MicroRNAs in the regulation of TLR and RIG-I pathways

Abstract

The innate immune system recognizes invading pathogens through germline-encoded pattern recognition receptors (PRRs), which elicit innate antimicrobial and inflammatory responses and initiate adaptive immunity to control or eliminate infection. Toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I) are the key innate immune PRRs and are tightly regulated by elaborate mechanisms to ensure a beneficial outcome in response to foreign invaders. Although much of the focus in the literature has been on the study of protein regulators of inflammation, microRNAs (miRNAs) have emerged as important controllers of certain features of the inflammatory process. Several miRNAs are induced by TLR and RIG-I activation in myeloid cells and act as feedback regulators of TLR and RIG-I signaling. In this review, we comprehensively discuss the recent understanding of how miRNA networks respond to TLR and RIG-I signaling and their role in the initiation and termination of inflammatory responses. Increasing evidence also indicates that both virus-encoded miRNAs and cellular miRNAs have important functions in viral replication and host anti-viral immunity.

Introduction

Microbial pathogen recognition is an essential step in the initiation of innate immune responses. However, the interaction between microbial pathogens and the host has been poorly understood for a long time. Recently, the discovery of certain pathogen recognition receptors (PRRs) and pathogen-associated molecular patterns has provided evidence on the nature of this interaction.^1,2 PRRs are microbial sensors of the host innate immune system that recognize relatively invariant molecular patterns broadly shared by most types of microorganisms. These recognized microorganismal structures are referred to as pathogen-associated molecular patterns. Several families of PRRs have been characterized over the past decades, including Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors and Nod-like receptors. TLRs represent the most studied PRRs, in terms of the known ligands, the downstream signal pathways and the effector molecules, and play critical roles in the host's defense against invading microbial pathogens.^1,3 After pathogen-associated molecular pattern recognition, TLRs initiate innate immune responses by activating signaling pathways that depend on the adaptor MyD88 or the adaptor TRIF and consequently induce pro-inflammatory cytokine and type I interferon (IFN) production.^4,5 Moreover, any dysregulation of this process has been associated with inflammatory diseases, autoimmune diseases or pathogen dissemination. Many molecules have been identified as positive or negative regulators of TLR signaling,^6,7 including phosphatases (SHP-1, SHP-2, SHIP-1, PTP1B,^8,9,10,11 etc.), protein kinases (calmodulin-dependent protein kinase II, Btk, MEKK3,^12,13,14 etc.), ubiquitin-related proteins (A20, Nrdp1, CHIP,^15,16,17 etc.), Nod-like family proteins (NLRX1, NLRC5,^18,19 etc.), membrane molecules (CD300F, CD11b, PECAM-1,^20,21 etc.), endosome/lysosome-localized molecules (Rab7b,^22,23 CLM-3²⁴), gene transcription coactivators (beta-catenin²⁵), antigen-presenting molecules (MHC I and MHC II^26,27), and even HSP70,²⁸ HSP70L1²⁹ and NGF.³⁰ Recently, RIG-I signal pathway regulation was extensively investigated;^31,32,33,34 however, the full anti-inflammatory response mechanisms and the precise fine-tuning of this process still remain incompletely elucidated.

MicroRNAs (miRNAs) have recently emerged as important regulators of gene expression essential for organ development, cell differentiation and tumor progression.^35,36 They are endogenous, single-stranded RNAs of approximately 22 nucleotides in length and are highly conserved in eukaryotes. MicroRNAs are encoded in genomic clusters and produced by an elaborate expression and processing mechanism. MiRNA biogenesis involves two processing steps by two RNaseIII enzymes, Drosha and Dicer. After the primary transcript is generated by RNA polymerase II or III, the primary miRNA is processed by Drosha in the nucleus to produce a short hairpin precursor miRNA transcript, which is shuttled into the cytoplasm.^37,38 The final mature miRNA requires further cytoplasmic processing by Dicer, producing the 22-base-pair dsRNA.³⁹ The mature miRNA duplex is then incorporated into the RNA-induced silencing complex in the cytoplasm, which uses the ‘seed sequence' to bind partially complementary sequences in the 3′-untranslated region (UTR) of target mRNA transcripts, resulting in mRNA degradation or translational repression.^40,41,42,43 Presently, more than 1000 miRNAs have been identified in the human genome, and as many as 60% of all mRNAs are predicted to be regulated by miRNAs.^44,45 In the past few years, accumulating evidence has shown that miRNAs affect mammalian immune cell differentiation, the outcome of immune responses to infection and the development of immunological diseases.^{46,47,48,49,50,51} Therefore, it is not surprising that miRNAs are involved in TLR and RIG-I signaling in the innate immune system.

