Cell Death Triggered by Yersinia enterocolitica Identifies Processing of the Proinflammatory Signal Adapter MyD88 as a General Event in the Execution of Apoptosis This information is current as of September 30, 2021. Lena Novikova, Nicole Czymmeck, Anne Deuretzbacher, Friedrich Buck, Kathleen Richter, Alexander N. R. Weber, Martin Aepfelbacher and Klaus Ruckdeschel J Immunol published online 20 December 2013 http://www.jimmunol.org/content/early/2013/12/20/jimmun Downloaded from ol.1203464 References This article cites 40 articles, 15 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/early/2013/12/20/jimmunol.1203464.ful l#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 30, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 20, 2013, doi:10.4049/jimmunol.1203464 The Journal of Immunology Cell Death Triggered by Yersinia enterocolitica Identifies Processing of the Proinflammatory Signal Adapter MyD88 as a General Event in the Execution of Apoptosis Lena Novikova,* Nicole Czymmeck,* Anne Deuretzbacher,*,† Friedrich Buck,‡ Kathleen Richter,x,{ Alexander N. R. Weber,‖ Martin Aepfelbacher,* and Klaus Ruckdeschel*,x Many pathogenic microorganisms have evolved tactics to modulate host cell death or survival pathways for establishing infection. The enteropathogenic bacterium Yersinia enterocolitica deactivates TLR-induced signaling pathways, which triggers apoptosis in macrophages. In this article, we show that Yersinia-induced apoptosis of human macrophages involves caspase-dependent cleavage of the TLR adapter protein MyD88. MyD88 was also cleaved when apoptosis was mediated by overexpression of the Toll–IL-1R Downloaded from domain–containing adapter inducing IFN-b in epithelial cells. The caspase-processing site was mapped to aspartate-135 in the central region of MyD88. MyD88 is consequently split by caspases in two fragments, one harboring the death domain and the other the Toll–IL-1R domain. Caspase-3 was identified as the protease that conferred the cleavage of MyD88 in in vitro caspase assays. In line with a broad role of caspase-3 in the execution of apoptosis, the processing of MyD88 was not restricted to Yersinia infection and to proapoptotic Toll–IL-1R domain–containing adapter inducing IFN-b signaling, but was also triggered by staur- osporine treatment. The cleavage of MyD88 therefore seems to be a common event in the advanced stages of apoptosis, when http://www.jimmunol.org/ caspase-3 is active. We propose that the processing of MyD88 disrupts its scaffolding function and uncouples the activation of TLR and IL-1Rs from the initiation of proinflammatory signaling events. The disruption of MyD88 may consequently render dying cells less sensitive to proinflammatory stimuli in the execution phase of apoptosis. The cleavage of MyD88 could therefore be a means of conferring immunogenic tolerance to apoptotic cells to ensure silent, noninflammatory cell demise. The Journal of Immunology, 2014, 192: 000–000. oll-like receptors are transmembrane receptors that play typic TIR–TIR domain interaction (3). MyD88 is a central TIR an essential role in the recognition of foreign, invading adapter that is implicated in TLR and IL-1R signaling (4). It is by guest on September 30, 2021 T microbial pathogens. They are activated by conserved, activated by the IL-1R family and by all TLRs with the exception pathogen-associated molecular patterns and trigger the induction of of TLR3. In contrast, TLR3, stimulated by microbial dsRNA, protective proinflammatory signaling pathways (1, 2). The human signals via the Toll–IL-1R domain–containing adapter inducing TLR family comprises . 10 TLRs, each detecting a specific set of IFN-b (TRIF). TRIF is recruited also by TLR4, which is the sole ligands. LPS from Gram-negative bacteria, for instance, activates TLR that can engage both the MyD88 and TRIF pathways. The TLR4, whereas bacterial lipoproteins signal through TLR2. The TIR adapter protein–dependent signals converge on NF-kB and TLR-triggered effects depend on the activation of signaling cas- MAPK activation, which mounts the inflammatory response. Mean- cades that are initiated by the recruitment of intracytoplasmic while, it is well established that the TLRs, in addition to promoting adapter proteins to the Toll–IL-1R (TIR) domain through homo- inflammation, are also implicated in controlling