ASC Controls IFN-γ Levels in an IL-18− Dependent Manner in Caspase-1−Deficient Mice Infected with Francisella novicida This information is current as Roberto Pierini, Magali Perret, Sophia Djebali, Carole Juruj, of September 27, 2021. Marie-Cécile Michallet, Irmgard Förster, Jacqueline Marvel, Thierry Walzer and Thomas Henry J Immunol 2013; 191:3847-3857; Prepublished online 23 August 2013; doi: 10.4049/jimmunol.1203326 Downloaded from http://www.jimmunol.org/content/191/7/3847 Supplementary http://www.jimmunol.org/content/suppl/2013/08/23/jimmunol.120332 Material 6.DC1 http://www.jimmunol.org/ References This article cites 60 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/191/7/3847.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 27, 2021 • No Triage! 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The Journal of Immunology ASC Controls IFN-g Levels in an IL-18–Dependent Manner in Caspase-1–Deficient Mice Infected with Francisella novicida Roberto Pierini,*,†,‡,x Magali Perret,*,†,‡,x Sophia Djebali,*,†,‡,x Carole Juruj,*,†,‡,x Marie-Ce´cile Michallet,*,†,‡,x Irmgard Fo¨rster,{,1 Jacqueline Marvel,*,†,‡,x Thierry Walzer,*,†,‡,x and Thomas Henry*,†,‡,x The inflammasome is a signaling platform that is central to the innate immune responses to bacterial infections. Francisella tularensis is a bacterium replicating within the host cytosol. During F. tularensis subspecies novicida infection, AIM2, an inflam- masome receptor sensing cytosolic DNA, activates caspase-1 in an ASC-dependent manner, leading to both pyroptosis and release of the proinflammatory cytokines IL-1b and IL-18. Activation of this canonical inflammasome pathway is key to limit F. novicida Downloaded from infection. In this study, by comparing the immune responses of AIM2 knockout (KO), ASCKO, and Casp1KO mice in response to F. novicida infection, we observed that IFN-g levels in the serum of Casp1KO mice were much higher than the levels observed in AIM2KO and ASCKO mice. This difference in IFN-g production was due to a large production of IFN-g by NK cells in Casp1KO mice that was not observed in ASCKO mice. The deficit in IFN-g production observed in ASCKO mice was not due to a reduced Dock2 expression or to an intrinsic defect of ASCKO NK cells. We demonstrate that in infected Casp1KO mice, IFN-g production is due to an ASC-dependent caspase-1–independent pathway generating IL-18. Furthermore, we present in vitro data suggesting http://www.jimmunol.org/ that the recently described AIM2/ASC/caspase-8 noncanonical pathway is responsible for the caspase-1–independent IL-18 releasing activity. To our knowledge, this study is the first in vivo evidence of an alternative pathway able to generate in a caspase-1–independent pathway bioactive IL-18 to boost the production of IFN-g, a cytokine critical for the host antibacterial response. The Journal of Immunology, 2013, 191: 3847–3857. he inflammasome (1) is an innate immune platform ac- masome receptor detecting DNA within the host cytosol (2–5). We tivated in response to danger signals and infections and and others have recently described that the AIM2 inflammasome T leading to caspase-1 activation. Caspase-1 is an inflam- is activated following infections with “cytosolic” bacteria such as by guest on September 27, 2021 matory caspase leading to the processing of pro–IL-1b and pro–IL- Listeria monocytogenes and Francisella tularensis (6–9). 18 and the release of the corresponding cytokines. Furthermore, F. tularensis is a highly infectious bacterium that causes tula- caspase-1 also triggers cell death in a process termed pyroptosis. remia in humans (10). Francisella novicida (also known as F. Caspase-1 activation takes place within a multiprotein complex, the tularensis subspecies novicida) is considered nonpathogenic for inflammasome, which includes a receptor, and an adaptor, ASC. humans and is used as a model to study highly virulent F. tular- Several inflammasomes have been described depending on the ensis infections. In mice, F. novicida is found mostly in myeloid detected ligand and on the receptor involved. AIM2 is an inflam- cells such as macrophages and neutrophils (11). Its ability to cause disease is tightly linked to its ability to rapidly escape from the phagosome into the host cytosol where it can replicate to very high *Centre International de Recherche en Infectiologie, Universite´ de Lyon, Lyon numbers (12, 13). 