Spotlight Review

Leukemia (2012) 26, 404–413; doi:10.1038/leu.2011.356; published online 20 December 2011

Spotlight on miRNA and Hematopoiesis

MicroRNAs in inflammation and immune responses

J Contreras1,2 and D S Rao2,3,4,5

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

Correspondence: Dr DS Rao, Department of Pathology and Laboratory Medicine, UCLA, 650 Charles E Young Drive South, Factor 12-272, Los Angeles CA, 90095, USA. E-mail: drao@mednet.ucla.edu

Received 4 September 2011; Revised 26 October 2011; Accepted 4 November 2011
Advance online publication 20 December 2011

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Abstract

MicroRNAs (miRNAs) are important regulators of gene expression in the immune system. In a few short years, their mechanism of action has been described in various cell lineages within the immune system, targets have been defined and their unique contributions to immune cell function have been examined. Certain miRNAs serve in important negative feedback loops in the immune system, whereas others serve to amplify the response of the immune system by repressing inhibitors of the response. Here, we review some of the better understood mechanisms as well as some emerging concepts of miRNA function. Future work will likely involve defining the function of specific miRNAs in specific immune cell lineages and to utilize them in the design of therapeutic strategies for diseases involving the immune system.

Keywords:

microRNA; inflammation; NF-κB

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MicroRNA biogenesis and mechanism of action

First discovered by the laboratories of Victor Ambros and Gary Ruvkun in Caenorhabditis elegans, microRNAs (miRNAs) are the first extensively studied class of noncoding RNA.1, 2 There are between 600 and 700 miRNAs that have been cataloged, with various degrees of conservation. It is now clear that there are likely species-specific miRNAs, and the most current listing in miRBase lists 1452 miRNAs in humans.3 miRNA biogenesis is a highly regulated process that allows for their expression in particular cellular and functional contexts. miRNAs are encoded within the cellular genome in three main ways: (1) as unique genes (2) as intronic sequences within protein-coding genes and (3) as polycistronic miRNAs, or a single transcript encoding multiple miRNAs.4, 5 This variable encoding renders complex possibilities for their regulation, through alternative processing or miRNA processing. The canonical miRNA biogenesis pathway involves transcription from these genes by RNA polymerase II, which allows for regulation by transcription factors.6 Following transcription, the primary miRNA transcript is processed by the endoribonucleases Drosha/DGCR8 in the nucleus before transport into the cytoplasm.7, 8 The resulting pre-miRNA is further processed by the endoribonuclease Dicer.9, 10 Dicer acts by cleaving the looped end of the miRNA precursor and ‘dicing’ the RNA at 20–25 base-pair intervals thereafter. This results in the formation of a short double-stranded RNA consisting of the miRNA and its complementary sequence. This is unwound and loaded onto the RNA-induced silencing complex (RISC) where the mature miRNA binds to target sequences (reviewed in Czech and Hannon11).

Recently, a second, non-Dicer-dependent pathway of miRNA biogenesis has been discovered.12, 13 This alternative pathway relies on a component of the RISC complex, namely the Argonaute protein Ago2, to mediate the final processing of the miRNA. Several miRNAs are thought to be processed by this pathway. Interestingly, the secondary structure of the pre-miRNA seems to be important in determining the pathway of biogenesis, with distinct loop and hairpin structures determining whether the miRNA precursor is cleaved by Dicer or Ago2. Of these, miR-451 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 proteins and RNAs. Here, the miRNA encounters and binds to a target mRNA sequence. The basis of targeting is thought to be Watson-Crick base-pairing via a six-nucleotide seed sequence located within the 3′-untranslated region of the target mRNA. This seems to be the most well-accepted model for targeting, and several target prediction algorithms are based on this model.14, 15, 16, 17 Usually, there are also areas of complementarity along the 3′ end of the miRNA, leading to a central region of noncomplementarity or ‘bulge’ sequence. In contrast to this, small interfering RNAs bind to target sequences with perfect complementarity, leading to endoribonucleolytic cleavage of the target mRNA via the Slicer function of Ago2.18, 19 The mechanism of miRNA repression of the targeted transcript is thought to involve a combination of mRNA destabilization and exoribonucleolytic activity as well as translational repression (reviewed in Nilsen20). Recent work suggests that mRNA levels are an accurate way to determine the degree of repression caused by miRNA expression.21, 22

