IL-18 Drives ILC3 Proliferation and Promotes IL-22 Production Via NF-Κb

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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/

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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 function 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 synthesized. 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). Purity analysis routinely revealed that sorted populations were $97% pure. 293T cells were maintained in DMEM supplemented with 10% FBS, penicillin G (100 mg/ml), and streptomycin (100 mg/ml) (Invitrogen). The Cell culture cell line was obtained from the American Type Culture Collection in 2008; ILC3s purified by FACS were cultured in a round-bottom 96-well plate it has not been authenticated since receipt. 293T cells were seeded into 24- 3 4 (Costar) at a starting density of 2.5 3 104 cells/ml in a-MEM containing well plates at a density of 2.5 10 cells per well and grown overnight. 10% FBS, penicillin G (100 mg/ml), and streptomycin (100 mg/ml) Cotransfection of the IL22-Luc construct with a vector expressing p50, or a (Invitrogen). Cells were cultured with the indicated recombinant human vector expressing p65, or a combination of both expression vectors, or an cytokines, including IL-15 (1 nM; Amgen), IL-18 (100 ng/ml; Medical and empty vector was performed using Lipofectamine 2000 with Plus reagent (Invitrogen) per the manufacturer’s protocol. The pGL3 basic reporter Biological Laboratories), IL-1b (10 ng/ml; PeproTech), IL-12 (10 ng/ml; Downloaded from Genetics Institute), IL-6 (20 ng/ml; R&D Systems), IL-27 (10 ng/ml; R&D vector was used as a control for basal promoter activity. A Renilla lucif- Systems), IL-21 (100 ng/ml; R&D Systems), IL-23 (20 ng/ml; Miltenyi erase vector, pRL-TK (Promega), was cotransfected to serve as an internal Biotec), IL-7 (10 ng/ml; Miltenyi Biotec), IL-10 (10 ng/ml; Schering- control for transfection efficiency. Cells were harvested after 48 h and Plough), IL-25 (100 ng/ml; R&D Systems), IFN-a (20 U/ml; Schering- assessed for luciferase activity as previously described (22). The experi- Plough), IFN-g (2 U/ml; PeproTech), TRAIL (10 ng/ml; R&D Systems), ment was performed with the wild-type IL22 promoter sequence and the and TGF-b (20 ng/ml; R&D Systems). Pam2CSK4 (TLR2/6 agonist) sequence carrying the mutated putative NF-kB binding sites. In mutation was obtained from InvivoGen (1 mg/ml).Allcellculturewasper- 1, the sequence corresponding to probe 1 (59-GAAAATTTCTGG- formed in the presence of IL-15 because it serves as a survival factor GATTTGTC-39) was changed to 59-GCACATCTCCGAGCTTCGAC-39. http://www.jimmunol.org/ for ILC3s (21). ILC3s were cultured for 14 d in proliferation assays, unless In mutation 2, the sequence corresponding to probe 2 (59-GGGAAA- otherwise indicated. Cells were enumerated by a trypan blue exclusion CACT-39) was changed to 59-TTGACACTCT-39. In the double mutation, assay. both sequences were changed accordingly. Flow cytometry Immunohistochemistry, imaging, and multispectral analysis of dual color immunohistochemistry Abs for human CD34, CD3, CD16, and CD117 were purchased from BD Biosciences; those for CD94, IL-18Ra, IL-18Rb, IL-6Ra, TGF-bRII, Paraffin-embedded tonsillar tissue sections (0.5 mm in thickness) were IL-27Ra, IL-10Ra, IL-17RB, TRAIL R1, and IL-22 were purchased from stained for immunohistochemistry (IHC) using a Bond Rx autostainer R&D Systems; those for CD14, CD19, CD20, IL-7R (CD127), IL-12R, (Leica). Briefly, slides were baked at 65˚C for 15 min, and automated IFNAR2, IL-10Rb, and IL-21R were purchased from Miltenyi Biotec. software performed dewaxing, rehydration, Ag retrieval, blocking, primary by guest on October 3, 2021 Unless otherwise indicated, Abs were used according to the manufacturers’ Ab incubation, postprimary Ab incubation, detection (diaminobenzidine or instructions. For survival