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5-Lipoxygenase Is a Direct Target of miR-19a-3p and miR-125b-5p Saskia Busch, Eileen Auth, Friederike Scholl, Sabine Huenecke, Ulrike Koehl, Beatrix Suess and Dieter This information is current as Steinhilber of October 2, 2021. J Immunol published online 14 January 2015 http://www.jimmunol.org/content/early/2015/01/14/jimmun ol.1402163 Downloaded from

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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 © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 14, 2015, doi:10.4049/jimmunol.1402163 The Journal of Immunology

5-Lipoxygenase Is a Direct Target of miR-19a-3p and miR-125b-5p

Saskia Busch,* Eileen Auth,† Friederike Scholl,* Sabine Huenecke,† Ulrike Koehl,‡ Beatrix Suess,x and Dieter Steinhilber*

5-Lipoxygenase (5-LO) is the key enzyme in leukotriene biosynthesis. Leukotrienes are mediators of the innate immune system and inflammatory processes, and they might also be involved in cancer development. MicroRNAs (miRNAs) are important translational regulators and have been shown to be involved in development, differentiation, and cancer. Unraveling the miRNA network is important for understanding the cellular regulation processes. We identified two new miRNAs, miR-19a-3p and miR-125b-5p, regulating 5-LO and confirmed direct interaction by reporter gene assays. Furthermore, we investigated the regulation of 5-LO by these two miRNAs in several cell types. Inhibition of both miRNAs by antagomirs during differentiation of the myeloid cell line Mono Mac 6 led to a significant increase in 5-LO protein expression. Stimulation of human T lymphocytes with PHA resulted in a strong down- Downloaded from regulation of 5-LO mRNA expression and in the induction of miR-19a-3p. The inhibition of miR-19a-3p with an antagomir led to a significant increase in 5-LO mRNA expression in T lymphocytes. Taken together, our data reveal that miR-19a-3p and miR-125b-5p target 5-LO in a cell type– and stimulus-specific manner. The Journal of Immunology, 2015, 194: 000–000.

eukotrienes are important mediators for inﬂammatory, al- transcribed by RNA polymerase II and metabolized in the nucleus lergic, and immunologic reactions. 5-Lipoxygenase (5-LO) by Drosha to pre-miRNA with a length of ∼70 nt. The pre- L is the key enzyme in leukotriene biosynthesis, catalyzing miRNAs, once in the cytoplasm, are further processed by Dicer http://www.jimmunol.org/ the conversion of arachidonic acid to leukotriene A4 (1). Depending to ∼21 nt short, double-stranded miRNA. Only one strand of the on the cell type, leukotriene A4 is either metabolized to leukotriene miRNA duplex, the guide strand, is then incorporated into the B4 or the cysteinyl leukotrienes (leukotrienes C4,D4,andE4). RNA-induced silencing complex and directs this complex to the 39 Leukocytes are the major producers of leukotrienes, leading to in- untranslated region (39UTR) of its target genes. duction and recruitment of macrophages and monocytes as well as Even though regulation of gene expression by miRNAs has been dendritic cell maturation and migration from the peripheral site of studied intensively in recent years, not much is known about the action to the lymphoid nodes (2). As part of the adaptive immune regulation of the 5-LO pathway by miRNAs. In 2010, miR-135a and

response, leukotriene B4 is a chemoattractant for T cells (3, 4). miR-199a-5p were the first two miRNAs identified to target the 5-LO by guest on October 2, 2021 Moreover, 5-LO is involved in the regulation of Ig production in activating protein (10). miR-219-2 was the first and only miRNA B cells (5). The expression of the 5-LO can mainly be found in the identified so far as a regulator of 5-LO in macrophages (11). myeloid lineage of the hematopoietic cells (monocytes, macro- Recently, it was shown that inhibition of the leukotriene receptors phages, granulocytes, neutrophils, mast cells, and dendritic cells) as cysteinyl leukotriene 1 or leukotriene B4 receptor-1 (BLT1) receptor or well as B lymphocytes (6, 7), where the expression and activity a knockdown of the BLT1 receptor led to a downregulation of several depend on stimulation and differentiation status of the cell (8). miRNAs (12). Among them were miR-19a-3p and miR-125b-5p. MicroRNAs (miRNAs) are small, noncoding, regulatory RNAs. Overexpression of miR-125b-5p in the myeloid cell line HL-60 pre- miRNAs can induce mRNA degradation or translational repression vented their differentiation into macrophages or granulocytes by ei- of their target genes (9). Their precursors, the pri-miRNAs, are ther DMSO or GM-CSF (13). Alternatively, it could be shown that high expression of 5-LO requires cell differentiation, for example, by DMSO, GM-CSF, or TGF-b (14–17), which provides a possible link *Institute of Pharmaceutical Chemistry, Goethe University, 60438 Frankfurt/Main, Germany; †Laboratory for Stem Cell Transplantation and Immunotherapy, Pediatric between miR-125 and the 5-LO pathway. Mir-19a-3p is a member of Hematology and Oncology, University Hospital Frankfurt, 60596 Frankfurt, Germany; the miR-17∼92 cluster, which has been shown to be expressed in ‡Integrated Research and Treatment Center Transplantation, Institute of Cellular Ther- apeutics, Hannover Medical School, 30625 Hannover, Germany; and xDepartment of B cells (18). Within B cell development, the 5-LO is downregulated Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany in germinal center cells (8, 19), whereas the miR-17∼92 cluster as well Received for publication August 22, 2014. Accepted for publication December 10, as miR-125b-5p are upregulated (20–23). Furthermore, it has been 2014. shown that the miR-17∼92 cluster regulates T cell proliferation (24, This work was supported by Merck, Deutsche Forschungsgemeinschaft Grant GRK 25) and that miR-19a-3p plays a role in promoting Th1 responses (26). 1172, and by the LOEWE Center for Cell and Gene Therapy. All of these data suggest a possible role of miR-19a-3p and miR- Address correspondence and reprint requests to Prof. Dieter Steinhilber or Prof. 125b-5p in the regulation of leukocyte functions and immune Beatrix Suess, Institute of Pharmaceutical Chemistry, Goethe University, Max-von-Laue- Strasse 9, 60438 Frankfurt/Main, Germany (D.S.) or Department of Biology, Technical responses by regulation of the 5-LO pathway. In this study, we show University of Darmstadt, 64287 Darmstadt, Germany (B.S.). E-mail addresses: that miR-19a-3p and miR-125b-5p directly regulate 5-LO ex- [email protected] (D.S.) or [email protected] (B.S.) pression in myeloid cells and in T lymphocytes. The online version of this article contains supplemental material.

