NF-{kappa}B-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses

Activation of mammalian innate and acquired immune responses must be tightly regulated by elaborate mechanisms to control their onset and termination. MicroRNAs have been implicated as negative regulators controlling diverse biological processes at the level of posttranscriptional repression. Expression profiling of 200 microRNAs in human monocytes revealed that several of them (miR-146a/b, miR-132, and miR-155) are endotoxin-responsive genes. Analysis of miR-146a and miR-146b gene expression unveiled a pattern of induction in response to a variety of microbial components and proinflammatory cytokines. By means of promoter analysis, miR-146a was found to be a NF-{kappa}B-dependent gene. Importantly, miR-146a/b were predicted to base-pair with sequences in the 3' UTRs of the TNF receptor-associated factor 6 and IL-1 receptor-associated kinase 1 genes, and we found that these UTRs inhibit expression of a linked reporter gene. These genes encode two key adapter molecules downstream of Toll-like and cytokine receptors. Thus, we propose a role for miR-146 in control of Toll-like receptor and cytokine signaling through a negative feedback regulation loop involving down-regulation of IL-1 receptor-associated kinase 1 and TNF receptor-associated factor 6 protein levels

Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder

Mammalian microRNAs are emerging as key regulators of the development and function of the immune system. Here, we report a strong but transient induction of miR-155 in mouse bone marrow after injection of bacterial lipopolysaccharide (LPS) correlated with granulocyte/monocyte (GM) expansion. Demonstrating the sufficiency of miR-155 to drive GM expansion, enforced expression in mouse bone marrow cells caused GM proliferation in a manner reminiscent of LPS treatment. However, the miR-155–induced GM populations displayed pathological features characteristic of myeloid neoplasia. Of possible relevance to human disease, miR-155 was found to be overexpressed in the bone marrow of patients with certain subtypes of acute myeloid leukemia (AML). Furthermore, miR-155 repressed a subset of genes implicated in hematopoietic development and disease. These data implicate miR-155 as a contributor to physiological GM expansion during inflammation and to certain pathological features associated with AML, emphasizing the importance of proper miR-155 regulation in developing myeloid cells during times of inflammatory stress

Regulation of APC development, immune response, and autoimmunity by Bach1/HO-1 pathway in mice

APCs are essential for innate and adaptive immunity as well as self-immune tolerance. Here, we show that the Cap’n’collar member Bach1 regulates the generation of APCs, specifically macrophages and dendritic cells, in mice. The impaired APC development in Bach1^(-/-) mice was accompanied by defects in downstream T-cell responses and partial protection from experimental autoimmune encephalomyelitis. Genomewide analyses identified a panel of Bach1 target genes and ablation of the direct Bach1 target gene HO-1 exacerbated the impaired APC development observed in Bach1^(-/-) mice. This was attributed to the impaired ability of HO-1^(-/-)Bach1^(-/-) double mutants to produce upstreamAPC progenitor cells, including common myeloid progenitor (CMP)–Flk2^+. By contrast, we observed an increase in hematopoietic stem-progenitor cells (HSPCs) in these mice, suggesting a developmental block in the progression of HSPCs to CMP-Flk2^+ and subsequently APCs

Identification of targets of tumor suppressor microRNA-34a using a reporter library system

miRNAs play critical roles in various biological processes by targeting specific mRNAs. Current approaches to identifying miRNA targets are insufficient for elucidation of a miRNA regulatory network. Here, we created a cell-based screening system using a luciferase reporter library composed of 4,891 full-length cDNAs, each of which was integrated into the 3′ UTR of a luciferase gene. Using this reporter library system, we conducted a screening for targets of miR-34a, a tumor-suppressor miRNA. We identified both previously characterized and previously uncharacterized targets. miR-34a overexpression in MDA-MB-231 breast cancer cells repressed the expression of these previously unrecognized targets. Among these targets, GFRA3 is crucial for MDA-MB-231 cell growth, and its expression correlated with the overall survival of patients with breast cancer. Furthermore, GFRA3 was found to be directly regulated by miR-34a via its coding region. These data show that this system is useful for elucidating miRNA functions and networks

Encoding NF-κB temporal control in response to TNF: distinct roles for the negative regulators IκBα and A20

TNF-induced NF-κB activity shows complex temporal regulation whose different phases lead to distinct gene expression programs. Combining experimental studies and mathematical modeling, we identify two temporal amplification steps—one determined by the obligate negative feedback regulator IκBα—that define the duration of the first phase of NF-κB activity. The second phase is defined by A20, whose inducible expression provides for a rheostat function by which other inflammatory stimuli can regulate TNF responses. Our results delineate the nonredundant functions implied by the knockout phenotypes of iκbα and a20, and identify the latter as a signaling cross-talk mediator controlling inflammatory and developmental responses

Myeloid cell-targeted miR-146a mimic alleviates NF-κB-driven cytokine storm without interfering with CD19-specific CAR T cell activity against B cell lymphoma

