title: Pro-inflammatory gene expression and neurotoxic effects of activated microglia are attenuated by absence of CCAAT/enhancer binding protein β.
abstract: BACKGROUND
  Microglia and astrocytes respond to homeostatic disturbances with profound changes of gene expression. This response, known as glial activation or neuroinflammation, can be detrimental to the surrounding tissue. The transcription factor CCAAT/enhancer binding protein β (C/EBPβ) is an important regulator of gene expression in inflammation but little is known about its involvement in glial activation. To explore the functional role of C/EBPβ in glial activation we have analyzed pro-inflammatory gene expression and neurotoxicity in murine wild type and C/EBPβ-null glial cultures.
  
  
  METHODS
  Due to fertility and mortality problems associated with the C/EBPβ-null genotype we developed a protocol to prepare mixed glial cultures from cerebral cortex of a single mouse embryo with high yield. Wild-type and C/EBPβ-null glial cultures were compared in terms of total cell density by Hoechst-33258 staining; microglial content by CD11b immunocytochemistry; astroglial content by GFAP western blot; gene expression by quantitative real-time PCR, western blot, immunocytochemistry and Griess reaction; and microglial neurotoxicity by estimating MAP2 content in neuronal/microglial cocultures. C/EBPβ DNA binding activity was evaluated by electrophoretic mobility shift assay and quantitative chromatin immunoprecipitation.
  
  
  RESULTS
  C/EBPβ mRNA and protein levels, as well as DNA binding, were increased in glial cultures by treatment with lipopolysaccharide (LPS) or LPS + interferon γ (IFNγ). Quantitative chromatin immunoprecipitation showed binding of C/EBPβ to pro-inflammatory gene promoters in glial activation in a stimulus- and gene-dependent manner. In agreement with these results, LPS and LPS+IFNγ induced different transcriptional patterns between pro-inflammatory cytokines and NO synthase-2 genes. Furthermore, the expressions of IL-1β and NO synthase-2, and consequent NO production, were reduced in the absence of C/EBPβ. In addition, neurotoxicity elicited by LPS+IFNγ-treated microglia co-cultured with neurons was completely abolished by the absence of C/EBPβ in microglia.
  
  
  CONCLUSIONS
  These findings show involvement of C/EBPβ in the regulation of pro-inflammatory gene expression in glial activation, and demonstrate for the first time a key role for C/EBPβ in the induction of neurotoxic effects by activated microglia.
authors.affiliation: Biochemistry and Molecular Biology Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.
authors.forename: Marco
authors.initials: M
authors.lastname: Straccia
authors.forename: Núria
authors.initials: N
authors.lastname: Gresa-Arribas
authors.forename: Guido
authors.initials: G
authors.lastname: Dentesano
authors.forename: Aroa
authors.initials: A
authors.lastname: Ejarque-Ortiz
authors.forename: Josep M
authors.initials: JM
authors.lastname: Tusell
authors.forename: Joan
authors.initials: J
authors.lastname: Serratosa
authors.forename: Carme
authors.initials: C
authors.lastname: Solà
authors.forename: Josep
authors.initials: J
authors.lastname: Saura
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D018920:Coculture Techniques; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D007249:Inflammation / Q000235:genetics* / Q000378:metabolism*; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017628:Microglia / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000166:cytology / Q000502:physiology; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D011247:Pregnancy
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D016207:Cytokines; D008070:Lipopolysaccharides; D009569:Nitric Oxide; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse
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