Modelling Neuroinflammation in vitro: a tool to test the potential neuroprotective effect of anti-inflammatory agents

Neuron-microglia co-cultures treated with pro-inflammatory agents are a useful tool to study neuroinflammation in vitro, where to test the potential neuroprotective effect of anti-inflammatory compounds. However, a great diversity of experimental conditions can be found in the literature, making difficult to select the working conditions when considering this approach for the first time. We compared the use of neuron-primary microglia and neuron-BV2 cells (a microglial cell line) co-cultures, using different neuron:microglia ratios, treatments and time post-treatment to induce glial activation and derived neurotoxicity. We show that each model requires different experimental conditions, but that both neuron-BV2 and neuron-primary microglia LPS/IFN-γ-treated co-cultures are good to study the potential neuroprotective effect of anti-inflammatory agents. The contribution of different pro-inflammatory parameters in the neurotoxicity induced by reactive microglial cells was determined. IL-10 pre-treatment completely inhibited LPS/IFN-γ-induced TNF-α and IL-6 release, and COX-2 expression both in BV2 and primary microglial cultures, but not NO production and iNOS expression. However, LPS/IFN-γ induced neurotoxicity was not inhibited in IL-10 pre-treated co-cultures. The inhibition of NO production using the specific iNOS inhibitor 1400 W totally abolished the neurotoxic effect of LPS/IFN-γ, suggesting a major role for NO in the neurotoxic effect of activated microglia. Consequently, among the anti-inflammatory agents, special attention should be paid to compounds that inhibit NO production

Pro-inflammatory gene expression and neurotoxic effects of activated microglia are attenuated by absence of CCAAT/enhancer binding protein β

Background. Microglia and astrocytes respond to homeostatic disturbances with profound changes of gene expression. This response, known as glial activation or neuroinflammation, can be detrimental to the surrounding tissue. The transcription factor CCAAT/enhancer binding protein ß (C/EBPß) is an important regulator of gene expression in inflammation but little is known about its involvement in glial activation. To explore the functional role of C/EBPß in glial activation we have analyzed pro-inflammatory gene expression and neurotoxicity in murine wild type and C/EBPß-null glial cultures. Methods. Due to fertility and mortality problems associated with the C/EBPß-null genotype we developed a protocol to prepare mixed glial cultures from cerebral cortex of a single mouse embryo with high yield. Wild-type and C/EBPß-null glial cultures were compared in terms of total cell density by Hoechst-33258 staining; microglial content by CD11b immunocytochemistry; astroglial content by GFAP western blot; gene expression by quantitative real-time PCR, western blot, immunocytochemistry and Griess reaction; and microglial neurotoxicity by estimating MAP2 content in neuronal/microglial cocultures. C/EBPß DNA binding activity was evaluated by electrophoretic mobility shift assay and quantitative chromatin immunoprecipitation. Results. C/EBPß mRNA and protein levels, as well as DNA binding, were increased in glial cultures by treatment with lipopolysaccharide (LPS) or LPS + interferon ¿ (IFN¿). Quantitative chromatin immunoprecipitation showed binding of C/EBPß to pro-inflammatory gene promoters in glial activation in a stimulus- and gene-dependent manner. In agreement with these results, LPS and LPS+IFN¿ induced different transcriptional patterns between pro-inflammatory cytokines and NO synthase-2 genes. Furthermore, the expressions of IL-1ß and NO synthase-2, and consequent NO production, were reduced in the absence of C/EBPß. In addition, neurotoxicity elicited by LPS+IFN¿-treated microglia co-cultured with neurons was completely abolished by the absence of C/EBPß in microglia

Inhibition of CD200R1 expression by C/EBP beta in reactive microglial cells

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

The transcription factor C/EBPd represses a-synuclein transcription: potential pathogenic effects of C/EBPd deficiency in Parkinson's disease

