Cutting Edge: Distinct Regulatory Mechanisms Control Proinflammatory Cytokines IL-18 and IL-1β This information is current as Qifan Zhu and Thirumala-Devi Kanneganti of October 1, 2021. J Immunol published online 3 May 2017 http://www.jimmunol.org/content/early/2017/05/03/jimmun ol.1700352 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/05/03/jimmunol.170035 Material 2.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 1, 2021 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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 3, 2017, doi:10.4049/jimmunol.1700352 Th eJournal of Cutting Edge Immunology Cutting Edge: Distinct Regulatory Mechanisms Control Proinflammatory Cytokines IL-18 and IL-1b Qifan Zhu*,† and Thirumala-Devi Kanneganti* Interleukin-18 and IL-1b, which are cytokines of the expressed in murine macrophages, dendritic cells, endothelial IL-1 family, are synthesized as precursor proteins and cells, intestinal epithelial cells, and keratinocytes under steady activated by the inflammasome via proteolytic process- state (8–10). However, IL-1b is not constitutively expressed ing. IL-1b is only induced in response to inflammatory under homeostasis. IL-1b expression is induced in blood stimuli, but IL-18 is constitutively expressed. However, mononuclear cells, macrophages, and dendritic cells during how IL-18 and IL-1b expression is regulated by differ- stimulation with TLR ligands and other cytokines (e.g., TNF) ent inflammatory signals remains poorly studied. In (1, 2, 4). Unlike IL-1b, a cellular pool for IL-18 already exists this study, we found that IL-18 and IL-1b are differ- before an inflammatory stimulus, and is ready to be activated Downloaded from entially regulated. Despite being constitutively expressed, and released by the inflammasome (11). However, it must be IL-18 expression was increased and sustained after stim- noted that LPS-mediated priming of NLRP3 is required for ulation of TLRs. In contrast, IL-1b was induced but not inflammasome activation and subsequent release of both sustained after chronic treatment. Furthermore, type I IL-18 and IL-1b (12). However, several studies suggest that IFN signaling was essential for induction of IL-18 and despite it being constitutively expressed, IL-18 can also be macrophages lacking type I IFN signaling were impaired induced under certain circumstances. Treatment with LPS http://www.jimmunol.org/ in their ability to promote IL-18 induction. Thus, our (TLR4 agonist) has been shown to upregulate the mRNA findings reveal a fundamental difference in IL-18 and levels of Il18 (7, 13). Incubation of CpG oligonucleotides IL-1b regulation and uncover novel mechanisms that (TLR9 agonist) with dendritic cells or infection of monocytes are relevant to the inflammatory settings where these proin- with the Sendai virus can also increase the expression of Il18 flammatory cytokines play a critical role. The Journal (14, 15). of Immunology, 2017, 198: 000–000. The mechanisms by which IL-18 and IL-1b levels are regulated by different inflammatory signals remain unclear. In this study, we showed that expression of IL-18 is induced and by guest on October 1, 2021 nterleukin-18 and IL-1b, members of the IL-1 cytokine that the increased level is sustained during TLR4, TLR2, or family, are important mediators of inflammatory diseases TLR7 ligand stimulation. In contrast, IL-1b expression de- I and play critical roles in infection and cancer (1–4). Unlike other cytokines, cellular IL-18 and IL-1b are synthe- clines soon after reaching its peak. The TLR3 and cyclic GMP–AMP synthase (cGAS)–stimulator of IFN genes (STING) sized as precursor proteins and need to be cleaved to generate b their biologically active forms. Inflammasome, a multimeric pathways induced IL-18 but were modest at inducing IL-1 protein complex, is a central regulator of this process by which expression. Importantly, type I IFN signaling was required to bioactive IL-18 and IL-1b are generated. The inflammasome upregulate IL-18 in response to all stimuli tested. Thus, in comprises an innate immune sensor that includes the nucleotide- macrophages lacking components of type I IFN signaling, binding domain, leucine-rich repeat–containing protein (NLR), IL-18 was not induced by any inflammatory stimuli. Together, AIM2-like receptor, and pyrin; the adaptor protein ASC; and the our findings demonstrate that IL-18 and IL-1b expression are cysteine protease caspase 1 (5). Inflammasome assembly induces differentially regulated. Expression of these inactive proin- the activation of