. 2017 Mar 16;14(1):54.

doi: 10.1186/s12974-017-0834-5.

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

Marta Pulido-Salgado^{1

2}, Jose M Vidal-Taboada^{1

2}, Gerardo Garcia Diaz-Barriga^{3

2}, Joan Serratosa⁴, Tony Valente^{1

4

2}, Paola Castillo⁵, Jonathan Matalonga⁶, Marco Straccia^{1

3

4

2}, Josep M Canals^{3

2}, Annabel Valledor⁶, Carme Solà⁴, Josep Saura^{7

8}

Affiliations

¹ Department of Biomedicine, Biochemistry and Molecular Biology Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.
² Institute of Neurosciences, University of Barcelona, Barcelona, Spain.
³ Department of Biomedicine, Histology Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.
⁴ Department of Cerebral Ischemia and Neurodegeneration, Institut d'Investigacions Biomèdiques de Barcelona, CSIC, IDIBAPS, Barcelona, Spain.
⁵ Department of Pathology, Hospital Clinic, ISGlobal, CRESIB, Barcelona, Spain.
⁶ Department of Physiology and Immunology, School of Biology, University of Barcelona, Barcelona, Catalonia, Spain.
⁷ Department of Biomedicine, Biochemistry and Molecular Biology Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain. [email protected].
⁸ Institute of Neurosciences, University of Barcelona, Barcelona, Spain. [email protected].

PMID: 28302135
PMCID: PMC5356255
DOI: 10.1186/s12974-017-0834-5

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

Marta Pulido-Salgado et al. J Neuroinflammation. 2017.

. 2017 Mar 16;14(1):54.

doi: 10.1186/s12974-017-0834-5.

Authors

Affiliations

¹ Department of Biomedicine, Biochemistry and Molecular Biology Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.
² Institute of Neurosciences, University of Barcelona, Barcelona, Spain.
³ Department of Biomedicine, Histology Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain.
⁴ Department of Cerebral Ischemia and Neurodegeneration, Institut d'Investigacions Biomèdiques de Barcelona, CSIC, IDIBAPS, Barcelona, Spain.
⁵ Department of Pathology, Hospital Clinic, ISGlobal, CRESIB, Barcelona, Spain.
⁶ Department of Physiology and Immunology, School of Biology, University of Barcelona, Barcelona, Catalonia, Spain.
⁷ Department of Biomedicine, Biochemistry and Molecular Biology Unit, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain. [email protected].
⁸ Institute of Neurosciences, University of Barcelona, Barcelona, Spain. [email protected].

PMID: 28302135
PMCID: PMC5356255
DOI: 10.1186/s12974-017-0834-5

Abstract

Background: 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.

Methods: Mice with myeloid C/EBPβ deficiency were generated by crossing LysMCre and C/EBPβ^fl/fl mice. Primary microglial cultures from C/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/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/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/fl mice treated systemically with lipolysaccharide or vehicle. Mice of LysMCre-C/EBPβ^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.

Results: LysMCre-C/EBPβ^fl/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/EBPβ^fl/fl mice showed robust attenuation of clinical signs in experimental autoimmune encephalitis.

Conclusion: 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.

Keywords: Interferon γ; Lipopolysaccharide; Neuroinflammation; RNA sequencing; Transcription factor.

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Figures

**Fig. 1**
Phenotypic characterization of LysMCre-C/EBPβ^fl/fl mice. a Body weight curves of female C/EBPβ^fl/fl (n = 10) and LysMCre-C/EBPβ^fl/fl (n = 6) mice shown as mean ± SEM. No significant differences in body weight are observed between both genotypes. b Representative microscopic images of haematoxylin and eosin staining of the mammary gland and spleen from 10-week-old C/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/fl mice. Normal ductal development and branching as well as unaltered adipose tissue in mammary gland from both genotypes are noted. Normal morphology of red and white pulps and megakaryocytes (*insets*) are also seen in both genotypes. No histological differences are observed also in the brain, lung, heart, liver, lung, kidney, femur bone and lumbar vertebrae (not shown). *Scale bars* 100 μm (*large images*) and 50 μm (*insets*)

**Fig. 2**
Reduced C/EBPβ protein in LysMCre-C/EBPβ^fl/fl microglia in culture. a Representative Western blot showing C/EBPβ LAP protein levels in primary microglial cultures from C/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/fl mice treated with vehicle (C) or LPS (100 ng/mL) + IFNγ (1 ng/mL) (L + I) for 24 h. Detection of the C/EBPβ isoforms Full and LIP in these samples required longer exposure. b Quantification of Western blot signals from four independent experiments as that shown in a. C/EBPβ protein levels are normalized using β-actin levels. Data are shown as mean + SEM. Significant effect of treatment is observed in C/EBPβ^fl/fl microglia (**p < 0.01), and significant effect of genotype is observed both in vehicle- and LPS + IFNγ-treated microglia (###p < 0.001). c Primary microglial cultures were treated as in a, fixed and C/EBPβ protein was analyzed by immunocytochemistry and nuclei were counterstained with DAPI. Note the nuclear C/EBPβ expression in virtually all microglial cells in C/EBPβ^fl/fl cultures which is enhanced by LPS + IFNγ and the complete absence of C/EBPβ immunoreactivity in LysMCre-C/EBPβ^fl/fl microglial cells in culture. *Scale bar* 100 μm. d, e Primary mixed glial cultures containing mainly astrocytes and microglia were treated as in a, fixed, immunostained for C/EBPβ (*red*) and the astroglial marker GFAP (*green* in d) or microglial marker Iba1 (*green* in e) and counterstained with DAPI. In C/EBPβ^fl/fl cultures, C/EBPβ immunostaining is observed in both GFAP-positive cells (astrocytes; *arrows*) and Iba1-positive cells (microglia; *arrowheads*), whereas in LysMCre-C/EBPβ^fl/fl cultures, C/EBPβ is only observed in astrocytes. *Scale bar* 50 μm

