By differential screening of tumor necrosis factor α (TNF-α) and lipopolysaccharide (LPS)- activated endothelial cells (ECs), we have identified a cDNA clone that turned out to be a member of the inhibitor of apoptosis (iap) gene family. iap genes function to protect cells from undergoing apoptotic death in response to a variety of stimuli. These iap genes, hiap1, hiap2, and xiap were found to be strongly upregulated upon treatment of ECs with the inflammatory cytokines TNF-α, interleukin 1β, and LPS, reagents that lead to activation of the nuclear transcription factor κB (NF-κB). Indeed, overexpression of IκBα, an inhibitor of NF-κB, suppresses the induced expression of iap genes and sensitizes ECs to TNF-α–induced apoptosis. Ectopic expression of one member of the human iap genes, human X-chromosome–linked iap (xiap), using recombinant adenovirus overrules the IκBα effect and protects ECs from TNF-α– induced apoptosis. We conclude that xiap represents one of the NF-κB–regulated genes that counteracts the apoptotic signals caused by TNF-α and thereby prevents ECs from undergoing apoptosis during inflammation.

SUMMARY Cytosolic pathogen- and damage-associated molecular patterns are sensed by pattern recognition receptors, including members of the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR), which cause inflammasome assembly and caspase-1 activation to promote maturation and release of the inflammatory cytokines interleukin (IL)-1β and IL-18 and induction of pyroptosis. However, the contribution of most of the NLRs to innate immunity, host defense, inflammasome activation and their specific agonists, are still unknown. Here we describe identification and characterization of an NLRP7 inflammasome in human macrophages, which is induced in response to microbial acylated lipopeptides. Activation of NLRP7 promoted ASC-dependent caspase-1 activation, IL-1β and IL-18 maturation and restriction of intracellular bacterial replication, but not caspase-1-independent secretion of the pro-inflammatory cytokines IL-6 and tumor necrosis factor-α. Our study therefore increases our currently limited understanding of NLR activation, inflammasome assembly and maturation of IL-1β and IL-18 in human macrophages.

The innate immune system responds to infections and tissue damage by activating cytosolic sensory complexes called inflammasomes. Cytosolic DNA is sensed by AIM2-like receptors (ALRs) during bacterial and viral infections and in autoimmune diseases. Subsequently, recruitment of the adaptor protein ASC links ALRs to the activation of caspase-1. A controlled immune response is crucial for maintaining homeostasis, but ALR inflammasome regulation is poorly understood. Here, we identified the PYRIN domain (PYD)-only protein 3 (POP3), which competes with ASC for recruitment to ALRs, as an inhibitor of DNA virus-induced ALR inflammasome activation in vivo. Using a mouse model with macrophage-specific POP3 expression, the data emphasizes the importance of ALR inflammasome regulation in the monocytic/macrophage.

Proteins containing PAAD [pyrin, AIM (absent-in-melanoma), ASC [apoptosis-associated speck-like protein containing a CARD (caspase-recruitment domain)] and DD (death domain)-like] (PYRIN, DAPIN) domains are involved in innate immunity, regulating pathways leading to nuclear-factor-kappa B (NF-kappa B) and pro-caspase-1 activation. Many PAAD-family proteins have structures reminiscent of Nod-1, a putative intracellular sensor of lipopolysaccharide. Hereditary mutations in some of the PAAD-family genes are associated with auto-inflammatory diseases. Several of these proteins utilize the bipartite PAAD- and CARD-containing adapter protein ASC/TMS-1 (target of methylation-induced silencing) for linking to downstream signalling pathways. In the present paper, we describe characterization of human PAAD-only protein-1 (POP1)/ASC2, which is highly homologous with the PAAD domain of ASC, and which probably originated by gene duplication on chromosome 16. We demonstrate that POP1/ASC2 associates with ASC via PAAD-PAAD interactions and modulates NF-kappa B and pro-caspase-1 regulation by this adapter protein. In gene transfer experiments, POP1/ASC2 suppressed cytokine-mediated NF-kappa B activation similar to other PAAD-family proteins previously tested. Immunohistochemical studies showed expression of POP1/ASC2 predominantly in macrophages and granulocytes. We propose that POP1/ASC2 functions as a modulator of multidomain PAAD-containing proteins involved in NF-kappa B and pro-caspase-1 activation and innate immunity.