In this review, we will discuss miRNAs induced by TLR and RIG-I signaling and speculate on the role of miRNAs in innate immunity, specifically focusing on TLR and RIG-I signaling pathway regulation. miRNAs are thought to regulate TLR signaling at different levels by targeting multiple molecules involved in the TLR pathway, such as the expression of TLRs themselves, TLR signal molecules, TLR-induced transcription factors, regulators of the TLR signaling pathway and even the final functional cytokines of TLR signaling. We have also provided an overview of miRNAs in RIG-I-mediated antiviral activities, which is an important aspect of miRNA function in innate immunity. In addition to virus-encoded miRNAs, viral infection has also been found to upregulate or downregulate miRNA expression in host cells. Thus, miRNA studies found novel targets for TLR and RIG-I signaling at the post-transcriptional level that function to manipulate the inflammatory response.

Regulation of miRNA expression by TLR and RIG-I signaling

Many studies have indicated that TLR signals can modulate miRNA expression by various techniques, such as microarray and deep sequencing. Although subtle changes have been found in most miRNA expression after TLR stimulation, a subset of strong miRNA targets of TLR signaling has emerged, especially miR-146, miR-155 and miR-21. Similar to other TLR-induced genes, this miRNA subset can further be classified as early- or late-response miRNAs, according to the response speed following TLR ligand stimulation.⁴⁹ Specifically, miR-146 and miR-155 are highly induced within 2 h after TLR treatment, and thus belong to the early-response miRNAs; however, miR-21 belongs to the late-response miRNAs.^52,53,54 There are also subtle differences in miRNA expression profiles depending on the TLR ligands used, stimulation time and specific cell types.

The first TLR-induced miRNA expression profiling study was performed in human monocytes in David Baltimore's lab in 2006.⁵³ In their study, miR-146a, miR-155 and miR-132 were upregulated after lipopolysaccharide (LPS) stimulation. miR-146 upregulation has also been confirmed by other independent studies.^55,56 miR-146a induction is controlled by nuclear factor kappaB (NF-κB), which is the most common transcription factor that triggers TLR-induced miRNA transcription. In contrast, miR-155 expression was observed to depend on MyD88- or TRIF-induced JNK activation after TLR stimulation.⁵⁷ Additionally, miR-132 was demonstrated to be controlled by cyclic AMP response element-binding protein and transcriptional coactivator p300.^58,59 Moreover, miR-21 is induced in an NF-κB-dependent manner at later times in macrophages after LPS treatment.⁵²

Other miRNAs are also induced after TLR treatment or pathogen infection, including miR-223, miR-147, miR-9, miR-27b and let-7e.^60,61,62,63 While the upregulation of these miRNAs by TLR activation has been identified to be mostly dependent on de novo primary miRNA transcription by certain transcription factors, we cannot exclude the possibility that the accelerated processing of primary miRNA/precursor miRNA or the retarded degradation of mature miRNA also results in increased miRNA concentration. For the most part, primary miRNA transcription is dependent on the NF-κB transcription factor.^64,65 Additionally, TLR signals can negatively regulate miRNAs, including miR125b, let-7i and miR-98,^66,67,68 in certain cell types. Little is known about how TLR signals decrease miRNA expression, which most likely occurs through transcriptional repression or post-transcriptional destabilization of miRNA.⁴⁹

The strong changes in miRNA expression during TLR stimulation indicate a functional biological or pathological role of these miRNAs. For example, miR-146 is increased only following surface TLR4 stimulation. The lack of responsiveness to TLR3, TLR7 or TLR9 treatment suggests that miR-146a plays a key role in the host innate responses to predominant bacterial pathogens, but not to viral infection.⁵³ In the following section, we will provide insights into how these TLR-induced miRNAs control TLR signaling.

