Micrornas in Inflammation and Immune Responses

MicroRNAs in inflammation and immune responses

J Contreras1,2 and DS Rao2,3,4,5

1Cellular and Molecular Pathology PhD Program, UCLA, Los Angeles, CA, USA; 2Department of Pathology and Laboratory Medicine, UCLA, Los Angeles, CA, USA; 3Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA, USA; 4Broad Stem Cell Research Center, UCLA, Los Angeles, CA, USA and 5Division of Biology, California Institute of Technology, Pasadena, CA, USA

MicroRNAs (miRNAs) are important regulators of gene expres- This results in the formation of a short double-stranded RNA sion in the immune system. In a few short years, their consisting of the miRNA and its complementary sequence. This mechanism of action has been described in various cell is unwound and loaded onto the RNA-induced silencing lineages within the immune system, targets have been defined complex (RISC) where the mature miRNA binds to target and their unique contributions to immune cell function have 11 SPOTLIGHT been examined. Certain miRNAs serve in important negative sequences (reviewed in Czech and Hannon ). feedback loops in the immune system, whereas others serve to Recently, a second, non-Dicer-dependent pathway of miRNA amplify the response of the immune system by repressing biogenesis has been discovered.12,13 This alternative pathway inhibitors of the response. Here, we review some of the better relies on a component of the RISC complex, namely the Argonaute understood mechanisms as well as some emerging concepts of protein Ago2, to mediate the final processing of the miRNA. miRNA function. Future work will likely involve defining the function of specific miRNAs in specific immune cell lineages Several miRNAs are thought to be processed by this pathway. and to utilize them in the design of therapeutic strategies for Interestingly, the secondary structure of the pre-miRNA seems to diseases involving the immune system. be important in determining the pathway of biogenesis, with Leukemia (2012) 26, 404–413; doi:10.1038/leu.2011.356; distinct loop and hairpin structures determining whether the published online 20 December 2011 miRNA precursor is cleaved by Dicer or Ago2. Of these, miR-451 Keywords: microRNA; inflammation; NF-kB is the best characterized, and it contains a very small, distinctive loop structure. Following its maturation via endoribonucleolytic cleavage, the miRNA is unwound and the thermodynamically more stable strand is loaded onto the RISC, a complex composed of multiple MicroRNA biogenesis and mechanism of action proteins and RNAs. Here, the miRNA encounters and binds to a target mRNA sequence. The basis of targeting is thought to be First discovered by the laboratories of Victor Ambros and Gary Watson-Crick base-pairing via a six-nucleotide seed sequence Ruvkun in Caenorhabditis elegans, microRNAs (miRNAs) are located within the 30-untranslated region of the target mRNA. the first extensively studied class of noncoding RNA.1,2 There This seems to be the most well-accepted model for targeting, are between 600 and 700 miRNAs that have been cataloged, and several target prediction algorithms are based on this with various degrees of conservation. It is now clear that there model.14 – 17 Usually, there are also areas of complementarity are likely species-specific miRNAs, and the most current listing along the 30 end of the miRNA, leading to a central region of in miRBase lists 1452 miRNAs in humans.3 miRNA biogenesis is noncomplementarity or ‘bulge’ sequence. In contrast to this, a highly regulated process that allows for their expression in small interfering RNAs bind to target sequences with perfect particular cellular and functional contexts. miRNAs are encoded complementarity, leading to endoribonucleolytic cleavage of within the cellular genome in three main ways: (1) as unique the target mRNA via the Slicer function of Ago2.18,19 The genes (2) as intronic sequences within protein-coding genes and mechanism of miRNA repression of the targeted transcript is (3) as polycistronic miRNAs, or a single transcript encoding thought to involve a combination of mRNA destabilization and 4,5 multiple miRNAs. This variable encoding renders complex exoribonucleolytic activity as well as translational repression possibilities for their regulation, through alternative processing (reviewed in Nilsen20). Recent work suggests that mRNA levels are or miRNA processing. The canonical miRNA biogenesis path- an accurate way to determine the degree of repression caused by way involves transcription from these genes by RNA polymerase 21,22 miRNA expression. II, which allows for regulation by transcription factors.6 Because repression is thought to be mediated through a short Following transcription, the primary miRNA transcript is ‘seed’ sequence, miRNAs are predicted to target numerous processed by the endoribonucleases Drosha/DGCR8 in the mRNAs within a cell, from tens to hundreds. This has been nucleus before transport into the cytoplasm.7,8 The resulting pre- partially validated by high-throughput techniques that show that miRNA is further processed by the endoribonuclease Dicer.9,10 miRNA gain and loss of function can lead to the repression or Dicer acts by cleaving the looped end of the miRNA precursor 21,22 derepression of numerous genes, respectively. This has led and ‘dicing’ the RNA at 20–25 base-pair intervals thereafter. investigators seeking targets for miRNAs to use a combination of high-throughput experimental techniques and bioinformatics- Correspondence: Dr DS Rao, Department of Pathology and Laboratory based predictive algorithms to delineate direct miRNA targets.23 Medicine, UCLA, 650 Charles E Young Drive South, Factor 12-272, Other recent methodological advances include crosslinking of Los Angeles CA, 90095, USA. E-mail: [email protected] miRNAs with the RISC complex followed by immunoprecipita- Received 4 September 2011; revised 26 October 2011; accepted 4 tion and high-throughput sequencing to identify mRNA targets November 2011; published online 20 December 2011 in the relevant biochemical context, the so-called Argonaute miRNAs in inflammation and immune responses J Contreras and DS Rao 405 HITS-CLIP.24 These technological advances have rendered specific role in a particular cellular/functional context. The first possible the identification of various pathways regulated by idea is that miRNAs may be able to act in a shorter time scale miRNAs. Overall, the multiplicity of targeting by miRNAs has than protein transcriptional repressors. miRNA transcription is led to the idea that they can regulate large sets of genes and thus induced by inflammation, like transcriptional regulators, but have profound effects on gene expression. miRNAs are processed and become active more quickly than protein transcription factors, as they require neither translation nor translocation back into the nucleus to begin repressing their MicroRNA regulation during inflammation targets.35 This may indeed be important in innate responses (for example, miR-155) where a short time frame is critical to begin Many miRNAs are co-regulated with protein-coding genes the fight against pathogenic organisms. The second idea is that during the inflammatory response. The regulation of miRNA although miRNAs can act more quickly than transcriptional biogenesis occurs at many levels. The first and perhaps best regulators, they may act more slowly than protein factors that studied is transcriptional regulation, and many miRNAs are could directly target and inactivate miRNA targets.36 As miRNAs induced as part of an inflammatory transcriptional program, as act at the RNA level, protein that is already produced and active we detail below for nuclear factor (NF)-kB. However, such will remain active until miRNA action downregulates the regulation is not limited to the immune system and forms a amount of new protein being produced. Here, miRNAs may general paradigm for regulation of miRNA, and regulation of act as ‘delay switches’ in negative feedback regulation of miR-17B92 and miR-34 family miRNAs by MYC and p53, immune responses. This is certainly true for the recently 25 – 29 respectively, are undoubtedly important in cancer. The described function of miR-21 in inflammation. miR-21 causes overall conclusions from various studies is that miRNAs are a delayed inhibition of PDCD4 (programmed cell death 4), a induced in much the same way as protein-coding genes, but the proinflammatory protein, following induction by NF-kB.37 time frame of their biogenesis and mode of action may confer miRNAs are also thought to have distinctive functions in unique regulatory properties to these small RNA molecules. different cell types. Although the mechanism of action of miRNA biogenesis is also regulated at the level of processing miRNAs is the same in different cell, the transcriptional program during inflammation. Several proteins that are induced during of a given cell type limits the number of relevant targets for a inflammatory responses can regulate the processing of miRNAs. given miRNA. Hence, a cell undergoing massive transcriptional These include the transforming growth factor-b-induced SMAD activation during inflammation will have a different set of targets proteins, which are recruited into a complex with the RNA regulated by a given miRNA than a quiescent cell. Additionally, helicase p68, a component of the Drosha endoribonucleolytic developmentally distinct lineages have distinct sets of target 30 complex. ADAR, an RNA-modifying enzyme that is upregu- genes for a given miRNA. For example, miR-146a seems to have lated during inflammation, can introduce mutations in double- distinct sets of targets in myeloid cells (TRAF6 (TNF receptor- stranded miRNA precursors, thereby changing the targeting associated factor 6) and IRAK1 (interleukin-1 receptor-asso- 31 specificity of the miRNA. The tumor-suppressor protein p53, ciated kinase 1)) versus regulatory T cells (STAT1 (signal which is upregulated during certain types of inflammatory transducer and activator of transcription 1)), and hence the responses, can facilitate processing of a number of miRNA functional consequences of miRNA expression can be quite 32 transcripts. In addition, interferon-g can directly repress the different in these two distinct cell lineages.38 – 40 33 expression of Dicer and other biogenesis factors. Some post- The last concept regarding miRNA regulation that is important transcriptional processing changes are highly specific for a given to understand is that their inhibition of target mRNAs does not miRNA; for example, the Lin28 protein tightly regulates the lead to a complete knockdown. This has been shown by both 34 processing of let-7 miRNA in a loop-specific manner. Whether gain- and loss-of-function experiments. These observations have other such regulatory factors, which are specific for a single or a led to the idea that miRNAs are ‘fine-tuners’ of gene expression few miRNAs, are induced during inflammation remains to be as opposed to transcription factors that act as ‘switches’. In determined. It also remains to be determined whether the cellular function, this would suggest that miRNAs serve to ‘fix’ alternative pathway of miRNA biogenesis plays any role in or ‘buffer’ cellular gene expression patterns, given that they regulating the immune response. cause small changes in many genes. However, it is also true that gene expression patterns can sometimes be changed drastically by small changes of a key transcription factor, which miRNAs General concepts regarding MicroRNA function in the can also regulate. Such is the case with miR-150, whose SPOTLIGHT immune system incremental repression of c-Myb leads to a dramatic change in the output of B lymphocytes from the hematopoietic system.41,42 The preceding discussion of miRNA biogenesis should hint at miRNAs, then, can perform various functions of either stabiliz- the idea that miRNAs may play distinctive regulatory roles in the ing cellular gene expression patterns (and phenotypes) or immune system. Several general hypotheses and theories have causing differentiation, depending on the context in which they been presented in the recent literature about how miRNAs may are expressed. function in gene regulation. This occurs as part of a combined The preceding paragraphs capture some of the current protein/miRNA gene expression program and there may be explanations and ideas for miRNA function in hematopoiesis some unique properties that a miRNA is able to confer to a gene and immunity. It is also apparent that there is a lot to understand regulatory pathway.35,36 These concepts include: (1) miRNAs and that the field is evolving rapidly. New reports about have a unique role in regulating time-sensitive aspects of gene miRNAs acting in a noncanonical manner (for example, by regulation in the immune system, (2) miRNAs have distinct upregulating gene expression, acting in a non-30-untranslated functions in distinct cell types and (3) miRNAs regulate gene region-dependent fashion and regulating noncoding RNA) are expression by incomplete repression of their targets. appearing on an ongoing basis.43,44 These will no doubt lead us First, miRNAs may have a particularly important function in to revisit some of these ideas and to further modify them as we cellular responses that are time dependent. Two apparently strive to understand the regulation of gene expression and divergent ideas have been proposed, but each may have a immunity.

