Inhibition of CD200R1 expression by C/EBP beta in reactive microglial cells
© Dentesano et al.; licensee BioMed Central Ltd. 2012
Received: 9 March 2012
Accepted: 9 July 2012
Published: 9 July 2012
In physiological conditions, it is postulated that neurons control microglial reactivity through a series of inhibitory mechanisms, involving either cell contact-dependent, soluble-factor-dependent or neurotransmitter-associated pathways. In the current study, we focus on CD200R1, a microglial receptor involved in one of these cell contact-dependent mechanisms. CD200R1 activation by its ligand, CD200 (mainly expressed by neurons in the central nervous system),is postulated to inhibit the pro-inflammatory phenotype of microglial cells, while alterations in CD200-CD200R1 signalling potentiate this phenotype. Little is known about the regulation of CD200R1 expression in microglia or possible alterations in the presence of pro-inflammatory stimuli.
Murine primary microglial cultures, mixed glial cultures from wild-type and CCAAT/enhancer binding protein β (C/EBPβ)-deficient mice, and the BV2 murine cell line overexpressing C/EBPβ were used to study the involvement of C/EBPβ transcription factor in the regulation of CD200R1 expression in response to a proinflammatory stimulus (lipopolysaccharide (LPS)). Binding of C/EBPβ to the CD200R1 promoter was determined by quantitative chromatin immunoprecipitation (qChIP). The involvement of histone deacetylase 1 in the control of CD200R1 expression by C/EBPβ was also determined by co-immunoprecipitation and qChIP.
LPS treatment induced a decrease in CD200R1 mRNA and protein expression in microglial cells, an effect that was not observed in the absence of C/EBPβ. C/EBPβ overexpression in BV2 cells resulted in a decrease in basal CD200R1 mRNA and protein expression. In addition, C/EBPβ binding to the CD200R1 promoter was observed in LPS-treated but not in control glial cells, and also in control BV2 cells overexpressing C/EBPβ. Finally, we observed that histone deacetylase 1 co-immunoprecipitated with C/EBPβ and showed binding to a C/EBPβ consensus sequence of the CD200R1 promoter in LPS-treated glial cells. Moreover, histone deacetylase 1 inhibitors reversed the decrease in CD200R1 expression induced by LPS treatment.
CD200R1 expression decreases in microglial cells in the presence of a pro-inflammatory stimulus, an effect that is regulated, at least in part, by C/EBPβ. Histone deacetylase 1 may mediate C/EBPβ inhibition of CD200R1 expression, through a direct effect on C/EBPβ transcriptional activity and/or on chromatin structure.
KeywordsNeuroinflammation Reactive microglia CD200R1 C/EBPβ Neuron-microglia communication In vitro
In the presence of neuronal damage, microglial cells acquire reactive phenotypes characterized by both morphological and functional changes [1, 2]. Microglial activation is a beneficial response, aimed at re-establishing brain homeostasis and avoiding or minimizing neuronal damage. However, reactive microglial cells produce several factors (pro-inflammatory cytokines, reactive oxygen and nitrogen species) that are typical of an inflammatory response, with potential neurotoxic effects. Consequently, the progression and resolution of microglial activation need to be tightly controlled to avoid the negative secondary effects of excessive or chronic microglial reactivity .
In the normal brain, it has been postulated that microglial reactivity is maintained under control by a series of inhibitory mechanisms, in which signals arising from neuronal cells are thought to play an important role (reviewed in ). Alterations in these inhibitory signals can result in microglial activation. In the presence of brain tissue injury, microglial cells become activated with a pro-inflammatory phenotype, suggesting that the inhibitory mechanisms have been overcome. In the present work, we focused on the study of one of these inhibitory mechanisms: the CD200-CD200R1 ligand-receptor system.
In the central nervous system (CNS), microglial cells express CD200R1 and CD200 is constitutively expressed mainly by neurons. Results from studies using CD200-deficient mice suggest that this protein plays an important role in the inhibition of the microglial pro-inflammatory phenotype in the normal brain [5–8]. Results from in vitro studies also suggest a role for CD200 in the control of microglial activation [9, 10]. CD200 expression is decreased in the human brain of patients with multiple sclerosis [11, 12], and both CD200 and CD200R1 expression are decreased in the brain of Alzheimer’s disease patients . These observations suggest that the CD200-CD200R1 inhibitory pathway is altered in neurodegenerative disorders affecting the human brain, in which glial activation/neuroinflammation has been suggested to play a role in progression of the neurodegeneration.
Little is known about the molecular mechanisms involved in the regulation of CD200 and CD200R1 expression in physiological and pathological conditions or on the mechanisms involved in the control of the microglial pro-inflammatory response in the presence of CD200R1 stimulation. In terms of CD200, Rosenblum et al.  described the presence of functional DNA binding sites for tumor suppressor protein p53, a critical regulator of the apoptotic program, in the promoters of the human and mouse CD200 gene and suggested a role for CD200 in the regulation of apoptosis. Gorczynski’s group detected a C/EBPβ binding site in the promoter of the human CD200 gene that is required for the constitutive expression of CD200 . The same group also reported that STAT1α, NF-κB p65 and IRF-1 play a role in the regulation of CD200 inducible expression in human T-cell lines . Lyons et al.  showed that the anti-inflammatory cytokine IL-4 induced an increase in CD200 expression in rat neurons both in vivo and in vitro. In contrast, molecular mechanisms controlling the expression of CD200R1 have yet to be identified.
The CCAAT/enhancer binding protein β (C/EBPβ) transcription factor is known to play a role in the control of the expression of genes encoding pro-inflammatory factors in reactive glial cells [17, 18]. However, little is known about its role in the regulation of genes encoding anti-inflammatory factors. The objective of the present work was to study whether C/EBPβ plays a role in the regulation of CD200R1 expression in microglial cells. Using glial cultures from wild-type and C/EBPβ-deficient mice and BV2 cells (microglial cell line) overexpressing C/EBPβ, we show that this transcription factor down-regulates the expression of CD200R1 in reactive microglial cells, an effect that is mediated, at least in part, by histone deacetylase (HDAC) 1.