Because repression is thought to be mediated through a short ‘seed’ sequence, miRNAs are predicted to target numerous mRNAs within a cell, from tens to hundreds. This has been partially validated by high-throughput techniques that show that miRNA gain and loss of function can lead to the repression or derepression of numerous genes, respectively.21, 22 This has led investigators seeking targets for miRNAs to use a combination of high-throughput experimental techniques and bioinformatics-based predictive algorithms to delineate direct miRNA targets.23 Other recent methodological advances include crosslinking of miRNAs with the RISC complex followed by immunoprecipitation and high-throughput sequencing to identify mRNA targets in the relevant biochemical context, the so-called Argonaute HITS-CLIP.24 These technological advances have rendered possible the identification of various pathways regulated by miRNAs. Overall, the multiplicity of targeting by miRNAs has led to the idea that they can regulate large sets of genes and thus have profound effects on gene expression.

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MicroRNA regulation during inflammation

Many miRNAs are co-regulated with protein-coding genes during the inflammatory response. The regulation of miRNA biogenesis occurs at many levels. The first and perhaps best studied is transcriptional regulation, and many miRNAs are induced as part of an inflammatory transcriptional program, as we detail below for nuclear factor (NF)-κB. However, such regulation is not limited to the immune system and forms a general paradigm for regulation of miRNA, and regulation of miR-17~92 and miR-34 family miRNAs by MYC and p53, respectively, are undoubtedly important in cancer.25, 26, 27, 28, 29 The overall conclusions from various studies is that miRNAs are induced in much the same way as protein-coding genes, but the time frame of their biogenesis and mode of action may confer unique regulatory properties to these small RNA molecules.

miRNA biogenesis is also regulated at the level of processing during inflammation. Several proteins that are induced during inflammatory responses can regulate the processing of miRNAs. These include the transforming growth factor-β-induced SMAD proteins, which are recruited into a complex with the RNA helicase p68, a component of the Drosha endoribonucleolytic complex.30 ADAR, an RNA-modifying enzyme that is upregulated during inflammation, can introduce mutations in double-stranded miRNA precursors, thereby changing the targeting specificity of the miRNA.31 The tumor-suppressor protein p53, which is upregulated during certain types of inflammatory responses, can facilitate processing of a number of miRNA transcripts.32 In addition, interferon-γ can directly repress the expression of Dicer and other biogenesis factors.33 Some post-transcriptional processing changes are highly specific for a given miRNA; for example, the Lin28 protein tightly regulates the processing of let-7 miRNA in a loop-specific manner.34 Whether other such regulatory factors, which are specific for a single or a few miRNAs, are induced during inflammation remains to be determined. It also remains to be determined whether the alternative pathway of miRNA biogenesis plays any role in regulating the immune response.

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General concepts regarding MicroRNA function in the immune system

The preceding discussion of miRNA biogenesis should hint at the idea that miRNAs may play distinctive regulatory roles in the immune system. Several general hypotheses and theories have been presented in the recent literature about how miRNAs may function in gene regulation. This occurs as part of a combined protein/miRNA gene expression program and there may be some unique properties that a miRNA is able to confer to a gene regulatory pathway.35, 36 These concepts include: (1) miRNAs have a unique role in regulating time-sensitive aspects of gene regulation in the immune system, (2) miRNAs have distinct functions in distinct cell types and (3) miRNAs regulate gene expression by incomplete repression of their targets.

First, miRNAs may have a particularly important function in cellular responses that are time dependent. Two apparently divergent ideas have been proposed, but each may have a specific role in a particular cellular/functional context. The first idea is that miRNAs may be able to act in a shorter time scale than protein transcriptional repressors. miRNA transcription is induced by inflammation, like transcriptional regulators, but 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 targets.35 This may indeed be important in innate responses (for example, miR-155) where a short time frame is critical to begin the fight against pathogenic organisms. The second idea is that although miRNAs can act more quickly than transcriptional regulators, they may act more slowly than protein factors that could directly target and inactivate miRNA targets.36 As miRNAs act at the RNA level, protein that is already produced and active will remain active until miRNA action downregulates the amount of new protein being produced. Here, miRNAs may act as ‘delay switches’ in negative feedback regulation of immune responses. This is certainly true for the recently described function of miR-21 in inflammation. miR-21 causes a delayed inhibition of PDCD4 (programmed cell death 4), a proinflammatory protein, following induction by NF-κB.37

