MicroRNA-15b Suppresses Th17 Differentiation and Is Associated with Pathogenesis of Multiple Sclerosis by Targeting O-GlcNAc This information is current as of September 28, 2021. Ruiqiong Liu, Xiaofeng Ma, Li Chen, Yang Yang, Yi Zeng, Jie Gao, Wei Jiang, Fang Zhang, Daojing Li, Bin Han, Ranran Han, Rongfang Qiu, Wei Huang, Yan Wang and Junwei Hao

J Immunol 2017; 198:2626-2639; Prepublished online 22 Downloaded from February 2017; doi: 10.4049/jimmunol.1601727 http://www.jimmunol.org/content/198/7/2626 http://www.jimmunol.org/

Supplementary http://www.jimmunol.org/content/suppl/2017/02/21/jimmunol.160172 Material 7.DCSupplemental References This article cites 74 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/198/7/2626.full#ref-list-1 by guest on September 28, 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

MicroRNA-15b Suppresses Th17 Differentiation and Is Associated with Pathogenesis of Multiple Sclerosis by Targeting O-GlcNAc Transferase

Ruiqiong Liu,*,†,1 Xiaofeng Ma,*,1 Li Chen,‡ Yang Yang,† Yi Zeng,† Jie Gao,† Wei Jiang,* Fang Zhang,* Daojing Li,* Bin Han,* Ranran Han,* Rongfang Qiu,† Wei Huang,† Yan Wang,† and Junwei Hao*

IL-17–producing Th17 cells have gradually become considered as key factors in the pathogenesis of many autoimmune diseases, including multiple sclerosis (MS). Although the involvement of certain microRNAs in the development of MS has been reported, their role in Th17-driven autoimmunity is still poorly understood. In this study, we identified microRNA (miR)-15b as an important factor in Th17-associated effects and determined that the expression of miR-15b is significantly downregulated in Downloaded from MS patients and in mice with experimental autoimmune encephalomyelitis. Overexpression of miR-15b alleviated experimental autoimmune encephalomyelitis, whereas knockdown of miR-15b aggravated it. We demonstrated that miR-15b suppressed Th17 differentiation both in vivo and in vitro. We also found that O-linked N-acetylglucosamine transferase is a potential target of miR-15b, enabling it to affect the transcriptional regulation of retinoic acid–related orphan receptor gT through O-linked N-acetylglucosamine glycosylation of NF-kB. These results contribute to the importance of miR-15b in Th17 differentiation and the pathogenesis of MS. The Journal of Immunology, 2017, 198: 2626–2639. http://www.jimmunol.org/

ultiple sclerosis (MS) is a prototypic inflammatory partial remission; this is the relapsing/remitting form of MS autoimmune disorder characterized by chronic demy- (RRMS) (3, 4). Although the specific details of MS pathogenesis M elination and neurodegeneration of the CNS. MS is remain poorly understood, CD4+ T cell–mediated autoimmunity among the most common causes of neurologic disability in young may be one of the most important components (5). Except for Th1 adults (1, 2). In almost 80% of these patients, the disease starts cells, which are characterized by their production of IFN-g, Th17 with episodes of neurologic dysfunction followed by complete or cells are likely the most critical pathogenic factor of human MS

and of its most widely used animal model, experimental autoim- by guest on September 28, 2021 mune encephalomyelitis (EAE) (6, 7). *Department of Neurology, Tianjin Neurological Institute, Tianjin Medical Univer- + sity General Hospital, Tianjin 300052, China; †Tianjin Key Laboratory of Medical Upon activation, naive CD4 T cells differentiate into several Epigenetics, Department of Biochemistry and Molecular Biology, Tianjin Medical subsets of cells with distinct functions. One of these subsets in- ‡ University, Tianjin 300070, China; Department of Neurology, Tianjin Huanhu Hos- cludes Th17 cells, producers of IL-17 (8, 9). These cells are pri- pital, Tianjin 300350, China marily involved in mediating inflammatory diseases and in 1R.L. and X.M. contributed equally to this work. immune defenses against extracellular bacteria. Received for publication October 6, 2016. Accepted for publication January 30, 2017. The orphan nuclear receptor, retinoic acid–related orphan re- ceptor (ROR)gT, has been described as the key transcription factor This work was supported by National Natural Science Foundation of China Grants 81571600, 81322018, 81273287, and 81100887 (to J.H.), 81322032 and 81472733 that orchestrates the differentiation of Th17 lineage (10, 11). (to Y.W.), and 81401361 (to X.M.), Ministry of Education of China Grants NCET-12- Recent studies have shown that NF-kB, especially c-Rel and p65, 1067, FANEDD-201231, and RFDP-20131202110012 (to Y.W.), and by grants from the Youth Top-Notch Talent Support Program (to J.H.) and from the National Key drives Th17 differentiation by binding to the promoter and tran- Clinical Specialty Construction Project of China (to J.H.). scriptional regulation of RORgT expression (12, 13). This makes R.L., X.M., and J.H. designed the research; Y.W. and J.H. supervised the research; NF-kB an important factor in Th17 differentiation and autoim- R.L. and X.M. conducted the experiments; R.L., X.M., Y.W., and J.H. wrote the mune disease progression. manuscript and revised the manuscript; and L.C., Y.Y., Y.Z., J.G., W.J., F.Z., D.L., B.H., R.H., R.Q., and W.H. helped with the experiments. MicroRNAs (miRs) comprise a large family of small, noncoding Address correspondence and reprint requests to Dr. Yan Wang or Dr. Junwei Hao, RNAs that play an important role in posttranscriptional regulation of Tianjin Key Laboratory of Medical Epigenetics, Department of Biochemistry and expression (14). Dysregulation of miRs leads to many kinds of Molecular Biology, Tianjin Medical University, Tianjin 300052, China (Y.W.) or human diseases, including cancer, diabetes mellitus, neurologic Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University, General Hospital, Anshan Road, 154 Heping, Tianjin 300052, China (J.H.). E-mail disorders, cardiovascular disease, autoimmune disease (15–19). addresses: [email protected] (Y.W.) or [email protected] (J.H.) Therefore, miRs appear to be good diagnostic markers, therapeutic The online version of this article contains supplemental material. targets, or prognostic markers (20, 21). Until now, many reports Abbreviations used in this article: B6, C57BL/6J; ChIP, chromatin immunoprecipi- have involved miRs in autoimmunity, especially as a central factor tation; DLN, draining lymph node; EAE, experimental autoimmune encephalomy- in MS and EAE, such as miR-23b, miR-132, miR-155, miR-301a, elitis; miR, microRNA; MOG, myelin oligodendrocyte glycoprotein; MRI, magnetic resonance imaging; MS, multiple sclerosis; O-GlcNAc, O-linked N-acetylglucos- and miR-326 (22–26). However, the possible role of miRs in the amine; O-GlcNAcylation, O-GlcNAc glycosylation; ROR, retinoic acid–related or- pathogenesis and treatment of MS is still ambiguous. phan receptor; RRMS, relapsing/remitting multiple sclerosis; Treg, regulatory T cell; O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) UTR, untranslated region; WT, wild-type. is a that catalyzes the addition of a single Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 N-acetylglucosamine in O-glycosidic linkage to serine or threonine www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601727 The Journal of Immunology 2627 residues (27) of peptides or proteins. Knockdown of OGT impairs pensions were first mixed with 10 ml of CD4 T cell MicroBeads (Miltenyi 7 + early T cell activation (28). Many proteins of importance in cel- Biotec) per 10 total cells. After incubation at 4˚C for 10 min, CD4 Tcells lular processes have been found to contain O-GlcNAc glycosyl- were purified in a suitable MACS column (Miltenyi Biotec) in the magnetic field. Subsequently, flow-through was mixed with CD8 T cell MicroBeads ation (O-GlcNAcylation) sites (29). NF-kB is one of them. (Miltenyi Biotec) and target cells were isolated as described above. The Reports have noted that c-Rel and p65 are O-GlcNAcylated at purity of isolated cells was routinely .90%, as determined by flow Ser350 and Thr352, respectively, and that O-GlcNAcylation modi- cytometry analysis, and these cells were considered suitable for downstream fication is important for the transcriptional activity of both (30, application, as the manufacturer instructed (35). After mice were sacrificed, their splenic monocytes were purified as previously described (36). Murine 31). Therefore, OGT is a vital factor for lymphocyte activation and CD4+ T cells were then isolated from the splenic monocytes by using mouse the ability to incite inflammation (28, 32, 33). CD4 MicroBeads (Miltenyi Biotec) as described above. In the present study, we demonstrated that miR-15b is an im- Induction and evaluation of EAE portant actor in the regulation of Th17 cell differentiation and MS pathogenesis. We observed that the expression of miR-15b was The mouse peptide autoantigen, myelin oligodendrocyte glycoprotein . downregulated in CD4+ T cells from patients with MS. The (MOG)35–55, was synthesized (purity 95%) by and obtained from Bio- Synthesis. To induce acute active EAE, we injected B6 mice s.c. in the hind overexpression of miR-15b alleviated EAE; however, EAE was flank with 200 mgofMOG35–55 peptide in CFA (BD Biosciences) containing aggravated by the knockdown of miR-15b. Finally, miR-15b 500 mg of nonviable desiccated Mycobacterium tuberculosis. On the day of, negatively regulated Th17 differentiation in vivo and in vitro by and 2 d after, immunization, the mice were also inoculated i.p. with 200 ng of targeting OGT, which in turn affected the transcriptional regula- (List Biological Laboratories). Adoptive transfer (passive) EAE 3 6 tion of RORgT through O-GlcNAcylation of NF-kB. was induced by injecting recipient mice with 5.0 10 polarized MOG- specific Th17 cells. On the day of, and 2 d after, transfer, the mice were also inoculated i.p. with 200 ng of pertussis toxin (List Biological Laboratories). To Downloaded from Materials and Methods prepare MOG-specific polarized Th17 cells, we immunized donor mice with Mice MOG35–55 s.c. and prepared draining lymph node cells from donor mice 12 d after immunization. We cultured cells for 5 d with MOG35–55 at a concentration C57BL/6J (B6; wild-type [WT]) mice were purchased from the Academy of of 25 mg/ml under Th17 cell–polarizing conditions. After induction, neurologic Military Medical Science (Beijing, China). All these experimental mice deficit was assessed with clinical scores according to clinical syndrome of mice, were maintained under specific pathogen-free conditions in the animal ranging between 0 and 5. Having tail paralysis was assigned a score of 1,

