micrornas: the fine-tuners of toll-like receptor signalling

It is well established that Toll-like receptors (TLRs) have important roles in detecting pathogens and in initiat-ing inflammatory responses that subsequently prime specific adaptive immune responses during infection1. It has also been recognized that dysregulation of this process is a hallmark of inflammatory and autoimmune diseases2. It is therefore important that TLR signalling pathways are tightly regulated. Although many mecha-nisms for the negative regulation of TLR signalling have been described3, the induction of an anti-inflammatory response and the processes by which inflammation is resolved remain incompletely understood.

MicroRNAs (miRNAs) have recently emerged as impor-tant regulators of gene expression. They were formerly thought to mainly repress the translation of target mRNAs, but it has recently been shown that the main function of miRNAs in mammalian systems is to decrease target mRNA levels4. Each highly conserved mammalian miRNA probably targets several hundred distinct mRNAs5, so it is probable that most mRNAs are controlled by miRNAs to some extent. miRNAs could therefore be as important as transcription factors in controlling the protein content of a cell. The expression of miRNAs is highly regulated and they are therefore well placed to function as immunomodulators. It is not surprising that given the dynamic nature of miRNAs, they are involved in regulating the components of TLR signalling and innate immune pathways.

The signalling molecules that comprise each TLR sig-nalling pathway are regulated by numerous mechanisms, including physical interactions, conformational changes, phosphorylation, ubiquitylation and proteasome- mediated degradation6. A more energy-efficient way to regulate the activity of TLR signalling molecules could

be to destabilize the mRNA molecules that encode them. miRNA-mediated control of the expression of these signalling molecules through either mRNA decay or translational inhibition might not be as rapid as control through proteasomal degradation; however, this might be an advantage during infection, as miRNA-mediated control of mRNA levels allows for a strong initial immune response that is gradually dampened down. However, it must be noted that for miRNA- mediated targeting of the mRNAs encoding TLR sig-nalling molecules to be effective, the signalling protein itself must also be targeted by a complementary method: either, it must be sufficiently unstable such that by the time it has passed one half-life no newly synthesized protein is available to take its place, or it must also be targeted for degradation by separate or complementary signalling mechanisms.

The end result of the TLR signalling pathways is the activation of pro-inflammatory transcription factors that enhance the transcription of RNA poly merase II- sensitive genes such as those encoding cytokines, chemo-kines and antimicrobial enzymes. Because miRNAs are also transcribed by RNA polymerase II7,8, it stands to reason that miRNAs themselves are targets of TLR signalling pathways.

In this Review, we examine how miRNAs regulate TLR signalling. We consider those miRNAs that are induced by TLR signalling and speculate that these miRNAs regulate the strength, location and timing of TLR responses. miRNAs might provide a link between innate and adaptive immune signalling pathways and they might also have a role in controlling the switch from a strong early pro-inflammatory response to the resolution phase of the inflammatory process.

*School of Biochemistry and Immunology, Trinity College Dublin, Ireland.School of Medicine, New York University Langone Medical Center, 550 First Avenue, New York 10016, USA.Monash Institute of Medical Research, Monash University, Clayton, Victoria 3168, Australia.Correspondence to L.A.ON. email: [email protected]:10.1038/nri2957Published online 18 February 2011

Toll-like receptors(TLRs). A family of pattern recognition receptors that detect conserved microbial components during infection and initiate an inflammatory response. They are commonly expressed by cells of the immune system including macrophages and dendritic cells, as well as other sentinel cells such as epithelial cells. TLRs have also been implicated in the recognition of endogenous danger signals that are present in the body during disease.

MicroRNAs: the fine-tuners of Toll-like receptor signallingLuke A. ONeill*, Frederick J. Sheedy and Claire E. McCoy

Abstract | Toll-like receptor (TLR) signalling must be tightly regulated to avoid excessive inflammation and to allow for tissue repair and the return to homeostasis after infection and tissue injury. MicroRNAs (miRNAs) have emerged as important controllers of TLR signalling. Several miRNAs are induced by TLR activation in innate immune cells and these and other miRNAs target the 3 untranslated regions of mRNAs encoding components of the TLR signalling system. miRNAs are also proving to be an important link between the innate and adaptive immune systems, and their dysregulation might have a role in the pathogenesis of inflammatory diseases.

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NATuRE REvIEWs | Immunology voLumE 11 | mARcH 2011 | 163

2011 Macmillan Publishers Limited. All rights reserved

MicroRNAs(miRNAs). Small (1822 nucleotide) RNA molecules that regulate gene expression by binding to the 3 untranslated regions of specific mRNAs. They are derived from larger precursor and primary transcript molecules and are themselves transcriptionally regulated in a manner similar to mRNAs.

Induction of miRNAs by TLR signallingmany studies have addressed the hypothesis that TLR signalling can modulate miRNA expression using various profiling techniques. Although a subset of miRNAs has emerged as strong targets of TLR signalling, subtle differences in miRNA expression profiles have been observed depending on the TLR stimulus used, treatment time, technology used and, importantly, the cell type. TABLE 1 summarizes the results of these pro-filing experiments and lists those miRNAs that have been confirmed to be regulated by TLR signalling in independent studies.

multiple miRNAs are induced in innate immune cells, with a consensus emerging that miR-155, miR-146 and miR-21 are particularly ubiquitous. There is also evidence that the expression of certain miRNAs can decrease following TLR activation. similar to other TLR-responsive genes, miRNAs can be clas-sified as early or late response genes: some miRNAs (for example, miR-155) are highly induced 2 hours after treatment, whereas other miRNAs (for example, miR-21) are induced at later times. The expression of most TLR-responsive miRNAs described so far depends on nuclear factor-B (NF-B) activity. In all cases of miRNA

induction by TLR activation that have been described so far, the transcription of miRNA primary tran-scripts is upregulated, although it remains possible that the processing of miRNA precursors could also be upregulated by TLR signalling9. similar to other TLR-responsive genes, it is also important that the induction of TLR-responsive miRNAs is regulated and mecha-nisms are now being discovered that negatively regu-late miRNA induction by TLR signalling; for example, miR-155 in particular is subject to negative regulation by interleukin-10 (IL-10)10. Less is known about how TLR signalling can decrease miRNA expression this could be through transcriptional repression or through post-transcriptional mechanisms that destabilize miRNA transcripts, and these areas are being actively investigated.

Despite the wealth of information regarding miRNA induction, there has been a tendency in the field of miRNA biology to document changes in miRNA levels without effectively analysing the functional consequences of these changes. New data are beginning to emerge from studies analysing the consequences of changes in miRNA expression for TLR biology that provide insights into the control of TLR signalling by miRNAs.

