MicroRNA let-7 in the spotlight : Role in innate immunity

Innate immunity forms the first line of host defense. It is quick and usually shapes the adaptive immune responses which clear off the pathogens. However, dysregulated innate immune responses can lead to excessive inflammation which causes significant damage to the host. As a result, innate immune responses are tightly regulated. In this review, we focus on mechanisms by which microRNA let-7 regulate innate immune responses and contribute in maintaining tissue homeostasis.


Introduction
The innate immune system is an ancient defense mechanism present in all forms of life.It represents the first line of defense system that is capable of differentiating between non-infectious self and infectious non-self.It relies on "pattern recognition" such that it can recognize and bind to certain signature motifs expressed exclusively by micro-organisms but not by the host [1].The receptors which can recognize patterns are called the pattern recognition receptors (PRRs) and they recognize pathogen associated molecular patterns (PAMPs).At present, PRRs consist of three main classes of proteins-Toll like Receptors (TLRs), Nod like Receptors (NLRs) and RIG like Receptors (RLRs).The members of these receptor families and their binding ligands have been summarized in Table 1 [2-22]   .PRR's initiate innate immune responses and set-off an inflammatory cascade.The pro-inflammatory cytokines and interferons secreted by cells upon ligation of PRRs result in activation of immune system, shaping of the adaptive immune response leading to clearance of invading pathogens.However, dysregulated signaling via PRRs can lead to excessive inflammation and can result in many diseases.For example, low grade infection by Helicobacter pylori has been shown to be one of the causative factors of gastric cancer [23] .Similarly, chronic inflammation is also involved in etiologies of other diseases like atherosclerosis, autoimmunity, diabetes and metabolic syndrome [23] .In these cases, inability of the body to resolve inflammation leads to tissue damage which impairs the functioning of the inflamed organ.Chronic inflammation also aids in malignant transformation as well as metastasis [23] .Just as chronic inflammation, acute inflammation could be fatal.For example, patients suffering from septic shock have high amounts of bacterial lipopolysaccharide (LPS) in their blood stream which activates TLR4 on immune cells such as macrophages and endothelial cells.TLR signaling can also induce clotting factors required for wound repair and formation of fibrin clots in blood vessels leading to organ REVIEW failures [24] .Hence, it is essential that innate immune responses be regulated so that excessive production of proinflammatory cytokines is prevented.Multiple mechanisms have evolved in the body which limits inflammation by fine tuning PRR signaling pathways.These include reduced expression of receptors [25,26] , abrogation of signaling via targeting adaptor molecules (MyD88s, SIGIRR/ST2), induction of defective kinase (IRAK-M) or deubiquitinating enzymes (A20 and CYLD), ubiquitinating enzymes (TRIM30α, RNF125).These regulators have been discussed in detail elsewhere [27,28] .A novel class of molecules known as microRNAs are known to regulate PRR signaling at posttranscriptional level.They affect many biological systems, including the mammalian immune system.MicroRNAs (miRNAs) are small, single-stranded, noncoding 19-22 nucleotide RNAs sequences, many of which have been highly conserved throughout evolution.They function by directly binding to the 3' untranslated region (3'UTR) of specific target mRNAs, leading to the repression of protein expression.Till date 2578 mature miRNAs have been identified in the human genome [29,30,31,32,33] .Each miRNA could have the potential to repress the expression of many, perhaps hundreds, of target genes, highlighting the extent of this form of regulation in mammals [34,35,36] .More than 100 different miRNAs are expressed by cells of the immune system; they have the potential to broadly influence the molecular pathways that control the development and function of innate and adaptive immune responses.Besides immune function disorders, miRNAs are also known to play a role in cancer, age-related diseases, heart diseases and neurological diseases [37] .