Abbreviations used in this article: BLT1, leukotriene B4 receptor-1; 5-LO, 5-lipoxy- Materials and Methods genase; miRNA, microRNA; MM6, Mono Mac 6; SAC, Staphylococcus aureus Cell lines Cowan strain protein A; 39UTR, 39 untranslated region. Cos-7 cells were cultivated in DMEM medium (Invitrogen) supplemented Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 with 10% FCS (Biochrom), 100 mg/ml streptomycin, and 100 U/ml

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402163 2 CONTROL OF 5-LIPOXYGENASE BY miRNAs penicillin (Invitrogen). The cells were detached using trypsin-EDTA For cDNA synthesis the high-capacity RNA-to-cDNA kit (Invitrogen) (Invitrogen) and split at a ratio of 5:1 twice a week. Mono Mac 6 was used according to the manufacturer’s protocol. All quantitative PCR (MM6) cells were cultivated in RPMI 1640 medium (Invitrogen) sup- experiments were carried out with a StepOne Plus device (Applied Bio- plemented with 10% FCS (Biochrom), 100 mg/ml streptomycin, 100 systems). The endogenous control used for the MM6 cells was b-actin and U/ml penicillin (Invitrogen), 1 mM sodium pyruvate (Invitrogen), for the primary cells RNU48. 13 nonessential amino acids (Sigma-Aldrich), 1 mM oxaloacetate For the specific cDNA synthesis of hsa-miR-19a-3p, 100 ng RNA was (AppliChem), and 10 mg/ml human insulin (Sanofi-Aventis). The cells were transcribed into specific cDNA using stem-loop primers, designed as pre- split twice a week to a density of 0.35 3 106/ml. For the time course ex- viously described (28). Consequently, the quantitative PCR was carried out periment, the cells were seeded at a density of 0.3 3 106/ml and were using a specific forward primer, a universal reverse primer, and Universal cultivated for 4 d. MM6 cells were seeded at a density of 0.3 3 106/ml for ProbeLibrary probe no. 21 (Roche Diagnostics). RNU48 served as the en- untreated time courses and 0.2 3 106/ml for differentiation experiments. For dogenous control. All primer sequences are available upon request. Hsa- differentiation, the cells were stimulated with 50 nM calcitriol (Sigma- miR-125b-5p was detected using the TaqMan miRNA assay kit according Aldrich) and 1 ng/ml TGF-b (isolated and purified in-house) for up to 4 d. to the manufacturer’s protocol. RNU48 served as the endogenous control. All used cell lines were kept at 37˚C, 5% CO2, and 100% humidity and were purchased from the German Collection of Microorganisms and Cell Western blot analysis Cultures. A minimum of 5 3 106 cells were lysed in 100 ml 0.1% Triton X-100 miRNA target predictions solution (Fluka BioChemika) in PBS (Invitrogen) supplemented with 1 mM PMSF (Fluka BioChemika) and frozen at 280˚C. After thawing, the Three online bioinformatic target prediction programs were used: TargetScan cells were centrifuged for 10 min at 10,000 3 g (4˚C) and the protein (http://www.targetscan.org/), MicroRNA.org (http://www.microrna.org/ concentration of the supernatant was determined by the Bradford colori- microrna/getMirnaForm.do), and MicroCosm Targets (http://www.ebi.ac. metric assay (Bio-Rad Laboratories) (29). The samples (50–100 mg pro- uk/enright-srv/microcosm/cgi-bin/targets/v5/search.pl). tein) were separated by SDS-PAGE on a 10% gel and then blotted onto

a nitrocellulose membrane (Whatman reinforced nitrocellulose; GE Life Downloaded from Plasmid construction Sciences). The membrane was blocked for 1 h at room temperature with Odyssey blocking buffer (LI-COR Biosciences) and afterward incubated All plasmids used are based on the pMIR-Report plasmid (Invitrogen). The with primary and secondary Abs. The Abs were diluted in PBS (Invi- SacI and HindIII restriction sites within the multiple cloning site, which are trogen) as follows: mouse anti–5-LO at 1:1,000 (6A12; Frankfurt Uni- located behind the ﬁreﬂy luciferase gene were used to insert the 5-LO versity), donkey anti-mouse at 1:10,000 (IRDye; LI-COR Biosciences), 39UTR sequences. The upper construct in Fig. 1B carries the complete goat b-actin at 1:5,000 (Santa Cruz Biotechnology), and donkey anti-goat 5-LO 39UTR cloned between SacI and HindIII. The so named “triplicate at 1:10,000 (IRDye; LI-COR Biosciences). The visualization was carried

