Dentesano et al. Journal of Neuroinflammation 2012, 9:165 JOURNAL OF http://www.jneuroinflammation.com/content/9/1/165 NEUROINFLAMMATION RESEARCH Open Access Inhibition of CD200R1 expression by C/EBP beta in reactive microglial cells Guido Dentesano1, Marco Straccia1,2, Aroa Ejarque-Ortiz1, Josep M Tusell1, Joan Serratosa1, Josep Saura2 and Carme Solà1* Abstract Background: 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. Methods: 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. Results: 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. Conclusions: 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. Keywords: Neuroinflammation, Reactive microglia, CD200R1, C/EBPβ, Neuron-microglia communication, In vitro * Correspondence: [email protected] 1Department of Cerebral Ischemia and Neurodegeneration, Institut d’Investigacions Biomèdiques de Barcelona-Consejo Superior de Investigaciones Científicas (CSIC), Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), C/ Rosselló 161, 6th Floor, Barcelona E-08036, Spain Full list of author information is available at the end of the article © 2012 Dentesano et al.; licensee 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. Dentesano et al. Journal of Neuroinflammation 2012, 9:165 Page 2 of 13 http://www.jneuroinflammation.com/content/9/1/165 Background group detected a C/EBPβ binding site in the promoter of In the presence of neuronal damage, microglial cells the human CD200 gene that is required for the constitu- acquire reactive phenotypes characterized by both mor- tive expression of CD200 [15]. The same group also phological and functional changes [1,2]. Microglial acti- reported that STAT1α, NF-κB p65 and IRF-1 play a role vation is a beneficial response, aimed at re-establishing in the regulation of CD200 inducible expression in brain homeostasis and avoiding or minimizing neuronal human T-cell lines [16]. Lyons et al. [9] showed that the damage. However, reactive microglial cells produce sev- anti-inflammatory cytokine IL-4 induced an increase in eral factors (pro-inflammatory cytokines, reactive oxygen CD200 expression in rat neurons both in vivo and and nitrogen species) that are typical of an inflammatory in vitro. In contrast, molecular mechanisms controlling response, with potential neurotoxic effects. Conse- the expression of CD200R1 have yet to be identified. quently, the progression and resolution of microglial The CCAAT/enhancer binding protein β (C/EBPβ) activation need to be tightly controlled to avoid the transcription factor is known to play a role in the control negative secondary effects of excessive or chronic micro- of the expression of genes encoding pro-inflammatory glial reactivity [3]. factors in reactive glial cells [17,18]. However, little is In the normal brain, it has been postulated that micro- known about its role in the regulation of genes encoding glial reactivity is maintained under control by a series of anti-inflammatory factors. The objective of the present inhibitory mechanisms, in which signals arising from work was to study whether C/EBPβ plays a role in the neuronal cells are thought to play an important role regulation of CD200R1 expression in microglial cells. (reviewed in [4]). Alterations in these inhibitory signals Using glial cultures from wild-type and C/EBPβ-deficient can result in microglial activation. In the presence of mice and BV2 cells (microglial cell line) overexpressing brain tissue injury, microglial cells become activated C/EBPβ, we show that this transcription factor down- with a pro-inflammatory phenotype, suggesting that the regulates the expression of CD200R1 in reactive micro- inhibitory mechanisms have been overcome. In the glial cells, an effect that is mediated, at least in part, by present work, we focused on the study of one of these histone deacetylase (HDAC) 1. inhibitory mechanisms: the CD200-CD200R1 ligand- receptor system. Methods In the central nervous system (CNS), microglial cells Animals express CD200R1 and CD200 is constitutively expressed A colony of C/EBPβ+/- mice [19] on a C57BL/6-129 S6/ mainly by neurons. Results from studies using CD200- SvEv background was used to obtain C/EBPβ+/+ and C/ deficient mice suggest that this protein plays an EBPβ-/- mixed glial cultures as previously described by important role in the inhibition of the microglial pro- Straccia et al. [18]. Experiments were carried out in ac- inflammatory phenotype in the normal brain [5-8]. cordance with the Guidelines of the European Union Results from in vitro studies also suggest a role for Council (86/609/EU) and following the Spanish regula- CD200 in the control of microglial activation [9,10]. tions (BOE 67/8509-12, 1988) for the use of laboratory CD200 expression is decreased in the human brain of animals, and were approved by the Ethics and Scientific patients with multiple sclerosis [11,12], and both CD200 Committees of Barcelona University and the Hospital and CD200R1 expression are decreased in the brain Clínic de Barcelona. of Alzheimer’s disease patients [13]. These observations suggest that the CD200-CD200R1 inhibitory pathway Cell cultures is altered in neurodegenerative disorders affecting the Mixed glial cultures were obtained from pools of cere- human brain, in which glial activation/neuroinflammation bral cortices of two- to four-day-old C57BL/6 wild-type hasbeensuggestedtoplayaroleinprogressionof mice as described by Gresa-Arribas et al. [20]. In the the neurodegeneration. experiments with C/EBPβ-deficient mice, C/EBPβ+/+ Little is known about the molecular mechanisms and C/EBPβ-/- mixed glial cultures were obtained from involved in the regulation of CD200 and CD200R1 single 19-day-old embryos from C/EBPβ+/- progenitors expression in physiological and pathological conditions as described by Straccia et al. [18], due to the infertility or on the mechanisms involved in the control of the of C/EBPβ-/- females and a perinatal death rate of microglial pro-inflammatory response in the presence of approximately 50% for C/EBPβ-/- neonates. The culture CD200R1 stimulation. In terms of CD200, Rosenblum medium consisted of Dulbecco’s modified Eagle medium et al. [14] described the presence of functional DNA (DMEM)/F-12 nutrient mixture (Invitrogen, Carlsbad, binding sites for tumor suppressor protein p53, a critical CA, USA) supplemented with 10% fetal bovine serum regulator of the apoptotic program, in the promoters of (FBS, Invitrogen), 0.1% penicillin-streptomycin (Invitro- W the human and mouse CD200 gene and suggested a role gen), and 0.5 mg/mL amphotericin B (Fungizone , Invi- ’ for CD200 in the regulation of apoptosis. Gorczynski s trogen). Cells were maintained at 37°C in a 5% CO2 Dentesano et al. Journal of Neuroinflammation 2012, 9:165 Page 3 of 13 http://www.jneuroinflammation.com/content/9/1/165 humidified atmosphere. Medium was replaced every five incubating cells with 10% normal donkey serum (Vector, to sevendays. Peterborough, UK) in PBS containing 1% BSA for Microglial cultures were prepared from 19 to 21 days 20 minutes at room temperature. Cells were then incu- in vitro (DIV) mixed