspase-1-mediated IL-1ß production is generally controlled by two pathways. Toll-like receptors (TLRs) recognize pathogen-derived products and induce NF-κB-dependent pro-IL-1ß transcription; NOD-like receptors (NLRs) assemble caspase-1-activating inflammasome complexes that sense bacterial products/danger signals. Through a targeted chemical screen, we identify bromoxone, a marine natural product, as a specific and potent inhibitor of the caspase-1 pathway. Bromoxone is effective over diverse inflammatory stimuli including TLR ligands plus ATP/nigericin, cytosolic DNA, flagellin and Bacillus anthracis lethal toxin. Bromoxone also efficiently suppresses caspase-1 activation triggered by several types of bacterial infection. Bromoxone acts upstream or at the level of the inflammasome in a transcription-independent manner. Bromoxone also inhibits pro-IL-1ß expression by targeting components upstream of IKK in the TLR-NF-κB pathway. The unique dual activities of bromoxone are shared by the known TAK1 inhibitor that specifically blocks Nalp3 inflammasome activation. Hinted from the mechanistic and pharmacological properties of bromoxone, we further discover that several known NF-κB inhibitors that act upstream of IKK, but not those targeting IKK or IKK downstream, are potent blockers of different NLRs-mediated caspase-1 activation. Our study uncovers a possible non-transcriptional molecular link between the NLR (Nalp3)-mediated inflammasome pathway and TLR-NF-κB signaling, and suggests a potential strategy to develop new anti-inflammatory drugs.