Leukemia miRNAs in inflammation and immune responses J Contreras and DS Rao 406 Innate immune cells and MicroRNA various immune lineages. The net effect of miR-146a deficiency at the molecular level is to cause activation of NF-kB, and the The immune system is usually divided into the innate and myeloproliferative phenotype can be corrected in these mice by adaptive arms. The innate immune system is a ‘first responder’ a genetic knockout of Nfkb1/p50, one of the NF-kB subunits.39 that also sets the stage for the adaptive immune response by This miRNA is also deleted in the 5q-syndrome, a myelodys- initiating the inflammatory process. The adaptive immune plastic syndrome, highlighting a similarity in pathways involved response culminates with the generation of antigen-specific in inflammatory hematopoiesis and in myeloid malignancies.52 lymphocytes that clear the invading pathogen with a great deal miR-146a is also important in mature immune cells. miR-146a- of specificity and provide long-lasting immunity against the deficient bone marrow-derived macrophages show increased same. In this section, we will consider the effects that three well- inflammatory cytokine production, including interleukin (IL)-6, studied miRNAs have on the innate immune system (miR-155, IL-1b and tumor necrosis factor-a, upon LPS stimulation.38 miR-146a and miR-223), although approximately a dozen miR-146a also negatively regulates endotoxin-induced toler- different miRNAs have been implicated in the innate immune ance in human monocytes by repressing TRAF6 and IRAK1.53,54 system by various studies. These studies have pointed out an Type I interferon production, which is important in the antiviral interesting connection between miRNA-modulated pathways response, is inhibited by miR-146a during vesicular stomatitis involved in the activation of mature innate immune cells (that is, virus infection of peritoneal macrophages.55 Components of the macrophages, dendritic cells and granulocytes) and in the TLR signaling pathway, STAT5 and IRF5, are additional putative developmental program in the bone marrow during inflamma- targets of miR-146a.56 tion. We further discuss the critical signaling pathways that are The last miRNA that we will discuss is miR-223. Originally SPOTLIGHT found in these immune cells in the next section about Toll-like described as a miRNA that was highly expressed in myeloid receptors (TLRs) and NF-kB. cells in the bone marrow, this miRNA is silenced in certain types miRNAs have developmental effects on the bone marrow of myeloid leukemia, with re-expression promoting differentia- and this has been reviewed elsewhere recently.45 However, tion of myeloid blast cells.57 – 59 However, miR-223À/À mice inflammation causes some dramatic changes in the develop- showed increased granulocytes with features of hyper activa- mental sequence of hematopoietic cells. It turns out that tion, which would not be expected if this miRNA only promoted hematopoietic stem and progenitor cells can respond to granulocytic differentiation.60 Hence, this miRNA may also inflammatory signals such as interferon and lipopolysaccharide connect myeloid development with inflammation. The exact (LPS).46,47 This, along with the conventionally accepted mechanisms of these changes remain to be worked out, but it mechanism of hematopoietic stem and progenitor cells sensing appears to involve the transcription factor Mef2c. depletion of downstream mature hematolymphoid cells, is Overall, these studies highlight the roles of miRNAs in thought to underlie a program of ‘inflammatory hematopoiesis’. activation of innate immune cells and inflammatory hematopoi- During inflammatory hematopoiesis, the output of the marrow is esis, which seem to be molecularly related processes in mature directed toward myeloid cell production and away from the and immature hematopoietic cells, respectively. The roles of production of B lymphocytes and erythroid cells, and can be these miRNAs depend on repression of a single or a few targets induced by LPS injection in mice. This change gradually wears and they can function as proinflammatory factors (miR-155) or off and homeostatic hematopoiesis returns after several days in as negative feedback regulators (miR-146a). mice. Constitutive activation of this program may result in chronic inflammatory conditions and may also be important in the pathogenesis of myeloid malignancies. MicroRNAs in the TLR and NF-jB pathways The first miRNA that is of importance in inflammatory hematopoiesis program is miR-155. This miRNA is upregulated The TLR pathway is an evolutionarily conserved pathway that in acute myeloid leukemia and causes a myeloproliferative recognizes pathogen-associated molecular patterns and is active disorder when overexpressed in bone marrow.48 miR-155 mainly in the immune lineages, including macrophages, targets the SHIP1 inositol phosphatase, and knockdown of Src dendritic cells and B and T cells.36,61 The TLR pathway is Homology-2 domain-containing inositol-5’-phosphatase 1 activated by the recognition of these pathogen-associated (SHIP1) in the bone marrow recapitulates the myeloproliferative molecular patterns by intracellular or cell surface TLRs that disorder.49 It is also noteworthy that miR-155 is rapidly and recognize different types of pathogens. This binding is followed effectively induced in intact mice that are given LPS. Hence, by the recruitment of various adapter proteins such as MyD88, miR-155 is a key proinflammatory miRNA that is involved in TRIF and Tirap. MyD88-mediated signaling leads to the changing the output of the bone marrow. Interestingly, recent elaboration of the ‘inflammatory program’, whereas TRIF- studies have found that miR-155 promotes expression of mediated signaling contributes to the ‘antiviral response’. Both inflammatory cytokines and the interferon response in primary pathways activate NF-kB, which leads to the transcriptional macrophages and dendritic cells. This likely occurs via upregulation of a large number of genes that mediate the repression of the negative regulators of inflammation, SOCS1 response of the immune cell (see Figure 1). In many of these (suppressor of cytokine signaling 1) and SHIP1, both direct lineages, specific miRNAs are produced after activation of the targets of miR-155.50,51 Through the downregulation of these TLR pathway. The first study examining miRNA production after anti-inflammatory pathways, miR-155 also promotes endotox- TLR stimulation identified three miRNAs that are induced by LPS emia in mice.50 miR-155-deficient dendritic cells are not in human macrophages, namely miR-155, miR-146a and miR- as effective at promoting inflammation as their wild-type 132.62 Although some miRNAs are produced immediately, as counterparts. soon as 2 h after treatment (for example, miR-155), others are The miR-146a is a feedback regulator of the TLR/NF-kB produced in a delayed time frame after TLR stimulation (for pathway, as further discussed in the next section. miR-146a- example, miR-21). This temporally regulated expression se- deficient mice show multiple abnormalities, with a spectrum of quence may distinguish the function of miRNAs in regulating immunoproliferative and autoimmune disease.38 These various inflammation. In addition to miRNA induction by TLR signaling, phenotypes are likely the result of miR-146a deficiency in recent studies also demonstrate inflammatory repression, such