69007, France; †INSERM, Unite´ 851, Lyon 69007, France; ‡Centre National de la Recherche Scientifique, Unite´ Mixte de Recherche 5308, Lyon 69007, France; xEcole Infection with wild-type (WT) F. novicida leads to the activation Normale Supe´rieure de Lyon, Lyon 69342, France; and {Department of Molecular of the AIM2 inflammasome (7–9). Mice deficient for AIM2, ASC, Immunology, Leibniz Research Institute for Environmental Medicine, 40225 Du¨ssel- or for both caspase-1 and caspase-11 (14) are very susceptible to F. dorf, Germany novicida infection. This high susceptibility of ASC knockout (KO) 1Current address: Immunology and Environment, Life and Medical Sciences Insti- tute, University of Bonn, Bonn, Germany. and Casp1/11 double KO mice is due to both caspase-1–mediated Received for publication December 4, 2012. Accepted for publication July 24, 2013. control of IL-1b and IL-18 and to caspase-1–mediated cell death (15–17). In contrast to other intracellular bacteria, detection of This work was supported by Marie Curie International Reintegration Grant PIRG07- GA-2010-268399 and by a Fondation Innovations en Infectiologie Young Inves- F. novicida by the inflammasome in murine cells seems to be ex- tigator grant. This work was performed within the framework of the Laboratoire clusively dependent on AIM2 (8). F. novicida infection is thus d’Excellence eco-evolutionary dynamics of infectious diseases (ECOFECT Grant ANR- 11-LABX-0042) of the Universite´ de Lyon, within the program “Investissements d’Avenir” emerging as a very good model to study both in vitro and in vivo this (Grant ANR-11-IDEX-0007) operated by the French National Research Agency. innate immune response (8, 9, 18). Caspase-11 is dispensable for Address correspondence and reprint requests to Dr. Thomas Henry, INSERM, Unite´ caspase-1 activation in response to F. novicida infection (14, 19). 851, 21 Avenue Tony Garnier, Lyon 69007, France. E-mail address: thomas.henry@ The canonical inflammasome pathway leads to caspase-1 acti- inserm.fr vation. However, we and others have shown that the inflammasome The online version of this article contains supplemental material. adaptor ASC could have caspase-1–independent activities. In vitro, Abbreviations used in this article: BMM, bone marrow macrophage; KO, knockout; ASC can recruit and activate apoptotic caspases leading to mac- PI, postinfection; WT, wild-type. rophage apoptosis (18, 20–23). In several noninfectious inflam- Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 matory models, ASC contributes to inflammation in a caspase-1– www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203326 3848 ASC/IL-18–DEPENDENT REGULATION OF IFN-g IN Casp1KO MICE dependent and caspase-1–independent manner (24–26). Similarly, in FACS buffer and Live/Dead fixable dead cell stain kit (Invitrogen) in during chronic Mycobacterium tuberculosis infection in mice, PBS containing 2.5 mM EDTA. Peripheral blood samples were collected ASC participates in granuloma formation mechanisms indepen- in EDTA-containing tubes and incubated for 30 min at 4˚C with mAbs cocktails. dently of caspase-1 (27). Recently, Dock2 expression was shown Phenotyping and analysis of NK cell maturation and function was to be deficient in several but not all ASC-deficient mouse lines, performed as previously described (32, 33). When applicable, spleen leading to a severe immune deficit in ASCKO mouse lines ex- single-cell suspensions were incubated for 4 h with IL-12 (20 ng/ml), IL- pressing low levels of Dock2 protein (28, 29). It is still unclear 12 plus IL-2 (3000 U/ml; PeproTech), IL-12 plus IL-18 (20 mg/ml; MBL International), or on coated Abs anti-Ly49 (4E5) or anti-NKp46 (29A1.4) whether some of the phenotypes described above relate to a deficit in the presence of monensin and anti-CD107a (Lamp-1) Ab (BD Bio- in Dock2. sciences). Cells were analyzed using a FACSCanto II flow cytometer In this study, using F. novicida infection as a model to engage (Becton Dickinson, San Jose, CA). Data were processed using FlowJo the AIM2/ASC inflammasome components, we compared the im- software (Tree Star, Asland, OR). mune responses of WT mice with the ones of mice deficient for RNA and quantitative real-time RT-PCR ASC (ASCKO) or for both caspase-1 and caspase-11 (termed Casp1KO). We found that ASC controls IFN-g secretion by NK Approximately a third of the spleen was ground in 1 ml TRIzol. RNA was extracted using chloroform followed by 70% ethanol-mediated precipitation. cells in a caspase-1–dependent and caspase-1–independent man- RNA was then isolated using the RNeasy Mini kit (Qiagen). Reverse tran- ner. This was independent of Dock2 because our ASCKO mouse scription and quantitative PCR were performed using Improm (Promega) line had normal Dock2 protein levels.