19a” or “triplicate 125b” plasmids carry a triplicate of the predicted miR http://www.jimmunol.org/ out with an Odyssey Classic infrared imaging system (LI-COR Bio- binding site within the 39UTR with a spacer of 15 nt in between and have sciences). the following sequences (the predicted miR binding site, as shown in Fig. 1A, is underlined): triplicate 125b, 59-TTTATTCTTTGATCTTCAGG- Isolation of B and T lymphocytes GAATAAAGCTTTGGGTCTTTATTCTTTGATCTTCAGGGAATAACGC- GTTGAGATTTTATTCTTTGATCTTCAGGGA-39; triplicate 19a, 59- The primary cells were isolated from buffy coats obtained from the German TGCTTAGTCCAATTCCTTGCACAATAAAGCTTTGGGTCTGCTTAGT- Red Cross Blood Donor Service Baden-Wurttemberg-Hessen€ in Frankfurt, CCAATTCCTTGCACAATAACGCGTTGAGATTGCTTAGTCCAATTC- Germany. A Ficoll separation was performed before enrichment of either B CTTGCACA-39. or T lymphocytes with the respective RosetteSep human enrichment kit The plasmids with the perfect match contain the complementary (StemCell Technologies). The magnetic separation was carried out sequences of the mature 19a and 125b miRNA behind the firefly luciferase according to the manufacturer’s protocol. The purity of the cells was gene. Mutagenesis of the seed regions, as shown in Fig. 1A, was performed checked by staining with CD45-FITC, CD3-PE, CD19-ECD, viability dye by guest on October 2, 2021 by overlap PCR, as previously described (27). All sequences were con- 7-aminoactinomycin D, and CD56-PC7 (Beckman Coulter). As an internal firmed by DNA sequencing. standard for quantification of the cell count accuracy, fluorosphere beads (Flow-Count; Beckman Coulter Immunotech) were added. The measure- Luciferase assay ments were carried out with an FC500 by Beckman Coulter, and analysis of the data was performed with the CXP v2.2 software (Beckman Coulter). The Cos-7 cells were seeded at a density of 40,000 cells/well into a 24-well The CD3+CD45+ T cells had a median purity of 97.1% with a SD of plate in a volume of 1 ml 1 d prior to transfection. The pRL-SV40 plasmid 1.8%; the remaining cells were ,1% monocytes. The CD19+CD32CD45+ from Promega, carrying the Renilla luciferase gene, was used as a trans- B cells had a median purity of 97.3% with a SD of 7.5%; the remaining fection control. The pre-miRNAs for hsa-19a-3p and hsa-miR125b-5p as cells were ,5% monocytes. The viability directly after the purification well as a negative control with a random sequence (Ambion Pre-miR step was .99%. miRNA precursor hsa-miR-19a-3p, hsa-miR-125b-5p, and negative control no. 2) were used to study miR overexpression. The mature miRNA for Stimulation of primary B lymphocytes hsa-miR-29a-5p as well as a negative control were used (MISSION miRNA negative control 1 and MISSION microRNA mimic hsa-miR- B lymphocytes were taken into culture in RPMI 1640 medium (Invitrogen) 29a*; Sigma-Aldrich). Every cotransfection was carried out with 150 ng supplemented with 10% FCS (Invitrogen), 2 mM glutamine (Invitrogen), pMIR reporter plasmid construct, 20 ng pRL-SV40, and 5 pmol precursor 50 mg/ml streptomycin, 50 U/ml penicillin (Invitrogen), and 25 mM HEPES 6 miRNA or mature miRNA. Cells were transfected with 1 ml Lipofectamine buffer (Invitrogen) at a density of 1 3 10 /ml (culture volume 4 ml). The 2000 (Invitrogen) in 100 ml, according to the manufacturer’s protocol. The cells were stimulated with 10 ng/ml protein A from Staphylococcus aureus medium change from Opti-MEM to DMEM was performed 3 h after the Cowan strain (SAC) (Sigma-Aldrich) and 20 U/ml IL-2 (PeproTech). transfection. Luciferase activity was determined 24 h after the medium change using the Dual-Glo luciferase assay system (Promega) and a Tecan Stimulation of primary T lymphocytes Infinite M200 reader. T lymphocytes were taken into culture in X-VIVO 10, without phenol red and gentamicin (Lonza), and supplemented with 10% human heat- RNA isolation, cDNA, and quantitative PCR inactivated plasma (Red Cross Baden-Wurttemberg-Hessen)€ at a density 3 6 RNA isolation of MM6 cells was performed with the phenol method. The of 1 10 /ml (culture volume 4 ml). The cells were stimulated with m harvested cell pellet of 7 3 106 cells was lysed with 200 ml buffer (150 mM 5 g/ml PHA (Sigma-Aldrich). saccharose [Merck], 10 mM sodium acetate [AppliChem], adjusted to pH 4.8 Antagomir experiment using acetic acid [AppliChem]) in the presence of 1% SDS (AppliChem) and 200 ml 6 M guanidine thiocyanate (Sigma-Aldrich). The extraction with The antagomirs for miR-19a-3p and miR-125b-5p as well as a control, with phenol and a 25:1 mixture of chloroform and isoamyl alcohol was carried a complementary sequence to GFP, were designed as previously described out twice. MaXtract high-density tubes (Qiagen) were used for phase sep- (30) and obtained from VBC Biotech (Vienna, Austria) with the following aration. TurboDNase (Ambion) was used for DNase digest and was handled sequences: antagomir-19a, 59- TCAGTTTTGCATAGATTTGCACA-39; according to the manufacturer’s protocol. The RNA integrity was analyzed antagomir-125b, 59-AGGGACTCTGGGATTGAACACT-39; control antagomir- after the DNase digest by agarose gel electrophoresis. The RNA of the Co, 59-AAGGCAAGCUGACCCUGAAGUU-39. primary B and T cells was extracted with the miRNeasy kit (Qiagen) and The antagomirs were added at a final concentration of 25 nM to the eluted in 30 ml MilliQ water, according to the manufacturer’s protocol. differentiating MM6 cells and at a final concentration of 150 nM to the The Journal of Immunology 3 primary T cells. The efficiency of the antagomir treatment was measured by (31). As positive control, we constructed a reporter plasmid con- determination of mRNA levels of the verified hsa-miR-19a-3p target cyclin D1. taining a sequence complementary to the respective miRNA Statistical analysis (perfect match). All constructs are shown in Fig. 1B. Cotransfection of the constructs containing the entire 5-LO GraphPad Prism 5.03 (GraphPad Sopftware) was used for statistical 39UTR with the pri-miRNAs 19a or 125b decreased luciferase analysis. A p value ,0.05 was considered significant. activity by 23 and 35%, respectively. Mutation of the predicted Results binding sites abolished the decrease, confirming a direct interaction. Reporter gene activity was even further reduced (by 35 and Target predictions 41%, respectively) when the miR-19a-3p and 125b-5p binding The search engines TargetScan, MicroRNA.org, and MicroCosm sites were inserted in triplicates without the 5-LO 39UTR context. Targets were used to identify potential miRNA binding sites in the Cotransfection of the 39UTR of the 5-LO with miR-29a-5p did not 5-LO 39UTR. Seven putative miRNA binding sites were obtained reduce the luciferase activity (Supplemental Fig. 1), hence miR- that were further filtered by the quality of their predicted target 29a-5p did not interact with the 39UTR and was therefore not site combined with their expression pattern in leukocytes. Taking further investigated. these criteria into account, the number of miRNAs was further Taken together, these results show that miR-19a-3p and miR- reduced from seven to three (i.e., miR-19a-3p, miR-125b-5p, and 125b-5p target the 5-LO 39UTR at the predicted binding sites. miR-29a-5p), which were further investigated. 5-LO is a target of miR-19a-3p and miR-125b-5p in MM6 cells The binding sites for these miRNAs within the context of the 39UTR of 5-LO are illustrated in Fig. 1A and Supplemental Fig. 1. Next we investigated the expression pattern of 5-LO and both All binding sites have a good seed region and complementary base miRNAs in a native cellular context. As a model system we chose Downloaded from pairing in the 39 end of the miRNAs. the human monocytic cell line MM6 to analyze a potential role of the miRNAs in monocytic cells. In this cell line, 5-LO mRNA and MiR-19a-3p and miR-125b-5p can regulate 5-LO expression protein expression are induced after differentiation with TGF-b 9 via two binding sites in the 5-LO 3 UTR and calcitriol (17, 32, 33). In contrast, untreated cells show a low To validate the predicted binding sites, we performed luciferase level of 5-LO mRNA expression but no detectable protein ex- reporter gene assays. The complete 5-LO 39UTR was inserted into pression. We found that miR-19a-3p is prominently expressed in http://www.jimmunol.org/ the luciferase reporter plasmid pMIR. Within the 39UTR we undifferentiated MM6 cells, but the obtained miR-19a-3p amounts mutated the seed regions to abolish miRNA binding (the mutated were rather variable (Fig. 2A). Interestingly, when the 5-LO bases are underlined in Fig. 1A). Additionally, a plasmid con- mRNA expression is plotted against miR-19a-3p expression in taining only the predicted binding site in triplicate was created MM6 cell samples, there is a negative correlation between both by guest on October 2, 2021

