The Interaction Between Two Worlds: MicroRNAs and Toll-Like Receptors

MicroRNAs (miRNAs) are critical mediators of posttranscriptional regulation via their targeting of the imperfect antisense complementary regions of coding and non-coding transcripts. Recently, researchers have shown that miRNAs play roles in many aspects of regulation of immune cell function by targeting of inflammation-associated genes, including Toll-like receptors (TLRs). Besides this indirect regulatory role of miRNAs, they can also act as physiological ligands of specific TLRs and initiate the signaling cascade of immune response. In this review, we summarize the potential roles of miRNAs in regulation of TLR gene expression and TLR signaling, with a focus on the ability of miRNAs bind to TLRs.

INTRODUCTION An efficient immune system is required for all multicellular organisms to detect and respond to pathogenic microorganisms or cells and/or tissue damage (1,2). After discovery of the Toll-like receptor (TLR) family of pattern recognition receptors in the late 1990s, investigators showed that they recognize specific and distinct conserved endogenous and exogenous molecular patterns (3,4). TLRs are crucial in recognition of microbial products, release of inflammatory mediators through the induction of transcription factors in immune response and inflammation and control of adaptive immune responses (5,6). Emerging evidence has demonstrated that non-coding RNAs such as microRNAs (miRNAs) are involved in almost all known cellular processes, including innate, and adaptive immune responses, via modulation of gene expression (7)(8)(9)(10)(11). MiRNAs are secreted by several cell types, including tumor cells and macrophages within extracellular vesicles, such as exosomes and microvesicles; act as cell-to-cell communication vectors; and are taken up by recipient cells (12)(13)(14)(15)(16)(17). Moreover, several miRNAs can bind to TLRs and initiate immune response by inducing immune and inflammatory gene expression. This review focuses on the inflammation-related miRNAs in the let-7 family, miR-21, miR-146b, and miR-155 and their involvement in TLR signaling pathways via regulation of TLRs and/or TLR signaling expression and binding to TLRs.

TLRS AND TLR SIGNALING
TLRs are evolutionarily conserved molecules that initiate the signaling cascade of immune response against a wide variety of pathogens (18). Moreover, TLRs are type I integral membrane proteins consisting of 10-30 leucine-rich repeats in the N-terminal portion of TLRs that participate in ligand recognition and a cytoplasmic domain of the Toll/interleukin (IL)-1 receptor (TIR) in the C-terminal portion of TLRs that is responsible for activation of downstream signaling. Both pathogen-associated and damage-associated molecular patterns can be recognized by different TLRs and subsequently trigger signaling transduction pathways through adaptor molecules (19,20). Damage-associated molecular patterns are endogenous molecules released from stressed or dying cells. Depending on the type of cells and tissues damaged, they can be classified as protein damage-associated molecular patterns, such as heat shock proteins, high-mobility group box 1 protein, or nonprotein damage-associated molecular patterns, such RNA and DNA (3,21,22). Pathogen associated-molecular patterns are exogenous molecules derived from pathogens such as bacteria, fungi, parasites, and viruses and can be recognized by TLRs, leading to activation of the TLR signaling cascade, which regulates the expression of inflammation-related genes such as IL-1 receptor-associated kinase (IRAK1), tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), and type I interferon (IFN) (3,21,22). Various TLRs are primarily or selectively expressed in specific cell types, including immune cells such as lymphocytes, dendritic cells (DCs), macrophages, and neutrophils and non-immune cells such as epithelial cells and fibroblasts (23)(24)(25)(26). Recent studies identified that TLRs are also expressed in tumor cells and their microenvironments that composed cancer-associated fibroblasts, tumor-associated macrophages, marrow-derived suppressive cells, and regulatory T cells, adipocytes, and immune cells (23,27). In mammals, the TLR protein family currently comprises 13 members (humans, TLR1-10; mice, TLR1-9, and TLR11-13), with the TLRs in humans and mice having some functional differences (28)(29)(30)(31). Based on the subcellular localization of TLRs, they can be broadly divided into two subgroups . Those in the first group, including  TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10, are expressed on  the surface of cells and recognize microbially derived ligands.  TLRs in the second group, including TLR3, TLR7, TLR8, and  TLR9, are expressed intracellularly in vesicles such as endosomes and lysosomes and recognize microbial nucleic acids (32,33). In contrast, TLR3 can be localized both on cell surfaces and in intracellular vesicles (34).