facility at Tianjin Medical University (Tianjin, China) and were used at quadriplegia was assigned a score of 4, and mortality was defined as a score of 5. http://www.jimmunol.org/ 6–10 wk of age. All animal procedures were approved by the Tianjin Medical University Experimental Animal Ethics Committee. Histology Human subjects For histological analysis, the EAE mice were fixed by transcardial perfusion with 4% (w/v) paraformaldehyde, and then the spinal cords were embedded Patients recruited from Tianjin Medical University General Hospital and in paraffin. Sections (6 mm thick) were then deparaffinized, rehydrated, and Tianjin Huanhu Hospital were enrolled, and all met the revised McDonald stained with H&E or Luxol fast blue for inflammation or demyelination criteria for MS (34). Patients with a history of additional CNS diseases, analysis, respectively. tumor or systemic hematologic diseases, or infections were excluded. Age- and gender-matched healthy volunteers were also recruited for control Flow cytometry subjects. Blood samples were obtained from each subject after an informed Mice were sacrificed and transcardially perfused with PBS. Draining lymph by guest on September 28, 2021 consent was signed. All experimental protocols were approved by the In- nodes (DLNs) were removed and disrupted by passing through wire mesh to stitutional Review Board at Tianjin Medical University General Hospital. obtain single-cell suspensions. Erythrocytes were lysed by 5-min incubation 3 Cell lines of 1 lysing solution (BD Biosciences). For analysis of CNS-infiltrated cells, spinal cords and brains were dissociated with a 40-mm cell strainer, The cell lines used were obtained from the American Type Culture Col- and mononuclear cell suspensions were obtained by centrifuge with Per- lection. HEK 293T cells were cultured in DMEM (HyClone; Thermo coll (GE Healthcare). Cells were cultured in the growth medium with the Fisher) with 10% (v/v) FBS. Jurkat cells were cultured in RPMI 1640 protein transport inhibitor, brefeldin A, ionomycin, and PMA for 6 h. For (HyClone; Thermo Fisher) with 10% (v/v) FBS and maintained in a hu- T cell activation and proliferation assays, anti-CD4 Ab and anti-Ki67 with midified incubator containing 5% CO2 at 37˚C. fluorochrome were incorporated with treated cells according to the man- ufacturer’s instructions. CFSE was also used in the proliferation assay, and Cell culture and Th17 cell differentiation in vitro labeling was performed as described previously (36). Anti–IFN-g Ab, anti–IL-17A Ab, anti-CD25 Ab, and anti-FOXP3 Ab conjugated with The isolated human and mouse CD4+ T cells were cultured in RPMI 1640, fluorochrome were also used in this study (all Abs were purchased from supplemented with 10% (v/v) FBS, 100 mM nonessential amino acid, BioLegend). Data were acquired on a FACSAria III (BD Biosciences) and 1 mM sodium pyruvate, 55 mM 2-ME, 100 U/ml penicillin, and 100 mg/ml analyzed with FlowJo (Tree Star). streptomycin (all purchased from Life Technologies) at 37˚C in the pres- + ence of 5% CO2. For assessing CD4 T cell activation and expansion, we Oligonucleotide and transfection used untreated cells or cells treated with miR mimics or scramble controls. These cells were cultured with anti-CD3 (1 mg/ml)/anti-CD28 (5 mg/ml) Lipofectamine RNAiMAX reagent was used according to the manufacturer’s mAbs (BioLegend) for 12 or 72 h. For the Th17 polarization assay, CD4+ protocol. Cells were seeded onto culture plates the day before transfection to T cells treated with miR mimics or scramble controls were cultured with ensure a suitable cell confluence on the day of transfection. For the transfection anti-CD3 (1 mg/ml)/anti-CD28 (5 mg/ml) mAbs (BioLegend) together with of miR, 50 nM negative control RNA, miR-15b mimics (59-UAGCAGCA- IL-6 (10 ng/ml), IL-1b (10 ng/ml), TGF-b1 (10 ng/ml), and IL-23 (10 ng/ml) CAUCAUGGUUUACA-39), and inhibitors (59-UGUAAACCAUGAUGUG- (all purchased from PeproTech), and the neutralizing Abs anti–IL-4 CUGCUA-39) (GenePharma, Shanghai, China) were used. Human and mouse (10 mg/ml) and anti–IFN-g (10 mg/ml) (BioLegend) for 6 d. miR-15b share the same conservative sequence. miR-15b agomir is a double- strand miR-15b mimic with chemical modification that can mimic mature T cell isolation and sorting endogenous miRs after transfection into cells. The antisense strand has two 9 Human PBMCs were isolated from blood samples of patients and healthy phosphorothioates at the 5 end, four phosphorothioates and four cholesterol + 9 9 controls via Ficoll density gradient centrifugation. For naive CD4 T cell groups at the 3 end, and full-length nucleotide 2 -methoxy modification isolation, PBMCs were suspended in PBS buffer containing 0.5% (v/v) (GenePharma). miR-15b antagomir is single-strand miR-15b inhibitor carrying BSA and 2 mM EDTA, then mixed with 10 ml of naive CD4+ T cell biotin– the same modifications and functions by blocking its regulation of target gene 7 expression efficiently (GenePharma). Ab cocktail II (Miltenyi Biotec) per 10 total cells. After incubation at 4˚C + for 5 min, 30 ml of buffer and 20 ml of naive CD4 T cell MicroBead Real-time quantitative RT-PCR cocktail II were added per 107 total cells and incubated at 4˚C for 10 min. Cell suspension was applied onto a suitable MACS column (Miltenyi Total RNA was isolated from samples with TRIzol reagent according to the Biotec) in the magnetic field, and flow-through containing unlabeled cells manufacturer’s instructions (Invitrogen). cDNA was prepared with Moloney was collected. For CD4+ T cell and CD8+ T cell isolations, PBMC sus- murine leukemia virus reverse transcriptase (Fermentas). Relative quantitation 2628 miR-15b REGULATES Th17 AND MS BY TARGETING OGT was determined using the ABI Prism 7500 sequence detection system Th1 and regulatory T cell polarization