miRNAs are also thought to have distinctive functions in different cell types. Although the mechanism of action of miRNAs is the same in different cell, the transcriptional program of a given cell type limits the number of relevant targets for a given miRNA. Hence, a cell undergoing massive transcriptional activation during inflammation will have a different set of targets regulated by a given miRNA than a quiescent cell. Additionally, developmentally distinct lineages have distinct sets of target genes for a given miRNA. For example, miR-146a seems to have distinct sets of targets in myeloid cells (TRAF6 (TNF receptor-associated factor 6) and IRAK1 (interleukin-1 receptor-associated kinase 1)) versus regulatory T cells (STAT1 (signal transducer and activator of transcription 1)), and hence the functional consequences of miRNA expression can be quite different in these two distinct cell lineages.38, 39, 40

The last concept regarding miRNA regulation that is important to understand is that their inhibition of target mRNAs does not lead to a complete knockdown. This has been shown by both gain- and loss-of-function experiments. These observations have led to the idea that miRNAs are ‘fine-tuners’ of gene expression as opposed to transcription factors that act as ‘switches’. In cellular function, this would suggest that miRNAs serve to ‘fix’ or ‘buffer’ cellular gene expression patterns, given that they 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 can also regulate. Such is the case with miR-150, whose incremental repression of c-Myb leads to a dramatic change in the output of B lymphocytes from the hematopoietic system.41, 42 miRNAs, then, can perform various functions of either stabilizing cellular gene expression patterns (and phenotypes) or causing differentiation, depending on the context in which they are expressed.

The preceding paragraphs capture some of the current explanations and ideas for miRNA function in hematopoiesis and immunity. It is also apparent that there is a lot to understand and that the field is evolving rapidly. New reports about miRNAs acting in a noncanonical manner (for example, by upregulating gene expression, acting in a non-3′-untranslated region-dependent fashion and regulating noncoding RNA) are appearing on an ongoing basis.43, 44 These will no doubt lead us to revisit some of these ideas and to further modify them as we strive to understand the regulation of gene expression and immunity.

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Innate immune cells and MicroRNA

The immune system is usually divided into the innate and adaptive arms. The innate immune system is a ‘first responder’ that also sets the stage for the adaptive immune response by initiating the inflammatory process. The adaptive immune response culminates with the generation of antigen-specific lymphocytes that clear the invading pathogen with a great deal of specificity and provide long-lasting immunity against the same. In this section, we will consider the effects that three well-studied miRNAs have on the innate immune system (miR-155, miR-146a and miR-223), although approximately a dozen different miRNAs have been implicated in the innate immune system by various studies. These studies have pointed out an interesting connection between miRNA-modulated pathways involved in the activation of mature innate immune cells (that is, macrophages, dendritic cells and granulocytes) and in the developmental program in the bone marrow during inflammation. We further discuss the critical signaling pathways that are found in these immune cells in the next section about Toll-like receptors (TLRs) and NF-κB.

miRNAs have developmental effects on the bone marrow and this has been reviewed elsewhere recently.45 However, inflammation causes some dramatic changes in the developmental sequence of hematopoietic cells. It turns out that hematopoietic stem and progenitor cells can respond to inflammatory signals such as interferon and lipopolysaccharide (LPS).46, 47 This, along with the conventionally accepted mechanism of hematopoietic stem and progenitor cells sensing depletion of downstream mature hematolymphoid cells, is thought to underlie a program of ‘inflammatory hematopoiesis’. During inflammatory hematopoiesis, the output of the marrow is directed toward myeloid cell production and away from the production of B lymphocytes and erythroid cells, and can be induced by LPS injection in mice. This change gradually wears off and homeostatic hematopoiesis returns after several days in mice. Constitutive activation of this program may result in chronic inflammatory conditions and may also be important in the pathogenesis of myeloid malignancies.

