Regulation of TLR signaling pathways by microRNAs: implications in inflammatory diseases.

In the past several decades, extensive studies have focused on how the host’s immune response discriminates between microorganisms. One of the most extensively studied recognition mechanisms is through the toll-like receptor (TLR) family. The activation of the TLR signaling pathways is necessary to initiate the immune response to eliminate an infection; however, inappropriate activation, such as a persistent infection (i.e., bacteria, virus, or other microorganisms), can compromise immunological homeostasis, leading to pathologies such as autoimmune diseases, chronic inflammation, tumor development and even cancer. TLRs identify microbe-associated molecular patterns (MAMPs), which in turn triggers an intracellular signaling cascade involving adaptor proteins and the activation of transcription factors that prompt the production of cytokines [ 1 ]. Once signaling has been initiated, transcription factors and message translation can be halted through post-transcriptional regulation of key proteins along the signaling cascade. This negative regulation can be achieved by the destabilization of encoding messenger RNA (mRNA) or by hampering the translation. One of the post-transcriptional regulatory mechanisms is through microRNAs (miRNAs), which are small non-coding RNAs, approximately 23 nucleotides that bind the seed region (2-7 nucleotides from the 5’ end) to the 3’ untranslated region (UTR) of the mRNA from target proteins [ 2 ]. The sequences of miRNAs are conserved between species; however, they are not specific for a single protein. The importance of miRNAs as a regulatory mechanism for protein expression has been the focus of several research studies in the past decade. Several of them imply diverse aspects of the immune system, particularly the inflammatory process. This review will discuss the role of some miRNAs in the regulation of TLRs and related signaling proteins, cytokines and their important roles in maintaining homeostasis, and the implications of this regulation in several diseases linked to the inflammatory response.

The MyD88-dependent pathway relies on the activation of a family of kinase proteins termed interleukin-1 receptor (IL-1R)-associated kinase (IRAK), composed of four proteins termed IRAK1, IRAK2, IRAK3 (or IRAKM) and IRAK4 [ 3 ]. IRAK1 also interacts with TNF receptor-associated factor 6 (TRAF6), and together they play important roles in signal transduction mediated by TLRs and IL-1Rs. IRAK4 and IRAKM, in conjunction with TRAF6, are key proteins in the TLR signaling pathway. However, several other important proteins with kinase activity or adapter function are involved in signaling that leads to activation of the transcription factor NF-κB. The IRAK family proteins are independently regulated by different miRNAs. Figure 2 shows some of the reported miRNAs that regulate proteins involved in the signaling cascade implicated in inflammatory diseases. For instance, miR-21-5p targets several cellular processes, which suggests a multifaceted role. In hepatitis C virus-infected PBMCs, miR-21-5p regulates IRAK1 and MyD88 expression [ 18 ]. The microRNAs miR-146a-5p and miR-155-5p are the most extensively implicated in the regulation of TLR downstream signaling [ 13 , 19 ]; in human DCs, miR-146a-5p and miR-146b-5p regulate apoptosis and cytokine production by targeting TRAF6 and IRAK1 [ 20 , 21 ]. Other miRNAs have been reported to regulate TLR downstream signaling, such as miR-133-5p and miR-142-3p, which target IRAK1 [ 22 , 23 ]. Mycobacterium bovis -infected mouse macrophages show high levels of IRAK1; however, miR-142-3p overexpression results in the downregulation of IRAK1 and the consequent downregulation of NF-κB, TNF-α and IL-6 [ 23 ]. This finding suggests that miR-142-3p contributes to the control of the exacerbated inflammatory response to Mycobacterium bovis and could be related to the induction of tolerance. NF-κB and IRF3 signaling pathway regulation by miRNAs implicated in inflammatory diseases Several studies have indicated the importance of IRAK4 in TLR4 signaling. Patients who carry IRAK4 mutations are extremely susceptible to bacterial infections. Infection with Pseudomonas aeruginosa , an important etiological agent in nosocomial infections, strongly induces miR-302b-5p expression in mouse alveolar macrophages, which targets IRAK4, affecting NF-κB activation and the associated inflammatory response [ 24 ]; thus, miR-302b-5p is a negative regulator of the innate immune response. Conversely, miR-93-5p exhibits reduced expression in a rat model of endotoxin-induced uveitis, as well as in LPS-treated RAW 264.7 cells, and its overexpression greatly reduced IL-1β, IL-6, and TNF-α. Further assays demonstrated that IRAK4 is the target of miR-93-5p, which thereby hampers NF-κB signaling, resulting