Abstract
BackgroundCCAAT/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.MethodsMice 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.ResultsLysMCre-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.ConclusionThis 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.
Highlights
CCAAT/enhancer binding protein β (C/EBPβ) is a transcription factor that regulates the expression of important pro-inflammatory genes in microglia
C/EBPβ proteins were not detected in primary microglia from LysMCre-C/EBPβfl/fl mice, neither in control nor after LPS + interferon γ (IFNγ) treatment (Fig. 2a, b)
C/EBPβ was present in most microglial cells in primary cultures from C/EBPβfl/fl mice, and the number of positive cells and the intensity of the staining was enhanced by LPS + IFNγ treatment (24 h)
Summary
CCAAT/enhancer binding protein β (C/EBPβ) is a transcription factor that regulates the expression of important pro-inflammatory genes in microglia. There is strong evidence showing that pro-inflammatory gene expression by activated microglia is regulated by the transcription factors NF-κB, AP-1, CREB, STATs, C/EBPβ and C/EBPδ, whereas PPARγ and Nrf are two of the most important transcription factors regulating anti-inflammatory programs in microglia [3]. Because of their bottleneck position, transcription factors are potential targets to pharmacologically modulate whole cellular programs such as the neuroinflammatory one in activated microglia
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