Nobuhiko Kayagaki

Non-canonical inflammasome activation targets caspase-11

Caspase-1 activation by inflammasome scaffolds comprised of intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and the adaptor ASC is believed to be essential for production of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 during the innate immune response. Here we show, with C57BL/6 Casp11 gene-targeted mice, that caspase-11 (also known as caspase-4) is critical for caspase-1 activation and IL-1β production in macrophages infected with Escherichia coli, Citrobacter rodentium or Vibrio cholerae. Strain 129 mice, like Casp11−/− mice, exhibited defects in IL-1β production and harboured a mutation in the Casp11 locus that attenuated caspase-11 expression. This finding is important because published targeting of the Casp1 gene was done using strain 129 embryonic stem cells. Casp1 and Casp11 are too close in the genome to be segregated by recombination; consequently, the published Casp1–/– mice lack both caspase-11 and caspase-1. Interestingly, Casp11–/– macrophages secreted IL-1β normally in response to ATP and monosodium urate, indicating that caspase-11 is engaged by a non-canonical inflammasome. Casp1–/–Casp11129mt/129mt macrophages expressing caspase-11 from a C57BL/6 bacterial artificial chromosome transgene failed to secrete IL-1β regardless of stimulus, confirming an essential role for caspase-1 in IL-1β production. Caspase-11 rather than caspase-1, however, was required for non-canonical inflammasome-triggered macrophage cell death, indicating that caspase-11 orchestrates both caspase-1-dependent and -independent outputs. Caspase-1 activation by non-canonical stimuli required NLRP3 and ASC, but caspase-11 processing and cell death did not, implying that there is a distinct activator of caspase-11. Lastly, loss of caspase-11 rather than caspase-1 protected mice from a lethal dose of lipopolysaccharide. These data highlight a unique pro-inflammatory role for caspase-11 in the innate immune response to clinically significant bacterial infections.

IAP Antagonists Induce Autoubiquitination of c-IAPs, NF-κB Activation, and TNFα-Dependent Apoptosis

Inhibitor of apoptosis (IAP) proteins are antiapoptotic regulators that block cell death in response to diverse stimuli. They are expressed at elevated levels in human malignancies and are attractive targets for the development of novel cancer therapeutics. Herein, we demonstrate that small-molecule IAP antagonists bind to select baculovirus IAP repeat (BIR) domains resulting in dramatic induction of auto-ubiquitination activity and rapid proteasomal degradation of c-IAPs. The IAP antagonists also induce cell death that is dependent on TNF signaling and de novo protein biosynthesis. Additionally, the c-IAP proteins were found to function as regulators of NF-kappaB signaling. Through their ubiquitin E3 ligase activities c-IAP1 and c-IAP2 promote proteasomal degradation of NIK, the central ser/thr kinase in the noncanonical NF-kappaB pathway.

BAFF/BLyS Receptor 3 Binds the B Cell Survival Factor BAFF Ligand through a Discrete Surface Loop and Promotes Processing of NF-κB2

The TNF-like ligand BAFF/BLyS is a potent survival factor for B cells. It binds three receptors: TACI, BCMA, and BR3. We show that BR3 signaling promotes processing of the transcription factor NF-kappaB2/p100 to p52. NF-kappaB2/p100 cleavage was abrogated in B cells from A/WySnJ mice possessing a mutant BR3 gene, but not in TACI or BCMA null B cells. Furthermore, wild-type mice injected with BAFF-neutralizing BR3-Fc protein showed reduced basal NF-kappaB2 activation. BR3-Fc treatment of NZB/WF1 mice, which develop a fatal lupus-like syndrome, inhibited NF-kappaB2 processing and attenuated the disease process. Since inhibiting the BR3-BAFF interaction has therapeutic ramifications, the ligand binding interface of BR3 was investigated and found to reside within a 26 residue core domain. When stabilized within a structured beta-hairpin peptide, six of these residues were sufficient to confer binding to BAFF.