Leukemia miRNAs in inflammation and immune responses J Contreras and DS Rao 407 Central to the propagation of the signal from TLRs is activation of NF-kB, a key transcriptional regulator of the inflammatory response. The regulation of NF-kB is very complex and thoroughly reviewed elsewhere.67 Briefly, NF-kB consists of four subunits that are independently transcribed and processed. These subunits can homo- or heterodimerize upon activation and translocate into the nucleus. In the inactive state, the subunits are maintained in the cytoplasm by the IkB proteins, which in turn are regulated by IkB kinases (IKKs). Several of these proteins are regulated by miRNAs, affecting NF-kB activity. miR-155 and miR-199 target the IKKs, upstream of IkBa.68,69 miR-223 also targets IKK-a, a component of the NF-kB pathway, during differentiation of monocytes into macrophages.70 miR-223 downregulation during macrophage activation had two effects: first, the cells had less baseline NF-kB activity and, at the same time, the cells were primed to be activated. miR-9 has been shown to target NF-kB itself, namely the Nfkb1/p50 subunit.71 Downstream of NF-kB are the numerous genes that it transcriptionally induces as part of the inflammatory program. One of these, the cytokine IL-6, is targeted by Let-7 family members. As Let-7 miRNAs themselves are negatively regulated by TLR/NF-kB, this allows for elaboration of the proinflammatory program.72 miRNAs can also regulate other transcription factors of importance to the inflammatory response, as with miR-155 downregulation of C/EBP-b.48 One last point that has emerged from recent studies is that miRNAs may act in concert with other mechanisms to cause mRNA downregulation. Previous studies have demonstrated the Figure 1 Schematic of the TLR pathway and miRNAs that are importance of AU-rich elements within mRNA sequences in involved in negative regulation of the pathway. directing mRNA degradation. These elements recruit RNA- binding proteins, such as tristetraprolin, whose binding leads to RNA degradation. For instance, the mRNA encoding tumor necrosis factor-a and IL-10 contains sites for binding by miR-16 as miR-155 repression, in response to IL-10.63 Despite this and miR-106, respectively, but also contains AU-rich elements. understanding of transcription-based production of miRNAs, Hence, RNA-binding proteins, such as tristetraprolin, may bind virtually nothing is known about how inflammatory transcrip- concurrently with miR-106/miR-16, leading to clearance of tion programs may affect miRNA degradation. The question of these mRNAs.73,74 In other studies, miR-221, miR-579 and miR- what regulates miRNA degradation could drastically change our 125b were found to interact with tumor necrosis factor-a mRNA, understanding of miRNA regulation of inflammation. indicating that many other miRNAs may also influence the In regulation of the TLR pathway, a central question has been degradation of this key inflammatory cytokine along with the whether miRNAs regulate TLRs themselves. Although the AU-rich elements.75 Hence, miRNAs regulate a variety of targets TargetScan algorithm (www.targetscan.org) shows very few in the TLR/NF-kB pathways and may interact with other factors miRNAs that target TLRs, alternative algorithms indicate that regulating mRNA stability to achieve the gene expression miR-223 is a regulator of TLR3 and TLR4. Supporting this is a patterns necessary for the immune response. We summarize finding that granulocytes that express miR-223 at high levels the effects of miRNAs on TLR signaling in Table 1. For a further have low levels of TLR3.64 In oral keratinocytes, miR-105 review of miRNAs in the TLR pathway, the reader is referred to regulates TLR2.65 In mouse peritoneal macrophages, Let-7 an excellent recent review.36 SPOTLIGHT family miRNAs target TLR4.50 Downstream of the TLRs, miR- 145 and miR-146a target the TLR adapter proteins TRAF6 and IRAK1.52 miR-147 has been shown to attenuate TLR2, TLR3 and MicroRNAs in B-cell activation TLR4-mediated production of inflammatory proteins such as tumor necrosis factor-a and IL-6.66 B cells are a central component of the adaptive immune system Recent studies have shown that TLRs can sequentially responsible for the production of long-lived antibodies, which upregulate different miRNAs to help guide the temporal are the main determinant of long-term specific immunity.76 B regulation of the immune response.37 In this study, the authors cells develop in the bone marrow and achieve a remarkable determined that the initial TLR signal is propagated by miR-155 diversity of antigen specificities by rearrangement of their that downregulates SHIP1, an inflammatory inhibitor, thereby immunoglobulin loci by V(D)J recombination. Once they have allowing for inflammation to proceed. Later, induction of miR- matured in the bone marrow, they seed secondary lymphoid 21 inhibits PDCD4, an IL-10 inhibitor, hence derepressing organs where they are activated by antigens that their B-cell IL-10. The time scale of PDCD4 inhibition was found to be receptors (surface immunoglobulin) recognize. Once they are delayed when compared with induction of miR-21, leading to activated, they undergo proliferation and further differentiation. the idea that this miRNA acts as a delayed negative feedback The outcome of antigenic activation is differentiation into either regulator. IL-10 then inhibits miR-155, allowing SHIP1 to be plasma cells that secrete highly specific antibodies or memory B derepressed and inhibit TLR signaling.36,37 cells that can be reactivated for future protection.77 Once an

