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Cutting Edge: MicroRNA Regulation of Fusion into Multinucleated Giant Cells

This information is current as James R. Sissons, Jacques J. Peschon, Frank Schmitz, Rosa of September 25, 2021. Suen, Mark Gilchrist and Alan Aderem J Immunol 2012; 189:23-27; Prepublished online 1 June 2012; doi: 10.4049/jimmunol.1102477

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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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cutting Edge: MicroRNA Regulation of Macrophage Fusion into Multinucleated Giant Cells James R. Sissons, Jacques J. Peschon, Frank Schmitz, Rosa Suen, Mark Gilchrist, and Alan Aderem Cellular fusion of into multinucleated gi- the Th2 IL-4 and IL-13 participate in wound re- ant cells is a distinguishing feature of the granulomatous pair, immune suppression, and defense against parasites (2, 4, response to inflammation, , and foreign bodies 5). In addition, IL-4 and IL-13 can induce macrophage fusion (Kawai and Akira. 2011. Immunity 34: 637–650). We into multinucleated giant cells that serve to isolate and/or observed a marked increase in fusion of macrophages eliminate foreign bodies and parasites and are a hallmark genetically deficient in Dicer, an enzyme required for of the granulomatous response (6, 7). Studies of molecular canonical microRNA (miRNA) biogenesis. Gene ex- mechanisms that regulate mammalian fusion have iden- Downloaded from pression profiling of miRNA-deficient macrophages re- tified several key fusogenic molecules (i.e., CD9, CD47, vealed an upregulation of the IL-4–responsive fusion CD44, Tm7sf4/DC-STAMP, and SIRPa) (8, 9). Whereas protein Tm7sf4, and analyses identified miR-7a-1 as a macrophage activation is generally considered a plastic and negative regulator of macrophage fusion, functioning reversible response, the formation of multinucleated cells rep- resents a terminally differentiated state and is thus likely subject

by directly targeting Tm7sf4 mRNA. miR-7a-1 is itself http://www.jimmunol.org/ to unique regulatory mechanisms. an IL-4–responsive gene in macrophages, suggesting MicroRNAs (miRNAs) are 21- to 23-nt noncoding RNAs feedback control of cellular fusion. Collectively, these generated by sequential nuclear and cytosolic processing of data indicate that miR-7a-1 functions to regulate IL-4– precursor transcripts by the RNAseIII enzymes Drosha and directed multinucleated giant cell formation. The Dicer, respectively (10). They negatively regulate gene expression Journal of Immunology, 2012, 189: 23–27. by binding to conserved, partially complementary sequences most often found in the 39-noncoding regions of target irculating and tissue-resident macrophages mRNAs. Several miRNAs are responsive to TLR agonists and

serve as sentinels of the immune system by sensing function to dampen or reshape innate immune responses by guest on September 25, 2021 C the presence of pathogens using a diverse array of within macrophages (11). In this study, we demonstrate a role membrane-anchored and cytosolic detectors. Upon pathogen for miR-7a-1 in regulating multinucleated giant cell forma- exposure, macrophages coordinate both intrinsic and extrinsic tion by targeting the fusogenic cell surface protein Tm7sf4. host defense mechanisms to eliminate the intruding organism and establish protective immunity against reinfection (1, 2). Materials and Methods Macrophages are remarkably adaptable in their response to Mice infection and activate distinct effector mechanisms commen- Mice in which myeloid cells were selectively deficient in Dicer were generated surate with the pathogen detected. For example, detection of by crossing C57BL/6.129 Dicerfl/fl (12) with the C57BL/6.129 Lyz2tm1(cre) fl/fl tm1(cre)/+ viruses through cytosolic sensors triggers a rapid type 1 IFN- deletor strain (13) to generate C57BL/6 Dicer Lyz2 mice. Through- out experiments, age- and sex-matched C57BL/6 Dicerfl/+Lyz2tm1(cre)/+ mice dominated response, whereas proinflammatory expres- were used as controls. Mice obtained from The Jackson Laboratory (Bar Har- sion is induced following TLR2 recognition of peptidoglycan, bor, ME) were housed under specific pathogen-free conditions with approval of a component of Gram-positive (1, 3). Institutional Animal Care and Use Committee at the Institute for Systems Macrophage responses are further regulated by leukocyte- Biology. derived soluble and cell surface molecules. Exposure of mac- Tissue culture rophages to TLR ligands in the presence of IFN-g induces a classically activated state characterized by expression of Bone marrow-derived macrophages (BMM) were cultured in complete DMEM (plus 10% FBS, 2 mM L-glutamine, penicillin, and streptomycin) with TNF-a and IL-12, which together promote Th1- or Th17- recombinant human CSF-1 (50 ng/ml) for 6 d. To generate giant cells, bone dominated responses. In contrast, macrophages activated by marrow cells were cultured as above for 4 d, and then, mouse IL-4 (50 ng/ml;

Seattle Biomedical Research Institute, Seattle, WA 98109 The online version of this article contains supplemental material.

