Published OnlineFirst September 12, 2019; DOI: 10.1158/2326-6066.CIR-18-0934 Research Article Cancer Immunology Research Mirc11 Disrupts Inflammatory but Not Cytotoxic Responses of NK Cells Arash Nanbakhsh1, Anupallavi Srinivasamani1, Sandra Holzhauer2, Matthew J. Riese2,3,4, Yongwei Zheng5, Demin Wang4,5, Robert Burns6, Michael H. Reimer7,8, Sridhar Rao7,8, Angela Lemke9,10, Shirng-Wern Tsaih9,10, Michael J. Flister9,10, Shunhua Lao1,11, Richard Dahl12, Monica S. Thakar1,11, and Subramaniam Malarkannan1,3,4,9,11 Abstract Natural killer (NK) cells generate proinflammatory cyto- g–dependent clearance of Listeria monocytogenes or B16F10 kines that are required to contain infections and tumor melanoma in vivo by NK cells. These functional changes growth. However, the posttranscriptional mechanisms that resulted from Mirc11 silencing ubiquitin modifiers A20, regulate NK cell functions are not fully understood. Here, we Cbl-b, and Itch, allowing TRAF6-dependent activation of define the role of the microRNA cluster known as Mirc11 NF-kB and AP-1. Lack of Mirc11 caused increased translation (which includes miRNA-23a, miRNA-24a, and miRNA-27a) of A20, Cbl-b, and Itch proteins, resulting in deubiquityla- in NK cell–mediated proinflammatory responses. Absence tion of scaffolding K63 and addition of degradative K48 of Mirc11 did not alter the development or the antitumor moieties on TRAF6. Collectively, our results describe a func- cytotoxicity of NK cells. However, loss of Mirc11 reduced tion of Mirc11 that regulates generation of proinflammatory generation of proinflammatory factors in vitro and interferon- cytokines from effector lymphocytes. Introduction TRAF2 and TRAF6 promote K63-linked polyubiquitination that is required for subcellular localization of the substrates (20), Natural killer (NK) cells generate proinflammatory factors and and subsequent activation of NF-kB (21) and AP-1 (22). Regu- mediate antitumor cytotoxicity (1, 2). Upon recognizing target lation of these signaling events is obligatory to prevent pathologic cells, activation receptors initiate unique signaling cascades in NK inflammation (23). One established mechanism is mediated cells (3). Adaptor proteins DAP10 (4), DAP12 (5), and CD3z (6) by lipids (SHIP and PTEN) and protein phosphatases (CD45, recruit key signaling proteins including Syk (7), Fyn (8), PI(3)- SHP1, and SHP2) that are recruited to inhibitory Ly49/KIR p110d/p85a (9–11), Grb2 (12), PLC-g2 (13, 14), ADAP (15, 16), receptors (24, 25). Carma1 (17), Bcl10 (18), and TAK1 (19). E3 ligases including In addition, ubiquitin regulators such as A20, Itch, Cbl-b, and Cyld synthesize and add polyubiquitin chains to lysine (K) 48 of 1Laboratory of Molecular Immunology and Immunotherapy, Blood Research ubiquitin molecules leading to the proteasomal degradation of Institute, BloodCenter of Wisconsin, Milwaukee, Wisconsin. 2Laboratory of substrates (26–29). Ubiquitin regulators can alter the activation Lymphocyte Signaling, Blood Research Institute, BloodCenter of Wisconsin, threshold of NF-kB and AP-1 by targeting multiple signaling Milwaukee, Wisconsin. 3Department of Medicine, Medical College of Wisconsin, proteins including TRAF6 (30). However, the regulatory role of 4 Milwaukee, Wisconsin. Department of Microbiology and Immunology, Medical small noncoding microRNAs (miRNA), which degrade mRNAs in 5 College of Wisconsin, Milwaukee, Wisconsin. Laboratory of B Cell Biology, other cell types, is unknown in NK cells. The Mirc11 (micro RNA Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, Wisconsin. 6Bioinformatics Core, Blood Research Institute, BloodCenter of Wisconsin, cluster 11, also known as miRNA-23a) locus encodes three Milwaukee, Wisconsin. 7Laboratory of Stem Cell Biology, Blood Research miRNAs, miR-23a, miR-27a, and miR-24-2, from a single primary Institute, BloodCenter of Wisconsin, Milwaukee, Wisconsin. 8Department of Cell mRNA transcript (31). The Mirc11 cluster is highly conserved in Biology, Medical College of Wisconsin, Milwaukee, Wisconsin. 9Genome mouse and human genomes. Mirc22 (micro RNA cluster 22, Sciences and Precision Medicine Center, Medical College of Wisconsin, Milwau- also known as miRNA-23b) is a paralog of Mirc11 and encodes 10 kee, Wisconsin. Department of Physiology, Medical College of Wisconsin, miR-23b, miR-27b, and miR-24-1 (31). Despite study of Mirc11 in Milwaukee, Wisconsin. 11Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin. 12Indiana University School of Medicine, South Bend, lymphocyte biology (32, 33), little functional or mechanistic Indiana. insight exists regarding its effect on NK cell development and function. Note: Supplementary data for this article are available at Cancer Immunology fi fl Research Online (http://cancerimmunolres.aacrjournals.org/). Here we de ne Mirc11 as an obligatory regulator of in amma- tory responses in NK cells. A lack of the Mirc11 cluster did not Corresponding Author: Subramaniam Malarkannan, Blood Research Institute, affect the development of NK cells. Ex vivo and in vitro antitumor 8727 Watertown Plank Road, Milwaukee, WI 53226. Phone: 414-937-3812; À À cytotoxicity of NK cells from Mirc11 / mice was mostly intact. Fax: 414-937-6811; E-mail: [email protected] À À However, in vitro stimulation of NK cells from Mirc11 / mice Cancer Immunol Res 2019;7:1647–62 with mitogenic antibodies revealed defective production of doi: 10.1158/2326-6066.CIR-18-0934 inflammatory cytokines. NK cell–produced IFNg-dependent Ó2019 American Association for Cancer Research. in vivo clearance of Listeria monocytogenes and B16F10 melanoma www.aacrjournals.org 1647 Downloaded from cancerimmunolres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst September 12, 2019; DOI: 10.1158/2326-6066.CIR-18-0934 Nanbakhsh et al. À À was defective in Mirc11 / mice. Transcriptome-wide analyses of release (MR) by lysing the cells with hydrochloric acid and NK cells after either anti-NKG2D–mediated in vitro stimulation or spontaneous release (SR) of the radioactivity in the media with in vivo Listeria challenge indicated a global defect in NF-kB and AP- target cells alone. Specific lysis: 100 Â [(experimental counts per 1–mediated gene transcription in the absence of Mirc11. We show minutes (CPM) À SR (average CPM)/(MR (average CPM) À SR that members of Mirc11 targeted and silenced the transcripts (average CPM)] ¼ % specific lysis. encoding A20, Itch, and Cbl-b, thereby reducing the TRAF6- fi mediated activation and nuclear translocation of NF-kB and Quanti cation of cytokines and chemokines AP-1 complexes. Our findings provide insight into microRNA- IL2-cultured NK cells were activated with plate-bound mito- mediated regulation of lymphocyte functions and identify targets genic antibodies (2.5 mg/mL) against NKG2D (A10), CD137 for containing pathologic inflammation. (17B5), or Ly49D (4E5) for 18 hours. Culture supernatants were analyzed in a Bioplex assay (Bio-Rad). As positive controls, IL2- cultured NK cells were stimulated with 1 ng/mL of IL12 (R&D Materials and Methods Systems) and 10 ng/mL of IL18 (MBL), and the supernatants were Mice and cell lines analyzed. Intracellular IFNg was quantified as previously C57BL/6 mice (wild type, WT) were obtained from The Jackson described (36). Cytokine encoding transcripts were quantified as À À Laboratory. Mirc11 / mice were backcrossed with C57BL/6 mice described earlier (15). À À À À for at least 10 generations (34). Spleens from Cyld / and Itch / mice were a gift from Dr. V.K. Poojary at Baylor Research Institute, Cytoplasmic and nuclear extracts À À 6 Dallas, TX. Spleens from Tnfaip3 / mice were a gift from Dr. D. IL2-cultured NK cells (3 Â 10 ) were stimulated with anti- Starczynowski at Cincinnati Children Hospital, Cincinnati, OH. All NKG2D mAb (2.5 mg/mL) for 40 minutes. Cells were suspended mice were maintained in pathogen-free conditions at the Biomed- in 100 mL cold buffer A (10 mmol/L HEPES pH 7.9, 10 mmol/L ical Resource Center-Medical College of Wisconsin. All animal and KCl, 0.2 mmol/L EDTA, 1 mmol/L DTT, 3 mg/mL aprotinin, human peripheral blood mononuclear cell (PBMC) usage was 2 mg/mL Pepstatin, and 1 mg/mL leupeptin). After incubation on approved by the Institutional Animal Care and Use Committee ice for 15 minutes, Nonidet P-40 was added to a final concen- (IACUC; AUA1512) and Institutional Review Board (IRB). EL4, tration of 0.5%. The mixtures were vortexed for 10 seconds and RMA, RMA/S, and YAC-1 cell lines were purchased from ATCC. spun at 16,000 Â g for 30 seconds. The supernatants were Generation of H60-expressing EL4 stable cell lines has been collected as the cytoplasmic extracts. The pellets were washed described (35). When needed, frozen aliquots of these cell lines with buffer A once and resuspended in 50 mL buffer B (20 mmol/L were thawed and used for a period of 3 weeks. Reauthentication of HEPES pH 7.9, 400 mmol/L NaCl, 2 mmol/L EDTA, 1 mmol/L RMA and RMA/S was performed through analysis of levels of MHC DTT, 3 mg/mL aprotinin, 2 mg/mL Pepstatin, and 1 mg/mL class I (H2-Kb and H2-Db). Expression of H60 was used to authen- leupeptin), followed by incubation on ice for 15 minutes. The ticate EL4-H60 and the parental EL4 cell lines. YAC-1 background mixtures were spun at 16,000 Â g for 5 minutes, and the super- was verified through lack of H-2b and the presence of H-2a.Allcell natants were collected as the nuclear extracts. lines are tested every 3 months for and are negative for Mycoplasma. Immunoblotting Antibodies and flow cytometry Whole-cell lysate (15–20 mg) or nuclear extracts (10 mg) were Antibodies for NK1.1 (PK136), CD3e (17A2, 145-2C11), resolved using 10% SDS-PAGE gels, transferred to polyvinylidene NKG2D (A10), CD137 (17b5), CD137L (TKS-1), and anti- difluoride (PVDF) membranes, and probed with indicated anti- human IFNg (MG1.2) were from e-Bioscience. Anti-Ly49D bodies. Antibodies for b-actin (ACTBD11B7), TRAF6 (H-274), and (4E5) and RIP1 were from BD Pharmingen.