THE EFFECTS OF MIRNAS ON TLR EXPRESSION AND SIGNALING
A growing number of reports have stated that specific epigenetic processes such as histone modifications, DNA methylation, and non-coding RNAs may regulate the transcriptional responses of TLRs (41)(42)(43). MiRNAs make up one of the wellcharacterized non-coding RNA families that generally bind to the 3' untranslated regions of their target messenger RNAs (mRNAs) to suppress translation or degradation of the mRNAs (44,45). MiRNAs have a fundamental role in many biological processes, including apoptotic cell death, cellcycle, tumorigenesis, and inflammation. Also, dysregulation of miRNAs has been associated with prognosis for and progression of multiple human diseases, including cancer (46)(47)(48)(49)(50)(51). An increasing number of studies have demonstrated that several miRNAs, including miR-21, miR-146, miR-155, and let-7 family, target TLRs or proteins in TLR signaling pathways (Figure 1) that are involved in the regulation of various processes, such as inflammation, T-cell activation, cellular infiltration, and immunity development (52,53). We have selectively listed recent miRNAs and their regulator roles on TLRs in Table 1.
In one of the first studies demonstrating that miRNAs regulate immune response, researchers found that let-7i binds directly to TLR4 and regulates its expression in human cholangiocytes (54). In that study, infection of cultured cholangiocytes with Cryptosporidium parvum and lipopolysaccharide (LPS) stimulation of the cholangiocytes led to decreased let-7 expression via a MyD88/NF-κB-dependent mechanism, and low expression of let-7 was associated with upregulation of TLR4 in cholangiocytes. In concordance with this, upon C. parvum infection in non-malignant human biliary epithelial cells, inhibits expression of let-7 family miRNAs, including let-7i, let-7d, let-7f, let-7e, and miR-98, whereas induces the protein content of total SNAP23 and enhances phosphorylation of SNAP23. Activation of TLR4 signaling may induce SNAP23 protein expression by modulation of let-7-mediated gene regulation (57). Subsequently, investigators showed that let-7 and miR-98 target the 3' untranslated region of the cytokine-inducible Src homology 2-containing protein, resulting in translational FIGURE 1 | Schematic of the regulatory mechanism of miRNAs in TLR signaling. Cell surface and cytoplasmic TLRs can be regulated by several miRNAs, including let-7 family members, miR-21, miR-146, and miR-155. First, miRNAs can bind directly to 3 ′ untranslated region of TLRs or TLR-related genes, leading to modulated expression of TLRs through posttranscriptional regulation of TLR signaling. Second, miRNAs serve as physiological ligands of TLRs, such as miR-21, let-7 family members, and miR-29a, which can activate TLR signaling and stimulate the release of inflammatory cytokines and IFN genes in some cell types. Functional studies have demonstrated that these miRNAs may participate in activation of TLR signaling through regulating the NF-κB pathway and the production of inflammatory cytokines, which are shown here.
repression of this protein in cholangiocytes, and that this may be associated with modulation of inflammatory responses in epithelial cells during microbial infection (76). In addition to this regulatory role of let-7 regarding TLR4 activation the inflammation-associated transcription factors NF-κB p50 and C/EBPβ can interact with the let-7 promoter region and repress transcription following microbial stimulus in human cholangiocytes (91).
MiR-155 has a well-characterized oncogenic role in tumorigenesis (92,93), and aberrant expression and function of miR-155 have been associated with inflammation and affect immune cell functions at various levels by targeting inflammation-related genes, including TLRs (87,94). This miRNA suppresses the expression of the adaptor protein TAB2 in the TLR/IL-1 signaling cascade, thereby regulating the feedback mechanism of IL-1β and other inflammatory cytokines produced during LPS-mediated DC activation (72). MiR-155 can also target suppressor of cytokine signaling 1 (SOCS1) and consequently modulate transcriptional expression of SOCS1 in LPS-activated Akt1 −/− murine macrophages (95). In addition, miR-155 and miR-M4 (virally encoded functional orthologs of miR-155) may target coding sequences of the TLR3 gene and regulate TLR3 expression in macrophages (61). In line with this, inhibition of miR-155 by antagomirs markedly increased  TLR3 expression, whereas ectopic overexpression of miR-155 decreased IFN-β production in primary chicken embryo fibroblast cells (61). In another study, researchers found not only that miR-155 regulates TLR expression but also that miR-155 and caspase 3 mRNA can interact with AGO2 in LPS-activated murine macrophages (60). MiR-21 is one of the multifunctional miRNAs and is mainly characterized by overexpression in many inflamed states, including lung inflammation in LPS-treated mice, allergic airway inflammation, and osteoarthritis (73,(96)(97)(98). Moreover, researchers detected high miR-21 expression in extracellular vesicles during simian immunodeficiency virus pathogenesis and increased miR-21 expression in mouse hippocampal neurons associated with neurotoxicity due to neuronal TLR7 expression (89). Furthermore, investigators showed that miR-21 expression was induced in murine macrophages by treatment with LPS, whereas proinflammatory protein PDCD4 expression was downregulated in these cells due to induction of miR-21 expression via the adaptor proteins MyD88 and NF-κB (73). In another study, miR-21 expression decreased in patients with primary graft dysfunction after lung transplantation, and incubation of human monocytes with bronchoalveolar lavage fluid obtained from patients with primary graft dysfunction induced miR-21 expression, suggesting that dysregulation of miR-21expression is a novel regulator of TLR signaling during development of lung injury (75). Another study demonstrated that activation of TLR4 by treatment with LPS induced miR-21expression in primary human lung cancer cells and reactive oxygen species production by these cells (74). A more recent study demonstrated that miR-21 was upregulated in plasmacytoid DCs and that miR-21 deficiency significantly impaired production of IFN-γ and IFN-α in response to HSV-1 infection through targeting of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signaling pathway in miR-21-knockout mice (86).