In addition to playing precise roles in regulating TLR signaling, miRNAs are also important in viral infection and host anti-viral immunity. Some DNA and RNA viruses express miRNAs, and these miRNAs may target viral or cellular gene expression. miRNA expression has been reported for various groups of viruses. By far, DNA viruses encode the highest number of miRNAs, especially the herpesvirus family, such as HSV, HCMV, EBV and KSHV.⁶⁹ Some small DNA viruses (adenovirus and polyomavirus families) and retroviruses have also been demonstrated to express miRNAs. Additionally, virus infection can induce the expression of certain miRNAs. For example, vesicular stomatitis virus (VSV) infection upregulates miR-146a expression in mouse macrophages in a TLR/MyD88-independent and RIG-I/NF-κB-dependent manner.⁷⁰ VSV infection has been recently shown to induce miR-155 expression in macrophages, which promotes type I IFN signaling and has an antiviral effect by targeting SOCS1.⁷¹

MiRNA-mediated regulation of TLR signaling pathway

Targeting TLR expression

To trigger an anti-bacterial immunity, the innate immune response needs to initially recognize the infecting pathogens by their PRRs. Thus, the level of receptor expression is the first, and most likely the most effective, point at which to manipulate the miRNA-mediated TLR signaling pathway. However, bioinformatic analysis of the human 3′-UTR TLR region revealed that TLR genes are targeted by miRNAs mainly through weaker, non-conserved sites.^49,72 There are very few highly conserved target sites in the 3′-UTR of TLR genes.⁴⁹ This less effective regulation by miRNAs may lead to the constitutive expression of certain TLRs in certain cell types, and provide the potential to respond to TLR activators.

The mRNA encoding TLR4 is regulated by let-7i and let-7e in different cell types. In mouse macrophages, let-7e is one of the TLR signaling-induced miRNAs.⁶⁸ After LPS stimulation, increased let-7e downregulates the surface TLR4 expression to avoid excessive inflammation and tissue damage. Suppression of let-7e action with an antisense miRNA probe leads to increased TLR4 expression and cytokine production after LPS challenge.⁶⁸ However, in epithelial cells, TLR4 mRNA is regulated by let-7i, which is decreased after TLR stimulation. Downregulation of let-7i increases the surface TLR4 level and the inflammatory response in these epithelial cells,⁷³ which may be due to the need of epithelial cells to enhance inflammation during infection, as they lack the professional capacity to recognize pathogens and express low amounts of TLR4 in a quiescent state. Recently, TLR4 was also demonstrated to be targeted by miR-146a, another TLR-induced miRNA in macrophages.⁷⁴

Other TLRs are also targets of certain miRNAs. A reciprocal relationship between TLR2 signaling and miR-105 was found in primary human oral keratinocytes, and TLR2 mRNA was shown to be regulated by miR-105.⁷⁵ However, in fibroblast-like synoviocytes from rheumatoid arthritis patients, miR-19a/b was decreased in response to TLR2 ligands and was responsible for the upregulated TLR2 expression in these cells.⁷⁶ miR-19a/b mimics reduced TLR2 level and thus inhibited TLR2-triggered cytokine production. Although these data suggest that miRNAs regulate TLR expression, it is more effective, economical and common in humans cells to regulate downstream TLR signal molecules by miRNAs than by completely shutting down the TLR signal pathway by eliminating receptor expression.