Leukemia miRNAs in inflammation and immune responses J Contreras and DS Rao 408 Table 1 miRNAs involved in the TLR pathway and their targets Bcells.82 It was also determined that these mice had an increased titer of autoimmune antibodies with autoimmune disease in miRNA Target Effect female mice. This study also hinted that the relevant target was Bruton’s tyrosine kinase, which is important in controlling B-cell miR-9 NF-kB1/p50 Transcription by NF-kB activation. is decreased In B-2 cells, miR-155 plays a major role in regulating the miR-16 TNF-a Decrease levels of inflammatory ctyokines response in germinal center cells, where B cells undergo a miR-17-5p, Transcription factor CBF Inhibition of monocyte second round of DNA rearrangement, followed by selec- 20a, 106a maturation tion.83,84 miR-155-deficient mice show marked defects in both miR-21 PDCD4, IL12p35 Derepression of IL-10 antibody secretion and class-switch recombination upon im- miR-27b PPARg munization.85,86 miR-155 represses over 60 target genes in B miR-105 TLR2 cells, including Pu.1, SHIP1 and AID.86 In fact, Pu.1 seems to be miR-106 IL-10 Cooperates with RNA binding proteins to at least partially responsible for the defects in B-cell activation 86 decrease IL-10 seen in these mice. The role of AID, which mediates class- miR-125b TNF-a switch recombination and somatic hypermutation, was further miR-145 MAL Inhibits TLR signaling explored by two groups who generated a highly specific miR-146a TRAF6, IRAK1, IRAK2 Negative feedback disruption of the miR-155 target site in the Aicda 30-untranslated regulator of TLR region.87,88 Disruption of the miR-155/AID interaction led to signaling persistent/increased somatic hypermutation, including abnormal SPOTLIGHT miR-155 AID, MyD88, TAB2, IKKe, Overall proinflammatory, SHIP1, SOCS1, C/.EBPBb some feedback translocations, and decreased high-affinity antibodies in im- regulation munized mutant mice. This result suggests that the majority of miR-199 IKKb miR-155-mediated phenotypic effects in B cells are not miR-221 TNF-a mediated by AID, but that the AID/miR-155 interaction has an miR-223 TLR3, TLR4, IKK Granulopoiesis and a important function. One possibility is that miR-155 inhibition of monocyte activation Let-7i, let-7e TLR4 Downregulate Aicda may represent a delayed negative regulatory switch, inflammatory signaling which allows for the proper temporal control of somatic hypermutation and positive selection. Interestingly, miR-181b Abbreviations: AID, activation induced deaminase; CBF, Core binding overexpression in B cells was found to reduce the class-switch Factor; C/EBPb, CCAAT/enhancer-binding protein beta; IKK, IkB 89 kinase; IL-10, interleukin-10; IRAK, interleukin-1 receptor-associated recombination rates, possibly by also downregulating AID. In kinase; MAL, myelin and lymphocyte protein; miRNA, microRNA; another study, miR-150-deficient mice show an expansion of B1 MyD88, Myeloid differentiation primary response gene (88); NF-kB; B cells accompanied by dramatic increases in steady-state nuclear factor-kB; PDCD4, programmed cell death 4; PPARg, antibodies.41 miR-150 deficient mice also showed an enhanced peroxisome proliferator-activated receptor-g; SOCS1, suppressor of response to immunization with T-dependent antigens, indicat- cytokine signaling 1; TAB2, TGF-beta activated kinase 1/MAP3K7 binding protein 2; TLR, Toll-like receptor; TNF-a, tumor necrosis ing an effect on follicular (B2) B cells. Together, these studies factor-a; TRAF6, TNF receptor-associated factor 6. have revealed an important function for miRNAs in B-cell development and activation. The finding of autoimmune or immunodysregulated phenotypes in miRNA-disrupted mice should provide an impetus to search for B-cell-mediated antigen comes into contact with the B-cell receptor, a mechanisms of autoimmune disease. signalosome consisting of the B-cell receptor and of intracellular signaling kinases such as Phospholipase C-2 gamma, phospha- tidyl inositol 3-kinase, Bruton’s tyrosine kinase and Vav as well MicroRNAs in T lymphocytes and autoimmunity as B-cell adaptor Blnk is assembled.77 Although conventional B cells (B2 B cells) require CD4 þ T-cell activation, B1 B cells do T lymphocytes orchestrate and effect some of the most potent not. It is thought that B1 B cells are activated by a TLR microbial responses against invading pathogenic organisms. In this role, detection pathway.78 they both activate and suppress various components of the The overall importance of miRNAs in hematopoiesis was immune system and, as such, dysregulation of their function established by a seminal study that showed that miR-181 was leads to alterations in immunity. In this context, we will discuss highly expressed in B cells and guided B-cell development when the function of specific miRNAs in T cells. Although a thorough constitutively expressed.57 Mice with an early B-cell-specific exposition of T-cell biology is well beyond the scope of this deletion of Dicer demonstrated a lack of B-cell development past review, we highlight a few important points that are subject to the pro-B-cell stage.79 This seemed to be mediated via increased miRNA regulation. First, the function of the T cell is dependent apoptosis, and the protein targets, bcl-2 interacting mediator of on its T-cell receptor, the surface molecule that mediates cell death and phosphatase and tensin homolog, were thought to recognition of major histocompatibility complex-bound anti- be responsible for the phenotype. miR-150 constitutive expres- genic peptides.90 The strength of binding is critical, as it sion has an effect on B-cell development, as does miR-34a determines whether the T cell survives through selection during constitutive expression.41,80,81 Both miRNAs must normally be development in the thymus.91 Indeed, T cells with autoreactive downregulated at the pro-B to pre-B transition and their receptors (that is, those that bind too strongly) are deleted during continued expression leads to inhibition of their respective development in the thymus. Second, once T cells are mature, targets, c-Myb and Foxp1 (forkhead box P1), which are required CD4 þ helper T cells differentiate further into specific types of for further B-cell development. helper cells. These include T helper (TH)1, TH2 and TH17 cells, miRNAs are now known to play a clear role in controlling the which activate various types of immune responses, as well as activation of mature B cells. At the global level, Dicer ablation in regulatory T cells (Treg), which suppress the immune response. mature B cells (as opposed to early precursor B cells) resulted miRNAs have now been found to play roles in several of these in an increase in marginal zone B cells and a decrease in follicular aspects of T-cell function.