studies, annexin V protein (conjugated to BV421 alkaline phosphatase red), and counterstaining using Bond reagents (Leica fluorochrome; BD Biosciences) was added at specific time points to cul- Biosystems). Samples were then removed from the machine, dehydrated tured ILC3s resuspended in annexin V binding buffer (103 solution di- through ethanol series and xylenes, mounted, and coverslipped. Abs for the luted in distilled water for 13 working concentration; BD Biosciences). following markers were diluted in Ab diluent (Leica Biosystems): rabbit Abs Following annexin V binding, cells were stained with propidium iodide CD3 (1:100; Dako) and IL-18 (1:1000; Sigma-Aldrich); mouse Abs CD11c (PI; BD Biosciences) immediately prior to analysis by flow cytometry. For (1:100; Leica Biosystems), IL-1b (1:100; Cell Signaling Technology), mast proliferation studies, 5 mM CellTrace Violet stain (tracer dye) from Life cell tryptase (MCT; 1:300; Leica Biosystems), and RORgt(1:800;Milli- Technologies was added to cells prior to culture and analyzed by flow pore); and goat Ab CD117 (1:50; R&D Systems). Images were digitally cytometry after 7 or 14 d. For 5-ethynyl-29-deoxyuridine (EdU) incorpo- captured using an Axiocam 105 color camera, an Observer.Z1 microscope, ration, cells were pulsed with 10 mM EdU for 30 min and cultured for 1 h and Plan Neofluar objectives (Zeiss). Broad-field high-resolution images in EdU-free medium before cytometric analysis. For intracellular IL-22 were collected as 5 3 5 grids and digitally stitched together using ZEN 2 staining, ILC3s were cultured for 14 d with either IL-15 alone or IL-15 (blue edition) software (Carl Zeiss Microscopy). Some dual-stained samples plus IL-18. Staining for intracellular IL-22 was then performed following a were imaged using the PerkinElmer Vectra multispectral slide analysis 4-h incubation in 2 mM GolgiPlug (BD Biosciences), and using the system. For visualization of the component images, the multispectral images Cytofix/Cytoperm Plus fixation/permeabilization kit (BD Biosciences) and were spectrally unmixed using Inform software. Briefly, red (CD117), brown anti–IL-22 PE (R&D Systems). Flow cytometry was performed on an LSR (MCT), and blue (DAPI) channels were defined using Nuance spectral li- II flow cytometer (BD Biosciences), and analysis was performed using braries. Individual component images were used to identify single- and dual- FlowJo Software (Tree Star). positive cells. Cell lysis and immunoblotting Protein lysate preparation and immunoblotting were performed as described Results previously (22). Primary Abs used were: rabbit polyclonal anti-human GAPDH IL-18 stimulation promotes ILC3 proliferation Ab (Santa Cruz Biotechnology), rabbit polyclonal anti-human p65 Ab To explore the role of IL-18 and other cytokines in the expansion (Rockland Immunochemicals), and rabbit monoclonal anti-human phospho- 2 2 2 + p65Ser536 (Cell Signaling Technology). of ILC3s, CD3 CD34 CD94 CD117 ILC3s were purified by FACS from fresh human tonsils (Supplemental Fig. 1A). ILC3s ELISA were then stimulated with cytokines as indicated for 14 d (Fig. ELISA was performed with the eBioscience human IL-22 ELISA Ready- 1A, Supplemental Fig. 1B). All ILC3 cultures were performed in SET-Go! kit (Thermo Fisher) and IL-22 standard (Thermo Fisher) the presence of IL-15, which served as a survival factor (21). according to the manufacturer’s protocols. Supernatant was collected ILC3s stimulated with IL-6, TGF-b, IL-21, IL-23, IL-27, IL-10, from sorted ILC3s cultured with IL-15 plus DMSO, IL-15 plus IL-18 IL-7, TRAIL, IL-25, IL-12, or TGF-b plus IL-6 demonstrated plus DMSO, or IL-15 plus IL-18 plus N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). TPCK was used at a concentration of 10 mM little to no increased expansion—and in one case (IFN-a) dem- (Sigma-Aldrich). onstrated decreased expansion—compared with controls cultured The Journal of Immunology 2335

