Jürg Tschopp, Full Professor

Apoptosis is a naturally occurring process of cell death. All mammalian cells constitutively express the basic machinery that mediates apoptotic cell death, including a family of cysteine proteases, designated the caspases. Pro-apoptotic caspases are generally activated by death-receptors or damaged mitochondria and are inhibited by a number of cytoplasmic proteins including the caspase-8 homologue FLIP. Modulators of caspase activation are aberrantly expressed in pathological processes such as neurodegenerative diseases or cancer.

In contrast to caspase-8, the inflammatory caspase-1 is not involved in apoptosis but in the proteolytic activation of IL-1β, which triggers the rapidly acting innate immune system. Caspase-1 is activated by a complex, called inflammasome, which comprises NALP3, ASC and caspase-1. The importance of the NALP3 inflammasome in the process of inflammation is underscored by the observation that mutations of the NALP3 gene are associated with several autoinflammatory diseases. While the inflammasome is activated by bacteria and danger signals such as uric acid crystals, another cytoplasmic complex, called PIDDosome, detects DNA damage and orchestrates DNA repair. If unsuccessful, PIDD, the scaffolding protein of the PIDDosome, recruits caspase-2 and initiates cell death. Yet another cytoplasmic complex which is studied in our group is formed by RIG-I and Cardif. RIG-I-Cardif senses the presence of viral RNA and triggers an innate anti-viral response including the synthesis of type I interferon.

Our goal is to understand the signaling networks that control these inflammatory and apoptotic responses to pathogens and danger signals. We believe that this will provide important insights into the genesis of various human diseases.

The inflammasome: A platform sensing PAMPs and danger-associated molecules triggering innate immunity

The inflammasome is a multiprotein complex responsible for the activation of caspase-1 and -5, thereby leading to the activation of the pro-inflammatory cytokines IL-1β and IL-18. Our group identified two types of inflammasomes; the NALP1-inflammasome, which is composed of NALP1/ASC/Caspase-1/Caspase-5 and the NALP2/3-inflammasomes that contain, in addition to NALP2 or NALP3, CARDINAL/ASC/Caspase-1 (Fig. 1). An alternative inflammasome is formed by IPAF that directly recruits caspase-1 without the need of an adaptor protein.

Figure 1: The inflammasomes
Structural organization of the typical NALP3 and IPAF inflammasomes. The core structure of the NALP3 inflammasome is formed by NALP3, the adaptor ASC, and caspase-1 (left panel). IPAF recruits caspase-1 directly via CARD-CARD interactions (right panel). The LRR of NALP3 or IPAF sense the activating signals leading to the oligomerization of the NACHT region and initiating the formation of the donut-shaped inflammasome. Based on the structure of the apoptosome, the caspases and IL-1β-processing activity most likely face the inside of the donut (lower panel).

The NALPs and IPAF are central proteins in the inflammasome complex. They belong to the NLR (NOD-like receptors) family of cytoplasmic proteins. Fourteen NALP proteins have been identified in humans. The role of most of these proteins remains to be determined. Little is also known about the natural stimuli that lead to the assembly and activation of the inflammasomes. Similar to Toll-like receptors, activation of the inflammasome is thought to occur through the recognition of pathogen-associated patterns (PAMPs) by the Leucine-Rich Repeats (LRR) present in the NALP proteins. For example muramyl dipeptide (MDP), a component of peptidoglycans, which is a cell wall component present in both Gram-positive and Gram-negative bacteria, activates the inflammasome.

While inflammasomes are emerging more and more as key players of the inflammatory and immune responses, a growing number of studies also reveals their function in the sensing of a controversial signal in immunology: danger. Indeed, activation of the NALP3 inflammasome is also induced by endogenous, microbe-independent stress signals. For instance, the exposure of macrophages to ATP, monosodiumurate (MSU) crystals or asbestos induces strong activation of caspase-1 in a NALP3 inflammasome-dependent manner.

