Differential TGF-β Signaling in Glial Subsets Underlies IL-6−Mediated Epileptogenesis in Mice

This information is current as Nitzan Levy, Dan Z. Milikovsky, Gytis Baranauskas, of October 1, 2021. Ekaterina Vinogradov, Yaron David, Maya Ketzef, Shai Abutbul, Itai Weissberg, Lyn Kamintsky, Ilya Fleidervish, Alon Friedman and Alon Monsonego J Immunol 2015; 195:1713-1722; Prepublished online 1 July 2015; doi: 10.4049/jimmunol.1401446 Downloaded from http://www.jimmunol.org/content/195/4/1713

References This article cites 50 articles, 13 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/195/4/1713.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on October 1, 2021

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Differential TGF-b Signaling in Glial Subsets Underlies IL-6–Mediated Epileptogenesis in Mice

Nitzan Levy,*,†,1 Dan Z. Milikovsky,†,1 Gytis Baranauskas,† Ekaterina Vinogradov,* Yaron David,† Maya Ketzef,† Shai Abutbul,* Itai Weissberg,† Lyn Kamintsky,† Ilya Fleidervish,† Alon Friedman,†,‡,2 and Alon Monsonego*,2

TGF-b1 is a master cytokine in immune regulation, orchestrating both pro- and anti-inflammatory reactions. Recent studies show that whereas TGF-b1 induces a quiescent microglia phenotype, it plays a pathogenic role in the neurovascular unit and triggers neuronal hyperexcitability and epileptogenesis. In this study, we show that, in primary glial cultures, TGF-b signaling induces rapid upregulation of the cytokine IL-6 in , but not in microglia, via enhanced expression, phosphorylation, and nuclear translocation of SMAD2/3. Electrophysiological recordings show that administration of IL-6 increases cortical excitability, cul-

minating in epileptiform discharges in vitro and spontaneous in C57BL/6 mice. Intracellular recordings from layer V Downloaded from pyramidal cells in neocortical slices obtained from IL-6–treated mice show that during epileptogenesis, the cells respond to repetitive orthodromic activation with prolonged after-depolarization with no apparent changes in intrinsic membrane properties. Notably, TGF-b1–induced IL-6 upregulation occurs in of FVB/N but not in brains of C57BL/6 mice. Overall, our data suggest that TGF-b signaling in the can cause activation whereby IL-6 upregulation results in dysregulation of astrocyte–neuronal interactions and neuronal hyperexcitability. Whereas IL-6 is epileptogenic in C57BL/6 mice, its upregulation

by TGF-b1 is more profound in FVB/N mice characterized as a relatively more susceptible strain to -induced cell death. http://www.jimmunol.org/ The Journal of Immunology, 2015, 195: 1713–1722.