Received for publication September 14, 2011. Accepted for publication May 1, 2012. Abbreviations used in this article: BMM, bone marrow-derived macrophage; HEK, This work was supported by National Institutes of Health Grants 5RO1AI032971-20, human embryonic kidney; miRNA, microRNA; qPCR, quantitative PCR; UTR, un- 5RO1AI025032-23, and HHSN272200700038C. translated region.

The array data presented in this article have been submitted to the Gene Expression Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE36585. Address correspondence and reprint requests to Dr. Alan Aderem, Seattle Biomedical Research Institute, 307 Westlake Avenue North, Suite 500, Seattle, WA 98109. E-mail address: [email protected] www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102477 24 CUTTING EDGE: miRNA REGULATION OF MACROPHAGE FUSION

PeproTech) was added for an additional 6 d. Human embryonic kidney cells assess a role for miRNAs in regulating macrophage fusion into (HEK 293) for luciferase assays were grown in complete DMEM. Macro- multinucleated giant cells, a specialized Th2-driven response of phages derived from conditionally immortalized Hoxb8 progenitors were generated from C57BL/6 mice essentially as previously described (14) and macrophages to intrusion by parasites or synthetic foreign maintained in their myeloid progenitor stage in RPMI 1640 medium, 10% material. Mice carrying a floxed allele of Dicer were crossed to a FBS, 2 mM L-glutamine, 10 ng/ml GM-CSF-1, and 1 mM b-estradiol. To myeloid restricted cre expressing strain (Lys2Cre). The resulting generate Hoxb8 macrophages from wild-type and miRNA-transduced lines, fl/fl, Lys2Cre progenitors were harvested, washed twice in 13 PBS, and resuspended in mice, Dicer , were born at the expected Mendelian fresh BMM culture medium containing CSF-1 and cultured for 6 d as per the frequencies from the appropriate crosses and were overtly in- BMM protocol. distinguishable from littermate controls heterozygous for the floxed Dicer allele, Dicerfl/+, Lys2Cre. Dicer mRNA levels were Gene expression arrays reduced ∼ 75% in Dicerfl/fl, Lys2Cre bone marrow macrophages Total RNA was isolated using TRIzol (Invitrogen), and mRNA was labeled and (BMM) relative to controls (Fig. 1). The extent of Dicer de- hybridized to an Affymetrix GeneChip Mouse Exon ST 1.0 array. Expression data were acquired from six microarray hybridization experiments comprising letion could not be further increased by using mice homozy- three Dicerfl/flLyz2tm1(cre)/+ mutant-derived macrophages and three Dicerfl/+ gous for Lyz2cre, prolonged culture or LPS stimulation (J.R. Lyz2tm1(cre)/+ controls. Data were background-subtracted and normalized Sissons and J.J. Peschon, unpublished observations) and are in and then averaged across biological replicates using the log2 intensities (15, agreement with previous reports using the Lyz2-cre deletor 16). Significance testing was performed using log2 intensities and expression cutoff value of 6 with a Student t test; p , 0.01wasregardedassignificant. strain (19). A corresponding decrease in the expression of Gene array data are deposited in National Center for Biotechnology miRNAs abundantly expressed in BMMs was observed (Fig. 1).

Information-Gene Expression Omnibus accession number GSE36585 (http: We used genome wide expression profiling to analyze the Downloaded from //www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSE36585) consequences of Dicer deficiency on macrophage responses Retroviral transduction of miR-7a-1 to IL4. IL4 programs macrophages to dampen immune Genomic sequences encoding pri-miR-7a-1 were cloned corresponding to responses, repair damaged tissue and participate in defense genetic coordinates Chr13:58493908 and Chr13:58494429. The product was against parasites (2, 4). The expression of classic markers of cloned into a pMSCV-Puro-IRES-GFP vector (17), and a control construct in IL4 macrophage activation including arginase 1, chitinase-3 the same backbone was generated encoding a pri-miR-30–formatted short like-3 (Chi3l3/Ym1), and resistin-like molecule-a (Retnl1a/ http://www.jimmunol.org/ hairpin RNA against Tlr5, a gene not expressed by mouse BMM. Retroviral constructs were packaged using the Phoenix ecotropic 293 line. Macrophages Fizz) is unaffected by Dicer depletion (Supplemental Fig. 1B). were transduced and cells selected in puromycin for 21 d prior to analysis. IL-4 additionally induces the expression of genes involved in macrophage fusion. Among this panel of genes, only Tm7sf4, Tm7sf4 39-untranslated region cloning, mutagenesis, and luciferase a molecule required for macrophage fusion (20–22), was reporter assays significantly altered in Dicer-deficient cells (Supplemental The 39-untranslated region (UTR) of Tm7sf4 was cloned from C57BL/6 Fig. 1A). These data were confirmed by quantitative RT-PCR mouse genomic DNA by PCR corresponding to Ensembl coordinates 15071 and 15621. The product was cloned 39 of the firefly luciferase ORF in and Western blot analyses of resting and IL-4–stimulated

