Macrophages activated by the gram negative bacterial product lipopolysaccharide (LPS) switch their core metabolism from oxidative phosphorylation to glycolysis1. Inhibition of glycolysis with 2-deoxyglucose (2DG) suppressed LPS-induced Interleukin-1 beta (IL-1β) but not Tumour necrosis factor alpha (TNFα) in macrophages. A comprehensive metabolic map of LPS-activated macrophages revealed up-regulation of glycolytic and down-regulation of mitochondrial genes, which correlated directly with the expression profiles of altered metabolites. LPS strongly increased the TCA cycle intermediate succinate. Glutamine-dependent anerplerosis was the major source of succinate with the ‘Gamma-Aminobutyric Acid (GABA)-shunt’ pathway also playing a role. LPS-induced succinate stabilized Hypoxia-inducible factor 1α (HIF-1α), an effect inhibited by 2DG, with IL-1β as an important target. LPS also increases succinylation of several proteins. Succinate is therefore identified as a metabolite in innate immune signalling which leads to enhanced IL-1β production during inflammation.

The response to an innate immune challenge is conditioned by the time of day, but the molecular basis for this remains unclear. In myeloid cells, there is a temporal regulation to induction by lipopolysaccharide (LPS) of the proinflammatory microRNA miR-155 that correlates inversely with levels of BMAL1. BMAL1 in the myeloid lineage inhibits activation of NF-κB and miR-155 induction and protects mice from LPS-induced sepsis. Bmal1 has two miR-155-binding sites in its 3′-UTR, and, in response to LPS, miR-155 binds to these two target sites, leading to suppression of Bmal1 mRNA and protein in mice and humans. miR-155 deletion perturbs circadian function, gives rise to a shorter circadian day, and ablates the circadian effect on cytokine responses to LPS. Thus, the molecular clock controls miR-155 induction that can repress BMAL1 directly. This leads to an innate immune response that is variably responsive to challenges across the circadian day.inflammation | sepsis | circadian clock | miR-155 | Bmal1

MiRNAs regulate gene expression at a post-transcriptional level and their dysregulation can play major roles in the pathogenesis of many different forms of cancer, including neuroblastoma, an often fatal paediatric cancer originating from precursor cells of the sympathetic nervous system. We have analyzed a set of neuroblastoma (n = 145) that is broadly representative of the genetic subtypes of this disease for miRNA expression (430 loci by stem-loop RT qPCR) and for DNA copy number alterations (array CGH) to assess miRNA involvement in disease pathogenesis. The tumors were stratified and then randomly split into a training set (n = 96) and a validation set (n = 49) for data analysis. Thirty-seven miRNAs were significantly over- or under-expressed in MYCN amplified tumors relative to MYCN single copy tumors, indicating a potential role for the MYCN transcription factor in either the direct or indirect dysregulation of these loci. In addition, we also determined that there was a highly significant correlation between miRNA expression levels and DNA copy number, indicating a role for large-scale genomic imbalances in the dysregulation of miRNA expression. In order to directly assess whether miRNA expression was predictive of clinical outcome, we used the Random Forest classifier to identify miRNAs that were most significantly associated with poor overall patient survival and developed a 15 miRNA signature that was predictive of overall survival with 72.7% sensitivity and 86.5% specificity in the validation set of tumors. We conclude that there is widespread dysregulation of miRNA expression in neuroblastoma tumors caused by both over-expression of the MYCN transcription factor and by large-scale chromosomal imbalances. MiRNA expression patterns are also predicative of clinical outcome, highlighting the potential for miRNA mediated diagnostics and therapeutics.

MicroRNAs function as negative regulators of post-transcriptional gene expression, playing major roles in cellular differentiation. Several neuroblastoma cell lines can be induced to undergo differentiation by all-trans-retinoic acid (ATRA) and are used for modelling signalling pathways involved in this process. To identify miRNAs contributing to differentiation, we profiled 364 loci following ATRA treatment of neuroblastoma cell lines and found miR-10a and miR-10b to be highly over-expressed in SK-N-BE, LAN5, and SHSY-5Y. Ectopic over-expression of these miRNAs led to a major reprogramming of the transcriptome and a differentiated phenotype that was similar to that induced by ATRA in each of these cell lines. One of the predicted down-regulated miR-10a/b targets was nuclear receptor co-repressor 2 (NCOR2), a co-repressor of gene transcription which is known to suppress neurite outgrowth. NCOR2 was experimentally validated as a direct target of miR-10a/b, and siRNA mediated inhibition of this mRNA alone resulted in neural cell differentiation. Moreover, induction of differentiation could be blocked by ectopic up-regulation of NCOR2 using an expression construct lacking the miR-10a/b 3’ UTR target site. We conclude that miR-10a/b play major roles in the process of neural cell differentiation through direct targeting of NCOR2, which in turn induces a cascade of primary and secondary transcriptional alterations, including the down-regulation of MYCN.