miRNAs: Biogenesis and mechanism of action
The processes of miRNA biogenesis and target mRNA repression have been intensively studied in recent years.miRNAs are initially transcribed as primary transcripts known as pri-miRNAs by RNA polymerase II (RNA pol II) and cropped into about 70-to 100-nucleotide-long hairpin precursors (termed pre-miRNAs) in the nucleus by the RNAse III, Drosha [38,39,40] .Pre-miRNAs are actively transported by exportin-5 to the cytoplasm where they are cleaved by the enzyme, Dicer, to form mature miRNAs [38,41,42] .This cleavage event gives rise to a double stranded, 22-nt product comprised of the mature miRNA guide strand and the miRNA* passenger strand [43] .The mature miRNA is then loaded onto the RNA-induced silencing complex (RISC), while the passenger strand is degraded.The RISC identifies target mRNA by base-pair complementarity resulting in mRNA cleavage and/or translational suppression (Figure 1) [44] .

The case of lin-4 and the discovery of microRNA
miRNAs were discovered during a developmental study in the nematode, Caenorhabditis elegans (C.elegans) [34] .The authors found that lin-4, a gene known to control the timing of C.elegans larval development does not code for a protein but instead produces a pair of small RNAs [34] .One RNA is approximately 22 nucleotides (nt) in length, and the other is approximately 61 nt long; the longer one was predicted to fold into a stem loop proposed to be the precursor of the shorter one.The authors then noticed that these lin-4 RNAs had antisense complementarity to multiple sites in the 3'UTR of the lin-14 gene [34] .This complementarity fell in a region of the 3'UTR previously proposed to mediate the repression of lin-14 by the lin-4 gene product [45] .The shorter lin-4 RNA is now recognized as the founding member of an abundant class of tiny regulatory RNAs called microRNAs [46] .Let-7 family of microRNAs was the next to be discovered.In C. elegans another gene, let-7, was found to encode for a ~22 nt regulatory RNA.Reinhart et al. [35] , reported let-7 to be involved in the regulation of development timing in C.elegans.let-7 is an important microRNA family consisting of 13 members.They are highly conserved across many animal species [47] .The role of let-7 family members in tumor suppression is well documented but their role in the regulation of innate immunity is slowly unfolding.

Physiological role of let-7 in innate immunity
MicroRNAs are important controllers of Toll-like receptor (TLR) signaling.It is well established that TLRs have important roles in detecting pathogens and in initiating inflammatory responses that subsequently prime specific adaptive immune responses during infection [48] .It has also been recognized that deregulation of this process is a hallmark of inflammatory and autoimmune diseases [49]   .It is therefore important that TLR signaling pathways are tightly regulated.
Chen et al. [50] have shown expression of let-7 family members in human biliary epithelial cells (cholangiocytes).During Cryptosporidium parvum infection, let-7i levels decrease which is associated with up-regulation of TLR4 in cholangiocytes.This suggests that let-7i contributes to epithelial defense responses by regulating C. parvuminduced up-regulation of TLR4 in cholangiocytes.The cytokine-inducible Src homology 2-containing protein (CIS) is an important negative regulator for inflammatory cytokine signaling.Expression of CIS is associated with an accelerated degradation of IκBα and enhanced NF-κB activation in cholangiocytes in response to LPS stimulation.LPS stimulation with the parasitic protozoan C.parvum induces expression of CIS protein by activating the TLR signaling pathway.Hu et al. [51] found that miR-98 Iliopoulos et al. [52] identified interleukin-6 (IL-6) as a potential gene target of let-7 resulting in inhibition of the IL-6 dependent signaling pathway in MCF10A cells.IL-6 treatment also inhibits let-7a microRNA expression in a manner that depends upon NF-κB.This finding indicates a negative feedback loop between let-7a and IL-6.κB-Ras2 is an inhibitor of NF-κB signaling which is down regulated when macrophages are activated with LPS.Murphy et al. [53] showed that LPS induces the expression of let-7a and inhibits the expression of miR-125b in macrophages.microRNAs let-7a and miR-125b regulate the stability of the κB-Ras2 3′UTR.This provides a net instability to the κB-Ras2 3′UTR.Estradiol reverses the effect of LPS on let-7a and miR-125b expression, for a net increase in κB-Ras2 stability.This was the first account of estradiol modulation of miRNA expression in human macrophages.
Schulte et al. [54] have shown that let-7a and let-7d levels fall after Salmonella infection in murine macrophages while those of IL-6 and IL-10 increase.They also found all let-7 family members to contain complementary sequences to the 3'UTR of IL-6 and IL-10.let-7 plays a role in cellular immune responses by regulating the levels of key cytokines: IL-6 and IL-10.TLR-4 is up-regulated in response to H. pylori infection in gastric epithelial cells.Teng et al. [55] have shown let-7b to regulate the expression of TLR4 via post-transcriptional suppression.Let-7b subsequently influences the activation of NF-κB and the expression of downstream genes.Let-7b may thus contribute to the initiation of innate immune responses and inflammation of gastric mucosa against H. pylori infection.
B lymphocyte-induced maturation protein-1 (Blimp-1) is an important transcriptional repressor in B cells and T cells.Expression of Blimp-1 can be induced by various stimuli including engagement of TLRs.Kim et al. [56] have identified a novel function of Blimp-1 as a transcriptional repressor of let-7c miRNA.Blimp-1 binds to the let-7c regulatory region and reduces the level of let-7c in dendritic cells (DCs).In the absence of Blimp-1, let-7 miRNA levels increase resulting in a broad spectrum of pro-inflammatory features in DCs, which is partly mediated through suppression of suppressor of cytokine signaling-1 (SOCS1) expression.This provides a novel mechanism of let-7 regulation mediated by Blimp-1 (Figure 2).