Inflammasome and diseases

Mutations in the gene coding for NALP3 have been associated with several autoinflammatory disorders such as Muckle-Wells syndrome, familial cold urticaria and CINCA (Chronic Infantile Neurological Cutaneous and Articular autoinflammatory disease). These disorders are characterized by recurrent episodes of fever and serosal inflammation, due to increased production of IL-1β. Based on the discovery of the function of the inflammasome, patients are now successfully treated with the natural IL-1 inhibitor IL-1ra (Anakinra). 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 (Fig. 2).

Although MSU crystals were first identified as the etiologic agent of gout in the 18th century and more recently as a "danger signal" released from dying cells, little is known concerning the molecular mechanisms underlying MSU- or CPPD-induced inflammation. We found that MSU and CPPD engage the NALP3 inflammasome, resulting in the production of active 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 further support a pivotal role of the inflammasome in several autoinflammatory diseases.

The RIG-I/CARDIF platform: Sensing viruses and activating type I interferons

Innate immunity against a pathogen is mounted upon recognition by the host of, for example, viral products. 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 and MDA5. Both helicases detect dsRNA, and through their protein-interacting CARD domains, relay a poorly defined signal resulting in the activation of the transcription factors interferon regulatory factor 3 (IRF3) and NF-κB. We have recently identified Cardif (also known as IPS-1, MAVS or VISA), a new CARD-containing adaptor protein that interacts with RIG-I and recruits IKKα, IKKβ and IKKε 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 inhibits RIG-I-dependent antiviral responses. Cardif is localized to mitochondria and 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.

Figure 3: Overview of TLR- and RIG-I-dependent antiviral pathways (Left) Upon ssRNA or dsRNA binding within endosomes, TLR7/8 and TLR3, respectively, recruit the adaptors MyD88 or Trif through a TIR-TIR interaction. MyD88 recruits IRAKs through DD-DD interactions, and also TRAF3 and TRAF6, to activate IRF and NF-kB. Trif recruits RIP1 through a RHIM-RHIM interaction to activate NF-κB. To activate IRF, Trif recruits TBK1 and possibly TRAF3. (Right) Upon dsRNA binding, RIG-I (and also MDA5) recruits the mitochondrial-bound adaptor Cardif, which in turn recruits appropriate IKKs to activate NF-κB and IRF. During an infection with HCV, both Trif and Cardif are cleaved by the HCV NS3-4A protease. As a consequence, Trif cannot recruit TBK1, which prevents IRF activation and type-I interferon production.

TNF receptor and Cardif-induced death and survival signals are similar

Binding of TNF to the TNFR1 results in the generation of two opposite signals leading to NF-kB activation and apoptosis.

The initial plasma membrane-bound complex (complex I) consists of TNFR1, TRADD, RIP1 and TRAF2, and rapidly signals activation of the transcription factor NF-κB (Fig. 4). In this complex, TRADD and RIP1 undergo important posttranlational modifications and subsequently dissociate from the receptor. In a second step, TRADD and RIP1 associate with FADD and caspase-8, thereby forming the cytoplasmic complex II. In surviving cells where NF-kB is activated by complex I, complex II harbors the caspase-8 inhibitor FLIPL. In apoptosis-sensitive, NF-κB signal-defective cells, substantial amounts of caspase-10 are found in complex II while FLIPL levels are highly reduced. Thus, TNFR1-triggered signal transduction includes a check-point, resulting in cell death (via signal complex II) in instances where the initial signal (via complex I, NF-κB) fails to be activated.

Figure 4: A schematic overview of the signaling pathways triggered by RLH-Cardif and TNFRI. Viral RNA binds to RIG-I or MDA5, which transmit the downstream signals via Cardif to activate IRF3 and NF-κB. A central signaling unit is the TRADDosome which consists of TRADD-RIP1 and FADD and which is also crucial for TNFR1-mediated proinflammatory responses.