FIGURE 1. Functional analysis of the miRNA binding sites within the 5-LO 39UTR. (A) Predicted binding sites within the 5-LO 39UTR for miR-19a-3p and miR-125b-5p. (B) Plasmid constructs for the in vitro analysis based on the pMIR-Report plasmid by Invitrogen. In the first construct the complete 5-LO 39UTR is cloned behind the luciferase gene. The second construct consists of a triplicate of the predicted binding site with a spacer of 15 nt and is named triplicate. As an internal control the third construct, the perfect match, carries the complementary sequence of the miRNA. The last construct has a mutated miR binding site as shown in the upper panel.(C) Results of the reporter gene assay performed in Cos-7 cells by cotransfection of pMIR-Reporter plasmid and the respective pre-miRNA. Results are shown as relative light units normalized to the used negative control no. 2 and represent the means 6 SEM (n = 2–5). **p , 0.001, *** p , 0.001 by one-way ANOVA and Bonferroni posttest. 4 CONTROL OF 5-LIPOXYGENASE BY miRNAs parameters (r2 = 0.6125), suggesting that high 5-LO mRNA ex- Treatment of the MM6 cells with the combination of TGF-b and pression corresponds to a low expression of the miR-19a-3p and calcitriol led, as already published, to a strong induction of 5-LO vice versa in undifferentiated MM6 cells (Fig. 2B). The expression mRNA by ∼50-fold on day 2. In contrast, the expression pattern of of miR-125b-5p increased from day 0 to day 3 to ∼1.3-fold. The both miRNAs did not change significantly (compare Fig. 2A with overall expression of hsa-miR-125b-5p in comparison with hsa- Fig. 2C). miR-19a-3p is much lower (22DCt values were 65.8 6 46.6 for During differentiation, the cells were treated with antagomirs miRNA-19a [n = 9] and 8.7 6 6.7 for miRNA-125b [n = 7]). against miR-19a-3p and miR-125b-5p at a final concentration of 25 nM. To study the effect of miR-19a-3p and miR-125b-5p on the 5-LO Each antagomir alone increased the protein only to a small extent, protein expression, MM6 cells were incubated with a combination but the combination of both antagomirs increased the protein level of 1 ng/ml TGF-b and 50 nM calcitriol for 4 d, which leads to significantly by ∼1.8-fold compared with the control antagomir a strong increase in 5-LO protein expression (17). The RNA ex- (Fig. 2D). Because the antagomir treatment led to an increase in 5-LO pression was determined for 5-LO mRNA and both miRNAs. protein expression, the effect of the antagomir treatment on 5-LO Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Analysis of 5-LO mRNA and miR-19a-3p/125b-5p expression in MM6 cells. (A) Time course of 5-LO mRNA, miR-19a-3p, and miR-125b- 5p expression in MM6 cells. Values for 5-LO mRNA and miR-19a-3p/miR-125b-5p are normalized to b-actin or U48, respectively, and are expressed as means 6 SEM (n = 4–7). 22DCt values for miR-19a-3p and miR-125b-5p expression were 65.8 6 46.6 and 8.7 6 6.7 (n = 9 and 7), respectively. (B) Correlation of miR-19a-3p and 5-LO expression in undifferentiated MM6 cells. Values were analyzed by linear regression (r2 = 0.6125, n = 35). (C) Time course of 5-LO mRNA, miR-19a-3p, and miR-125b-5p expression in MM6 cells stimulated with 1 ng/ml TGF-b and 50 nM calcitriol. Values of 5-LO mRNA are normalized to b-actin and miR-19a-3p and miR-125b-5p expression to U48. Values are expressed as means 6 SEM (n = 3). (D) Western blot analysis of 5-LO expression in MM6 cells. The cells were differentiated for 4 d with 1 ng/ml TGF-b and 50 nM calcitriol in the absence or presence of 25 nM antagomir as indicated and analyzed for 5-LO and b-actin expression by Western blot using anti-5-LO and anti–b-actin Abs and an Odyssey Classic infrared imaging system. Results are expressed as means 6 SEM of four to six independent experiments. *p , 0.05 by one-way ANOVA and a Dunnett multiple comparison test. (E) 5-LO mRNA expression in antagomir-treated MM6 cells. The cells were differentiated for 4 d with 1 ng/ml TGF-b and 50 nM calcitriol in the presence of 25 nM antagomir. Results are given as means 6 SEM of the relative 5-LO mRNA expression compared with the control antagomir. 5-LO values are normalized to actin expression and are the results of four independent experiments. The Journal of Immunology 5 mRNA expression was also determined. Both antagomirs alone or in 5-LO protein expression was determined by Western blot. As combination did not change 5-LOmRNAexpression(Fig.2E). shown in Fig. 3C, 5-LO protein expression was detectable on all This, together with the reporter gene experiments, shows that miR- 4 d. Slightly stronger bands were observed in stimulated cells, 19a-3p and miR-125b-5p directly regulate 5-LO protein expression but this coincided with increased b-actin expression, suggesting but do not interfere with 5-LO mRNA expression in MM6 cells. that the relative 5-LO protein expression is rather constant in B cells regardless of cell stimulation. Thus, taken together our 5-LO, miR-19a-3p, and miR-125b-5p expression in results in B cells suggest that 5-LO expression seems not to be B lymphocytes stimulated with SAC and IL-2 regulated by miR-19a-3p and miR-125b-5p under these cell The 5-LO pathway plays an important role in T and B lymphocyte culture conditions using these stimuli. responses. Therefore, we investigated the expression of both 5-LO and the miRNAs miR-19a-3p and miR-125b-5p in peripheral B and MiR-19a-3p regulates 5-LO in activated T lymphocytes T cells. B lymphocytes were either cultivated in the absence or Next, we analyzed the influence of the miRNAs miR-19a-3p and presence 10 ng/ml SAC in combination with 20 U/ml IL-2 for the miR-125b-5p on 5-LO expression in activated T cells. Freshly indicated times (Fig. 3). SAC leads to BCR-mediated activation isolated T cells were stimulated with the lectin PHA, which leads to and proliferation of naive B cells (CD272, CD19+) (34, 35), and a strong polyclonal proliferation (37). T cell stimulation by PHA IL-2 was used as costimulus (36). can be divided into three phases, that is, an increase in volume, Fig. 3A illustrates the expression pattern of both 5-LO mRNA followed by an onset of RNA synthesis, marking in G1b phase, as well as miR-19a-3p and miR-125b-5p in untreated B cells and finally the DNA synthesis and cell proliferation leading to after cultivation of the cells for up to 4 d. The expression of a 5-fold increase in cell counts after 96 h (38). the 5-LO mRNA varied at ∼60–80% of the peak expression. The Fig. 4A shows the expression pattern of 5-LO mRNA and the two Downloaded from miRNAs showed a slight downregulation toward day 2 and an miRNAs in untreated cells during 4 d. Probably as a response to cell upregulation on day 4 to the level of the initial expression. When isolation and cultivation under cell culture conditions, 5-LO ex- we stimulated the B cells with SAC and IL-2, there was a slight pression decreased on day 1 to ∼20% of its peak expression and decrease in 5-LO mRNA as well as miR-19a-3p and miR-125b- remained constant around this level during the following days. The 5p expression (Fig. 3B). To analyze an influence on translation, miRNAs did not significantly change their expression during the 4 d. http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. Expression of 5-LO and miR-19a-3p and 125b-5p in human B cells stimulated with SAC plus IL-2. Untouched B cells were isolated as described in Materials and Methods.(A) Isolated B lymphocytes were cultivated for the indicated times and expression of 5-LO mRNA and miR-19a-3p and miR-125b-5p was determined by quantitative PCR. The values are normalized to U48 and expressed as means 6 SEM of ﬁve different donors. (B)B lymphocytes were stimulated with 10 ng/ml SAC plus 20 U/ml IL-2 and expression of 5-LO mRNA and miR- 19a-3p and miR-125b-5p was determined after the indicated times. Results were normalized to U48 and are expressed as means 6 SEM of three different donors. (C) Western blot analysis of 5-LO protein expression in untreated and SAC/IL-2–stimulated B cells. Samples (cor- responding to 3 3 106 cells) were collected after the indicated times and analyzed for 5-LO and b-actin expression by Western blot using anti–5-LO and anti–b-actin Abs and an Odyssey Classic infrared imaging system. 6 CONTROL OF 5-LIPOXYGENASE BY miRNAs