in low production of the pro-inflammatory cytokines [ 25 ]. TRAF6 is another important protein in the NF-κB signaling cascade, and is a target for miRNAs. The microRNA miR-124-5p also targets TRAF6, together with TLR6, MyD88, and TNF-α in epithelial cells and alveolar macrophages in response to mycobacterial infection [ 16 ]. The miRNA miR-125b-5p is downregulated after LPS-treatment in mouse macrophages; its overexpression led to decrease in pro-inflammatory cytokines by targeting TRAF6 [ 26 ]. Interestingly, miR-125b-5p has a dual inhibitory effect in the immune response, since it not only represses the transcription of pro-inflammatory cytokines by inhibiting TRAF6 but also has the ability to transcriptionally regulate TNF-α, by binding to TNF-α mRNA, inhibiting its production [ 12 ]. In addition, tissue from patients with autoimmune diseases such as SLE and RA exhibit reduced expression of miR-23b-5p and consequently higher expression of TAB2, TAB3 and IKK-α, and increased production of TNF-α, IL-1β and IL-17 [ 27 ]. On the other hand, the increased expression of miR-381-5p in LPS-stimulated human lung epithelial carcinoma cells reduces the expression of the inhibitor IκBα, which allows the release of NF-κB and the subsequent production of pro-inflammatory cytokines [ 28 ]. The TRIF-dependent pathway culminates in the activation of both IRF3 and NF-κB [ 3 ]. TRIF recruits TRAF6, TRADD and TRAF3. TRAF6 in turn initiates the signaling cascade described above. On the other hand, TRADD recruits RIP1 and activates TAK1 (transforming growth factor beta-activated kinase 1), which in turns activates NF-κB for the production of inflammatory cytokines. Conversely, TRAF3 activates the kinases TBK1 and IKKε, which phosphorylate and activate IRF3, inducing the production of type I interferons (mainly IFN-α and β), with anti-viral activity [ 3 ]. Figure 2 shows some of the reported miRNAs that regulate proteins involved in the signaling cascade implicated in inflammatory diseases. Patients with Helicobacter pylori -positive gastric cancer had downregulation of miR-3178-5p and elevated levels of TRAF3 and in IL-6 and IL1β; further experiments found TRAF3 as the target for miR-3178-5p. Treatment with miR-3178-5p mimic impeded the proliferation of gastric cancer cells via inhibition of TRAF3 and the concomitant production of inflammatory cytokines [ 29 ]. Other microorganisms also use miRNAs to target TRAF3 and induce an inflammatory response, for example, Burkholderia pseudomallei , the causative agent of melioidosis, induces a strong inflammatory response mediated by TNF-α, IL-10, IL-1β, IL-8, IL-6 and IFN-γ that has been associated with mortality among patients. Fang et al . (2016) demonstrated that B. pseudomallei -infected mouse macrophages had elevated levels of miR-3473-5p and its target was TRAF3. Inhibition of miR-3473-5p resulted in reduced levels of TNF-α; however, the in vivo administration of miR-3473 did not reduce the death rate of infected mice [ 30 ].

MAMPs’ recognition by TLRs triggers signaling pathways ending with the activation of transcription factors for cytokine production. Very precise regulation of this signaling occurs from start to finish; hence, the mRNA of cytokines can also be post-transcriptionally regulated. Cytokines act as soluble mediators of the innate and adaptive immune system. Nonetheless, a response to pathogens or debris from damaged host cells could cause elevated and uncontrolled cytokine production, and also inadequately resolved chronic inflammation such as chronic gastritis, inflammatory bowel diseases (IBD), prostatitis or endometriosis, which in turn may increase the risk of cancer. Some of the miRNAS implicated in the regulation of the cytokines described herein are shown in Figure 2 . In addition to the pathological implications related to the overproduction of TNF-α mentioned above, this cytokine has been considered the main effector associated with septic shock syndrome. In chronic inflammatory diseases, such as psoriasis, it has been observed that miR-203-5p and TNF-α are abundant in cells in psoriatic lesions. However, in primary keratinocytes and a keratinocyte cell line, the overexpression of miR-203-5p inhibits TNF-α transcript and protein production. The increase in TNF-α and miR-203-5p in psoriatic lesions skin is contradictory [ 44 ], since miR-203 also directly targets SOCS3 (suppressor of cytokine signaling 3) and SOCS6 [ 45 ], negative regulators of cytokine signaling. Hence, miR-203 acts as both an anti- and a pro-inflammatory regulator. Due to the key role that TNF-α plays in the pathogenesis of IBD, miR-19a-5p may also regulate the production of TNF-α in cells from patients with ulcerative colitis and colitis experimentally induced in mice; this finding is of great importance since many therapies for this condition include anti-TNF-α treatments [ 