cellular life and death. Hence, the engagement of TLRs may enable cell survival, but may also elicit cell death through either apoptosis or necrosis *Institute for Medical Microbiology, Virology and Hygiene, University Medical (5, 6). The signaling networks that control the differential out- † Center Eppendorf, 20246 Hamburg, Germany; Division of Hematology, Department come are not yet completely understood. However, it seems that of Internal Medicine II, University Hospital of Wu¨rzburg, 97080 Wu¨rzburg, Ger- many; ‡Institute of Clinical Chemistry, University Medical Center Eppendorf, 20246 the type of cell, as well as the composition of the signals induced x Hamburg, Germany; Max von Pettenkofer-Institute for Hygiene and Medical Mi- by the microbial challenge, determines the fate of the stimulated crobiology, 80336 Munich, Germany; {Stiefel/GlaxoSmithKline Early Development, ‖ Durham, NC 27709; and Department of Immunology, University of Tu¨bingen, cell. 72076 Tu¨bingen, Germany Several pathogenic bacteria have evolved mechanisms to mod- Received for publication December 19, 2012. Accepted for publication November ify TLR signaling pathways for establishing infection (7, 8). One 20, 2013. example is given by the Gram-negative bacterial pathogen Yersi- This work was supported by Deutsche Forschungsgemeinschaft Grants Ru788/3-1, 3-2. nia, which deregulates TLR-induced immune responses by en- Address correspondence and reprint requests to Dr. Klaus Ruckdeschel, Institute for gaging Yersinia outer protein (Yop) P in Y. enterocolitica, or the Medical Microbiology, Virology and Hygiene, University Medical Center Eppendorf, homologous YopJ protein in Y. pestis and Y. pseudotuberculosis. Martinistrasse 52, 20246 Hamburg, Germany. E-mail address: [email protected] YopP/YopJ is injected with several other Yops by Yersinia type III Abbreviations used in this article: DD, death domain; HEK, human embryonic kid- ney; IKK, inhibitory kB kinase; IRAK, IL-1R–associated kinase; MOI, multiplicity protein secretion into host cells to modify critical signaling path- of infection; PVDF, polyvinylidene difluoride; TAK1, TGF-b–activated kinase-1; ways of innate immunity (9, 10). YopP/YopJ acts as an acetyltrans- TIR, Toll–IL-1R; TRIF, Toll–IL-1R domain–containing adapter inducing IFN-b; ferase on the NF-kB–activating IkB kinase (IKK) b and on mem- Yop, Yersinia outer protein. bers of the MAPK kinase superfamily (11, 12). The acetylation of Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 these kinases deactivates the NF-kB and MAPK pathways and www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203464 2 TARGETING AND REPROGRAMMING OF MyD88 IN APOPTOSIS downregulates the proinflammatory host cell response. Because micin (100 mg/ml) after 90 min of infection to prevent bacterial over- these pathways also promote cell survival, the action of YopP/YopJ growth. Staurosporine (Sigma-Aldrich, Munich, Germany) was applied sensitizes macrophages to undergo apoptosis following Yersinia to the cells at a concentration of 5 mM. Where indicated, the cells were pretreated with 40 mM of the caspase inhibitor zVAD-fmk (Bachem, infection (13, 14). The proapoptotic response thereby is accelerated Bubendorf, Switzerland) 30 min prior to infection. by the activation of TLR4 and TRIF, whereas the MyD88 pathway Eukaryotic expression plasmids, cell transfection, and seems to be dispensable for Yersinia-induced cell death (15, 16). measurement of NF-kB activation TRIF is connected to the cell death machinery by the adapter’s receptor-interacting protein 1 and Fas-associated death domain Transfections of HEK293 cells were performed by the calcium phosphate (DD) to initiate apoptosis through caspase-8 (16–18). The signal- method using ProFection according to the manufacturer’s instructions (Promega, Mannheim, Germany). For the detection of transiently over- ing of apoptosis following TLR4 or TLR3 activation seems to be expressed Myc-tagged MyD88 (24), the cells were transfected with 3 mg a prominent function of TRIF, which is substantiated by the finding expression plasmid in 6-cm cell culture dishes. Where indicated, plas- that the overexpression of TRIF significantly compels transfected mids for TRIF (25), the caspase-8 inhibitor CrmA (26), or IL-1R–as- cells to undergo apoptosis (16–18). sociated kinase (IRAK) 4 (27) were included.