Background: NF-κB is a key regulator of inflammation, myeloproliferation and cancer progression, with an important role in leukemogenesis. Despite therapeutic potential, targeting NF-κB proved challenging. However, in non-malignant myeloid cells NF-κB activity is tightly regulated through many molecular mechanisms, including miRNA. Methods: Here, we describe an original approach to NF-κB inhibition using miR146a, which targets upstream regulators of NF-κB signaling. We generated a myeloid cell-targeted NF-κB inhibitor by tethering a chemically-modified miR146a mimic oligonucleotide to a scavenger receptor (SR)/Toll-like receptor 9 (TLR9) ligand (C-miR146a). Results: Unlike an unconjugated miR-146a molecule, C-miR146a was rapidly internalized and delivered to cytoplasm of target myeloid cells such as macrophages or myeloid leukemia cells. C-miR146a reduced protein levels of classic miR-146a targets, IRAK1 and TRAF6, thereby efficiently blocking NF-κB activation in target cells. Intravenous injections of C-miR146a mimic to miR-146-deficient mice prevented excessive NF-κB activation in myeloid cells, thereby alleviating myeloproliferation and exaggerated inflammatory responses to bacterial challenge. The NF-κB-driven release of IL-1 and IL-6 from monocytes is known to be responsible for cytokine release syndrome (CRS), which can occur in response to bacterial infections, antibody-based therapies and relatively frequently as a serious adverse effect of chimeric antigen receptor (CAR) T-cell therapies. While low expression of miR146a has not yet been implicated in CRS, C-miR146a treatments did reduce pro-inflammatory activity of human monocytes, at the level of IL-1 and IL-6 production, induced by the CD19-specific but not by the naive CAR T cells in vitro. Repeated systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent CRS in xenotransplanted B-cell lymphoma model without impeding the on-target therapeutic effects of CAR T-cells against lymphoma cells. Conclusions: Our results demonstrate potential of using myeloid cell-targeted miR146a mimics for treatment of inflammatory diseases and prevention of potential side effects of immunotherapies. The SR/TLR9-targeted miR-146a mimic design provides an outline for the development of miRNA therapeutics for a variety of myeloid cell-related diseases

STAT3 Induction of miR-146b Forms a Feedback Loop to Inhibit the NF-kB to IL-6 Signaling Axis and STAT3-Driven Cancer Phenotypes

Interleukin-6 (IL-6)–mediated activation of signal transducer and activator of transcription 3 (STAT3) is a mechanism by which chronic inflammation can contribute to cancer and is a common oncogenic event. We discovered a pathway, the loss of which is associated with persistent STAT3 activation in human cancer. We found that the gene encoding the tumor suppressor microRNA miR-146b is a direct STAT3 target gene, and its expression was increased in normal breast epithelial cells but decreased in tumor cells. Methylation of the miR-146b promoter, which inhibited STAT3-mediated induction of expression, was increased in primary breast cancers. Moreover, we found that miR-146b inhibited nuclear factor kB (NF-kB)–dependent production of IL-6, subsequent STAT3 activation, and IL-6/STAT3–driven migration and invasion in breast cancer cells, thereby establishing a negative feedback loop. In addition, higher expression of miR-146b was positively correlated with patient survival in breast cancer subtypes with increased IL6 expression and STAT3 phosphorylation. Our results identify an epigenetic mechanism of crosstalk between STAT3 and NF-kB relevant to constitutive STAT3 activation in malignancy and the role of inflammation in oncogenesis

miR-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice

Excessive or inappropriate activation of the immune system can be deleterious to the organism, warranting multiple molecular mechanisms to control and properly terminate immune responses. MicroRNAs (miRNAs), ~22-nt-long noncoding RNAs, have recently emerged as key posttranscriptional regulators, controlling diverse biological processes, including responses to non-self. In this study, we examine the biological role of miR-146a using genetically engineered mice and show that targeted deletion of this gene, whose expression is strongly up-regulated after immune cell maturation and/or activation, results in several immune defects. Collectively, our findings suggest that miR-146a plays a key role as a molecular brake on inflammation, myeloid cell proliferation, and oncogenic transformation

miR-146a controls the resolution of T cell responses in mice

T cell responses in mammals must be tightly regulated to both provide effective immune protection and avoid inflammation-induced pathology. NF-κB activation is a key signaling event induced by T cell receptor (TCR) stimulation. Dysregulation of NF-κB is associated with T cell–mediated inflammatory diseases and malignancies, highlighting the importance of negative feedback control of TCR-induced NF-κB activity. In this study we show that in mice, T cells lacking miR-146a are hyperactive in both acute antigenic responses and chronic inflammatory autoimmune responses. TCR-driven NF-κB activation up-regulates the expression of miR-146a, which in turn down-regulates NF-κB activity, at least partly through repressing the NF-κB signaling transducers TRAF6 and IRAK1. Thus, our results identify miR-146a as an important new member of the negative feedback loop that controls TCR signaling to NF-κB. Our findings also add microRNA to the list of regulators that control the resolution of T cell responses