caspase-1, which mediates the proteolytic flammatory cytokines is an essential step for their maturation processing of pro–IL-18 and pro–IL-1b to generate their bio- into bioactive forms by caspase-1 inflammasomes. Overall, our active forms and their release from the cell (5). data demonstrating differential regulation of IL-18 and IL-1b IL-18 is constitutively expressed in the blood monocytes and will be fundamental to understanding several inflammatory intestinal epithelial cells of healthy humans (6, 7). It is also disease settings. *Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN E7057, 262 Danny Thomas Place, Memphis, TN 38105-3678. E-mail address: 38105; and †Integrated Biomedical Sciences Program, University of Tennessee Health [email protected] Science Center, Memphis, TN 38163 The online version of this article contains supplemental material. ORCIDs: 0000-0001-7174-7943 (Q.Z.); 0000-0002-6395-6443 (T.-D.K.). Abbreviations used in this article: BMDM, bone marrow–derived macrophage; cGAMP, Received for publication March 9, 2017. Accepted for publication April 9, 2017. cyclic GMP-AMP; cGAS, cyclic GMP–AMP synthase; IFNAR, IFN-a/b receptor; IRF, IFN regulatory factor; ISG, IFN-stimulated gene; ISGF3, IFN stimulated gene factor 3; This work was supported by grants from the National Institutes of Health (AI101935, NLR, leucine-rich repeat–containing protein; STING, stimulator of IFN genes; TRIF, AI124346, AR056296, and CA163507) and the American Lebanese Syrian Associated TIR-domain–containing adapter-inducing IFN-b; WT, wild type. Charities (to T.-D.K.). Address correspondence and reprint requests to Dr. Thirumala-Devi Kanneganti, De- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 partment of Immunology, St. Jude Children’s Research Hospital, MS #351, Room www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700352 2 CUTTING EDGE: DIFFERENTIAL REGULATION OF IL-18 AND IL-1b Materials and Methods hand, IL-1b was upregulated 4 h after treatment but di- Mice minished dramatically at later time points (Fig. 1B). Gar- 2 2 2 2 2 2 2 2 2 2 diquimod induces the MyD88 pathway downstream of Ifnar1 / (16), Ifnar2 / (17), Irf1 / (18), Irf3 / (19), Irf7 / (18), and 2 2 Irf9 / mice (20) were generated as described previously. C57BL/6J mice TLR7. Induction kinetics of mRNA expression and protein [wild type (WT)], Irf4tm1Rdf/J mice (Stock Number 009380), Irf8 tm1.2Hm/J levels of IL-18 and IL-1b in WT BMDMs treated with 2/2 tm1Ppr mice (also known as Irf8 ; Stock Number 018298) and Irf5 /J mice gardiquimod were similar to those treated with Pam3CSK4 (Stock Number 017311) were purchased from The Jackson Laboratory. Irf4tm1Rdf/J mice and Irf5tm1Ppr/J mice were crossed with mice expressing (Supplemental Fig. 1A). Poly(I:C) is sensed by TLR3 to LsyM-Cre and Cre recombinase, respectively, to generate Irf4fl/fl-Lysm-Cre activate the TRIF pathway. Poly(I:C) treatment induced 2 2 2 2 mice and Irf5 / mice. Stat1 / mice were provided by Dr. A. Satoskar Il18 expression in WT BMDMs. It also induced Il1b ex- (Ohio State University). All mice were bred at the Animal Resource Center at pression, although at a much lower fold induction than by St. Jude Children’s Research Hospital. Animal studies were conducted according to protocols approved by the St. Jude Animal Care and Use LPS, Pam3CSK4, and gardiquimod (Fig. 1C). These find- Committee. ings suggest that the TRIF pathway moderately induces Il1b expression, and both the MyD88 and TRIF pathways in- Cell culture and stimulation duce Il18 expression. Taken together, our results show that Bone marrow–derived macrophages (BMDMs) were prepared as described IL-18 and IL-1b can be induced by various TLR ligands. previously (21). Cells were stimulated with LPS (500 ng/ml; InvivoGen), m m Further, during chronic TLR stimulation, IL-18 expression Pam3CSK4 (1 g/ml), gardiquimod (1 g/ml; InvivoGen), poly(I:C) (10 b mg/ml; InvivoGen), or IFN-b (400 U/ml; PBL Assay Science). For trans- is sustained but IL-1 expression is downregulated. These fection of DNA or 2939-cyclic GMP-AMP (cGAMP), 0.25 mg of poly(dA: datasuggestdistinctregulatorypathwaysthatcontrolthe dT) (InvivoGen), or 1 mgof2939-cGAMP (InvivoGen) was mixed with 0.3 expression of pro–IL-18 and pro–IL-1b levels during chronic Downloaded from ml of Xfect polymer in Xfect reaction buffer (Clontech Laboratories) for 10 min, and then added to BMDMs in Opti-MEM (Thermo Fisher Scientific). TLR stimulation. One of the prominent mediators induced by the TRIF Quantitative real-time PCR pathway are type I IFNs, including IFN-b (23).