The first miRNA that is of importance in inflammatory hematopoiesis program is miR-155. This miRNA is upregulated in acute myeloid leukemia and causes a myeloproliferative disorder when overexpressed in bone marrow.48 miR-155 targets the SHIP1 inositol phosphatase, and knockdown of Src Homology-2 domain-containing inositol-5′-phosphatase 1 (SHIP1) in the bone marrow recapitulates the myeloproliferative disorder.49 It is also noteworthy that miR-155 is rapidly and effectively induced in intact mice that are given LPS. Hence, miR-155 is a key proinflammatory miRNA that is involved in changing the output of the bone marrow. Interestingly, recent studies have found that miR-155 promotes expression of inflammatory cytokines and the interferon response in primary macrophages and dendritic cells. This likely occurs via repression of the negative regulators of inflammation, SOCS1 (suppressor of cytokine signaling 1) and SHIP1, both direct targets of miR-155.50, 51 Through the downregulation of these anti-inflammatory pathways, miR-155 also promotes endotoxemia in mice.50 miR-155-deficient dendritic cells are not as effective at promoting inflammation as their wild-type counterparts.

The miR-146a is a feedback regulator of the TLR/NF-κB pathway, as further discussed in the next section. miR-146a-deficient mice show multiple abnormalities, with a spectrum of immunoproliferative and autoimmune disease.38 These various phenotypes are likely the result of miR-146a deficiency in various immune lineages. The net effect of miR-146a deficiency at the molecular level is to cause activation of NF-κB, and the myeloproliferative phenotype can be corrected in these mice by a genetic knockout of Nfkb1/p50, one of the NF-κB subunits.39 This miRNA is also deleted in the 5q-syndrome, a myelodysplastic syndrome, highlighting a similarity in pathways involved in inflammatory hematopoiesis and in myeloid malignancies.52 miR-146a is also important in mature immune cells. miR-146a-deficient bone marrow-derived macrophages show increased inflammatory cytokine production, including interleukin (IL)-6, IL-1β and tumor necrosis factor-α, upon LPS stimulation.38 miR-146a also negatively regulates endotoxin-induced tolerance in human monocytes by repressing TRAF6 and IRAK1.53, 54 Type I interferon production, which is important in the antiviral response, is inhibited by miR-146a during vesicular stomatitis virus infection of peritoneal macrophages.55 Components of the TLR signaling pathway, STAT5 and IRF5, are additional putative targets of miR-146a.56

The last miRNA that we will discuss is miR-223. Originally described as a miRNA that was highly expressed in myeloid cells in the bone marrow, this miRNA is silenced in certain types of myeloid leukemia, with re-expression promoting differentiation of myeloid blast cells.57, 58, 59 However, miR-223/ mice showed increased granulocytes with features of hyper activation, which would not be expected if this miRNA only promoted granulocytic differentiation.60 Hence, this miRNA may also connect myeloid development with inflammation. The exact mechanisms of these changes remain to be worked out, but it appears to involve the transcription factor Mef2c.

Overall, these studies highlight the roles of miRNAs in activation of innate immune cells and inflammatory hematopoiesis, which seem to be molecularly related processes in mature and immature hematopoietic cells, respectively. The roles of these miRNAs depend on repression of a single or a few targets and they can function as proinflammatory factors (miR-155) or as negative feedback regulators (miR-146a).

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MicroRNAs in the TLR and NF-κB pathways

The TLR pathway is an evolutionarily conserved pathway that recognizes pathogen-associated molecular patterns and is active mainly in the immune lineages, including macrophages, dendritic cells and B and T cells.36, 61 The TLR pathway is activated by the recognition of these pathogen-associated molecular patterns by intracellular or cell surface TLRs that recognize different types of pathogens. This binding is followed by the recruitment of various adapter proteins such as MyD88, TRIF and Tirap. MyD88-mediated signaling leads to the elaboration of the ‘inflammatory program’, whereas TRIF-mediated signaling contributes to the ‘antiviral response’. Both pathways activate NF-κB, which leads to the transcriptional upregulation of a large number of genes that mediate the response of the immune cell (see Figure 1). In many of these lineages, specific miRNAs are produced after activation of the TLR pathway. The first study examining miRNA production after TLR stimulation identified three miRNAs that are induced by LPS in human macrophages, namely miR-155, miR-146a and miR-132.62 Although some miRNAs are produced immediately, as soon as 2h after treatment (for example, miR-155), others are produced in a delayed time frame after TLR stimulation (for example, miR-21). This temporally regulated expression sequence may distinguish the function of miRNAs in regulating inflammation. In addition to miRNA induction by TLR signaling, recent studies also demonstrate inflammatory repression, such as miR-155 repression, in response to IL-10.63 Despite this understanding of transcription-based production of miRNAs, virtually nothing is known about how inflammatory transcription programs may affect miRNA degradation. The question of what regulates miRNA degradation could drastically change our understanding of miRNA regulation of inflammation.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Schematic of the TLR pathway and miRNAs that are involved in negative regulation of the pathway.