Leukemia miRNAs in inflammation and immune responses J Contreras and DS Rao 409 Global knockout of Dicer in the T-cell lineage led to an arrest in the inflammatory response. Identifying these factors will help set T-cell development and abnormal development of T-cell sub- the stage for developing therapeutic avenues for autoimmune sets.92,93 Early studies with miR-181a, a miRNA that has disease, which remains a very serious problem in human significant function in B cells as well as T cells, revealed that its patients. knockdown allowed for T-cell reactivity against self-antigens, as a consequence of modulating the expression of several phospha- MicroRNAs in leukemia tases that are responsible for determining the strength of the transduced T-cell receptor signal.94 These targets were distinct Many profiling studies have led to a wealth of information about from the targets described for miR-181a in the B-cell lineage dysregulated miRNA expression in various tumors of the where it targets BCL2 (B-cell lymphoma 2) and TCL1 (T-cell hematolymphoid system. This has led to the concept of leukemia/lymphoma 1).95 This highlights targeting distinctions ‘oncomiRs’, or oncogenic miRNAs, and tumor-suppressor in different cell lineages, which adds to the regulatory capacity miRNAs. We have already discussed some of the molecular of miRNAs. These studies highlight the need to study miRNA similarities between inflammatory hematopoiesis and certain targeting in a given cell lineage. clonal hematopoietic neoplasms. However, there are no The recent description of T has revolutionized the field of reg unifying themes that emerge from these observations. Several autoimmunity research.96 These cells, which depend on the miRNAs can cause leukemic transformation in mouse models, transcription factor Foxp3 for their development, have the but their roles in immunology can be divergent or incompletely capacity to suppress the activity of the immune system in an understood. antigen-specific manner. The miRNAs seem to play important Extensive profiling studies of miRNAs in acute and chronic roles in regulating the function of these cells. Dicer deletion in leukemia are reported and reviewed elsewhere.104,105 When Foxp3 þ cells led to a lethal and severe autoimmune disease in overexpressed in mice, miR-155 causes a myeloproliferative mice, characterized by decreased numbers of mature T in the reg disorder; miR-125b leads to a chronic myeloproliferative periphery, inappropriate activation and impaired maturation.97 disease that evolves into an acute leukemia.48,106,107 miR-29a These findings led to a search for specific miRNAs that may be was also recently reported to cause an acute myeloid leukemia responsible for this phenotype. Further studies with miR-155- when overexpressed in mice, whereas overexpression of miR-21 deficient mice showed that miR-155-deficient T showed reg resulted in an acute lymphoblastic leukemia.108,109 These impaired survival compared with wild-type T .98 Using a reg studies highlight that several oncomiRs are capable of trans- competitive assay, the Rudensky group found that miR-146a- forming hematopoietic cells. Despite these similarities in deficient T were unable to rescue a hyperactive immune reg induction of malignant phenotypes, the functions of these system that was generated by knocking out Foxp3.40 These miRNAs in immune cells may be quite different; for example, findings are concordant with the findings of a hyperinflamma- TLR signaling induces miR-155 and miR-21 but it downregulates tory-immunoproliferative phenotype in miR-146aÀ/À mice.38,39 miR-125b. Together, these studies highlight significant alterations in T reg In addition to oncomiRs, several tumor-suppressor miRNAs function as a consequence of miRNA activity, and correlate well may have important roles in leukemogenesis. Perhaps the most with studies from human patients that demonstrate miRNA elegantly studied tumor-suppressor miRNA is the miR-15a/16 dysregulation in several autoimmune diseases. Specifically, it polycistron, which was initially identified as being located at the has been found that miR-155 and miR-146a are upregulated in 13q14 chromosomal locus deleted in chronic lymphocytic lymphocytes from patients with rheumatoid arthritis.99 – 101 leukemia.110 Initial studies identified several important targets of The functional effects of miRNAs, however, are not limited to miR-15a/16 including oncogenes such as BCL2 and the T lineage, and autoimmune disease has been observed in reg TCL1.95,111,112 Experimentally, deletion of (1) the miRNA- other T-cell contexts as well. T-cell directed overexpression of containing segment of the gene, (2) the larger noncoding RNA the oncogenic miRNA locus, miR-17B92, caused an immuno- that contained the miRNAs and of (3) the entire homologous proliferative disorder and autoimmune disease when expressed locus in mice led to successively more severe phenotypes, and in a lymphocyte-specific manner.102 Although compromised all recapitulated chronic lymphocytic leukemia to some T lineage function was implicated in miR-146a-deficient reg degree.113 Overall, the observations made regarding the mice, other mechanisms are also operant in the autoimmune functional role of miRNAs in leukemogenesis suggest that pathologies. In addition to the myeloid proliferations, T cells

replacement of tumor-suppressor miRNAs and inhibition of SPOTLIGHT from miR-146a-deficient mice showed features of activation and oncomiRs may have an important role in future therapeutic contributed to the autoimmune sequelae in these mice.38 On the approaches. other hand, miR-155-deficient mice were less prone to induction of experimental autoimmune encephalomyelitis.51 These findings were attributed to the decreased function of TH1 Conclusions and speculations and TH17 cells, and partially to dendritic cell function, thus implicating miRNA function in the activation of all CD4 þ T-cell The field of miRNA research has undergone a tremendous lineages. Like miR-155, miR-326 also appears to promote expansion in the past decade, and these studies have changed autoimmune inflammation, and acts via repression of the Ets-1 fundamentally how we look at gene expression. For example, transcription factor.103 the unique temporal regulation properties that are possible with Although nonexhaustive, this examination of miRNAs pro- miRNAs would not be possible with transcription factors. vides an idea of the breadth of miRNA functions in T-cell Furthermore, although not reviewed here, coexpression of a biology. The many facets of T-cell biology all seem to involve host protein coding gene and an intronic miRNA can produce a miRNA functions at some level. Perhaps most intriguingly, unique regulatory module.114 Further insights into miRNA dysregulation of several different miRNAs leads to pathologic function continue to be made–a recent study that examined inflammation and autoimmune disease. At least in the T-cell gene expression using single-cell imaging studies reveal that the lineage, this seems to reflect the function of miRNAs in fine- changes in target gene expression are highly dependent on tuning the expression of protein factors that may ‘set’ the level of abundance in a miRNA-expressing cell.115 There seems to be a

Leukemia miRNAs in inflammation and immune responses J Contreras and DS Rao 410 threshold level below which repression by miRNA can be close growing field because of space limitations. We thank David to complete. Around the threshold value, miRNAs can modulate Baltimore and Ryan O’Connell for helpful discussions over the significant but subtotal repression of the target. Well above the years. DSR is a Kimmel Scholar of the Sidney Kimmel Foundation threshold value, miRNAs do not change target gene expression for Cancer Research and has received a career development at all. At the population level, such effects would amplify award from the NIH (5K08-CA133251). JC is a recipient of the differences in gene expression between low- and high-expres- Eugene V. Cota-Robles Fellowship from UCLA. sing cells. In the immune system, we have reviewed the centrality of miRNAs regulating TLR and other signaling pathways. Acting in References positive feedback loops (such as miR-155 promoting inflammation) and in delayed negative feedback regulatory loops (such as 1 Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic miR-21 and miR-146a), miRNAs may confer a level of gene lin-4 encodes small RNAs with antisense complementarity regulation that is not possible with transcription factors. to lin-14. Cell 1993; 75: 843–854. However, the function of a single miRNA in a single-cell 2 Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern lineage is still incompletely understood, as highlighted by miR- formation in C. elegans. Cell 1993; 75: 855–862. 155, which can repress components of the TLR signaling 3 Kozomara A, Griffiths-Jones S. miRBase: integrating microRNA pathway in addition to having proinflammatory effects by annotation and deep-sequencing data. Nucleic Acids Res 2011; repressing SHIP1 (Figure 1 and Tang et al.116). Adding to this 39 (Database issue): D152–D157. complexity, miRNAs may have different functions in different 4 Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. SPOTLIGHT cell lineages, because of the exquisite sensitivity of miRNA Nat Rev 2009; 10: 126–139. 5 Winter J, Jung S, Keller S, Gregory RI, Diederichs S. Many roads regulation to the concentration of its target mRNA. Future to maturity: microRNA biogenesis pathways and their regula- research with exacting loss of function of specific miRNAs in tion. Nat Cell Biol 2009; 11: 228–234. subsets of immune cells, as well as deletions of miRNA target 6 Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH et al. MicroRNA sites, will be required to precisely understand the function of genes are transcribed by RNA polymerase II. EMBO J 2004; 23: miRNAs during immune responses. 4051–4060. In addition to these detailed mechanistic studies to delineate 7 Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 2003; miRNA function, research tapping into the therapeutic applica- 425: 415–419. tions of these miRNAs has begun. Indeed, small RNA delivery 8 Gregory RI, Yan KP, Amuthan G, Chendrimada T, Doratotaj B, has already been demonstrated in a few human trials, and small Cooch N et al. The microprocessor complex mediates the RNA inhibition in animal models can be achieved with anti- genesis of microRNAs. Nature 2004; 432: 235–240. miRNAs or antagomirs.117,118 These studies demonstrate the 9 Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a potential of these therapeutics in tuning down miRNA function. bidentate ribonuclease in the initiation step of RNA interference. Nature 2001; 409: 363–366. A related theme of research, replacing miRNAs, is also gaining 10 Ketting RF, Fischer SE, Bernstein E, Sijen T, Hannon GJ, Plasterk traction. For example, delivery of miR-146a or miR-21 may be RH. Dicer functions in RNA interference and in synthesis of able to tune down inappropriate immune responses. The small RNA involved in developmental timing in C. elegans. technology for such delivery may entail using double-stranded Genes Dev 2001; 15: 2654–2659. small RNA molecules, but delivery into the right cell types and 11 Czech B, Hannon GJ. Small RNA sorting: matchmaking for tissues remains a challenge. One method is suggested by the Argonautes. Nat Rev Genet 2011; 12: 19–31. 12 Cheloufi S, Dos Santos CO, Chong MM, Hannon GJ. A dicer- recent observation that some miRNAs can pass between cells independent miRNA biogenesis pathway that requires Ago 119 via exosome-mediated transfer. It is also tempting to catalysis. Nature 2010; 465: 584–589. speculate that such cell-to-cell communications may regulate 13 Cifuentes D, Xue H, Taylor DW, Patnode H, Mishima Y, immune cell function. Cheloufi S et al. A novel miRNA processing pathway indepen- miRNA research in the immune system has progressed a great dent of Dicer requires Argonaute2 catalytic activity. Science deal in just a few short years. Their evolutionary conserva- 2010; 328: 1694–1698. F 14 Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. tion these mechanisms are present in simple multicellular Prediction of mammalian microRNA targets. Cell 2003; 115: animals and plants in addition to mammalsFis remarkable and 787–798. suggests an ancient function. Some have suggested that they 15 Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often may have arisen as a primitive antiviral response and that these flanked by adenosines, indicates that thousands of human genes miRNAs may be particularly important in the immune system. are microRNA targets. Cell 2005; 120: 15–20. Regardless of their origin, it is clear that miRNAs have diverse 16 Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP. MicroRNA targeting specificity in mammals: deter- and important functions in the immune system. Exciting minants beyond seed pairing. Mol Cell 2007; 27: 91–105. developments and unexpected twists undoubtedly remain as 17 Bartel DP. MicroRNAs: target recognition and regulatory we continue our efforts to understand the function and develop functions. Cell 2009; 136: 215–233. the therapeutic potential of these enigmatic regulators of gene 18 Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ expression. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 2004; 305: 1437–1441. 19 Song JJ, Smith SK, Hannon GJ, Joshua-Tor L. Crystal structure of Conflict of interest Argonaute and its implications for RISC slicer activity. Science 2004; 305: 1434–1437. 20 Nilsen TW. Mechanisms of microRNA-mediated gene regula- The authors declare no conflict of interest. tion in animal cells. Trends Genet 2007; 23: 243–249. 21 Baek D, Villen J, Shin C, Camargo FD, Gygi SP, Bartel DP. The impact of microRNAs on protein output. Nature 2008; 455:64– Acknowledgements 71. 22 Guo H, Ingolia NT, Weissman JS, Bartel DP. Mammalian Although this review is meant to be comprehensive, we acknowl- microRNAs predominantly act to decrease target mRNA levels. edge that we may not have included all papers in this large and Nature 2010; 466: 835–840.

