Jürg Tschopp

Gout-associated uric acid crystals activate the NALP3 inflammasome

Development of the acute and chronic inflammatory responses known as gout and pseudogout are associated with the deposition of monosodium urate (MSU) or calcium pyrophosphate dihydrate (CPPD) crystals, respectively, in joints and periarticular tissues. Although MSU crystals were first identified as the aetiological agent of gout in the eighteenth century and more recently as a ‘danger signal’ released from dying cells, little is known about the molecular mechanisms underlying MSU- or CPPD-induced inflammation. Here we show that MSU and CPPD engage the caspase-1-activating NALP3 (also called cryopyrin) inflammasome, resulting in the production of active interleukin (IL)-1β and IL-18. Macrophages from mice deficient in various components of the inflammasome such as caspase-1, ASC and NALP3 are defective in crystal-induced IL-1β activation. Moreover, an impaired neutrophil influx is found in an in vivo model of crystal-induced peritonitis in inflammasome-deficient mice or mice deficient in the IL-1β receptor (IL-1R). These findings provide insight into the molecular processes underlying the inflammatory conditions of gout and pseudogout, and further support a pivotal role of the inflammasome in several autoinflammatory diseases.

The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta.

Generation of Interleukin (IL)-1beta via cleavage of its proform requires the activity of caspase-1 (and caspase-11 in mice), but the mechanism involved in the activation of the proinflammatory caspases remains elusive. Here we report the identification of a caspase-activating complex that we call the inflammasome. The inflammasome comprises caspase-1, caspase-5, Pycard/Asc, and NALP1, a Pyrin domain-containing protein sharing structural homology with NODs. Using a cell-free system, we show that proinflammatory caspase activation and proIL-1beta processing is lost upon prior immunodepletion of Pycard. Moreover, expression of a dominant-negative form of Pycard in differentiated THP-1 cells blocks proIL-1beta maturation and activation of inflammatory caspases induced by LPS in vivo. Thus, the inflammasome constitutes an important arm of the innate immunity.

Inhibition of death receptor signals by cellular FLIP

The widely expressed protein Fas is a member of the tumour necrosis factor receptor family which can trigger apoptosis. However, Fas surface expression does not necessarily render cells susceptible to Fas ligand-induced death signals,, indicating that inhibitors of the apoptosis-signalling pathway must exist. Here we report the characterization of an inhibitor of apoptosis, designated FLIP (for FLICE-inhibitory protein), which is predominantly expressed in muscle and lymphoid tissues. The short form, FLIPS, contains two death effector domains and is structurally related to the viral FLIP inhibitors of apoptosis, whereas the long form, FLIPL, contains in addition a caspase-like domain in which the active-centre cysteine residue is substituted by a tyrosine residue. FLIPS and FLIPL interact with the adaptor protein FADD, and the protease FLICE,, and potently inhibit apoptosis induced by all known human death receptors. FLIPL is expressed during the early stage of T-cell activation, but disappears when T cells become susceptible to Fas ligand-mediated apoptosis. High levels of FLIPL protein are also detectable in melanoma cell lines and malignant melanoma tumours. Thus FLIP may be implicated in tissue homeostasis as an important regulator of apoptosis.

A role for mitochondria in NLRP3 inflammasome activation

An inflammatory response initiated by the NLRP3 inflammasome is triggered by a variety of situations of host ‘danger’, including infection and metabolic dysregulation. Previous studies suggested that NLRP3 inflammasome activity is negatively regulated by autophagy and positively regulated by reactive oxygen species (ROS) derived from an uncharacterized organelle. Here we show that mitophagy/autophagy blockade leads to the accumulation of damaged, ROS-generating mitochondria, and this in turn activates the NLRP3 inflammasome. Resting NLRP3 localizes to endoplasmic reticulum structures, whereas on inflammasome activation both NLRP3 and its adaptor ASC redistribute to the perinuclear space where they co-localize with endoplasmic reticulum and mitochondria organelle clusters. Notably, both ROS generation and inflammasome activation are suppressed when mitochondrial activity is dysregulated by inhibition of the voltage-dependent anion channel. This indicates that NLRP3 inflammasome senses mitochondrial dysfunction and may explain the frequent association of mitochondrial damage with inflammatory diseases.

Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.

Antiviral immunity against a pathogen is mounted upon recognition by the host of virally associated structures. One of these viral ‘signatures’, double-stranded (ds) RNA, is a replication product of most viruses within infected cells and is sensed by Toll-like receptor 3 (TLR3) and the recently identified cytosolic RNA helicases RIG-I (retinoic acid inducible gene I, also known as Ddx58) and Mda5 (melanoma differentiation-associated gene 5, also known as Ifih1 or Helicard). Both helicases detect dsRNA, and through their protein-interacting CARD domains, relay an undefined signal resulting in the activation of the transcription factors interferon regulatory factor 3 (IRF3) and NF-κB. Here we describe Cardif, a new CARD-containing adaptor protein that interacts with RIG-I and recruits IKKα, IKKβ and IKKɛ kinases by means of its C-terminal region, leading to the activation of NF-κB and IRF3. Overexpression of Cardif results in interferon-β and NF-κB promoter activation, and knockdown of Cardif by short interfering RNA inhibits RIG-I-dependent antiviral responses. Cardif is targeted and inactivated by NS3-4A, a serine protease from hepatitis C virus known to block interferon-β production. Cardif thus functions as an adaptor, linking the cytoplasmic dsRNA receptor RIG-I to the initiation of antiviral programmes.