Noncanonical Inflammasome Activation by Intracellular LPS Independent of TLR4

Move Over, TLR4 The innate immune system senses bacterial lipopolysaccharide (LPS) through Toll-like receptor 4 (TLR4) (see the Perspective by Kagan). However, Kayagaki et al. (p 1246, published online 25 July) and Hagar et al. (p. 1250) report that the hexa-acyl lipid A component of LPS from Gramnegative bacteria is able to access the cytoplasm and activate caspase-11 to signal immune responses independently of TLR4. Mice that lack caspase-11 are resistant to LPS-induced lethality, even in the presence of TLR4. Cytoplasmic lipopolysaccharide from Gram-negative bacteria can activate the innate immune system directly.[Also see Perspective by Kagan] Gram-negative bacteria including Escherichia coli, Citrobacter rodentium, Salmonella typhimurium, and Shigella flexneri are sensed in an ill-defined manner by an intracellular inflammasome complex that activates caspase-11. We show that macrophages loaded with synthetic lipid A, E. coli lipopolysaccharide (LPS), or S. typhimurium LPS activate caspase-11 independently of the LPS receptor Toll-like receptor 4 (TLR4). Consistent with lipid A triggering the noncanonical inflammasome, LPS containing a divergent lipid A structure antagonized caspase-11 activation in response to E. coli LPS or Gram-negative bacteria. Moreover, LPS-mutant E. coli failed to activate caspase-11. Tlr4–/– mice primed with TLR3 agonist polyinosinic:polycytidylic acid [poly(I:C)] to induce pro-caspase-11 expression were as susceptible as wild-type mice were to sepsis induced by E. coli LPS. These data unveil a TLR4-independent mechanism for innate immune recognition of LPS.

DUBA: A Deubiquitinase That Regulates Type I Interferon Production

Production of type I interferon (IFN-I) is a critical host defense triggered by pattern-recognition receptors (PRRs) of the innate immune system. Deubiquitinating enzyme A (DUBA), an ovarian tumor domain-containing deubiquitinating enzyme, was discovered in a small interfering RNA–based screen as a regulator of IFN-I production. Reduction of DUBA augmented the PRR-induced IFN-I response, whereas ectopic expression of DUBA had the converse effect. DUBA bound tumor necrosis factor receptor–associated factor 3 (TRAF3), an adaptor protein essential for the IFN-I response. TRAF3 is an E3 ubiquitin ligase that preferentially assembled lysine-63–linked polyubiquitin chains. DUBA selectively cleaved the lysine-63–linked polyubiquitin chains on TRAF3, resulting in its dissociation from the downstream signaling complex containing TANK-binding kinase 1. A discrete ubiquitin interaction motif within DUBA was required for efficient deubiquitination of TRAF3 and optimal suppression of IFN-I. Our data identify DUBA as a negative regulator of innate immune responses.

GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes.

Significance Pyroptosis is a form of cell death that is critical for eliminating innate immune cells infected with intracellular bacteria. Microbial products such as lipopolysaccharide, which is a component of Gram-negative bacteria, trigger activation of the inflammatory caspases 1, 4, 5, and 11. These proteases cleave the cytoplasmic protein Gasdermin-D into two pieces, p20 and p30. The p30 fragment is cytotoxic when liberated from the p20 fragment. Our work suggests that p30 induces pyroptosis by associating with cell membranes and forming pores that perturb vital electrochemical gradients. The resulting imbalance causes the cell to lyse and release intracellular components that can alert other immune cells to the threat of infection. Gasdermin-D (GsdmD) is a critical mediator of innate immune defense because its cleavage by the inflammatory caspases 1, 4, 5, and 11 yields an N-terminal p30 fragment that induces pyroptosis, a death program important for the elimination of intracellular bacteria. Precisely how GsdmD p30 triggers pyroptosis has not been established. Here we show that human GsdmD p30 forms functional pores within membranes. When liberated from the corresponding C-terminal GsdmD p20 fragment in the presence of liposomes, GsdmD p30 localized to the lipid bilayer, whereas p20 remained in the aqueous environment. Within liposomes, p30 existed as higher-order oligomers and formed ring-like structures that were visualized by negative stain electron microscopy. These structures appeared within minutes of GsdmD cleavage and released Ca2+ from preloaded liposomes. Consistent with GsdmD p30 favoring association with membranes, p30 was only detected in the membrane-containing fraction of immortalized macrophages after caspase-11 activation by lipopolysaccharide. We found that the mouse I105N/human I104N mutation, which has been shown to prevent macrophage pyroptosis, attenuated both cell killing by p30 in a 293T transient overexpression system and membrane permeabilization in vitro, suggesting that the mutants are actually hypomorphs, but must be above certain concentration to exhibit activity. Collectively, our data suggest that GsdmD p30 kills cells by forming pores that compromise the integrity of the cell membrane.

Interleukin-22 induces interleukin-18 expression from epithelial cells during intestinal infection.