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FIGURE 1. ILC3 proliferation in response to treatment with cytokines in the presence of IL-15. (A) Relative fold change was calculated and averaged from six donors as the absolute number of cells enumerated (by trypan blue exclusion) after 14 d of culture in IL-15 plus cytokine divided by the absolute number of cells enumerated after 14 d of culture in IL-15 alone (n = 6). Data are expressed as mean 6 SD. **p , 0.01, IL-15 plus IL-18 versus other treatments. (B) ILC3s were sorted, labeled with tracer dye, and cultured in IL-15 alone or IL-15 plus IL-18 for 14 d. Cells were analyzed by flow cytometry at 7 and 14 d for retention of tracer dye. Histograms of a representative experiment are shown (n = 3). in IL-15 alone. However, when ILC3s were cultured in IL-15 plus with those cultured in IL-15 alone (at 14 d, IL-15 alone mean IL-18 they demonstrated significant expansion compared with fluorescence intensity of 5746 versus IL-15 plus IL-18 mean ILC3s cultured in IL-15 alone, as measured by both fold change fluorescence intensity of 992, p = 0.04) (Fig. 1B). These data were and absolute numbers (Fig. 1A, Supplemental Fig. 1B). We also supported by an EdU incorporation assay, in which greater EdU in- compared cultures of ILC3s with IL-18 to its combination with corporation was observed in ILC3s cultured in IL-15 plus IL-18 IL-12 or TGF-b to determine whether IL-12 or TGF-b enhanced compared with those cultured in IL-15 alone (Supplemental Fig. or impaired ILC3 proliferation induced by IL-18. The addition of 2C). We assessed survival between the two conditions by annexin/ IL-12 or TGF-b did not significantly alter the proliferation of PI staining. We found no difference of the percentage of live ILC3s compared with stimulation with IL-18 (Supplemental (annexin2/PI2) or apoptotic cells (annexin+/PI2) on treatment days 1, Fig. 2A). Additionally, treatment of ILC3s with IFN-g ledtoa 3, 7, and 14 (data not shown). These data suggest that the quantitative decrease in cell number compared with controls (Supplemental expansion of ILC3s following culture in IL-15 plus IL-18 is due to an Fig. 2B). increase in proliferation rather than enhanced survival. We also ob- To determine whether the increase in cell number in the presence served that treatment of ILC3s with the TLR2/6 agonist Pam2CSK4 of IL-15 plus IL-18 compared with IL-15 alone could be attributed did not augment ILC3 proliferation in the presence of IL-15 alone or to proliferation, we performed a fluorescent dye proliferation assay. in the presence of IL-15 plus IL-18 (data not shown). ILC3s were labeled with tracer dye prior to culture, and after 7 and 14 d cultured cells were harvested, counted, and examined for Expression of IL-18Ra and IL-18Rb on the cell surface of retained fluorescence intensity by flow cytometry. Whereas ILC3s ILC3s cultured in IL-15 alone as well as those cultured in IL-15 plus IL- Human IL-18 signals through a heterodimeric receptor composed 18 both proliferated during 7 d and 14 d, those cultured in IL-15 of the IL-18Ra and IL-18Rb subunits (24). To validate that IL-18 plus IL-18 showed significantly increased proliferation compared can signal through its receptor on ILC3s, we examined the ex- 2336 REGULATION OF ILC3s BY IL-18