A colony of C/EBPβ+/- mice  on a C57BL/6-129 S6/SvEv background was used to obtain C/EBPβ+/+ and C/EBPβ-/- mixed glial cultures as previously described by Straccia et al.. Experiments were carried out in accordance with the Guidelines of the European Union Council (86/609/EU) and following the Spanish regulations (BOE 67/8509-12, 1988) for the use of laboratory animals, and were approved by the Ethics and Scientific Committees of Barcelona University and the Hospital Clínic de Barcelona.
Mixed glial cultures were obtained from pools of cerebral cortices of two- to four-day-old C57BL/6 wild-type mice as described by Gresa-Arribas et al. . In the experiments with C/EBPβ-deficient mice, C/EBPβ+/+ and C/EBPβ-/- mixed glial cultures were obtained from single 19-day-old embryos from C/EBPβ+/- progenitors as described by Straccia et al. , due to the infertility of C/EBPβ-/- females and a perinatal death rate of approximately 50% for C/EBPβ-/- neonates. The culture medium consisted of Dulbecco’s modified Eagle medium (DMEM)/F-12 nutrient mixture (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 0.1% penicillin-streptomycin (Invitrogen), and 0.5 mg/mL amphotericin B (Fungizone®, Invitrogen). Cells were maintained at 37°C in a 5% CO2 humidified atmosphere. Medium was replaced every five to sevendays.
Microglial cultures were prepared from 19 to 21 days in vitro (DIV) mixed glial cultures using the mild trypsinization method as previously described by our group . Briefly, the cultures were treated for 30 minutes with 0.06% trypsin in the presence of 0.25 mM ethylenediamine tetraacetic acid (EDTA) and 0.5 mM Ca2+. This resulted in the detachment of an intact layer of cells containing virtually all the astrocytes, leaving a population of firmly attached cells identified as >98% microglia. The microglial cultures were used 24 hours after isolation. Flow cytometry studies, qRT-PCR assays, quantitative chromatin immunoprecipitation (qChIP) and co-immunoprecipitation experiments were performed using primary mixed glial cultures due to the limited amount of primary microglial cells usually obtained.
Astroglia-enriched cultures were obtained as described by Saura et al. .
The mouse microglial cell line BV2 (generated from primary mouse microglia transfected with a v-raf/v-myc oncogene, Blasi et al. ) was kindly provided by Dr. Elisabetta Blasi (Dip. Scienze Igienistiche, Microbiologiche e Biostatistiche, Modena, Italy). Cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen), supplemented with 0.1% penicillin-streptomycin (Invitrogen) and 10% heat-inactivated FBS. Cells were maintained at 37°C in a 5% CO2 humidified atmosphere. Stable clones overexpressing the C/EBPβ LAP isoform were obtained. Briefly, BV2 cells were transfected with 1 μg pCDNA (empty vector) or pCDNA-LAP expression plasmids (Dr. Steven Smale, UCLA, USA) using Lipofectamine 2000 (Invitrogen). Positive clones were selected with Zeocin™ (150 μg/mL) (Invitrogen) associated resistance. Cells were seeded at a density of 5 × 104 cells/mL (1.6 × 104 cells/cm2) for immunocytochemistry and at 105 cells/mL (2.4 × 106 cells/cm2) for protein and RNA extraction. One day after seeding, the culture medium was replaced with fresh RPMI medium, and the cells were used one day later.
Cells were exposed to 100 ng/mL LPS from Escherichia coli (026:B6, Sigma-Aldrich, St. Louis, MO, USA) for different lengths of time.
The HDAC inhibitors suberoylanilide hydroxamic acid (SAHA) and MS-275 (Cayman Chemicals, Ann Arbor, MI, USA) were used at 100 nM, 500 nM, 1 μM and 10 μM. They were added to the cultures one hour before LPS treatment.
Cultured cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 20 minutes at room temperature. Non-specific staining was blocked by incubating cells with 10% normal donkey serum (Vector, Peterborough, UK) in PBS containing 1% BSA for 20 minutes at room temperature. Cells were then incubated overnight at 4°C with polyclonal goat anti-CD200R1 (1:50, R&D, Abingdon, UK), alone or in combination (mixed glial cultures) with monoclonal rat anti-CD11b (1:200, Serotec, Oxford, UK) or polyclonal rabbit anti-GFAP (1:1000, DAKO, Glostrub, DK) primary antibodies. After rinsing in PBS, cells were incubated for one hour at room temperature with donkey anti-goat ALEXA 488 (1:500) or ALEXA 594 (1:500), alone or in combination with donkey anti-rat ALEXA 594 (1:500) or donkey anti-rabbit ALEXA 546 (1:1000) secondary antibodies (Molecular Probes, Eugene, OR, USA). In the case of mixed glial cultures, cells were permeated with 0.3% Triton X-100 in PBS containing 1% BSA and 10% normal donkey serum for 20 minutes at room temperature following fixation. Cell nuclei were stained with Hoechst 33258 (Sigma). Microscopy images were obtained with an Olympus IX70 microscope (Olympus, Okoya, Japan) and a digital camera (CC-12, Olympus Soft Imaging Solutions GmbH, Hamburg, Germany).
Isolation of total and nuclear proteins
Total protein extracts were obtained from mixed glial cultures and BV2 cells. One well on a six-well plate was used for each experimental condition. After a cold PBS wash, cells were scraped off and recovered in 100 μL of radioimmunoprecipitation assay (RIPA) buffer per well (1% Igepal CA-630, 5 mg/mL sodium deoxycholate, 1 mg/mL SDS, protease inhibitor cocktail Complete® -Roche Diagnostics, Mannheim, Germany- in PBS). Samples were sonicated, centrifuged for five minutes at 10,400 g and stored at -20°C. The amount of protein was determined using the Lowry assay (Total protein kit micro-Lowry, Sigma-Aldrich).