46 ]. Interleukin 6 is a cytokine involved in the acute phase response to infection and injury but, in addition to its role in the immune system, it plays a crucial role in hematopoiesis, neuronal and liver regeneration, embryonic development and fertility. Increased levels of circulating IL-6 have been implicated in several autoimmune diseases such as systemic lupus erythematosus (SLE) and RA, and the control of IL-6 production is a therapeutic target for these diseases. In addition to RA, IL-6 dysregulation contributes to the onset of other diseases such as inflammatory bowel disease (IBD), osteoporosis, multiple sclerosis, multiple myeloma, Hodgkin’s lymphoma, epithelial cancer and other several cancers. There is evidence that the let-7 family is involved in the regulation of tumorigenesis since low expression of the let - 7 family has been found in several cancers [ 47 , 48 ]. The overexpression of let-7g in a mouse model of lung cancer reduces tumorigenesis [ 49 ], while let-7a inhibits IL-6 transcript in the epithelial tissue of breast and prostate cancers [ 50 ]. However, it was found that miR-365-5p is a more potent regulator, since its overexpression in cervical cancer cells induced greater inhibition of IL-6 production than let-7a [ 51 ]. IL-6 can also be suppressed by miR-9-5p in cervical cancer cells, as inhibiting this cytokine also inhibits the activation pathways involved in progression of cervical adenocarcinoma [ 52 ]. Another miRNA related to the evolution of IL-6 and cancer is miR-26a-5p, as it is implicated in apoptosis induction and its low expression causes metastasis and recurrence of hepatocellular carcinoma (HCC); transfection of HCC cells with miR-26a-5p resulted in reduced proliferation, migration and invasion [ 53 ]. Interleukin 10 is an anti-inflammatory cytokine and plays a crucial role in the prevention of inflammatory and autoimmune disorders. Mice deficient in IL-10 develop IBD and show an exacerbated response to bacteria challenges; thus, it is suggested that IL-10 may play a protector role and may be used as a therapy for IBD. Bioinformatics analysis followed by experimental validation revealed that miR-106a-5p is a post-transcriptional regulator of IL-10 in several cell lines [ 54 ]. In general, members of the let-7 family have been shown to participate in the regulation of IL-10. In T cells, this microRNA family responds to infection by human immunodeficiency virus (HIV). IL-10 levels in plasma of HIV-infected patients are increased, which contributes to an abnormal response of cytotoxic T cells to infection; meanwhile, let-7 family levels are repressed by the infection [ 55 ]. In LPS-stimulated macrophages, miR-98-5p expression decreases, which contributes to the production of IL-10. Similarly, overexpression of miR-98 in LPS-stimulated macrophages promotes the generation of pro-inflammatory cytokines such as IL-6 and TNF-α. Therefore, miR-98-5p may play a role in inflammatory diseases since it might be involved in IL-10 deficiency and exaggerated pro-inflammatory responses in the intestine [ 56 ]. Given that cytokines share pathways and transcription factors, the role of miRNAs in the post-transcriptional regulation of cytokines further clarifies the fine mechanism that manages the immune response to maintain control of homeostasis.

The importance of miRNAs as a regulatory mechanism for protein expression has been the focus of several research studies in the past decade, several of which imply diverse aspects of the immune system. On the other hand, the TLR signaling cascade has been implicated in several inflammatory disorders. Hence, it is plausible that miRNAs play a regulatory function as well. In fact, the microRNA screening or microtranscriptome profile has been applied to explore the mechanism of immune system disorders. It is noteworthy that a single disease could harbor overexpression of several genes and discrepant miRNA values. While many proteins participate in the TLR signaling cascade, few are known targets of miRNA. In this review, we have presented key aspects of cellular signaling regulation by miRNAs, emphasizing those inflammatory disorders in which their dysregulation has been described. It may be possible to regulate the inflammatory processes associated with these diseases through the manipulation of key miRNAs. However, given that miRNAs can regulate several proteins, further studies are needed to elucidate whether they could really be used therapeutically. In the near future, the knowledge of how to modify the microtranscriptome could be useful for designing new therapies that target miRNAs, which in turn could restore or maintain immune system homeostasis altered by an excessive inflammatory response. Caution should be taken, as their application would not only affect the gene of interest but also other genes not involved in the immune response.