pilepsy is one of the most common neurologic disorders signaling cascade (through SMAD2/3 phosphorylation), and in- and is estimated to affect up to 1% of the population duces an astrocytic transcriptional response with proinflammatory E worldwide (1). Postinjury often develops fol- characteristics (10, 14–16). Furthermore, losartan, previously lowing brain insults, including ischemic or traumatic injury, as identified as a blocker of peripheral TGF-b signaling, effectively well as following infectious and inflammatory diseases (2). Recent blocks albumin-induced brain TGF-b signaling and prevents ep- studies suggest that vascular injury, and specifically blood–brain ilepsy (16). barrier (BBB) dysfunction and the extravasation of serum albu- Although accumulating experimental data from animal models by guest on October 1, 2021 min, plays a key role in postinjury epilepsy (3–7) (for reviews, see strongly support a role for inflammatory responses in epilepto- Refs. 8, 9). genesis (17, 18), several important questions remain unanswered: Epileptic brains often show glial activation, which was suggested What triggers the epileptogenic inflammatory process? Which to play an important role in neuronal hyperexcitability (10), specific cell populations are involved? What inflammatory medi- synaptogenesis (11), and epileptogenesis (12, 13). Under BBB ators are critical to the epileptogenesis process? And what are the breakdown, serum albumin binds to TGF-bR2, activates TGF-b mechanisms by which inflammatory cytokines affect neuronal excitability? Because activation of TGF-b signaling in glial sub- sets has been implicated in both pro- and anti-inflammatory pro- *Shraga Segal Department of Microbiology, Immunology, and Genetics, Faculty of Health Sciences, Zlotowski Center for Neuroscience, Ben-Gurion University of the cesses (14, 19–25), this study aims to uncover the molecular and Negev, Beer-Sheva 84105, Israel; †Department of Physiology and Cell Biology, cellular mechanisms promoting a proinflammatory TGF-b1 sig- Faculty of Health Sciences, Zlotowski Center for Neuroscience, Ben-Gurion Univer- ‡ naling in non-neuronal populations and their potential role in the sity of the Negev, Beer-Sheva 84105, Israel: and Department of Medical Neurosci- ence, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 4R2, induction of epilepsy. Canada 1N.L. and D.Z.M. contributed equally to this work. Materials and Methods 2A.F. and A.M. contributed equally to this work as cosenior authors. Mice Received for publication June 5, 2014. Accepted for publication June 2, 2015. C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, This work was supported by Israel Science Foundation Grants 713/11 (to A.F.) and ME). FVB/N mice were purchased from Harlan Laboratories (Rehovot, 531/11 (to A.M.), German Israeli Foundation Grant 124/2008 (to A.F.), National Israel). Mice were bred and maintained in a local specific pathogen-free Institute for Neurological Disorders and Grant 1rO1N5066005 (to A.F.), animal facility. All surgical and experimental procedures were approved by European Union’s Seventh Framework Program FP7/2007–2013 Grant 602102 (EPI- the Institutional Animal Care and Use Committee of Ben-Gurion Uni- TARGET, to A.F.), and by a local institutional grant (Ben-Gurion University of the Negev, Faculty of Health) (to A.M. and A.F.). versity of the Negev, Israel. Address correspondence and reprint requests to Prof. Alon Monsonego, Ben-Gurion University of the Negev, Faculty of Health Sciences, Beer-Sheva 84105, Israel. E-mail address: [email protected] Primary glial cultures were prepared from the of 1-d-old Abbreviations used in this article: ACSF, artificial cerebrospinal fluid; BBB, blood– C57BL/6 mice as previously described (19). Briefly, cortices were ex- brain barrier; GFAP, glial fibrillary acidic ; ICC, immunocytochemistry; ICV, cised, cleaned from meningeal tissues, and digested with 2.5% trypsin intracerebroventricular; qPCR, quantitative PCR. (solution C, Biological Industries, Beit Haemek, Israel) supplemented with 0.5 mg/ml DNase I (Worthington Biochemical, Lakewood, NJ) for 5 min at Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 37˚C. DNase I (5 mg/ml) was then added prior to passing the digested www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401446 1714 TGF-b SIGNALING–INDUCED EPILEPTOGENESIS tissue through a thin pipette several times. The cells were then suspended Little Chalfont, U.K.] or anti-mouse [Jackson ImmunoResearch Labora- in DMEM (Life Technologies, Paisley, U.K.) supplemented with 10% FBS tories, West Grove, PA]) for 1.5 h at room temperature. Detection of im- (Thermo Fisher Scientific, Logan, UT), 4 mM L-glutamine, 100 U/ml munoreactive bands was carried out with ImageQuant LAS 4000 (GE penicillin, 1 mg/ml streptomycin, 2.5 U/ml nystatin, 10 mM HEPES, Healthcare Life Sciences) using ECL. 1 mM sodium pyruvate, 10 mM nonessential amino acids, and 50 mM 2-ME (all purchased from Biological Industries) and seeded onto poly-D-lysine Immunocytochemistry (Sigma-Aldrich, Jerusalem, Israel)–coated flasks and kept at 37˚C, 5% CO2 Primary glial cells or purified astrocytes were cultured on eight-well Lab- and 95% humidity. Growth medium was replenished after 24 h and every Tek culture slides (1.5 3 105 cells/well) treated with 10 ng/ml TGF-b1 for 2–3 d thereafter. The culture reaches confluence after 12–14 d and is then 1 h, fixed in 4% buffered paraformaldehyde for 15 min, washed in PBS, subjected to quantitative PCR (qPCR), ELISA, and Western blot analyses as and permeabilized in methanol for 10 min at 220˚C. After PBS washing, whole or purified cell subsets as described below. cells were blocked for 2 h at room temperature with Dyna Ab diluent (GBI Cell separation and flow cytometry Labs, Bothell, WA) and were then incubated for 24 h at 4˚C with primary anti-CD68 diluted 1:250 (BioLegend) or anti–glial fibrillary acidic protein Cell separation procedure was conducted according to the standard man- (GFAP) diluted 1:500 (Invitrogen) together with anti-SMAD2/3 and anti– ufacturer’s protocol. In brief, glial cells were harvested between days 14 p-SMAD3 diluted 1:200 (Cell Signaling Technologies). Anti-CNPase was and 20 of the culture using a 0.25% trypsin solution (solution A, Biological diluted 1:150 (Abcam). Cells were then washed with PBS and incubated Industries), washed, and incubated with PE-conjugated anti-CD11b mag- with Alexa Fluor 488, 546, or 633 Abs (Invitrogen) diluted 1:500. TO- netic beads (Stemcell Technologies, Vancouver, BC, Canada). Cells were PRO-3 (Invitrogen) diluted 1:1500 was used for counterstaining. All then placed onto a magnet for separation. Subsequently, cells were stained images were obtained with an Olympus FluoView FV1000 confocal mi- with PE-conjugated anti–GLAST-1 Ab (Milteny Biotec, Bergisch Glad- croscope (Olympus, Hamburg, Germany). Z-stack images were taken at bach, Germany) and analyzed with FACSCalibur (BD Biosciences, 0.5-mm intervals across a 20-mm sample thickness. Confocal images were Franklin Lakes, NJ). Glial cultures were further analyzed using PE- analyzed with Volocity image analysis software (Impovision, Waltham, conjugated anti-CX3CR1, allophycocyanin-Cy7–conjugated anti-F4/80, MA). Fluorescence intensities of SMAD2/3 or phospho-SMAD3 staining Downloaded from PE/Dazzle-conjugated anti-CD11c, Alexa Fluor 700–conjugated anti– were obtained from the TO-PRO-3–labeled area of GFAP+ or CD68+ cells. MHC class II, Brilliant Violet 421–conjugated anti-Ly6C (BioLegend, San In each experiment, image acquisition settings were initially adjusted to Diego, CA), Pacific Orange–conjugated anti-CD45.2 (Invitrogen, Grand avoid saturation and were then used for all experimental groups. At least Island, NY), and FITC-Alexa Fluor 488–conjugated anti-CCR2 (R&D 10 images were obtained for each experimental group from randomly Systems, Minneapolis, MN). Isotype control staining was used for all Abs selected fields. (BioLegend). Samples were analyzed with Gallios (Beckman Coulter, Brea, CA). Brain qPCR analysis http://www.jimmunol.org/ Cell culture for ELISA and qPCR Hippocampi and cortices were dissected from the brain and lysed in a buffer containing 3 mM NaCl, 250 mM HEPES, 2% Triton X-100, and cocktails of Mixed glia, purified astrocytes, and microglia were seeded in 6-well (1 3 protease (Sigma-Aldrich) and phosphatase (Santa Cruz Biotechnology) 106 cells/well) or 48-well (1 3 105 cells/well) plates and were treated with inhibitors. Total RNA was extracted with TRI Reagent (Sigma-Aldrich) 10 ng/ml TGF-b1 (PeproTech, Rocky Hill, NJ) for 8–48 h as indicated in according to the manufacturer’s instructions and stored at 280˚C. RNA the figure legends. Cells were cultured in the absence or presence of 10 quality was analyzed using Bioanalyzer (Agilent Technologies, Santa mM SB431542 (an ALK4/5/7 inhibitor; Sigma-Aldrich), 10–20 mM SIS3 Clara, CA). Two micrograms RNA was reverse transcribed with a high- (a SMAD3 inhibitor; Santa Cruz Biotechnology, Dallas, TX), or 10 mM capacity cDNA reverse transcription kit (Applied Biosystems/Invitrogen) SJN2511 (an ALK5 inhibitor; Tocris Bioscience, Bristol, UK). Super- and IL-6, IL-1b, and TNF-a gene expression was quantified with TaqMan natants were then analyzed by ELISA array (for IL-1a, IL-1b, IL-2, IL-3, real-time PCR (Roche). Samples were run in triplicates. The GAPDH gene IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, MCP-1, IFN-g, TNF-a, MIP-1a, was used as an endogenous control to normalize gene expression. by guest on October 1, 2021 RANTES, and GM-CSF) (Quansys Bioscience, Logan, UT) or with a single cytokine sandwich ELISA (for IL-6 and TNF-a) (BioLegend) Electrophysiological recordings ex vivo according to the manufacturers’ instructions. Total RNA was extracted with For extracellular electrophysiological experiments, 12- to 14-wk-old TRI Reagent (Sigma-Aldrich) according to the manufacturer’s instructions. 2 C57BL/6 mice were anesthetized with isoflurane, brains were removed, RNA was stored at 80˚C. Two micrograms RNA was reverse transcribed and transverse cortico-hippocampal slices (400 mm thick) were prepared with a high-capacity cDNA reverse transcription kit (Applied Biosystems/ with a vibratome according to established methods (5). Slices were Invitrogen, Carlsbad, CA). IL-6, IL-1b,andTNF-a gene expression was maintained in a humidified, carbogenated (5% CO2 and 95% O2) gas at- analyzed with TaqMan real-time PCR (Applied Biosystems/Invitrogen). mosphere at 36.1˚C and perfused with artificial cerebrospinal fluid (ACSF; TGF-bR gene expression was quantified with SYBR Green real-time PCR 124 mM NaCl, 26 mM NaHCO , 1.25 mM NaH PO , 2 mM MgSO ,2 (Roche, Basel, Switzerland) using the following primers: forward, 59- 3 2 4 4 mM CaCl2, 3 mM KCl, 10 mM glucose [pH 7.4]) in a standard interface CCTCGAGACAGGCCATTTGTA-39,reverse,59-GCTGACTGCTTTTCTG- chamber (3, 5). Slices were incubated with either ACSF or IL-6 (100 TAGTTGG-39;TGF-bR2, forward, 59-TTTGCGATGTGAGACTGTCC-39, ng/ml) for 2–3 h before transfer to the perfusion chamber. Recordings were reverse, 59-GAGTGAAGCCGTGGTAGGTG-39. Samples were run in trip- performed with a constant ACSF perfusion. Glass microelectrodes (3 MV, licates. The GAPDH gene was used as an endogenous control to normalize 154 mM NaCl) were positioned in layers four through five of the sensory- gene expression. motor neocortex. Slices were stimulated with brief (0.1 ms) pulses using Immunoblot analysis bipolar stimulation electrodes placed at the border between white and gray matter in the same cortical column. Mixed glia and purified astrocytes were seeded in six-well plates at a density Somatic whole-cell recordings were obtained from layer five pyramidal of 6 3 105 cells/well. Cultures were treated with 10 ng/ml TGF-b1 for 1 h in somatosensory neocortical coronal slices prepared from C57BL/ in the presence or absence of 10 mM SB431542. Cell lysates were prepared 6 mice implanted either with ACSF (control) or IL-6 (5 ng/h using a stock using a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, solution of 5 mg/ml) containing osmotic pumps (Alzet osmotic pumps, 1 mM EGTA, 1% Nonidet P-40, and cocktails of protease (Sigma-Aldrich) Cupertino, CA) for 3–4 d (26). Mice were anesthetized with Nembutal (60 and phosphatase (Santa Cruz Biotechnology) inhibitors. Cell lysates were mg kg21) and decapitated. Coronal slices (300 mm) from primary so- quantified with the Bradford protein assay (Bio-Rad, Hercules, CA) using matosensory cortex were sectioned with a vibratome (VT1200, Leica, BSA (Sigma-Aldrich) as standard. Forty-microgram samples were then Wetzlar, Germany) and placed in a holding chamber containing oxygen- separated by SDS-PAGE (10% acrylamide) followed by a gel transfer to ated ACSF at room temperature; they were transferred to a recording 0.45 mM nitrocellulose membrane (Cole-Parmer, Vernon Hills, IL). Blots chamber after .1 h of incubation. were blocked with 5% nonfat dry milk in TBS-T (10 mM Tris, 135 mM Cells were viewed with a 3603 water-immersion lens (Olympus) in an NaCl [pH 7.4], 0.05% Tween 20) and incubated overnight at 4˚C with Olympus BX51WI microscope mounted on an X–Y translation stage primary anti-SMAD2/3 diluted 1:1000 (Cell Signaling Technology), anti– (Luigs and Neumann, Ratingen, Germany). Whole-cell recordings were phospho-(Ser465/467)-SMAD2 diluted 1:1000 (Cell Signaling Technology, conducted using patch pipettes pulled from thick-walled borosilicate glass Beverly, MA), or anti–TGF-bR2 diluted 1:1000 (Abcam, Cambridge, capillaries (1.5 mm outside diameter, Hilgenberg, Malsfeld, Germany). U.K.). Anti–b-actin, diluted 1:10,000 (MP Biomedicals, Solon, OH), was The pipette solution contained 130 mM potassium gluconate, 6 mM KCl, used to control protein load. Blots were then incubated with peroxidase- 2 mM MgCl2, 4 mM NaCl, and 10 mM HEPES (pH adjusted to 7.25 with conjugated secondary Abs (anti-rabbit [GE Healthcare Life Sciences, KOH). Pipettes had resistances of 5–7 MV when filled with this solution. The Journal of Immunology 1715