Importantly, an expression signature analysis of 200 miRNAs demonstrated that miR-146a/b, miR-132, and miR-155 were highly expressed in the acute monocytic leukemia cell line THP-1 after treatment with LPS as well as other microbial components and proinflammatory mediators (87). This finding suggests that miR-146 directly targets IRAK1 and TRAF6, which are key adapter molecules in the TLR4/NF-κB pathway (87). Furthermore, researchers found that miR-146 was significantly upregulated in hepatic stellate cells in mice infected with Schistosoma japonicum (99), is a negative regulator of NF-κB signaling in hepatic stellate cells, and acts by targeting TRAF6. Moreover, ectopic overexpression of the miR-146b-5p mimic significantly attenuated LPS-induced inflammatory responses and IRAK1 and TRAF6 expression in human umbilical vein endothelial cells (88).
Furthermore, miR-195 can regulate TLR2 expression through an indirect mechanism, as TLR2 expression was significantly reduced in miR-195-transfected THP-1 macrophages polarized toward the M1 phenotype (100). Treatment with LPS, synthetic lipid A, IL-2, IL-15, IL-1β, IFN-γ, and TNF-α similarly induced TLR2 gene expression in murine macrophages (101,102), and acts is a key player in inflammation and atherosclerosis progression (103). Besides the role of cellular miRNAs in TLR signaling, authors recently reported that multiple viral miRNAs can activate production of proinflammatory mediators. For instance, Kaposi sarcoma herpes virus miRNAs such as miR-K-10b and miR-K12-12 * are involved in sepsis as agonists of TLR8 through secretion of IL-6 and IL-10 (104, 105). Furthermore, Epstein-Barr virus miRNAs such as BHRF1-1 are expressed at higher levels in patients with chronic lymphocytic leukemia than in healthy individuals, and these viral miRNAs can serve as prognostic biomarkers for cancer (106,107). Overall, these studies highlight miRNAs as central drivers of the TLR expression through transcriptional regulation of them, as indicated in Table 1.

HOW TLR SIGNALING MAY MODULATE MIRNA EXPRESSION
Initiation of the signaling cascade of immune response induced by TLR signaling can drive transcription of miRNAs during infection and inflammation. This is demonstrated by the fact that aberrant activation of TLR signaling after infection with microbial pathogens leads to dysregulation of miRNAs. Researchers have shown that infection of human peripheral blood monocytes (PBMCs) with Francisella tularensis, which is a highly pathogenic gram-negative bacterium that infects macrophages, induces expression of miR-155 in a TLR-dependent manner through downregulation of Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) (84). In concordance with this, authors reported significant differential expression of several miRNAs, including miR-155, after F. tularensis infection in primary human monocyte-derived macrophages and that F. tularensis infection leads to downmodulation of MyD88 and SHIP1 through an miR-155-dependent mechanism (64). Moreover, Leishmania RNA virus 1 was recognized by TLR3, and Leishmania infection induced miR-155 expression in murine bone-marrow macrophages (59). Concurrently, in that study, the pathogenesis of LRV1+ Leishmania infection decreased drastically in miR-155-deficient mice. In another study, let-7 and miR-98 were downregulated in murine macrophages upon Salmonella infection, whereas miR-155, miR-146a, and miR-21 were upregulated (77).
