Distinct Requirements of Micrornas in NK Cell Activation, Survival, and Function

The Journal of Immunology Distinct Requirements of MicroRNAs in NK Cell Activation, Survival, and Function Natalie A. Bezman,*,† Elizabeth Cedars,*,†,‡ David F. Steiner,*,†,‡ Robert Blelloch,x,{ David G. T. Hesslein,*,† and Lewis L. Lanier*,†,‡ MicroRNAs (miRNAs) are small noncoding RNAs that have recently emerged as critical regulators of gene expression within the immune system. In this study, we used mice with conditional deletion of Dicer and DiGeorge syndrome critical region 8 (Dgcr8) to dissect the roles of miRNAs in NK cell activation, survival, and function during viral infection. We developed a system for deletion of either Dicer or Dgcr8 in peripheral NK cells via drug-induced Cre activity. We found that Dicer- and Dgcr8-deficient NK cells were significantly impaired in survival and turnover, and had impaired function of the ITAM-containing activating NK cell receptors. We further demonstrated that both Dicer- and Dgcr8-dependent pathways were indispensable for the expansion of Ly49H+ NK cells during mouse cytomegalovirus infection. Our data indicate similar phenotypes for Dicer- and Dgcr8-deficient NK cells, which strongly suggest that these processes are regulated by miRNAs. Thus, our findings indicate a critical role for miRNAs in controlling NK cell homeostasis and effector function, with implications for miRNAs regulating diverse aspects of NK cell biology. The Journal of Immunology, 2010, 185: 3835–3846. atural killer cells are a key component of the innate im- complexes results in recruitment and activation of protein tyrosine mune system, providing early cellular defense against kinases, which initiate the signaling cascade, resulting in secretion N viruses and intracellular pathogens, and contributing to of effector cytokines, such as IFN-g, and target cell cytotoxicity. the early detection and destruction of transformed cells (1). NK cells Lack of NK cells renders both human and mice susceptible to develop from a common lymphoid precursor in the bone marrow certain infections, particularly the herpesviruses, including human and undergo terminal maturation in the periphery, acquiring opti- cytomegalovirus and MCMV (5). Therefore, experimental in- mal cytolytic and effector functions (2). Mature NK cells are rela- fection of mice with MCMV provides a useful model for studying tively static in terms of proliferative capacity until transferred into factors that are important for NK cell activation in vivo. Although a lymphopenic environment (3, 4) or challenged with pathogens (5). much has been learned about the signaling molecules and tran- NK cell activation and function are determined by a balance of scription factors that control activation of NK cells, less is known signals transmitted by inhibitory and activating NK cell receptors about the posttranscriptional mechanisms that regulate NK cell (NKRs) (1). Many of the inhibitory NKRs, including Ly49 recep- function. tors in mice and inhibitory killer cell Ig-like receptors in humans, MicroRNAs (miRNAs) are short (∼22 nt) noncoding RNAs, recognize self-ligands, such as MHC class I molecules. Activating expressed from endogenous genes, that act on protein-encoding NKRs recognize host-encoded molecules induced by transfor- mRNAs, targeting them for translational repression or degrada- mation or viral infection (e.g., NKG2D ligands) (6), or nonself- tion (9). The biogenesis of miRNAs involves two processing steps. ligands, including the mouse cytomegalovirus (MCMV) m157 gp, Primary miRNA transcripts are first cleaved by the nuclear “micro- which is recognized by Ly49H (7, 8). Some activating receptors processor” complex containing the RNAse III enzyme Drosha and associate with adapter proteins that contain ITAMs, including its dsRNA-binding partner DiGeorge syndrome critical region 8 DAP12, FcεRIg, and CD3z. Ligation of ITAM-containing receptor (Dgcr8), resulting in short hairpin structures (precursor miRNAs) (10, 11). These precursor miRNAs are then exported to the cyto- plasm, and are further processed by another RNAse III enzyme, *Department of Microbiology and Immunology, †Cancer Research Institute, ‡Bio- medical Sciences Graduate Program, xEli and Edythe Broad Center of Regeneration Dicer, to generate the mature duplex miRNAs (12). Finally, a single Medicine and Stem Cell Research, and {Department of Urology, University of Cali- strand of the mature miRNA duplex, loaded into the RNA-induced fornia San Francisco, San Francisco, CA 94143 silencing complex, targets specific sequences within mRNA 39 Received for publication March 26, 2010. Accepted for publication July 19, 2010. untranslated regions resulting in mRNA degradation and/or trans- This work was supported by National Institutes of Health Grant AI068129. L.L.L. is lational