FIGURE 2. Expression of IL-18Ra and IL-18Rb on HPC progenitors and ILC3s freshly isolated from human tonsil. Histograms show a representative donor stained ex vivo with isotype (unfilled) or Ab specific for the indicated Ag (filled). CD34+CD1172 HPCs 2

were more specifically defined as CD3 Downloaded from CD192CD34+CD1172CD942.CD34+CD117+ HPCs were more specifically defined as CD32CD192CD34+CD117+CD942.ILC3swere defined as CD32CD192CD342CD117+CD942 . http://www.jimmunol.org/ by guest on October 3, 2021

pression of IL-18Ra and IL-18Rb on the surface of ILC3s as well sought to characterize the in vitro–derived cells generated in re- as in early and late hematopoietic progenitor cells (HPCs) by flow sponse to each treatment. Following culture, CD94 expression and cytometry. Whereas surface IL-18Rb was expressed by most early IL-22 production were mutually exclusive, demonstrating that (CD34+CD1172) HPCs, late (CD34+CD117+) HPCs, and mature only ILC3s, and not in vitro–derived mature NK cells, were ca- ILC3s, robust surface expression of IL-18Ra was restricted to pable of producing IL-22 in response to cultures containing IL-18 mature ILC3s (Fig. 2). Of note, mature NK cells derived from (Fig. 3A). Whereas ILC3s cultured with IL-18 acquired CD94 peripheral blood also express both IL-18R subunits (Supplemental surface expression at a rate similar to that of ILC3s cultured in the Fig. 3A). The constitutive expression of the heterodimeric re- presence of IL-15 alone, the inclusion of IL-18 in the cultures led ceptor on the mature ILC3s is consistent with the above prolif- to a significantly greater fraction of cells expressing IL-22 (1.1% eration data and suggests that IL-18 signaling may have a unique versus 12.3%) (Fig. 3B). Thus, IL-18 expands ILC3s and main- role in regulating differentiation or homeostasis of ILC3s. We also tains their production of IL-22. assessed for cytokine receptor expression for the other cytokines used in the proliferation assay (Supplemental Fig. 3B). Freshly Inhibition of NF-kB blocks IL-18–induced IL-22 production isolated ILC3s showed expression of the receptor for IL-7, To determine a potential mechanism by which IL-18 may be reg- whereas receptors for IL-6, TGF-b, IL-21, IL-27, IFN-a, IL-10, ulating IL-22 production in ILC3s, we considered the downstream TRAIL, IL-17, IL-25, and IL-12 were either absent or negligibly intermediates through which IL-18 signals (31, 32), that is, NF-kB expressed following flow cytometric staining. (33) and MAPKs (34, 35). To test whether NF-kB signaling mediates IL-22 production in response to IL-18, we employed the NF- IL-18 sustains IL-22 protein expression in ILC3s kB antagonist TPCK. ILC3s were treated with IL-15 plus DMSO, Previous studies have demonstrated that freshly isolated ILC3s IL-15 plus IL-18 plus DMSO, or IL-15 plus IL-18 plus TPCK. We constitutively produce high levels of IL-22 (8–10, 25–30). To confirmed that the addition of IL-18 stimulated IL-22 production determine whether IL-18 plays a role in regulating IL-22 pro- compared with IL-15 plus DMSO (Fig. 4). Furthermore, we found duction, ILC3s were isolated from human tonsils by FACS and that treatment of ILC3s with TPCK in the presence of IL-15 plus cultured with IL-15 in the presence or absence of IL-18 for 14 d. A IL-18 almost completely blocked this response. This suggests that minor fraction of cultured cells expressed CD94 at day 14 (which the stimulatory effect of IL-18 on IL-22 production by ILC3s is identifies mature NK cells) under both culture conditions. We then mediated mainly by NF-kB signaling. The Journal of Immunology 2337