Total protein extracts (50 μg) or immunoprecipitated samples were denatured (120 mM Tris HCl pH 6.8, 10% glycerol, 3% SDS, 20 mM dithiothreitol (DTT), and 0.4% bromophenol blue, 100°C for five minutes) and subjected to SDS-PAGE on a 10% polyacrylamide minigel, together with a molecular weight marker (Fullrange Rainbow Molecular Weight Marker, GE Healthcare, Little Chalfont, UK), and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA) for 90 minutes at 1 mA/cm2. The detection of the proteins of interest was performed as described by Gresa-Arribas et al. . Monoclonal mouse anti-C/EBPβ (1:500, Abcam, Cambridge, UK) and monoclonal mouse anti-β-actin (1:50000, Sigma-Aldrich) were used as primary antibodies. Horseradish peroxidase-labelled goat anti-mouse was used as the secondary antibody (1:5000, Santa Cruz Biotechnology, Temecula, CA, USA). The signal was developed with ECL-Plus (GE) and images were obtained using a VersaDoc System (Bio-Rad Laboratories, Hercules, CA, USA). The pixel intensities of the immunoreactive bands were quantified using the % adjusted volume feature of Quantity One 5.4.1 software (Bio-Rad Laboratories). Data are expressed as the ratio between the intensity of the C/EBPβ band and the loading control protein band (β-actin).
Quantitative real-time PCR
CD200R1 and C/EBPβ mRNA expression was determined in primary glial cultures and BV2 cells six hours after treatments. For isolation of total RNA, one well from six-well culture plates was used per experimental condition, with the exception of primary microglial cultures, where one 75 cm2 flask was considered. Total RNA was isolated using a High Pure RNA Isolation Kit (Roche Diagnostics) and 1.5 μg of RNA for each condition was reverse transcribed with random primers using Transcriptor Reverse Transcriptase (Roche Diagnostics). Three nanograms of cDNA were used to perform quantitative real-time PCR. The following primers (Integrated DNA Technology, IDT) were used: 5′-AGGAGGATGAAATGCAGCCTTA-3′ (Forward) and 5′-TGCCTCCACCTTAGTCACAGTATC-3′ (Reverse) to amplify mouse CD200R1 mRNA; 5′-AAGCTGAGCGACGAGTACAAGA-3′ (Forward) and 5′-GTCAGCTCCAGCACCTTGTG-3′ (Reverse) for mouse C/EBPβ. For the normalization of cycle threshold (Ct) values to an endogenous control, the following primers were used: 5′-CAACGAGCGGTTCCGATG-3′ (Forward) and 5′-GCCACAGGATTCCATACCCA-3′ (Reverse) for mouse β-actin mRNA and 5′-GTAACCCGTTGAACCCCATT-3′ (Forward) and CCATCCAATCGGTAGTAGCG (Reverse) for mouse Rn18s mRNA; β-actin and Rn18s mRNA levels were not altered by cell treatment. Real-time PCR was carried out using the IQ SYBR Green SuperMix (Bio-Rad Laboratories) in 15 μL of final volume using an iCycler MyIQ apparatus (Bio-Rad Laboratories). Samples were run for 50 cycles (95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 15 seconds). Relative gene expression values were calculated using the comparative Ct or ΔΔCt method  and iQ5 2.0 software (Bio-Rad Laboratories). Ct values were corrected according to the amplification efficiency of the respective primer pair which was estimated from standard curves generated by dilution of a cDNA pool.
CD200R1 protein expression was analyzed in control and LPS-treated (24 hours) mixed glial cultures and BV2 cells. For each experimental condition, 5 × 105 cells were collected and labelled with 10 μg/mL of polyclonal goat anti-CD200R1 (R&D, Abingdon, UK) or goat immunoglobulin G (IgG) (isotype control) in PBS for 20 minutes. Cells were washed once in PBS and incubated with 1 μg/mL of donkey anti-goat ALEXA 488 secondary antibody (Molecular Probes) for 20 minutes. Cell viability was evaluated by incubation with 10 μg/mL propidium iodide (Molecular Probes). CD200R1 expression was analyzed with gating on live cells, using the BD FACSCalibur™ platform (BD, Franklin Lakes, NJ, USA).
Quantitative chromatin immunoprecipitation (qChIP)
C/EBPβ putative binding sites in CD200R1 gene promoter and primers used in quantitative ChIP assay
Genomic localization with respect to ATG
Rev: 5′- GCCCTTACGCTTAACATCCA-3′
Fifty microliters of Dynabeads® protein A (Invitrogen, No.100.01D) were washed three times in PBS with 0.02% Tween-20 and incubated for two hours at 4°C with 4 μg of polyclonal rabbit anti-C/EBPβ (Santa Cruz Biotechnology), or with 4 μg of rabbit IgG (Santa Cruz Biotechnology) as negative control. Two hundred micrograms of protein obtained from chromatin extracts from control and LPS-treated (six hours) mixed glial cultures, as described above in the qChIP protocol, were incubated overnight with the antibody-Dynabeads® complex at 40 rpm on a rotating wheel at 4°C. The immuno complexes were pelleted using a magnetic rack and washed three times with PBS. Beads were removed from the samples by boiling in sample buffer (120 mM Tris HCl pH 6.8, 10% glycerol, 3% SDS, 20 mM DTT, and 0.4% bromophenol blue) for five minutes. Input (1/5 dilution) and immunoprecipitated samples were subjected to SDS-PAGE on a 10% polyacrylamide minigel. Western blot analysis was conducted as described above using monoclonal anti-HDAC1 antibody (1:2500, clone 2E10, Millipore) and monoclonal mouse anti-C/EBPβ antibody (1:500, Abcam). Samples immunoprecipitated with isotype-Dynabeads® or incubated with unlabeled beads were used as negative controls to demonstrate the specificity of the signal obtained.