FIGURE 4. Expression of 5-LO mRNA and miR-19a- 3p and 125b-5p in primary human T lymphocytes stimulated with PHA. Untouched T cells were isolated as described in Materials and Methods.(A) T cells were cultivated for the indicated times and expression of 5-LO mRNA and miR-19a-3p and miR-125b-5p was determined by quantitative PCR. Values are given as means 6 SEM obtained from six different donors. (B) T cells were stimulated with 5 mg/ml PHA for the indicated times and expression of 5-LO mRNA and miR-19a-3p and miR- 125b-5p was determined by quantitative PCR and normalized to U48. Results are given as means 6 SEM of ﬁve Downloaded from to six different donors. http://www.jimmunol.org/

In contrast, stimulation with PHA decreased 5-LO expression was to identify miRNAs that target 5-LO in immune-competent dramatically on day 3 to ∼5% and reincreased to ∼20%onday4as cells. Among several bioinformatically predicted miRNAs, we shown in Fig. 4B. The two miRNAs showed different expression experimentally validated the interaction between 5-LO and the patterns compared with untreated T lymphocytes. MiR-125b-5p miR-19a-3p and miR-125b-5p. We used the monocytic cell line by guest on October 2, 2021 expression decreased to ∼40% on day 2 and remained on that MM6 where 5-LO is expressed on a low basal mRNA level and level. In contrast, miR-19a-3p expression increased ∼5-fold on day 3 treatment with the differentiation stimuli TGF-b and calcitriol and slightly decreased again on day 4. Unfortunately, 5-LO protein leads to a strong increase in 5-LO mRNA, protein, and activity expression in T cells varied between the different patients. Interest- (17). In undifferentiated cells, we could detect a distinct negative ingly, low 5-LO expression could be detected by Western blot, which correlation between the expression of 5-LO mRNA and miR-19a- rapidly disappeared after 3 h in culture (data not shown). Taken to- 3p. Interestingly, miR-19a-3p belongs to the miR-17∼92 cluster, gether, there is an inverse regulation of 5-LO mRNA and miR-19a- which was reported to inhibit TGF-b signaling (44). Specifically, 3p expression in PHA-activated T lymphocytes, which suggested members of this cluster (miR-17 and miR-20) reduce the expres- that 5-LO might be a target of miR-19a-3p in activated T cells. sion of the type II TGF-b receptor and miR-18, another member, To prove the regulation of 5-LO by miR-19a-3p, PHA-stimulated limits the expression of Smad4, suggesting that regulation of 5-LO T cells were incubated with 150 nM of the antagomir against hsa- miR-19a-3 for 4 d. This treatment led to an increase in 5-LO mRNA expression on days 3 and 4 compared with the treatment with a control antagomir (Fig. 5). The efficiency of the antagomir treatment was verified by mRNA expression analysis of the proven miR-19a-3p target gene cyclin D1 (Supplemental Fig. 2) (39). Taken together, the inverse expression of 5-LO and miR-19a- 3p as well as the antagomir results suggest that 5-LO is a target of miR-19a-3p also in PHA-stimulated T lymphocytes. Discussion Arachidonic acid metabolism by 5-LO plays a role in immune responses and inflammatory processes. 5-LO has also been shown to be involved in the development of prostate cancer as well as FIGURE 5. Effects of antagomir against miR-19a-3p on 5-LO mRNA expression in PHA-stimulated primary human T lymphocytes. Untouched chronic myeloid leukemia in BCR-ABL+ mice (40, 41). Further- T cells were treated with 5 mg/ml PHA and 150 nM control antagomir or more, 5-LO has been shown to be highly expressed in mantle cell antagomir-19a for the indicated times. Results are given as means 6 SEM lymphoma (42), B lymphocytes, as well as in differentiated my- of the relative 5-LO mRNA expression compared with the respective eloid cells such as granulocytes, monocytes, or dendritic cells. control antagomir sample at days 3 and 4. 5-LO values were normalized Cell differentiation as well as leukocyte proliferation have been to U48 expression. Samples were obtained from three different donors. shown to be regulated by miRNAs (43). Within this study, our aim ***p , 0.001 by one-way ANOVA and Bonferroni posttest. The Journal of Immunology 7 expression by the miR-17∼92 cluster can occur at multiple steps miR-19a-3p by PHA is in line with the observation that the miR- either by modulation of TGF-b signaling or directly by targeting 17∼92 cluster regulates T cell proliferation (24, 25) and that 5-LO (44). miR-19a-3p plays a role in promoting Th1 responses (26). Our study now revealed that miR-19a-3p and miR-125b-5p are Different mechanisms are discussed for the control of the gene involved in the direct regulation of 5-LO expression. We were able expression by miRNAs. They can either switch off or only fine tune to show that knocking down both miRNAs with antagomirs led to target gene expression (48). The latter case leads to a slight de- an increase in 5-LO protein expression in differentiated MM6 cells crease in translation but still allows protein expression (49). From (Fig. 2D). The miRNA knockdown did not affect 5-LO mRNA our data it can be concluded that the relationship between 5-LO expression. According to these data, the mechanism involved in and miR-19a-3p in MM6 cells as well as human peripheral T cells the regulation of 5-LO in MM6 cells by the two investigated stimulated with PHA is an example for the target tuning mecha- miRNAs is an inhibition of translation rather than mRNA decay. nism, because a knockdown leads only to a small effect on 5-LO Interestingly, miR-19a-3p and miR-125b-5p have previously been mRNA or protein expression. discussed in conjunction with the leukotriene pathway. A recent Interestingly, the effects of both miRNAs seem to be cell study showed that inhibition of either the BLT1 receptor or the type– and stimulus-specific. Because our reporter assay data cysteinyl leukotriene 1 receptor decreases expression of both suggest a direct binding of both miRNAs to the 39UTR of 5-LO miRNAs in macrophages. Additionally, miR-125b-5p expression mRNA, one possible explanation for this phenomenon could be 2/2 was decreased in BLT1 mice compared with wild-type mice the cell-specific expression of mRNA or miRNA binding proteins (12) and is upregulated by resolvin D1 (45). These observations that prevent interaction of the miRNAs with the 5-LO mRNA. and our data suggest a possible negative feedback regulation of In conclusion, we were able to find two miRNAs regulating 5-LO via the 5-LO products by induction of these two miRNAs. 