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Identifier 000433400
Title Understanding transcriptional and epigenetic regulation of macrophage activation in neonatal mice
Alternative Title Κατανόηση της μεταγραφικής και επιγενετικής ρύθμισης της ενεργοποίησης μακροφάγων στα νεογνά ποντικών
Author Εφραίμογλου, Χριστίνα Β.
Thesis advisor Τσατσάνης, Χρήστος
Abstract Macrophages are key regulators of innate immunity through their different activation phenotypes, M1 and M2, that they acquire by responding to diverse signals. Differentiation and activation of macrophages require the timely regulation of gene expression including transcription factors and epigenetic modifications. The M1 macrophage phenotype, that is, the state of activated pro-inflammatory macrophages, is characterized by increased glycolysis, induction of the pentose phosphate pathway, conversion of arginine to nitric acid by the inducible Nitric Oxide Synthase (iNOS), fatty acid synthesis and oxidation. Anti-inflammatory M2 macrophages exhibit increased glutamine uptake and catabolism, Arginase-1 metabolism of arginine, oxidative phosphorylation, enhanced fatty acid oxidation and glycolysis. Metabolism also regulates macrophage cell activation, mainly through the PI3K / AKT / mTOR pathway, whereas in recent years, epigenetic regulators appear to play an important role in regulating metabolism and therefore in macrophage activation. Previously performed siRNA screen, identified histone modifying enzymes being important for M1/M2 polarization. Earlier experiments in the laboratory have shown the different metabolic profiles of AKT1 - / - and AKT2 - / - macrophages as well as the possible differential role of PHF2 and PHF8, two histone demethylases, in macrophage activation. The purpose of this thesis was to study the role of PHF2, PHF8 and other epigenetic factors, in the regulation of macrophage metabolism in both adult and neonatal mice. The expression and role of histone modifying enzymes in the metabolic profile of macrophage-deficient mice for AKT1 or AKT2 kinase was also studied. In addition, other epigenetic factors were screened at the mRNA level to determine potential differential expression between adults and neonates. In the present work, primary macrophages from adult and neonatal mice were used, whose metabolic profile was examined using molecular techniques such as Elisa, real time PCR, Western Blot. We showed that neonatal macrophages inefficiently induced key components of proinflammatory reaction upon infection, such as Inducible Nitric Oxide Synthase, Tumor Necrosis Factor alpha and Interleukin-12 while they maintained higher basal levels for the first two. We demonstrated that neonatal macrophages induced much higher expression of Interleukin-10, an anti-inflammatory cytokine, upon TLR4 stimulation compared to adult macropahges. However, this difference was not evident when other anti-inflammatory regulators were examined, such IRAKm, Arginase 1, Fizz1, while CCAAT Enhancer Binding Protein beta, which appeared to be slightly higher in neonates upon LPS treatment. Transcription factors of the Interferon Regulatory Factors family such as IRF5 and IRF4 were identified to be higher in adults in unstimulated macrophages. IRF5, a regulator of M1 pro-inflammatory activation, was induced only in adults after TLR4 triggering. IRF4, a key regulator of M2 macrophages, was induced at later stages of activation and at comparable levels between adults and neonates. We also identified differences in the post-transcriptional regulators miR-155, which is higher in neonates both basally and 6 hours after LPS stimulation, and miRNA-147a, which was slightly elevated in neonatal macrophages compared to adult 6 hours following LPS stimulation. Furthermore, we showed that epigenetic regulation of innate immune response might vary due to the differential expression of the methyltransferases MLL1 and SETD7 as well as the histone demethylases JMJD3, PHF8 and PHF2. Finally, we demonstrated that the epigenetic factors SETD7, MLL1, MLL3, HDAC1, SIRT6 were regulated by the AKT pathway since they were differentially expressed in Akt1 or Akt2 deficient primary murine macrophages. In this master thesis, we propose some transcriptional, post-transcriptional and epigenetic regulators that might be responsible for the insufficient innate immune response of neonates.
Language English
Subject Innate immunity
Έμφυτη ανοσία
Issue date 2020-11-27
Collection   School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses
  Type of Work--Post-graduate theses
Permanent Link https://elocus.lib.uoc.gr//dlib/4/b/6/metadata-dlib-1603367448-635399-17732.tkl Bookmark and Share
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