Upon detection of viral RNA, the helicases RIG-I and/or MDA5 trigger, via their adaptor Cardif, the activation of the transcription factors NF-κB and IRF3, which collaborate to induce an antiviral type I interferon response (see above). Interestingly, FADD, TRADD and RIP1 are also implicated in the antiviral pathway triggered by Cardif and RIG-I (see above). TRADD is recruited to Cardif and orchestrates complex formation with the E3 ubiquitin ligase TRAF3 and TANK, and with FADD and RIP1, leading to the activation of IRF3 and NF-kB. Loss of TRADD prevents Cardif-dependent activation of interferon-b, reduces the production of interferon-β in response to RNA viruses, and enhances vesicular stomatitis virus replication. Thus, TRADD is not only an essential component of pro-inflammatory TNFRI signaling, but is also required for RLH-Cardif-dependent antiviral immune responses.

The PIDDosome: Detecting DNA damage and activating caspase-2

Activation of initiatior caspases is a key event in apoptosis execution. Prototypically, activation is triggered upon complex-mediated clustering of the inactive zymogen such as in the caspase-9-activating apoptosome complex. Likewise, caspase-2, which is involved in stress-induced apoptosis, is recruited into a large protein complex that contains the death-domain containing protein PIDD. Increased amounts of PIDD expression result in spontaneous activation of caspase-2 and sensitization to apoptosis by genotoxic stimuli. Because PIDD functions in p53-mediated apoptosis, the complex assembled by PIDD and caspase-2 is likely to have a crucial role in the regulation of apoptosis induced by genotoxins. We found that PIDD also plays a critical role in DNA damage-induced NF-κB activation. Upon genotoxic stress, a complex between PIDD, RIP1 and NEMO is formed. Cells stably expressing PIDD show enhanced genotoxic stress-induced NF-κB activation, through augmented sumoylation and ubiquitination of NEMO. Knock-down of PIDD and RIP1 expression abrogates DNA damage-induced NEMO-modification and hence NF-κB activation.

PIDD is constitutively processed giving rise to a 48-kDa N-terminal fragment and a 51-kDa C-terminal fragment (PIDD-C). The latter undergoes further cleavage resulting in a 37-kDa fragment (PIDD-CC). Processing occurs at S446 (generating PIDD-C) and S588 (generating PIDD-CC) by an auto-processing mechanism. Auto-cleavage of PIDD determines the outcome of the downstream signaling events. Whereas initially formed PIDD-C mediates the activation of NF-κB via the recruitment of RIP1 and NEMO, subsequent formation of PIDD-CC causes caspase-2 activation and thus cell death. In this way, auto-proteolysis of PIDD might participate in the orchestration of the DNA damage-induced life and death signaling pathways (Fig. 5).

Figure 5: PIDD acts as a molecular switch, controlling the balance
between life and death upon DNA damage.