Recordings were made using an Axoclamp-2A amplifier equipped with an with an unpaired Student t test. Graphs indicate mean 6 SD, with n rep- HS-2-x0.01MU headstage (Molecular Devices, Sunnyvale, CA) in bridge resenting the number of mice, slices, or cultures used for analysis, as in- mode; data were low pass filtered at 30 kHz (23 dB, single-pole Bessel dicated in the figure legend. A x2 test was used to compare the frequency filter) and digitized at 100 kHz. Brief (0.1 ms) orthodromic stimuli were of epileptic slices in extracellular recording, and a Fisher exact test was delivered via a bipolar tungsten electrode (resistance ∼0.5 MV) placed at used to compare the frequency of epileptic animals in treated and control the border between gray and . Trains of pulses, 10-fold the groups. Seizure duration and amplitude was compared by two-tailed Stu- threshold intensity, were applied at 50 Hz for 1 s. Input resistance was dent t test. measured by injecting long (150–500 ms) low amplitude (,25 pA) hyperpolarizing current pulses. Three control and six IL-6–injected (5 ng/h) Results mice were used for these experiments. All recordings were made at room b temperature (22 6 2˚C). Electrophysiological data analysis was accom- TGF- 1 upregulates IL-6 expression in cultured glia plished using pCLAMP 7.0 (Axon Instruments) and Igor Pro 5.05 (Wave- To explore the role of TGF-b signaling in glial inflammatory metrics, Lake Oswego, OR). response, we treated primary glial cultures with TGF-b1and Electroencephalogram electrodes and osmotic pump quantified cytokine expression at the mRNA and protein levels implantation procedure using qPCR and ELISA, respectively. Of all cytokines and che- Experiments were performed on 12- to 14-wk-old C57BL/6 mice. For elec- mokines analyzed (see Materials and Methods), only IL-6 and trode implantation, animals were deeply anesthetized (isoflurane, 1.5–2%) and MCP-1 (CCL2) are significantly increased 24 h after treatment positioned in a stereotactic frame. Two screw electrodes were implanted (Fig. 1A, 1B). A time kinetics experiment showed that the up- 3 mm posterior and 2 mm lateral to bregma and were fixed to the skull with regulation of IL-6 mRNA is evident already 8 h after treating the bone cement. Alzet osmotic pumps were implanted in position 0.5 mm cells with TGF-b1 and peaked 24 h after treatment (12.19- posterior, 1 mm lateral, and 3 mm deep relative to bregma and contained p , ACSF only (1 ml/h, n = 6) or ACSF supplemented with IL-6 (1 mg/ml and 22.87-fold increases, respectively; F = 20.62, 0.001) Downloaded from [1 ng/h], n = 3 or 5 mg/ml [5 ng/h], n = 8). Perfusion lasted 1 wk. The (Fig. 1C). The transcriptional response is associated with in- pump and transmitter (Data Science International, Saint Paul, MN) were creased secreted levels of IL-6 in the supernatant at 8, 24, and 48 h placed s.c. in the dorsal part of the animal’s neck. Following the proce- after treatment (379.5 6 59.5, 825.1 6 25.3, and 1473.6 6 153.7 dure, the incision was sutured and the animals were s.c. injected with buprenorphine (0.1 mg/kg) and then allowed to recover from the anesthesia. pg/ml, respectively) (Fig. 1D). A qPCR analysis shows a milder increase in the mRNA levels of IL-1b and TNF-a at 24 h after Electroencephalogram recording and analysis treatment (F = 13.57, p , 0.01; Fig. 1C); however, ELISA does http://www.jimmunol.org/ After recovery, the animals were moved to a behavior room and maintained not demonstrate significantly elevated protein levels (data not on a 12-h circadian cycle with ad libitum access to food and water. Single- shown). channel electrographic signals were acquired from freely moving mice for 2 wk using a telemetric electroencephalogram monitoring system (Data TGF-b1–induced IL-6 expression is astrocyte-specific and Science International), as previously described (27). SMAD2/3-dependent At the end of the monitoring period, the animals were sacrificed and their brains were extracted. Two mice were excluded due to wound infection To determine the glia cell subsets that express IL-6 following ex- (n = 1) or a gross inflamed lesion on the surface of the cortex (n = 1). For posure to TGF-b1, we enriched astrocyte and microglia cultures unbiased detection of seizures, recordings were analyzed retrospectively with anti-CD11b magnetic beads as described in Materials and using an in-house written algorithm, based on feature extraction and ar- Methods. Both microglia and astrocytes were used at purities .95% tificial neural network classification as previously described (16). by guest on October 1, 2021 (Fig. 2A). The purified astrocytic culture contained ∼2.07 6 0.07% Statistical analysis CNPase+ oligodendrocyte precursors (data not shown). The + + + For ELISA, Western blot, immunocytochemistry (ICC), and qPCR anal- microglial subset contained primarily CD11b F4/80 CX3CR1 yses, statistical significance was tested with unpaired one-way ANOVA. cells and were negative for CD11c, CD45.2, CCR2, Ly6C, Comparison of amplitude and duration of seizure-like events were made and MHC class II staining (data not shown). The cultures were