a pVitro-blasti (Invivogen) backbone carrying a control Renilla luciferase macrophages (Fig. 2A, 2B) and suggests a unique role for by guest on September 25, 2021 gene. Point mutations in the Tm7sf4 miR-7a target sequence were introduced Dicer, and hence miRNAs, in the control of Tm7sf4 ex- by mutagenesis PCR. HEK 293 cells were cotransfected with luciferase and pression. Extended culture of BMMs in IL-4 containing miRNA expressing vectors and assayed using a luminometer. media leads to the generation of multinucleated giant cells. DNA staining to determine giant cell ploidy Under these conditions, we noticed a marked increase in the fl/fl, Lys2Cre Ploidy staining was performed as described previously (18). Flow cytometry number of multinucleated cells in Dicer relative to was performed using a BD FACSAria II, and ploidy classes for giant cells were control cells (Fig. 2C, 2D). This suggests a direct link be- determined. tween the expression of the fusogenic molecule, Tm7sf4, and Gene expression analysis the fusion phenotype. Considering that IL-4 highly induces Tm7sf4 in Dicerfl/fl, Lys2Cre cultures relative to controls, we To analyze recombination of Dicer1 alleles, expression of genes and miRNAs, examined whether Dicerfl/fl, Lys2Cre macrophages were globally total RNA was isolated from macrophages using TRIzol (Invitrogen), treated with DNAase (Ambion), and used as template for reverse transcription (Su- hyperresponsive to IL-4. This was not the case. For example, perscript II; Invitrogen). Quantitative PCRs (qPCR) were performed using an IL-4–induced Stat6 phosphorylation and dephosphorylation ABI 2900 HT thermocycler, and expression levels were calculated using the were identical in Dicerfl/fl, Lys2Cre and control macrophages DCT method relative to control Ef1a gene. miRNA-specific reverse tran- scription was performed for each miRNA using gene-specific primers (Ap- plied Biosystems). miRNA levels were normalized to small-nucleolar-RNA sno202. The levels of Sno202 were unchanged by CSF-1 and IL-4 or by a variety of TLR agonists in the primary BMM or Hox macrophage systems. Western blotting analyses Western blotting analyses were performed using standard techniques, and membranes were probed with relevant primary Abs: rabbit anti-Stat6 (Cell Signaling Technology), rabbit anti–phospho-Stat6 (Abcam), rabbit anti- mouse-Tm7sf4 (Santa Cruz Biotechnology), rabbit anti-mouse b-actin 1- HRP (Jackson ImmunoResearch Laboratories). Statistical analysis Significance was determined using an unpaired two-tailed Student t test. FIGURE 1. Deletion of Dicer mRNA in BMM reduces miRNA levels. Quantification of Dicer mRNA levels in control (Dicerfl/+,Lys2cre) and Dicer- Results and Discussion deficient (Dicerfl/fl,Lys2cre) macrophages and analysis of miRNAs let-7a, miR- miRNAs have established roles in regulating classic innate 125a-5p and miR-7a expression in control and Dicerfl/fl,Lys2cre macrophages immune responses. We took a genetic approach to globally by qPCR. The Journal of Immunology 25 Downloaded from http://www.jimmunol.org/

FIGURE 2. IL-4 induces Tm7sf4 expression and excess giant cell formation in Dicerfl/fl,Lys2cre macrophages. (A) qPCR of resting and IL-4–induced (24 h) Tm7sf4 mRNA in control relative to Dicerfl/fl,Lys2cre macrophages. Error bars denote SEM; *p , 0.05, representative of five independent experiments. (B)

Immunoblotting of Tm7sf4 protein expression in macrophages (BMM; CSF-1 derived) or IL-4–derived multinucleated giant cells (MGC; CSF-1/IL-4 derived) by guest on September 25, 2021 in control or Dicerfl/fl,Lys2cre cells; representative of n = 3 experiments. (C) Dicer deficiency increases cellular fusion and multinucleated giant cell formation. Hematoxylin staining of control or Dicerfl/fl,Lys2cre macrophages stimulated with IL-4 for 144 h; representative of five experiments. Original magnification 3100. (D) Flow cytometric enumeration of the frequency of multinucleated cells in control or Dicerfl/fl,Lys2cre-multinucleated giant cells. Cells were stained with propidium iodide. Bars indicate the percentage of cells against chromosomal copy number. *p , 0.05. (E) Immunoblotting analysis of Dicerfl/fl,Lys2cre cultures for total Stat6 protein and phospho-Stat6 response to IL-4; representative of n = 5 experiments.

