Features of bilirubin-induced reactive microglia: From phagocytosis to inflammation

Neurobiology of Disease 40 (2010) 663–675 Contents lists available at ScienceDirect Neurobiology of Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n b d i Features of bilirubin-induced reactive microglia: From phagocytosis to inflammation Sandra L. Silva, Ana R. Vaz, Andreia Barateiro, Ana S. Falcão, Adelaide Fernandes, Maria A. Brito, Rui F.M. Silva, Dora Brites ⁎ Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Avenida Professor Gama Pinto, 1649-003 Lisbon, Portugal a r t i c l e i n f o Article history: Received 11 May 2010 Revised 26 July 2010 Accepted 11 August 2010 Available online 19 August 2010 Keywords: Microglial activation Phagocytic activity Inflammatory signalling pathways Mitogen activated protein kinases Nuclear factor-κB Hyperbilirubinemia Cyclooxygenase-2 Matrix metalloproteinases a b s t r a c t Microglia constitute the brain's immunocompetent cells and are intricately implicated in numerous inflammatory processes included in neonatal brain injury. In addition, clearance of tissue debris by microglia is essential for tissue homeostasis and may have a neuroprotective outcome. Since unconjugated bilirubin (UCB) has been proven to induce astroglial immunological activation and neuronal cell death, we addressed the question of whether microglia acquires a reactive phenotype when challenged by UCB and intended to characterize this response. In the present study we report that microglia primary cultures stimulated by UCB react by the acquisition of a phagocytic phenotype that shifted into an inflammatory response characterized by the secretion of the proinflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, upregulation of cyclooxygenase (COX)-2 and increased matrix metalloproteinase (MMP)-2 and -9 activities. Further investigation upon upstream signalling pathways revealed that UCB led to the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB at an early time point, suggesting that these pathways might underlie both the phagocytic and the inflammatory phenotypes engaged by microglia. Curiously, the phagocytic and inflammatory phenotypes in UCB-activated microglia seem to alternate along time, indicating that microglia reacts towards UCB insult firstly with a phagocytic response, in an attempt to constrain the lesion extent and comprising a neuroprotective measure. Upon prolonged UCB exposure periods, either a shift on global microglia reaction occurred or there could be two distinct sub-populations of microglial cells, one directed at eliminating the damaged cells by phagocytosis, and another that engaged a more delayed inflammatory response. In conclusion, microglial cells are relevant partners to consider during bilirubin encephalopathy and the modulation of its activation might be a promising therapeutic target. © 2010 Elsevier Inc. All rights reserved. Introduction Abbreviations: BSA, Bovine serum albumin; CNS, Central nervous system; COX-2, Cyclooxygenase-2; DMEM-Ham's F-12, Dulbecco's modified Eagle's medium-Ham's F12 medium; DTT, Dithiothreitol; EDTA, Ethylenediamine tetraacetic acid; ELISA, Enzyme-linked immune sorbent assay; ERK1/2, Extracellular signal regulated kinase 1/ 2; FBS, Fetal bovine serum; FITC, Fluorescein isothiocyanate; GFAP, Glial fibrillary acidic protein; HIV, Human immunodeficiency virus; HSA, Human serum albumin; Iba1, Ionized calcium-binding adaptor molecule 1; IgG, Immunoglobulin G; IL-1β, Interleukin-1β; IL-6, Interleukin-6; iNOS, Inductible nitric oxide synthase; JNK1/2, c-Jun N-terminal kinase 1/2; LPS, Lipopolysaccharide; MAPK, Mitogen-activated protein kinase; MMP, Matrix metalloproteinase; NEA, Non-essential aminoacids; NF-κB, Nuclear factor-κB; NMDA, N-methyl-D-aspartic acid; NO, Nitric oxide; PBS, Phosphate-buffered saline; P-ERK1/2, Phosphorylated ERK1/2; PGE2, Prostaglandin E2; PI, Propidium iodide; PJNK1/2, Phosphorylated JNK1/2; pNA, P-nitroaniline; P-p38, Phosphorylated p38; SDS-PAGE, Sodium dodecyl sulphate-polyacrylamide gel electrophoresis; TNF-α, Tumour necrosis factor-α; TREM2, Triggering receptor expressed on myeloid cells-2; TRITC, Tetramethylrhodamine isothiocyanate; UCB, Unconjugated bilirubin. ⁎ Corresponding author. Fax: + 351 217946491. E-mail address: dbrites@ff.ul.pt (D. Brites). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2010.08.010 Hyperbilirubinemia is a common condition in the neonatal period and results from a limited ability of the newborns to excrete an over produced bilirubin (Dennery et al., 2001; Watson, 2009). Physiological to pathological transition is driven by the multifocal deposition of unconjugated bilirubin (UCB) in selected regions of the brain leading to encephalopathy and kernicterus (Hansen, 2002; Porter and Dennis, 2002). This event is directly correlated with death, as well as with impairments of neural development and hearing (Oh et al., 2003). Additionally, moderate hyperbilirubinemia has been proven to be associated with a significant increase in minor neurologic dysfunction throughout the first year of life (Soorani-Lunsing et al., 2001) and has also been related to the outcome of mental disorders such as schizophrenia (Miyaoka et al., 2000). The cytotoxic effects of UCB in the central nervous system (CNS) have been broadly studied and comprise several features such as: perturbation of nerve cell and mitochondria membranes (Rodrigues et al., 2002b,c); inhibition of glutamate uptake prolonging its presence in the synaptic cleft (Silva et al., 1999, 2002); N-methyl-D-aspartic acid (NMDA)- 664 S.L. Silva et al. / Neurobiology of Disease 40 (2010) 663–675 mediated excitotoxicity (Brito et al., 2010; Grojean et al., 2000, 2001; McDonald et al., 1998); and increase in intracellular calcium (Brito et al., 2004). All these events may culminate in cell death by both necrosis and apoptosis (Rodrigues et al., 2002a; Silva et al., 2001) being neurons more susceptible to death mechanisms than astrocytes (Falcão et al., 2006; Silva et al., 2002). Some of the injurious effects of UCB on astrocytes are an elevated glutamate secretion and the activation of inflammatory pathways that lead to cytokine release (Falcão et al., 2006; Fernandes et al., 2006, 2004). Furthermore, our group was the first to demonstrate that UCB activates and damages microglial cells (Gordo et al., 2006). Indeed microglia showed to be the most reactive brain cells when compared to astrocytes and neurons, as they evidence increased UCB-induced cell death, release of glutamate and cytokine production (Brites et al., 2009). Microglial cells reside within the CNS parenchyma (Streit, 2002) and engage several important roles in the developing brain (Cuadros and Navascues, 1998; Kim and de Vellis, 2005) as well as in pathological conditions (Block and Hong, 2005; Nakajima and Kohsaka, 2004). In response to injury, microglia turn into an activated state and display a complexity of phenotypic alterations that illustrate what is called “reactive microglia.” This activation entails several features such as: dramatic morphologic changes by the acquisition of an amoeboid phenotype (Kreutzberg, 1996); upregulation of intracellular enzymes and cell surface markers, release of pro-inflammatory mediators, oxygen radicals and proteases (Kim and de