Full figure and legend (107K)

In regulation of the TLR pathway, a central question has been whether miRNAs regulate TLRs themselves. Although the Target Scan algorithm (www.targetscan.org) shows very few miRNAs that target TLRs, alternative algorithms indicate that miR-223 is a regulator of TLR3 and TLR4. Supporting this is a finding that granulocytes that express miR-223 at high levels have low levels of TLR3.64 In oral keratinocytes, miR-105 regulates TLR2.65 In mouse peritoneal macrophages, Let-7 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 TLR4-mediated production of inflammatory proteins such as tumor necrosis factor-α and IL-6.66

Recent studies have shown that TLRs can sequentially upregulate different miRNAs to help guide the temporal regulation of the immune response.37 In this study, the authors determined that the initial TLR signal is propagated by miR-155 that downregulates SHIP1, an inflammatory inhibitor, thereby allowing for inflammation to proceed. Later, induction of miR-21 inhibits PDCD4, an IL-10 inhibitor, hence derepressing IL-10. The time scale of PDCD4 inhibition was found to be delayed when compared with induction of miR-21, leading to the idea that this miRNA acts as a delayed negative feedback regulator. IL-10 then inhibits miR-155, allowing SHIP1 to be derepressed and inhibit TLR signaling.36, 37

Central to the propagation of the signal from TLRs is activation of NF-κB, a key transcriptional regulator of the inflammatory response. The regulation of NF-κB is very complex and thoroughly reviewed elsewhere.67 Briefly, NF-κB 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 IκB proteins, which in turn are regulated by IκB kinases (IKKs). Several of these proteins are regulated by miRNAs, affecting NF-κB activity. miR-155 and miR-199 target the IKKs, upstream of IκBα.68, 69 miR-223 also targets IKK-α, a component of the NF-κB pathway, during differentiation of monocytes into macrophages.70 miR-223 downregulation during macrophage activation had two effects: first, the cells had less baseline NF-κB activity and, at the same time, the cells were primed to be activated. miR-9 has been shown to target NF-κB itself, namely the Nfkb1/p50 subunit.71 Downstream of NF-κB 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-κB, 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-β.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 importance of AU-rich elements within mRNA sequences in 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-α and IL-10 contains sites for binding by miR-16 and miR-106, respectively, but also contains AU-rich elements. Hence, RNA-binding proteins, such as tristetraprolin, may bind concurrently with miR-106/miR-16, leading to clearance of these mRNAs.73, 74 In other studies, miR-221, miR-579 and miR-125b were found to interact with tumor necrosis factor-α mRNA, indicating that many other miRNAs may also influence the degradation of this key inflammatory cytokine along with the AU-rich elements.75 Hence, miRNAs regulate a variety of targets in the TLR/NF-κB pathways and may interact with other factors regulating mRNA stability to achieve the gene expression patterns necessary for the immune response. We summarize the effects of miRNAs on TLR signaling in Table 1. For a further review of miRNAs in the TLR pathway, the reader is referred to an excellent recent review.36


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MicroRNAs in B-cell activation

B cells are a central component of the adaptive immune system responsible for the production of long-lived antibodies, which are the main determinant of long-term specific immunity.76 B cells develop in the bone marrow and achieve a remarkable diversity of antigen specificities by rearrangement of their immunoglobulin loci by V(D)J recombination. Once they have matured in the bone marrow, they seed secondary lymphoid organs where they are activated by antigens that their B-cell receptors (surface immunoglobulin) recognize. Once they are activated, they undergo proliferation and further differentiation. The outcome of antigenic activation is differentiation into either plasma cells that secrete highly specific antibodies or memory B cells that can be reactivated for future protection.77 Once an antigen comes into contact with the B-cell receptor, a signalosome consisting of the B-cell receptor and of intracellular signaling kinases such as Phospholipase C-2 gamma, phosphatidyl inositol 3-kinase, Bruton's tyrosine kinase and Vav as well as B-cell adaptor Blnk is assembled.77 Although conventional B cells (B2 B cells) require CD4+ T-cell activation, B1 B cells do not. It is thought that B1 B cells are activated by a TLR microbial detection pathway.78

