IL-18 Drives ILC3 Proliferation and Promotes IL-22 Production via NF- κB Aaron R. Victor, Ansel P. Nalin, Wenjuan Dong, Susan McClory, Min Wei, Charlene Mao, Raleigh D. Kladney, This information is current as Youssef Youssef, Wing Keung Chan, Edward L. Briercheck, of October 3, 2021. Tiffany Hughes, Steven D. Scoville, Jason R. Pitarresi, Charlie Chen, Sarah Manz, Lai-Chu Wu, Jianying Zhang, Michael C. Ostrowski, Aharon G. Freud, Gustavo W. Leone, Michael A. Caligiuri and Jianhua Yu J Immunol 2017; 199:2333-2342; Prepublished online 25 Downloaded from August 2017; doi: 10.4049/jimmunol.1601554 http://www.jimmunol.org/content/199/7/2333 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2017/08/24/jimmunol.160155 Material 4.DCSupplemental References This article cites 76 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/199/7/2333.full#ref-list-1 by guest on October 3, 2021 Why The JI? Submit online. • 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 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. The Journal of Immunology IL-18 Drives ILC3 Proliferation and Promotes IL-22 Production via NF-kB Aaron R. Victor,*,1 Ansel P. Nalin,*,1 Wenjuan Dong,†,1 Susan McClory,*,† Min Wei,† Charlene Mao,† Raleigh D. Kladney,†,‡,x Youssef Youssef,{ Wing Keung Chan,† Edward L. Briercheck,*,† Tiffany Hughes,† Steven D. Scoville,*,† Jason R. Pitarresi,†,‡ Charlie Chen,† Sarah Manz,† Lai-Chu Wu,† Jianying Zhang,‖ Michael C. Ostrowski,†,‡ Aharon G. Freud,†,{ Gustavo W. Leone,†,‡,x Michael A. Caligiuri,†,# and Jianhua Yu†,# Group 3 innate lymphoid cells (ILC3s) are important regulators of the immune system, maintaining homeostasis in the presence of commensal bacteria, but activating immune defenses in response to microbial pathogens. ILC3s are a robust source of IL-22, a cytokine critical for stimulating the antimicrobial response. We sought to identify cytokines that can promote proliferation and induce or maintain IL-22 production by ILC3s and determine a molecular mechanism for this process. We identified IL-18 as Downloaded from a cytokine that cooperates with an ILC3 survival factor, IL-15, to induce proliferation of human ILC3s, as well as induce and maintain IL-22 production. To determine a mechanism of action, we examined the NF-kB pathway, which is activated by IL- 18 signaling. We found that the NF-kB complex signaling component, p65, binds to the proximal region of the IL22 promoter and promotes transcriptional activity. Finally, we observed that CD11c+ dendritic cells expressing IL-18 are found in close proximity to ILC3s in human tonsils in situ. Therefore, we identify a new mechanism by which human ILC3s proliferate and produce IL-22, and identify NF-kB as a potential therapeutic target to be considered in pathologic states characterized by overproduction of IL- http://www.jimmunol.org/ 18 and/or IL-22. The Journal of Immunology, 2017, 199: 2333–2342. nnate lymphoid cells (ILCs) play many roles in protective orphan receptor g isoform b (RORgt) and aryl hydrocarbon re- immunity and disease (1). There are several types of ILCs, ceptor, and they produce the cytokines IL-17A and IL-22 (3–7). I each of which produces a characteristic cytokine profile and ILC3s are enriched within human secondary lymphoid tissues is regulated by unique transcription factors (2). Group 3 ILCs (SLTs), such as the tonsils and lymph nodes (8–10). (ILC3s) are composed of ILC3s and lymphoid tissue–inducer ILC3s are critical regulators of homeostasis and immunity (9– cells. They are dependent on the transcription factors RAR-related 12). ILC3s are the primary steady-state source of IL-22, a cytokine by guest on October 3, 2021 critical for tissue regeneration and for maintenance of barrier func- tion in the gut, skin, oral mucosa, and lung (3, 13). IL-22 signaling *Medical Scientist Training Program, Ohio State University, Columbus, OH 43210; †The James Cancer Hospital and Solove Research Institute, The Ohio State Univer- in epithelial cells drives genes involved in proliferation and wound sity Comprehensive Cancer Center, Columbus, OH 43210; ‡Department of Molecular healing (14, 15). In addition to bolstering the physical barrier of the Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, OH 43210; xDepartment of Molecular Genetics, College of Biological Sciences, The epithelium, IL-22 stimulates epithelial cells to produce antimicro- Ohio State University, Columbus, OH 43210; {Department of Pathology, The Ohio bial peptides necessary for barrier maintenance and prevention of ‖ State University, Columbus, OH 43210; Center for Biostatistics, Department of infection by