Leukemia miRNAs in inflammation and immune responses J Contreras and DS Rao 411 23 O’Connell RM, Taganov KD, Boldin MP, Cheng G, Baltimore D. innate immune system replenishment. Immunity 2006; 24: MicroRNA-155 is induced during the macrophage inflammatory 801–812. response. Proc Natl Acad Sci USA 2007; 104: 1604–1609. 48 O’Connell RM, Rao DS, Chaudhuri AA, Boldin MP, Taganov 24 Chi SW, Zang JB, Mele A, Darnell RB. Argonaute HITS-CLIP decodes KD, Nicoll J et al. Sustained expression of microRNA-155 in microRNA-mRNA interaction maps. Nature 2009; 460: 479–486. hematopoietic stem cells causes a myeloproliferative disorder. 25 He L, He X, Lim LP, de Stanchina E, Xuan Z, Liang Y et al. A J Exp Med 2008; 205: 585–594. microRNA component of the p53 tumour suppressor network. 49 O’Connell RM, Chaudhuri AA, Rao DS, Baltimore D. Inositol Nature 2007; 447: 1130–1134. phosphatase SHIP1 is a primary target of miR-155. Proc Natl 26 He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Acad Sci USA 2009; 106: 7113–7118. Goodson S et al. A microRNA polycistron as a potential human 50 Androulidaki A, Iliopoulos D, Arranz A, Doxaki C, Schworer S, oncogene. Nature 2005; 435: 828–833. Zacharioudaki V et al. The kinase Akt1 controls macrophage 27 Bommer GT, Gerin I, Feng Y, Kaczorowski AJ, Kuick R, Love RE response to lipopolysaccharide by regulating microRNAs. et al. p53-mediated activation of miRNA34 candidate tumor- Immunity 2009; 31: 220–231. suppressor genes. Curr Biol 2007; 17: 1298–1307. 51 O’Connell RM, Kahn D, Gibson WS, Round JL, Scholz RL, 28 Chang TC, Wentzel EA, Kent OA, Ramachandran K, Mullendore Chaudhuri AA et al. MicroRNA-155 promotes autoimmune M, Lee KH et al. Transactivation of miR-34a by p53 broadly inflammation by enhancing inflammatory T cell development. influences gene expression and promotes apoptosis. Mol Cell Immunity 2010; 33: 607–619. 2007; 26: 745–752. 52 Starczynowski DT, Kuchenbauer F, Argiropoulos B, Sung S, 29 Raver-Shapira N, Marciano E, Meiri E, Spector Y, Rosenfeld N, Morin R, Muranyi A et al. Identification of miR-145 and miR- Moskovits N et al. Transcriptional activation of miR-34a con- 146a as mediators of the 5q- syndrome phenotype. Nat Med tributes to p53-mediated apoptosis. Mol Cell 2007; 26:731–743. 2010; 16: 49–58. 30 Davis BN, Hilyard AC, Lagna G, Hata A. SMAD proteins control 53 Nahid MA, Pauley KM, Satoh M, Chan EK. miR-146a is critical DROSHA-mediated microRNA maturation. Nature 2008; 454: for endotoxin-induced tolerance: IMPLICATION IN INNATE 56–61. IMMUNITY. J Biol Chem 2009; 284: 34590–34599. 31 Yang W, Chendrimada TP, Wang Q, Higuchi M, Seeburg PH, 54 Nahid MA, Satoh M, Chan EK. Mechanistic role of microRNA- Shiekhattar R et al. Modulation of microRNA processing and 146a in endotoxin-induced differential cross-regulation of TLR expression through RNA editing by ADAR deaminases. Nat signaling. J Immunol 2011; 186: 1723–1734. Struct Mol Biol 2006; 13: 13–21. 55 Hou J, Wang P, Lin L, Liu X, Ma F, An H et al. MicroRNA-146a 32 Suzuki HI, Yamagata K, Sugimoto K, Iwamoto T, Kato S, feedback inhibits RIG-I-dependent Type I IFN production in Miyazono K. Modulation of microRNA processing by p53. macrophages by targeting TRAF6, IRAK1, and IRAK2. J Immunol Nature 2009; 460: 529–533. 2009; 183: 2150–2158. 33 Wiesen JL, Tomasi TB. Dicer is regulated by cellular stresses and 56 Tang Y, Luo X, Cui H, Ni X, Yuan M, Guo Y et al. MicroRNA- interferons. Mol Immunol 2009; 46: 1222–1228. 146A contributes to abnormal activation of the type I interferon 34 Viswanathan SR, Daley GQ, Gregory RI. Selective blockade of pathway in human lupus by targeting the key signaling proteins. microRNA processing by Lin28. Science (New York, NY) 2008; Arthritis Rheum 2009; 60: 1065–1075. 320: 97–100. 57ChenCZ,LiL,LodishHF,BartelDP.MicroRNAsmodulate 35 Hobert O. Gene regulation by transcription factors and hematopoietic lineage differentiation. Science 2004; 303:83–86. microRNAs. Science 2008; 319: 1785–1786. 58 Fazi F, Racanicchi S, Zardo G, Starnes LM, Mancini M, 36 O’Neill LA, Sheedy FJ, McCoy CE. MicroRNAs: the fine-tuners Travaglini L et al. Epigenetic silencing of the myelopoiesis of Toll-like receptor signalling. Nat Rev Immunol 2011; 11: regulator microRNA-223 by the AML1/ETO oncoprotein. 163–175. Cancer Cell 2007; 12: 457–466. 37 Sheedy FJ, Palsson-McDermott E, Hennessy EJ, Martin C, 59 Fazi F, Rosa A, Fatica A, Gelmetti V, De Marchis ML, Nervi C O’Leary JJ, Ruan Q et al. Negative regulation of TLR4 via et al. A minicircuitry comprised of microRNA-223 and targeting of the proinflammatory tumor suppressor PDCD4 by transcription factors NFI-A and C/EBPalpha regulates human the microRNA miR-21. Nat Immunol 2010; 11: 141–147. granulopoiesis. Cell 2005; 123: 819–831. 38BoldinMP,TaganovKD,RaoDS,YangL,ZhaoJL,KalwaniM 60 Johnnidis JB, Harris MH, Wheeler RT, Stehling-Sun S, Lam MH, et al. miR-146a is a significant brake on autoimmunity, myelopro- Kirak O et al. Regulation of progenitor cell proliferation and liferation, and cancer in mice. J Exp Med 2011; 208: 1189–1201. granulocyte function by microRNA-223. Nature 2008; 451: 39 Zhao JL, Rao DS, Boldin MP, Taganov KD, O’Connell RM, 1125–1129. Baltimore D. NF-\{kappa\}B dysregulation in microRNA-146a- 61 Kawai T, Akira S. Signaling to NF-kappaB by Toll-like receptors. deficient mice drives the development of my. Proc Natl Acad Sci Trends Mol Med 2007; 13: 460–469. USA 2011; 108: 9184–9189. 62 Taganov KD, Boldin MP, Chang KJ, Baltimore D. NF-kappaB- 40 Lu LF, Boldin MP, Chaudhry A, Lin LL, Taganov KD, Hanada T dependent induction of microRNA miR-146, an inhibitor et al. Function of miR-146a in controlling Treg cell-mediated targeted to signaling proteins of innate immune responses. Proc