Vellis, 2005), antigen presentation (Aloisi, 2001) and phagocytosis (Chew et al., 2006). Moreover, the implication of microglia to neonatal pathologic conditions has been acknowledged (McRae et al., 1995; Vexler and Yenari, 2009), since the production of inflammatory mediators by these cells is a major contributor to hypoxic–ischemic injury in the neonatal brain (Doverhag et al., 2010). Microglial activation must not be viewed as an “on/off” process, but rather as a shift between activity states, altering between a surveying and an effector status. In fact, microglia's activation process is an adaptive one but, depending on the circumstances in which it occurs, may have neuroprotective or neurotoxic outcomes (Hanisch and Kettenmann, 2007). Indeed, microglial involvement in various neurodegenerative disorders is notorious, namely in Parkinson's disease (Tansey et al., 2008), Alzheimer's disease (Kim and Joh, 2006), multiple sclerosis (Jack et al., 2005; Muzio et al., 2007), and human immunodeficiency virus (HIV)-associated dementia (Gonzalez-Scarano and Baltuch, 1999), mostly owing to its inflammatory character. Yet, microglial phagocytic role in numerous neurodegenerative diseases, as well as in acute brain injury, is essential for tissue debris removal and contributes for a pro-regenerative environment (Neumann et al., 2009). Moreover, phagocytic clearance of debris may be considered a protective measure as it constitutes an attempt to restrain further detrimental inflammatory responses (Napoli and Neumann, 2009). In this study we characterize the microglial response to UCB stimulation by evaluating both its phagocytic properties and the inflammatory mechanisms engaged upon activation. We observed, for the first time, that UCB induces an increase in the phagocytic properties of microglia, followed by a shift into a rather inflammatory response with prolonged exposure time. Moreover, this inflammatory response triggered by UCB follows different temporal profiles of interleukin (IL)-1β, tumour necrosis factor (TNF)-α and IL-6 secretion. Remarkably, an increase in TNF-α and IL-1β release is observed prior to the secretion of IL-6. Our findings also point to mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB as probable signalling pathways involved in microglial reactivity to UCB as they might be entailed either in inflammation (Hanisch et al., 2001) or phagocytosis (Sun et al., 2008; Tanaka et al., 2009). In fact, we demonstrate that MAPKs phosphorylation is an essential step for NF-κB nuclear translocation. Furthermore, our results reveal the induction of cyclooxygenase (COX)-2 expression and matrix metalloproteinase (MMP)-2 and -9 activity in a later phase of microglial response to UCB, implicating these events in the overall deleterious and inflammatory response. We provide evidence that UCB- induced cytokine secretion may participate in MMP activation and hypothesize that these events might be reciprocally regulated, further contributing to the complex network of microglia activation process. Taken together, these results strongly imply a multiple response of microglia to UCB, suggesting that those cells are relevant partners to consider during bilirubin encephalopathy. Materials and methods Chemicals Dulbecco's modified Eagle's medium-Ham's F12 medium (DMEMHam's F-12), Opti-MEM medium, fetal bovine serum (FBS), L-glutamine, sodium pyruvate and non-essential aminoacids (NEA) were purchased from Biochrom AG (Berlin, Germany). Antibiotic antimycotic solution (20×), human serum albumin (HSA; Fraction V, fatty acid free), bovine serum albumin (BSA), Hoechst 33258 dye, biotinylated tomato lectin (Lycopersicon esculentum), avidin-fluorescein isothiocyanate (FITC), avidin-tetramethylrhodamine isothiocyanate (TRITC), fluorescent latex beads 1 μm (2.5%), mouse anti-β-actin, FITC-labelled goat antirabbit IgG, rabbit anti-glial fibrillary acidic protein (GFAP), TRITClabelled goat anti-rabbit IgG, Coomassie Brilliant Blue R-250 and propidium iodide (PI) were from Sigma Chemical Co. (St. Louis, MO). UCB was also obtained from Sigma and purified according to the method of McDonagh and Assisi, 1972. Trypsin/Ethylenediamine tetraacetic acid (EDTA) solution (0.25% trypsin, 1 mM EDTA in Hank's balanced salt solution) and Alexa Fluor 594 chicken anti-goat IgG were purchased from Invitrogen Corporation (Carlsbad, CA). FuGENE HD Transfection Reagent was acquired from Roche Molecular Biochemicals (Mannheim, Germany); Dual Luciferase reporter assay system was from Promega (Madison, WI, USA); and Caspase-3, -8 and -9 substrates, Ac-DEVD-pNA, Ac-IETD-pNA and Ac-LEHD-pNA, respectively, were purchased from Calbiochem (San Diego, CA, USA). Concentrated solutions (10 mM) of MAPK pathways inhibitors SB203580 (p38 MAPK inhibitor; Calbiochem, San Diego, CA, USA), and U0126 [Extracellular signal regulated kinase (ERK1/2)-upstream inhibitor; Promega, Madison, WI, USA], were prepared in dimethylsulfoxide. Recombinant rat IL-1β and DuoSet® ELISA kits were from R&D Systems, Inc. (Minneapolis, MN, USA). Nitrocellulose membrane, Hyperfilm ECL and Horseradish peroxidase-labelled goat anti-mouse IgG were obtained from Amersham Biosciences (Piscataway, NJ, USA). LumiGLO®, Cell lysis buffer®, rabbit anti-phosphorylated-p38 (P-p38) and rabbit anti-phosphorylated-ERK 1/2 (P-ERK1/2) were from Cell Signalling (Beverly, MA, USA). Mouse anti-phosphorylated-c-Jun N-terminal kinase (P-JNK1/2), rabbit anti-p65 NF-κB subunit and horseradish peroxidase-labelled goat anti-rabbit IgG were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat anti-ionized calcium-binding adaptor molecule 1(Iba1) was from Abcam (Cambridge, UK). All other chemicals were of analytical grade and were purchased from Merck (Darmstadt, Germany). Primary culture of microglia Animal care followed the recommendations of European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes (Council Directive 86/609/EEC) and National Law 1005/92 (rules for protection of experimental animals). All animal procedures were approved by the Institutional Animal Care and Use Committee. Every effort was made to minimize the number of animals used and their suffering. Mixed glial cultures were prepared from 1- to 2-day-old Wistar rats as previously described (McCarthy and de Vellis, 1980), with minor modifications (Gordo et al., 2006). Cells (4× 105 cells/cm2) were plated on uncoated 12- or 6-well tissue culture plates (Corning Costar Corp., Cambridge, MA) in culture medium (DMEM-Ham's F-12 medium S.L. Silva et al. / Neurobiology of Disease 40 (2010) 663–675 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, nonessential amino acids 1×, 10% FBS, and 1% antibiotic-antimycotic solution) and maintained at 37 °C in a humidified atmosphere of 5% CO2. Microglia were isolated as previously described by Saura et al., 2003. Briefly, after 21 days in culture, microglia were obtained by mild trypsinization with a trypsin-EDTA solution diluted 1:3 in DMEM-F12 for 20–45 min. The trypsinization resulted in detachment of an upper layer of cells containing all the astrocytes, whereas the microglia remained attached to the bottom of the well. The medium containing detached cells was removed and replaced with initial mixed glialconditioned medium. Twenty-four hours after trypsinization, the