commensal bacteria (16, 17). ILC3s are also necessary Bioinformatics, The Ohio State University, Columbus, OH 43210; and #Division of Hematology, Department of Internal Medicine, The Ohio State University, Colum- for the formation of cryptopatches and isolated lymphoid follicles in bus, OH 43210 the intestinal lamina propria (4) as well as repair of lymph nodes 1A.R.V., A.P.N., and W.D. contributed equally to this work. following infection (18). IL-22 and IL-18 have recently been shown ORCIDs: 0000-0003-4118-3088 (A.R.V.); 0000-0002-5517-7183 (C.M.); 0000- to cooperatively contribute to murine intestinal immunity to mul- 0002-5257-1521 (W.K.C.); 0000-0003-1224-470X (T.H.); 0000-0002-1798- tiple infectious agents. IL-18 induces IL-22 during Toxoplasma 9113 (C.C.); 0000-0002-0489-9645 (S.M.); 0000-0002-1348-269X (L.-C.W.). gondii infection in the murine ileum, whereas IL-22 induces IL-18 Received for publication September 20, 2016. Accepted for publication July 27, 2017. during Citrobacter rodentium infection (17). A combination of This work was supported by National Institutes of Health Grants AI129582, IL-18 and IL-22 was shown to be critical for clearance of rotavirus CA095426, CA185301, and CA097189, American Cancer Society Research Scholar in infected mice (19). Grant RSG-14-243-01-LIB, a grant from Gabrielle’s Angel Foundation for Cancer In this study, we identified IL-18 as a cytokine that can induce Research, and by a grant from the Leukemia and Lymphoma Society. proliferation of and sustain IL-22 production by human ILC3s. Address correspondence and reprint requests to Dr. Michael A. Caligiuri and Dr. Jianhua Yu, Division of Hematology, Department of Internal Medicine, College of IL-18 signals through the IL-18 receptor to activate NF-kBsignaling, Medicine, Ohio State University, 320 West 10th Avenue, Columbus, OH 43210. E-mail which acts at the IL22 promoter. In the tonsil, ILC3s reside in close addresses: [email protected] (M.A.C.) and [email protected] (J.Y.) proximity to dendritic cells (DCs), a source of IL-18. Taken to- The online version of this article contains supplemental material. gether, these data support the hypothesis that DC-derived IL-18 Abbreviations used in this article: DC, dendritic cell; EdU, 5-ethynyl-29-deoxyuri- stimulates ILC3 function by maintaining the population through dine; HPC, hematopoietic progenitor cell; IHC, immunohistochemistry; ILC, innate lymphoid cell; ILC3, group 3 ILC; MCT, mast cell tryptase; PI, propidium iodide; proliferation and by sustaining production of IL-22 through an NF- RORgt, RAR–related orphan receptor g isoform b; SLT, secondary lymphoid tissue; kB–dependent mechanism. Our study further clarifies the role of TPCK, N-tosyl-L-phenylalanine chloromethyl ketone. DCs in ILC3 function and identifies NF-kB as a potential target for Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 future therapies against IL-22–mediated diseases. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601554 2334 REGULATION OF ILC3s BY IL-18 Materials and Methods EMSA and Ab supershift assays Isolation of human ILC3s and developmental precursors Nuclear extracts were isolated using a nuclear extraction kit (Active Motif). Complementary oligonucleotides—probe 1 (2248 to 2216 bp, referred to All procedures were performed with approval of the Ohio State Uni- 2 2 versity Institutional Review Board. Normal human pediatric tonsils were as transcription start site) and probe 2 ( 191 to 163 bp)—containing a obtained following routine tonsillectomy from Nationwide Children’s putative NF-kB binding site from the human IL22 promoter were syn- thesized. EMSAs were performed as described previously (23). In brief, Hospital (Columbus, OH). ILC3s and developmental precursors were 32 isolated as previously described (20). Briefly, total mononuclear cells probes 1 and 2 were P labeled and incubated with the nuclear extracts were depleted of CD19+ and/or CD3+ cells via magnetic negative se- (2 mg) in the presence of poly(deoxyinosinic-deoxycytidylic) acid (1 mg). lection (Miltenyi Biotec). For some experiments, B and/or T cell– For the Ab gel supershift assays, nuclear extracts were incubated with Abs depleted mononuclear cells were used immediately for flow cytometric for p65 (Rockland Immunochemicals) or p50 (Millipore) overnight at 4˚C analysis. Alternatively, ILC3s were sorted directly from the depleted fraction before the addition of the IL22 promoter probes. 2 2 2 2 2 2 2 + by gating on CD3 CD14 CD19 CD20 CD34 CD16 CD94 CD117 Transient transfection and luciferase assay events on a FACSAria II cell sorter (BD Biosciences).