(Applied Biosystems), which measures real-time SYBR Green fluorescence, + and then relative gene expression was calculated by means of the compar- The isolated human naive CD4 T cells were cultured in RPMI 1640, ative Ct method (22DDCt) with the expression of GAPDH as an inter- supplemented with 10% (v/v) FBS, 100 mM nonessential amino acid, nal control. The primers used are listed as follows: mmu-Il17 (forward, 1 mM sodium pyruvate, 55 mM 2-ME, 100 U/ml penicillin, and 100 mg/ml streptomycin (all purchased from Life Technologies) at 37˚C in the pres- 59-GGCCCTCAGACTACCTCAAC-39; reverse, 59-TCTCGACCCTGAAA- + GTGAAGG-39), mmu-Il21 (forward, 59-GGACCCTTGTCTGTCTGGTAG-39; ence of 5% CO2. For Th1 polarization assay, CD4 T cells treated with reverse, 59-TGTGGAGCTGATAGAAGTTCAGG-39), mmu-Il22 (forward, miR mimics or scramble controls were cultured with anti-CD3 (1 mg/ml)/ 59-ATGAGTTTTTCCCTTATGGGGAC-39;reverse,59-GCTGGAAGTTG- anti-CD28 (5 mg/ml) mAbs (BioLegend) together with IL-2 (5 ng/ml) and GACACCTCAA-39), mmu-Il23r (forward, 59-TTCAGATGGGCATGAAT- IL-12 (10 ng/ml) (both purchased from PeproTech) and the neutralizing Ab anti–IL-4 (10 mg/ml) (BioLegend) for 6 d. For regulatory T cell (Treg) GTTTCT-39;reverse,59-CCAAATCCGAGCTGTTGTTCTAT-39), mmu-Il6 + (forward, 59-CTGCAAGAGACTTCCATCCAG-39;reverse,59-AGTGGT- polarization assays, CD4 T cells treated with miR mimics or scramble ATAGACAGGTCTGTTGG), mmu-Il4 (forward, 59-GGTCTCAACCCC- controls were cultured with anti-CD3 (1 mg/ml)/anti-CD28 (5 mg/ml) CAGCTAGT-39;reverse,59-GCCGATGATCTCTCTCAAGTGAT-39), mAbs (BioLegend) together with IL-2 (5 ng/ml) and TGF-b1 (10 ng/ml) mmu-Il10 (forward, 59-GCTGGACAACATACTGCTAACC-39; reverse, (both purchased from PeproTech) for 6 d. 59-ATTTCCGATAAGGCTTGGCAA-39), mmu-Ifng (forward, 59-GCCAC- Cell sorting and culture GGCACAGTCATTGA-39;reverse,59-TGCTGATGGCCTGATTGTCTT-39), mmu-Tgfb (forward, 59-CTCCCGTGGCTTCTAGTGC-39;reverse,59-GCC- At day 12 after immunization, mice were sacrificed and transcardially TTAGTTTGGACAGGATCTG-39 ), mmu-Ogt (forward, 59-GACGCAACCA- perfused with PBS. To obtain single-cell suspensions, DLNs were removed AACTTTGCAGT-39; reverse, 59-TCAAGGGTGACAGCCTTTTCA-39), and disrupted by passing through wire mesh. CD11c+ dendritic cells and mmu-Gapdh (forward, 59-TGACCTCAACTACATGGTCTACA-39;reverse, F4/80+ macrophages were enriched by using indicated Ab-conjugated 59-CTTCCCATTCTCGGCCTTG-39), hsa-OGT (forward, 59-CCATTCA- magnetic beads (MACS; Miltenyi Biotec) according to the manufac- TAAGGATTCAGGGA-39;reverse,59-GAGCCAAGTTACAATAAGCA-39), turer’s instructions. For cytokine assay, dendritic cells were cultured in Downloaded from hsa-IL-2 (forward, 59-AACTCCTGTCTTGCATTGCAC-39;reverse,59-GC- 96-well plates for 24 h in the presence of LPS (1 mg/ml; Sigma-Aldrich). TCCAGTTGTAGCTGTGTTT-39), hsa-CSF2 (forward, 59-TCCTGAACCT- For the sorting of CNS-infiltrated cells, spinal cords and brains were GAGTAGAGACAC-39; reverse, 59-TGCTGCTTGTAGTGGCTGG-39), dissociated with a 40-mm cell strainer, and mononuclear cell suspensions hsa-COX2 (forward, 59-TAAGTGCGATTGTACCCGGAC-39; reverse, were obtained by centrifuge with Percoll (GE Healthcare). CD11b+CD45int 59-TTTGTAGCCATAGTCAGCATTGT-39), hsa-RORgT (forward, 59-CCG- microglia in the CNS were sorted by using a FACSAria III cell sorter (BD CTGAGAGGGCTTCAC-39; reverse, 59-TGCAGGAGTAGGCCACATT- Biosciences) with indicated gating strategy. For cytokine assays, macro- ACA-39), hsa-GAPDH (forward, 59-GGAGCGAGATCCCTCCAAAAT-39;

phages and microglia were cultured in 96-well plates for 24 h in the http://www.jimmunol.org/ reverse, 59-GGCTGTTGTCATACTTCTCATGG-39), miR-15b (reverse tran- presence of LPS (100 ng/ml; Sigma-Aldrich). scription, 59-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCAC- TGGATACGACTGTAAACC-39;forward,59-GTAGCAGCACATCATGGT- Cytokine assay TTACA-39;reverse,59-CAGTGCGTGTCGTGGAGT-39), U6 (reverse tran- scription, 59-AAAATATGGAACGCTTCACGAATTTGC-39;forward,59- After 24 cultivation, the cell culture medium was collected and then CCTGCTTCGGCAGCACA-39; reverse, 59-TGGAACGCTTCACGAA-39). centrifuged at 1500 rpm for 10 min. The supernatants were harvested for assay. Cytokines in supernatants, including IL-1b, IL-6, IL-10, IL-12, Immunoprecipitation and Western blotting TNF-a, and TGF-b, were analyzed by using a multi-analyte ELISArray kit (Qiagen) according to the instructions of manufacturer as previously Jurkat cellular extracts were incubated with cold lysis buffer (150 mM NaCl, described (38). 1 mM EDTA, 50 mM Tris-HCl [pH 7.4], 0.25% [w/v] sodium deoxycholate, by guest on September 28, 2021 1% [w/v] Nonidet P-40 and protease inhibitor mixture) at 4˚C for 30 min. For immunoprecipitation, 500 mg of protein was incubated by constant Results rotation at 4˚C with specific Abs (2–3 mg) overnight. Then, 60 mlof50% Expression of miR-15b is downregulated in patients with MS (v/v) protein A or G agarose beads were added and incubated for an addi- tional 2 h. After that, beads were washed five times with the lysis buffer. The To determine whether the expression of miR-15b is altered in patients beads were resuspended in 23 SDS-PAGE loading buffer and boiled for with MS, we first searched Gene Expression Omnibus (http://www. 10 min to elute the precipitated proteins. The resultant products from im- ncbi.nlm.nih.gov/geo/) for raw data from miR expression profiles of munoprecipitation or cell lysates were resolved using 10% SDS-PAGE gel MS patients and healthy control subjects and analyzed the differential electrophoresis and transferred onto acetate cellulose membranes. For expression of some miRs. We found that miR-15b was downregu- Western blotting, membranes were incubated at 4˚C overnight with appro- , priate Abs followed by incubation with a secondary Ab. Immunoreactive lated (p 0.01) in clinical datasets GSE21079 (39) and GSE43590 bands were visualized by using Western blotting luminol reagent (Santa (40) (Fig. 1A–D). To verify the downregulation of miR-15b in pa- Cruz Biotechnology) following the manufacturer’s recommendations. tients with MS, we collected blood samples from healthy control subjects and MS patients and isolated PBMCs for miR expression Luciferase assay analysis (Table I). These analyses confirmed the significantly lower The WT and mutant OGT 39–untranslated region (UTR) were amplified by miR-15b expression in PBMCs of patients with RRMS compared PCR and cloned in pMIR-REPORT (Ambion) with firefly luciferase. with healthy control subjects (Fig. 1E). More remarkably, miR-15b Jurkat cells were transfected with WT or mutants of OGT 39-UTR lucif- expression was even more significantly lower in the CD4+ T cells but erase reporters and Renilla plasmid after treatment with control miR, miR- + 15b mimics, or miR-15b inhibitors. The firefly and Renilla luciferases were not in the CD8 T cells or non–T cells of MS patients (Fig. 1F). assayed following the manufacturer’s instructions (Promega) after 48 h, and firefly luciferase activity was normalized to that of Renilla luciferase. miR-15b inhibits T cell activation and proliferation, as well as Each experiment was repeated in triplicate. Th17 differentiation in vitro Because miR-15b proved to be downregulated in patients with MS, Chromatin immunoprecipitation we next investigated the potential role of miR-15b in T cell activation Chromatin immunoprecipitation (ChIP) assays were performed in Jurkat and proliferation, which are critical steps in T cell–mediated cells as described previously (37). Briefly, after having been cross-linked 3 7 responses. For that purpose, we transfected Jurkat cells and human with 1% (v/v) formaldehyde, 1 10 cells were sonicated and diluted + 10-fold with ChIP dilution buffer, following incubation with 3–5 mgof CD4 T cells with either miR-15b mimics or a scrambled miR specific Ab overnight. After adding beads, complexes were washed with control. After stimulation with anti-CD3 and anti-CD28 mAbs for low- and high-salt buffers. The final elution step was performed by using 12 h, T cell activation was analyzed by flow cytometry. Membrane Tris-EDTA buffer (pH 8) and 1% SDS solution. Primers specific for each expression of CD69, an early activation marker of CD4+ Tcells, target gene promoter were used to analyze the enrichment of the DNA increased markedly after stimulation, whereas miR-15b inhibited template by conventional PCR. The primers used are listed as follows: + RORgT promoter forward primer, 59-TCCACTGTCAATGACCCTAGG-39; that response in both Jurkat cells and primary human CD4 Tcells RORgTpromoterreverseprimer,59-GTCTCGTCTCGCCATCCTC-39. (Fig. 2A, 2B). The Journal of Immunology 2629

FIGURE 1. Downregulation of miR-15b A D in patients with MS. ( – ) Analysis of Downloaded from two published clinical datasets GSE21079 and GSE43590 showed downregulation of miR-15b in patients with MS. (E) Quanti- tative PCR analysis of miR-15b expression in PBMCs from healthy control subjects (n = 40) and patients with RRMS (n = 37). + (F) Expression of miR-15b in CD4 Tcells, http://www.jimmunol.org/ CD8+ T cells, and non–T cell fraction of PBMCs from MS patients and controls (n = 10 per group). Results are normalized to those of controls and are presented rela- tive to expression of the small nuclear RNA RNU6B. *p , 0.05, **p , 0.01, ***p , 0.001, versus healthy control subjects (Student t test). HC, healthy control; MS, RMMS. by guest on September 28, 2021