Data presentation and statistical analysis
The results are presented as the means + standard error of the mean (SEM). Statistical analyses were performed using one-way or two-way analysis of variance (ANOVA) followed by the Bonferroni post-test when three or more experimental groups were compared. Student’s t-test was used to compare two experimental groups. Values of P <0.05 were considered statistically significant.
CD200R1 expression in microglial cells decreases in response to the pro-inflammatory stimulus LPS
The absence of C/EBPβ prevents the inhibition of CD200R1 expression in response to LPS in microglial cells
C/EBPβ overexpression inhibits CD200R1 expression
C/EBPβ binds to the CD200R1 promoter in response to LPS
Using our BV2 cell clones, we also assessed whether C/EBPβ overexpression resulted in increased binding of the transcription factor to the CD200R1 gene promoter. As in mixed glial cultures, significant C/EBPβ binding to the CD200R1 gene promoter in BV2-pCDNA cells was only detected after LPS treatment, at the binding site closest to the ATG translation start site (Figure 7B). Nevertheless, significant C/EBPβ binding at the same site was observed in untreated BV2-LAP cells. No differences in C/EBPβ binding were observed between control and LPS-treated BV2-LAP cells.
C/EBPβ interacts with HDAC1 and determines the inhibition of CD200R1 expression in microglial cells
The results demonstrate that C/EBPβ is critically involved in the regulation of CD200R1 gene expression in reactive microglial cells. CD200R1 expression decreases in microglia in response to the pro-inflammatory stimulus LPS. However, this effect is not observed in the absence of C/EBPβ and, in contrast, C/EBPβ overexpression results in a decrease in CD200R1 expression in microglial cells in basal conditions. We also show that, in response to LPS, C/EBPβ binds the CD200R1 promoter and that C/EBPβ interacts with HDAC1. These observations suggest that the decrease in CD200R1 expression induced by LPS in microglial cells is due, at least in part, to C/EBPβ transcriptional regulation through a mechanism involving histone deacetylation.
In the CNS, it has been suggested that the CD200-CD200R1 interaction is one of the cell-contact mechanisms involved in the regulation of microglial activity by neurons. Results from studies using CD200-deficient mice and experimental models of inflammatory diseases show that the CD200-CD200R1 interaction keeps microglial cells in a quiescent/surveying phenotype in which the pro-inflammatory response is inhibited, and that microglial cells show increased reactivity when the CD200-CD200R1 signal is impaired [6, 8, 25–28]. However, the mechanisms of inhibition of pro-inflammatory activity triggered by CD200-CD200R1 signaling in microglial cells have not been characterized. The signal transduction pathways responsible for the inhibitory effects of CD200R1 engagement have only been partially described in mouse mast cells overexpressing CD200R1 differentiated in vitro and in the human lymphoma cell line U937 . On the other hand, little is known about the molecular mechanisms regulating CD200 and CD200R1 expression in the CNS. We observed that microglial CD200R1 expression decreases in response to a pro-inflammatory stimulus. This effect would result in decreased interaction between CD200 and CD200R1 in the presence of neurons, which in turn would reduce the inhibitory input microglia receive from neurons in normal conditions. Consequently, one of the mechanisms contributing to the induction of a reactive microglia phenotype by pro-inflammatory factors may be the down-regulation of inhibitory pathways such as CD200-CD200R1 signaling.
Glial activation results in significant changes in the expression of a large number of genes, among them those encoding pro-inflammatory and anti-inflammatory molecules. The expression of these genes must be tightly regulated in order to orchestrate a controlled inflammatory response lasting no longer than necessary. Various transcription factors are involved in the regulation of this expression (NF-κB, AP-1, STATs, PPARs, C/EBPs), resulting in fine regulation of the inflammatory response from start to finish. The transcriptional regulation of CD200R1 has not been studied. We show here that C/EBPβ is one of the transcription factors that is activated by pro-inflammatory stimuli playing a role in the regulation of CD200R1 transcription. C/EBPβ does not appear to play a role in the constitutive expression of CD200R1 in microglial cells, given that we did not detect any alteration in basal levels of CD200R1 mRNA expression in control C/EBPβ-deficient glial cultures. Nevertheless, increased levels of C/EBPβ down-regulate CD200R1 expression, as observed in LPS-treated wild-type glial cultures and not in C/EBPβ-deficient cultures, as well as in BV2 cells overexpressing C/EBPβ. These results suggest that C/EBPβ upregulation in response to LPS contributes to the development of a pro-inflammatory phenotype in microglial cells through the inhibition of CD200R1 transcription.
In recent years, we have been studying the involvement of C/EBPβ in glial activation. Using in vitro and in vivo experimental approaches we have reported the expression of C/EBPβ in astroglial and microglial cells, and an increase in C/EBPβ expression in reactive glial cells in response to pro-inflammatory stimuli and neuronal death [18, 31–33]. This increase was further accentuated in reactive microglial cells of G93A-SOD1 mice (animal model of amyotrophic lateral sclerosis) and also observed in microglial cells in the spinal cord of amyotrophic lateral sclerosis patients . All these results suggest an active role for C/EBPβ in glial activation. C/EBP binding sites have been found in the promoters of many genes encoding pro-inflammatory molecules [34–36]. C/EBPβ regulates the LPS and LPS/IFNγ-induced transcription of IL-6, IL-1β, TNF-α, COX-2 and iNOS genes [18, 37–41]. Interestingly, C/EBPβ deficiency has a neuroprotective effect following ischemic  and excitotoxic injuries , as well as in an in vitro model of neuroinflammation . Nevertheless, little is known about the involvement of C/EBPβ in the transcriptional regulation of genes encoding anti-inflammatory molecules, and even less so in CNS cells. Some authors have reported a role for C/EBPβ in induction of the expression of the anti-inflammatory cytokine IL-10 in response to LPS [43, 44] or other stimuli [45, 46] in macrophages. These observations, together with the results of the present study, suggest that, apart from its role in the expression of pro-inflammatory molecules, C/EBPβ plays a role in the control of the expression of anti-inflammatory molecules, either activating or inhibiting their expression. This constitutes an additional point of regulation of the inflammatory response in glial cells by C/EBPβ.