Induction of TNF Receptor I-Mediated Apoptosis via Two Sequential Signaling Complexes

Apoptosis induced by TNF-receptor I (TNFR1) is thought to proceed via recruitment of the adaptor FADD and caspase-8 to the receptor complex. TNFR1 signaling is also known to activate the transcription factor NF-kappa B and promote survival. The mechanism by which this decision between cell death and survival is arbitrated is not clear. We report that TNFR1-induced apoptosis involves two sequential signaling complexes. The initial plasma membrane bound complex (complex I) consists of TNFR1, the adaptor TRADD, the kinase RIP1, and TRAF2 and rapidly signals activation of NF-kappa B. In a second step, TRADD and RIP1 associate with FADD and caspase-8, forming a cytoplasmic complex (complex II). When NF-kappa B is activated by complex I, complex II harbors the caspase-8 inhibitor FLIP(L) and the cell survives. Thus, TNFR1-mediated-signal transduction includes a checkpoint, resulting in cell death (via complex II) in instances where the initial signal (via complex I, NF-kappa B) fails to be activated.

Innate Immune Activation Through Nalp3 Inflammasome Sensing of Asbestos and Silica

The inhalation of airborne pollutants, such as asbestos or silica, is linked to inflammation of the lung, fibrosis, and lung cancer. How the presence of pathogenic dust is recognized and how chronic inflammatory diseases are triggered are poorly understood. Here, we show that asbestos and silica are sensed by the Nalp3 inflammasome, whose subsequent activation leads to interleukin-1β secretion. Inflammasome activation is triggered by reactive oxygen species, which are generated by a NADPH oxidase upon particle phagocytosis. (NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate.) In a model of asbestos inhalation, Nalp3–/– mice showed diminished recruitment of inflammatory cells to the lungs, paralleled by lower cytokine production. Our findings implicate the Nalp3 inflammasome in particulate matter–related pulmonary diseases and support its role as a major proinflammatory “danger” receptor.

Fas triggers an alternative, caspase-8–independent cell death pathway using the kinase RIP as effector molecule

Cell death is achieved by two fundamentally different mechanisms: apoptosis and necrosis. Apoptosis is dependent on caspase activation, whereas the caspase-independent necrotic signaling pathway remains largely uncharacterized. We show here that Fas kills activated primary T cells efficiently in the absence of active caspases, which results in necrotic morphological changes and late mitochondrial damage but no cytochrome c release. This Fas ligand–induced caspase-independent death is absent in T cells that are deficient in either Fas-associated death domain (FADD) or receptor-interacting protein (RIP). RIP is also required for necrotic death induced by tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL). In contrast to its role in nuclear factor κB activation, RIP requires its own kinase activity for death signaling. Thus, Fas, TRAIL and TNF receptors can initiate cell death by two alternative pathways, one relying on caspase-8 and the other dependent on the kinase RIP.

NALP3 Forms an IL-1β-Processing Inflammasome with Increased Activity in Muckle-Wells Autoinflammatory Disorder

Mutations within the NALP3/cryopyrin/CIAS1 gene are responsible for three autoinflammatory disorders: Muckle-Wells syndrome, familial cold autoinflammatory syndrome, and CINCA. The NALP3 protein is homologous to NALP1, which is a component of the inflammasome, a molecular platform that activates the proinflammatory caspases-1 and -5. NALP3 (and other members of the NALP family) lacks the C-terminal, CARD-containing sequence of NALP1, and its role in caspase activation is unclear. Here, we report that NALP2 and NALP3 associate with ASC, the CARD-containing protein Cardinal, and caspase-1 (but not caspase-5), thereby forming an inflammasome with high proIL-1beta-processing activity. Macrophages from Muckle-Wells patients spontaneously secrete active IL-1beta. Increased inflammasome activity is therefore likely to be the molecular basis of the symptoms associated with NALP3-dependent autoinflammatory disorders.

Melanoma cell expression of Fas(Apo-1/CD95) ligand : Implications for tumor Immune escape

Malignant melanoma accounts for most of the increasing mortality from skin cancer. Melanoma cells were found to express Fas (also called Apo-1 or CD95) ligand (FasL). In metastatic lesions, Fas-expressing T cell infiltrates were proximal to FasL+ tumor cells. In vitro, apoptosis of Fas-sensitive target cells occurred upon incubation with melanoma tumor cells; and in vivo, injection of FasL+ mouse melanoma cells in mice led to rapid tumor formation. In contrast, tumorigenesis was delayed in Fas-deficient lpr mutant mice in which immune effector cells cannot be killed by FasL. Thus, FasL may contribute to the immune privilege of tumors.