Table 1 | miRNAs regulated by TLRs

miRnA TlRs Signalling molecules Cell type other miRnA inducers Refs

Upregulated

miR-155* TLR2, TLR3, TLR4, TLR9

MYD88, TRIF, JNK, AP1, NF-B, KSRP

BMDMs, THP1 cells, monocytes, macrophages, DCs, B cells, T

Reg cells

Helicobacter pylori, KSHV, EBV, oxidized LDL, TNF, IFN

9,10,22,25,27,29, 40,41,50,60,61,

124127

miR-146 TLR2, TLR3, TLR4, TLR5

MYD88, NF-B THP1 cells, macrophages, BMDMs, T cells

EBV, VSV, RIG-I, TNF, IL-1 25,26,27,41,61,62, 74,77,80,82,124,126

miR-132 TLR4, TLR9 ND THP1 cells, human monocytes and macrophages, BMDMs, splenocytes

KSHV 9,25,41,45,61

miR-21 TLR4 MYD88, TRIF, NF-B Inflamed lung tissue, RAW264.7 cells, BMDMs, B cells, H69 cholangiocytes

Cryptosporidium parvum, EBV (LMP1)

62,76,126, 128132

miR-223 TLR4 ND Inflamed lung tissue, DCs ND 27,128

miR-147 TLR2, TLR3, TLR4 MYD88, TRIF, NF-B, IRF3

BMDMs, RAW264.7 cells, THP1 cells, alveolar macrophages

ND 80

miR-9 TLR2, TLR4, TLR7TLR8

MYD88, NF-B Human monocytes and granulocytes

IL-1 41

miR-125b TLR4 NF-B H69 cholangiocytes, rheumatoid arthritis synovial fibroblasts, LPS-tolerized THP1 cells

Cryptosporidium parvum 32,57,131

let-7e TLR4 AKT1 Peritoneal macrophages ND 22

miR-27b TLR4 NF-B Human macrophages ND 44Downregulated

miR-125b TLR4 NF-B, AKT1 Splenocytes, BMDMs, DCs ND 22,27,50 let-7i|| TLR4 NF-B, C/EBP H69 cholangiocytes Cryptosporidium parvum 23,48,133miR-98 TLR4 ND H69 cholangiocytes Cryptosporidium parvum 134

*Expression inhibited by IL-10 and AKT. High basal level of expression in this cell type. The differences in miR-125b expression after TLR activation are currently unknown. ||Expression inhibited by the miRNA lin-8. AP1, activator protein 1; BMDM, bone marrow-derived macrophage; C/EBP, CCAAT/enhancer-binding protein-; DC, dendritic cell; EBV, EpsteinBarr virus; IFN, interferon-; IL, interleukin; IRF3, IFN-regulatory factor 3; JNK, JUN N-terminal kinase; KSHV, Kaposis sarcoma-associated herpesvirus; KSRP, KH-type splicing regulatory protein; LDL, low-density lipoprotein; LMP1, latent membrane protein 1; LPS, lipopolysaccharide; miRNA, microRNA; MYD88, myeloid differentiation primary-response protein 88; ND, not determined; NF-B, nuclear factor-B; RIG-I, retinoic acid-inducible gene I; TLR, Toll-like receptor; TNF, tumour necrosis factor; TRIF, TIR domain-containing adaptor protein inducing IFN; TReg cell, regulatory T cell; VSV, vesicular stomatitis virus.

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Nuclear factor-B(NF-B). A highly pro-inflammatory transcription factor that is activated by many stimuli, including TLR activation. NF-B complexes are held inactive in the cytoplasm by inhibitor of NF-B (IB) proteins. Degradation and removal of IB is a common NF-B-activating process and TLR signalling pathways converge on this mechanism. NF-B-responsive genes include those encoding cytokines, chemokines and antimicrobial enzymes.

3 untranslated region(3 UTR). The RNA sequence found 3 (downstream) of the stop codon in the open reading frame of a mRNA before the poly(A) tail sequence. 3 UTR sequences vary in length and nucleotide content. It is now recognized that 3 UTR sequences contain regulatory RNA sequences that determine the translation efficiency and stability of the mRNA, including miRNA target sites.

Foam cellsMacrophages that localize at sites of early vascular inflammation and that subsequently ingest oxidized low-density lipoprotein and slowly become overloaded with lipids. Foam cells eventually die and attract more macrophages, further propagating inflammation in blood vessels.

Targeting of TLR signalling pathways by miRNAsTargeting TLR expression. The most obvious point at which to manipulate the TLR signalling pathway is at the level of receptor expression. Because TLR signal-ling induces a strong pro-inflammatory response, the expression of these receptors is restricted to certain cell lineages, including macrophages, dendritic cells (Dcs) and B and T cells1114. Furthermore, the expres-sion of particular TLRs is restricted to specific cell types to adapt these cells for particular functions15. In addition, the expression of particular miRNAs in mammals has been shown to be limited to particular cell types, which indicates that miRNAs might have a role in controlling cell differentiation and cell-specific functions16,17.

An attractive hypothesis is that the differences in TLR distribution between different immune cell types could be the result of differential miRNA expression. However, so far, there is little evidence that TLRs them-selves are directly targeted by miRNAs. Bioinformatic analysis of the 3 untranslated regions (3 UTRs) of human TLR mRNAs using the prediction program Targetscan shows that TLR-encoding genes have very few highly conserved target sites for miRNAs (F.J.s., c.E.m. and L.A.oN., unpublished observations). These observa-tions might indicate that TLR genes are constitutively expressed and that any regulation occurs at the tran-scriptional or post-translational level. It is also possi-ble that post-transcriptional control of TLRs might be species specific, as a result of differential selective pres-sures that have affected the evolution of mammalian immune systems18,19. miRNA-mediated control might add to this diversity. If this is the case, it is entirely possible that TLR genes can be targeted by miRNAs through weaker, non-conserved sites. A recent study20 that used a refined bioinformatic algorithm to predict active miRNA target sites in the 3 uTRs of TLR and related genes showed that the myeloid-specific miRNA miR-223 (REF. 17) which has an important role in granulopoiesis17,21 is a strong candidate for regulat-ing both TLR4 and TLR3 expression; TLR3 has been shown to be expressed at low mRNA levels in granulo-cytes12 possibly owing to increased levels of miR-223 in these cells. This implies that in resting cells, miRNA activity might regulate the potential of innate immune cells to respond to TLR activators.