FIGURE 3. ILC3s treated with IL-18 maintain expression of IL-22. (A) Representative staining for surface CD94 and intracellular IL-22 protein at day 14 of culture. (B) Percentage of ILC3s staining positively for IL-22 was assessed via flow cytometry after a 4-h culture with either IL-15 alone or IL-15 plus IL-18 (*p , 0.05; n = 4). Data are expressed as mean 6 SD. Downloaded from http://www.jimmunol.org/ IL-18 activates NF-kB via phosphorylation of p65 and induces assayinwhichtheIL22 promoter was cloned immediately NF-kB binding to the IL22 promoter upstream of the luciferase gene in the pGL3 firefly luciferase To further support that NF-kB mediates IL-18 signaling in ILC3s, construct. The IL22-Luc promoter construct was cotransfected we identified three NF-kB binding sites in the proximal promoter with an empty vector or an expression vector containing p65 or region of the human IL22 gene, two of which are immediately p50 or with both expression vectors. Transfections with the adjacent to one another (Fig. 5A). NF-kB dimers bind target gene p65 vector (p65 and p65/p50) demonstrated strong increases regulatory regions through binding sites that generally match the in luciferase activity compared with the empty vector control consensus sequence 59-GGGRNTY(C/T)C-39 (R indicates A or G, by guest on October 3, 2021 Y indicates C or T, and N indicates any nucleotide) (36). The promoter sequence corresponding to probe 1 contains the two adjacent putative NF-kB binding sites, and the promoter sequence corresponding to probe 2 contains the third binding site. The probe 2 site has high homology with the consensus binding sequence, whereas the probe 1 site has more mismatched bases. The addition of IL-18 to sorted ILC3s cultured in IL-15 revealed increased phosphorylation of NF-kB p65 at serine 536 in ILC3s (Fig. 5B). To test whether nuclear transcription factors in ILC3s bind to these sites, we synthesized two DNA probes containing the NF-kB binding sites in the IL22 promoter. In EMSAs, more DNA–protein complexes were observed in nuclear lysates prepared from ILC3s treated with IL-18 in the presence of the survival cytokine IL-15 than from ILC3s treated with IL-15 alone (Fig. 5C). Furthermore, an Ab supershift experiment showed the presence of NF-kB subunits p65 and p50 in the DNA–protein complexes (Fig. 5D). Taken together, these data demonstrate that phosphorylated NF-kBiscapableofspe- cific yet faint binding to the IL22 promoter in ILC3s stimulated with IL-15 alone, in contrast to specific and avid binding to the IL22 promoter in ILC3s stimulated with IL-15 plus IL-18. Thus, IL-18 phosphorylates NF-kB, and activated NF-kB binds to the IL22 promoter, suggesting it participates in the regulation of FIGURE 4. The NF-kB antagonist TPCK inhibits IL-22 production by ILC3s in the presence of IL-18. ILC3s were cultured for 14 d in the IL-22 expression. presence of IL-15 plus DMSO, IL-15 plus IL-18 plus DMSO, or IL-15 plus NF-kB subunit p65 positively regulates transcription at the IL-18 plus TPCK. At day 14, the supernatants were harvested for ELISA to IL22 promoter measure production of IL-22. Data are expressed at mean 6 SEM (n = 8). For statistical analysis, data were transformed by log base 2 to stabilize the Because we established that NF-kB can bind avidly to the IL22 variance. A linear mixed model was performed to account for the covariance promoter in an IL-18–dependent manner, we next set out to de- structure due to repeated measures on the same donor. The p values were termine whether NF-kB is also capable of regulating IL-22 tran- adjusted for multiple comparisons by the Holm procedure. A p value #0.05 scription. To this end, we performed a luciferase reporter gene was considered significant. **p , 0.01. 2338 REGULATION OF ILC3s BY IL-18 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 5. NF-kB is activated by IL-18 and binds to the IL22 promoter. (A) IL22 promoter sequence. Boxes indicate predicted NF-kB binding sites. Oligonucleotide probes were synthesized using the indicated ranges of the IL22 promoter sequence. (B) ILC3s were treated with either IL-15 alone or IL-15 plus IL-18 and subjected to immunoblotting with the indicated Abs. (C) EMSA was performed with 32P-labeled predicted NF-kB binding sites (probe 1 or probe 2) and nuclear extract (NE) prepared from ILC3s treated with IL-15 alone or IL-15 plus IL-18 or no NE (2). The DNA–protein complexes are indicated with arrows. (D) Ab gel supershift assay. Probe 1 was incubated with NE from ILC3s treated with IL-15 plus IL-18 and preincubated with no Ab, p65 Ab, p50 Ab, or without NE. The DNA–protein complexes and Ab gel supershifted complexes are indicated with arrows.