To explore the effect of miR-15b on T cell proliferation, we used pressed the early activation and proliferation of CD4+ T cells and a proliferation assay with anti-CD3 and anti-CD28 added as decreased the expression of IL-17A. stimuli. Flow cytometry analysis indicated that miR-15b treatment impaired the proliferation of Jurkat cells and primary CD4+ T cells miR-15b suppresses the pathogenesis and development of EAE (Fig. 2C, 2D). The differentiation of primary human naive CD4+ Because EAE is the most widely used animal model for MS, we T cells in the presence of vehicles, miR-15b mimics, or scramble first sought published raw data of miR expression profiles of EAE controls was also determined by using flow cytometry. Transfec- mice in the Gene Expression Omnibus. However, we found only tion of the expression of miR-15b reduced IL-17A expression one dataset. miR-15b expression in CD4+ T cells from mice at 16 compared with scramble controls (Fig. 2E), whereas the polari- or 32 d after the inception of EAE was only about half that from zation of CD4+ T cells into the Th1 or Treg lineage were controls in dataset GSE66621 (Fig. 3A). We next purified CD4+ not remarkably altered by miR-15b intervention (Supplemental T cells from the spleens of mice with EAE or sham control mice at Fig. 1). These results suggest that miR-15b intervention sup- day 18 after immunization to determine the expression of miR-15b. 2630 miR-15b REGULATES Th17 AND MS BY TARGETING OGT

Table I. Characteristics of healthy controls and multiple sclerosis patients

Healthy Control Multiple Sclerosis p Value Sample number 40 37 — Age, y 6 SD 37.7 6 6.9 40.3 6 5.4 0.07 Gender 1.0 Female 21 (52.5%) 19 (51.4%) — Male 19 (47.5%) 18 (48.6%) — Clinical characteristics Age at onset, y, median (range) — 37.4 (28–47) — Disease duration from first clinical — 2.9 (1–12) — symptom, y, median (range) OCBs positive/tested (%) — 28/37 (75.7%) — Brain MRI abnormalities (%) — 37/37 (100%) — EDSS score, median (range) — 1.9 (0–6) — Sample number is the total number of subjects; gender is total number (with percentage of group in parentheses). EDSS, expanded disability status score; OCB, oligoclonal band.

Similar to the outcomes in patients with MS, our results using a cells, macrophages from the DLNs, as well as in microglia from the model animal revealed that significantly less miR-15b was present CNS. Moreover, we also analyzed the expression of MHC class II and in CD4+ T cells of EAE mice (Fig. 3B). cytokine production in dendritic cells, macrophages, and microglia, and Downloaded from Instead of recombinant lentivirus, we selected the chemically no remarkable difference was found (Supplemental Fig. 2). We did not engineered oligonucleotides agomir and antagomir to inject into mice, detect any significant differences in flow cytometry of T cells, B cells, which are more easily synthesized, less harmful, and much safer for use CD4+ T cells, or CD8+ T cells from DLNs (Fig. 4A, Supplemental in animals and humans. Agomir and antagomir are improved from miR Fig. 3). However, CD4 surface staining of CNS leukocyte infiltrates mimics and inhibitors, because of their methylation, phosphorothioate, revealed that fewer CD4+ T cells accumulated in the CNS of agomir- and cholesterol modifications. These multiple modifications make them injected mice compared with those in those control mice, whereas more http://www.jimmunol.org/ moreaffinitiveandstable;thus,theyareeasiertobeusedfortrans- CD4+ T cells were detected in antagomir-injected mice (Fig. 4B). fection and long-term expression. We chose to use an miR injection Next, we examined effector T cells from the DLNs and CNS. method referenced from published research (41) and, thereby, delivered Intracellular cytokine staining showed that the frequencies of Th17 miR to mice i.v. through the tail vein on days 7, 11, 15, and 18 after the cells, but not Th1, Th2, or Tregs, in the CD4+ population was lower induction of EAE. During the intervention, we assessed the expression in agomir-injected mice and higher in antagomir-injected mice of miR-15b in various organs by quantitative PCR analysis of miR-15b (Fig. 4C, 4D, Supplemental Fig. 3). Additionally, when we con- agomir–injected, miR-15b antagomir–injected, and control miR– sulted published data for miR expression profiles between mouse injected mice. Consistent with our prediction, the expression level of naive T cells and Th17 cells (42), those results were consistent with by guest on September 28, 2021 miR-15b continuously increased in several organs of miR-15b agomir– our findings. That is, the expression of miR-15b was higher in naive injected mice whereas that of miR-15b in antagomir-injected mice was T cells than that in Th17 cells (p , 0.05), supporting the conclusion unchanged compared with the control mice (Fig. 3C). The miR-15b that miR-15b suppresses Th17 differentiation (Fig. 4E, 4F). antagomir was a reverse complement of miR-15b, which specifically We next examined the expression of cytokines related to the bound to and inhibited endogenous miR molecules. Therefore, the Th cell subsets. specific for the Th17 lineage, including Il17, miR-15b antagomir blocked miR regulation of target gene expression Il22, Il21, and Il23r, were significantly downregulated in agomir- efficiently but the expression level of miR-15b was not affected. injected mice and upregulated in antagomir-injected mice, To investigate the role of miR-15b in MS pathogenesis, we first whereas there was no significant difference between the three induced EAE by immunizing mice with an encephalitogenic groups in the expression of genes related to other Th cell subsets, peptide, MOG, consisting of aa 35–55. These mice were then such as Ifng, Il4, Il10, and Tgfb (Fig. 4G). injected with agomir and antagomir, as described above. The re- To validate the effect of miR-15b on Th17 lineage, we induced sults showed that the agomir-injected mice developed mild EAE, passive EAE by means of adoptive transfer into naive recipients with whereas the antagomir-injected mice had severe EAE, both MOG-specific Th17 cells treated or not with miR-15b agomir or compared with the control mice (Fig. 3D). antagomir. We found that WT recipients receiving cells treated with Histological analysis of spinal cord sections revealed that miR- miR-15b agomir developed mild EAE, whereas the WT recipients 15b–injected mice developed mild inflammatory infiltration and receiving cells treated with miR-15b antagomir had severe EAE demyelination, whereas the antagomir-injected mice showed (Fig. 4H). Cytokine production of CNS-infiltrating CD4+ Tcellsby prominent pathologic effects (Fig. 3E, 3H, 3I). T2-weighted images flow cytometry analysis further supported that miR-15b suppresses of brain or spinal cord were obtained with a 7T magnetic resonance Th17 differentiation in EAE (Fig. 4I, 4J). imaging (MRI) scanner as previously reported (36). MRI results OGT is a downstream target of miR-15b were consistent with the histological analysis (Fig. 3F, 3J, 3K). We performed immunofluorescence to detect the expression of myelin Subsequently, we applied the miR target prediction algorithms basic protein, which confirmed that the demyelination in agomir- PicTar, TargetScan, and miRanda to identify the potential down- injected mice was slight and that fewer inflammatory cells had stream targets of miR-15b. In that context, we found a putative miR- infiltrated their spinal cords (Fig. 3G). 15b binding site within 39-UTR of OGT and demonstrated that this binding site is evolutionarily conserved (Fig. 5A). To evaluate our miR-15b suppresses Th17 differentiation in EAE hypothesis that OGT is a downstream target of miR-15b, we used To identify the immune cell subsets predominantly associated with the reporter assays in HEK 293T cells with luciferase report vectors differing outcomes in groups of sham control– and miR-injected mice, that contain the putative normal or mutant miR-15b binding sites we examined different cell subsets from their DLNs and CNS. There within the 39-UTR (Fig. 5B). miR-15b mimics inhibited the lucif- were no significant differences in flow cytometry counts of dendritic erase activity of the reporter containing the WT OGT 39-UTR (WT) The Journal of Immunology 2631

FIGURE 2. miR-15b inhibits T cell activation and proliferation, as well as Th17 differentiation in vitro. (A and B) Jurkat cells and human CD4+ T cells were activated with CD3 and CD28 Abs for 12 h after transfection with miR-15b mimics or control. CD69 expression was detected by flow cytometry. n =6 per group for experiments both in Jurcat cells and human CD4+ T cells. (C) Jurkat cells were acti- vated with CD3 and CD28 Abs for 72 h after transfection with miR-15b mimics or control. Ki67 expression Downloaded from was detected by flow cytometry. n =6 per group. (D) CFSE-labeled human CD4+ T cells were activated with CD3 and CD28 Abs for 72 h after transfection with miR-15b mimics or control. n = 6 per group. (E) Naive http://www.jimmunol.org/ human CD4+ T cells were activated with CD3 and CD28 Abs for 6 d in the culture medium containing Th17 polarization cytokines after trans- fection with miR-15b mimics or control. Cells were intracellularly stained by fluorochrome-conjugated Ab against IL-17A and IFN-g and analyzed by flow cytometry. n =6 per group. *p , 0.05, **p , 0.01 by guest on September 28, 2021 (Student t test). ns, not significant.