AB

FIGURE 1. TGF-b1 induces rapid IL-6 upregulation in primary glial cultures. Cultures prepared from new- born C57BL/6 mice were treated with 10 ng/ml TGF-b1 or left untreated. Cytokine and chemokine expression and secretion were quantified by qPCR and ELISA. (A and B) Multiplex ELISA analysis performed 24 h following TGF-b1 treatment. (C and D) qPCR analysis of IL-1b, CD TNF-a, and IL-6 (C), and ELISA analysis of secreted IL- 6(D), at 8, 24, and 48 h following TGF-b1 treatment. Bars and data points represent means 6 SD obtained from one experiment out of at least three performed. *p , 0.05, **p , 0.01, ***p , 0.001 compared with untreated cells; (A and B) unpaired two-tailed Student t test, (C and D) one-way ANOVA test. 1716 TGF-b SIGNALING–INDUCED EPILEPTOGENESIS Downloaded from http://www.jimmunol.org/

FIGURE 2. TGF-b1 induces IL-6 upregulation primarily in astrocytes. Primary glial cultures were first purified to astrocytic and microglial cultures, and cells were then treated with 10 ng/ml TGF-b1. The kinetics of IL-6 expression were evaluated by qPCR and ELISA at 8, 24, and 48 h. (A) FACS analysis of mixed glia, purified microglia, and purified astrocytes. Microglia were labeled with and anti-CD11b, anti-F4/80, and anti-CX3CR1. Astrocytes were labeled with anti–GLAST-1. (B and C) qPCR analysis of IL-6 mRNA (B) and ELISA analysis of secreted IL-6 (C) in astrocyte (As.) and microglia (MG) cultures. Datum points represent the mean 6 SD fold change (RQ) or IL-6 levels at each time point compared with untreated controls (UT). (D) ELISA analysis of secreted IL-6 in mixed glia, astrocyte, and microglia cultures treated with LPS. (E) ELISA analysis of secreted IL-6 in mixed glia and astrocyte cultures treated with TGF-b1 in the absence or presence of SB431542 (SB), SJN2511 (SJN), or SIS3, and in microglia cultures treated with TGF-b1 in the absence or presence of SB431542. The results shown represent one experiment out of at least three performed. *p , 0.05, **p , 0.01, ***p , 0.001 (one-way by guest on October 1, 2021 ANOVA test). incubated with TGF-b1 for 8, 24, or 48 h. Compared with un- TGF-b1 induces SMAD2/3 phosphorylation and nuclear treated astrocytes, IL-6 is significantly upregulated in TGF-b1– translocation in astrocytes significantly more than in microglia treated astrocytes at both the mRNA (17.4-, 42.2-, and 34.3-fold To explore the mechanisms underlying the differential signaling , increase at 8, 24, and 48 h, respectively, F = 44.6, p 0.001 of TGF-b1 in astrocytes and microglia, we first evaluated basal , for 24 and 48 h, p 0.05 for 8 h; Fig. 2B) and protein levels expression levels of TGF-bR1 and TGF-bR2 in cultured cells. (396.2 6 9.2, 722.9 6 32.6, and 1459.5 6 188.1 pg/ml at 8, 24, qPCR analysis shows that the levels of both units of the TGF-bR and48h,respectively,F=64.7,p , 0.001 for each time point; are higher in microglia compared with astrocytes (5.175 6 0.6- Fig. 2C), with kinetics similar to those of mixed glia (Fig. 1). and 6.154 6 0.59-fold increase of TGF-bR1 and TGF-bR2, This is in contrast to purified microglia, which show no signif- respectively, p , 0.001; Fig. 3A) and similarly higher is the icant increase in IL-6 mRNA levels (Fig. 2B) and only a mild TGR-bR2 protein in microglia (Fig. 3B). We then exposed accumulation of secreted IL-6 (64 6 0.5, 165.3 6 4.7, and 266.2 cultures of mixed glia, purified astrocytes, and purified microglia 6 23.5 pg/ml at 8, 24, and 48 h after treatment, respectively, F = 324.8, p , 0.01; Fig. 2C). LPS stimulation of glia or the to TGF-b1 and quantified the expression, phosphorylation, and astrocyte- and microglia-purified subsets acting via the TLR nuclear translocation of SMAD2/3 (compared with actin as pathway show, as expected, a more profound secretion of IL-6 a control) using Western blot and ICC analysis. In the presence by microglia than by astrocytes (Fig. 2D) along with increased of TGF-b1, no change is observed in total SMAD2/3 levels, secreted levels of IL-1b and TNF-a (data not shown). Two which are higher in cultures of mixed glia and purified astrocytes separate blockers of SMAD2/3 phosphorylation, namely ALK 4/ compared with cultures of purified microglia (Fig. 3C, 3D). 5/7 (SB431542) and ALK5 (SJN2511), inhibit TGF-b1–induced Concomitantly, significantly increased levels of p-SMAD2 are IL-6 secretion in cultures of mixed glia (F = 29.56, p , 0.01), observed in purified astrocyte cultures than in purified microglia enriched astrocytes (F = 51.26, p , 0.01), or enriched microglia cultures (Fig. 3E). (F = 35.25, p , 0.01) (Fig. 2E). A SMAD3-specific inhibitor ICC analysis of glial cultures shows that the TGF-b1treatment (SIS3) decreases IL-6 secretion although to a lesser extent, promotes nuclear translocation of SMAD2/3 that is significantly namely by 60 and 57% in cultures of mixed glia and astrocytes, more colocalized with the astrocytic marker (GFAP) than with the respectively (p , 0.01; Fig. 2E). Taken together, these data in- microglial marker CD68 (Fig. 3F, 3H). Additionally, immunore- dicate that TGF-b1 induces a SMAD2/3-dependent upregulation activity of p-SMAD3 is evident only upon TGF-b1treatment and secretion of IL-6, as the main proinflammatory cytokine, (Fig. 3G) and is significantly more intense in astrocytes than in preferentially in astrocytes. microglia (Fig. 3I, p , 0.001). The Journal of Immunology 1717 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Astrocytes show more pronounced SMAD2/3 phosphorylation and nuclear translocation after TGF-b1 treatment compared with microglia. (A and B) Primary glial cultures were purified to astrocytes and microglia as described in Fig. 2 and analyzed for TGF-bR1 and TGF-bR2 expression levels using qPCR and Western blot. RNA and whole-cell lysates were prepared from purified astrocytes and purified microglia after CD11b separation. (A) qPCR analysis performed for TGF-bR1 and TGF-bR2 in purified astrocytes and microglia. (B) Western blot analysis performed for TGF-bR2 in purified astrocytes and microglia. The black line indicates joined parts of the image. Bars display TGF-bR2 to actin ratio in both astrocytes and microglia. (C and D) Primary glial cultures were treated with TGF-b1 and analyzed for SMAD2 and SMAD3 phosphorylation and nuclear translocation with Western blot and ICC. Whole-cell lysates were prepared following 1 h treatment with TGF-b1 of mixed glia, astrocyte, and microglia cultures. Untreated cultures were used as controls. (C) Western blot analysis performed for total SMAD2/3 and p-SMAD2 in mixed glia, purified astrocyte, and purified microglia cell cultures. (D and E) Quantification analysis of total SMAD2 (D) and p-SMAD2 (E) as their ratio (mean 6 SD) to actin in each culture. (F–I) Immunocytochemical analysis of mixed glia. (F and G) Cultures were treated with TGF-b1 for 1 h and then coimmunolabeled with anti-CD68 or anti-GFAP together with anti- SMAD2/3 (F) or anti–p-SMAD3 (G). Scale bars in each set represent 20 mm and apply to the entire set. (H and I) Quantification analysis of nuclear SMAD2/3 (H) and p-SMAD3 (I) in CD68+ microglia and GFAP+ astrocytes. Bars represent nuclear fluorescence intensity of SMAD2/3 in microglia and astrocytes (H) and fold change of p-SMAD3 (I) in TGF-b1–treated compared with untreated glial cultures. The results shown represent one experiment out of at least three performed. **p , 0.01, ***p , 0.001 (two-tailed Student t test). 1718 TGF-b SIGNALING–INDUCED EPILEPTOGENESIS