Trabajo presentado en el XI Simposi de Neurobiologia: Future technical advances, organizado por la Socitat Catalana de Biologia, en Barcelona, los días 12 y 13 de noviembre de 2018α-Synuclein, one of the most abundant proteins in neuronal cytosol, plays an ill-defined role in neurotransmitter release and synaptic vesicle trafficking and is the main component of Lewy bodies, the intracellular protein aggregates that are considered the histological hallmark of Parkinson’s disease. High α-synuclein levels are associated with increased risk for Parkinson's disease. Surprisingly little is known about the regulation of transcription of the human αsynuclein (SNCA) gene. CCAAT/enhancer binding protein δ (C/EBPδ) is a b-zip transcription factor expressed in the CNS that plays distinct roles in neurons and glial cells. C/EBPδ binding boxes are present in the SNCA genomic region, suggesting that this transcription factor could regulate SNCA transcription. The aim of this study was to determine if C/EBPδ regulates the expression of SNCA. We first observed that α-synuclein expression was markedly increased in C/EBPδ-deficient mice in several brain regions, both at mRNA and protein level. α-synuclein levels were also increased in C/EBPδ-deficient primary neuronal, but not glial, cultures. In accordance, C/EBPδ overexpression in neuroblastoma cells and in primary neuronal cultures markedly reduced α-synuclein expression. ChIP experiments demonstrated C/EBPδ binding to the SNCA genomic region of mice and humans. Finally, decreased C/EBPδ expression was observed in the substantia nigra and in iPSC-derived dopaminergic neurons from Parkinson patients resulting in a significant negative correlation between α-synuclein and C/EBPδ levels. This study demonstrates for the first time that C/EBPδ is a potent repressor of SNCA transcription. These findings suggest that reduced C/EBPδ neuronal levels could be a pathogenic factor in Parkinson’s disease and other synucleinopathies and C/EBPδ activity a potential pharmacological target to treat these neurological disordersSupported by: PI14/302 from the Instituto de Salud Carlos III, Spain, cofinanced with FEDER funds.Peer reviewe

Temporal and regional pattern of expression of pro-inflammatory and anti-inflammatory genes in mouse experimental autoimmune encephalomyelitis

Póster presentado en el XI European Meeting on Glial Cells in Health and Disease, celebrado los días 3 al 6 de julio de 2013 en Berlín (Alemania)The role of reactive glia in the etiology and progress of neurological diseases is still unknown. Reactive glia produce several factors, typical of an inflammatory response, with a potential neurotoxic effect, highlighting the relevance of a strict control of the progression and resolution of glial activation. Under some circumstances glial activation does not resolve, but it exacerbates or becomes chronic resulting in detrimental secondary effects. It is necessary to develop strategies to halt the negative outcome of glial activation in these situations, by controlling the pro-inflammatory phenotype of reactive glial cells and potentiating their beneficial effects. We studied the temporal pattern of inflammatory reaction in spinal cord and brain regions in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Special attention was paid to the involvement of members of the C/EBP family of transcription factors, which regulate the expression of pro-inflammatory genes in reactive glia, and CD200, CD200R1 and TREM-2, membrane-associated inhibitors of the pro-inflammatory response in resting/surveillant microglia. Mice were scored for signs of EAE for up to 28 days postimmunization (DPI). EAE symptoms were present from 10 DPI, reaching score peak at 14 DPI. In the spinal cord, we observed a sustained increase in C/EBPα and a transient increase in C/EBPβ and C/EBPδ expression peaking at 14 DPI. CD200 expression decreased at 9 DPI, remaining below control values at 28 DPI. CD200R1 and TREM-2 expression increased at 14 DPI, thereafter this effect progressively attenuated. No alterations were observed in the brain. An acute increase in the expression of pro-inflammatory genes (IL-1β, IL-6, TNF-α, iNOS, COX2) occurred in the spinal cord at 14 DPI. Our results suggest that CD200 expression decreases before onset of EAE symptoms resulting in downregulation of the microglia inhibitory signal mediated by CD200-CD200R1, which would facilitate the pro-inflammatory response observed after onset of the EAE symptoms. The increase in CD200R1 expression observed after the onset of EAE symptoms suggests a concomitant intend of microglia/macrophages to resolve the inflammatory response. The transient increase in C/EBPβ and C/EBPδ expression could be related to the acute increase in pro-inflammatory gene expression, while the sustained increases of C/EBPα expression and TREM-2 could be involved in the resolution of the inflammatory responseSupported by grants PI10/378 and PI12/00709 (Instituto de Salud Carlos III, Spain)Peer Reviewe

The microglial inhibitory receptor CD200R1 as a candidate target to control neuroinflammation