Targeting TLR signaling proteins

TLRs mainly signal through the MyD88-dependent pathway, which leads to pro-inflammatory cytokine production, and the TRIF-dependent pathway, which is responsible for the antiviral TLR effect that will be discussed later. Many TLR signal pathway molecules and adaptor proteins are identified as strong miRNA targets, especially the TLR-induced miRNAs.

Accumulating evidence demonstrates that miR-155, a central TLR-induced miRNA, can negatively regulate TLR signaling pathway by targeting different key signal molecules. MyD88 has been identified as a miR-155 target in both mouse and human cells.^77,78 miR-155 overexpression leads to significantly suppressed IL-8 production during Helicobacter pylori infection. In human monocyte-derived DCs, miR-155 targets TAK1-binding protein 2,⁷⁹ a signal molecule downstream of TNFR-associated factor 6 that activates MAPK kinases. Additionally, miR-155 inhibition leads to elevated p38 pathway activation.

microRNA-146, one of the central TLR-induced miRNAs, is also a strong negative regulator of the TLR signaling pathway. IL-1R-associated kinase 1 (IRAK1) and TNFR-associated factor 6 are important components of the MyD88-dependent pathway for NF-κB activation downstream of several TLRs and are also direct targets of miR-146.⁵³ Moreover, miR-146 is induced by TLR signaling through NF-κB activation and involved in a feedback mechanism that suppresses TLR-triggered NF-κB activation, which suggests a role for miR-146 in TLR signal termination. Recently, IRAK2, a kinase that compensates for IRAK1 to activate NF-κB, has also been confirmed as a miR-146 target.⁷⁰

Other key TLR signaling pathway components have also been demonstrated as miRNA targets. MyD88 adaptor-like protein, which functions as a bridge for the TLR2 and TLR4-mediated MyD88-dependent pathway, has emerged as a miR-145 target,⁸⁰ although the miR-145 expression profile following TLR agonist stimulation remains undetermined. Bruton's tyrosine kinase is a critical tyrosine kinase in TLR4, TLR7 and TLR9 signaling for activating NF-κB.⁸¹ miR-346 is strongly induced by LPS treatment and targets Btk mRNA in the synovial fibroblasts of rheumatoid arthritis patients.⁸² Whether miR-346 targets Btk in macrophages needs to be further studied. Finally, IKKα, one of the IκB kinases in the canonical NF-κB pathway, has also been confirmed as a miR-223 targets.⁵⁰

While many molecules are involved in the TLR signaling pathway, few have been identified as miRNA targets. However, these miRNA targets are key components of several TLR pathways. Furthermore, an individual TLR-induced miRNA may target several TLR signaling pathway molecules, and there are many TLR-induced miRNAs. Thus, these miRNAs work together to appropriately and effectively control or terminate the TLR signaling pathway.

Targeting TLR-induced transcriptional factors

Activation of certain transcriptional factors is a key functional step in TLR signaling. Previous studies have suggested an interplay between transcription factors and miRNAs in genome-wide regulatory networks.⁸³ Targeting of transcription factors by miRNAs could have a global impact on TLR-induced gene expression. NF-κB is the most important transcription factor in the TLR signaling pathway. MiR-9, the TLR-responsive miRNA, is shown to directly target Nfkb1 mRNA.⁶² NFKB1 is the NF-κB p50 subunit precursor, which acts to transactivate the NF-κB p65 subunit. Thus, TLR agonists induce miR-9 in a MyD88/NF-κB-dependent pathway, while miR-9-mediated feedback controls NF-κB responses by fine-tuning the expression of a key NF-κB family member.⁶² More recently, miR-210 has also been demonstrated to be induced by LPS and target Nfkb1 mRNA.⁸⁴