attached cells were subjected to the different treatments. The use of 21-days-in-vitro cells intents to achieve the maximal yield and purity of the cultures. In fact, astrocyte contamination was less than 2%, as assessed by immunocytochemical staining with a primary antibody against GFAP followed by a species-specific fluorescent-labelled secondary antibody. Microglia were counterstained with a biotinylated tomato lectin (Lycopersicon esculentum), using a 1:166 dilution in 1% Triton X-100® in phosphate-buffered saline (PBS) overnight at 4 °C followed by 1 h incubation at room temperature with avidin-TRITC in a 1:100 dilution in PBS; the nuclei were immunostained with Hoechst 33258 dye. Thus, the high purity level of microglia cultures excludes interference of contaminating astroglial cells. Cell treatment Microglial cells were incubated in the absence (control) or in the presence of 50 μM UCB plus 100 μM HSA, from 5 min to 48 h, at 37 °C. A UCB stock solution (10 mM) was prepared in 0.1 M NaOH immediately before use and the pH of the incubation medium was restored to 7.4 by addition of equal amounts of 0.1 M HCl. All the experiments with UCB were performed under light protection to avoid photodegradation. To study the role of MAPK pathways in microglial response to UCB, cells were pretreated for 20 min with 10 μM of the MAPK inhibitors prior to UCB stimulation: SB203580, a selective inhibitor of p38 MAPK and U0126, a selective inhibitor of the MAPK kinases (MEK)1/2, upstream kinases in the ERK1/2 pathway. The involvement of IL-1β in MMP activation was investigated by treating microglia with 2 ng/mL recombinant rat IL-1β or vehicle alone, in the presence of 100 μM HSA, for 30 min and 1 h at 37 °C. The selected concentration of cytokine was based on the maximal levels obtained in our culture model upon UCB stimulation. Measurement of cytokine release Aliquots of the cell culture media were collected at the end of the incubations and, after removal of cellular debris by short centrifugation, placed in a 96-well microplate and assessed in triplicate for TNF-α, IL-1β and IL-6 with specific DuoSet® ELISA Development kits from R&D Systems, according to the manufacturer's instructions. Results were expressed as pg/mL. Western blot assay Western blot assay was carried out as usual in our lab (Fernandes et al., 2006). Briefly, total protein was extracted from primary microglia using Cell lysis buffer®. Protein extracts were separated on a 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Following electrophoretic transfer onto a nitrocellulose membrane and blocking with 5% milk solution, the blots were incubated with primary antibody overnight at 4 °C [anti-P-p38 MAPK (1:1000), anti-P-ERK1/2 (1:1000), anti-P-JNK1/2 (1:200), anti-COX-2 (1:1000) or anti-β-actin (1:10000) in 5% (w/v) BSA] and with horseradish peroxidase-labelled secondary antibody [anti-mouse (1:5000) or anti-rabbit (1:5000)] for 1 h at room temperature. Protein bands were detected by LumiGLO® and visualized by autoradiography with Hyperfilm ECL. 665 Detection of NF-κB activation To assay the transcriptional activity of NF-κB, reporter gene analysis was applied. A reporter plasmid under control of NF-κB binding sites was provided by Dr. Guy Haegeman (Flanders Interuniversity Institute for Biotechnology and University of Gent, Belgium). NF-κB-dependent reporter plasmids, p(IL6κB)350hu.IL6P-luc+, contain three NF-κB binding sites in the promoter region, while NF-κB-independent plasmids, p50hu.IL6P-luc+, do not (Vanden Berghe et al., 1998). These reporter genes were introduced into microglial cells using FuGENE HD Transfection Reagent. After 24 h of transfection, cells were treated with 50 μM UCB plus 100 μM HSA from 30 min to 4 h, at 37 °C. Luciferase assays were carried out using a Dual Luciferase Reporter Assay System (Promega), according to the instructions in manufacturer's manual. Firefly and renilla luciferase activities were measured using a luminometer (Berthold Technologies, Wildbad, Germany). Firefly luciferase activity value was normalized to renilla luciferase activity value from pSV-Sport-Rluc plasmid. Readings of promoter activities of NF-κB-independent plasmids, p50hu.IL6P-luc+ and p1168hIL6m NF-κB-luc (plasmid presenting a mutation in NF-κB binding sites), were also performed. Results were presented as fold change of the relative luciferase activity compared to the respective control. For immunofluorescence detection of NF-κB nuclear translocation, cells were fixed for 20 min with freshly prepared 4% (w/v) paraformaldehyde in PBS and a standard immunocytochemical technique was performed using a polyclonal rabbit anti-p65 NF-κB subunit antibody (1:200) as the primary antibody and a FITC-labelled goat anti-rabbit antibody (1:160) as the secondary antibody. To identify the total number of cells, microglial nuclei were stained with Hoechst 33258 dye as previously described. Fluorescence was visualized using a Leica DFC490 camera adapted to an Axioskop® microscope (Zeiss). Pairs of U. V. and green-fluorescence images of ten random microscopic fields (original magnification: 400×) were acquired per sample. NF-κBpositive nuclei (identified by localization of the NF-κB p65 subunit staining exclusively at the nucleus) and total cells were counted (N500 cells per sample) to determine the percentage of NF-κB-positive nuclei. Results were expressed as fold change versus respective control. Morphological analysis For morphological analysis, cells were fixed as described above and a standard indirect immunocytochemical technique was carried out using a primary antibody raised against Iba-1 (goat, 1:500) and a secondary Alexa Fluor 594 chicken anti-goat antibody (1:200). Fluorescent images were acquired using a Leica DFC490 camera attached to an Axioskop® microscope (Zeiss). Assessment of microglial phagocytic properties After treatment with UCB, cells were incubated with 0.0025% (w/w) 1 μm fluorescent latex beads for 75 min at 37 °C and fixed with freshly prepared 4% (w/v) paraformaldehyde in PBS. Labelling with tomato lectin was performed followed by avidin-TRITC and the nuclei counterstained with Hoechst 33258 dye. U.V., green and red-fluorescence images of fifteen random microscopic fields (original magnification: 630×) were acquired per sample. The number of ingested beads per cell was counted in approximately 250 cells per sample. Gelatin zymography Aliquots of culture supernatant were analyzed by SDS-PAGE zymography in 0.1% gelatine–10% acrylamide gels under non-reducing conditions. After electrophoresis, gels were washed for 1 h with 2.5% Triton X-100 (in 50 mM Tris pH7.4; 5 mM CaCl2; 1 μM ZnCl2) to remove SDS and renature the MMP species in the gel. Then the gels were 666 S.L. Silva et al. / Neurobiology of Disease 40 (2010) 663–675 incubated in the developing buffer (50 mM Tris pH7.4; 5 mM CaCl2; 1 μM ZnCl2) overnight to induce gelatine lysis. For enzyme activity analysis, the gels were stained with 0.5% Coomassie Brilliant Blue R-250 and destained in 30% ethanol/10% acetic acid/H2O. Gelatinase activity, detected as a white band on a blue background, was quantified by computerized image analysis and normalized with total cellular protein. Evaluation of microglial cell death Necrotic-like cell death was assessed by monitoring