Additional support for SMAD2/3-mediated IL-6 upregulation neuronal activity. We performed extracellular recordings in acute comes from analyzing the promoter region of IL-6 for SMAD- brain slices incubated for 2–4 h with IL-6 (100 ng/ml). Indeed, binding elements (see review in Ref. 28). We identified several a brief white matter electrical stimulation induces hyper- putative SMAD3/4-binding sequences such as GTCT, AGAC, and synchronous propagating epileptiform activity in 85% of all IL-6– CAGAC up to 2000 bp upstream to the IL-6 coding sequence. treated slices but in none of the control slices (n = 7 and 5, re- Whether these sequences indeed serve as SMAD3/4 binding sites spectively; p = 0.003; Fig. 4A, 4B). We next tested whether an that promote TGF-b1–induced IL-6 transcription in astrocytes is intraventricular application of IL-6 can induce epileptiform ac- yet to be determined. tivity in vivo. Either low (1 ng/h) or high (5 ng/h) concentrations Overall, our data show that both astrocytes and microglia express of IL-6 (IL-6 low and IL-6 high, respectively) were injected to TGF-bR1 and TGF-bR2 as well as SMAD2 and SMAD3, which mice (n = 3 and 8, respectively) with an osmotic pump, and undergo phosphorylation following TGF-b1 treatment. However, cortical activity was continuously monitored for 14 d thereafter. although higher levels of TGF-bR are expressed in microglia, Controls were injected with ACSF (n = 6). Seizure-like activity the expression, phosphorylation, and nuclear translocation of (.5 s duration) was detected with a custom-built semiautomatic SMAD2/3 are more abundant in astrocytes than in microglia and algorithm (see Materials and Methods). may thus be directly associated with the upregulation of IL-6 via Significantly more IL-6–treated mice show distinct paroxysmal the TGF-b1 signaling pathway. seizure-like activity compared with control mice (82 and 16.67%, respectively; p , 0.05). Moreover, the number and duration of IL-6 induces ex vivo and in vivo epileptiform activity seizures depend on IL-6 dose. In comparison with control mice Because both albumin and TGF-b1 induce epileptiform activity (n = 6; three seizures overall, each duration was limited to 5 s), when applied onto brain slices (14), and because IL-6 was the recordings from mice treated with a low dose of IL-6 (1 ng/h) Downloaded from primary cytokine upregulated by TGF-b1 in cultured astrocytes, revealed overall 20 seizures with a mean duration of 16.6 s (n =3; we tested whether IL-6 is sufficient to induce hypersynchronous median duration, 5 s; range, 5–60 s; p , 0.05), whereas recordings http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. IL-6 induces ex vivo and in vivo epileptiform activity. (A) Evoked cortical field potentials were recorded in response to a single brief electrical stimulation. Slices were incubated for 2–4 h in ACSF (n = 6, top trace) in or 100 ng/ml IL-6 (n = 7, bottom trace). (B) The integral of the field potentials was calculated for 0- to 50-ms and for 50- to 200-ms intervals after stimulation. (C) Electroencephalogram showing a seizure-like event detected by the seizure detection algorithm in IL-6–treated mouse (5 ng/hr). Inserts show magnification of a region indicated by an asterisk. (D) Histograms describing the distribution of durations of detected seizures during 14 recording days. Mice were injected ICV with IL-6 at 1 ng/h (left panel) or 5 ng/h (right panel). (E) Mean 6 SD duration of seizures detected in mice injected with low (1 ng/h) (n = 20) or high (5 ng/h) (n = 156) doses of IL-6 compared with control (ACSF-injected) mice (n = 3). (F) A temporal analysis showing the number of seizure-like events (SLEs) occurring at different days after surgery (day 0) in the different groups. *p , 0.05, **p , 0.01, ***p , 0.001 (two-tailed Student t test). The Journal of Immunology 1719 from high-dose IL-6–treated mice (5 ng/h) revealed overall 156 150-pA, 300-ms-long current pulses elicit, on average, 5.3 6 1.4 seizures with a mean duration of 30.4 s (n = 8; median duration, and 4.7 6 0.9 action potentials in neurons from control (n = 7) and 16 s; range, 5–60 s; p , 0.01) (Fig. 4C–E). Additionally, seizure in IL-6–treated mice (n = 9), respectively (p . 0.25). Current amplitude is higher in IL-6 high compared with IL-6 low mice clamp recordings likewise reveal no gross difference in the am- (3.72 6 3.47 and 1.52 6 1.42 mV2, respectively; p , 0.01). A plitude and frequency of spontaneous synaptic events at the level temporal analysis (Fig. 4F) shows that the earliest seizures are of resting potential. The minimal stimulus intensity required to detected 3 d following IL-6 injection, after which their frequency elicit an orthodromic action potential is significantly lower in increases during the first week and decreases during the second neurons from IL-6–treated mice compared with controls (10 6 4 week after injection. and 15 6 5V,n = 7 and 10, respectively; p , 0.02). Notably, a robust difference is found in the membrane potential time course Poststimulation depolarization is prolonged in neurons following repetitive (50 Hz, 1 s) afferent extracellular stimulation obtained from IL-6–treated mice (Fig. 5A–C). While membrane potential in neurons from control We next used somatic whole-cell recordings from neocortical py- mice rapidly returns to resting values, often with a tendency to- ramidal neurons to test the direct effect of an intracerebroventricular ward a small after-hyperpolarization, a prolonged (up to 4 s) (ICV) injection of IL-6 on neuronal activity. Recordings were made poststimulus depolarization is observed in most neurons from during epileptogenesis, that is, 72–88 h after implanting the ICV IL-6–treated cortices following extracellular (Fig. 5C) but not pumps and prior to the appearance of the first seizure. intracellular stimulation (Fig. 5D–F). Passive membrane properties are similar between ACSF-treated b controls and IL-6–injected mice (n = 8 and 10 neurons, respec- ICV administration of TGF- 1 induces upregulation of IL-6 in tively), including resting membrane potential (260.6 6 1.8 the brain of FVB/N mice Downloaded from and 261.1 6 2.9 mV, respectively; p . 0.05) and input resistance To determine whether IL-6 is upregulated in brains of mice during (133 6 29 and 149 6 66 MV, respectively; p . 0.5). Action the induction of epileptogeneis, both mouse strains FVB/N and potential properties are also similar between controls (n = 8) and C57BL6 exhibiting a general high and low sensitivity to seizure IL-6–injected mice (n = 11), including the threshold (247.4 6 3.8 induction, respectively, were ICV injected with TGF-b1. FVB/N and 247.0 6 5.9 mV, respectively; p . 0.85), the width at 220 mice (8–10 wk of age) were implanted with TGF-b1–containing mV (1.41 6 0.09 and 1.42 6 0.12 ms, respectively), and the (100 ng/ml, 0.1 ng/h) or ACSF-containing osmotic pumps. After http://www.jimmunol.org/ amplitude (99 6 5 and 95 6 7 mV, respectively). No difference 72 h, brains were dissected into hippocampi and cortices and between the groups is observed also in repetitive firing properties: examined for mRNA levels of IL-6, IL-1b, and TNF-a with qPCR by guest on October 1, 2021