The mRNA encoding TLR4 is regulated by members of the let-7 miRNA family. In mouse peritoneal mac-rophages, the induction of let-7e expression decreases cell surface expression of TLR4, the mRNA of which contains a let-7 target site22. Furthermore, transfection of macrophages with antisense miRNA to let-7e leads to an increased lipopolysaccharide (LPs)-induced cytokine response22. The mRNA encoding TLR4 can also be targeted by other isoforms of the let-7 family, such as let-7i. Downregulation of let-7i expression was shown to increase TLR4 expression by human cholangiocytes (biliary epithelial cells) after Cryptosporidium parvum infection or LPs treatment23. The differential regulation of TLR4 expression in these cell types (cholangiocytes compared with peritoneal macrophages) by members

of the let-7 family might be due to the fact that epi-thelial cells would need to be activated and to enhance their pro-inflammatory properties during infection, whereas macrophages need to constantly survey the environment for bacteria and therefore require consti-tutive expression of TLR4. The expression of TLR4 by macrophages would need to be turned off at later times after ligand sensing to avoid excessive inflammation during infection. This regulation of TLR expression by miRNAs might also explain the differences observed in the induction patterns of miRNAs by TLRs in different cell types. TLR2 mRNA has been shown to be regulated by miR-105, the expression of which is higher in oral keratinocytes derived from patients who respond weakly to TLR2 activation with low levels of cytokine induction, presumably owing to decreased TLR2 expres-sion24; this indicates that there might be a reciprocal relationship between TLR2 signalling and miR-105 expression. Although these data point to the regulation of certain TLRs by miRNAs, the absence of data in sup-port of other TLRs being targeted by miRNAs under-scores the importance of constitutive TLR expression. As discussed below, rather than shutting down the TLR signalling pathway completely by eliminating recep-tor expression, the trend for miRNA activity seems to be to tone down TLR activity through targeting key intracellular signalling proteins.

Targeting TLR signalling proteins. The molecular tar-gets of miR-146 are IL-1R-associated kinase 1 (IRAK1) and TNFR-associated factor 6 (TRAF6)25. These proteins are important components of the myeloid differentiation primary-response protein 88 (mYD88)-dependent path-way for NF-B activation downstream of TLR2, TLR4, TLR5, TLR7TLR8 and TLR9, which are the same TLRs that induce expression of miR-146 in the THP1 cell line. It was postulated that miR-146 can negatively regulate the mYD88NF-B signalling pathway after bacterial infection25. Recently, IRAK2, a kinase that is required for the persistence of NF-B activation, has also been shown to be targeted by miR-146 (REF. 26), although the relevance of this observation for TLR signalling remains unclear.

There is mounting evidence that miR-155 can nega-tively regulate TLR signalling pathways by targeting key signalling proteins. Inhibition of miR-155 in Dcs resulted in upregulated expression of components of the p38 mitogen-activated protein kinase (mAPK) pathway27. TAK1-binding protein 2 (TAB2), a signal-ling molecule downstream of TRAF6 that activates mAPK kinases, was confirmed as a direct target of miR-155 (REF. 27). mYD88 has also been identified as a target of miR-155 in studies of miR-155 induction by Helicobacter pylori 28. Furthermore, mYD88 is a target of miR-155 in foam cells, which induce the expression of miR-155 when loaded with oxidized low-density lipoprotein29.

Another Toll/IL-1R domain-containing adaptor pro-tein, mYD88 adaptor-like protein (mAL; also known as TIRAP), which functions as a bridging adaptor for TLR2- and TLR4-mediated mYD88-dependent signalling, has

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emerged as a target of miR-145 (REF. 30). It remains to be determined whether the expression of miR-145 is also regulated during TLR2 or TLR4 signalling. However, it is known that mAL undergoes proteasomal degradation following TLR2 and TLR4 stimulation31. Therefore, per-haps an additional level of control of mAL expression exists through miR-145.

Finally, Brutons tyrosine kinase (BTK) participates in the TLR4, TLR7TLR8 and TLR9 signalling path-ways to NF-B activation, and Btk mRNA is a target of miR-348 (REF. 32), which is strongly induced by LPs treatment of rheumatoid arthritis synovial fibrob-lasts. It remains to be determined whether miR-348- mediated regulation of Btk mRNA also occurs in macrophages and Dcs.

What is interesting with regard to the TLR signalling molecules that are targeted by miRNAs is that relatively few proteins have been confirmed as direct targets of miRNAs (specifically, mYD88, mAL, IRAK1, IRAK2, TRAF6, BTK and TAB2; TABLE 2). However, these pro-teins are components of several TLR signalling pathways, which indicates that once one TLR is triggered, miRNA-mediated targeting of common signalling proteins could silence signalling through multiple TLRs. Because most pathogens can engage several TLRs, miRNAs could help to avoid excessive pro-inflammatory responses after pathogen exposure by shutting down several TLR pathways. It is probable that miRNAs work together with multiple other mechanisms to control the expres-sion of TLR signalling components. The combination of these mechanisms could result in timely and appropriate toning down and termination of the pro-inflammatory response (FIG. 1).

Targeting transcription factors. The targeting of transcription factors by miRNAs would have a global impact on TLR-induced gene expression. many stud-ies have highlighted the fact that miRNA-mediated targeting of transcription factors is an important aspect of miRNA function33,34. For example, a miRNA that is induced by a particular signalling pathway can often feedback to inhibit the transcription factor that is required for its induction34. Higher basal levels of miRNAs in certain cell types might function as impor-tant epigenetic switches required for the functional maintenance of the cell type35,36. For example, forkhead box P3 (FoXP3), a transcription factor that is required for the maintenance of regulatory T cells, was shown to drive the high level of miR-155 expression found in these cells; miR-155 then feeds back and targets FoXP3 to decrease its expression35. more generally, transcription factors are usually expressed at low levels in cells, which might be the result of strict control by miRNAs. Evidence is emerging that the pro-inflammatory transcription factors activated by TLR signalling are targeted directly by miRNAs.

NF-B activity is mainly controlled by inhibitor of NF-B kinases (IKKs). IKK was recently shown to be targeted by a subset of miRNAs including miR-223 (REF. 37) and IKK is targeted by miR-199 in human ovarian cancer cells38. However, an effect of these miRNAs on TLR signalling was not directly examined in these studies. Analysis of both miR-155 and its Kaposis sarcoma-associated herpesvirus (KsHv) homologue has identified IKK as a potential target, which sup-ports the notion that miR-155 can negatively regulate innate immune signalling39,40. more recently, the TLR-responsive miRNA miR-9 was shown to directly target

Table 2 | Verified targets of miRNAs in TLR signalling

Target mRnA miRnA(s) Refs

Receptors

TLR4 miR-223, let-7i, let-7e 20,22,23

TLR3 miR-223 20

TLR2 miR-105 24

Signalling molecules

MYD88 miR-155 28,29

MAL miR-145 30

IRAK1 miR-146 25

IRAK2 miR-146 26

TRAF6 miR-146 25

BTK miR-348 32

TAB2 miR-155 27

IKK miR-223 37

IKK miR-199 38

IKK miR-155 39,40Transcription factors

NF-B1 miR-9 41FOXP3 miR-155 35

C/EBP miR-155 42,43

PPAR miR-27b 44p300 miR-132 45

Cytokines

IL-6 let-7 48

TNF miR-16, miR-125b, miR-155, miR-221, miR-579, miR-369-3

50,56,57

IL-10 miR-106, miR-466l 51,58

IL-12p35 miR-21 52

Regulators

ACHE miR-132 61

PDCD4 miR-21 62

SHIP1 miR-155 66,67

SOCS1 miR-155 82

ACHE, acetylcholinesterase; BTK, Brutons tyrosine kinase; C/EBP, CCAAT/enhancer-binding protein-; FOXP3, forkhead box P3; IL, interleukin; IKK, inhibitor of NF-B kinase; IRAK, IL-1R-associated kinase; MAL, MYD88 adaptor-like protein; miRNA, microRNA; MYD88, myeloid differentiation primary-response protein 88; NF-B1, nuclear factor-B1; PDCD4, programmed cell death 4; PPAR, peroxisome proliferator-activated receptor-; SHIP1, Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1; SOCS1, suppressor of cytokine signalling 1; TAB2, TAK1-binding protein 2; TLR, Toll-like receptor; TNF, tumour necrosis factor; TRAF6, TNFR-associated factor 6.