the cellular uptake of the fluorescent dye propidium iodide [PI; 3,8-diamino-5-(3-(diethylmethylamino)propyl)-6-phenyl phenanthridinium diiodide]. PI readily enters and stains non-viable cells, but cannot cross the membrane of viable cells. This dye binds to double-stranded DNA and emits red fluorescence (630 nm; absorbance 493 nm). Unpermeabilized adherent cells cultured on coverslips were incubated with a 75 μM PI solution for 15 min in the absence of light. Subsequently, cells were fixed with freshly prepared 4% (w/v) paraformaldehyde in PBS and the nuclei immunostained with Hoechst 33258 dye. Red-fluorescence and U.V. images of ten random microscopic fields (original magnification: 400×) were acquired per sample and the percentage of PI positive cells was counted and expressed as fold versus respective control. Activities of caspase-3, -8 and -9 were measured by a commercial colorimetric method. Cells were harvested, washed with ice-cold PBS and lysed for 30 min on ice in the lysis buffer [50 mM HEPES (pH 7.4); 100 mM NaCl; 0.1% (w/v) CHAPS; 1 mM dithiothreitol (DTT); 0.1 mM EDTA]. The activities of caspase-3, -8 and -9 were determined in cell lysates by enzymatic cleavage of chromophore p-nitroanilide (pNA) from the substrate Ac-DEVD-pNA for caspase-3, Ac-IETD-pNA for caspase-8 and Ac-LEHD-pNA for caspase-9, according to manufacturer's instructions. The proteolytic reaction was carried out in protease assay buffer [50 mM HEPES (pH 7.4); 100 mM NaCl; 0.1% (w/v) CHAPS; 10 mM DTT; 0.1 mM EDTA; 10% (v/v) glicerol], containing 2 mM specific substrate. Following incubation of the reaction mixtures for 1 to 2 h at 37 °C, the formation of pNA was measured in a microplate reader (PR 2100, BioRad Laboratories, Inc.) at λ = 405 nm with a reference filter at 620 nm. Readings were normalized to total protein content determined using a protein assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's specification, and expressed as fold change of respective control. dependent manner but with different temporal profiles. In fact, TNF-α and IL-1β seem to be the first to be upregulated, as an increase in those cytokine levels in culture supernatants can be observed as early as 2 h after the addition of 50 μM UCB. However, while peak values for TNF-α are reached at 4 h of UCB exposure (changing from 365 pg/mL for control conditions to 520 pg/mL for UCB, p b 0.05) and decline gradually along time of exposure (although an additional increase is noticed at 24 h), the maximum release of IL-1β was only achieved at 12 h, but in a much higher amount (ranging from 980 pg/mL for control conditions to 1700 pg/mL for UCB, p b 0.05). On the other hand, IL-6 secretion was only noticed from 2 h onwards, reaching peak levels at 8 h of UCB exposure (shifting from 1650 pg/mL for control conditions to 2050 pg/ mL for UCB, p b 0.01) and decreasing thereafter. These results seem to indicate that microglia responds to UCB stimulus with a rather inflammatory profile which is manifested for prolonged incubation periods. As earlier results on astrocytes demonstrated that UCB-induced cytokine secretion involves MAPK and NF-κB activations (Fernandes et al., 2007, 2006) we sought to verify if the same inflammatory signalling pathways are maintained by microglia. p38 and ERK1/2 phosphorylation is elicited by UCB in microglia at an early time point MAPKs have been reported by several studies to be involved in the production of inflammatory mediators by microglia (Bhat et al., 1998; Lee et al., 2000; Waetzig et al., 2005), but their involvement on UCB microglial stimulation is still not known. So, we assessed the phosphorylated (activated) forms of all three MAPKs (p38, ERK1/2 and JNK1/2) in total cell lysates of UCB-exposed microglia, by western blot, using specific antibodies. As shown in Fig. 2, upon UCB stimulation, P-p38 and P-ERK1/2 expression were significantly upregulated in a rapid but transient manner. A 1.4-fold induction (p b 0.05 vs. control) was observed for P-p38 as early as 15 min after UCB exposure, and this activation was sustained until 30 min of incubation (1.3-fold, p b 0.05 vs. control), fading afterwards. A second activation peak was observed after 2 h of exposure (1.4-fold, p b 0.05), declining to control levels at 6 h. In regard to P-ERK1/2, a 1.2-fold increase (p b 0.05 vs. control) was observed at 15 min of exposure and, as for P-p38 a second peak, although smaller, was noticed at 1 h of exposure (1.1-fold, pb0.05) with values that diminish from then on. A weaker increase in P-JNK1/2 was perceived at 15 and 30 min of UCB exposure; however, this effect was not Statistical analysis Results of, at least, three different experiments were expressed as mean ± S.E.M. Significant differences between two groups were determined by the two-tailed t-test performed on the basis of equal and unequal variance as appropriate. Comparison of more than two groups was done by ANOVA using Instat 3.05 (GraphPad Software, San Diego, CA, USA) followed by multiple comparisons Bonferroni post-hoc correction. Statistical significance was considered for a p value less than 0.05. Results UCB triggers IL-1β, TNF-α and IL-6 secretion following different temporal profiles Supporting evidence reports reactive microglia as one of the main sources of pro-inflammatory cytokines in the brain (Hanisch, 2002), which are known to exert deleterious effects in nerve cells (Rothwell, 1999). Previous results suggested that UCB is able to induce an inflammatory response by microglia (Gordo et al., 2006). Thus, we intended to further characterize those inflammatory events by the evaluation of the temporal secretion profile of IL-1β, TNF-α and IL-6. In Fig. 1 it can be observed that UCB stimulates cytokine release in a time- Fig. 1. UCB induces the release of TNF-α, IL-1β and IL-6 by microglia following different temporal profiles. Rat cortical microglial cells were treated with 50 μM UCB in the presence of 100 μM HSA for the indicated time periods. TNF-α, IL-1β and IL-6 concentrations in the media were determined by ELISA and expressed as mean ± SEM cytokine release from four independent experiments performed in triplicate, after deduction of cytokine values in control assays. *p b 0.05 and **p b 0.01 vs. respective control. S.L. Silva et al. / Neurobiology of Disease 40 (2010) 663–675 significantly different from the respective controls. Prolonging UCB incubation to longer periods did not modify the pattern of MAPK activation (data not shown). These results indicate that MAPKs activation is a rather early event on microglial activation, seeming to involve two activation peaks. Next we found interesting to check if this activation could be followed by the engagement of NF-κB nuclear translocation. NF-κB signalling pathway is triggered in UCB-activated microglia NF-κB is described as a convergent point of signalling for microglial activation by cytokines and other stressors (Glezer et al., 2007) and its implication in the inflammatory response induced by UCB in astrocytes has already been established (Fernandes et al., 2006). Hence, we examined the effect of UCB on NF-κB transactivation in microglial cells by gene reporter assay (Fig. 3A). The results indicated that UCB markedly induced NF-κB activation at 15 and 30 min of exposure (1.4-fold, pb 0.01 