FIGURE 5. White matter stimulation induces prolonged after- depolarization in pyramidal neurons from IL-6–treated mice. (A) Responses to white matter stimulation (50 Hz, 1 s) recorded in whole-cell current clamp from representative layer V cortical neurons obtained from one IL-6–treated mouse (5 mg/ml) (top) and one control (ACSF-treated) (bottom) mouse. (B) Magnifica- tion of the dashed regions shown in (A) (black, IL-6 treated; gray, ACSF treated). The broken gray line corresponds to the resting membrane potential (Erest). (C) The average poststimulus po- tential (PSP) between 0.2 and 0.3 s after termination of synaptic stimulation in neurons obtained from control and IL-6–treated mice. The thick horizontal bar indicates the median, the box limits the 25th and 75th percentiles, and the whiskers indicate the range. (D) Responses to intracellular stimulation (50 Hz, 1 nA, 1 s overall, 5-ms pulses) recorded in whole-cell current clamp from representative layer V cortical neurons obtained from an IL-6 and a control mouse (top and bottom traces, respectively). (E) Magnification of the dashed regions shown in D (black, IL-6 treated; gray, ACSF treated). The broken gray line corresponds to the resting membrane potential (Erest). (F) The average post- stimulus potential (PSP) between 0.2 and 0.3 s after termination of synaptic stimulation in neurons obtained from control and IL-6–treated mice. Annotations same as in (C). **p , 0.01 (two- tailed Student t test). 1720 TGF-b SIGNALING–INDUCED EPILEPTOGENESIS

FIGURE 6. TGF-b1–induced IL-6 secretion is genetic background–dependent. (A–C)FVBNmice (8-10 wk old) were implanted with 100 ng/ml TGF-b1–containing (n = 10) or ACSF-containing (n = 10) osmotic pumps. (D and E)C57BL/6mice were implanted with osmotic pumps containing ei- ther 100 ng/ml TGF-b1(D, n = 4), 1000 ng/ml TGF-b1(E, n =4),orACSF(n = 4). Untreated mice served as control (FVB/N, n = 10; C57BL/6, n =3). At 72 h after surgery, brains were removed and hippocampi were dissected. RNA was extracted from the tissues and analyzed for IL-6, IL-1b,and TNF-a gene expression using qPCR. *p , 0.05, **p , 0.01, ***p , 0.001 (one-way ANOVA test).