The immune-regulatory cytokine IL-10 may regulate transcription of miR-155 from the BIC gene in a signal transducer and activator of transcription 3-dependent manner in immortalized bone marrow-derived macrophages, and downmodulation of miR-155 expression leads to increased expression of SHIP1, which is one of the targets of miR-155 (71). Furthermore, investigators found that Ets2 is a critical transcription factor for the induction of miR-155 expression by LPS, and downmodulation of Ets2 leads to suppression of miR-155 by IL-10 (81). Another example miRNA signature is involved in human plasmacytoid DC activation (70), and miR-155 and its star form, miR-155 * , were the most upregulated miRNAs in in primary human plasmacytoid DCs after TLR7 stimulation. MiR-155 * induced IFN-α/β expression by suppressing IRAK-M expression, whereas miR-155 suppressed TAB2 expression (70). TLR3-dependent antiviral as well as inflammatory activity can be regulated by IL-10, transforming growth factor-β, and miR-155 in non-parenchymal liver cells in vitro (67). Other studies of macrophages demonstrated that chronic alcohol exposure induces TNF-α secretion through increased miR-155 expression both in vitro and in vivo (108), and miR-155 deficiency can protect against alcohol-induced liver injury, oxidative stress, steatosis, and inflammation in miR-155-knockout mice (79). In a similar study, after chronic ethanol feeding, miR-155 induced TNF-α and MCP1 expression in the cerebellum in a TLR4dependent manner (68). Researchers have also observed aberrant expression of miR-155 in macrophages after stimulation by poly(I:C) and IFN-β and that miR-155 expression is induced by other TLR ligands through MyD88-or TRIF-dependent signaling pathways (109). However, investigators found TLR-independent upregulation of mature miR-155 in the murine macrophage cell line J774A and murine primary bone marrow-derived macrophages during Helicobacter pylori infection (110). Authors reported that treatment with progesterone augmented LPS-and poly(I:C)-induced miR-155 expression in macrophages through inhibition of NF-κB activation and led to downmodulation of IL-6 and IFN-β production in TLR-activated macrophages by increasing SOCS1 expression (85). A recent study identified that miR-155 expression increased in monocyte-derived macrophages upon TLR3/4 but not TLR7 stimulation and that inhibition of miR-155 expression partially restored infectivity in poly(I:C)treated monocyte-derived macrophages (69). Furthermore, miR-155 in PBMCs of systemic lupus erythematosus patients was specifically upregulated by high-mobility group box 1 protein in a MyD88-dependent manner during induction of antidouble-stranded DNA antibody, which is the central pathogenic autoantibody involved in pathogenesis of systemic lupus erythematosus (82). Authors reported that cold exposure (32 • C) induced miR-155 expression in human monocytes and that increased miR-155 expression was associated with suppressed SOCS1 and SHIP1expression (65). Additionally, miR-155 was upregulated in ischemic cerebral tissue and promoted TNF-α and IL-1β expression by upregulating TLR4 and downregulating SOCS1 and MyD88 (63). Negative regulator proteins for the TLR4 pathway (IRAK-M, SHIP1, and SOCS1) were upregulated in Kupffer cells isolated from miR-155-deficient mice (58). Researchers have shown that miR-155-3p and miR-155-5p (the two mature miRNAs processed from the precursor miR-155 transcript) were highly expressed in mice after treatment with LPS, whereas expression of both was decreased in the lungs of triggering receptor expressed on myeloid cells 1-(TREM-1) knockout mice. Deficiency of TREM-1 significantly inhibited neutrophils and proinflammatory chemokines and cytokines, particularly IL-1β, TNF-α, and IL-6 (80). Researchers also identified that protein kinase Akt1 activated by LPS positively regulates the expression of let-7e and miR-181c but negatively regulates that of miR-155 and miR-125b revealing that let-7e inhibits the expression of TLR4, whereas miR-155 inhibits the expression of SOCS1; both proteins TLR4 and SOCS1 are critical for TLR signaling after LPS stimulation (95). In another study, investigators showed that exposure to angiopoietin-1 significantly decreased IRAK1 and TRAF6 protein expression but did not affect TLR4, MYD88, IRAK4, or TAK1 expression in human umbilical vein endothelial cells (88).

THE ABILITY OF MIRNAS TO BIND TO TLRS
MiRNAs may bind to TLRs and activate TLRs involved in intercellular communication in the tumor microenvironment (12,13,111). Authors reported that guanosine-and uridinerich single-stranded RNA oligonucleotides derived from HIV-1 and influenza virus are recognized by murine TLR7 and human TLR8. Subsequently, activation of DCs and macrophages lead to the production of proinflammatory mediators such as IFN-α and cytokines (112,113). Recently reported evidence demonstrated that extracellular vesicles such as exosomes and shed microvesicles isolated from different cell types may be novel mediators of cell-cell communication these vesicles can contain mRNAs, miRNAs, long non-coding RNAs, lipids, and DNA fragments. These active cargo molecules are packaged and released in exosome-derived cells and taken up by neighbor cells, where they are functionally active (114)(115)(116). MiRNAs are ubiquitously expressed in exosomes and are involved in modulation of the host immune response, expression of some activated molecules, enhanced tumor cell invasion, and mediation of intercellular communication (12,117).