for both time points). It should be stated that readings of the promoter activities of p50hu.IL6P-luc+ and p1168hIL6m NF-κB-luc plasmids (empty and mutated vectors, respectively) showed no change in the presence or absence of UCB (data not shown), thus validating the assay. To further confirm the activation of this signalling pathway we investigated NF-κB activation in microglia exposed to UCB at various time points by the immunochemical assessment of the cytoplasmic or nuclear localization of p65 NF-κB subunit. Interestingly we found NF-κB translocation into the nucleus to be significantly increased from 15 min to 2 h of exposure when compared to the respective controls (Fig. 3B and C), which is in line with our previous observations regarding NF-κB transcriptional activation by UCB. Maximum levels of nuclear NF-κB were observed at 30 min (2.2-fold, p b 0.01), while from 4 h onwards 667 NF-κB was mostly localized in the cellular cytoplasm. These results follow the ones from MAPK activation, suggesting that, as previously verified in astrocytes, both events are connected. UCB-induced NF-κB translocation depends on both ERK1/2 and p38 To assess whether MAPKs phosphorylation is an essential step for UCB-evoked NF-κB translocation, we investigated this event after exposure of microglia to UCB alone or in combination with the MAPK inhibitors SB203580 (p38 selective inhibitor) and UO126 [(MEK)1/2 selective inhibitor, upstream kinases in the ERK1/2 pathway]. The use of 30 min and 1 h incubation periods was based on previous results showing that maximal translocation of NF-κB to the nucleus in UCB-challenged microglia occurs between 30 min and 4 h. As expected, pre-incubation with SB203580 and U0126 completely abrogated UCB-stimulated NF-κB nuclear translocation after 30 min (p b 0.01) and 1 h (p b 0.05) of UCB stimulation (Fig. 4), thus providing proof of concept that p38 and ERK1/2 phosphorylation are required for the engagement of NF-κB pathway upon UCB exposure. So far our results have described the engagement of early inflammatory signalling pathways that culminate in the achievement of an inflammatory phenotype characterized by the release of proinflammatory mediators. We next intended to characterize UCBstimulated microglia at a morphological level, in order to verify the achievement of this activated state. Microglia depict morphological changes upon UCB stimulation Modification of microglial morphology is one of the hallmarks of its activation profile and has been widely used to categorize different Fig. 2. MAPKs activation is elicited by UCB in microglia. Rat cortical microglia were exposed to 50 μM UCB in the presence of 100 μM HSA for the indicated time periods. Total cell lysates were analysed by western blotting with antibodies specific for the phosphorylated forms of the three MAPKs, P-p38, P-ERK1/2 and P-JNK1/2. (A) Representative results of one experiment are shown. Similar results were obtained in three independent experiments. (B) The intensity of the bands was quantified by scanning densitometry, standardized with respect to β-actin protein expression and expressed as mean ± SEM fold change compared with control conditions. *p b 0.05 vs. respective control. 668 S.L. Silva et al. / Neurobiology of Disease 40 (2010) 663–675 Fig. 3. UCB activates NF-κB signalling pathway in microglia. Rat cortical microglia were exposed to 50 μM UCB in the presence of 100 μM HSA for the indicated time periods. (A) Microglial cells were transiently transfected with κB-dependent luciferase plasmids and control plasmids. Relative luciferase activities were plotted as fold change of respective controls. To further confirm NF-κB activation, cells were fixed and immunostained with an antibody against p65 NF-κB subunit. (B) Representative results of one experiment are shown. Scale bar, 20 μm. (C) The percentage of NF-κB-positive nuclei was calculated and expressed as fold change versus respective control. Results are mean ± SEM from three independent experiments performed in triplicate. *p b 0.05 and **p b 0.01 vs. respective control. activation states (Chew et al., 2006; Kim and de Vellis, 2005; Lynch, 2009; Raivich et al., 1999). For that reason, we evaluated the morphology and reactivity of UCB-stimulated microglia by immunocytochemistry after 4 and 24 h incubation periods. Our results indicate that, after a short UCB exposure, microglia shifted from an elongated morphology to a large and amoeboid shape (Fig. 5). This phenotype is characteristic of activated or reactive microglia (Nakajima and Kohsaka, 2004). In contrast, for longer exposure periods, microglia display fragmented and condensed cytoplasm, a feature described by other authors (Fendrick et al., 2007; HasegawaIshii et al., 2010) as cytorrhexis, indicative of microglia degeneration and senescence. Interestingly, the inflammatory phenotype previously described occurs after prolonged UCB incubation periods. However, activation features were also observed for shorter stimulations. For that matter, we sought to evaluate the phagocytic properties of UCB-challenged microglia and, more importantly, to verify whether this reactive phenotype occurred prior, simultaneously or after the inflammatory response triggered by UCB, in order to further characterize the chronologic events of UCB microglial activation. UCB differently modulates microglial phagocytosis depending on exposure time Phagocytosis is one of the main features of microglial activation dumping cell debris prior to cell regeneration, and can be involved in the pathogenesis of several brain dysfunctions (Neumann et al., 2009). However, this microglial property was never investigated under UCB stimulation. As can be seen in Fig. 6, a sharp increase in the uptake of fluorescent latex beads by UCB-stimulated microglia occurs from 2 h on, reaching a maximum peak after 4 h of exposure, (~50%, p b 0.05). This is a strong evidence that UCB is able to induce microglial phagocytic properties in a rather short-term exposure. Unspecific entry of latex beads due to UCB-induced cell permeabilization was excluded by performing the phagocytosis assay in microglial cells incubated with UCB for 4 h and simultaneously determining PI uptake. Dead cells did not appear to engulf particles (data not shown). Interestingly, prolonging UCB incubation until 8 and 12 h slightly reverts this phagocytic ability although it approached control values upon 24 h of exposure. This may indicate that, after an initial phagocytic reaction, microglial cells shifted to the previously observed S.L. Silva et al. / Neurobiology of Disease 40 (2010) 663–675 669 Fig. 4. Phosphorylation of p38 and ERK1/2 is essential for UCB-evoked NF-κB activation. Rat cortical microglia were exposed for 30 min and 1 h to 50 μM UCB alone or in combination with 10 μM of the p38 inhibitor SB203580 or the ERK1/2 inhibitor U0126. Cells were fixed and immunostained with an antibody against p65 NF-κB subunit. (A) Representative results of one experiment are shown. Scale bar, 20 μm. (B) The percentage of NF-κB-positive nuclei was calculated and expressed as fold change vs. respective control. Results are mean ± SEM from three independent experiments performed in triplicate. **p b 0.01 vs. respective control, §p b 0.05 and §§p b 0.01 vs. UCB alone. inflammatory