as described in Materials and Methods. As shown in Fig. 6A–C, latory cytokine (19, 20, 25, 31–34), it can also exert autoimmu- Downloaded from compared with untreated mice, all three proinflammatory genes nity, glial activation, and brain inflammation (22, 23, 35, 36). In are significantly upregulated in the hippocampi of TGF-b1– the present study, we demonstrate that glial exposure to TGF-b1 injected mice (IL-6, 3.53 6 2.39, p , 0.01; IL-1b, 19.46 6 17.12, induces a rapid and robust IL-6 upregulation at both the mRNA p , 0.01; and TNF-a,46 2.11, p , 0.001). The slight upregu- and protein levels. Such upregulation of IL-6 by TGF-b1 seems to lation of IL-6, IL-1b, and TNF-a obtained in the hippocampi of be a specific inflammatory response: it differs from the effect of the ACSF group at 3 d after injection resembles the normal in- LPS, occurs primarily in astrocytes (and not in microglia), and http://www.jimmunol.org/ flammatory response to injury (Fig. 6A–C). At 3 d after injection, involves only a slight consequent upregulation of IL-1b and TNF- TGF-b1 does not upregulate IL-6, IL-1b, and TNF-a in the cortex a. Our data demonstrate that both microglia and astrocytes ex- over the levels induced by ACSF (data not shown). Notably, in press the TGF-b as well as the trans-signaling molecules contrast to FVB/N mice, C57BL/6 mice were resistant to TGF- SMAD2 and SMAD3. However, whereas both subunits of the b1–induced IL-6 upregulation at both low (100 ng/ml, 0.1 ng/h for TGF-bR are expressed at higher levels in microglia, the expres- 24–48 and 72 h) and high (1000 ng/ml, 1 ng/h for 72 h) doses (Fig sion, phosphorylation, and nuclear translocation of SMAD2/3 are 6D, 6E), suggesting that immunoregulatory mechanisms control- significantly more abundant in astrocytes. Together with our ling the proinflammatory role of TGF-b1 are more stringent in findings that the SMAD3-specific inhibitor SIS3 markedly sup-