(p , 0.01) (Fig. 6A). Transfection with p65/p50 showed a par- luciferase activity beyond the effects of mutation 2 alone. These tially attenuated effect on luciferase activity compared with p65. data demonstrate that the NF-kB binding site from probe 2 Of note, a similar mechanism has been reported for other target mediated IL22 expression through interaction with the NF-kB genes (37). The p50 subunit is not thought to be activating and, as subunit p65. The promoter activity of the NF-kB binding site expected, did not stimulate luciferase activity. To further charac- from probe 2 showed a greater contribution than did those from terize the NF-kB binding site, additional IL22 promoter constructs probe 1, which corresponds with their relative sequence ho- were generated, which contained mutations at the putative NF-kB mologies to the NF-kB consensus binding site. These ﬁndings binding sites around which probes 1 and 2 were designed. Mutation in combination with above EMSA showing occupancy of both 1 corresponds to the putative binding sites found in probe 1, and NF-kB sites show that although NF-kB signaling is involved, it mutation 2 corresponds to the putative binding site found in probe likely works in concert with other yet undetermined cofactors. 2. Loss of the putative binding site due to mutation 2, but not Collectively, these data further support the hypothesis that IL-18 mutation 1, resulted in decreased luciferase activity in response positively regulates IL-22 production by ILC3s through an NF- to p65 (p , 0.01) (Fig. 6B). Double mutation did not impair kB–mediated mechanism. The Journal of Immunology 2339

FIGURE 6. NF-kB subunit p65 positively regulates transcription through the IL22 promoter. (A) 293T cells were transfected with the IL22-Luc reporter construct with empty vector Downloaded from (NC), p50, p65, or cotransfected with both p50 and p65 vectors (p65/50) (n = 4). (B) 293T cells were transfected with the IL22-Luc reporter construct or IL22-Luc mutation 1, IL22-Luc mutation 2, and IL22-Luc double mutation together with p65 vector (n = 3). A linear mixed model was used to http://www.jimmunol.org/ compare the four mutation groups by taking into account the block factor experiment. The p value was adjusted for multiple comparisons by the Holm procedure. A p value #0.05 was considered signiﬁcant. The ratio of each protein versus

“nc” was log2 transformed before entering the linear mixed model. Data are expressed as mean 6 SEM. *p , 0.05, **p , 0.01. mut, mutation. by guest on October 3, 2021