The overall importance of miRNAs in hematopoiesis was established by a seminal study that showed that miR-181 was highly expressed in B cells and guided B-cell development when constitutively expressed.57 Mice with an early B-cell-specific deletion of Dicer demonstrated a lack of B-cell development past the pro-B-cell stage.79 This seemed to be mediated via increased apoptosis, and the protein targets, bcl-2 interacting mediator of cell death and phosphatase and tensin homolog, were thought to be responsible for the phenotype. miR-150 constitutive expression has an effect on B-cell development, as does miR-34a constitutive expression.41, 80, 81 Both miRNAs must normally be downregulated at the pro-B to pre-B transition and their continued expression leads to inhibition of their respective targets, c-Myb and Foxp1 (forkhead box P1), which are required for further B-cell development.

miRNAs are now known to play a clear role in controlling the activation of mature B cells. At the global level, Dicer ablation in mature B cells (as opposed to early precursor B cells) resulted in an increase in marginal zone B cells and a decrease in follicular B cells.82 It was also determined that these mice had an increased titer of autoimmune antibodies with autoimmune disease in female mice. This study also hinted that the relevant target was Bruton's tyrosine kinase, which is important in controlling B-cell activation.

In B-2 cells, miR-155 plays a major role in regulating the response in germinal center cells, where B cells undergo a second round of DNA rearrangement, followed by selection.83, 84 miR-155-deficient mice show marked defects in both antibody secretion and class-switch recombination upon immunization.85, 86 miR-155 represses over 60 target genes in B cells, including Pu.1, SHIP1 and AID.86 In fact, Pu.1 seems to be at least partially responsible for the defects in B-cell activation seen in these mice.86 The role of AID, which mediates class-switch recombination and somatic hypermutation, was further explored by two groups who generated a highly specific disruption of the miR-155 target site in the Aicda 3′-untranslated region.87, 88 Disruption of the miR-155/AID interaction led to persistent/increased somatic hypermutation, including abnormal translocations, and decreased high-affinity antibodies in immunized mutant mice. This result suggests that the majority of miR-155-mediated phenotypic effects in B cells are not mediated by AID, but that the AID/miR-155 interaction has an important function. One possibility is that miR-155 inhibition of Aicda may represent a delayed negative regulatory switch, which allows for the proper temporal control of somatic hypermutation and positive selection. Interestingly, miR-181b overexpression in B cells was found to reduce the class-switch recombination rates, possibly by also downregulating AID.89 In another study, miR-150-deficient mice show an expansion of B1 B cells accompanied by dramatic increases in steady-state antibodies.41 miR-150 deficient mice also showed an enhanced response to immunization with T-dependent antigens, indicating an effect on follicular (B2) B cells. Together, these studies 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 mechanisms of autoimmune disease.

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MicroRNAs in T lymphocytes and autoimmunity

T lymphocytes orchestrate and effect some of the most potent responses against invading pathogenic organisms. In this role, they both activate and suppress various components of the immune system and, as such, dysregulation of their function leads to alterations in immunity. In this context, we will discuss the function of specific miRNAs in T cells. Although a thorough exposition of T-cell biology is well beyond the scope of this review, we highlight a few important points that are subject to miRNA regulation. First, the function of the T cell is dependent on its T-cell receptor, the surface molecule that mediates recognition of major histocompatibility complex-bound antigenic peptides.90 The strength of binding is critical, as it determines whether the T cell survives through selection during development in the thymus.91 Indeed, T cells with autoreactive receptors (that is, those that bind too strongly) are deleted during development in the thymus. Second, once T cells are mature, CD4+ helper T cells differentiate further into specific types of helper cells. These include T helper (TH)1, TH2 and TH17 cells, which activate various types of immune responses, as well as regulatory T cells (Treg), which suppress the immune response. miRNAs have now been found to play roles in several of these aspects of T-cell function.

Global knockout of Dicer in the T-cell lineage led to an arrest in T-cell development and abnormal development of T-cell subsets.92, 93 Early studies with miR-181a, a miRNA that has significant function in B cells as well as T cells, revealed that its knockdown allowed for T-cell reactivity against self-antigens, as a consequence of modulating the expression of several phosphatases that are responsible for determining the strength of the transduced T-cell receptor signal.94 These targets were distinct from the targets described for miR-181a in the B-cell lineage where it targets BCL2 (B-cell lymphoma 2) and TCL1 (T-cell leukemia/lymphoma 1).95 This highlights targeting distinctions in different cell lineages, which adds to the regulatory capacity of miRNAs. These studies highlight the need to study miRNA targeting in a given cell lineage.

