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Myeloid C/EBP? deficiency reshapes microglial gene expression and is protective in experimental autoimmune encephalomyelitis.

ABSTRACT: CCAAT/enhancer binding protein ? (C/EBP?) is a transcription factor that regulates the expression of important pro-inflammatory genes in microglia. Mice deficient for C/EBP? show protection against excitotoxic and ischemic CNS damage, but the involvement in this neuroprotective effect of the various C/EBP?-expressing cell types is not solved. Since C/EBP?-deficient microglia show attenuated neurotoxicity in culture, we hypothesized that specific C/EBP? deficiency in microglia could be neuroprotective in vivo. In this study, we have tested this hypothesis by generating mice with myeloid C/EBP? deficiency.Mice with myeloid C/EBP? deficiency were generated by crossing LysMCre and C/EBP?fl/fl mice. Primary microglial cultures from C/EBP?fl/fl and LysMCre-C/EBP?fl/fl mice were treated with lipopolysaccharide ± interferon ? (IFN?) for 6 h, and gene expression was analyzed by RNA sequencing. Gene expression and C/EBP? deletion were analyzed in vivo in microglia isolated from the brains of C/EBP?fl/fl and LysMCre-C/EBP?fl/fl mice treated systemically with lipolysaccharide or vehicle. Mice of LysMCre-C/EBP?fl/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/EBP?fl/fl mice showed an efficiency of C/EBP? 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/EBP? deficiency. Transcriptomic analysis of C/EBP?-deficient primary microglia revealed C/EBP?-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/EBP? deletion. CNS expression of C/EBP? was upregulated in experimental autoimmune encephalitis and in multiple sclerosis samples. Finally, LysMCre-C/EBP?fl/fl mice showed robust attenuation of clinical signs in experimental autoimmune encephalitis.This study provides new data that support a central role for C/EBP? in the biology of activated microglia, and it offers proof of concept for the therapeutic potential of microglial C/EBP? inhibition in multiple sclerosis.

SUBMITTER: Pulido-Salgado M

PROVIDER: S-EPMC5356255 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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

Pulido-Salgado Marta M Vidal-Taboada Jose M JM Garcia Diaz-Barriga Gerardo G Serratosa Joan J Valente Tony T Castillo Paola P Matalonga Jonathan J Straccia Marco M Canals Josep M JM Valledor Annabel A Solà Carme C Saura Josep J

Journal of neuroinflammation 20170316 1

<h4>Background</h4>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 ...[more]

PMID: 28302135

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Project description:BackgroundLeucine rich repeat containing 4 (LRRC4), also known as netrin-G ligand-2 (NGL-2), belongs to the superfamily of LRR proteins and serves as a receptor for netrin-G2. LRRC4 regulates the formation of excitatory synapses and promotes axon differentiation. Mutations in LRRC4 occur in Autism Spectrum Disorder (ASD) and intellectual disability. Multiple sclerosis (MS) is a chronic neuroinflammatory disease with spinal cords demyelination and neurodegeneration. Here, we sought to investigate whether LRRC4 is involved in spinal cords neuron-associated diseases.MethodsLRRC4 was detected in the CNS of experimental autoimmune encephalomyelitis (EAE) mice by the use of real-time PCR and western blotting. LRRC4-/- mice were created and immunized with myelin oligodendrocyte glycoprotein peptide (MOG)35-55. Pathological changes in spinal cords of LRRC4-/- and WT mice 15 days after immunization were examined by using hematoxylin and eosin (H&E), Luxol Fast Blue (LFB) staining and immunohistochemistry. The number of Th1/Th2/Th17/Treg cells in spleens and blood were measured with flow cytometry. Differential gene expression in the spinal cords from WT and LRRC4-/- mice was analyzed by using RNA sequencing (RNA-seq). Adeno-associated virus (AAV) vectors were used to overexpress LRRC4 (AAV-LRRC4) and were injected into EAE mice to assess the therapeutic effect of AAV-LRRC4 ectopic expression on EAE.ResultsWe report that LRRC4 is mainly expressed in neuron of spinal cords, and is decreased in the spinal cords of the EAE mice. Knockout of LRRC4 have a disease progression quickened and exacerbated with more severe myelin degeneration and infiltration of leukocytes into the spinal cords. We also first found that Rab7b is high expressed in EAE mice, and the deficiency of LRRC4 induces the elevated NF-κB p65 by up-regulating Rab7b, and up-regulation of IL-6, IFN-γ and down-regulation of TNF-α, results in more severe Th1 immune response in LRRC4-/- mice. Ectopic expression of LRRC4 alleviates the clinical symptoms of EAE mice and protects the neurons from immune damages.ConclusionsWe identified a neuroprotective role of LRRC4 in the progression of EAE, which may be used as a potential target for auxiliary support therapeutic treatment of MS.

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Myeloid C/EBP? deficiency reshapes microglial gene expression and is protective in experimental autoimmune encephalomyelitis.

Publications

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

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