Other transcription factors downstream of the TLR signaling pathway have been identified as miRNA targets. miR-17-5p and miR-20a are expressed at low levels in myeloid-derived suppresser cells and alleviate the suppressive function of myeloid-derived suppresser cells by targeting the transcription factor STAT3.⁸⁵ Peroxisome proliferator-activated receptor γ (PPARγ) expression is reduced after LPS stimulation due to miR-27b induction, which directly targets PPARγ mRNA.⁶³ Inhibition of miR-27b increased PPARγ expression and thus resulted in the reduced production of pro-inflammatory cytokines from LPS-stimulated macrophages. The transcriptional coactivator p300, which often associates with CREB, is targeted by miR-132 in endothelial cells.⁵⁹ Additionally, the transcriptional corepressor C/EBPβ is targeted by miR-155 as well.⁸⁶ Thus, miRNAs can directly target several transcription factors and control the transcription of TLR-induced genes.

Targeting TLR regulatory molecules

In addition to directly regulating TLR signal molecules, recent data have indicated that miRNAs can target new molecules that have important roles in TLR signaling regulation, thus ‘regulating the regulators'. While miRNA-21 has long been recognized to have many targets and play important roles in various types of cancer, its anti-inflammatory role in innate immunity has only been identified recently.⁵² After induction by TLR signals, miR-21 is shown to target PDCD4, which negatively regulates TLR signaling by inhibiting eukaryotic translation initiation factor 4F,⁸⁷ which eventually attenuates inflammatory cytokine production. Furthermore, IL-10 production is found to be dependent on miR-21 and its target PDCD4 in the later phase of TLR stimulation.⁵² MiRNA-21 can promote IL-10 secretion by decreasing PDCD4 expression, suggesting another anti-inflammatory miR-21 role in TLR signaling. SHIP1 has been well characterized as a negative regulator of TLR induced signals, and identified as a primary target of miR-155.^10,88,89,90 miR-155 inhibition by IL-10 leads to increased SHIP1 expression and results in the termination of TLR signals.⁹¹ Thus, we can propose that in the early phase of TLR signaling, miR-155 is upregulated and inhibits the negative regulator SHIP1, allowing TLR signal transduction and function; in the later phase, the increased miR-21 induces IL-10 production through PDCD4 inhibition. IL-10 then suppresses miR-155 expression, leading to increased SHIP1 expression and termination of TLR signaling.⁴⁹ Additionally, studies focusing on miR-146a-mediated repression of IL-12p70 production in TLR9-triggered DCs identified Notch1 as a target of miR-146a.⁹² However, there may be other proteins and miRNAs involved in this process, which requires further investigation.

In addition to two key TLR-induced miRNAs, miR-21 and miR-155, many other miRNAs can also target proteins involved in regulating TLR signaling. For example, miR-148 and miR-152 impair TLR signaling by targeting calcium/calmodulin-dependent protein kinase II.^12,93 MicroRNA-132 was shown to target acetylcholinesterase, which attenuates TLR-induced responses in macrophages by blocking NF-κB nuclear translocation.⁹⁴ Inhibition of miR-181 can also inhibit TNF-induced cytokine production in epithelial cells by targeting p300/cyclic AMP response element binding protein-associated factor, a coactivator and an acetyltransferase that promotes histone acetylation and gene transcription.⁹⁵ Thus, miRNAs regulate TLR signaling pathway regulators in combination with regulating the signal proteins themselves to fine-tune TLR signaling and downstream events.

Targeting TLR-induced functional cytokines

The major consequence of TLR signaling is to produce inflammatory cytokines and chemokines, which are important in pathogen clearance and inflammatory cell recruitment to the infection site. TNF, IL-6, IL-12 and IL-10 are the key cytokines produced from myeloid cells after TLR agonist stimulation. Tnf mRNA can be directly targeted by miR-125b.⁶⁷ Moreover, miR-125b is one of the miRNAs decreased after TLR ligand treatment, thus ensuring that TNF mRNA is stabilized. Recently, miR-365 and miR-142-3p were demonstrated to target Il6 mRNA, reducing the endotoxin-induced mortality.^96,97 TLR signaling can induce miR-365 through the Erk pathway, which restricts TLR signals by a feedback mechanism. Bio-informatic analysis also has revealed a let-7-binding site in the 3′-UTR of Il6 mRNA.⁹⁸ Considering that many let-7 family members are downregulated after TLR stimulation, let-7 may contribute to the increased IL-6 production. Il-12p35 mRNA can be targeted by miR-21 in macrophages and DCs, leading to restricted adaptive Th1 responses.^99,100,101 miR-106 has been demonstrated to target Il-10 mRNA,¹⁰² while miR-29 suppresses immune responses against intracellular pathogens by targeting IFN-γ.¹⁰³