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+4#-+4#- +4#-p50p65

p50p65

p50p65

NF-B

NF-B

NF-B

6.4KPFWEGFIGPGUNFKB1 mRNA41. NF-B1 is cleaved to form the NF-B p50 subunit, which has an important role in transactiva-tion of the NF-B p65 subunit. Therefore, the finding that miR-9 can target the NFKB1 gene identifies a key control point for TLR signalling.

other transcription factors downstream of TLR signalling have been identified as miRNA targets. The transcriptional co-repressor ccAAT/enhancer- binding protein- (c/EBP) has been identified as a target of miR-155 by the analysis of B cells that consti-tutively express miR-155 or mice that had received anti-sense miR-155 (REFS 42,43). The targeting of c/EBP by miR-155 was shown in these studies to decrease expres-sion levels of granulocyte colony-stimulating factor (G-csF), and possibly IL-6, in splenocytes. Peroxisome proliferator-activated receptor- (PPAR) has anti-inflammatory effects and its expression is decreased after LPs treatment. This was shown to be the result of NF-B-dependent induction of miR-27b, which directly targets Pparg mRNA; the inhibition of miR-27b resulted in increased PPAR expression and blunted LPs-induced secretion of tumour necrosis factor (TNF)44. Finally, the transcriptional co-activator p300, which is often associ-ated with cAmP-responsive element-binding protein (cREB) and is required for the induction of antiviral genes, was shown to be a direct target of miR-132 in KsHv-infected lymphatic endothelial cells45.

multiple transcription factors can therefore be controlled by miRNAs, providing a direct mechanism to control the transcription of TLR-responsive genes.

Targeting cytokine mRNAs. Bioinformatic analysis has identified several miRNA-binding sites in cytokine- and chemokine-encoding mRNAs46,47 (FIG. 2a). of note, IL6 mRNA contains a binding site for let-7 (REF. 48); given the fact that let-7 family members can be negatively regulated by TLR signalling and NF-B activation48,49, this could potentially contribute to the increased IL-6 expression observed following TLR stimulation, although this has not been examined directly. similarly, the 3 uTR of TNF mRNA contains a binding site for the LPs-downregulated miRNA miR-125b, which indi-cates a mechanism by which TLR signalling might sta-bilize TNF expression50. IL10 mRNA contains binding sites for 8 miRNAs in its 3 uTR and overexpression of two of these miR-106a and miR-106b resulted in decreased IL-10 protein expression in a human Burkitts lymphoma Raji cell line51. However, a role for TLR signal-ling in the induction of miR-106a and miR-106b expres-sion was not explored in this study. mRNA encoding the IL-12p35 subunit contains a target site for miR-21 (REF. 52), as confirmed by reporter assays in which the 3 uTR of the gene encoding p35 was linked to the luciferase gene, although the extent to which this might contribute to TLR responses remains undetermined.

Although evidence for the direct targeting of cytokine mRNAs by miRNAs is limited, it is increasingly apparent that miRNAs can function together with RNA-binding proteins to regulate mRNA expression through the Au-rich elements (AREs) that are found in numer-ous cytokine-encoding mRNAs (FIG. 2a). For example,

TNF and IL10 mRNAs both contain long AREs that are targeted by the RNA-binding protein tristetraprolin (TTP), which has a key role in mRNA destabilization downstream of TLR signalling5355. miR-16 cooperates with TTP to mediate TNF destabilization, although this

Figure 1 | miRnAs function together with other mechanisms to control the expression of TlR signalling components. The canonical Toll-like receptor 4 (TLR4) signalling pathway uses the adaptor molecules myeloid differentiation primary-response protein 88 (MYD88) and MYD88 adaptor-like protein (MAL) to propagate nuclear factor-B (NF-B)-dependent gene transcription, the products of which are required for initiating an inflammatory response. However, it is crucial that mechanisms exist to switch this pathway off to prevent over-amplification of this signal. One such mechanism is mediated by K48-linked ubiquitylation, which targets specific TLR signalling components for degradation by the proteasome. Another mechanism is mediated by TLR-induced microRNAs (miRNAs), several of which are regulated by NF-B. miRNAs bind to the 3 untranslated region of specific mRNA target sequences to inhibit the de novo synthesis of gene products, such as cytokines and components of the TLR signalling pathways. The TLR signalling molecules that are currently known to be targeted by miRNAs include MYD88, MAL, Brutons tyrosine kinase (BTK), IL-1R-associated kinase 1 (IRAK1), IRAK2, TNFR-associated factor 6 (TRAF6), TAK1-binding protein 2 (TAB2), inhibitor of NF-B (IB) kinase- (IKK) and IKK. In this manner, TLR-induced signals can tailor the levels of protein in the cell upon infection. Pri-miRNA, primary miRNA.

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LPS toleranceA transient state of hyporesponsiveness to subsequent stimulation with lipopolysaccharide (LPS) after TLR activation.

miRNA has not yet been shown to be TLR responsive56. more relevant was a recent study57 that showed that miR-221, miR-579 and miR-125b are expressed follow-ing the induction of a state of LPS tolerance, during which TNF mRNA is degraded. miR-221 was found to associ-ate with TTP and to accelerate TNF mRNA decay, and miR-579 and miR-125b seemed to block TNF transla-tion, possibly through recruitment of the translational inhibitor TIAR. However, it should also be noted that some of these effects could be mediated directly by the upregulation of miR-125b expression, which might destabilize TNF by direct binding50.

conversely, miRNAs can also compete with RNA-binding proteins to protect mRNA from destabilization. For example, miR-466l contains a seed region that is complementary to the canonical ARE AuuuA sequence. Transfection of LPs-stimulated RAW264.7 macrophages with miR-466l resulted in the upregulation of Il10 mRNA and protein expression by competing with TTP for bind-ing to the ARE sequence in Il10 mRNA, which protected the mRNA from TTP-mediated degradation58. In addi-tion to the recruitment of specific RNA-binding proteins, mRNA stability can also depend on environmental fac-tors. For example, miR-369-3, which associates directly with the ARE in TNF mRNA by base-pairing, could mediate translational activation of TNF only under

conditions of serum starvation, and this activation depended on recruitment of the RNA-binding proteins fragile-X mental retardation-related protein 1 (FXR1) and argonaute 2 (AGo2)59. By contrast, miR-369-3 could repress TNF when the cells were actively proliferating59. It has not been yet been investigated whether the activation of TLRs could affect the ability of a miRNA to degrade or stabilize mRNA sequences, but this possibility warrants further investigation.