RORgt+ ILC3s reside near IL-18–producing DCs in human SLTs sections were used in Fig. 7 to demonstrate the proximity of RORgt+CD32 cells with IL-18–expressing cells. This shows that ILC3s have previously been defined by IHC procedures as + + lymphoid-shaped CD117+ cells residing within the interfollicular CD11c IL-18 DCs exist in close proximity to ILC3s in the interfollicular areas and may serve as a source of IL-18 in situ. and lamina propria regions of the human tonsil (10, 25, 38). To ILC3s can also be phenotypically defined by expression of refine our identification of ILC3s by IHC, we defined ILC3s as + 2 CD117 and the absence of MCT. Therefore, we also sought to use RORgt CD3 cells with lymphoid morphology. We hypothesized + 2 + CD117 /MCT cells of lymphoid morphology as another way to that CD11c DCs within the human tonsil might be a physiolog- + + + identify ILC3s by IHC. Most of the CD117 cells costained with ical source of IL-18 for ILC3s. We observed that CD11c IL-18 MCT, but we did identify several CD117+MCT2 cells (Supple- cells with DC morphology were present in the interfollicular areas mental Fig. 4A–D). Most of these CD117+MCT2 cells stained + proximal to clusters of RORgt cells with lymphoid morphology weakly for CD117 compared with double-positive cells. This is (Fig. 7A, 7B). Most of the latter lymphoid cells coexpressed CD3, consistent with the expectation that CD117 expression on ILC3s is 2 consistent with Th17 cells; however, occasional RORgt+CD3 lower than on mast cells. Furthermore, weakly staining CD117+ cells were also present, consistent with ILC3s (Fig. 7C). Serial cells were smaller and more consistent with lymphoid morphology 2340 REGULATION OF ILC3s BY IL-18 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 7. ILC3s reside in proximity to IL-18+CD11c+ DCs within the human tonsil. Immunohistochemical staining (diaminobenzidine 5 brown; alkaline phosphatase red 5 red) depicted in sections of human tonsil. Original magnification, 3100 (top row) and 3400 (middle and bottom rows). (A) Brown indicates CD11c staining and red indicates IL-18 staining. White arrows indicate examples of costained cells. (B) Brown indicates RORgt staining and red indicates IL-18 staining. White arrows indicate examples of RORgt+ cells associating with IL-18+ cells. (C) Brown indicates RORgt staining and red indicates CD3 staining. White arrows indicate examples of RORgt+CD32 cells. (B) and (C) represent consecutive, or serial, sections. GC, germinal center; IF, interfollicular region. than strongly staining CD117+ cells, which were predominantly IL-18 is an important regulator of ILC function. ILC1s and NK larger in size. We similarly observed that IL-18+ cells were found cells produce IFN-g in response to IL-18 stimulation (39–41). proximally to CD117+ cells in the interfollicular region (Supple- Furthermore, IL-12 stimulation induces the IL-18 receptor on NK mental Fig. 4E). IL-18+ cells were found in close proximity to cells whereas IL-18 reciprocally induces the IL-12 receptor (39, RORgt+, CD3+, and MCT+ cells, in part due to the abundance of 42–45). This is hypothesized to be the mechanism by which the IL-18+ cells (Fig. 7B, Supplemental Fig. 4F, 4G). However, RORgt+ combination of IL-12 and IL-18 stimulation synergizes to activate and MCT+ cells were mutually exclusive (Supplemental Fig. 4H). NK cells. Recent work in a murine rotavirus model showed that These data support the notion that ILC3s colocalize with IL-18+ IL-18, IL-22, and ILCs are involved in clearing infection fol- DCs in the interfollicular regions and that CD11c+ DCs are a primary lowing flagellin challenge (19). source of IL-18 protein in human SLTs. Taken together, these find- In this study, we show that human ILC3s are responsive to ings suggest that CD11c+ DCs are capable of stimulating RORgt+ IL-18 stimulation. In addition to driving ILC3 proliferation, we ILC3s resulting in quantitative expansion and production of IL-22. found that IL-18 can stimulate and sustain production of IL-22. IL-23 has been reported to stimulate IL-22 production in human Discussion (9) and murine (46) ILC3s in the short term (,5 d); however, Human ILC3s play critical roles in antimicrobial immunity and this effect does not appear to be sustained in human ILC3s (after homeostasis. As such, it is important to define the mechanisms by 14 d), nor does IL-23 drive ILC3 proliferation (25). Our earlier which these cells proliferate and regulate cytokine production. Of work showed that IL-1b induced ILC3 homeostasis (25), which is the 13 cytokine conditions tested, only 1 (IL-18) resulted in sig- consistent with the data presented in the present study, as both nificant expansion of ILC3s when cultured in combination with cytokines belong to the same cytokine superfamily and are medi- IL-15, a survival factor for ILC3s (25). Interestingly, IL-18 was ated by similar signaling pathways. However, in this study we also able to override the antiproliferative effects of TGF-b. provide a molecular mechanism (the NF-kB signaling pathway) by The Journal of Immunology 2341 which IL-18 induces the production of IL-22, and we speculate that 3. Montaldo, E., K. Juelke, and C. Romagnani. 2015. 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Expression of ADAM33 is a novel regula- 1595–1603. Supplemental Figure 1 A

