title: Myeloid C/EBPβ deficiency reshapes microglial gene expression and is protective in experimental autoimmune encephalomyelitis.
abstract: CCAAT/enhancer binding protein β (C/EBPβ) is a transcription factor that regulates the expression of important pro-inflammatory genes in microglia. Mice deficient for C/EBPβ show protection against excitotoxic and ischemic CNS damage, but the involvement in this neuroprotective effect of the various C/EBPβ-expressing cell types is not solved. Since C/EBPβ-deficient microglia show attenuated neurotoxicity in culture, we hypothesized that specific C/EBPβ deficiency in microglia could be neuroprotective in vivo. In this study, we have tested this hypothesis by generating mice with myeloid C/EBPβ deficiency.
  
  
  
  Mice with myeloid C/EBPβ deficiency were generated by crossing LysMCre and C/EBPβfl/flmice. Primary microglial cultures from C/EBPβfl/fland LysMCre-C/EBPβfl/flmice were treated with lipopolysaccharide ± interferon γ (IFNγ) for 6 h, and gene expression was analyzed by RNA sequencing. Gene expression and C/EBPβ deletion were analyzed in vivo in microglia isolated from the brains of C/EBPβfl/fland LysMCre-C/EBPβfl/flmice treated systemically with lipolysaccharide or vehicle. Mice of LysMCre-C/EBPβfl/flor control genotypes were subjected to experimental autoimmune encephalitis and analyzed for clinical signs for 52 days. One- or two-way ANOVA or Kruskal-Wallis with their appropriate post hoc tests were used.
  
  
  
  LysMCre-C/EBPβfl/flmice showed an efficiency of C/EBPβ deletion in microglia of 100 and 90% in vitro and in vivo, respectively. These mice were devoid of female infertility, perinatal mortality and reduced lifespan that are associated to full C/EBPβ deficiency. Transcriptomic analysis of C/EBPβ-deficient primary microglia revealed C/EBPβ-dependent expression of 1068 genes, significantly enriched in inflammatory and innate immune responses GO terms. In vivo, microglial expression of the pro-inflammatory genes Cybb, Ptges, Il23a, Tnf and Csf3 induced by systemic lipopolysaccharide injection was also blunted by C/EBPβ deletion. CNS expression of C/EBPβ was upregulated in experimental autoimmune encephalitis and in multiple sclerosis samples. Finally, LysMCre-C/EBPβfl/flmice showed robust attenuation of clinical signs in experimental autoimmune encephalitis.
  
  
  
  This study provides new data that support a central role for C/EBPβ in the biology of activated microglia, and it offers proof of concept for the therapeutic potential of microglial C/EBPβ inhibition in multiple sclerosis.
authors.affiliation: Department of Biomedicine, Biochemistry and Molecular Biology Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.|Institute of Neurosciences, University of Barcelona, Barcelona, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Pulido-Salgado
authors.affiliation: Department of Biomedicine, Biochemistry and Molecular Biology Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.|Institute of Neurosciences, University of Barcelona, Barcelona, Spain.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Vidal-Taboada
authors.affiliation: Department of Biomedicine, Histology Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.|Institute of Neurosciences, University of Barcelona, Barcelona, Spain.
authors.forename: Gerardo
authors.initials: G
authors.lastname: Garcia Diaz-Barriga
authors.affiliation: Department of Cerebral Ischemia and Neurodegeneration, Institut d'Investigacions Biomèdiques de Barcelona, CSIC, IDIBAPS, Barcelona, Spain.
authors.forename: Joan
authors.initials: J
authors.lastname: Serratosa
authors.affiliation: Department of Biomedicine, Biochemistry and Molecular Biology Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.|Department of Cerebral Ischemia and Neurodegeneration, Institut d'Investigacions Biomèdiques de Barcelona, CSIC, IDIBAPS, Barcelona, Spain.|Institute of Neurosciences, University of Barcelona, Barcelona, Spain.
authors.forename: Tony
authors.initials: T
authors.lastname: Valente
authors.affiliation: Department of Pathology, Hospital Clinic, ISGlobal, CRESIB, Barcelona, Spain.
authors.forename: Paola
authors.initials: P
authors.lastname: Castillo
authors.affiliation: Department of Physiology and Immunology, School of Biology, University of Barcelona, Barcelona, Catalonia, Spain.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Matalonga
authors.affiliation: Department of Biomedicine, Biochemistry and Molecular Biology Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.|Department of Biomedicine, Histology Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.|Department of Cerebral Ischemia and Neurodegeneration, Institut d'Investigacions Biomèdiques de Barcelona, CSIC, IDIBAPS, Barcelona, Spain.|Institute of Neurosciences, University of Barcelona, Barcelona, Spain.
authors.forename: Marco
authors.initials: M
authors.lastname: Straccia
authors.affiliation: Department of Biomedicine, Histology Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.|Institute of Neurosciences, University of Barcelona, Barcelona, Spain.
authors.forename: Josep M
authors.initials: JM
authors.lastname: Canals
authors.affiliation: Department of Physiology and Immunology, School of Biology, University of Barcelona, Barcelona, Catalonia, Spain.
authors.forename: Annabel
authors.initials: A
authors.lastname: Valledor
authors.affiliation: Department of Cerebral Ischemia and Neurodegeneration, Institut d'Investigacions Biomèdiques de Barcelona, CSIC, IDIBAPS, Barcelona, Spain.
authors.forename: Carme
authors.initials: C
authors.lastname: Solà
authors.affiliation: Department of Biomedicine, Biochemistry and Molecular Biology Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain. josepsaura@ub.edu.|Institute of Neurosciences, University of Barcelona, Barcelona, Spain. josepsaura@ub.edu.
authors.forename: Josep
authors.identifier: 0000-0002-5601-4466
authors.initials: J
authors.lastname: Saura
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D000831:Animals, Newborn; D064229:Biological Ontologies; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000172:deficiency* / Q000235:genetics; D039481:CD11b Antigen / Q000378:metabolism; D002478:Cells, Cultured; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000209:etiology / Q000473:pathology* / Q000628:therapy; D005260:Female; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D008822:Mice, Transgenic; D017628:Microglia / Q000378:metabolism*; D008875:Middle Aged; D009103:Multiple Sclerosis / Q000473:pathology; D063308:Myelin-Oligodendrocyte Glycoprotein / Q000633:toxicity; D009569:Nitric Oxide / Q000378:metabolism; D010446:Peptide Fragments / Q000633:toxicity; D010587:Phagocytosis / Q000187:drug effects / Q000235:genetics
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D039481:CD11b Antigen; D008070:Lipopolysaccharides; D063308:Myelin-Oligodendrocyte Glycoprotein; D010446:Peptide Fragments; C445081:myelin oligodendrocyte glycoprotein (35-55); D009569:Nitric Oxide; D007371:Interferon-gamma
keywords: Interferon γ; Lipopolysaccharide; Neuroinflammation; RNA sequencing; Transcription factor