(Fig. 2E). Furthermore, another IL-4–responsive fusogenic miR-7a-1 on chromosome 13, whereas there was no detectable molecule, E-cadherin (Cdh1), was unaffected in Dicerfl/fl, Lys2Cre histone acetylation in the vicinity of miR-7a-2 on chromosome macrophages (Fig. 1), demonstrating the specificity of regu- 7. This suggests that miR-7a-1 is the IL-4–responsive species lation of Tm7sf4 by miRNA. Finally, the expression of (Supplemental Fig. 2). We therefore considered miR-7a-1 to be a number of macrophage fusogenic molecules was unaffected a candidate miRNA regulating both Tm7sf4 expression and IL- in Dicerfl/fl, Lys2Cre macrophages (Supplemental Fig. 1A). 4–induced multinucleated giant cell formation. Examination of the Tm7sf4 39-UTR using various target To directly examine a role for miR-7a-1 in regulating prediction algorithms (23) demonstrated a number of miRNA Tm7sf4 expression, we transduced immortalized BMMs with target sites. However, only the predicted miR-7a target se- retroviruses encoding either pri-miR-7a-1 or a control pri- quence was conserved among mammalian species. There are miR-30. Decreased Tm7sf4 expression was specifically ob- three isoforms of miR-7, miR-7a-1, miR-7a-2, and miR-7b; served in miR-7a-1–overexpressing BMMs (Fig. 3B). More- miR-7a and 7b can be distinguished by PCR based on their over, IL-4–induced multinucleated giant cell formation was mature form, but the miR-7a-1 and miR-7a-2 isoforms cannot. attenuated in miR-7a-1–overexpressing BMMs (Fig. 3C). To miR-7b is not expressed in macrophages, whereas miR-7a-(1/2) determine whether the ability of miR-7a-1 to regulate Tm7sf4 are (data not shown). Moreover, miR-7a-(1/2) are strongly expression is through direct targeting of the 39-UTR, we fused induced by IL-4 (Fig. 3A), suggesting a potential role in regu- these sequences downstream of a firefly-luciferase reporter. lating Tm7sf4 (Fig. 3A). To distinguish between miR-7a-1 and The resulting construct was tested in HEK 293 cells cotrans- miR-7a-2, we turned to epigenetic mapping (Supplemental Fig. fected with either miR-7a-1 or a control miRNA. As shown in 2). These two miRNAs are found on chromosomes 13 and 7, Fig. 3D, the Tm7sf4 39-UTR imparted miR-7a-1–specific respectively. Histone acetylation results in an open chromosome suppression of luciferase expression. Transfection of the structure that facilitates transcription. Chromatin immunopre- Tm7sf4 39-UTR containing a point mutation in the miR-7a-1 cipitation-sequencing experiments in IL-4–treated macrophages target sequence had no effect on luciferase activity (Fig. 3D). demonstrated substantial histone acetylation in the vicinity of These experiments demonstrate that miR-7a-1 interacts directly 26 CUTTING EDGE: miRNA REGULATION OF MACROPHAGE FUSION Downloaded from http://www.jimmunol.org/ FIGURE 3. miR-7a is regulated by IL-4, and ectopic expression of miR-7a regulates Tm7sf4 mRNA and protein levels, leading to restricted giant cell formation. (A) qPCR analysis of miR-7a response to IL-4 in macrophages. Representative of three independent experiments. *p , 0.05, **p , 0.01. (B) Immunoblotting of Tm7sf4 in macrophages expressing a control miRNA or miR-7a; cells were stimulated with IL-4 for 24 h and examined for Tm7sf4 expression relative to b-actin. (C) Flow cytometric analysis of multinucleated cells from control miRNA or miR-7a–expressing cells. *p , 0.05. (D) HEK 293 cells cotransfected with wild-type (WT) or mutated Tm7sf4 39-UTR and miR-7a and then assessed for luciferase activity. *p , 0.01. and specifically with the Tm7sf4 39-UTR to regulate its ex- References

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