B Supplemental Figure 1. Purity analysis of sorted ILC3s and ILC3 proliferation in response to cytokine stimulation. (A) Representative plots showing purity analysis of ILC3s used in culture experiments. ILC3s were sorted as live CD3─CD14─CD19─CD20─CD34─CD16─CD94─CD117+ lymphocytes. (B) Absolute numbers of ILC3s were enumerated (by Trypan blue exclusion) after 14 days of culture (** denotes p < 0.01, * denotes p < 0.05. Cytokine vs other treatments; n=6). Supplemental Figure 2 A

C Supplemental Figure 2. ILC3 proliferation in response to cytokine stimulation. (A and B) Relative fold change was calculated and averaged from 5 donors as the absolute number of cells enumerated (by Trypan blue exclusion) after 14 days of culture in IL-15 plus indicated cytokine divided by the absolute number of cells enumerated after 14 days of culture in IL-15 alone. * denotes p < 0.05. Data are expressed as mean ± SD. (C) Proliferation was assessed via 5-ethynyl-2’-deoxyuridine (EdU) incorporation in total ILC3s treated with IL-15 plus IL-18 or IL- 15 alone. One of three experiments with similar results is presented. Supplemental Figure 3

B Supplemental Figure 3. Surface expression of cytokine receptors on freshly isolated NK cells and ILC3s. (A) Human NK cells were isolated from peripheral blood and were defined as CD3─CD56+ . Histograms show a representative donor stained ex vivo with isotype (unfilled) or antibody specific for the indicated antigen (filled). (B) Human ILC3s were isolated from tonsil. ILC3s were sorted as live CD3─CD14─CD19─CD20─CD34─CD16─CD94─CD117+ lymphocytes. Histograms show representative plots of surface expression of cytokine receptors in freshly isolated ILC3s compared to isotype controls. Supplemental Figure 4

E F

G H Supplemental Figure 4. Phenotype and distribution of immune cells within human tonsil. (A-D) IHC stains of human tonsil. Original magnification of images is 200x. CD117+ cells are labeled in red and MCT+ cells are labeled in brown. Red arrow identifies an example of a cell co-staining for both CD117 and MCT. Black arrows indicate examples of cells which stain only for CD117. (A) Raw image of co-stained tonsil section. (B) Image analysis software was used to separate the colors by wavelength. Here is a composite image containing wavelengths for blue (nuclei), red, and brown. (C) Red wavelengths only image of human tonsil. (D) Brown wavelengths only image of human tonsil. (E-H) Tonsil sections were co-stained with the indicated antibodies with the designated colors. Original magnification of images is 400x. (E) Co-staining with CD117 (in place of RORγt) with IL-18 to show that ILC3s are found in proximity to DCs in the interfollicular region. White arrows indicate CD117+ cells in close proximity to IL-18+ cells. (F) Co- staining with CD3 and IL-18 to show the distribution of T cells and DCs in the tonsil. White arrows indicate CD3+ cells in close proximity to IL-18+ cells. (G) Co-staining with MCT and IL-18 to show the distribution of mast cells and DCs. White arrows indicate MCT+ cells in close proximity to IL-18+ cells. (H) Co- staining with RORγt and MCT to show that cells staining with these markers are mutually exclusive.