Numerous cytokine encoding mRNAs are regulated by RNA-binding proteins (RBPs) through the AU-rich elements in their 3′-UTR, which may play a vital role in mRNA stabilization. MiRNAs can also cooperate with these RBPs to regulate mRNA stability or translation.¹⁰⁴ For example, miR-16 cooperates with tristetraprolin to mediate Tnf mRNA destabilization,¹⁰⁵ miR-221 associates with tristetraprolin and can accelerate Tnf mRNA decay, and miR-579 and miR-125b inhibit Tnf mRNA translation through translational inhibitor recruitment.¹⁰⁶

In addition to cooperating with RBPs to destabilize mRNA, miRNAs can also interact with RBPs to protect mRNAs from degradation. For example, the seed sequence of miR-466l is complementary to the AU-rich element sequence ‘AUUUA' of Il-10 mRNA and, thus, can compete with tristetraprolin for the AU-rich element sequence binding, resulting in elevated IL-10 production from macrophages.¹⁰⁷ Thus, it is interesting to identify miRNAs involved in the direct mRNA regulation of TLR-induced cytokine expression.

MiRNA-mediated regulation of RIG-I signaling pathway and viral infection

The innate immune system initially recognizes RNA virus infection and evokes antiviral responses by producing type I IFNs. TLRs and cytoplasmic RIG-I-like helicases are the two major receptor systems employed by the host for detecting RNA viruses.^108,109 RIG-I-like helicase signaling pathways play essential roles in the recognition of RNA viruses in various cells.¹¹⁰ Although many proteins are known to modify the RIG-I pathway and the subsequent IFN signal transduction,^31,32,33,34 the role of miRNA-mediated regulation is only beginning to emerge.

Some miRNAs can be induced by RIG-I signaling and employ feedback mechanisms to regulate viral replication by modulating RIG-I pathway and type I IFN expression/function. VSV Infection in macrophages induces miR-146a expression in a TLR/MyD88-independent/RIG-I-dependent manner, and miR-146a sequentially suppresses VSV-triggered type I IFN production by targeting IRAK1, IRAK2 and TNFR-associated factor 6, thus promoting VSV replication.⁷⁰ Recently, we have demonstrated that miR-466l can directly bind to the 3′-UTR of IFN-α and reduce its expression during VSV infection.¹¹¹ There is mounting evidence that type I IFN can manipulate miRNA expression. A recent study that used an in-depth miRNome analysis of resting and IFN-α-activated human natural killer cells revealed that IFN-α activation suppressed two abundantly expressed miRNAs, miR-378 and miR-30e, which allowed the translation of cytolytic mRNAs, resulting in augmented natural killer cell cytotoxicity.¹¹² Furthermore, miRNAs can also regulate antiviral immunity by modulating IFN downstream signals. For example, in the absence of a miR-29a cluster in the thymic epithelium, IFN-α receptor expression by the thymic epithelium was higher, thus allowing suboptimal signals to trigger a rapid loss of thymic cellularity.¹¹³