**Fig. 3**
LysMCre-C/EBPβ^fl/fl microglial function in culture. a Primary microglial cultures from C/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/fl mice were treated with vehicle (C), LPS (100 ng/mL) ± IFNγ (0.1, 1, 10, 30 ng/mL) for 48 h. NO production was estimated by measuring nitrites in the conditioned medium by the Griess reaction. Data show mean + SEM of six independent experiments. *Asterisks* show the significance of the treatment effect (**p < 0.01; ***p < 0.001 compared with the respective control conditions) and *number sign* shows the significance of the genotype effect (##p < 0.01; ###p < 0.001 compared with the respective C/EBPβ^fl/fl condition). b Mixed glial cultures from C/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/fl mice were treated for 24 h with vehicle (C) or LPS (100 ng/mL) + IFNγ (1 ng/mL) and processed for immunocytochemistry for NOS2 (*green*), the microglial marker CD11b (*red*) and the nuclear counterstain DAPI (*blue*). LPS-induced NOS2 expression in C/EBPβ^fl/fl cultures is markedly attenuated in LysMCre-C/EBPβ^fl/fl cultures. NOS2 expression is always microglial as revealed by CD11b colocalization. *Arrows* show NOS2-positive cells. *Scale bar*, 100 μm. c Primary glial cultures from C/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/fl mice were treated with vehicle (C), LPS (100 ng/mL) ± IFNγ (1 and 30 ng/mL). Five days after treatment, Iba1-positive microglial cells were counted as indicated in the “Methods” section. Data represent the percentage of microglial cells on day 0 shown as mean + SEM of three independent experiments. Note the marked AICD effect induced by LPS and particularly by LPS + IFNγ and the absence of differences between the two genotypes. d Primary microglial cell cultures from C/EBPβ^fl/fl and LysMCre C/EBPβ^fl/fl mice were treated with vehicle (C), LPS (100 ng/mL) or LPS + IFNγ (1 ng/mL) for 24 h prior to infection with fluorescent *Salmonella* for 30 min. Microglial cultures were fixed immediately (*white bars*) or 4 h later (*black bars*), and the percentage of infected microglial cells and the number of bacteria phagocytosed were analyzed. Data show mean + SEM of three independent experiments. The *asterisk* shows the significance of the differences between 30 min and 4 h (*p < 0.05; **p < 0.01 compared with the respective 30-min value) and the *number sign* shows the significance of the genotype effect (#p < 0.05; ##p < 0.01; ###p < 0.001 compared with the respective C/EBPβ^fl/fl condition)

**Fig. 4**
Treatment with LPS ± IFNγ (6 h) evidences differential transcriptomic changes between C/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/fl microglia. a Hierarchical clustering of total RNASeq RPKM normalized values separate samples neatly by treatment. Remarkably, C/EBPβ^fl/fl and LysMCre C/EBPβ^fl/fl groups are mixed in the control condition, but separate into subgroups once any treatment is applied to microglia (Euclidean distance). b Linear model fitting of filtered genes (RPKM sum of genes through samples >2) detects 10,888 genes as differentially expressed (DEGs) (lfc > 1, adjusted p value <0.01 Benjamini–Hochberg correction). Contrasts between groups are shown on the Venn diagram. More specifically, 1068 genes are detected as DEGs for the C/EBPβ^fl/fl vs LysMCre-C/EBPβ^fl/fl contrast. c Heatmap of C/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/fl DEGs (n = 1068) with applied hierarchical clustering (average linkage, Euclidean distance). Values are represented as SD from the normalized average for each gene. Sample clustering evidences three main groups; with cluster 1 (*pink*) corresponding to the control condition which contain subclusters separating C/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/fl. Remarkably, clusters 2 (*magenta*) and 3 (*purple*) correspond to C/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/fl conditions and contain subclusters organized by treatment. On the other hand, gene cluster classification (A–M) evidences the combination of patterns of expression among samples. In this figure, WT and KO refer to C/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/fl, respectively