Activation of caspase 1 is essential for the maturation and release of IL-1β and IL-18 and occurs in multiprotein complexes, referred to as inflammasomes. The apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is the essential adaptor protein for recruiting pro-caspase 1 into inflammasomes, and consistently gene ablation of ASC abolishes caspase 1 activation and secretion of IL-1β and IL-18. However, distribution of endogenous ASC has not yet been examined in detail. In the present study, we demonstrated that ASC localized primarily to the nucleus in resting human monocytes/macrophages. Upon pathogen infection, ASC rapidly redistributed to the cytosol, followed by assembly of perinuclear aggregates, containing several inflammasome components, including caspase 1 and Nod-like receptors. Prevention of ASC cytosolic redistribution completely abolished pathogen-induced inflammasome activity, which affirmed that cytosolic localization of ASC is essential for inflammasome function. Thus, our study characterized a novel mechanism of inflammasome regulation in host defense.

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/target of methylation-induced silencing/PYCARD represents one of only two proteins encoded in the human genome that contains a caspase recruitment domain (CARD) together with a pyrin, AIM, ASC, and death domain-like (PAAD)/PYRIN/DAPIN domain. CARDs regulate caspase family proteases. We show here that ASC binds by its CARD to procaspase-1 and to adapter proteins involved in caspase-1 activation, thereby regulating cytokine pro-IL-1β activation by this protease in THP-1 monocytes. ASC enhances IL-1β secretion into the cell culture supernatants, at low concentrations, while suppressing at high concentrations. When expressed in HEK293 cells, ASC interferes with Cardiak/Rip2/Rick-mediated oligomerization of procaspase-1 and suppresses activation this protease, as measured by protease activity assays. Moreover, ASC also recruits procaspase-1 into ASC-formed cytosolic specks, separating it from Cardiak. We also show that expression of the PAAD/PYRIN family proteins pyrin or cryopyrin/PYPAF1/NALP3 individually inhibits IL-1β secretion but that coexpression of ASC with these proteins results in enhanced IL-1β secretion. However, expression of ASC uniformly interferes with caspase-1 activation and IL-1β secretion induced by proinflammatory stimuli such as LPS and TNF, suggesting pathway competition. Moreover, LPS and TNF induce increases in ASC mRNA and protein expression in cells of myeloid/monocytic origin, revealing another level of cross-talk of cytokine-signaling pathways with the ASC-controlled pathway. Thus, our results suggest a complex interplay of the bipartite adapter protein ASC with PAAD/PYRIN family proteins, LPS (Toll family receptors), and TNF in the regulation of procaspase-1 activation, cytokine production, and control of inflammatory responses.

SUMMARY In response to infections and tissue damage, ASC-containing inflammasome protein complexes are assembled that promote caspase-1 activation, IL-1β and IL-18 processing and release, pyroptosis, and the release of ASC particles. However, excessive or persistent activation of the inflammasome causes inflammatory diseases. Therefore, a well-balanced inflammasome response is crucial to maintain homeostasis. We show that the PYD-only protein POP1 inhibited ASC-dependent inflammasome assembly by preventing inflammasome nucleation, and consequently interfered with caspase-1 activation, IL-1β and IL-18 release, pyroptosis and the release of ASC particles. There is no mouse ortholog for the POP1 gene, but transgenic expression of human POP1 in monocytes, macrophages and dendritic cells protected mice from systemic inflammation triggered by molecular PAMPs, inflammasome component NLRP3 mutation and ASC danger particles. POP1 expression was regulated by TLR- and IL-1R–signalling, and we propose that POP1 provides a regulatory feedback loop that shuts down excessive inflammatory responses and thereby prevents systemic inflammation.

Exposure of endothelial and many other cell types to tumor necrosis factor ␣ generates both apoptotic and anti-apoptotic signals. The anti-apoptotic pathway leads to activation of the transcription factor NF-B that regulates the expression of genes such as A20 or members of the IAP gene family that protect cells from tumor necrosis factor ␣-mediated apoptosis. In turn, some anti-apoptotic genes have been shown to modulate NF-B activity. Here we demonstrate that XIAP, a NF-Bdependent member of the IAP gene family, is a strong stimulator of NF-B. Expression of XIAP leads to increased nuclear translocation of the p65 subunit of NF-B via a novel signaling pathway that involves the mitogen-activated protein kinase kinase kinase TAK1. We show that TAK1 physically interacts with NIK and with IKK2, and both XIAP or active TAK1 can stimulate IKK2 kinase activity. Thus, XIAP may be part of a system of regulatory loops that balance a cell's response to environmental stimuli.