response to UCB stimulus. To add on the characterization of microglial behaviour we advanced to the evaluation of other markers of its inflammatory response such as (MMPs activity and COX2 expression. Release of active MMPs is enhanced upon UCB stimulation of microglia MMPs are a family of proteases with many important roles in normal development although they may also participate in several neuronal diseases such as multiple sclerosis, ischemia and neuroinflammation given their ability to degrade the basal lamina surrounding the blood– brain barrier allowing infiltration of immune cells, and thus aggravating inflammatory reactions in the CNS (Michaluk and Kaczmarek, 2007; Rosenberg, 2002). Since microglia have been reported to secrete active MMPs further contributing to neuronal injury (Kauppinen and Swanson, 2005; Woo et al., 2008), and given the fact that cytokines are reported to stimulate MMPs secretion and activity (Gottschall and Yu, 1995; Lin et al., 2009; Vincenti and Brinckerhoff, 2007), we intended to evaluate the levels of active MMPs secreted by these cells in response to UCB and to verify if this activation could be ascribed to UCB-induced IL-1β. Cell supernatants collected after UCB incubation were subjected to gelatin zymography for the assessment of MMP-2 and MMP-9 activity 670 S.L. Silva et al. / Neurobiology of Disease 40 (2010) 663–675 significantly increased at a relatively later time points (from 4 to 12 h of exposure). It is interesting to notice that cell death seems to occur on the onset of inflammatory response and when phagocytic activity declines, again suggesting a possible double response from microglia to UCB. Discussion Fig. 5. UCB-stimulated microglia show reactive morphological changes. Rat cortical microglia were exposed to 50 μM UCB in the presence of 100 μM HSA for the indicated time periods. Cells were fixed and immunostained with an antibody against Iba1 reactivity marker. Representative results of one experiment are shown. Scale bar, 20 μm. levels. As can be seen in Fig. 7 there is a slight but significant increase in the activity of MMP-2 and MMP-9 (1.1-fold, p b 0.05) after a prolonged exposure time (24 h) to UCB. Since this event occurs after the onset of cytokine secretion elicited by microglia, a possible regulation of this event by inflammatory mediators could be occurring. In fact, stimulation of microglia with 2 ng/mL of IL-1β (which correspond to maximal levels obtained in our culture model upon UCB stimulation) increases significantly MMP-2 and MMP-9 activity at both 12 (1.3-fold and 1.2-fold, p b 0.05 and p b 0.01, respectively) and 24 h of exposure (1.2-fold and 1.3-fold, p b 0.01, respectively). UCB-stimulated microglia evidence enhanced COX-2 expression COX-2 is the enzyme responsible for the production of prostanoids such as prostaglandin E2 (PGE2), which is known to be involved in the initiation and propagation of the immune response (de Oliveira et al., 2008). The expression of this enzyme can be induced by lipopolysaccharide (LPS) and cytokines (Levi et al., 1998). It has been proven to be elevated in Alzheimer's disease (Yokota et al., 2003) and ischemia (Iadecola et al., 1999). As depicted in Fig. 8, a significant upregulation of COX-2 expression was only noticed after 12 and 24 h of UCB exposure (1.1-fold, p b 0.01 and 1.2-fold, p b 0.05, respectively), when compared to the respective controls, again suggesting a later response of microglia to UCB, in line with the previous results. Finally, we were interested in examining how UCB interaction affected microglial cell survival. UCB reduces microglial viability leading to loss of membrane integrity and increased caspase activity To evaluate the necrotic-like cell death we used the uptake of the fluorescent dye PI as an indicator of membrane integrity and cell damage since this polar substance can only enter dead or dying cells. To address the possible involvement of the apoptotic pathways in microglial demise we determined the relative levels of caspase activity, since these proteases have been traditionally viewed as central regulators of apoptosis (Fink and Cookson, 2005). As depicted in Fig. 9, UCB stimulation only arouses increased PI uptake from 4 to 12 h of exposure, reaching maximum significance at 8 h. Accordingly, the activities of the initiator caspase-8 and -9 were found to be significantly elevated in response to UCB from 2 to 12 h of exposure reaching maximum the activities 6 h (Fig. 10), while effector caspase-3 was In this paper we describe, for the first time, different activation states of microglia in the presence of UCB, since these cells display both phagocytic and inflammatory phenotypes. Indeed, this study is original in depicting the increased phagocytic properties of microglia upon UCB stimulation. In addition, our group was also the first to implicate microglial cells in the inflammatory response elicited by UCB (Gordo et al., 2006). Thus, in this study we further investigated the activation profile of microglia under UCB stimulation, by the evaluation of some of its characteristical features, and the signalling events involved in cell response. In fact, previous studies performed by our group have proven that UCB induces immunological responses in astrocytes by the activation of inflammatory pathways and secretion of glutamate (Fernandes et al., 2006, 2004), and also that UCB is neurotoxic (Falcão et al., 2007; Silva et al., 2001). Accordingly, it is conceivable that increased microglia reactivity may further contribute to neuronal injury during hyperbilirubinemia. Microglia contribute to both innate and adaptive immune responses in the brain (Chew et al., 2006). As innate immune cells, they constitute the first line of defence against invading microorganisms. The hallmark indicators of such response are the production of pro-inflammatory cytokines, the upregulation of cell surface antigens and phagocytosis (Town et al., 2005). In addition, phagocytosis of debris by microglia can be beneficial in several pathological conditions, such as multiple sclerosis (Takahashi et al., 2007) and Alzheimer's disease (Simard et al., 2006), as it restricts lesion extension and facilitates tissue recovery. The fact that UCB may alter the function of various cells of the immune system (both in vivo and in vitro) seems to be firmly established and a wide range of immunosuppressive effects on peripheral immune cells are summarized by Vetvicka et al., 1991, such as alterations on antigen expression, chemotaxis, bactericidal activity, proliferative response of T lymphocytes, or antibody production. On the other hand, an increase in phagocytosis of both peripheral blood granulocytes and monocytes after UCB treatment was reported by Miler et al., 1985. Thus, a rather contradictory immunosuppressive–immunostimulant status seems to be observed upon UCB challenge that might be explained by dose- or time-dependent effect (Vetvicka et al., 1991). Our findings demonstrate that, in conditions that intend to mimic a mild hyperbilirubinemia, enhancing of microglial phagocytic properties by UCB is an early, but transient, event that seems to be lost with increased time of exposure. Thus, we may assume that phagocytosis is the first response towards UCB insult and may constitute