Póster presentado en el IX Simposi de Neurobiologia Experimental, celebrado los días 22 y 23 de octubre de 2014 en Barcelona y organizado por la Societat Catalana de Biologia del Institut d'Estudis CatalansMicroglia are the main endogenous immune cells of the central nervous system. In response to noxious stimuli, they develop reactive phenotypes to re-establish cerebral homeostasis and minimize neuronal damage. However, reactive microglia produce pro-inflammatory factors with potential neurotoxic effects. Consequently, progress and resolution of microglial activation have to be tightly controlled to avoid negative secondary effects. Neuronal signals play a relevant role in the control of microglial activation. Among them, inhibitory mechanisms such as CD200 ligand (neuronal)-CD200R1 receptor (microglial) interaction, maintain under control the microglia inflammatory phenotype in physiological conditions. Alterations in CD200 and CD200R1 expression have been described in neurodegenerative diseases. The aim of the present work was to study the effect of CD200R1 modulation on microglia reactive phenotype using experimental in vitro approaches. Glial cell cultures were treated with a proinflammatory stimulus (LPS+IFN-γ) and the pattern of expression of pro- and anti-inflammatory molecules was determined in the absence and presence of CD200R1 overexpression, CD200R1 stimulation and CD200R1 inhibition. CD200R1 overexpression resulted in a reduced induction of the expression of pro-inflammatory cytokines and enzymes and a strong induction of IL-10 expression in BV2 microglial cells. The induction of the expression of pro-inflammatory molecules was inhibited in reactive primary microglial cells treated with a CD200R1 agonist, while the expression of antiinflammatory molecules was enhanced. Finally, inhibition of CD200-CD200R1 interaction in mixed glial cultures potentiated the pro-inflammatory response. Thus, the reactive phenotype of glial cells can be modulated through an action on CD200R1 expression or stimulation, suggesting CD200R1 as a candidate target to act against neuroinflammation in neurodegenerative diseasesSupported by La Marató de TV3 Foundation and Instituto de Salud Carlos III, Spain-FEDER funds, EU (grants PI10/378 and PI12/00709). GD and TV are recipients of IDIBAPS and JAE-CSIC-FSE contracts respectivelyPeer Reviewe

C/Ebpß regulates microglial ptges expression and pge2 production

Trabajo presentado al 8th Forum of Neuroscience (FENS) celebrado en Barcelona del 14 al 18 de julio de 2012.We have recently described that the transcription factor C/EBPß regulates pro-inflammatory gene expression in glial activation. Since the production of the inflammatory lipid mediator PGE2 is increased in activated glial cells, we have undertaken a study to analyze whether C/EBPß participates in the regulation of PGE2 synthesis enzymes in these cells. To this end gene expression and PGE2 production was compared between wild-type and C/EBPß -/- mice, both in vitro and in vivo. We have observed a robust effect of C/EBPß deficiency in the expression of PTGES (=mPGES1) in glial activation. Thus, PTGES expression and PGE2 production induced by LPS (100 ng/ml) +IFN?(0.1 ng/ml) in primary mixed glial cultures were abolished in the absence of C/EBPß. qChIP experiments revealed C/EBPß binding to the PTGES promoter after LPS+IFN? treatment and double immunofluorescence revealed that PTGES expression was of microglial origin. In line with this, marked LPS+IFN?-induced upregulation of PTGES expression was observed in microglial-enriched cultures and this was also abolished in the absence of C/EBPß. Systemic LPS injection (100 µg/mouse, i.p.) was used then to study neuroinflammation in vivo. This treatment upregulated PTGES mRNA levels in cerebral cortex and, as seen in vitro, the increase in PTGES expression was abolished in C/EBPß -/- mice. Interestingly, the expression of COX-2, another key enzyme in PGE2 synthesis, was also upregulated in glial activation in vitro and in vivo, but in contrast to PTGES, COX2 expression was barely affected by C/EBPß absence. Altogether these findings suggest that in microglial activation C/EBPß binds to the PTGES promoter, inducing its transcription. This leads to increased PTGES mRNA and protein levels and subsequent PGE2 accumulation. These findings strengthen the proposed role of C/EBPß as a key player in the orchestration of gene response in neuroinflammation.Supported by PI08/1396 and PI10/378 from ISCIII.Peer reviewe

Mecanismos implicados en el control de la expresión del receptor inmune inhibitorio microglial CD200R1 en presencia de neuroinflamación

Trabajo presentado en la VI Reunión de la Red Glial Española, celebrada en Oviedo, en septiembre de 2013Peer Reviewe

The neuroprotective effect of PPAR-y agonist in microglia-induced neuronal death is abrogated if CD200-CD200R1 interaction is disrupted

Póster presentado en el XI European Meeting on Glial Cells in Health and Disease, celebrado los días 3 al 6 de julio de 2013 en Berlín (Alemania)Peer Reviewe

C/EBPbeta inhibits the expression of the inhibitory immune receptor CD200R1 in microglial cells in response to pro-inflammatory stimulus

Trabajo presentado en la 10th European meeting on Glial Cells in Health and Disease, celebrada en Praga, República Checa, del 13 al 17 de septiembre de 2011Peer Reviewe