Several mechanisms may be responsible for the inhibition of CD200R1 gene transcription by C/EBPβ. In vitro studies using different cell types have shown that the transactivating activity of C/EBPβ in the transcription of target genes can be inhibited by post-translational modifications such as sumoylation [47, 48], phosphorylation [49, 50], methylation , deacetylation [52, 53] and glycosylation . Interestingly, C/EBPβ has been shown to associate with co-repressor complexes containing HDACs in a gene promoter and inhibit gene transcription [55–57]. The acetylation status of histones is a key determinant of transcriptional activity (reviewed in ). Histone acetyltransferases and HDACs are the enzymes that reversibly catalyze histone acetylation. Recruitment of histone acetyltransferases to gene promoters is usually associated with the facilitation of gene transcription, while that of HDACs is associated with gene repression. However, it has been shown that a dynamic equilibrium between acetylation and deacetylation is also necessary for transcriptional activity. Using glial cultures, we found that C/EBPβ interacts with HDAC1 and that HDAC1 binds the CD200R1 promoter at a C/EBPβ consensus sequence following LPS treatment. These results, together with the observation that HDAC inhibitors reverse the LPS-induced reduction in CD200R1 expression, suggest that HDAC1 plays a role in the C/EBPβ-mediated repression of CD200R1 transcription observed in microglial cells in response to LPS treatment. However, the possible involvement of other HDACs cannot be ruled out. This mechanism of transcriptional repression could involve both histone deacetylation [55–57], resulting in changes in chromatin structure and consequently in transcriptional activity, and C/EBPβ deacetylation [52, 53], resulting in direct changes in transcriptional activity.
In recent years, several in vitro studies have shown that HDAC inhibitors down-regulate pro-inflammatory gene expression in macrophages and glial cells , as well as in other cell types [60–62], in response to inflammatory stimuli. HDAC inhibitors also attenuate the pro-inflammatory response in experimental models of cerebral ischemia  and endotoxemia in vivo. Our results suggest that the anti-inflammatory effects of HDAC inhibitors can be mediated, at least in part, by potentiating the transcription of genes involved in keeping the pro-inflammatory response under control, such as CD200R1.
We describe, for the first time, a molecular mechanism involved in the regulation of the expression of CD200R1 in microglial cells, in which the transcription factor C/EBPβ plays a role. CD200R1 expression decreases in microglial cells in response to a pro-inflammatory stimulus, reducing the input that inhibits the microglial pro-inflammatory phenotype in physiological conditions. We show that C/EBPβ, in addition to its known role in the regulation of the expression of pro-inflammatory genes, can also negatively regulate the expression of genes involved in the inhibition of the pro-inflammatory response, such as CD200R1, thus contributing to the development of the pro-inflammatory phenotype in microglial cells. HDAC1 may mediate the inhibition of CD200R1 expression by C/EBPβ through changes in chromatin structure and transcriptional activity.
analysis of variance
bovine serum albumin
CCAAT/enhancer binding protein β
central nervous system
days in vitro
Dulbecco’s modified Eagle’s medium
experimental autoimmune encephalomyelitis
fetal bovine serum
phosphate buffered saline
polymerase chain reaction
quantitative chromatin immunoprecipitation
quantitative reverse transcriptase-polymerase chain reaction
suberoylanilide hydroxamic acid
standard error of the mean
tumor necrosis factor.
We thank Colleen Croniger and Valeria Poli for the generous gift of C/EBPβ knockout mice, Teresa Domingo and colleagues at the animal facilities of the School of Pharmacy(University of Barcelona) for their professional care of the C/EBPβ knockout mice, Steven Smale for providing pCDNA and pCDNA-LAP expression plasmids, Isabel Crespo and Cristina López from the Cytomics Platform of IDIBAPS for their technical assistance in the flow cytometry experiments, and Peter Podlesniy and Joana Figueiro-Silva for advice in Co-IP experiments. GD and MS are recipients of contracts from IDIBAPS and JAE-CSIC, respectively. This study was supported by grants PI081396 and PI10/378 of the Instituto de Salud Carlos III, Spain, and La Marató de TV3 foundation.