5-LO. We determined the expression pattern of these miRNAs in Downloaded from Another interesting aspect is the observation that overexpression stimulated B and T cells and were able to show that antagonizing of miR-125b-5p blocks the differentiation of the promyeloic cell miR-19a-3p in PHA-stimulated T cells leads to an increase in 5-LO line HL-60 by DMSO (13) and induces myeloid leukemia in mice mRNA expression. Furthermore, treatment of differentiated (13, 46). Alternatively, differentiation of HL-60 cells by DMSO monocytic MM6 cells with antagomirs for miR-19a-3p and miR- strongly induces the 5-LO pathway in these cells (14). In B cell 125b-5p increases 5-LO protein expression. The data suggest that development miR-125b-5p seems to play an important role in the both miRNAs modulate the leukotriene pathway via regulation of http://www.jimmunol.org/ germinal center reaction, and it has been shown that miR-125b-5p the key enzyme 5-LO. This observation unravels previously un- is upregulated in germinal center cells (20). In our study, expres- recognized roles for both miRNAs by linking 5-LO with B and sion of miR-125b-5p in myeloid cells and in isolated lymphocytes T cell responses, and it indicates that 5-LO control by miRNAs is was much lower compared with miR-19a-3p, which might be due to a key component in the complex cellular signaling network of the culture conditions. innate immune responses. Regarding miR-19a-3p, it has been reported that its expression in B cells increases during differentiation from naive to germinal Acknowledgments center cells with an abrupt downregulation when the cells leave the We thank Olga Zimmermann (University Hospital Frankfurt, Frankfurt, by guest on October 2, 2021 germinal center (22). Alternatively, 5-LO is downregulated in Germany) for technical support and FACS measurements of the primary germinal center cells (8) and this downregulation is stronger on cells, Christoph Lehmann for HPLC measurements, Ann-Kathrin Ha¨fner protein than on the mRNA level, which supports a possible reg- and Sven George for TGF-b supply, and Paul Liles for proofreading the ulation by a miRNA. When human B cells were activated under manuscript. cell culture conditions, we did not observe strong changes in either 5-LO or miR-19a-3p and miR-125b-5p expression, indicating that regulation of 5-LO expression in B lymphocytes by the respective Disclosures miRNAs is restricted to certain physiological conditions and The authors have no financial conflicts of interest. might not be apparent under the usual cell culture conditions. The presence of the 5-LO in T cells has been controversially References discussed over the years (6, 47). We were able to detect 5-LO in 1. Borgeat, P., M. Hamberg, and B. Samuelsson. 1976. Transformation of arachidonic freshly isolated T lymphocytes on the mRNA level as well as on acid and homo-gamma-linolenic acid by rabbit polymorphonuclear leukocytes. Monohydroxy acids from novel lipoxygenases. J. Biol. Chem. 251: 7816–7820. the protein level (data not shown). However, in contrast to Cook- 2. Di Gennaro, A., and J. Z. Haeggstro¨m. 2012. The leukotrienes: immune- Moreau et al. (47), who detected 5-LO protein by Western blot modulating lipid mediators of disease. Adv. Immunol. 116: 51–92. even after stimulation, we saw a strong decrease in the 5-LO 3. Goodarzi, K., M. Goodarzi, A. M. Tager, A. D. Luster, and U. H. von Andrian. 2003. Leukotriene B4 and BLT1 control cytotoxic effector T cell recruitment to mRNA and protein levels within 3 h of cell culture. Because we inflamed tissues. Nat. Immunol. 4: 965–973. could not detect concomitant changes in the respective miRNAs 4. Tager, A. M., S. K. Bromley, B. D. Medoff, S. A. Islam, S. D. Bercury, E. B. Friedrich, A. D. Carafone, R. E. Gerszten, and A. D. Luster. 2003. Leu- and because the t1/2 of the 5-LO protein in PMNL and HL60 cells kotriene B4 receptor BLT1 mediates early effector T cell recruitment. Nat. was shown to be 24 h, this 5-LO downregulation seems to be due Immunol. 4: 982–990. to active degradation of the 5-LO protein when T cells are cul- 5. Irvin, C. G., Y. P. Tu, J. R. Sheller, and C. D. Funk. 1997. 5-Lipoxygenase products are necessary for ovalbumin-induced airway responsiveness in mice. tured. Interestingly, when we stimulated the T cells, we observed Am. J. Physiol. 272: L1053–L1058. a further downregulation on the mRNA level, which, at least is in 6. Jakobsson, P. J., D. Steinhilber, B. Odlander, O. Ra˚dmark, H. E. Claesson, and part, is due to the strong upregulation of miR-19a. In PHA- B. Samuelsson. 1992. On the expression and regulation of 5-lipoxygenase in human lymphocytes. Proc. Natl. Acad. Sci. USA 89: 3521–3525. stimulated T cells, we observed a strong decrease in 5-LO 7. Ra˚dmark, O., O. Werz, D. Steinhilber, and B. Samuelsson. 2007. 5-Lip- mRNA expression down to 5% after 4 d. Concomitantly, we saw oxygenase: regulation of expression and enzyme activity. Trends Biochem. Sci. a 5-fold increase in miR-19a-3p expression after 3 d of stimu- 32: 332–341. 8. Mahshid, Y., M.-R. Lisy, X. Wang, R. Spanbroek, J. Flygare, B. Christensson, lation (Fig. 4B). As expected, application of antagomirs against M. Bjo¨rkholm, B. Sander, A. J. R. Habenicht, and H.-E. Claesson. 2009. High miR-19a-3p upregulated 5-LO mRNA expression (Fig. 5). Im- expression of 5-lipoxygenase in normal and malignant mantle zone B lymphocytes. BMC Immunol. 10: 2. portantly, cyclin D1, another target gene of miR-19a, was up- 9. Bartel, D. P. 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. regulated too (Supplemental Fig. 2). The strong induction of Cell 116: 281–297. 8 CONTROL OF 5-LIPOXYGENASE BY miRNAs