regulation of Th1 responses. Cell 2010; 142: 914–929. Natl Acad Sci USA 2006; 103: 12481–12486. SPOTLIGHT 41 Xiao C, Calado DP, Galler G, Thai TH, Patterson HC, Wang J 63 McCoy CE, Sheedy FJ, Qualls JE, Doyle SL, Quinn SR, Murray PJ et al. MiR-150 controls B cell differentiation by targeting the et al. IL-10 inhibits miR-155 induction by toll-like receptors. transcription factor c-Myb. Cell 2007; 131: 146–159. J Biol Chem 2010; 285: 20492–20498. 42 Xiao C, Rajewsky K. MicroRNA control in the immune system: 64 Muzio M, Bosisio D, Polentarutti N, D’Amico G, Stoppacciaro A, basic principles. Cell 2009; 136: 26–36. Mancinelli R et al. Differential expression and regulation of toll-like 43 Lal A, Navarro F, Maher CA, Maliszewski LE, Yan N, O’Day E receptors (TLR) in human leukocytes: selective expression of TLR3 in et al. miR-24 Inhibits cell proliferation by targeting E2F2, MYC, dendritic cells. JImmunol2000; 164: 5998–6004. and other cell-cycle genes via binding to ‘‘seedless’’ 30UTR 65 Benakanakere MR, Li Q, Eskan MA, Singh AV, Zhao J, Galicia JC microRNA recognition elements. Mol Cell 2009; 35: 610–625. et al. Modulation of TLR2 protein expression by miR-105 44 Vasudevan S, Tong Y, Steitz JA. Switching from repression to in human oral keratinocytes. J Biol Chem 2009; 284: activation: microRNAs can up-regulate translation. Science 23107–23115. 2007; 318: 1931–1934. 66 Liu G, Friggeri A, Yang Y, Park YJ, Tsuruta Y, Abraham E. miR- 45 O’Connell RM, Zhao JL, Rao DS. MicroRNA function in myeloid 147, a microRNA that is induced upon Toll-like receptor biology. Blood 2011; 118: 2960–2969. stimulation, regulates murine macrophage inﬂammatory re- 46 Baldridge MT, King KY, Boles NC, Weksberg DC, Goodell MA. sponses. Proc Natl Acad Sci USA 2009; 106: 15819–15824. Quiescent haematopoietic stem cells are activated by IFN- 67 Hoffmann A, Baltimore D. Circuitry of nuclear factor kappaB gamma in response to chronic infection. Nature 2010; 465: signaling. Immunol Rev 2006; 210: 171–186. 793–797. 68 Chen R, Alvero AB, Silasi DA, Kelly MG, Fest S, Visintin I et al. 47 Nagai Y, Garrett KP, Ohta S, Bahrun U, Kouro T, Akira S et al. Regulation of IKKbeta by miR-199a affects NF-kappaB activity in Toll-like receptors on hematopoietic progenitor cells stimulate ovarian cancer cells. Oncogene 2008; 27: 4712–4723.

Leukemia miRNAs in inﬂammation and immune responses J Contreras and DS Rao 412 69 Eis PS, Tam W, Sun L, Chadburn A, Li Z, Gomez MF et al. Immunobiology: The Immune System in Health and Accumulation of miR-155 and BIC RNA in human B cell Disease, 6th edn. Garland Science Publishing: New York, 2005, lymphomas. Proc Natl Acad Sci USA 2005; 102: 3627–3632. pp 203–236. 70 Li T, Morgan MJ, Choksi S, Zhang Y, Kim YS, Liu ZG. 92 Cobb BS, Nesterova TB, Thompson E, Hertweck A, O’Connor E, MicroRNAs modulate the noncanonical transcription factor Godwin J et al. T cell lineage choice and differentiation in the NF-kappaB pathway by regulating expression of the kinase absence of the RNase III enzyme Dicer. J Exp Med 2005; 201: IKKalpha during macrophage differentiation. Nat Immunol 1367–1373. 2010; 11: 799–805. 93 Muljo SA, Ansel KM, Kanellopoulou C, Livingston DM, Rao A, 71 Bazzoni F, Rossato M, Fabbri M, Gaudiosi D, Mirolo M, Mori L Rajewsky K. Aberrant T cell differentiation in the absence of et al. Induction and regulatory function of miR-9 in human Dicer. J Exp Med 2005; 202: 261 – 269. monocytes and neutrophils exposed to proinﬂammatory signals. 94 Li QJ, Chau J, Ebert PJ, Sylvester G, Min H, Liu G et al. miR-181a Proc Natl Acad Sci USA 2009; 106: 5282–5287. is an intrinsic modulator of T cell sensitivity and selection. 72 Asirvatham AJ, Magner WJ, Tomasi TB. miRNA regulation of Cell 2007; 129: 147–161. cytokine genes. Cytokine 2009; 45: 58–69. 95 Pekarsky Y, Santanam U, Cimmino A, Palamarchuk A, Efanov A, 73 Carballo E, Lai WS, Blackshear PJ. Feedback inhibition of Maximov V et al. Tcl1 expression in chronic lymphocytic macrophage tumor necrosis factor-alpha production by triste- leukemia is regulated by miR-29 and miR-181. Cancer Res traprolin. Science (New York, NY) 1998; 281: 1001–1005. 2006; 66: 11590–11593. 74 Lai WS, Carballo E, Strum JR, Kennington EA, Phillips RS, 96 Rudensky AY. Regulatory T cells and Foxp3. Immunol Rev 2011; Blackshear PJ. Evidence that tristetraprolin binds to AU-rich 241: 260–268. elements and promotes the deadenylation and destabilization of 97 Liston A, Lu LF, O’Carroll D, Tarakhovsky A, Rudensky AY. Dicer- tumor necrosis factor alpha mRNA. Mol Cell Biol 1999; 19: dependent microRNA pathway safeguards regulatory T cell