a neuroprotective measure. Various conditions have been shown to greatly modify microglial phagocytic activity, such as cytokines (Koenigsknecht-Talboo and Landreth, 2005) and LPS (Sun et al., 2008), among others. Interestingly, the study performed by von Zahn et al., 1997 reports an induction of nearly two-fold increase in the uptake of uncoated latex particles by TNF-α-stimulated microglia, substantiating this cytokine as an autocrine activator of microglial immune functions. Indeed, UCB-activated microglia are reportedly one of the main sources of TNF-α, even when compared to astrocytes (Brites et al., 2009). Similarly to what we already observed for astrocytes (Fernandes et al., 2006), our results point to TNF-α as the first cytokine to be released by microglia upon UCB challenge and, remarkably, its temporal profile of secretion is rather paralleled by the observed phagocytic alterations. TNF-α secretion reaches a maximum level at 4 h of UCB exposure, when microglial phagocytic properties are significantly increased and a decline in UCB-induced TNF-α release is observed from this point on, coinciding with the decreased phagocytosis elicited by UCB. S.L. Silva et al. / Neurobiology of Disease 40 (2010) 663–675 671 Fig. 6. Microglial phagocytosis is differently modulated by UCB. Rat cortical microglia were exposed to 50 μM UCB in the presence of 100 μM HSA for the indicated time periods and incubated with 1 μm fluorescent latex beads as described in Materials and methods. (A) Representative results of one experiment are shown. Scale bar, 20 μm. (B) Results are expressed as number of ingested beads per cell (± SEM) from three independent experiments. *p b 0.05 vs. respective control. Besides its role as microglial phagocytosis inducer, several experiments have also implicated TNF-α in demyelination (Akassoglou et al., 1998) and neuronal degeneration (Allan and Rothwell, 2003; Silva et al., 2006). This cytokine, along with IL-1β, participates in astrogliosis (Hanisch, 2002). IL-1β is involved in fever induction and edema, stimulation of COX-2, release of nitric oxide (NO) and free radicals (Rothwell, 1999), also participating in the recruitment of circulating leukocytes into the CNS due to its ability to upregulate the expression of adhesion molecules and chemokine synthesis (Lee and Benveniste, 1999; Sedgwick et al., 2000). IL-6 can have both pro- and anti-inflammatory functions and is produced in the early phases of CNS insult (Raivich et al., 1999). Our results clearly imply microglia as an important player in the 672 S.L. Silva et al. / Neurobiology of Disease 40 (2010) 663–675 Fig. 8. COX-2 expression is upregulated by UCB in microglia. Rat cortical microglia were exposed to 50 μM UCB in the presence of 100 μM HSA for the indicated time periods. Total cell lysates were analysed by western blotting. (A) Representative results of one experiment are shown. Similar results were obtained in three independent experiments. (B) The intensity of the bands was quantified by scanning densitometry, standardized with respect to β-actin protein and expressed as mean ± SEM fold change compared with control conditions. *p b 0.05 vs. respective control. Fig. 7. MMP-2 and MMP-9 activities are enhanced upon UCB stimulation. Culture supernatants from rat cortical microglial cells were harvested after incubation with 50 μM UCB in the presence of 100 μM HSA, or with 2 ng/mL IL-1β, for the indicated time periods and subjected to zymography as described in Materials and methods. (A) Representative gels of one experiment are reported. MMP-2 and MMP-9 were identified by their apparent molecular mass of 67 and 92 kDa, respectively. (B) The intensity of the bands was quantified by scanning densitometry, standardized with respect to total protein content and expressed as mean ± SEM fold change compared with control conditions. *p b 0.05 and **p b 0.01 vs. respective control. cause of microglia-induced neuron death (Kauppinen and Swanson, 2005). In addition, inhibition of gelatinases (MMP-2 and -9) showed efficacy in reducing neural injury and dampening neuroinflammation (Leonardo et al., 2008). Thus, these proteases seem to actively participate in inflammatory events and their activity is very tightly regulated (Sternlicht and Werb, 2001). Cytokines are firmly established inducers of MMP expression and secretion (Gottschall and Yu, 1995; Ito et al., 1996), and the induction of MMPs has been shown to be mediated by MAPKs, NF-κB and activator protein-1 signalling pathways (Lin et al., 2009; Shakibaei et al., 2007; Vincenti and Brinckerhoff, 2007; Woo et al., 2008). Interestingly, in our study model, MMPs enhanced activity occurs at a later time of exposure, when MAPKs and NF-κB activation as well as cytokine secretion have already taken place, suggesting that these events might be involved in the activation of MMPs induced by UCB. Moreover, active MMPs may also participate in the regulation of cytokine activity by promoting the secretion and activation of these molecules (Chauvet et al., 2001; Kim et al., 2005; Nuttall et al., 2007; inflammatory response instigated by UCB, since the observed early release of TNF-α, previously discussed, is followed by a later but intense secretion of IL-6 and an even stronger induction of IL-1β. Strikingly, UCB seems to induce a major release of pro-inflammatory cytokines in a time period in which phagocytosis is already absent. Recent reports have, in fact, substantiated the existence of a nonphlogistic (non-inflammatory) phagocytic response from microglia (Neumann et al., 2009), triggered by apoptotic stimuli and potentially mediated by phosphatidylserine receptors and triggering receptor expressed on myeloid cells-2 (TREM2) (Hsieh et al., 2009; Takahashi et al., 2005). Additionally, IL-1β and PGE2 were shown to suppress microglial ability to phagocytise insoluble fibrillar β-amyloid deposits, suggesting that a pro-inflammatory milieu inhibits this type of phagocytosis (Koenigsknecht-Talboo and Landreth, 2005). Our findings also suggest a role for MMPs in UCB-induced microglia reactivity, since their activity is enhanced upon prolonged exposure periods to this molecule. MMP-9 has been associated with glutamate dysfunction (Michaluk and Kaczmarek, 2007) and its release can be a Fig. 9. UCB induces microglial decreased viability and membrane disruption. Rat cortical microglia were exposed to 50 μM UCB in the presence of 100 μM HSA for the indicated time periods and incubated with 75 μM PI as described in Materials and methods. The percentage of PI-positive cells was calculated and expressed as fold vs. respective control. Results are mean ± SEM from three independent experiments performed in triplicate *p b 0.05 and **p b 0.01 vs. respective control. S.L. Silva et al. / Neurobiology of Disease 40 (2010) 663–675 Fig. 10. Microglial apoptotic cell death is elicited by UCB. Rat cortical microglia were exposed to 50 μM UCB in the presence of 100 μM HSA for the indicated time periods. The activities of caspase-3, -8 and -9 were determined in cell lysates by enzymatic cleavage of chromophore pNA from specific substrates. Results are expressed as fold of respective control at each time point. Data are means ± SEM from at least three independent experiments. *p b 0.05 and **p b 0.01 