Although a direct binding site for miR-155 in the TNF mRNA has not been identified, miR-155 might be required for its stabilization, as miR-155-deficient B cells fail to produce TNF60. Furthermore, a role for miR-155 in TNF mRNA stabilization has been shown in HEK293 cells and miR-155-transgenic mice have increased lev-els of circulating TNF after LPs injection50. Although an exact mechanism has yet to be elucidated, it is possible that RNA-binding proteins might be involved. It will be interesting to determine whether other TLR-responsive cytokines are regulated by miRNAs.

Targeting TLR signalling regulators. In addition to the insights discussed above, studies of the miRNAs that are induced by and control TLR signalling have provided us with a tool to uncover new molecules that have important and unexpected roles in TLR signalling pathways.

Figure 2 | miRnA-mediated control of cytokines induced by TlRs and Il-1 signalling. a |The microRNA (miRNA) let-7 has been shown to directly target and destabilize interleukin-6 (IL6) mRNA. miR-155 might have a role in the stabilization of tumour necrosis factor (TNF) mRNA. miR-369-3 mediates TNF stabilization only under conditions of serum starvation, which was shown to depend on the recruitment of fragile-X mental retardation-related protein 1 (FXR1) and argonaute 2 (AGO2). By contrast, miR-221 in association with tristetraprolin (TTP) can accelerate TNF destabilization in lipopolysaccharide (LPS)-tolerized cells. miR-16, miR-125b and miR-579 also have an important role in TNF destabilization, and miR-16 is thought to mediate its effect through association with TTP. miR-21 has been shown to positively regulate IL-10 expression through its targeted repression of the mRNA encoding programmed cell death 4 (PDCD4). miR-446l can stabilize IL10 mRNA by competing with TTP for association with the AU-rich elements (AREs) in IL10. miR-106 has been shown to directly target the 3 untranslated region of IL10, leading to gene repression. b | Overexpression of miR-146a, miR-155 and miR-9 modulates IL-1 receptor (IL-1R) signalling, which might be the result of direct targeting of IL-1R-associated kinase 1 (IRAK1) and TNFR-associated factor 6 (TRAF6) by miR-146a, the targeting of TAK1-binding protein 2 (TAB2) by miR-155 and/or the targeting of the p50 subunit of nuclear factor-B (NF-B) by miR-9. IL-6-induced signalling can be modulated through the targeted repression of suppressor of cytokine signalling 1 (SOCS1) by miR-155 (not shown). IB, inhibitor of NF-B; MYD88, myeloid differentiation primary-response protein 88.

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0CVWTG4GXKGYU^+OOWPQNQI[6.4 OK4OK45*+2 2&%&+.O40#+.Cholinergic anti-inflammatory pathwayThis pathway fine-tunes cytokine production during inflammation in a highly regulated and reflexive manner. Interaction of acetylcholine with the 7-nicotinic acetylcholine receptor expressed by macrophages results in the suppression of pro-inflammatory cytokine production. The main component of this pathway is the vagus nerve of the parasympathetic branch of the autonomic nervous system.

Luciferase reporter assayA method to measure the transcriptional response. This assay uses a regulatory sequence from a gene of interest fused to the gene that encodes luciferase to determine the effect of the regulatory sequence on gene expression. It is commonly used to determine promoter sequences and transcription factor-binding sites, but can also be used to determine miRNA targeting through the fusion of a 3 UTR sequence containing miRNA target sites to the luciferase gene.

studies of miR-132 have identified that its target, the gene encoding acetylcholinesterase (AcHE), is a key regulator of TLR signalling and have provided links between TLRs and neuroinflammation. AcHE hydrolyses acetylcholine (an important component of the cholinergic anti-inflammatory pathway that is released by efferent vagus nerve fibres) and acetylcholine can block NF-B nuclear translocation in macrophages and thus attenuate TLR-induced innate immune responses. so, an increase in miR-132 levels in response to TLR stimulation will result in the repression of AcHE and increased acetylcholine-mediated negative regulation of TLR-induced signals. Indeed, LPs-treated macrophages from mice in which the 3 uTR of Ache mRNA has been deleted and therefore cannot bind miR-132 overproduce IL-6, IL-12 and TNF61.

In our studies of the induction of miR-21 by LPs, we identified the tumour suppressor protein programmed cell death 4 (PDcD4) as a key target that is down-regulated by miR-21 during TLR4 signalling in macro-phages62. PDcD4 functions as an inhibitor of translation by inhibiting eukaryotic translation-initiation factor 4F (EIF4F)63,64, which is required for the initiation of trans-lation at the 5 uTR of mRNA sequences. Therefore, mRNAs with complex 5 uTRs which are longer, have a higher Gc content and contain the potential for sec-ondary RNA structures are more sensitive to EIF4F inhibition65 and therefore will also be more sensitive to the levels of PDcD4 in a cell. Interestingly, it has been proposed that mRNAs encoding growth factors and cytokines can be considered as complex mRNAs65 and would therefore be susceptible to PDcD4 activity. We found that the production of IL-10, which is increased at later time points after LPs treatment, depended on the levels of both miR-21 and PDcD4 (REF. 62). Although we have yet to show that PDcD4 inhibits IL10 mRNA translation directly at the 5 uTR, it is clear that miR-21 promotes an anti-inflammatory response by increas-ing IL-10 production, which highlights a previously unappreciated level of control for IL-10 expression and might explain the differences in cytokine production downstream of many TLRs.

src homology 2 (sH2) domain-containing inositol-5-phosphatase 1 (sHIP1) has been well characterized as a primary target of miR-155 (REF. 66). many studies have shown that increased miR-155 expression in response to LPs stimulation or pathogen infection in macrophages can lead to decreased sHIP1 expression10,43,66,67. This might be an important mechanism that is required for the propagation of a pro-inflammatory response as it has previously been shown that sHIP1 can function as a nega-tive regulator of TLR-induced responses6870. We showed that the inhibition of miR-155 expression by IL-10 led to an increase in sHIP1 expression, identifying a new tar-get for IL-10-mediated anti-inflammatory responses10. Furthermore, AKT has been shown to negatively regulate miR-155, which indicates that a negative feedback loop might exist whereby sustained AKT expression can switch off miR-155 expression, allowing sHIP1 levels to increase and subsequently inhibit the AKT signalling pathway22. We propose that the initial increase in miR-155 expression

in response to TLR4 activation downregulates expression of the negative regulator sHIP1, allowing TLR4 signal-ling to proceed. Later in the response, IL-10 is induced in response to the increased level of miR-21, which decreases the expression of PDcD4, an inhibitor of IL10 translation. IL-10 then feeds back on the pathway to decrease miR-155 levels, thereby restoring sHIP1 levels and limiting TLR4 signalling (FIG. 3).