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• Muruve, D.A., Petrilli, V., Zaiss, A.K., White, L.R., Clark, S.A, Ross, J., Parks, R.A., Tschopp, J. (2008). The inflammasome recognizes cytosolic microbial and host DNA and triggers an innate immune response. Nature, 452, 103-107. PubMed
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• Park, H.H., Logette, E., Raunser, S., Cuenin, S., Walz, T., Tschopp, J., Wu, H. (2007). Death Domain Assembly Mechanism Revealed by Crystal Structure of the Oligomeric PIDDosome Core Complex. Cell 128, 533-546. PubMed
• Mayor, A., Martinon, F., De Smedt, T., Petrilli, V., Tschopp, J. (2007). A crucial function of SGT1 and HSP90 in inflammasome activity links mammalian and plant innate immune responses. Nat. Immunol. 8, 497-503. PubMed
• Papin, S., Cuenin, S., Agostini, L., Martinon, F., Werner, S., Beer, H., Grütter, C., Grütter, M., Tschopp, J. (2007). The SPRY domain of Pyrin, mutated in familial Mediterranean fever patients, interacts with inflammasome components and inhibits proIL-1beta processing. Cell Death Differ. 14, 1457-66. PubMed
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• Tinel, A., Janssens, S., Lippens, S., Cuenin, S., Logette, E., Jaccard, B., Quadroni, M., Tschopp, J. (2007). Autoproteolysis of PIDD marks the bifurcation between pro-death caspase-2 and pro-survival NF-kappaB pathway. EMBO J. 26, 197-208. PubMed
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• Kummer, J.A., Micheau, O., Schneider, P., Bovenschen, N., Broekhuizen, R., Quadir, R., Strik, M.C.M., Hack, C. E., Tschopp, J. (2007). Ectopic expression of the serine protease inhibitor PI9 modulates death receptor-mediated apoptosis. Cell Death and Differentiation. 14, 1486-1496. PubMed
• Grutter, C., Briand, C., Capitani, G., Mittl, P.R., Papin, S., Tschopp, J., Grutter, M.G. (2006). Structure of the PRYSPRY-domain: Implications for autoinflammatory diseases. FEBS Lett. 580, 99-106. PubMed
• Martinon, F., Petrilli, V., Mayor, A., Tardivel, A., Tschopp, J. (2006). Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440, 237-241. PubMed
• Brissoni, B., Agostini, L., Kropf, M., Martinon, F., Swoboda, V., Lippens, S., Everett, H., Aebi, N., Janssens, S., Meylan, E., Felberbaum-Corti, M., Hirling, H., Gruenberg, J., Tschopp, J., Burns, K. (2006). Intracellular trafficking of interleukin-1 receptor I requires Tollip. Curr. Biol. 16, 2265-2270. PubMed
• Didierlaurent, A., Brissoni, B., Velin, D., Aebi, N., Tardivel, A., Kaslin, E., Sirard, J. C., Angelov, G., Tschopp, J., Burns, K. (2006). Tollip regulates proinflammatory responses to interleukin-1 and lipopolysaccharide. Mol. Cell. Biol. 26, 735-742. PubMed
• Gurcel, L., Abrami, L., Girardin, S., Tschopp, J., van der Goot, F.G. (2006). Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival. Cell 126, 1135-1145. PubMed
• Munding, C., Keller, M., Niklaus, G., Papin, S., Tschopp, J., Werner, S., Beer, H. D. (2006). The estrogen-responsive B box protein: a novel enhancer of interleukin-1beta secretion. Cell Death Differ. 13, 1938-1949. PubMed
• Budd, R.C., Yeh, W.C., Tschopp, J. (2006). cFLIP regulation of lymphocyte activation and development. Nat. Rev. Immunol. 6, 196-204. PubMed
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• Dohrman, A., Kataoka, T., Cuenin, S., Russell, J.Q., Tschopp, J., Budd, R.C. (2005). Cellular FLIP (long form) regulates CD8+ T cell activation through caspase-8-dependent NF-kappa B activation. J. Immunol. 174, 5270-5278. PubMed
• Ingold, K., Zumsteg, A., Tardivel, A., Huard, B., Steiner, Q.G., Cachero, T.G., Qiang, F., Gorelik, L., Kalled, S.L., Acha-Orbea, H., Rennert, P.D., Tschopp, J., Schneider, P. (2005). Identification of proteoglycans as the APRIL-specific binding partners. J. Exp. Med. 201, 1375-1383. PubMed
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• Tinel, A., and Tschopp, J. (2004). The PIDDosome, a Protein Complex Implicated in Activation of Caspase-2 in Response to Genotoxic Stress. Science 304, 843-846. PubMed
• Burns, K., Janssens, S., Brissoni, B., Olivos, N., Beyaert, R., Tschopp, J. (2003). Inhibition of Interleukin 1 Receptor/Toll-like Receptor Signaling through the Alternatively Splices, Short Form of MyD88 Is Due to Its Failure to Recruit IRAK-4. J. Exp. Med. 197, 263-268. PubMed
• Jaattela, M., Tschopp, J. (2003). Caspase-independent cell death in T lymphocytes. Nat. Immunol. 4, 416-423. PubMed
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• Hiller, S., Kohl, A., Fiorito, F., Herrmann, T., Wider, G., Tschopp, J., Grutter, M. G., Wuthrich, K. (2003). NMR structure of the apoptosis- and inflammation-related NALP1 pyrin domain. Structure 11, 1199-1205. PubMed