whereas miR-15b inhibitors promoted that activity. However, nei- OGT is involved in miR-15b–regulated EAE ther of them influenced the luciferase activity of the reporter with a We evaluated the involvement of OGT in miR-15b–regulated EAE mutant 39-UTR that was unable to bind to miR-15b (Fig. 5C, 5D). mice. CD4+ T cells from the spleens of WT naive mice and miR- Because the ability of miRs to silence genes by mRNA desta- injected EAE mice were tested for the expression of OGT and miR- bilization or translational inhibition has been reported (43), we next 15b. The results showed that the expression of OGT was upregulated researched the impact of miR-15b on the expression levels of in EAE mice and was negatively correlated with miR-15b expression OGT mRNA and protein. For this purpose, we transfected HEK (Fig. 6A, 6B). Then, to demonstrate directly that OGT was a func- 293T and Jurkat cells with miR-15b mimics and then measured the tional target of miR-15b in vivo, we performed rescue experiments expression of OGT mRNA and protein using real-time quantita- with an OGT-expressing lentivirus containing Ogt coding sequence tive PCR and Western blotting, respectively. miR-15b over- but not 39-UTR, which was resistant to the regulation mediated by expression led to the downregulation of mRNA and protein levels miR-15b. Consistent with the previous results, miR-15b agomir- of OGT. Consistently, treatment with miR-15b inhibitors resulted injected mice developed mild EAE, whereas that effect was res- in an increase of OGT expression (Fig. 5E, 5F). cued by coinfection with lentivirus-expressing OGT (Fig. 6C). Although these data supported our hypothesis in model cell lines Clearly, this lentivirus-expressing OGT protein reversed the influence that OGT is a downstream target of miR-15b, we sought to confirm of miR-15b (Fig. 6D). Cytokine production of CNS-infiltrating CD4+ whether the inhibition of miR-15b to OGT occurs in healthy people T cells by flow cytometry analysis further supported our hypothesis and in patients with MS. As our previous outcomes led us to expect, that OGT is a functional target of miR-15b during Th17 differenti- we found that the mRNA and protein levels of OGT were higher in ation and in the pathogenic effects of MS (Fig. 6E, 6F). the patients than that in the healthy control subjects, whereas those of miR-15b were lower in the patients (Fig. 5G, 5H). Particularly, the miR-15b suppresses the transcription of RORgT through NF-kB mRNA expression of OGT correlated negatively with that of miR- Considering that NF-kB p65 and c-Rel are O-GlcNAcylated and 15b in the patients with RRMS (Fig. 5I). that the modification is necessary for their nuclear translocation 2632 miR-15b REGULATES Th17 AND MS BY TARGETING OGT Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. miR-15b suppresses the pathogenesis and development of EAE. (A) Analysis of published dataset GSE66621 shows downregulation of miR-15b + in EAE. (B) Gender- and age-matched B6 WT mice were immunized with MOG35–55 (EAE) or not (naive), as described in Materials and Methods.CD4 T cells were sorted from the spleens of naive or EAE mice at 14 d postimmunization. The expression of miR-15b was analyzed by quantitative RT-PCR. (C) Quantitative PCR analysis of miR-15b expression in the brain, lung, liver, heart, lymph nodes, and spleen of mice injected with miR-15b agomir, antagomir, or control as described in the text. Results are normalized to those from control-injected mice and are presented relative to RNU6B results. (D) Clinical scores for EAE in mice injected with miR-15b agomir, miR-15b antagomir, or control (n = 5 mice per group). (E, H,andI) Histology of paraffin sections of spinal cords isolated from miR-injected mice on day 21 after immunization. Scale bars, 100 mm. (F, J,andK) T2-weighted periventricular images were obtained with a 7T MRI scanner. Arrows indicate focal lesions. (G) Myelin basic protein (MBP) was detected by immunofluorescence of paraffin sections of spinal cords isolated from miR-injected mice on day 21 after immunization. Scale bars, 100 mm. *p , 0.05, **p , 0.01, ***p , 0.001 (Student t test). and transcriptional activity (30, 31), we next sought to probe the miR-15b, we performed coimmunoprecipitation in Jurkat cells to possible effect of miR-15b on NF-kB. First, we identified that detect the influence of miR-15b on the O-GlcNAcylation of miR-15b had no effect on the basal expression of p65 and c-Rel NF-kB. The results showed that, under the intervention of miR- (Supplemental Fig. 4). Because OGT was a downstream target of 15b, p65 and c-Rel were less physically associated with OGT and The Journal of Immunology 2633 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. miR-15b suppresses Th17 differentiation in EAE. (A and B) Quantification of CD4+ T cells isolated from DLNs or CNS of miR-injected mice on day 21 after immunization. (C and D) Intracellular staining of IL-17 and IFN-g in the cells in (A). Numbers in quadrants indicate percentage cells in the CD4+ gate. (E and F) Analysis of published datasets show downregulation of miR-15b in Th17 cells. (G) Quantitative PCR analysis of gene expression in the spleen cells in (B), presented relative to GAPDH expression. (H) Clinical scores for EAE induced by adoptive transfer of MOG-specific Th17 cells (n = 5 mice per group). (I) Intracellular cytokine staining of CNS infiltrates from the mice in (H). Numbers in quadrants indicate percentage IL-17+ IFN-g2 cells (top left) or IL-172IFN-g+ cells (bottom right) in the CD4+ gate. (J) Quantification of CNS Th17 cells from the mice in (I). *p , 0.05, **p , 0.01, ***p , 0.001 (Student t test). the O-GlcNAcylation was reduced accordingly, all of which was sion of a luciferase reporter gene as described previously (44). attributed to the downregulation of OGT (Fig. 7A, 7B). Next, we Consistent with the reduced O-GlcNAcylation, the luciferase fused p65 or c-Rel to Gal4 DNA-binding domain (Gal4-p65 or reporter assays revealed that miR-15b suppressed the tran- Gal4–c-Rel) and tested the effect of the construct on the expres- scriptional activity of p65 and c-Rel (Fig. 7C). Therefore, miR-15b 2634 miR-15b REGULATES Th17 AND MS BY TARGETING OGT Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. OGT is a downstream target of miR-15b. (A) Sequence alignment of OGT 39-UTR between human (Homo sapiens), chimpanzee (Pan troglodytes), dog (Canis familiaris), rat (Rattus norvegicus), and mouse (Mus musculus). (B) Luciferase reporter constructs for full-length WT and mutant (Mut) of OGT 39-UTR and analysis of OGT as a downstream target of miR-15b. Sequences of Wt and Mut target sites for miR-15b in OGT 39-UTR are shown. (C and D) Luciferase reporter assays were used to identify the binding and the site of miR-15b to OGT 39-UTR. After treatment with negative control miR (NC), miR-15b mimics (miR-15b), or miR-15b inhibitors (In-miR-15b), HEK 293T cells were transfected with WT or mutants of OGT 39-UTR luciferase reporters and Renilla plasmid, including the WT OGT 39-UTR as well as its Mut by electrotransfection. Each bar represents the mean 6 SD for triplicate experiments. The p values were determined by a Student t test. (E and F) miR-15b represses the expression of OGT mRNA and protein. En- dogenous OGT mRNA (top panel) and protein expression (bottom panel) were measured by real-time quantitative PCR and Western blotting, respectively. HEK 293T and Jurkat cells were transfected with control miR, miR-15b mimics, or miR-15b inhibitors for 48 h. The relative level of OGT mRNA was normalized to GAPDH. Each bar represents the mean 6 SD for triplicate experiments. (G) Real-time quantitative PCR analysis (Figure legend continues) The Journal of Immunology 2635 Downloaded from

+ FIGURE 6. OGT is involved in miR-15b–regulated EAE. (A) Immunoblot analysis of OGT protein expression in CD4 T cells sorted from the spleens http://www.jimmunol.org/ from miR-injected mice. Naive, B6 WT mice. (B) Linear correlation between the expression of miR-15b and that of OGT in CD4+ T cells from EAE mice (n = 10). The mRNA expression was measured by real-time quantitative PCR. (C) Clinical scores of EAE in mice injected with miR-15b agomir, lentivirus- expressing (LV) OGT, or control (n = 5 mice per group). (D) Immunoblot analysis of OGT expression in splenocytes from the mice in (C). (E) Intracellular cytokine staining of CNS infiltrates from the mice in (C). Numbers in quadrants indicate percentage IL-17+IFN-g2 cells (top left) or IL-172IFN-g+ cells (bottom right) in the CD4+ gate. (F) Quantification of CNS Th17 cells from the mice in (E). *p , 0.05, **p , 0.01, ***p , 0.001 (Student t test). suppressed the expression of NF-kB dependent genes, including inhibiting the expression of RORgT, which is a key factor that IL2, COX2, CSF2,andRORgT (12, 45–47) (Fig. 7D). directs differentiation of the Th17 lineage (Fig. 8).