10. Gonsalves, C. S., and V. K. Kalra. 2010. Hypoxia-mediated expression of 29. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of 5-lipoxygenase-activating protein involves HIF-1a and NF-kB and microRNAs microgram quantities of protein utilizing the principle of protein-dye binding. 135a and 199a-5p. J. Immunol. 184: 3878–3888. Anal. Biochem. 72: 248–254. 11. Fredman, G., Y. Li, J. Dalli, N. Chiang, and C. N. Serhan. 2012. Self-limited 30. Krutzfeldt,€ J., N. Rajewsky, R. Braich, K. G. Rajeev, T. Tuschl, M. Manoharan, versus delayed resolution of acute inﬂammation: temporal regulation of pro- and M. Stoffel. 2005. Silencing of microRNAs in vivo with “antagomirs”. Nature resolving mediators and microRNA. Sci Rep 2: 639. 438: 685–689. 12. Wang, Z., L. R. Filgueiras, S. Wang, A. P. M. Serezani, M. Peters-Golden, 31. Mohri, T., M. Nakajima, S. Takagi, S. Komagata, and T. Yokoi. 2009. MicroRNA S. Jancar, and C. H. Serezani. 2014. Leukotriene B4 enhances the generation of regulates human vitamin D receptor. Int. J. Cancer 125: 1328–1333. proinﬂammatory microRNAs to promote MyD88-dependent macrophage acti- 32. Seuter, S., B. L. Sorg, and D. Steinhilber. 2006. The coding sequence mediates vation. J. Immunol. 192: 2349–2356. induction of 5-lipoxygenase expression by Smads3/4. Biochem. Biophys. Res. 13. Bousquet, M., C. Quelen, R. Rosati, V. Mansat-De Mas, R. La Starza, Commun. 348: 1403–1410. C. Bastard, E. Lippert, P. Talmant, M. Lafage-Pochitaloff, D. Leroux, et al. 2008. 33. Ha¨rle, D., O. Ra˚dmark, B. Samuelsson, and D. Steinhilber. 1998. Calcitriol and Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome transforming growth factor-b upregulate 5-lipoxygenase mRNA expression and acute myeloid leukemia with the t(2;11)(p21;q23) translocation. J. Exp. by increasing gene transcription and mRNA maturation. Eur. J. Biochem. 254: Med. 205: 2499–2506. 275–281. 14. Brungs, M., O. Ra˚dmark, B. Samuelsson, and D. Steinhilber. 1994. On the in- 34. Graille, M., E. A. Stura, A. L. Corper, B. J. Sutton, M. J. Taussig, duction of 5-lipoxygenase expression and activity in HL-60 cells: effects of J. B. Charbonnier, and G. J. Silverman. 2000. Crystal structure of a Staphylo- vitamin D3, retinoic acid, DMSO and TGFb. Biochem. Biophys. Res. Commun. coccus aureus protein A domain complexed with the Fab fragment of a human 205: 1572–1580. IgM antibody: structural basis for recognition of B-cell receptors and super- 15. Ring, W. L., C. A. Riddick, J. R. Baker, D. A. Munafo, and T. D. Bigby. 1996. antigen activity. Proc. Natl. Acad. Sci. USA 97: 5399–5404. Lymphocytes stimulate expression of 5-lipoxygenase and its activating protein in 35. Bekeredjian-Ding, I., S. Inamura, T. Giese, H. Moll, S. Endres, A. Sing, monocytes in vitro via granulocyte macrophage colony-stimulating factor and U. Za¨hringer, and G. Hartmann. 2007. Staphylococcus aureus protein A triggers interleukin 3. J. Clin. Invest. 97: 1293–1301. T cell-independent B cell proliferation by sensitizing B cells for TLR2 ligands. 16. Ring, W. L., C. A. Riddick, J. R. Baker, C. K. Glass, and T. D. Bigby. 1997. J. Immunol. 178: 2803–2812. Activated lymphocytes increase expression of 5-lipoxygenase and its activating 36. Xia, X., H. K. Lee, S. C. Clark, and Y. S. Choi. 1989. Recombinant interleukin protein in THP-1 cells. Am. J. Physiol. 273: C2057–C2064. (IL) 2-induced human B cell differentiation is mediated by autocrine IL6. Eur. J. 17. Brungs, M., O. Ra˚dmark, B. Samuelsson, and D. Steinhilber. 1995. Sequential Immunol. 19: 2275–2281. Downloaded from induction of 5-lipoxygenase gene expression and activity in Mono Mac 6 cells by 37. Trickett, A., and Y. L. Kwan. 2003. T cell stimulation and expansion using anti- transforming growth factor beta and 1,25-dihydroxyvitamin D3. Proc. Natl. CD3/CD28 beads. J. Immunol. Methods 275: 251–255. Acad. Sci. USA 92: 107–111. 38. Poulton, T. A., A. Gallagher, R. C. Potts, and J. S. Beck. 1988. Changes in 18. Ventura, A., A. G. Young, M. M. Winslow, L. Lintault, A. Meissner, activation markers and cell membrane receptors on human peripheral blood S. J. Erkeland, J. Newman, R. T. Bronson, D. Crowley, J. R. Stone, et al. 2008. T lymphocytes during cell cycle progression after PHA stimulation. Immunology Targeted deletion reveals essential and overlapping functions of the miR-17 64: 419–425. through 92 family of miRNA clusters. Cell 132: 875–886. 39. Qin, X., X. Wang, Y. Wang, Z. Tang, Q. Cui, J. Xi, Y.-S. J. Li, S. Chien, and