Fine-tuning of TLR signalling by miRNAsAlthough there are accumulating data about the expres-sion, induction and mRNA targeting of miRNAs in TLR signalling, there are currently surprisingly few studies that illustrate the global biological impact of this mRNA targeting by miRNAs and the full extent to which miRNAs actually control innate immune responses (BOX 1). many studies have illustrated the induction of a particular miRNA and used 3 uTR luciferase reporter assays to identify targets of the miRNA within the TLR pathways, but few studies have actually inhibited this tar-geting process and assessed the effects on the immune response. Therefore, it is difficult to determine whether an individual miRNA has an important pro-inflammatory or anti-inflammatory role in TLR signalling.

Influencing inflammatory cytokine signalling. The examination of cytokine expression when TLR-induced miRNAs are overexpressed or inhibited, as well as stud-ies of miRNA-deficient mice, has helped to determine

Figure 3 | Fine-tuning of TlR4 signalling by miR-155 and miR-21. Recent evidence indicates that the microRNAs (miRNAs) miR-155 and miR-21 are important for the regulation of Toll-like receptor 4 (TLR4) signalling. TLR4 signalling increases miR-155 levels, which leads to the degradation of Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1), a negative regulator of TLR4 signalling; thereby, this process increases TLR4 signalling. However, TLR4 activation also increases the level of miR-21, which targets the mRNA encoding programmed cell death 4 (PDCD4), leading to increased production of interleukin-10 (IL-10) (as PDCD4 is an inhibitor of IL10 translation). IL-10 then feeds back on the pathway and specifically inhibits the induction of miR-155. This, in turn, leads to an increase in SHIP1 and the inhibition of TLR4 signalling. This process might be important for the inhibitory effect of IL-10 on TLR signalling and could be relevant for lipopolysaccharide (LPS) tolerance or for the resolution of TLR4-induced inflammatory responses.

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Morpholino-modified oligonucleotideA nucleic acid analogue in which the base and phosphate linkages structurally differ from regular DNA or RNA. They are commonly 25 nucleotides in length and they function by blocking access of RNA-binding proteins or RNAs to target sites in mRNAs to which they are antisense. They can be used to protect a mRNA from miRNA activity by targeting the morpholino-modified oligonucleotide to a miRNA target site in a specific mRNA.

Langerhans cellsProfessional antigen-presenting dendritic cells that are localized in the skin epidermis.

whether specific miRNAs are pro-inflammatory or anti-inflammatory. Enforced expression of miR-146a decreases the expression of various pro-inflammatory chemokines and cytokines, such as cXc-chemokine ligand 8 (cXcL8) and cc-chemokine ligand 5 (ccL5) by epithelial cells71, IL-6 and cXcL8 by fibroblasts and TNF by osteoarthritic tissue after IL-1 stimulation72,73. miR-146a has also been shown to decrease the expres-sion of TNF, IL-1 and IL-6 by THP1 monocytes dur-ing LPs tolerance74; the expression of IL-2 by activated T cells75; and the production of type I interferons (IFNs) by TLR7-stimulated peripheral blood mononuclear cells and by EpsteinBarr virus- or vesicular stomatitis virus-infected cell lines26,76,77. These effects are probably medi-ated by the targeted repression of IRAK1 and TRAF6 by miR-146a. These data, together with the observation that miR-146a-deficient mice develop an exaggerated pro-inflammatory response when exposed to LPs (mark Boldin and David Baltimore, personal communication), strongly support a role for miR-146a as a negative regu-lator of TLR4 signalling in vivo. Interestingly, miR-146a has also recently been shown to have a crucial role in mediating protective tolerance in intestinal epithelial cells of neonatal mice78. Furthermore, Langerhans cells constitutively express high levels of miR-146a compared with monocytes and interstitial Dcs and therefore have decreased sensitivity to TLR activation as a result of IRAK1 and TRAF6 repression by miR-146a; this renders Langerhans cells tolerant to inappropriate activation by commensal bacteria79.

similarly, enforced expression of other TLR-induced miRNAs, such as miR-9, miR-132 and miR-147, has been shown to alter cytokine production profiles, which indicates that these miRNAs also function to negatively regulate pro-inflammatory responses. overexpression of miR-9 in human primary chondro-cytes decreased IL-1-induced expression of TNF and matrix metalloprotease 13 (REF. 73). miR-132 functions to inhibit the TLR-induced production of pro-inflammatory cytokines61 and KsHv-mediated induction of miR-132 in lymphatic endothelial cells impaired expression of the antiviral genes IFN and IFN-stimulated gene of 15 kDa (IsG15), as well as IL-1 and IL-6 (REF. 45). This indicates that viruses can induce host miRNAs to their advantage by manipulat-ing the expression of host cytokines. overexpression of miR-147 attenuates TLR2-, TLR3- and TLR4-induced production of TNF and IL-6, as well as of miR-147 itself 80. LPs challenge of miR-223-deficient mice results in exaggerated lung inflammation, which indicates that miR-223 might also target proteins to control and limit excess inflammation21.

The role of miR-155 in TLR responses is more com-plex. on the one hand, inhibition or overexpression of miR-155 has shown that miR-155 negatively regulates the expression of cytokines and chemokines such as IL-1 and cXcL8 (REFS 27,28,40,81). on the other hand, miR-155 is required for the expression of TNF, IL-6, IL-23 and type I IFNs22,43,50,60,66,8284, an effect that is probably mediated by the targeted suppression of sup-pressor of cytokine signalling 1 (socs1) and/or sHIP1, two negative regulators of cytokine-mediated and TLR signalling pathways22,82,85. The differences in cytokine regulation by miR-155 require careful consideration. They might simply be due to experimental design (for example, comparing overexpression and gene knockout studies), the cell types used and/or the time of miRNA induction that was analysed. From studies carried out in mouse models and the requirement for miR-155 in priming an adaptive immune response (see below), it seems that miR-155 might promote pro-inflammatory responses rather than being inhibitory. miR-155 might therefore function as a brake or a molecular rheostat that represses the overactivation of a pro-inflammatory response without completely suppressing it.

Another aspect of TLR-induced miRNAs is the feed-forward and regulatory effects they can have on signal-ling pathways downstream of cytokine receptors (FIG. 2b). However, this has been reviewed elsewhere86 and is not discussed in detail here.

Priming the adaptive immune response. Altered cytokine production profiles will obviously affect the ability of the innate immune system to prime adaptive immune responses. However, few studies have directly addressed the effect of miRNAs on this process. Indirect evidence from miRNA-deficient mice indicates that this might be an important feature of the TLR-induced miRNA response, particularly for miR-155; miR-155-deficient mice have global immune defects characterized by decreased Dc function and defective B and T cell responses60,87.

Box 1 | Identification of miRNA targets

The identification of microRNA (miRNA) targets is highly problematic. First, bioinformatic analysis is used to predict target mRNAs through sequence analysis of their 3 untranslated regions (3 UTRs), but many of these mRNAs might not even be expressed under the same conditions as the miRNA of interest. Second, once a correct mRNA target has been identified, the effects of the miRNA can be subtle, at least at the protein level. Twofold changes in mRNA levels are typically observed by microarray analysis after endogenous miRNA knockdown122. Changes in mRNA levels can be even more difficult to observe when you consider the problems of transfection, overexpression and immunodetection. Compounding this, at least for the study of Toll-like receptors (TLRs), can be the strong effects induced by TLR activation, which might be difficult to regulate by a single miRNA.