- Hanish UK, Kettenmann H: Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci 2007, 10:1387–1394.View ArticleGoogle Scholar
- Ransohoff RM, Perry VH: Microglial physiology: unique stimuli, specialized responses. Annu Rev Immunol 2009, 27:119–145.View ArticlePubMedGoogle Scholar
- Gao H-M, Hong J-S: Why neurodegenerative diseases are progressive: uncontrolled inflammation drives disease progression. Trends Immunol 2008, 29:357–365.View ArticlePubMedPubMed CentralGoogle Scholar
- Ransohoff RM, Cardona AE: The myeloid cells of the central nervous system parenchyma. Nature 2010, 468:253–262.View ArticlePubMedGoogle Scholar
- Boudakov I, Liu J, Fan N, Gulay P, Wong K, Gorczynski RM: Mice lacking CD200R1 show absence of suppression of lipopolysaccharide-induced tumor necrosis factor-α and mixed leukocyte culture responses by CD200. Transplantation 2007, 84:251–257.View ArticlePubMedGoogle Scholar
- Broderick C, Hoek RM, Forrester JV, Liversidge J, Sedgwick JD, Dick AD: Constitutive retinal CD200 expression regulates resident microglia and activation state of inflammatory cells during experimental autoimmune uveoretinitis. Amm J Pathol 2002, 161:1669–1677.View ArticleGoogle Scholar
- Copland DA, Calder CJ, Raveney BJ, Nicholson LB, Phillips J, Cherwinski H, Jenmalm M, Sedgwick JD, Dick AD: Monoclonal antibody-mediated CD200 receptor signalling supresses macrophage activation and tissue damage in experimental autoimmune uveoretinitis. Am J Pathol 2007, 171:396–398.View ArticleGoogle Scholar
- Hoek RM, Ruuls SR, Murphy CA, Wright GJ, Goddard R, Zurawski SM, Blom B, Homola ME, Streit WJ, Brown MH, Barclay AN, Sedgwick JD: Down-regulation of the macrophage lineage through interaction with OX2 (CD200). Science 2000, 290:1768–1771.View ArticlePubMedGoogle Scholar
- Lyons A, Downer EJ, Crotty S, Nolan YM, Mills KH, Lynch MA: CD200 ligand-receptor interaction modulates microglial activation in vivo and in vitro: a role for IL-4. J Neurosci 2007, 27:8309–8313.View ArticlePubMedGoogle Scholar
- Lyons A, McQuillan A, Deighan BF, O’Reilly J-A, Downer EJ, Murphy AC, Watson M, Piazza A, O’Connell F, Griffin R, Mills KHG, Lynch MA: Decreased neuronal CD200 expression in IL-4-deficient mice results in increased neuroinflammation in response to lipopolysaccharide. Brain Behav Immun 2009, 23:1020–1027.View ArticlePubMedGoogle Scholar
- Koning N, Bö L, Hoek RM, Huitinga I: Downregulation of macrophage inhibitory molecules in multiple sclerosis lesions. Ann Neurol 2007, 62:504–514.View ArticlePubMedGoogle Scholar
- Koning N, Swaab DF, Hoek RM, Huitinga I: Distribution of the immune inhibitory molecules CD200 and CD200R in the normal central nervous system and multiple sclerosis lesions suggests neuron-glia and glia-glia interactions. J Neuropathol Exp Neurol 2009, 68:159–167.View ArticlePubMedGoogle Scholar
- Walker DG, Dalsing-Hernandez JE, Campbell NA, Lue L-F: Decreased expression of CD200 and CD200 receptor in Alzheimer’s disease: a potential mechanism leading to chronic inflammation. Exp Neurol 2009, 215:5–19.View ArticlePubMedGoogle Scholar
- Rosenblum MD, Olasz E, Woodliff JE, Johnson BD, Konkol MC, Gerber KA, Orentas RJ, Sandford G, Truitt RL: CD200 is a novel p53-target gene involved in apoptosis-associated immune tolerance. Blood 2008, 103:2691–2698.View ArticleGoogle Scholar
- Chen Z, Marsden PA, Gorczynski RM: Cloning and characterization of the human CD200 promoter region. Mol Immunol 2006, 43:579–587.View ArticlePubMedGoogle Scholar
- Chen Z, Marsden PA, Gorczynski RM: Role of a distal enhancer in the transcriptional responsiveness of the human CD200 gene to interferon-γ and tumor necrosis factor-α. Mol Immunol 2009, 46:1951–1963.View ArticlePubMedGoogle Scholar
- Cortes-Canteli M, Luna-Medina R, Sanz-Sancristobal M, Alvarez-Barrientos A, Santos A, Perez-Castillo A: CCAAT/enhancer binding protein beta deficiency provides cerebral protection following excitotoxic injury. J Cell Sci 2008, 121:1224–1234.View ArticlePubMedGoogle Scholar
- Straccia M, Gresa-Arribas N, Dentesano G, Ejarque-Ortiz A, Tusell JM, Serratosa J, Solà C, Saura J: Pro-inflammatory gene expression and neurotoxic effects of activated microglia are attenuated by absence of CCAAT/enhancer binding protein beta. J Neuroinflam 2011, 8:156–170.View ArticleGoogle Scholar
- Screpanti I, Romani L, Musiani P, Modesti A, Fattori E, Lazzaro D, Sellitto C, Scarpa S, Bellavia D, Lattanzio G, et al.: Lymphoproliferative disorder and imbalanced T-helper response in C/EBP beta-deficient mice. EMBO J 1995, 14:1932–1941.PubMedPubMed CentralGoogle Scholar
- Gresa-Arribas N, Serratosa J, Saura J, Solà C: Inhibition of CCAAT/enhancer binding protein δ expression by chrysin in microglial cells results in anti-inflammatory and neuroprotective effects. J Neurochem 2010, 115:526–536.View ArticlePubMedGoogle Scholar
- Saura J, Tusell JM, Serratosa J: High-yield isolation of murine microglia by mild trypsinization. Glia 2003, 44:183–189.View ArticlePubMedGoogle Scholar
- Saura J, Angulo E, Ejarque A, Casado V, Tusell JM, Moratalla R, Chen J-F, Schwarzschild MA, Lluis C, Franco R, Serratosa J: Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia. J Neurochem 2005, 95:919–929.View ArticlePubMedGoogle Scholar
