Myeloid C/EBPβ deficiency reshapes microglial gene expression and is protective in experimental autoimmune encephalomyelitis

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-017-0834-5) contains supplementary material, which is available to authorized users.

RNA-Seq transcriptomic profiling of primary murine microglia treated with LPS or LPS + IFNγ

“…4D ). In fact, previous work by our group has demonstrated reduced microglial capacity to phagocytose bacteria after LPS + IFNγ treatment 11 , which is in accordance with this result. Meanwhile, analysis of genes downregulated by both LPS and LPS + IFNγ, clustered in the “turquoise” module, revealed “Huntington’s, Alzheimer’s and Parkinson’s disease” to be among the most significant affected KEGG pathways (Fig.…”

“…Microglial primary cultures were prepared as described previously 11 . Briefly, cortices from P1-P3 mice were dissected, carefully stripped of their meninges and digested with 0.25% trypsin for 30 min at 37 °C.…”

“…LPS + IFNγ treatment induces the production of pro-inflammatory mediators in the BV2 microglial cell line and in primary microglial cells 10 . However, whereas in some cases IFNγ potentiates the effect of LPS, e.g., NO production; in others, IFNγ counteracts it, e.g., Ptges and Csf3 expression 11 . These results indicate that two stimuli are capable of activating different pathways and they are not redundant.…”

Development of a Chimeric Model to Study and Manipulate Human Microglia In Vivo

“…Many studies regarding microglia responses to LPS have been performed in vitro, by treating murine or human microglia directly with LPS (Abud et al, 2017;Pulido-Salgado et al, 2018). We therefore decided to recapitulate this approach by treating iMGLs made from the same GFP + cell line as the LPS-exposed xMGs, with LPS in vitro.…”

Angiotensin AT1 and AT2 receptor heteromer expression in the hemilesioned rat model of Parkinson’s disease that increases with levodopa-induced dyskinesia

“…To prepare mouse striatal primary microglial cultures, brain was removed from C57BL/6 mice of 2-4 days of age. Microglial cells were isolated following protocols described elsewhere (Newell et al, 2015;Pulido-Salgado et al, 2017;Saura et al, 2003) and grown in DMEM medium supplemented with 2 mM Lglutamine, 100 U/ml penicillin/streptomycin, MEM Non-Essential amino acids preparation (1/100) and 5% (v/v) heat inactivated Fetal Bovine Serum (FBS) (Invitrogen, Paisley, Scotland, UK). Brie y, striatum tissue was dissected, carefully stripped of its meninges and digested with 0.25% trypsin for 20 min at 37 ºC.…”