Let-7f: Novel regulator of inflammation
The epithelial cells of female reproductive tract (FRT) play a crucial role in the initiation, regulation and resolution of both innate and adaptive immune functions in response to microbial infection [57,58] .Previously, we demonstrated TLRs may play an important role in mounting innate immune responses in cervicovaginal epithelial cells [59] .However, very little is known about the regulatory mechanisms that limit inflammation in the FRT.They are constantly exposed to lactobacilli commensals and yet impart protection from invading pathogens.Therefore, we hypothesized that genital epithelial cells must possess mechanisms which can fine tune the immune responses.
Using synthetic ligands of TLR9 and RIG-I, we demonstrated that stimulation of endocervical cells with TLR9 ligand induced a state of tolerance to subsequent stimulation with either TLR9/RIG-I [60] .Many TLR ligands have been shown previously to induce such phenomenon.For example, Sun et al. [61] demonstrated tolerance in intestinal epithelial cells in response to stimulation with flagellin.Tolerance to LPS is well documented [28] .Hence, it was not surprising that TLR9 stimulated epithelial cells showed very little cytokine response upon re-stimulation [60]   .In contrast, stimulation with RIG-I ligand or TLR3 ligand did not induce tolerance.One of the causes responsible for tolerance is down-regulation of receptors [25, 26] .However, stimulation of endocervical cells with ligands of TLR9 and RIG-I enhanced expression of their cognate receptors [62] ruling out diminished expression of receptors as a reason for induction of tolerance.Previously, Harada et al. [63] reported that stimulation of biliary epithelial cells with poly (I:C)a TLR3 liganddoes not induce tolerance.However, no mechanistic reason was attributed for prevention of tolerance.We speculated that analysis of transcriptome of cells after stimulation with TLR9 and RIG-I ligand would unravel possible molecules involved in tolerance.
Our microarray studies revealed stimulation with ligands of TLR9 and RIG-I significantly up-regulated cytokines/chemokines (Table 2).We also observed that negative regulators of PRR/ NF-κB signaling were differentially induced (Table 3).We also observed modulation of miRNAs in response to TLR9 and RIG-I stimulation (Table 4).Interestingly, we observed that RIG-I stimulation increases the levels of let-7f, while TLR9 decreases them.So far, this was the only instance where PRR stimulation was up-regulating let-7 expression.In conjunction, the levels of Blimp-1, a protein known to be targeted by let-7f was decreased on RIG-I stimulated cells.
Blimp-1 is a transcriptional repressor which plays a key role in plasma cell differentiation [64] .Blimp-1 is highly expressed in plasma cells and controls expression of genes such as Xbp1 (which induces secretory apparatus) and inhibits expression of genes favouring cell division (Myc and Bcl6) [65,66,67,68] .Blimp-1 is known to inhibit expression of pro-inflammatory cytokines such as IFN-γ, TNF-α, TNF-β [69] and IL-6 [70] .It recruits histone deacetylases namely HDAC1 and HDAC2 to the promoter regions of the target genes [71] which deacteylate histones, switching off transcription at those loci.
Knockdown of let-7f lead to increased expression of Blimp-1 and stimulation of these cells gave not only significantly lower amounts of cytokines after stimulation with RIG-I ligand, but also the cells displayed tolerance like phenomenon.In contrast, over-expression of let-7f decreased expression of Blimp-1 and those cells did not display state of tolerance even after re-stimulation with TLR9 ligand.Thus, let-7f primarily modulates tolerance via Blimp-1 in human endocervical cells.We also validated IL-8 and HDAC2 as a target of let-7f.We identified at least 4 let-7f target sites in 3'-UTR of HDAC2 mRNA and a single target site in IL-8 mRNA.Thus, Blimp-1 targets pro- inflammatory (IL-8 and IL-6) as well as anti-inflammatory (Blimp-1) genes to maintain a balance between inflammation and homeostasis.