In 2012, researchers discovered that tumor-secreted exosomes in supernatants of lung cancer cells and exosomes loaded with miRNAs are physiological ligands for TLR7 and TLR8 (9,12,13). Expression of miR-21, miR-27b, and miR-29a was higher in exosomes derived from lung cancer cells than in those derived from HEK-293 cells (13). Upon co-culture of HEK-293 and RAW macrophages in vitro, labeled exosomes released from HEK-293 cells were incorporated with RAW macrophages, and miR-29a co-localized with TLR7 and TLR8 in the RAW macrophages (9,12,13). In addition, these investigators reported that cancer cell-derived exosomal miRNAs can bind to and activate TLR8 in macrophages and stimulate TLR8-mediated activation of NF-κB and NF-κB-mediated release of the proinflammatory and prometastatic cytokines IL-6 and TNF-α (13). Therefore, malignant cells release signals via exosomes loaded with miRNAs to the surrounding cells in their microenvironments that promote tumorigenesis and dissemination by different TLRs in humans (TLR8) and mice (TLR7) (13,118). In our recent study, we demonstrated that exosomal miR-1246 released in abundance from ovarian cancer cells and miR-1246 transmit molecular signals to M2-type macrophages but not M0-type macrophages by shuttling exosomes (119).
Moreover, treatment with liposome-encapsulated miR-21 significantly induced human TLR8 expression in hematopoietic cell lines and PBMCs obtained from patients with systemic lupus erythematosus (90). Single-stranded RNAs containing twentynucleotide guanosine-and uridine-rich regions derived from human immunodeficiency virus and the influenza virus are physiological ligands for TLR7 and TLR8 that bind to TLR and activate TLR signaling (113,120). Furthermore, the let-7 family contains a specific GU-rich motif GUUGUGU, which is present in the core of single-stranded RNA40 and responsible for murine TLR7 activation (15,113,120). Researchers recently discovered that let-7 may interact with TLR7 and activate TLR signaling in murine macrophages and microglia (15). They found that six nucleotide exchanges in the seed sequence of let-7b dramatically diminished induction of TNF-α expression in microglia and macrophages. In addition to let-7b, let-7a, −7c, and −7g induced a dose-and time-dependent cytokine response in wild-type immortalized bone marrow-derived macrophages but not TLR7-deficient cells (15). Consistent with these findings in macrophages, these investigators showed that neuronal loss was induced by let-7a, let-7c, let-7g, and miR-599 through TLR7. To further understand the role of let-7 in neurodegeneration in vivo, Lehmann et al. (15) showed that treatment with let-7b significantly induced marked axonal injury and neuronal loss in wild-type mice, whereas mutant let-7b rescued this phenotype. In contrast, TLR7 deficiency in Tlr7 knockout mice can protect against let-7b's induced neurotoxic effects. These findings suggest that endogenous miRNAs such as let-7b can be released during neuroinflammation and may cause further spread of central nervous system damage in patients with neurodegenerative disorders such as Alzheimer disease (15,121).

CONCLUSION AND FUTURE PERSPECTIVES
MiRNAs are strongly implicated to have roles in the development and progression of inflammation-related diseases. Increasing numbers of studies have identified that miRNAs can act as physiological ligands for TLRs. The next challenges are to understand the complex mechanisms behind these integrated networks of interactions and, more importantly, determine whether therapeutic modulation of TLR-regulated and TLRregulating miRNAs is beneficial for patients with cancer or inflammatory diseases. At present, MRG-106 therapy, is an oligonucleotide inhibitor of miR-155, for being tested in phase 1 clinical studies in Cutaneous T-cell Lymphoma, Mycosis Fungoides, Chronic Lymphocytic Leukemia, Diffuse Large B-Cell Lymphoma and Adult T-Cell Leukemia/Lymphoma (clinicaltrials.gov identifier NCT02580552). Therefore, researchers expect more preclinical and clinical studies regarding this new therapeutic avenue (93,122,123).

AUTHOR CONTRIBUTIONS
RB, MB, and GC wrote the first draft of the manuscript and contributed to the writing of the manuscript.