- Blasi E, Barluzzi R, Bocchini V, Mazzolla R, Bistoni F: Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus. J Neuroimmunol 1990, 27:229–237.View ArticlePubMedGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Methods 2001, 25:402–408.View ArticlePubMedGoogle Scholar
- Wright GJ, Puklavec MJ, Willis AC, Hoek RM, Sedgwick JD, Brown MH, Barclay AM: Lymphoid/neuronal cell surface glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function. Immunity 2000, 13:233–242.View ArticlePubMedGoogle Scholar
- Meuth SG, Simon OJ, Grimm A, Melzer N, Herrmann AM, Spitzer P, Landgraf P, Wiendl H: CNS inflammation and neuronal degeneration is aggravated by impaired CD200-CD200R-mediated macrophage silencing. J Neuroimmunol 2008, 194:62–69.View ArticlePubMedGoogle Scholar
- Liu Y, Bando Y, Vargas-Lowy D, Elyaman W, Khoury SJ, Huang T, Reif K, Chitnis T: CD200R1 agonist attenuates mechanisms of chronic disease in a murine model of multiple sclerosis. J Neurosci 2010, 30:2025–2038.View ArticlePubMedPubMed CentralGoogle Scholar
- Chitnis T, Imitola J, Wang Y, Elyaman W, Chawla P, Sharuk M, Raddassi K, Bronson RT, Koury SJ: Elevated neuronal expression of CD200 protects Wlds mice from inflammation-mediated neurodegeneration. Am J Pathol 2007, 170:1695–1712.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang S, Cherwinski H, Sedgwick JD, Phillips JH: Molecular mechanisms of CD200 inhibition of mast cell activation. J Immunol 2004, 173:6786–6793.View ArticlePubMedGoogle Scholar
- Mihrshahi R, Barclay AN, Brown MH: Essential roles for Dok2 and RasGAP in CD200 receptor-mediated regulation of human myeloid cells. J Immunol 2009, 183:4879–4886.View ArticlePubMedPubMed CentralGoogle Scholar
- Ejarque-Ortiz A, Medina MG, Tusell JM, Pérez-González AP, Serratosa J, Saura J: Upregulation of CCAAT/enhancer binding protein β in activated astrocytes and microglia. Glia 2007, 55:178–188.View ArticlePubMedGoogle Scholar
- Valente T, Mancera P, Tusell JM, Serratosa J, Saura J: C/EBPβ expression in activated microglia in amyotrophic lateral sclerosis. Neurobiol Aging in press
- Pérez-Capote K, Saura J, Serratosa J, Solà C: Expression of C/EBPalpha and C/EBPbeta in glial cells in vitro after inducing glial activation by different stimuli. Neuroscience Lett 2006, 410:25–30.View ArticleGoogle Scholar
- Lowenstein CJ, Alley EW, Raval P, Snowman AM, Snyder SH, Russell SW, Murphy WJ: Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide. Proc Natl Acad Sci U S A 1993, 90:9730–9734.View ArticlePubMedPubMed CentralGoogle Scholar
- Wadleigh DJ, Reddy ST, Kopp E, Ghosh S, Herschman HR: Transcriptional activation of the cyclooxygenase-2 gene in endotoxin-treated RAW 264.7 macrophages. J Biol Chem 2000, 275:6259–6266.View ArticlePubMedGoogle Scholar
- Caivano M, Gorgoni B, Cohen P, Poli V: The induction of cyclooxygenase-2 mRNA in macrophages is biphasic and requires both CCAAT enhancer-binding protein beta (C/EBP beta) and C/EBP delta transcription factors. J Biol Chem 2001, 276:48693–48701.View ArticlePubMedGoogle Scholar
- Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T, Akira S: Transcription factors NF-IL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci U S A 1993, 90:10193–10197.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu H, Sidiropoulos P, Song G, Pagliari LJ, Birrer MJ, Stein B, Anrather J, Pope RM: TNF-alpha gene expression in macrophages: regulation by NF-kappa B is independent of c-Jun or C/EBP beta. J Immunol 2000, 164:4277–4285.View ArticlePubMedGoogle Scholar
- Gorgoni B, Maritano D, Marthyn P, Righi M, Poli V: C/EBP beta gene inactivation causes both impaired and enhanced gene expression and inverse regulation of IL-12 p40 and p35 mRNAs in macrophages. J Immunol 2002, 168:4055–4062.View ArticlePubMedGoogle Scholar
- Chen J, Ivashkiv LB: IFN-γ abrogates endotoxin tolerance by facilitating toll-like receptor-induced chromatin remodeling. Proc Natl Acad Sci U S A 2010, 107:19438–19443.View ArticlePubMedPubMed CentralGoogle Scholar
- Yan C, Cao J, Wu M, Zhang W, Jiang T, Yoshimura A, Gao H: Suppressor of cytokine signaling 3 inhibits LPS-induced IL-6 expression in osteoblasts by suppressing CCAAT/enhancer-binding protein beta activity. J Biol Chem 2010, 285:37227–37239.View ArticlePubMedPubMed CentralGoogle Scholar
- Kapadia R, Tureyen K, Bowen KK, Kalluri H, Johnson PF, Vemuganti R: Decreased brain damage and curtailed inflammation in transcription factor CCAAT/enhancer binding protein beta knockout mice following transient focal cerebral ischemia. J Neurochem 2006, 98:1718–1731.View ArticlePubMedGoogle Scholar
- Liu YW, Tseng HP, Chen LC, Chen BK, Chang WC: Functional cooperation of simian virus 40 promoter factor 1 and CCAAT/enhancer-binding protein beta and delta in lipopolysaccharide-induced gene activation of IL-10 in mouse macrophages. J Immunol 2003, 171:821–828.View ArticlePubMedGoogle Scholar
- Liu YW, Chen CC, Tseng HP, Chang WC: Lipopolysaccharide-induced transcriptional activation of interleukin-10 is mediated by MAPK- and NF-kappaB-induced CCAAT/enhancer-binding protein delta in mouse macrophages. Cell Signal 2006, 18:1492–1500.View ArticlePubMedGoogle Scholar