To evaluate our hypothesis that miR-15b affects the tran- miR-15b has been widely studied in many fields, particularly as a by guest on September 28, 2021 scriptional regulation of RORgT through NF-kB transcription participant in numerous cancers (49–52), cardiac dysfunction (53), factors, ChIP assays were performed in Jurkat cells with Abs neocortical development (54), neonatal sepsis (55), angiogenesis against p65 or c-Rel. These assays were designed to detect the (56), and cerebral ischemic injury (57). We report in this study, to occupation of p65 or c-Rel on the promoter of RORgT, which our knowledge for the first time, the involvement of miR-15b in directs the differentiation of Th17 cells. Both semiquantitative MS and T cell responses. Generally, miRs are thought to function analysis and quantitative ChIP analysis showed an enrichment of by targeting proteins, which results in mRNA degradation or in- p65 or c-Rel on the promoter of RORgT but a reduction of that hibition of mRNA translation (43, 58). We documented in the under the intervention of miR-15b (Fig. 7E, 7F). Collectively, present study that OGT is a potential target of miR-15b. these results signify that miR-15b diminished the O-GlcNAcylation As a glycosyltransferase, OGT doubtlessly participates in many of p65 and c-Rel, which suppressed the transcriptional activation of cellular processes, including immune responses (33, 59). Recently, RORgT (Fig. 8). the remarkable impact of O-GlcNAc modification on immunity and inflammation was identified (32). In fact, several studies Discussion provided preliminary clues that O-GlcNAc modification is a par- MS, a demyelinating disease of obscure origin (48), is one of the ticipant in the pathogenesis of autoimmune diseases (60–62). crucial causes of neurologic disability in young adults. In this However, no further studies have been performed to reveal the study, we report that the pathogenesis of MS and EAE involves precise role of O-GlcNAc modification in CNS inflammation. In miR-15b as a regulator of T cell activation and proliferation as T lymphocytes, O-GlcNAc modification is dramatically increased well as for Th17 differentiation. We have established that the after activation by mitogen (63), whereas active OGT is requisite expression of miR-15b was downregulated in CD4+ T cells of for activating TCRs (28, 64), and T cell–specific deletion of OGT patients with MS. We found that intervention with miR-15b af- leads to a reduction in the total T cell number due to increased fected the progression of EAE and Th17 differentiation both apoptosis (64, 65). Alteration of O-GlcNAc modification is also a in vivo and in vitro. For a mechanistic explanation, we docu- component of T cell expression of cytokines and regulation of the mented that, by targeting OGT, a O-GlcNAcylation transferase, cell volume (66, 67). Later studies put a strong emphasis on the miR-15b reduced the transcriptional activity of NF-kB, thereby relationship between OGT-mediated protein O-GlcNAcylation and

of OGT mRNA level in PBMCs from clinical samples of controls (n = 40) and patients with MS (n = 37). The p values were determined by a Student t test. (H) Immunoblot analysis of OGT protein in PBMCs from clinical samples of controls and patients with MS. (I) Linear correlation between the expression of miR-15b and that of OGT in PBMCs from patients with RRMS (n = 37). The mRNA expression was measured by real-time quantitative PCR. *p , 0.05, **p , 0.01, ***p , 0.001. 2636 miR-15b REGULATES Th17 AND MS BY TARGETING OGT Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. miR-15b suppresses the transcription of RORgT through NF-kB. (A and B) Whole-cell lysates of Jurkat cells transfected with miR-15b mimics or control miR were immunoprecipitated with Abs against p65 or c-Rel. Immunocomplexes were then immunoblotted using Abs against the indicated proteins. IgG served as the negative control. TMG, thiamet-G. (C) Gal4-DBD-p65 or Gal4–DBD–c-Rel construct or a control vector (containing Gal4-DBD only) was transfected into Jurkat cells stably expressing the Gal4-UAS reporter. After transfection with miR-15b mimics or control, Gal4 luciferase reporter activity was measured. (D) Cells were transfected with miR-15b mimics or control for 24 h and then were treated with plate-bound anti- CD3 and anti-CD28 Abs for 3 h. Samples were analyzed by quantitative PCR to determine the abundance of IL-2, CSF2, COX2, or RORgT mRNA levels. (E and F) Semiquantitative ChIP analysis and quantitative ChIP analysis of RORgT promoter in the Jurkat cells after transfection with miR-15b mimics or controls using the indicated Abs. Results are represented as fold change over IgG. *p , 0.05, **p , 0.01, ***p , 0.001 (Student t test).

T cell functions (68). Interestingly, elevated OGT mRNA ex- the pathogenesis of MS and EAE (data not shown). Moreover, pression was identified by a microarray screening of PBMCs from several reported targets of miR-15b, such as BCL2 (57), BMI1 MS patients (69). Our work revealed that OGT is a key factor in (70), MMP-344, FUT2 (51), and TET3 (54), function in multiple The Journal of Immunology 2637 Downloaded from http://www.jimmunol.org/

FIGURE 8. Hypothetic scheme of the findings that miR-15b suppresses Th17 differentiation by targeting OGT. As discussed in the text, overexpression of miR-15b results in mRNA degradation of OGT by targeting its 39-UTR. The downregulation of OGT protein diminishes O-GlcNAcylated p65 or c-Rel, which are active forms of NF-kB as reported before. As a result, the transcriptional activity of NF-kB decreases on its target genes, especially RORgT, which is a key factor that directs differentiation of the Th17 lineage. Therefore, miR-15b suppresses Th17 differentiation and the pathogenesis of multiple sclerosis. In a word, our findings demonstrate that miR-15b negatively regulates Th17 differentiation by targeting OGT, which affects the transcriptional regulation of RORgT through O-GlcNAcylation of NF-kB. by guest on September 28, 2021 cellular processes. Of course, OGT is not the only target involved from the Th17 lineage (IL-17A, IL-17F, and IL-22). Furthermore, in miR-15b–regulated immune responses. we analyzed the relationship between NF-kB signaling and Th17 Th17 cells play a key role in CNS inflammation and autoim- polarization. Results showed that miR-15b treatment decreased munity (71). Myelin-reactive CD4+ T cells secreting IFN-g were the expression of OGT as well as the O-GlcNAc moiety on p65 usually considered to be the main effector subset causing in- and c-Rel proteins. Meanwhile, the transcriptional activities of flammation in MS, until recent research assigned Th17 key roles p65 and c-Rel were suppressed by miR-15b treatment; however, (72, 73). Th17 cells secrete proinflammatory cytokines, including ChIP assays indicated that RORgT expression is regulated by p65 IL-17A, IL-17F, GM-CSF, IL-22, and TNF-a. IL-6, IL-23, and and c-Rel. The foregoing results suggest that miR-15b modulates IL-1b are involved in the differentiation of Th17 cells. Th17 cell Th17 polarization by decreasing OGT expression in a NF-kB production of IL-17 and IL-22 could efficiently disrupt the tight p65– and c-Rel–dependent manner. junction of the blood–brain barrier, express high levels of the Because this report is an initial study on the role of miR-15b in cytolytic granzyme B, and promote T cells and other cells T cell immunity during the course of MS, a limitation should be of the immune system to migrate into the CNS (74). An effect of noted. The underlying mechanism of OGT, the miR-15 targeting the O-GlcNAcase inhibitor thiamet-G in mice with collagen- protein involved in the pathology of MS, is still ambiguous. induced arthritis was later identified as yielding an increase in Notwithstanding that limitation, this study does correlate miR-15b the proportions of Th1 and Th17 cells (62). and nutrient-driven O-GlcNAcylation with CD4+ T cell activation Our study examined the previously untouched area of the effect and differentiation in MS. Our study sheds considerable light on of miR-15b on Th17 differentiation in vivo and in vitro. Experi- the direction for developing a therapeutic approach for alleviating ments performed in vitro showed that miR-15b targeting of OGT MS and provides a potential target. selectively reduces the expansion of Th17 cells, although no noteworthy alteration in Th1 proportion was observed. To validate Disclosures that result, we confirmed an enhancement of Th17 differentiation The authors have no financial conflicts of interest. by using the mouse EAE model with miR interventions. The outcome suggested that the miR-15b–mediated downregulation of OGT may be involved in the pathogenesis of MS, mainly by References inhibiting the differentiation of Th17 cells. 1. Compston, A., and A. Coles. 2002. Multiple sclerosis. Lancet 359: 1221–1231. 2. Trapp, B. D., and K. A. Nave. 2008. Multiple sclerosis: an immune or neuro- T lymphocyte differentiation is a complicated process and in- degenerative disorder? Annu. Rev. Neurosci. 31: 247–269. volves a variety of transcription factors. The sustained activation of 3. Kutzelnigg, A., C. F. Lucchinetti, C. Stadelmann, W. Bruck,€ H. Rauschka, M. Bergmann, M. Schmidbauer, J. E. Parisi, and H. Lassmann. 2005. Cortical STAT3 induces the expression of RORgT and related epigenetic demyelination and diffuse white matter injury in multiple sclerosis. Brain 128: alterations to activate the distinguishing expression of cytokines 2705–2712. 2638 miR-15b REGULATES Th17 AND MS BY TARGETING OGT

