The online version of this article (doi:10.1186/s12974-017-0834-5) contains supplementary material, which is available to authorized users.
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/fl mice. Primary microglial cultures from C/EBPβfl/fl and LysMCre-C/EBPβfl/fl mice 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/fl and LysMCre-C/EBPβfl/fl mice treated systemically with lipolysaccharide or vehicle. Mice of LysMCre-C/EBPβfl/fl or 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/fl mice 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/fl mice 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.
Additional file 1: Tables S1-S6. List the genes significantly up-regulated (tables 1, 3 and 5) or down-regulated (tables 2, 4 and 6) by the absence of C/EBPβ in control (tables 1, 2), LPS-treated (tables 3, 4) and LPS+IFNγ-treated (tables 5, 6) primary microglial cultures. These data were obtained by RNAseq as described in Methods. (ZIP 253 kb)12974_2017_834_MOESM1_ESM.zip
Additional file 2: Figure S1. Weighted Gene Correlation Network Analyses of microglial activation, treatment and LysMCre-C/EBPβfl/fl phenotype. A) Correlation dendrogram of genes. WGCNA algorithm was applied to filtered expression of all samples, a soft-threshold for the similarity matrix of β = 9 was used, and module detection was obtained with a dynamic tree cut; color bar down of the dendrogram shows module pertainance of genes with a large turquoise module corresponding to genes downregulated upon treatment with either LPS or LPS + IFNγ. B) Hierarchical clustering of detected modules by WGCNA and genotype and treatment traits; module MEyellow is the closest group of genes related to the treatment trait, whereas MEviolet is for genotype. C) Module violet heatmap (top) and eigengene expression graph (bottom): module violet is the closest group of genes related to genotype effect, contains C/EBPβ and Lyz2. D) Metacore™ network obtained by literature described interactions among the genes in module violet evidences a network of genes described to interact with C/EBPβ as a central hub. E) Correlation network obtained with a threshold for distance of 0.3, with C/EBPβ as a central hub (node size relative to degree of node). (TIF 1462 kb)12974_2017_834_MOESM2_ESM.tif
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- Myeloid C/EBPβ deficiency reshapes microglial gene expression and is protective in experimental autoimmune encephalomyelitis
Jose M. Vidal-Taboada
Gerardo Garcia Diaz-Barriga
Josep M. Canals
- BioMed Central