Many studies use 3 UTR luciferase reporter assays to confirm that a particular mRNA is targeted by a particular miRNA. However, true functional targets of miRNAs need to be confirmed through the use of antisense or overexpression assays both in vitro and in vivo. Antisense technology might be more physiologically relevant, as it inhibits the expression of endogenous miRNAs, compared with overexpression studies, which risk off-target effects. Target protection using morpholino-modified oligonucleotides can be used to confirm the specificity of a particular miRNAmRNA interaction. However, ultimately, it will be the generation of genetically modified mice that are deficient for, or overexpress, candidate miRNAs or that have mutated miRNA target sites in specific genes that will prove most informative.

It must also be pointed out that the concept of a miRNome has emerged123, whereby the concentration of miRNAs in most immune cells, including dendritic cells and macrophages, was analysed. As only a few miRNAs were enriched in cells and a minimum threshold amount must be reached for miRNAs to repress target mRNAs124, the abundance of miRNAs and their ratios in the entire miRNome of a specific cell or tissue might be extremely important for their functions. Therefore, it is possible that the miRNAs that are most abundantly expressed and those that are highly induced in innate immune cells are of importance. Equally, the expression and abundance of mRNA targets in these cells will have a role in determining the outcome and must be considered.

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Class switchingThe somatic recombination process by which immunoglobulin isotypes are switched from IgM to IgG, IgA or IgE.

Experimental autoimmune encephalomyelitis(EAE). An animal model of human multiple sclerosis. EAE develops in susceptible rodents and primates after immunization with antigens derived from the central nervous system.

Germinal centreA lymphoid structure that arises within B cell follicles after immunization with, or exposure to, a T cell- dependent antigen. It is specialized for facilitating the development of high-affinity, long-lived plasma cells and memory B cells.

ExosomesSmall lipid bilayer vesicles that are released from dendritic cells and other cells. They are composed of cell membranes or are derived from the membranes of intracellular vesicles. They might contain peptideMHC complexes and directly interact with antigen-specific lymphocytes, or they might be taken up by other antigen-presenting cells.

Resolvin D1A lipid mediator that is induced in the resolution phase following acute inflammation. Resolvins are synthesized from the essential omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

specifically, the failure of miR-155-deficient mice to acquire protective immunity after Salmonella typh-imurium infection was found to be owing to a failure of Dcs to efficiently activate T cells; however, this observa-tion was not examined further87. The adaptive immune response in these mice was also found to be skewed towards a T helper 2 (TH2)-type immune response, which indicates that miR-155 is normally required for a TH1-type response and for the polarization of T cells towards a pro-inflammatory phenotype87. In a later study88, immunization of miR-155-deficient mice with dinitrophenylated LPs, a T cell-independent antigen, resulted in defective B cell class switching. However, it was not determined whether this effect was the result of a defective innate immune response. Induction of miR-155 expression upon Dc maturation by LPs resulted in the downregulation of expression of Dc-specific inter-cellular adhesion molecule-3-grabbing non-integrin (Dc-sIGN), which limited pathogen uptake by these cells; the authors postulated that this effect could have an important role in balancing T cell polarization89.

Recently, it has been shown that miR-155 is required to promote TH17 cell polarization83. TH17 cells are potent drivers of experimental autoimmune encephalomyelitis (EAE), and mYD88 deficiency has been shown to pro-tect against EAE in mouse models through the decreased induction of IL-17 (REF. 90). Interestingly, deficiency of miR-155 also protected against EAE83. using microarray analysis, miR-155-deficient Dcs were shown to express lower levels of the TH17 cell-inducing cytokines IL-6 and IL-23 than wild-type Dcs; it was postulated that miR-155 has an important role in promoting a pro-inflammatory response mediated by Dcs to prime TH17-type adaptive immune responses.

It is therefore crucial that tissue-specific and/or inducible miRNA-deficient mice are generated to fully understand the role of miR-155 and other TLR-inducible miRNAs in determining the type of immune response. The generation of mice with mutations in the 3 uTR of specific mRNA targets will also help to decipher the specific role of miRNAs. For example, mice with a mutated miR-155 target site in the 3 uTR of the mRNA encoding activation-induced cytidine deaminase (AID), the key antibody class switching enzyme in B cells, have allowed investigators to specify the role of miR-155 in B cell responses. In this case, the induction of miR-155 in B cells following stimulation with LPs and IL-4 is crucial for controlling the germinal centre response and therefore the appropriate stimulation of humoral immune responses91,92.

It is interesting that IL-10, which is an important reg-ulator of both innate and adaptive immune responses, can inhibit LPs-induced expression of miR-155 (REF. 10). IL-10 is known to dampen the innate immune response by downregulating TLR-induced pro-inflammatory gene expression in macrophages and Dcs after patho-gen infection and to inhibit the proliferation of and cytokine production by cD4+ T cells. By contrast, IL-10 seems to have a stimulatory effect on B cells, resulting in enhanced proliferation, differentiation and class-switch recombination93,94. The obvious differences in the effects

of IL-10 on these cells might be partly owing to its inhib-itory effect on miR-155 (REF. 10). An increase in the level of the miR-155 target sHIP1 was shown to contribute to some of the anti-inflammatory effects of IL-10 on macrophages10, whereas an increase in the expression of other miR-155 targets might explain some of the respec-tive regulatory and stimulatory effects of IL-10 on B and T cells, an area which we are actively pursuing.

However, it remains possible that TLR-inducible miRNAs have a more direct effect on the adaptive immune response that is not solely dependent on cytokine expres-sion. For example, miRNAs have been shown to be con-tained in exosomes9598. Exosomes are membrane vesicles released by various cell types, including immune and tumour-derived cells, that are often present in biological fluids99,100. They contain common sets of molecules such as chaperone proteins, cytoskeletal proteins and proteins involved in transport and fusion, as well as cell type- specific molecules that reflect the cell of origin and the target cell. For example, exosomes derived from Dcs can transfer mHc class II molecules to recipient Dcs and neighbouring cells, in addition to priming T cell responses101103. It is therefore possible that exo-somes derived from antigen-presenting cells might contain miRNAs that have an important role in priming the adaptive immune response, an area that is worthy of further investigation.