The recent description of Treg has revolutionized the field of autoimmunity research.96 These cells, which depend on the transcription factor Foxp3 for their development, have the capacity to suppress the activity of the immune system in an antigen-specific manner. The miRNAs seem to play important roles in regulating the function of these cells. Dicer deletion in Foxp3+ cells led to a lethal and severe autoimmune disease in mice, characterized by decreased numbers of mature Treg in the periphery, inappropriate activation and impaired maturation.97 These findings led to a search for specific miRNAs that may be responsible for this phenotype. Further studies with miR-155-deficient mice showed that miR-155-deficient Treg showed impaired survival compared with wild-type Treg.98 Using a competitive assay, the Rudensky group found that miR-146a-deficient Treg were unable to rescue a hyperactive immune system that was generated by knocking out Foxp3.40 These findings are concordant with the findings of a hyperinflammatory-immunoproliferative phenotype in miR-146a/ mice.38, 39 Together, these studies highlight significant alterations in Treg function as a consequence of miRNA activity, and correlate well with studies from human patients that demonstrate miRNA dysregulation in several autoimmune diseases. Specifically, it has been found that miR-155 and miR-146a are upregulated in lymphocytes from patients with rheumatoid arthritis.99, 100, 101

The functional effects of miRNAs, however, are not limited to the Treg lineage, and autoimmune disease has been observed in other T-cell contexts as well. T-cell directed overexpression of the oncogenic miRNA locus, miR-17~92, caused an immunoproliferative disorder and autoimmune disease when expressed in a lymphocyte-specific manner.102 Although compromised Treg lineage function was implicated in miR-146a-deficient mice, other mechanisms are also operant in the autoimmune pathologies. In addition to the myeloid proliferations, T cells from miR-146a-deficient mice showed features of activation and contributed to the autoimmune sequelae in these mice.38 On the 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 and TH17 cells, and partially to dendritic cell function, thus implicating miRNA function in the activation of all CD4+ T-cell lineages. Like miR-155, miR-326 also appears to promote autoimmune inflammation, and acts via repression of the Ets-1 transcription factor.103

Although nonexhaustive, this examination of miRNAs provides an idea of the breadth of miRNA functions in T-cell biology. The many facets of T-cell biology all seem to involve miRNA functions at some level. Perhaps most intriguingly, dysregulation of several different miRNAs leads to pathologic inflammation and autoimmune disease. At least in the T-cell lineage, this seems to reflect the function of miRNAs in fine-tuning the expression of protein factors that may ‘set’ the level of the inflammatory response. Identifying these factors will help set the stage for developing therapeutic avenues for autoimmune disease, which remains a very serious problem in human patients.

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MicroRNAs in leukemia

Many profiling studies have led to a wealth of information about dysregulated miRNA expression in various tumors of the hematolymphoid system. This has led to the concept of ‘oncomiRs’, or oncogenic miRNAs, and tumor-suppressor miRNAs. We have already discussed some of the molecular similarities between inflammatory hematopoiesis and certain clonal hematopoietic neoplasms. However, there are no unifying themes that emerge from these observations. Several miRNAs can cause leukemic transformation in mouse models, but their roles in immunology can be divergent or incompletely understood.

Extensive profiling studies of miRNAs in acute and chronic leukemia are reported and reviewed elsewhere.104, 105 When overexpressed in mice, miR-155 causes a myeloproliferative disorder; miR-125b leads to a chronic myeloproliferative disease that evolves into an acute leukemia.48, 106, 107 miR-29a was also recently reported to cause an acute myeloid leukemia when overexpressed in mice, whereas overexpression of miR-21 resulted in an acute lymphoblastic leukemia.108, 109 These studies highlight that several oncomiRs are capable of transforming hematopoietic cells. Despite these similarities in induction of malignant phenotypes, the functions of these miRNAs in immune cells may be quite different; for example, TLR signaling induces miR-155 and miR-21 but it downregulates miR-125b.