repression. an American Cancer Society Professor. N.A.B. is an American Cancer Society Post- Genetic studies showed a critical requirement for Dicer in vivo. doctoral Fellow and was supported by National Institutes of Health Training Grant T32. D.G.T.H. is a special fellow of the Leukemia and Lymphoma Society. R.H.B. Ablation of Dicer in the mouse germline produces a lethal pheno- was supported by National Institutes of Health/National Institute of Neurological type (13), and conditional deletion of Dicer in various hemato- Disorders and Stroke Grants K08 NS048118 and R01 NS057221. poietic lineages has been shown to have detrimental effects, such as Address correspondence and reprint requests to Dr. Lewis L. Lanier, University of impaired cell differentiation, proliferation, and survival (14–21). California, San Francisco, 513 Parnassus Avenue, HSE 1001G, Box 0414, San Fran- cisco, CA 94143. E-mail address: [email protected] The function of Dicer, however, is not limited to miRNA biogenesis. The online version of this article contains supplemental material. Dicer is also required for the processing of small inhibitory RNAs Abbreviations used in this paper: Dgcr8, DiGeorge syndrome critical region 8; LNA, (siRNAs) derived from endogenous dsRNA transcripts or exoge- locked nucleotide acid; MCMV, mouse cytomegalovirus; MFI, mean fluorescence nous sources, including viral dsRNAs (22). Endogenous siRNAs intensity; miRNA, microRNA; NKR, NK cell receptor; PI, postinfection; siRNA, were discovered in mammalian oocytes and embryonic stem cells small inhibitory RNA; YFP, yellow fluorescent protein. (23–25) and were shown to be essential for oocyte maturation Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 (26). Thus, although the loss of miRNA-dependent regulation has www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000980 3836 DICER- AND DGCR8-DEPENDENT PATHWAYS REGULATE NK CELL FUNCTION been implicated in the phenotypes caused by Dicer deficiency, at layer were isolated from PBMCs (Stanford Blood Center, Palo Alto, CA), + 2 least in some cases they are the consequence of the loss of other stained with anti-CD56 and anti-CD3 mAbs, and CD56 CD3 lympho- . small RNA classes. Unlike Dicer, Dgcr8 is essential for the bio- cytes were sorted. The purity of the recovered NK cells was typically 98%. genesis of canonical miRNAs, but not in the generation of siRNAs In vivo BrdU labeling (23, 27). In this study, we developed a system for the deletion of Mice were injected i.p. with 200 mg BrdU (Sigma-Aldrich) every 24 h for either Dicer or Dgcr8 in NK cells via drug-induced Cre activity to 3 consecutive days (Fig. 3B) or once for 2 h (Fig. 3E) and then sacrificed. dissect the roles of miRNAs in NK cell activation, survival, and For the detection of incorporated BrdU, cells were first stained for surface function during infection. Ags, then fixed, permeabilized, treated with DNase I, and stained with allophycocyanin-conjugated anti-BrdU mAb (BD Pharmingen). Materials and Methods CFSE labeling and adoptive transfer Mice and infections A 1:1 splenocyte mixture of CD45.1+ (from wild-type C57BL/6 mice) and + D/+ D/D The Cre-ERT2 C57BL/6 transgenic mice were kindly provided by Dr. CD45.2 (from either Dgcr8 or Dgcr8 C57BL/6 mice) cells was M. Matloubian (University of California San Francisco, San Francisco, treated with ACK lysis buffer to remove RBCs, labeled for 8 min with 3 7 CA) (28), R26R-yellow fluorescent protein (YFP) knockin C57BL/6 mice 0.5 mM CFSE, and washed twice in PBS. A total of 1 10 labeled cells were provided by Dr. N. Killeen (University of California San Francisco) were transferred i.v. into Ly49H-deficient C57BL/6 mice. Twenty-four (29), and Ly49H-deficient (Klra82/2) mice were from Dr. S. Vidal (McGill hours later, recipient mice were infected with MCMV and sacrificed University, Montreal, Quebec, Canada). Mice carrying the conditional 4 d later. Adoptively transferred NK cells were analyzed for Ly49H ex- floxed alleles of Dicer (DicerF/F) (14) and Dgcr8 (Dgcr8F/F) (30) were pression and CFSE dilution by flow cytometry. backcrossed onto a C57BL/6 background for eight generations. To gen- MiRNA array profiling erate bone marrow chimeras, 6- to 8-wk-old CD45.1-congenic C57BL/6 mice (National Cancer Institute, Frederick, MD) were lethally irradiated Total RNA was extracted from sorted mouse splenic and human peripheral with 1000 rad. Donor bone marrow was harvested from DicerF/+, DicerF/F, blood NK cells by using TRIzol (Invitrogen). RNA quality was verified by an Dgcr8F/+, or Dgcr8F/F mice carrying Cre-ERT2 transgene and mixed 1:1 Agilent 2100 Bioanalyzer profile. A mixture of equal amounts of RNA from with bone marrow harvested from CD45.1 C57BL/6 mice. Cells were then mouse and human NK cells were made as reference.

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