Together, these observations indicate that miRNAs derived from innate immune cells might be impor-tant regulators of adaptive immune responses through the modulation of cytokine expression and in a cell autonomous manner through exosomes.

miRNAs and resolution of inflammationIt is clear from the above data that the miRNAs induced by TLRs have an important role in regulating the immune response. For example, an initial pro-inflammatory response might be mediated by the early TLR-induced expression of miR-155, which could limit and repress the expression of key negative regulators of TLR signal-ling, thereby promoting cytokine expression and adaptive immune responses. Furthermore, let-7i and miR-125b are downregulated early in the immune response to allow for the production of the pro-inflammatory mediators TLR4 and TNF, respectively. The induction of miR-146 expression later in the response has an important function as a negative regulator of innate immune responses, feed-ing back to turn off TLR signalling pathways. Following this, miR-21 is induced to promote the anti-inflammatory response and facilitate the production of IL-10. IL-10 can then feed back to inhibit the expression of miR-155, thereby further contributing to the anti-inflammatory effect. It is thought that both miR-9 and miR-147 contrib-ute to the negative regulation of innate immune responses at later times by targeting NF-B and affecting cytokine production. These data and the more recent finding that miRNAs, including miR-21 and miR-146b, are induced by the anti-inflammatory lipid mediator resolvin D1 in human macrophages104 imply that miRNAs are key players in the resolution of inflammation by mediating the removal of components of the TLR signalling pathways (FIG. 4).

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miRNAs in diseaseAlthough miRNAs are required for the induction and the resolution of innate immune responses, it is clear that if they themselves are not controlled, they can contribute to inflammatory disorders and the progression of cancer. most evidence for this has come from profiling studies that investigate whether aberrant expression of particu-lar miRNAs correlates with particular disease states, such as ulcerative colitis105. The aberrant expression of many miRNAs has been found to overlap between inflamma-tion and cancer, in support of the age-old link that has been thought to exist between these diseases. As there are several recent reviews on this topic106114, we only discuss a few key points.

multiple miRNA profiling studies have been carried out in patients with rheumatoid and osteoarthriits, pso-riasis and atopic eczema, for which increased expres-sion of the TLR-induced miRNAs miR-21, miR-132, miR-146a and miR-155 is commonly detected73,81,115119. By contrast, miRNA profiling of samples from patients with systemic lupus erythematosus has detected a lack of miR-146a120. It is possible that differences in cytokine expression and NF-B activity might contribute to the varying levels of miR-146 expression found in inflam-matory diseases, owing to the inhibition of IRAK1 and TRAF6 by miR-146a.

It is possible that the constant low-grade inflam-mation associated with inflammatory diseases leads to dysregulated miRNA expression, which in turn con-tributes to disease pathogenesis. In the case of the pro-inflammatory miRNA miR-155, it is easy to see how increased expression of this miRNA could result in the inappropriate activation of inflammatory pathways. However, for the other miRNAs that are upregulated in these diseases (miR-21, miR-132 and miR-146a), which have strong anti-inflammatory effects, it is more difficult to appreciate how their upregulation might contribute to the excessive inflammation character-ized by these disorders. Again, the context-specific mRNA expression profiles in diseased cells must be considered. It might be that different miRNA tar-gets are expressed in these cells or that the respective miRNA targets are no longer expressed. Alternatively, the anti-inflammatory effects of these miRNAs might result in disease because the cells are unable to mount appropriate immune responses. This could be a pos-sible explanation for the development of cancer, as the TLR-inducible miRNAs have also been found to be upregulated in several types of cancer106. Again, there is mounting evidence in support of TLR activation in cancer107 and the dysregulation of miRNA expression is likely to have a role in this pathogenic process.

Finally, another interesting aspect of the role of miRNAs in disease is how mutation or deletion of the 3 uTR of mRNAs that are normally targeted by miRNAs could contribute to disease. For example, a polymorphism in the 3 uTR of the mRNA encod-ing IRAK1 (a target of miR-146a) is associated with susceptibility to rheumatoid arthritis121. similarly, loss of miRNA expression has been shown to con-tribute to disease. For example, loss of miR-145 and miR-146a transcripts within the 5q locus identified these miRNAs as key mediators of 5q syndrome, a haematopoietic malignancy that progresses to acute myeloid leukaemia30. Loss of non-coding transcripts in the 5q locus identified that the genes encoding both miR-145 and miR-146a were absent in this dis-ease. As a result, the upregulation of mRNA targets for these miRNAs such as the mRNAs encoding mAL, IRAK1 and TRAF6 could be contributing to the disease. This finding emphasizes how the dysregu-lation in haematopoeitic cells of miRNAs that target key innate immune processes can cause leukaemia, emphasizing further the link between innate immunity and cancer.

Figure 4 | The role of miRnAs in the resolution of inflammation. Toll-like receptor (TLR) activation seems to result in the sequential induction of important microRNAs (miRNAs) that can control the strength and longevity of an inflammatory response. TLR signalling strongly induces miR-155, which has been proposed to enhance the pro-inflammatory response through the inhibition of negative regulators, such as Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1) and suppressor of cytokine signalling 1 (SOCS1), the promotion of cytokine expression and the subsequent induction of an adaptive immune response. The downregulation of let-7i and miR-125b miRNAs (indicated by dashed lines) could contribute to this effect owing to a lack of repression of their respective targets TLR4 and tumour necrosis factor (TNF). Later, the induction of miR-146a and miR-9 resolves the pro-inflammatory response by targeting TNFR-associated factor 6 (TRAF6) and IL-1R-associated kinase 1 (IRAK1), which are key components of TLR signalling pathways, and the nuclear factor-B (NF-B) subunit p50, respectively. miR-132 expression has also been shown to be increased by TLR signalling and would limit the antiviral response by targeting p300. At later times, miR-21 is induced and increases IL-10 expression by repressing programmed cell death 4 (PDCD4), which might contribute to the induction of an anti-inflammatory response through the inhibition of miR-155. miR-147 also promotes an anti-inflammatory response by repressing cytokine production.

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Together, these studies are of huge therapeutic rel-evance as they not only provide useful diagnostic infor-mation but also help to define which miRNAs might be most relevant for therapeutic intervention in particular disease states.

Future directionsThe study of miRNAs has opened up many new areas in the regulation of TLR signalling. The targeting of key proteins in the TLR signalling pathways highlights the important role of miRNAs and also illustrates new nega-tive feedback loops that can control the outcome of TLR responses through quantitatively regulating cytokine production but also, qualitatively, by switching from pro-inflammatory to anti-inflammatory responses and by the induction of adaptive immune responses in a timely and orchestrated manner. Although many data have been obtained on the induction of miRNAs by TLRs, true

functional data showing the exact effects of miRNAs on TLR responses are still required. These studies have also revealed novel targets for TLR signalling and have illustrated new processes that are activated downstream of TLRs. Importantly, they have highlighted the impor-tance of post-transcriptional control of the inflammma-tory response. The finding that several miRNAs work together with RNA-binding proteins and regulate the translation of TLR-responsive mRNAs in different ways opens up an exciting new avenue in TLR research. These extra levels of previously unappreciated gene regulation provide new targets for the therapeutic manipulation of these key innate immune signalling pathways. Given the modest fine-tuning and cell type-specific effects of TLR-responsive miRNAs, these might provide attractive drug targets. If this proves to be the case, then one must hope that the application of such RNA-based therapies does not get lost in translation.

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micrornas: the fine-tuners of toll-like receptor signalling

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