19. Spanbroek, R., M. Hildner, D. Steinhilber, N. Fusenig, K. Yoneda, O. Ra˚dmark, N. Wang. 2010. MicroRNA-19a mediates the suppressive effect of laminar flow http://www.jimmunol.org/ B. Samuelsson, and A. J. Habenicht. 2000. 5-lipoxygenase expression in den- on cyclin D1 expression in human umbilical vein endothelial cells. Proc. Natl. dritic cells generated from CD34+ hematopoietic progenitors and in lymphoid Acad. Sci. USA 107: 3240–3244. organs. Blood 96: 3857–3865. 40. Chen, Y., Y. Hu, H. Zhang, C. Peng, and S. Li. 2009. Loss of the Alox5 gene 20. Gururajan, M., C. L. Haga, S. Das, C.-M. Leu, D. Hodson, S. Josson, M. Turner, impairs leukemia stem cells and prevents chronic myeloid leukemia. Nat. Genet. and M. D. Cooper. 2010. MicroRNA 125b inhibition of B cell differentiation in 41: 783–792. germinal centers. Int. Immunol. 22: 583–592. 41. Gupta, S., M. Srivastava, N. Ahmad, K. Sakamoto, D. G. Bostwick, and 21. Malumbres, R., K. A. Sarosiek, E. Cubedo, J. W. Ruiz, X. Jiang, R. D. Gascoyne, H. Mukhtar. 2001. Lipoxygenase-5 is overexpressed in prostate adenocarcinoma. R. Tibshirani, and I. S. Lossos. 2009. Differentiation stage-specific expression of Cancer 91: 737–743. microRNAs in B lymphocytes and diffuse large B-cell lymphomas. Blood 113: 42. Boyd, R. S., R. Jukes-Jones, R. Walewska, D. Brown, M. J. S. Dyer, and K. Cain. 3754–3764. 2009. Protein profiling of plasma membranes defines aberrant signaling path- 22. Thapa, D. R., X. Li, B. D. Jamieson, and O. Martıńez-Maza. 2011. Over- ways in mantle cell lymphoma. Mol. Cell. Proteomics 8: 1501–1515.

expression of microRNAs from the miR-17-92 paralog clusters in AIDS-related 43. Ochs, M. J., D. Steinhilber, and B. Suess. 2011. MicroRNA involved in in- by guest on October 2, 2021 non-Hodgkin’s lymphomas. PLoS ONE 6: e20781. flammation: control of eicosanoid pathway. Front Pharmacol 2: 39. 23. Zhang, J., D. D. Jima, C. Jacobs, R. Fischer, E. Gottwein, G. Huang, P. L. Lugar, 44. Dews, M., J. L. Fox, S. Hultine, P. Sundaram, W. Wang, Y. Y. Liu, E. Furth, A. S. Lagoo, D. A. Rizzieri, D. R. Friedman, et al. 2009. Patterns of microRNA G. H. Enders, W. El-Deiry, J. M. Schelter, et al. 2010. The myc-miR-17∼92 axis expression characterize stages of human B-cell differentiation. Blood 113: 4586– blunts TGFb signaling and production of multiple TGFb-dependent anti- 4594. angiogenic factors. Cancer Res. 70: 8233–8246. 24. Wu, T., A. Wieland, K. Araki, C. W. Davis, L. Ye, J. S. Hale, and R. Ahmed. 45. Rius, B., E. Titos, E. Morań-Salvador, C. Lo´pez-Vicario, V. Garcıá-Alonso, 2012. Temporal expression of microRNA cluster miR-17-92 regulates effector A. Gonza´lez-Pe´riz, V. Arroyo, and J. Cla`ria. 2014. Resolvin D1 primes the and memory CD8+ T-cell differentiation. Proc. Natl. Acad. Sci. USA 109: 9965– resolution process initiated by calorie restriction in obesity-induced steatohe- 9970. patitis. FASEB J. 28: 836–848. 25. Khan, A. A., L. A. Penny, Y. Yuzefpolskiy, S. Sarkar, and V. Kalia. 2013. 46. Chaudhuri, A. A., A. Y. So, A. Mehta, A. Minisandram, N. Sinha, V. D. Jonsson, MicroRNA-17∼92 regulates effector and memory CD8 T-cell fates by modu- D. S. Rao, R. M. O’Connell, and D. Baltimore. 2012. Oncomir miR-125b reg- lating proliferation in response to infections. Blood 121: 4473–4483. ulates hematopoiesis by targeting the gene Lin28A. Proc. Natl. Acad. Sci. USA 26. Jiang, S., C. Li, V. Olive, E. Lykken, F. Feng, J. Sevilla, Y. Wan, L. He, and 109: 4233–4238. Q.-J. Li. 2011. Molecular dissection of the miR-17-92 cluster’s critical dual 47. Cook-Moreau, J. M., Y. El-Makhour Hojeij, G. Barrie`re, H. C. Rabinovitch- roles in promoting Th1 responses and preventing inducible Treg differenti- Chable, K. S. Faucher, F. G. Sturtz, and M. A. Rigaud. 2007. Expression of ation. Blood 118: 5487–5497. 5-lipoxygenase (5-LOX) in T lymphocytes. Immunology 122: 157–166. 27. Heckman, K. L., and L. R. Pease. 2007. Gene splicing and mutagenesis by PCR- 48. Bartel, D. P., and C.-Z. Chen. 2004. Micromanagers of gene expression: the driven overlap extension. Nat. Protoc. 2: 924–932. potentially widespread influence of metazoan microRNAs. Nat. Rev. Genet. 5: 28. Varkonyi-Gasic, E., R. Wu, M. Wood, E. F. Walton, and R. P. Hellens. 2007. 396–400. Protocol: a highly sensitive RT-PCR method for detection and quantification of 49. Hornstein, E., and N. Shomron. 2006. Canalization of development by micro- microRNAs. Plant Methods 3: 12. RNAs. Nat. Genet. 38(Suppl.): S20–S24.