SPOTLIGHT 4311–4323. function. J Exp Med 2008; 205: 1993–2004. 75 El Gazzar M, McCall CE. MicroRNAs distinguish translational 98 Lu LF, Thai TH, Calado DP, Chaudhry A, Kubo M, Tanaka K from transcriptional silencing during endotoxin tolerance. J Biol et al. Foxp3-dependent microRNA155 confers competitive Chem 2010; 285: 20940–20951. fitness to regulatory T cells by targeting SOCS1 protein. 76 Hardy RR, Hayakawa K. B cell development pathways. Annu Immunity 2009; 30: 80–91. Rev Immunol 2001; 19: 595–621. 99 Li J, Wan Y, Guo Q, Zou L, Zhang J, Fang Y et al. Altered 77 Harwood NE, Batista FD. Early events in B cell activation. Annu microRNA expression profile with miR-146a upregulation in Rev Immunol 2010; 28: 185–210. CD4+ T cells from patients with rheumatoid arthritis. Arthritis 78 Montecino-Rodriguez E, Dorshkind K. New perspectives in B-1 Res Ther 2010; 12: R81. B cell development and function. Trends Immunol 2006; 27: 100 Pauley KM, Satoh M, Chan AL, Bubb MR, Reeves WH, Chan EK. 428–433. Upregulated miR-146a expression in peripheral blood mono- 79 Koralov SB, Muljo SA, Galler GR, Krek A, Chakraborty T, nuclear cells from rheumatoid arthritis patients. Arthritis Res Ther Kanellopoulou C et al. Dicer ablation affects antibody diversity 2008; 10: R101. and cell survival in the B lymphocyte lineage. Cell 2008; 132: 101 Stanczyk J, Pedrioli DM, Brentano F, Sanchez-Pernaute O, 860–874. Kolling C, Gay RE et al. Altered expression of MicroRNA in 80 Zhou B, Wang S, Mayr C, Bartel DP, Lodish HF. miR-150, a synovial fibroblasts and synovial tissue in rheumatoid arthritis. microRNA expressed in mature B and T cells, blocks early B cell Arthritis Rheum 2008; 58: 1001–1009. development when expressed prematurely. Proc Natl Acad Sci 102 Xiao C, Srinivasan L, Calado DP, Patterson HC, Zhang B, Wang J USA 2007; 104: 7080–7085. et al. Lymphoproliferative disease and autoimmunity in mice 81 Rao DS, O’Connell RM, Chaudhuri AA, Garcia-Flores Y, Geiger with increased miR-17-92 expression in lymphocytes. Nat TL, Baltimore D. MicroRNA-34a perturbs B lymphocyte devel- Immunol 2008; 9: 405–414. opment by repressing the forkhead box transcription factor 103 Du C, Liu C, Kang J, Zhao G, Ye Z, Huang S et al. MicroRNA Foxp1. Immunity 2010; 33: 48–59. miR-326 regulates T(H)-17 differentiation and is associated with 82 Belver L, de Yebenes VG, Ramiro AR. MicroRNAs prevent the the pathogenesis of multiple sclerosis. Nat Immunol 2009; 10: generation of autoreactive antibodies. Immunity 2010; 33: 713–722. 1252–1259. 83 Cozine CL, Wolniak KL, Waldschmidt TJ. The primary germinal 104 Marcucci G, Mrozek K, Radmacher MD, Garzon R, center response in mice. Curr Opin Immunol 2005; 17: Bloomfield CD. The prognostic and functional role of 298–302. microRNAs in acute myeloid leukemia. Blood 2011; 117: 84 Wolniak KL, Shinall SM, Waldschmidt TJ. The germinal center 1121–1129. response. Crit Rev Immunol 2004; 24: 39–65. 105 Calin GA, Croce CM. MicroRNA signatures in human cancers. 85 Thai TH, Calado DP, Casola S, Ansel KM, Xiao C, Xue Y et al. Nat Rev Cancer 2006; 6: 857–866. Regulation of the germinal center response by microRNA-155. 106 O’Connell RM, Chaudhuri AA, Rao DS, Gibson WS, Balazs AB, Science 2007; 316: 604–608. Baltimore D. MicroRNAs enriched in hematopoietic stem cells 86 Vigorito E, Perks KL, Abreu-Goodger C, Bunting S, Xiang Z, differentially regulate long-term hematopoietic output. Proc Natl Kohlhaas S et al. microRNA-155 regulates the generation of Acad Sci USA 2010; 107: 14235–14240. immunoglobulin class-switched plasma cells. Immunity 2007; 107 Bousquet M, Harris MH, Zhou B, Lodish HF. MicroRNA miR- 27: 847–859. 125b causes leukemia. Proc Natl Acad Sci USA 2010; 107: 87 Teng G, Hakimpour P, Landgraf P, Rice A, Tuschl T, Casellas R 21558–21563. et al. MicroRNA-155 is a negative regulator of activation- 108 Han YC, Park CY, Bhagat G, Zhang J, Wang Y, Fan JB et al. induced cytidine deaminase. Immunity 2008; 28: 621–629. microRNA-29a induces aberrant self-renewal capacity in hema- 88 Dorsett Y, McBride KM, Jankovic M, Gazumyan A, Thai TH, topoietic progenitors, biased myeloid development, and acute Robbiani DF et al. MicroRNA-155 suppresses activation- myeloid leukemia. J Exp Med 2010; 207: 475–489. induced cytidine deaminase-mediated Myc-Igh translocation. 109 Medina PP, Nolde M, Slack FJ. OncomiR addiction in an in vivo Immunity 2008; 28: 630–638. model of microRNA-21-induced pre-B-cell lymphoma. Nature 89 de Yebenes VG, Belver L, Pisano DG, Gonzalez S, Villasante A, 2010; 467: 86–90. Croce C et al. miR-181b negatively regulates activation- 110 Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, induced cytidine deaminase in B cells. J Exp Med 2008; 205: Noch E et al. Frequent deletions and down-regulation of 2199–2206. micro- RNA genes miR15 and miR16 at 13q14 in chronic 90 Fahnestock ML, Tamir I, Narhi L, Bjorkman PJ. Thermal stability lymphocytic leukemia. Proc Natl Acad Sci USA 2002; 99: comparison of purified empty and peptide-filled forms of a 15524–15529. class I MHC molecule. Science (New York, NY) 1992; 258: 111 Calin GA, Liu CG, Sevignani C, Ferracin M, Felli N, Dumitru CD 1658–1662. et al. MicroRNA profiling reveals distinct signatures in B cell 91 Janeway CA, Travers P, Walport M, Schlomchik MJ. Signaling chronic lymphocytic leukemias. Proc Natl Acad Sci USA 2004; through immune system receptors In: Janeway CA (ed) 101: 11755–11760.

Leukemia miRNAs in inflammation and immune responses J Contreras and DS Rao 413 112 Cimmino A, Calin GA, Fabbri M, Iorio MV, Ferracin M, Shimizu regulation of Helicobacter pylori-induced inflammation. FEBS M et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Lett 2010; 584: 1481–1486. Proc Natl Acad Sci USA 2005; 102: 13944–13949. 117 DeVincenzo J, Lambkin-Williams R, Wilkinson T, Cehelsky J, 113 Klein U, Lia M, Crespo M, Siegel R, Shen Q, Mo T et al. The Nochur S, Walsh E et al. A randomized, double-blind, placebo- DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its controlled study of an RNAi-based therapy directed against deletion leads to chronic lymphocytic leukemia. Cancer Cell respiratory syncytial virus. Proc Natl Acad Sci USA 2010; 107: 2010; 17: 28–40. 8800–8805. 114 Small EM, Olson EN. Pervasive roles of microRNAs in 118 Krutzfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, cardiovascular biology. Nature 2011; 469: 336–342. Manoharan M et al. Silencing of microRNAs in vivo with 115 Mukherji S, Ebert MS, Zheng GX, Tsang JS, Sharp PA, van ‘antagomirs’. Nature 2005; 438: 685–689. Oudenaarden A. MicroRNAs can generate thresholds in target 119 Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. gene expression. Nat Genet 2011; 43: 854–859. Exosome-mediated transfer of mRNAs and microRNAs is a novel 116 Tang B, Xiao B, Liu Z, Li N, Zhu ED, Li BS et al. Identification of mechanism of genetic exchange between cells. Nat Cell Biol MyD88 as a novel target of miR-155, involved in negative 2007; 9: 654–659. SPOTLIGHT

Leukemia