In addition to targeting RIG-I and IFN signaling pathways, miRNAs can interfere with viral replication or modify the host antiviral immunity in many other manners. Viral miRNAs may target viral or cellular proteins, and function in the control of lytic and viral replication, in the limitation of antiviral responses, in the inhibition of apoptosis and in the stimulation of cellular growth.⁶⁹ For instance, miR-UL112 is encoded by HCMV and targets transcriptional factor IE1, which controls the expression of many viral genes and is vital for viral replication at the onset of infection.^114,115,116 HCMV miR-US4-1 specifically downregulates ERAP1, which participates in trimming MHC class I-presented peptide precursors to mature epitopes.¹¹⁷ Accordingly, the trimming of HCMV-derived peptides is inhibited, leading to decreased susceptibility of infected cells to HCMV-specific cytotoxic T lymphocytes, thus helping identify a previously unknown viral miRNA-based cytotoxic T lymphocyte-evasion mechanism. MiRNAs encoded by simian virus 40, accumulate at late times and perfectly target viral T antigen mRNAs without reducing the infectious viral yield.¹¹⁸ Thus, viruses lacking the expression of these viral antigens display decreased sensitivity to cytotoxic T cell lysis and decreased cytokine production. Viral BamHI A rightward transcripts miRNA, encoded by EBV, targets the EBV latency associated membrane protein 1, a transforming protein, which resulted in decreased apoptosis of infected cells.¹¹⁹ Thus, viral evolution has taken advantage of the miRNA pathway to generate effectors that enhance the probability of successful infection.

Virus-encoded miRNAs can also target cellular gene expression. miR-K12-11 is the most characterized miRNA and targets cellular proteins, derived from KSHV.¹²⁰ It has 100% seed sequence homology with the human miR-155, which is essential for B-cell development, while its overexpression leads to B-cell lymphoma.^121,122 Recently, miR-K12-11, the oncogenic miR-155 equivalent, has been shown to contribute to malignant viral transformation by downregulating tumor suppressor and anti-angiogenic factor thrombospondin 1¹¹² and a transcriptional repressor BACH-1.^120,123,124

Cellular miRNAs are highly induced or regulated by viral infection, and can affect both the viral replication and host anti-viral immunity. Repression of Dicer increases the replication of HIV-1, VSV and influenza A virus,^125,126,127 suggesting that cellular miRNAs play a role in the restriction of viral biology. Individual miRNAs that target viral genes responsible for this restriction have been identified. The HIV mRNA is targeted by a cluster of human miRNAs, including miR-28, miR-125b, miR-150, miR-233 and miR-382.¹²⁸ Hypersusceptibility to VSV infection in Dicer1-deficient mice is due to impaired miR-24 and miR-93 expression, which target VSV L and P proteins.¹²⁵ Furthermore, human miR-32 has been shown to repress replication-essential viral proteins and restrict the retrovirus primate foamy virus type 1 replication.¹²⁹ However, miR-122, which is specifically expressed and highly abundant in human liver, targets 5′-UTR of HCV RNA. In addition, rather than repressing replication, miR-122 appears to augment intracellular HCV production.¹³⁰

Perspective

miRNAs are known to target key proteins in the TLR and RIG-I signaling pathways, and like their regulatory protein counterparts, are also involved in the extensive network of inflammatory responses. For example, early after TLR stimulation, miR-155 is induced and promotes pro-inflammatory responses and subsequent adaptive immunity by repressing key negative regulators of TLR signaling. Meanwhile, miR-125b and let-7i are downregulated to allow the production of pro-inflammatory mediators TLR4 and TNF. Subsequently, miR-146 and miR-21 are induced as negative regulators in a feedback mechanism to turn off the TLR signaling pathway, facilitate the anti-inflammatory cytokine IL-10 production, and finally terminate the TLR responses. The regulatory mechanisms of the RIG-I signaling pathway are similar to the ones described above, although the regulation has not yet been clearly defined.⁷⁰ These data indicate that the immune system utilizes multiple miRNAs to properly regulate its functional capacity, creating a finely tuned balance between activation and repression. Nonetheless, the precise regulatory roles of miRNAs in TLR and RIG-I responses are still not fully understood, especially how these miRNA networks collaborate together to optimize immune responses. A better understanding of miRNA-mediated TLR and RIG-I signaling regulation would also reveal novel drug targets against inflammatory diseases.

The authors declare no competing financial interests.