MiR-146a and ADAPTIVE IMMUNITY: A Novel Player in the Regulation of Normal Immune function and Inflammation

MicroRNAs (miRNAs) have been shown to affect haematopoietic differentiation, acting as important determinants of cellular fate specification. The aim of this work is to investigate the role of miRNAs in the activation of T lymphocytes. By microarray analysis, we observed differential expression of miRNAs in distinct subclasses of T lymphocytes: CD8+ naïve, CD8+ memory effector, CD8+central memory; CD4+ naïve, CD4+ memory effector and CD4+ central memory. We found that miR-146a is expressed in separated primary T cells, in both CD4+ and CD8+ cells, but not in naïve T cells. Furthermore, a significative up-regulation of miR-146a expression levels was also obtained in the Jurkat T cell line, stimulated with αCD3-αCD28 antibodies, or with PMA and ionomycin, which are stimuli that mimic antigen recognition by TCR. Therefore, we attempted to discover the mechanisms of miR-146a transcriptional regulation. Some indications about the pathways possibly involved were obtained by treatment of induced Jurkat cells with specific pharmacological inhibitors of pathways triggered by TCR signalling.. We found that Cyclosporine (CsA), a Calcineurin inhibitor, significantly impaired miR-146a up-regulation, thus suggesting that the pathways affected by CsA play a key role in the induction of miR-146a upon TCR engagement. This observation prompted us to further investigate with molecular tools the transcription factors which are responsible for miR-146a expression in T lymphocytes. A deeper investigation to carefully characterize the cis regulatory elements involved in miR-146a regulation was performed through a bioinformatic analysis of miR-146a promoter and a luciferase assay. These approaches allowed us to identify conserved consensus sites for T lymphocytes-specific transcription factors, which are involved in pathways triggered by TCR signalling and are required for miR-146a induction. The results obtained indicate that NF-κB and c- ETS are involved in miR-146a induction in T cell activation pathway. Moreover, we also demonstrated that miR-146a is also induced in Jurkat cells by an inflammatory stimulus, TNF-α treatment, and NF-κB is the main transcription factor responsible of miR-146a induction. Another intriguing aspect of miRNAs study is the identification of miRNAs biological role in a cell. We identified miR-146a targets 5 taking advantage of a 3’-untranslated region (UTR) luciferase reporter assay. We tested a number of predicted miR-146a targets, relevant for T cell physiology. In particular, we report here that miR-146a downmodulates the apoptosis process via translational inhibition of FADD and it reduces AP-1 transcriptional activity, through the downregulation of c-FOS Altogether, our findings reveal miR-146a involvement in T cell activation process, significantly affecting key properties of lymphocytes

MicroRNAs as Molecular Switches in Macrophage Activation

The efficacy of macrophage- mediated inflammatory response relies on the coordinated expression of key factors, which expression is finely regulated at both transcriptional and post-transcriptional level. Several studies have provided compelling evidence that microRNAs play pivotal roles in modulating macrophage activation, polarization, tissue infiltration, and resolution of inflammation. In this review, we highlight the essential molecular mechanisms underlying the different phases of inflammation that are targeted by microRNAs to inhibit or accelerate restoration to tissue integrity and homeostasis. We further review the impact of microRNA-dependent regulation of tumor-associated macrophages and the relative implication for tumor biology

Multi-Step Regulation of the TLR4 Pathway by the miR-125a~99b~let-7e Cluster

An appropriate immune response requires a tight balance between pro- and anti-inflammatory mechanisms. IL-10 is induced at late time-points during acute inflammatory conditions triggered by TLR-dependent recognition of infectious agents and is involved in setting this balance, operating as a negative regulator of the TLR-dependent signaling pathway. We identified miR-125a~99b~let-7e as an evolutionary conserved microRNA cluster late-induced in human monocytes exposed to the TLR4 agonist LPS as an effect of this IL-10-dependent regulatory loop. We demonstrated that microRNAs generated by this cluster perform a pervasive regulation of the TLR signaling pathway by direct targeting receptors (TLR4, CD14), signaling molecules (IRAK1), and effector cytokines (TNFα, IL-6, CCL3, CCL7, CXCL8). Modulation of miR-125a~99b~let-7e cluster influenced the production of proinflammatory cytokines in response to LPS and the IL-10-mediated tolerance to LPS, thus identifying this gene as a previously unrecognized major regulatory element of the inflammatory response and endotoxin tolerance

Multi-Step Regulation of the TLR4 Pathway by the miR-125a~99b~let-7e Cluster

An appropriate immune response requires a tight balance between pro- and anti-inflammatory mechanisms. IL-10 is induced at late time-points during acute inflammatory conditions triggered by TLR-dependent recognition of infectious agents and is involved in setting this balance, operating as a negative regulator of the TLR-dependent signaling pathway. We identified miR-125a~99b~let-7e as an evolutionary conserved microRNA cluster late-induced in human monocytes exposed to the TLR4 agonist LPS as an effect of this IL-10-dependent regulatory loop. We demonstrated that microRNAs generated by this cluster perform a pervasive regulation of the TLR signaling pathway by direct targeting receptors (TLR4, CD14), signaling molecules (IRAK1), and effector cytokines (TNFα, IL-6, CCL3, CCL7, CXCL8). Modulation of miR-125a~99b~let-7e cluster influenced the production of proinflammatory cytokines in response to LPS and the IL-10-mediated tolerance to LPS, thus identifying this gene as a previously unrecognized major regulatory element of the inflammatory response and endotoxin tolerance

MiRNAs at the Crossroads between Innate Immunity and Cancer: Focus on Macrophages

Innate immune cells form an integrative component of the tumor microenvironment (TME), which can control or prevent tumor initiation and progression, due to the simultaneous processing of both anti- and pro-growth signals. This decision-making process is a consequence of gene expression changes, which are in part dependent on post-transcriptional regulatory mechanisms. In this context, microRNAs have been shown to regulate both recruitment and activation of specific tumor-associated immune cells in the TME. This review aims to describe the most important microRNAs that target cancer-related innate immune pathways. The role of exosomal microRNAs in tumor progression and microRNA-based therapeutic strategies are also discussed

The epigenetic landscape of innate immunity

The inflammatory response is the first line of defense against infectious agents or tissue damage. Innate immune cells are the crucial effectors regulating the different phase of inflammation. Their ability to timely develop an immune response is tightly controlled by the interplay of transcriptional and epigenetic mechanisms. The immunological imprinting elicited by exposure to different concentrations and types of infectious agents determine the functional fate of immune cells, forming the basis of innate immune memory. In this review we highlight the best-characterized examples of gene reprogramming occurring during different phases of inflammation with particular emphasis on the epigenetic marks that determine the specificity of the immune response. We further review the potential of cutting edge experimental techniques that have recently helped to reveal the deep complexity of epigenetic regulation during the inflammatory response