T helper 1 (Th1) cell-associated immunity exacerbates ileitis induced by oral Toxoplasma gondii infection. We show here that attenuated ileitis observed in interleukin-22 (IL-22)-deficient mice was associated with reduced production of Th1-cell-promoting IL-18. IL-22 not only augmented the expression of Il18 mRNA and inactive precursor protein (proIL-18) in intestinal epithelial cells after T. gondii or Citrobacter rodentium infection, but also maintained the homeostatic amount of proIL-18 in the ileum. IL-22, however, did not induce the processing to active IL-18, suggesting a two-step regulation of IL-18 in these cells. Although IL-18 exerted pathogenic functions during ileitis triggered by T. gondii, it was required for host defense against C. rodentium. Conversely, IL-18 was required for the expression of IL-22 in innate lymphoid cells (ILCs) upon T. gondii infection. Our results define IL-18 as an IL-22 target gene in epithelial cells and describe a complex mutual regulation of both cytokines during intestinal infection.

Phosphorylation-dependent activity of the deubiquitinase DUBA

Addition and removal of ubiquitin or ubiquitin chains to and from proteins is a tightly regulated process that contributes to cellular signaling and protein stability. Here we show that phosphorylation of the human deubiquitinase DUBA (OTUD5) at a single residue, Ser177, is both necessary and sufficient to activate the enzyme. The crystal structure of the ubiquitin aldehyde adduct of active DUBA reveals a marked cooperation between phosphorylation and substrate binding. An intricate web of interactions involving the phosphate and the C-terminal tail of ubiquitin cause DUBA to fold around its substrate, revealing why phosphorylation is essential for deubiquitinase activity. Phosphoactivation of DUBA represents an unprecedented mode of protease regulation and a clear link between two major cellular signal transduction systems: phosphorylation and ubiquitin modification.

Caspase-11: arming the guards against bacterial infection.

As a front line of defense against pathogenic microbes, our body employs a primitive, yet highly sophisticated and potent innate immune response pathway collectively referred to as the inflammasome. Innate immune cells, epithelial cells, and many other cell types are capable of detecting infection or tissue injury and mounting a coordinated molecular defense. For example, Gram‐negative bacteria are specifically detected via a surveillance mechanism that involves activation of extracellular receptors such as Toll‐like receptors (TLRs) followed by intracellular recognition and activation of pathways such as caspase‐11 (caspase‐4/5 in humans). Importantly, lipopolysaccharide (LPS), the major component of the outer membrane of Gram‐negative bacteria, is a strong trigger of these pathways. Extracellular LPS primarily stimulates TLR4, which can serve as a priming signal for expression of inflammasome components. Intracellular LPS can then trigger caspase‐11‐dependent inflammasome activation in the cytoplasm. Here, we briefly review the burgeoning caspase‐11‐dependent non‐canonical inflammasome field, focusing mainly on the innate sensing of LPS.

The Yersinia pestis Effector YopM Inhibits Pyrin Inflammasome Activation

Type III secretion systems (T3SS) are central virulence factors for many pathogenic Gram-negative bacteria, and secreted T3SS effectors can block key aspects of host cell signaling. To counter this, innate immune responses can also sense some T3SS components to initiate anti-bacterial mechanisms. The Yersinia pestis T3SS is particularly effective and sophisticated in manipulating the production of pro-inflammatory cytokines IL-1β and IL-18, which are typically processed into their mature forms by active caspase-1 following inflammasome formation. Some effectors, like Y. pestis YopM, may block inflammasome activation. Here we show that YopM prevents Y. pestis induced activation of the Pyrin inflammasome induced by the RhoA-inhibiting effector YopE, which is a GTPase activating protein. YopM blocks YopE-induced Pyrin-mediated caspase-1 dependent IL-1β/IL-18 production and cell death. We also detected YopM in a complex with Pyrin and kinases RSK1 and PKN1, putative negative regulators of Pyrin. In contrast to wild-type mice, Pyrin deficient mice were also highly susceptible to an attenuated Y. pestis strain lacking YopM, emphasizing the importance of inhibition of Pyrin in vivo. A complex interplay between the Y. pestis T3SS and IL-1β/IL-18 production is evident, involving at least four inflammasome pathways. The secreted effector YopJ triggers caspase-8- dependent IL-1β activation, even when YopM is present. Additionally, the presence of the T3SS needle/translocon activates NLRP3 and NLRC4-dependent IL-1β generation, which is blocked by YopK, but not by YopM. Taken together, the data suggest YopM specificity for obstructing the Pyrin pathway, as the effector does not appear to block Y. pestis-induced NLRP3, NLRC4 or caspase-8 dependent caspase-1 processing. Thus, we identify Y. pestis YopM as a microbial inhibitor of the Pyrin inflammasome. The fact that so many of the Y. pestis T3SS components are participating in regulation of IL-1β/IL-18 release suggests that these effects are essential for maximal control of innate immunity during plague.