Silencing of microRNA-155 in mice during acute inflammatory response leads to derepression of c/ebp Beta and down-regulation of G-CSF

microRNA-155 (miR-155) has been implicated as a central regulator of the immune system, but its function during acute inflammatory responses is still poorly understood. Here we show that exposure of cultured macrophages and mice to lipopolysaccharide (LPS) leads to up-regulation of miR-155 and that the transcription factor c/ebp Beta is a direct target of miR-155. Interestingly, expression profiling of LPS-stimulated macrophages combined with overexpression and silencing of miR-155 in murine macrophages and human monocytic cells uncovered marked changes in the expression of granulocyte colony-stimulating factor (G-CSF), a central regulator of granulopoiesis during inflammatory responses. Consistent with these data, we show that silencing of miR-155 in LPS-treated mice by systemically administered LNA-antimiR results in derepression of the c/ebp Beta isoforms and down-regulation of G-CSF expression in mouse splenocytes. Finally, we report for the first time on miR-155 silencing in vivo in a mouse inflammation model, which underscores the potential of miR-155 antagonists in the development of novel therapeutics for treatment of chronic inflammatory diseases

Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates

The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1–2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non-high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates

Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver

MicroRNA-122 (miR-122) is an abundant liver-specific miRNA, implicated in fatty acid and cholesterol metabolism as well as hepatitis C viral replication. Here, we report that a systemically administered 16-nt, unconjugated LNA (locked nucleic acid)-antimiR oligonucleotide complementary to the 5′ end of miR-122 leads to specific, dose-dependent silencing of miR-122 and shows no hepatotoxicity in mice. Antagonism of miR-122 is due to formation of stable heteroduplexes between the LNA-antimiR and miR-122 as detected by northern analysis. Fluorescence in situ hybridization demonstrated uptake of the LNA-antimiR in mouse liver cells, which was accompanied by markedly reduced hybridization signals for mature miR-122 in treated mice. Functional antagonism of miR-122 was inferred from a low cholesterol phenotype and de-repression within 24 h of 199 liver mRNAs showing significant enrichment for miR-122 seed matches in their 3′ UTRs. Expression profiling extended to 3 weeks after the last LNA-antimiR dose revealed that most of the changes in liver gene expression were normalized to saline control levels coinciding with normalized miR-122 and plasma cholesterol levels. Combined, these data suggest that miRNA antagonists comprised of LNA are valuable tools for identifying miRNA targets in vivo and for studying the biological role of miRNAs and miRNA-associated gene-regulatory networks in a physiological context

Inflammatory response in the immature brainafter hypoxia-ischemia. Application of microarray and transgenic technology

Hypoxic-ischemic (HI) brain injury remains a common problem encountered during theneonatal period, and it is a major cause of perinatal mortality and long term neurologicalimpairments. After HI, an inflammatory reaction is elicited in the brain, which is believedto contribute to the secondary progression of brain injury.The aim of this thesis was to investigate the inflammatory response and its possiblepathophysiological role after HI in the immature brain.Using a model of cerebral HI in 7-day-old rats, the expression of caspase-1 and IL-18 wasinvestigated by RT-PCR, Western blot and immunohistochemistry and the expression ofIL-1beta by ELISA and immunohistochemistry at various time points after HI. Theinvolvement of different members of the IL-1 family in the development of HI injury wasinvestigated in 9-day-old mice by using different transgenic mice and comparing theirsusceptibility to HI injury with wild-type mice. Global changes in gene expression atvarious time points after HI was investigated with Affymetrix Gene Chips (MG-U74Av2).After HI, caspase-1 and IL-18 mRNA and protein were increased in a similar pattern,starting at 1 day of reperfusion and reaching maximal levels 14 days after the insult. IL-1betawas expressed maximally at 8h after HI, when a 7-fold increase was detected comparedwith contralateral hemispheres. IL-18 deficient mice showed reduced injury after HIcompared with wild-type mice in both gray (-21% overall injury) and white matter(remaining amount MBP and NF was 92% and 78% higher, respectively, in IL-18-deficient compared with control mice). Mice deficient for IL-1beta or the combination ofIL-1alfabeta showed no reduced injury after HI compared with wild-type. When studyingchanges in global gene expression, a total of 491 genes were differentially expressed 2h to72h after HI, using the Significance analysis of microarray (SAM) statistical program withthe following criteria (FDR <10%, fold change 1.5). More than 90% of the differentiallyexpressed genes were previously unreported after HI in the immature brain. Real-timePCR confirmed the expression of 11 upregulated genes. Of the differentially expressedgenes, 29% belonged to the group of immune-inflammatory related genes. Our dataindicate that microglia/macrophages, T- and B- cells, NK-cells, mast cells, dendritic cells,and polymorphonuclear leukocytes may participate in the response to HI. In addition,novel cytokines/chemokines, complement-related, IFN-regulated, components of theTIR/NF-kB pathway and a number of immuno-modulatory genes were induced.In conclusion, the results from this thesis indicate that IL-18 is involved in HI injury inboth gray and white matter, while IL-1 does not seem to be a contributor. In addition,this study has provided novel information about changes in inflammatory gene expressionafter HI, and several inflammatory mediators which may be of pathophysiologicalsignificance after neonatal HI have been identified