4. Noseworthy, J. H., C. Lucchinetti, M. Rodriguez, and B. G. Weinshenker. 2000. 34. Polman, C. H., S. C. Reingold, B. Banwell, M. Clanet, J. A. Cohen, M. Filippi, Multiple sclerosis. N. Engl. J. Med. 343: 938–952. K. Fujihara, E. Havrdova, M. Hutchinson, L. Kappos, et al. 2011. Diagnostic 5. Sospedra, M., and R. Martin. 2005. Immunology of multiple sclerosis. Annu. criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann. Rev. Immunol. 23: 683–747. Neurol. 69: 292–302. 6. Bettelli, E., Y. Carrier, W. Gao, T. Korn, T. B. Strom, M. Oukka, H. L. Weiner, 35. Jonuleit, H., E. Schmitt, G. Schuler, J. Knop, and A. H. Enk. 2000. Induction of and V. K. Kuchroo. 2006. Reciprocal developmental pathways for the generation interleukin 10-producing, nonproliferating CD4+ T cells with regulatory prop- of pathogenic effector TH17 and regulatory T cells. Nature 441: 235–238. erties by repetitive stimulation with allogeneic immature human dendritic cells. 7. Patel, D. D., and V. K. Kuchroo. 2015. Th17 cell pathway in human immunity: J. Exp. Med. 192: 1213–1222. lessons from genetics and therapeutic interventions. Immunity 43: 1040–1051. 36. Hao, J., R. Liu, W. Piao, Q. Zhou, T. L. Vollmer, D. I. Campagnolo, R. Xiang, 8. Korn, T., E. Bettelli, M. Oukka, and V. K. Kuchroo. 2009. IL-17 and Th17 cells. A. La Cava, L. Van Kaer, and F.-D. Shi. 2010. Central nervous system (CNS)– Annu. Rev. Immunol. 27: 485–517. resident natural killer cells suppress Th17 responses and CNS autoimmune pa- 9. Dong, C. 2008. TH17 cells in development: an updated view of their molecular thology. J. Exp. Med. 207: 1907–1921. identity and genetic programming. Nat. Rev. Immunol. 8: 337–348. 37. Wang, Y., H. Zhang, Y. Chen, Y. Sun, F. Yang, W. Yu, J. Liang, L. Sun, X. Yang, 10. Ivanov, I. I., B. S. McKenzie, L. Zhou, C. E. Tadokoro, A. Lepelley, J. J. Lafaille, L. Shi, et al. 2009. LSD1 is a subunit of the NuRD complex and targets the D. J. Cua, and D. R. Littman. 2006. The orphan nuclear receptor RORgt directs metastasis programs in breast cancer. Cell 138: 660–672. the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126: 38. Ding, Y., R. Han, W. Jiang, J. Xiao, H. Liu, X. Chen, X. Li, and J. Hao. 2016. 1121–1133. Programmed death ligand 1 plays a neuroprotective role in experimental auto- 11. Yang, X. O., B. P. Pappu, R. Nurieva, A. Akimzhanov, H. S. Kang, Y. Chung, immune neuritis by controlling peripheral nervous system inflammation of rats. L. Ma, B. Shah, A. D. Panopoulos, K. S. Schluns, et al. 2008. T helper 17 lineage J. Immunol. 197: 3831–3840. differentiation is programmed by orphan nuclear receptors RORa and RORg. 39. Cox, M. B., M. J. Cairns, K. S. Gandhi, A. P. Carroll, S. Moscovis, G. J. Stewart, Immunity 28: 29–39. S. Broadley, R. J. Scott, D. R. Booth, and J. Lechner-Scott, ANZgene Multiple 12. Ruan, Q., V. Kameswaran, Y. Zhang, S. Zheng, J. Sun, J. Wang, J. DeVirgiliis, Sclerosis Genetics Consortium. 2010. MicroRNAs miR-17 and miR-20a inhibit H. C. Liou, A. A. Beg, and Y. H. Chen. 2011. The Th17 immune response is T cell activation genes and are under-expressed in MS whole blood. PLoS One 5: controlled by the Rel–RORg–RORg T transcriptional axis. J. Exp. Med. 208: e12132. 2321–2333. 40. Jernas,˚ M., C. Malmestro¨m, M. Axelsson, I. Nookaew, H. Wadenvik, J. Lycke, 13. Chen, G., K. Hardy, E. Pagler, L. Ma, S. Lee, S. Gerondakis, S. Daley, and and B. Olsson. 2013. MicroRNA regulate immune pathways in T-cells in mul- Downloaded from M. F. Shannon. 2011. The NF-kB transcription factor c-Rel is required for Th17 tiple sclerosis (MS). BMC Immunol. 14: 32. effector cell development in experimental autoimmune encephalomyelitis. J. 41. Ponomarev, E. D., T. Veremeyko, N. Barteneva, A. M. Krichevsky, and Immunol. 187: 4483–4491. H. L. Weiner. 2011. MicroRNA-124 promotes microglia quiescence and sup- 14. Ambros, V. 2001. microRNAs: tiny regulators with great potential. Cell 107: presses EAE by deactivating macrophages via the C/EBP-a-PU.1 pathway. Nat. 823–826. Med. 17: 64–70. 15. Croce, C. M. 2009. Causes and consequences of microRNA dysregulation in 42. Qu, X., J. Zhou, T. Wang, J. Han, L. Ma, H. Yu, D. Geng, H. Fan, Q. Zhang, cancer. Nat. Rev. Genet. 10: 704–714. F. Hua, and R. Yao. 2016. MiR-30a inhibits Th17 differentiation and demye-