vs. respective control. Woo et al., 2008) or, on the other hand, by negatively regulating their biological activities (Ito et al., 1996). As stated above, MMPs increased activity in UCB-stimulated microglia takes place after the peak of cytokine secretion, suggesting a possible reciprocal regulation between pro-inflammatory cytokines and MMPs, since the latter molecules could be involved in the termination of the inflammatory response by means of the degradation of IL-1β. Our results further address this issue since IL-1β demonstrated to intensely elevate MMP-2 and -9 activation providing proof of concept that IL-1β secretion produced upon UCB stimulation, is at least in part, responsible for MMP activation. Discrepancy between the activation levels observed for UCB or IL-1β alone may be a result of UCB-multiple cytokine activation which results in a pleiotropic regulatory loop, absent in the second condition. COX-2 expression can also be induced in microglia by several inflammatory conditions. As can be seen in our results, UCB is able to induce upregulation of COX-2 in microglial cells in a profile very similar to that observed for IL-6 and IL-1β, thus contributing to the overall inflammatory environment described so far and again pointing to an inflammatory response secondary to phagocytosis. Therefore, our studies suggest a dual role for microglia upon UCB stimulation, shifting from a phagocytic and possibly neuroprotective phenotype towards an inflammatory and deleterious one. This is consistent with our findings demonstrating that microglia portray altered morphological features after a prolonged UCB exposure, typical of an activated state. 673 As previously observed for astrocytes (Fernandes et al., 2006, 2004) we show here that UCB stimulation of microglial cells also involves the activation of MAPKs and NF-κB. MAPKs can be activated by a variety of different stimuli (Roux and Blenis, 2004), and the engagement of this signalling pathway can lead to the phosphorylation of several substrates, including transcription factors such as NF-κB, which may ultimately lead to the enhanced transcription of genes encoding for proinflammatory cytokines (Koj, 1996). Activation of p38 and ERK1/2 are regarded as essential steps for cytokine induction since their involvement in TNF-α, IL-1β, IL-6, COX-2 and inductible nitric oxide synthase (iNOS) expression in microglia has been widely established (Bhat et al., 1998; Hanisch et al., 2001; Lee et al., 2000). Intriguingly, MAPKs activation, particularly p38, seems to be also involved in the induction of microglia phagocytosis (Sun et al., 2008; Tanaka et al., 2009). In this regard our data indicate a rapid activation of p38 and ERK1/2 by UCB in microglial cells, which occurs prior to the production of inflammatory mediators previously reported. In fact, MAPKs activation by UCB in microglia is triggered at a much earlier stage than in astrocytes (Fernandes et al., 2006), reinforcing the greater responsiveness of these glial cells during hyperbilirubinemia, but also suggesting that the early phagocytic response of microglial cells to UCB may be under the control of p38 and ERK1/2 activation. In this case, the latter activation peak observed would engage the pro-inflammatory cascade that results in IL-1β and IL-6 enhanced secretion, as well as the induction of COX-2 and MMPs, a feature already observed for other immune cells (Gong et al., 2008; Hwang et al., 1997). Moving downstream on the intracellular signalling pathways is the original observation that, as in astrocytes (Fernandes et al., 2007, 2006), NF-κB activation is also present in microglia exposed to UCB. Interestingly, maximum activation of NF-κB takes place during and after the early MAPKs phosphorylation and again prior to the production of IL-1β, TNF-α and IL-6, postulating a possible involvement of NF-κB in both phagocytic and inflammatory responses elicited by UCB in microglia. The observations that NF-κB nuclear translocation in UCBstimulated microglia is completely abrogated when microglia are pretreated with p38 and ERK1/2 inhibitors provided an unequivocal proof of MAPKs involvement in NF-κB engagement in UCB-challenged microglia which had already been previously established by other authors in different disease models (Wilms et al., 2003). It is worthwhile to mention that cell viability and membrane integrity are compromised from 4 h onwards, indicating increased cell damage induced by UCB on microglial cells from this point onwards. In fact, UCB-induced apoptotic and necrotic microglial cell death have already been established (Gordo et al., 2006). Our results further indicate that both the extrinsic and intrinsic apoptotic pathways are triggered culminating in the activation of effector caspase-3 and consequently causing cell death. However, cell death phenomena reach maximum peaks between 6 to 8 h but decrease for longer incubation periods. Together with the findings described above these data portray an interesting hypothesis for microglia response to UCB stimulus. So, it is conceivable that, either a shift on global microglia reaction occurs, or there are two distinct sub-populations of microglial cells displaying complementary activation features, one directed at eliminating the damaged cells by phagocytosis, that died after engulfment of beads, and another engaging a more delayed inflammatory response. Actually, fragmentation of cytoplasm (cytorrhexis) which is suggested in our 24 h morphological observations, has been pointed to be indicative of widespread microglial degeneration in amyotrophic lateral sclerosis models (Fendrick et al., 2007). Degenerative changes in microglia such as beading and clusters of fragmented twigs have also been demonstrated in the aged brain (Hasegawa-Ishii et al., 2010). Which of the above mentioned hypotheses is the more valid demands further elucidation and will clarify the multifaceted profile of microglia activation under UCB stimulation. The complex network of UCB-induced events in microglia, as well as the proposed interactions between them, is depicted in Fig. 11. 674 S.L. Silva et al. / Neurobiology of Disease 40 (2010) 663–675 Fig. 11. Schematic representation of time-dependent microglial activation induced by UCB. Upon UCB stimulation of microglial cells, MAPK and NK-κB signalling pathways are engaged, culminating in the generation of a phagocytic response followed by an inflammatory profile. Both phenotypes might alternate due to a reciprocal regulatory effect or to the existence of two different subpopulations engaging both types of response, being the phagocytic subpopulation firstly extinguished and replaced by a rather inflammatory subpopulation. This inflammatory profile is characterized by the increased release of pro-inflammatory cytokines TNF-α, IL-1β and IL-6, the upregulation of COX-2 and enhanced activities of MMP-2 and MMP-9. Regulatory interactions between the UCB-induced events are portrayed in the figure. In conclusion, our experiments evidence that phagocytosis is differently modulated by UCB depending on the time of exposure, prevailing at an early time point, which is followed by the release of inflammatory cytokines, and activation of MMP-2 and -9, as well as of COX-2. 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