The Suitability of BV2 Cells as Alternative Model System for Primary Microglia Cultures or for Animal Experiments Examining Brain Inflammation

The role of microglia in neurodegeneration, toxicology and immunity is an expanding area of biomedical research requiring large numbers of animals. Use of a microglia-like cell line would accelerate many research programmes and reduce the necessity of continuous cell preparations and animal experimentation, provided that the cell line reproduces the in vivo situation or primary microglia (PM) with high fidelity. The immortalised murine microglial cell line BV-2 has been used frequently as a substitute for PM, but recently doubts were raised as to their suitability. Here, we re-evaluated strengths and potential short-comings of BV-2 cells. Their response to lipopolysaccharide was compared with the response of microglia in vitro and in vivo. Transcriptome (480 genes) and proteome analyses after stimulation with lipopolysaccharide indicated a reaction pattern of BV-2 with many similarities to that of PM, although the average upregulation of genes was less pronounced. The cells showed a normal regulation of NO production and a functional response to IFN-gamma, important parameters for appropriate interaction with T cells and neurons. BV-2 were also able to stimulate other glial cells. They triggered the translocation of NF-kappaB, and a subsequent production of IL-6 in astrocytes. Thus, BV-2 cells appear to be a valid substitute for PM in many experimental settings, incuding complex cell-cell interaction studies

Galectin-3 contributes to neonatal hypoxic-ischemic brain injury.

International audienceInflammation induced by hypoxia-ischemia (HI) contributes to the development of injury in the newborn brain. In this study, we investigated the role of galectin-3, a novel inflammatory mediator, in the inflammatory response and development of brain injury in a mouse model for neonatal HI. Galectin-3 gene and protein expression was increased after injury and galectin-3 was located in activated microglia/macrophages. Galectin-3-deficient mice (gal3-/-) were protected from injury particularly in hippocampus and striatum. Microglia accumulation was increased in the gal3-/- mice but accompanied by decreased levels of total matrix metalloproteinase (MMP)-9 and nitrotyrosine. The protection and increase in microglial infiltration was more pronounced in male gal3-/- mice. Trophic factors and apoptotic markers did not significantly differ between groups. In conclusion, galectin-3 contributes to neonatal HI injury particularly in male mice. Our results indicate that galectin-3 exerts its effect by modulating the inflammatory response

The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and in vivo conditions

Overall, the inflammatory potential of lipopolysaccharide (LPS) in vitro and in vivo was investigated using different omics technologies. We investigated the hippocampal response to intracerebroventricular (i.c.v) LPS in vivo, at both the transcriptional and protein level. Here, a time course analysis of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) showed a sharp peak at 4 h and a return to baseline at 16 h. The expression of inflammatory mediators was not temporally correlated with expression of the microglia marker F4/80, which did not peak until 2 days after LPS injection. Of 480 inflammation-related genes present on a microarray, 29 transcripts were robustly up-regulated and 90% of them were also detected in LPS stimulated primary microglia (PM) cultures. Further in vitro to in vivo comparison showed that the counter regulation response observed in vivo was less evident in vitro, as transcript levels in PM decreased relatively little over 16 h. This apparent deficiency of homeostatic control of the innate immune response in cultures may also explain why a group of genes comprising tnf receptor associated factor-1, endothelin-1 and schlafen-1 were regulated strongly in vitro, but not in vivo. When the overall LPS-induced transcriptional response of PM was examined on a large Affymetrix chip, chemokines and cytokines constituted the most strongly regulated and largest groups. Interesting new microglia markers included interferon-induced protein with tetratricopeptide repeat (ifit), immune responsive gene-1 (irg-1) and thymidylate kinase family LPS-inducible member (tyki). The regulation of the former two was confirmed on the protein level in a proteomics study. Furthermore, conspicuous regulation of several gene clusters was identified, for instance that of genes pertaining to the extra-cellular matrix and enzymatic regulation thereof. Although most inflammatory genes induced in vitro were transferable to our in vivo model, the observed discrepancy for some genes potentially represents regulatory factors present in the central nervous system (CNS) but not in vitro