Conclusions and future directions
In conclusion, microRNAs function as efficient finetuners of protein expression, rather than as "on-off" switches and they represent a mechanism to tone down the inflammatory response, which when left unchecked, can have catastrophic effects.Furthermore, our results underline the complexity of TLRs and miRNA in the modulation of innate immune functions regulation, revealing they are the fundamental players of this process.Although, it is now clear that reduced levels of let-7f increases levels of Blimp-1 which leads to tolerance.However, it is not clear whether this effect is brought about solely by targeting Blimp-1/HDAC2.It may be possible that other targets of let-7f may also be involved in induction of tolerance.Since there is overlap between targets of different isoforms of let-7, whether modulation of other isoforms of let-7 can induce tolerance is presently unknown.Future studies should also focus on transcription factors which up-regulate or repress expression of let-7 molecules.At present, only a few proteins such C/EBPβ [72] and YY1 [73] are known to bind to the upstream regions of let-7 genes.However, since the promoter regions of let-7 genes have not been defined, even bioinformatics approach to predict transcription factors regulating them has limited use.Finally, studies should also be undertaken to find out whether similar transcription factors/pathways are utilized in most tissues to modulate expression of let-7 molecules.Given the importance of let-7 miRNAs in processes such as inflammation, cancer and development; understanding of these factors would translate in therapeutic strategies to combat human diseases.

Fig 1 .
Fig 1. miRNA biogenesis and mechanism of action.MiRNA biogenesis is initiated with the processing of primary miRNA transcripts in the nucleus by the Drosha /DGCR8, to generate a 70 nt pre-miRNA, which are further processed into a miRNA duplex by Dicer, which is transported from nucleus to cytoplasm by exportin-5.In the cytoplasm, the pre-miRNA is further trimmed to 21-25 nt double stranded RNA by Dicer into an miRNA:miRNA* duplex.Assembled into the RISC, one of the strands negatively regulates gene expression by either translational repression or mRNA degradation.

Fig 2 .
Fig 2. Schematic representation of role of let-7 in Innate Immunity.

Table 1 .
PRRs and their respective ligands involved in innate immunity.