Abstract
BackgroundMicroglia 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.MethodsDue 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.ResultsC/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.ConclusionsThese 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.
Highlights
Microglia and astrocytes respond to homeostatic disturbances with profound changes of gene expression
Neither CD11b nor GFAP immunocytochemistry revealed differences between wild-type or C/ EBPb-null cultures in morphology of microglial cells or astrocytes, respectively (Figure 1C, F). These results indicate that wild-type and CCAAT/enhancer binding protein b (C/EBPb)-null mixed glial cultures do not differ in total cell density or in proportions or morphology of their two major cell types, astrocytes and microglia
Differential C/EBPb activation is triggered by LPS and LPS +interferon g (IFNg) Since the mRNA or protein levels of a transcription factor are of relative importance to study its functionality, we studied the DNA binding activity of C/EBPb in LPSor LPS+IFNg-treated glial cells
Summary
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 b (C/EBPb) 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/EBPb in glial activation we have analyzed pro-inflammatory gene expression and neurotoxicity in murine wild type and C/EBPb-null glial cultures. Glial activation is an inflammatory process that occurs in astrocytes and microglia to re-establish homeostasis of the CNS after a disequilibrium of normal physiology. CCAAT/enhancer binding protein b (C/EBPb) is a candidate to regulate pro-inflammatory gene expression in glial activation. C/EBPb deficiency provides neuroprotection following ischemic [26] or excitotoxic injuries [27]
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