- Brenner S, Prösch S, Schenke-Layland K, Riese U, Gausmann U, Platzer C: cAMP-induced interleukin-10 promoter activation depends on CCAAT/enhancer-binding protein expression and monocytic differentiation. J Biol Chem 2003, 278:5597–5604.View ArticlePubMedGoogle Scholar
- Csóka B, Németh ZH, Virág L, Gergely P, Leibovich SJ, Pacher P, Sun CX, Blackburn MR, Vizi ES, Deitch EA, Haskó G: A2A adenosine receptors and C/EBPbeta are crucially required for IL-10 production by macrophages exposed to Escherichia coli. Blood 2007, 110:2685–2695.View ArticlePubMedPubMed CentralGoogle Scholar
- Eaton EM, Sealy L: Modification of CCAAT/enhancer-binding protein-beta by the small ubiquitin-like modifier (SUMO) family members, SUMO-2 and SUMO-3. J Biol Chem 2003, 278:33416–33421.View ArticlePubMedGoogle Scholar
- Wang W-L, Lee Y-C, Yang W-M, Chang W-M, Chang W-C, Wang J-M: Sumoylation of LAP1 is involved in the HDAC4-mediated repression of COX-2 transcription. Nucleic Acid Res 2008, 36:6066–6079.View ArticlePubMedPubMed CentralGoogle Scholar
- Ghosh AK, Bhattacharyya S, Mori Y, Varga J: Inhibition of collagen gene expression by interferon-gamma: novel role of the CCAAT/enhancer binding protein beta (C/EBPbeta). J Cell Physiol 2006, 207:251–260.View ArticlePubMedGoogle Scholar
- Dong L-Y, Sun G, Jiang L, Shao L, Hu Y, Jiang Y, Wang Y, An W: Epidermal growth factor down-regulates the expression of human hepatic stimulator substance via CCAAT/enhancer-binding protein β in HepG2 cells. Biochem J 2010, 431:277–287.View ArticlePubMedGoogle Scholar
- Pless O, Kowenz-Leutz E, Knoblich M, Lausen J, Beyermann M, Walsch MJ, Leutz A: G9a-mediated lysine methylation alters the function of CCAAT/enhancer-binding protein-beta. J Biol Chem 2008, 283:26357–26363.View ArticlePubMedPubMed CentralGoogle Scholar
- Ceseña TI, Cui TX, Subramanian L, Fulton CT, Iñiguez-Lluhí JA, Kwok RPS, Schwartz J: Acetylation and deacetylation regulate CCAAT/enhancer binding protein β at K39 in mediating gene transcription. Mol Cell Endocrinol 2008, 289:94–101.View ArticlePubMedGoogle Scholar
- Xu M, Nie L, Kim SH, Sun XH: STAT5-induced Id-1 transcription involves recruitment of HDAC1 and deacetylation of C/EBPbeta. EMBO J 2003, 22:893–904.View ArticlePubMedPubMed CentralGoogle Scholar
- Li X, Molina H, Huang H, Zhang YY, Liu M, Qian SW, Slawson C, Dias WB, Pandey A, Hart GW, Lane MD, Tang QQ: O-linked N-acetylflucosamine modification on CCAAT enhancer-binding protein beta: role during adipocyte differentiation. J Biol Chem 2009, 284:19248–19254.View ArticlePubMedPubMed CentralGoogle Scholar
- Di-Poï N, Desvergne B, Michalik L, Wahli W: Transcriptional repression of peroxisome proliferator-activated receptor β/δ in murine keratinocytes by CCAAT/enhancer-binding proteins. J Biol Chem 2005, 280:38700–38710.View ArticlePubMedGoogle Scholar
- Wiper-Bergeron N, Wu D, Pope L, Schild-Poulter C, Haché RJ: Stimulation of preadipocyte differentiation by steroid through targeting of an HDAC1 complex. EMBO J 2003, 22:2135–2145.View ArticlePubMedPubMed CentralGoogle Scholar
- Zuo Y, Qiang L, Farmer SR: Activation of CCAAT/enhancer-binding protein (C/EBP) expression by C/EBPβ during adipogenesis requires a peroxisome proliferators-activated receptor-γ-associated repression of HDAC1 at the C/ebpα gene promoter. J Biol Chem 2006, 281:7960–7967.View ArticlePubMedGoogle Scholar
- Shahbazian MD, Grunstein M: Functions of site-specific histone acetylation and deacetylation. Annu Rev Biochem 2007, 76:75–100.View ArticlePubMedGoogle Scholar
- Faraco G, Pittelli M, Cavone L, Fossati S, Porcu M, Mascagni P, Fossati G, Moroni F, Chiarugi A: Histone deacetylase (HDAC) inhibitors reduce the glial inflammatory response in vitro and in vivo. Neurobiol Dis 2009, 36:269–279.View ArticlePubMedGoogle Scholar
- Bode KA, Schroder K, Hume DA, Ravasi T, Heeg K, Sweet MJ, Dalpke AH: Histone deacetylase inhibitors decrease toll-like receptor-mediated activation of proinflammatory gene expression by impairing transcription factor recruitment. Immunology 2007, 122:596–606.View ArticlePubMedPubMed CentralGoogle Scholar
- Choi Y, Park SK, Kim HM, Kang JS, Yoon YD, Han SB, Han JW, Yang JS, Han G: Histone deacetylase inhibitor KBH-A42 inhibits cytokine production in RAW 264.7 macrophage cells and in in vivo endotoxemia model. Exp Mol Med 2008, 40:574–581.View ArticlePubMedPubMed CentralGoogle Scholar
- Leoni F, Fossati G, Lewis EC, Lee JK, Porro G, Pagani P, Modena D, Moras ML, Pozzi P, Reznikov LL, Siegnund B, Fantuzzi G, Dinarello CA, Mascagni P: The histone deacetylase inhibitor ITF2357 reduces production of pro-inflammatory cytokines in vitro and systemic inflammation in vivo. Mol Med 2005, 11:1–15.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim HJ, Rowe M, Ren M, Hong JS, Chen PS, Chiang DM: Histone deacetylase inhibitors exhibit anti-inflammatory and neuroprotective effects in a rat permanent ischemic model of stroke: multiple mechanisms of action. J Pharmacol Exp Ther 2007, 321:892–901.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.