16. Small, E. M., R. J. Frost, and E. N. Olson. 2010. MicroRNAs add a new di- lination of EAE mice by targeting the IL-21R. Brain Behav. Immun. 57: 193–199 http://www.jimmunol.org/ mension to cardiovascular disease. Circulation 121: 1022–1032. 43. Bartel, D. P. 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. 17. Zampetaki, A., S. Kiechl, I. Drozdov, P. Willeit, U. Mayr, M. Prokopi, A. Mayr, Cell 116: 281–297. S. Weger, F. Oberhollenzer, E. Bonora, et al. 2010. Plasma microRNA profiling 44. Hu, H., Y. Yang, Q. Ji, W. Zhao, B. Jiang, R. Liu, J. Yuan, Q. Liu, X. Li, Y. Zou, reveals loss of endothelial miR-126 and other microRNAs in type 2 diabetes. et al. 2012. CRL4B catalyzes H2AK119 monoubiquitination and coordinates Circ. Res. 107: 810–817. with PRC2 to promote tumorigenesis. Cancer Cell 22: 781–795. 18. Pauley, K. M., S. Cha, and E. K. Chan. 2009. MicroRNA in autoimmunity and 45. Ko¨ntgen, F., R. J. Grumont, A. Strasser, D. Metcalf, R. Li, D. Tarlinton, and autoimmune diseases. J. Autoimmun. 32: 189–194. S. Gerondakis. 1995. Mice lacking the c-rel proto-oncogene exhibit defects 19. Singh, R. P., I. Massachi, S. Manickavel, S. Singh, N. P. Rao, S. Hasan, D. K. Mc in lymphocyte proliferation, humoral immunity, and interleukin-2 expression. Curdy, S. Sharma, D. Wong, B. H. Hahn, and H. Rehimi. 2013. The role of Genes Dev. 9: 1965–1977. miRNA in inflammation and autoimmunity. Autoimmun. Rev. 12: 1160–1165. 46. Ulivi, V., P. Giannoni, C. Gentili, R. Cancedda, and F. Descalzi. 2008. p38/NF- 20. Tricoli, J. V., and J. W. Jacobson. 2007. MicroRNA: potential for cancer de- kB-dependent expression of COX-2 during differentiation and inflammatory tection, diagnosis, and prognosis. Cancer Res. 67: 4553–4555. response of chondrocytes. J. Cell. Biochem. 104: 1393–1406. by guest on September 28, 2021 21. Cho, W. C. 2010. MicroRNAs: potential biomarkers for cancer diagnosis, 47. Schreck, R., and P. A. Baeuerle. 1990. NF-kappa B as inducible transcriptional prognosis and targets for therapy. Int. J. Biochem. Cell Biol. 42: 1273–1281. activator of the granulocyte-macrophage colony-stimulating factor gene. Mol. 22. Zhu, S., W. Pan, X. Song, Y. Liu, X. Shao, Y. Tang, D. Liang, D. He, H. Wang, Cell. Biol. 10: 1281–1286. W. Liu, et al. 2012. The microRNA miR-23b suppresses IL-17-associated autoim- 48. Frohman, E. M., M. K. Racke, and C. S. Raine. 2006. Multiple sclerosis—the mune inflammation by targeting TAB2, TAB3 and IKK-a. Nat. Med. 18: 1077–1086. plaque and its pathogenesis. N. Engl. J. Med. 354: 942–955. 23. Du, C., C. Liu, J. Kang, G. Zhao, Z. Ye, S. Huang, Z. Li, Z. Wu, and G. Pei. 49. Zheng, X., M. Chopp, Y. Lu, B. Buller, and F. Jiang. 2013. MiR-15b and miR- 2009. MicroRNA miR-326 regulates TH-17 differentiation and is associated 152 reduce glioma cell invasion and angiogenesis via NRP-2 and MMP-3. with the pathogenesis of multiple sclerosis. Nat. Immunol. 10: 1252–1259. Cancer Lett. 329: 146–154. 24. Shaked, I., A. Meerson, Y. Wolf, R. Avni, D. Greenberg, A. Gilboa-Geffen, and 50. Zhong, G., X. Cheng, H. Long, L. He, W. Qi, T. Xiang, Z. Zhao, and B. Zhu. H. Soreq. 2009. MicroRNA-132 potentiates cholinergic anti-inflammatory sig- 2013. Dynamically expressed microRNA-15b modulates the activities of CD8+ naling by targeting acetylcholinesterase. Immunity 31: 965–973. T lymphocytes in mice with Lewis lung carcinoma. J. Transl. Med. 11: 71. 25. O’Connell, R. M., D. Kahn, W. S. Gibson, J. L. Round, R. L. Scholz, 51. Wu, C. S., C. J. Yen, R. H. Chou, J. N. Chen, W. C. Huang, C. Y. Wu, and A. A. Chaudhuri, M. E. Kahn, D. S. Rao, and D. Baltimore. 2010. MicroRNA- Y. L. Yu. 2014. Downregulation of microRNA-15b by hepatitis B virus X en- 155 promotes autoimmune inflammation by enhancing inflammatory T cell hances hepatocellular carcinoma proliferation via 2-induced development. Immunity 33: 607–619. Globo H expression. Int. J. Cancer 134: 1638–1647. 26. Mycko, M. P., M. Cichalewska, A. Machlanska, H. Cwiklinska, 52. Kedmi, M., N. Ben-Chetrit, C. Ko¨rner, M. Mancini, N. B. Ben-Moshe, M. Mariasiewicz, and K. W. Selmaj. 2012. MicroRNA-301a regulation of a M. Lauriola, S. Lavi, F. Biagioni, S. Carvalho, H. Cohen-Dvashi, et al. 2015. T-helper 17 immune response controls autoimmune demyelination. Proc. Natl. EGF induces microRNAs that target suppressors of cell migration: miR-15b Acad. Sci. USA 109: E1248–E1257. targets MTSS1 in breast cancer. Sci. Signal. 8: ra29. 27. Lazarus, M. B., Y. Nam, J. Jiang, P. Sliz, and S. Walker. 2011. Structure of 53. Roy,S.,J.Banerjee,S.C.Gnyawali,S.Khanna,G.He,D.Pfeiffer,J.L.Zweier,and human O-GlcNAc transferase and its complex with a peptide substrate. Nature C. K. Sen. 2013. Suppression of induced microRNA-15b prevents rapid loss of car- 469: 564–567. diac function in a dicer depleted model of cardiac dysfunction. PLoS One 8: e66789. 28. Golks, A., T. T. Tran, J. F. Goetschy, and D. Guerini. 2007. Requirement for 54. Lv, X., H. Jiang, Y. Liu, X. Lei, and J. Jiao. 2014. MicroRNA-15b promotes O-linked N-acetylglucosaminyltransferase in lymphocytes activation. EMBO J. neurogenesis and inhibits neural progenitor proliferation by directly repressing 26: 4368–4379. TET3 during early neocortical development. EMBO Rep. 15: 1305–1314. 29. Hanover, J. A., M. W. Krause, and D. C. Love. 2012. Bittersweet memories: 55. Wang, X., X. Wang, X. Liu, X. Wang, J. Xu, S. Hou, X. Zhang, and Y. Ding. 2015. linking metabolism to epigenetics through O-GlcNAcylation. Nat. Rev. Mol. Cell miR-15a/16 are upreuglated in the serum of neonatal sepsis patients and inhibit the Biol. 13: 312–321. LPS-induced inflammatory pathway. Int. J. Clin. Exp. Med. 8: 5683–5690. 30. Ramakrishnan, P., P. M. Clark, D. E. Mason, E. C. Peters, L. C. Hsieh-Wilson, 56. Xu, J., C. Zgheib, J. Hu, W. Wu, L. Zhang, and K. W. Liechty. 2014. The role of and D. Baltimore. 2013. Activation of the transcriptional function of the NF-kB microRNA-15b in the impaired angiogenesis in diabetic wounds. Wound Repair protein c-Rel by O-GlcNAc glycosylation. [Published erratum appears in 2014 Regen. 22: 671–677. Sci. Signal. 7: er3.] Sci. Signal. 6: ra75. 57. Shi, H., B. L. Sun, J. Zhang, S. Lu, P. Zhang, H. Wang, Q. Yu, R. A. Stetler, 31. Yang, W. H., S. Y. Park, H. W. Nam, D. H. Kim, J. G. Kang, E. S. Kang, P. S. Vosler, J. Chen, and Y. Gao. 2013. miR-15b suppression of Bcl-2 contributes Y. S. Kim, H. C. Lee, K. S. Kim, and J. W. Cho. 2008. NFkB activation is as- to cerebral ischemic injury and is reversed by sevoflurane preconditioning. CNS sociated with its O-GlcNAcylation state under hyperglycemic conditions. Proc. Neurol. Disord. Drug Targets 12: 381–391. Natl. Acad. Sci. USA 105: 17345–17350. 58. He, L., and G. J. Hannon. 2004. MicroRNAs: small RNAs with a big role in gene 32. Baudoin, L., and T. Issad. 2015. O-GlcNAcylation and inflammation: a vast regulation. Nat. Rev. Genet. 5: 522–531. territory to explore. Front. Endocrinol. (Lausanne) 5: 235. 59. Kearse, K. P., and G. W. Hart. 1991. Topology of O-linked N-acetylglucosamine 33. Golks, A., and D. Guerini. 2008. The O-linked N-acetylglucosamine modifica- in murine lymphocytes. Arch. Biochem. Biophys. 290: 543–548. tion in cellular signalling and the immune system. “Protein modifications: be- 60. Hewagama, A., G. Gorelik, D. Patel, P. Liyanarachchi, W. J. McCune, yond the usual suspects” review series. EMBO Rep. 9: 748–753. E. Somers, T. Gonzalez-Rivera, F. Strickland, and B. Richardson, Michigan The Journal of Immunology 2639

Lupus Cohort. 2013. Overexpression of X-linked genes in T cells from women 68. Swamy, M., S. Pathak, K. M. Grzes, S. Damerow, L. V. Sinclair, D. M. van with lupus. J. Autoimmun. 41: 60–71. Aalten, and D. A. Cantrell. 2016. Glucose and glutamine fuel protein 61. Zhang, D., Y. Cai, M. Chen, L. Gao, Y. Shen, and Z. Huang. 2015. OGT- O-GlcNAcylation to control T cell self-renewal and malignancy. Nat. Immunol. mediated O-GlcNAcylation promotes NF-kB activation and inflammation in 17: 712–720. acute pancreatitis. Inflamm. Res. 64: 943–952. 69. Kemppinen, A. K., J. Kaprio, A. Palotie, and J. Saarela. 2011. Systematic 62. Kim, H. B., S. W. Lee, C. H. Mun, J. Y. Yoon, J. Pai, I. Shin, Y. B. Park, S. K. Lee, review of genome-wide expression studies in multiple sclerosis. BMJ Open 1: and J. W. Cho. 2015. O-linked N-acetylglucosamine glycosylation of p65 aggravated e000053. the inflammation in both fibroblast-like synoviocytes stimulated by tumor necrosis 70. Sun, L., Y. Yao, B. Liu, Z. Lin, L. Lin, M. Yang, W. Zhang, W. Chen, C. Pan, factor-a and mice with collagen induced arthritis. Arthritis Res. Ther. 17: 248. Q. Liu, et al. 2012. MiR-200b and miR-15b regulate chemotherapy-induced 63. Kearse, K. P., and G. W. Hart. 1991. Lymphocyte activation induces rapid epithelial-mesenchymal transition in human tongue cancer cells by targeting changes in nuclear and cytoplasmic glycoproteins. Proc. Natl. Acad. Sci. USA BMI1. Oncogene 31: 432–445. 88: 1701–1705. 71. Langrish, C. L., Y. Chen, W. M. Blumenschein, J. Mattson, B. Basham, 64. O’Donnell, N., N. E. Zachara, G. W. Hart, and J. D. Marth. 2004. Ogt-dependent J. D. Sedgwick, T. McClanahan, R. A. Kastelein, and D. J. Cua. 2005. IL-23 X--linked protein glycosylation is a requisite modification in so- drives a pathogenic T cell population that induces autoimmune inflammation. J. matic cell function and embryo viability. Mol. Cell. Biol. 24: 1680–1690. Exp. Med. 201: 233–240. 65. Johnson, B., M. Opimba, and J. Bernier. 2014. Implications of the O-GlcNAc 72. Jadidi-Niaragh, F., and A. Mirshafiey. 2011. Th17 cell, the new player of neu- modification in the regulation of nuclear apoptosis in T cells. Biochim. Biophys. roinflammatory process in multiple sclerosis. Scand. J. Immunol. 74: 1–13. Acta 1840: 191–198. 73. Durelli, L., L. Conti, M. Clerico, D. Boselli, G. Contessa, P. Ripellino, 66. Huang, J. B., A. J. Clark, and H. R. Petty. 2007. The hexosamine biosynthesis B. Ferrero, P. Eid, and F. Novelli. 2009. T-helper 17 cells expand in multiple pathway negatively regulates IL-2 production by Jurkat T cells. Cell. Immunol. sclerosis and are inhibited by interferon-b. Ann. Neurol. 65: 499–509. 245: 1–6. 74. Kebir, H., K. Kreymborg, I. Ifergan, A. Dodelet-Devillers, R. Cayrol, 67. Nagy, T., A. Balasa, D. Frank, A. Rab, O. Rideg, G. Kotek, T. Magyarlaki, M. Bernard, F. Giuliani, N. Arbour, B. Becher, and A. Prat. 2007. Human P. Bogner, G. L. Kova´cs, and A. Miseta. 2010. O-GlcNAc modification of proteins TH17 lymphocytes promote blood-brain barrier disruption and central nervous affects volume regulation in Jurkat cells. Eur. Biophys. J. 39: 1207–1217. system inflammation. Nat. Med. 13: 1173–1175. Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021