In addition to oncomiRs, several tumor-suppressor miRNAs may have important roles in leukemogenesis. Perhaps the most elegantly studied tumor-suppressor miRNA is the miR-15a/16 polycistron, which was initially identified as being located at the 13q14 chromosomal locus deleted in chronic lymphocytic leukemia.110 Initial studies identified several important targets of miR-15a/16 including oncogenes such as BCL2 and TCL1.95, 111, 112 Experimentally, deletion of (1) the miRNA-containing segment of the gene, (2) the larger noncoding RNA that contained the miRNAs and of (3) the entire homologous locus in mice led to successively more severe phenotypes, and all recapitulated chronic lymphocytic leukemia to some degree.113 Overall, the observations made regarding the functional role of miRNAs in leukemogenesis suggest that replacement of tumor-suppressor miRNAs and inhibition of oncomiRs may have an important role in future therapeutic approaches.

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Conclusions and speculations

The field of miRNA research has undergone a tremendous expansion in the past decade, and these studies have changed fundamentally how we look at gene expression. For example, the unique temporal regulation properties that are possible with miRNAs would not be possible with transcription factors. Furthermore, although not reviewed here, coexpression of a host protein coding gene and an intronic miRNA can produce a unique regulatory module.114 Further insights into miRNA function continue to be made–a recent study that examined gene expression using single-cell imaging studies reveal that the changes in target gene expression are highly dependent on abundance in a miRNA-expressing cell.115 There seems to be a threshold level below which repression by miRNA can be close to complete. Around the threshold value, miRNAs can modulate significant but subtotal repression of the target. Well above the threshold value, miRNAs do not change target gene expression at all. At the population level, such effects would amplify differences in gene expression between low- and high-expressing cells.

In the immune system, we have reviewed the centrality of miRNAs regulating TLR and other signaling pathways. Acting in positive feedback loops (such as miR-155 promoting inflammation) and in delayed negative feedback regulatory loops (such as miR-21 and miR-146a), miRNAs may confer a level of regulation that is not possible with transcription factors. However, the function of a single miRNA in a single-cell lineage is still incompletely understood, as highlighted by miR-155, which can repress components of the TLR signaling pathway in addition to having proinflammatory effects by repressing SHIP1 (Figure 1 and Tang et al.116). Adding to this complexity, miRNAs may have different functions in different cell lineages, because of the exquisite sensitivity of miRNA regulation to the concentration of its target mRNA. Future research with exacting loss of function of specific miRNAs in subsets of immune cells, as well as deletions of miRNA target sites, will be required to precisely understand the function of miRNAs during immune responses.

In addition to these detailed mechanistic studies to delineate miRNA function, research tapping into the therapeutic applications of these miRNAs has begun. Indeed, small RNA delivery has already been demonstrated in a few human trials, and small RNA inhibition in animal models can be achieved with anti-miRNAs or antagomirs.117, 118 These studies demonstrate the potential of these therapeutics in tuning down miRNA function. A related theme of research, replacing miRNAs, is also gaining traction. For example, delivery of miR-146a or miR-21 may be able to tune down inappropriate immune responses. The technology for such delivery may entail using double-stranded small RNA molecules, but delivery into the right cell types and tissues remains a challenge. One method is suggested by the recent observation that some miRNAs can pass between cells via exosome-mediated transfer.119 It is also tempting to speculate that such cell-to-cell communications may regulate immune cell function.

miRNA research in the immune system has progressed a great deal in just a few short years. Their evolutionary conservation—these mechanisms are present in simple multicellular animals and plants in addition to mammals—is remarkable and suggests an ancient function. Some have suggested that they may have arisen as a primitive antiviral response and that these miRNAs may be particularly important in the immune system. Regardless of their origin, it is clear that miRNAs have diverse and important functions in the immune system. Exciting developments and unexpected twists undoubtedly remain as we continue our efforts to understand the function and develop the therapeutic potential of these enigmatic regulators of gene expression.

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Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

Although this review is meant to be comprehensive, we acknowledge that we may not have included all papers in this large and growing field because of space limitations. We thank David Baltimore and Ryan O’Connell for helpful discussions over the years. DSR is a Kimmel Scholar of the Sidney Kimmel Foundation for Cancer Research and has received a career development award from the NIH (5K08-CA133251). JC is a recipient of the Eugene V. Cota-Robles Fellowship from UCLA.