**Fig. 5**
Ontology enrichment analyses reveal biological implication of obtained DEGs with microglial processes. C/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/fl DEGs where fed into the Genecodis platform for ontology enrichment. a p value and gene percentage annotation graph of the biological process (BP) category from Gene Ontology database (hypergeometric distribution). b p value and gene percentage annotation graph from Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses (hypergeometric distribution). c–e Heatmap of genes annotated with the enriched categories shown above (hierarchical clustering, average linkage, Euclidean distance, scale in SD over normalized gene expression in log2 values). Note the existence of gene clusters with distinct patterns of gene expression changes, e.g. cluster B in f corresponds to lysosome-related genes with reduced expression upon LPS ± IFNγ treatments, in C/EBPβ^fl/fl microglia that is attenuated in LysMCre-C/EBPβ^fl/fl microglia. In this figure, WT and KO refer to C/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/fl, respectively

**Fig. 6**
Reduced C/EBPβ expression in LysMCre-C/EBPβ^fl/fl microglia in vivo. a Representative Western blot showing C/EBPβ protein levels in microglia acutely isolated in vivo from the brains of C/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/fl mice treated i.p. with vehicle (C) or LPS (4 mg/kg) for 16 h. Two C/EBPβ isoforms are detected, Full (~38 kDa) and LAP (~35 kDa). b Quantification of Western blot signals from experiments as that shown in a (n = 4 mice/condition. C/EBPβ protein levels are normalized using β-actin levels. Data are shown as mean + SEM. LPS induces a significant increase in C/EBPβ protein levels in C/EBPβ^fl/fl microglia (***p < 0.001, compared with respective vehicle) and not in LysMCre-C/EBPβ^fl/fl microglia (###p < .001, compared with C/EBPβ^fl/fl). c Immunocytochemistry for C/EBPβ (*red*) and the microglial marker CD68 (*green*) in cytospin preparations of microglia isolated from the brains of C/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/fl mice treated as in a. Note the absence of C/EBPβ immunoreactivity in microglia from vehicle-treated mice, the nuclear C/EBPβ staining in virtually all microglial cells from LPS-treated C/EBPβ^fl/fl mice and the presence of C/EBPβ in a small fraction of microglial cells from LPS-treated LysMCre-C/EBPβ^fl/fl mice. *Scale bar* 25 μm. d Quantification of the proportion of C/EBPβ immunoreactive microglial cells in cytospin preparations as that shown in c. Data are expressed as percentage of C/EBPβ immunoreactive microglial cells and are shown as mean + SEM (n = 3 mice/condition). ***p < 0.001, compared with respective vehicle; ###p < .001, compared with respective C/EBPβ^fl/fl

**Fig. 7**
Reduced pro-inflammatory gene expression in C/EBPβ-deficient microglia in vivo and in vitro. Expression of C/EBPβ and five pro-inflammatory genes (Tnfa, Il23a, Csf3, Ptges and Cybb) was analyzed by qRT-PCR in microglia isolated from the brains of C/EBPβ^fl/fl and LysMCre-C/EBPβ^fl/fl mice treated i.p. with vehicle (C) or LPS (4 mg/kg) for 16 h (n = 4 mice/condition). *Insets* show the expression of the same genes in primary microglial cultures from both genotypes treated with vehicle (C), LPS (100 ng/mL) or LPS + IFNγ (1 ng/mL) for 6 h (n = 4 independent experiments). In all graphs, data are shown as mean + SEM. The *asterisk* shows the significance of the treatment effect, *p < 0.05; **p < 0.01; ***p < 0.001 compared with the respective control conditions. The *number sign* shows the significance of the genotype effect; #p < 0.05; ##p < 0.01; ###p < 0.001 compared with the respective C/EBPβ^fl/fl condition

**Fig. 8**
Neuroprotective effect of microglial C/EBPβ deficiency in EAE. a–d Time course analysis of C/EBPβ expression in the CNS in EAE. Expression was analyzed in vehicle-treated (CFA) mice and in EAE at 9, 14, 21 and 28 days post-injection (DPI). a, b C/EBPβ expression at mRNA level by qRT-PCR in the thoracic spinal cord (a) and hindbrain (b). c Representative Western blot image of C/EBPβ protein expression in the thoracic spinal cord in EAE. d Quantification of the C/EBPβ protein isoform LAP (n = 4–6 mice/condition). *p < 0.05; ** p < 0.01 compared with CFA. e EAE clinical scores of mice with myeloid C/EBPβ deficiency (LysMCre-C/EBPβ^fl/fl mice, n = 10) and the two parental control mouse lines (LysMCre, n = 12; C/EBPβ^fl/fl n = 12). Data show mean ± SEM. ***p < 0.001. f Western blot showing C/EBPβ protein levels in temporal cortex postmortem samples from three non-neurological controls (CT) and three multiple sclerosis (MS) patients. The three C/EBPβ isoforms, Full (~38 kDa), LAP (~35 kDa) and LIP (~21 kDa), are detected in these samples. g Quantification of C/EBPβ Western blots as that in f. Data show mean + SEM, n = 4 controls, n = 5 MS, *p < 0.05; ***p < 0.001

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

Affiliations

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

Authors

Affiliations

Abstract

Figures

References

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LinkOut - more resources

Full Text Sources

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