C57BL/6 mice than in FVB/N mice and may partially underlie pressed the upregulation of IL-6 in astrocytes, we suggest that by guest on October 1, 2021 findings demonstrating the relative resistance of C57BL/6, but not SMAD3 is required for the enhanced expression of IL-6 in astro- FVB/N mice, to seizure-induced cell death (29, 30). cytes presumably via direct binding to SMAD-binding elements in the IL-6 promoter region (28, 37, 38). The signals triggering Discussion SMAD3 expression and the molecular pathway of p-SMAD3– The present study sought to elucidate the mechanisms underlying induced IL-6 upregulation, alone or with additional cofactors (37, TGF-b signaling–induced epileptogenesis in mice. Using glial 39), are yet to be characterized. cultures, we demonstrate that TGF-b1 induces an astrocyte- Of the milieu of proinflammatory cytokines tested in the present specific response that includes a differential SMAD2/3 phos- study, we found TGF-b1 to induce first, and most prominently, the phorylation and nuclear translocation and a rapid upregulation and upregulation of IL-6. Numerous studies demonstrated significant secretion of IL-6. Interestingly, glial response to LPS is signifi- IL-6 levels in the CSF and blood serum of patients suffering from cantly different, with a robust, TLR4–mediated IL-6 secretion traumatic brain injuries or neurodegenerative processes (40). In- from microglia. Finally, electrophysiological experiments support terestingly, in most of these disorders, BBB dysfunction has been a direct epileptogenic effect of IL-6 both ex vivo and in vivo. documented together with increasing likelihood for seizures. In IL-6–induced neuronal hyperexcitability was associated with a this study, we show that IL-6 is sufficient to promote network prolonged depolarizing afterpotential following repetitive ortho- hypersynchronization and epileptiform activity both ex vivo and dromic activation with no apparent changes in intrinsic membrane in vivo and thus highlight IL-6 with the potential to play a key role properties in the recorded neurons. Thus, overall, we suggest that in the development of seizures following brain insults and under conditions in which vascular insult and BBB dysfunction breakdown of the BBB. Expressed in the brain with receptors on occur, TGF-b signaling–induced early upregulation of IL-6 in all neural lineages, IL-6 was reported to be involved in neuro- astrocytes is sufficient to trigger epileptogenesis. Owing to the genesis and synaptic activity (see reviews in Refs. 40, 41). A resistance to TGF-b1–induced IL-6 upregulation in C57BL/6 mice significant body of evidence also shows that, similar to TGF-b1, in vivo, this pathway may be limited to certain genetic backgrounds IL-6 is upregulated in neurodegenerative processes with functions and/or genetic predisposition. ranging from protective to detrimental. As such, IL-6 has been We previously demonstrated TGF-b signaling and a robust in- shown to either protect from in several in vitro and flammatory response in the brain of rats and FVB/N mice that in vivo models, or to enhance N-methyl-D-asparate–induced were exposed to serum albumin (10, 11, 14, 16). Furthermore, excitotoxicity in cerebellar granule neurons (40). Notably, tran- TGF-b1 pathway blockers efficiently blocked epileptogenesis in sient inhibition of STAT3 phosphorylation (a signaling cascade BBB breakdown and albumin models of epilepsy in mice and rats induced by IL-6) in a rat model of pilocarpine-induced status (11, 16). Although TGF-b1 is a well-characterized immunoregu- epilepticus has recently been shown to significantly reduce disease The Journal of Immunology 1721 severity along with downregulation of STAT3-regulated genes References (42). Our current investigation demonstrates that brain exposure to 1. Engel, J. J., andPedley, T. A.. 2007. Introduction: what is epilepsy? In Epilepsy: IL-6 is sufficient to induce seizures in vivo with duration and A Comprehensive Textbook. J. Engel, Jr., and T. Pedley, eds. Lippincot Williams & Wilkins, Philadelphia, p. 54–66. amplitude depending on the dose of injected IL-6. Interestingly, 2. Hesdorffer, D. C. 2007. Risk factors. In Epilepsy: A Comprehensive Textbook. J. ICV administration of TGF-b1 induces a significant IL-6 upreg- Engel, Jr., and T. Pedley, eds. Lippincot Williams & Wilkins, Philadelphia, p. 156–170. ulation in FVB/N but not in C57BL/6 mice. These results may at 3. Ivens, S., D. Kaufer, L. P. Flores, I. Bechmann, D. Zumsteg, O. Tomkins, least partially underlie previous studies showing that C57BL/6 E. Seiffert, U. Heinemann, and A. Friedman. 2007. TGF-b receptor-mediated mice exhibit a general low sensitivity to seizure induction (43– albumin uptake into astrocytes is involved in neocortical epileptogenesis. Brain 130: 535–547. 45) and that FVB/N mice have a lower threshold than do C57BL/6 4. Janigro, D. 1999. Blood-brain barrier, homeostatis and epilepsy: possible mice for seizure-induced cell death (29, 30, 46), further suggesting implications towards the understanding of ketogenic diet mechanisms. Epilepsy that IL-6 upregulation plays an important role in the brain inflam- Res. 37: 223–232. 5. Seiffert, E., J. P. Dreier, S. Ivens, I. Bechmann, O. Tomkins, U. Heinemann, and matory response associated with TGF-b–induced epileptogenesis. A. Friedman. 2004. Lasting blood-brain barrier disruption induces epileptic fo- The mechanisms underlying neuronal hypersynchronization and cus in the rat somatosensory cortex. J. Neurosci. 24: 7829–7836. 6. Tomkins, O., O. Friedman, S. Ivens, C. Reiffurth, S. Major, J. P. Dreier, epileptogenesis following exposure to IL-6 are not yet fully under- U. Heinemann, and A. Friedman. 2007. Blood-brain barrier disruption results in stood. Increase in intrinsic excitability (47), selective excitatory delayed functional and structural alterations in the rat neocortex. Neurobiol. Dis. synaptogenesis (48), and reduction in inhibitory transmission (14) 25: 367–377. 7. Tomkins, O., I. Shelef, I. Kaizerman, A. Eliushin, Z. Afawi, A. Misk, M. Gidon, were all reported to occur during epileptogenesis. Our recordings A. Cohen, D. Zumsteg, and A. Friedman. 2008. Blood-brain barrier disruption in from neocortical pyramidal neurons indicate activity-dependent in- post-traumatic epilepsy. J. Neurol. Neurosurg. Psychiatry 79: 774–777. crease in excitability that does not require a prominent change in 8. Abbott, N. J., and A. Friedman. 2012. Overview and introduction: the blood- brain barrier in health and disease. Epilepsia 53(Suppl. 6): 1–6. Downloaded from intrinsic properties and/or spontaneous synaptic transmission, and is 9. Shlosberg, D., M. Benifla, D. Kaufer, and A. Friedman. 2010. Blood-brain probably astrocyte mediated (10). Indeed, recent findings from hu- barrier breakdown as a therapeutic target in . Nat Rev Neurol 6: 393–403. man epileptic tissue and animal models of epilepsy suggest a key 10. David, Y., L. P. Cacheaux, S. Ivens, E. Lapilover, U. Heinemann, D. Kaufer, and role for astrocytic dysfunction in epileptogenesis, seizure generation, A. Friedman. 2009. Astrocytic dysfunction in epileptogenesis: consequence of and seizure propagation (10, 12, 49,50).Inparticular,asignificant altered potassium and glutamate homeostasis? J. Neurosci. 29: 10588–10599. 11. Weissberg, I., L. Wood, L. Kamintsky, O. Vazquez, D. Z. Milikovsky, role has been suggested for the downregulation of glial inward A. Alexander, H. Oppenheim, C. Ardizzone, A. Becker, F. Frigerio, et al. 2015. http://www.jimmunol.org/ rectifying potassium (Kir) channel 4.1, which underlies impaired Albumin induces excitatory synaptogenesis through astrocytic TGF-b/ALK5 signaling in a model of acquired epilepsy following blood-brain barrier dys- buffering of extracellular potassium (10, 12, 13). Because our pre- function. Neurobiol. Dis. 78: 115–125. vious molecular and physiological data in mouse and rat models of 12. Heinemann, U., D. Kaufer, and A. Friedman. 2012. Blood-brain barrier dys- albumin- and TGF-b–induced seizures showed early activation of function, TGFb signaling, and astrocyte dysfunction in epilepsy. Glia 60: 1251– 1257. astrocytes (3, 10), and specifically a robust excessive extracellular 13. Steinha¨user, C., G. Seifert, and P. Bedner. 2012. Astrocyte dysfunction in tem- potassium accumulation upon repetitive stimulation at physiologi- poral lobe epilepsy: K+ channels and gap junction coupling. Glia 60: 1192–1202. 14. Cacheaux, L. P., S. Ivens, Y. David, A. J. Lakhter, G. Bar-Klein, M. Shapira, cally relevant frequencies (10–50 Hz) (10), we tested a potential role U. Heinemann, A. Friedman, and D. Kaufer. 2009. Transcriptome profiling of such stimulation on neuronal excitability in the IL-6–treated reveals TGF-b signaling involvement in epileptogenesis. J. Neurosci. 29: 8927– 8935. cortices. Our recordings demonstrate a long-lasting depolarization by guest on October 1, 2021 ∼ 15. Frigerio, F., A. Frasca, I. Weissberg, S. Parrella, A. Friedman, A. Vezzani, and ( 8 mV) upon afferent but not intracellular stimulation, predicting F. M. Noe´. 2012. Long-lasting pro-ictogenic effects induced in vivo by rat brain a 25% increase in the accumulation of extracellular potassium during exposure to serum albumin in the absence of concomitant pathology. Epilepsia 53: 1887–1897. neuronal activation, consistent with the notion of astrocytic dys- 16. Bar-Klein, G., L. P. Cacheaux, L. Kamintsky, O. Prager, I. Weissberg, function and reduced potassium buffering after insult (51, 52) or K. Schoknecht, P. Cheng, S. Y. Kim, L. Wood, U. Heinemann, et al. 2014. during epileptogenesis (3, 10). Overall, although additional experi- Losartan prevents acquired epilepsy via TGF-b signaling suppression. Ann. Neurol. 75: 864–875. ments in different neuronal populations and experimental conditions 17. Vezzani, A., J. French, T. Bartfai, and T. Z. Baram. 2011. The role of inflam- (e.g., voltage clamp experiments under different holding membrane mation in epilepsy. Nat Rev Neurol 7: 31–40. potentials) are required to rule out additional changes in synaptic 18. Vezzani, A., and B. Viviani. 2015. Neuromodulatory properties of inflammatory cytokines and their impact on neuronal excitability. Neuropharmacology 96 properties, our experiments at an early time point during epilepto- (Part A): 70–82. genesis suggest that IL-6 is sufficient to facilitate stimulus-dependent 19. Abutbul, S., J. Shapiro, I. Szaingurten-Solodkin, N. Levy, Y. Carmy, R. Baron, S. Jung, and A. Monsonego. 2012. TGF-b signaling through SMAD2/3 induces neuronal depolarization and hyperexcitability, likely due to failure in the quiescent microglial phenotype within the CNS environment. Glia 60: 1160– buffering of extracellular potassium. 1171. In summary, our study highlights a novel proinflammatory re- 20. Brionne, T. C., I. Tesseur, E. Masliah, and T. Wyss-Coray. 2003. Loss of TGF-b1 leads to increased neuronal cell death and microgliosis in mouse brain. action, which does not essentially involve microglial activation but, 40: 1133–1145. rather, is executed via TGF-b1 signaling in astrocytes to promote 21. Lanz, T. V., Z. Ding, P. P. Ho, J. Luo, A. N. Agrawal, H. Srinagesh, R. Axtell, H. Zhang, M. Platten, T. Wyss-Coray, and L. Steinman. 2010. Angiotensin II the release of IL-6. This astrocytic response may be dependent on sustains brain inflammation in mice via TGF-b. J. Clin. Invest. 120: 2782–2794. genetic background and is further associated with impaired ex- 22. Luo, J., P. P. Ho, M. S. Buckwalter, T. Hsu, L. Y. Lee, H. Zhang, D. K. Kim, tracellular homeostasis upon neuronal activation and altered S. J. Kim, S. S. Gambhir, L. Steinman, and T. Wyss-Coray. 2007. Glia-dependent TGF-b signaling, acting independently of the TH17 pathway, is critical for ini- neuronal–astrocytic interactions, leading to spontaneous seizures. tiation of murine autoimmune encephalomyelitis. J. Clin. Invest. 117: 3306– Because increased CSF or blood serum levels of IL-6 have been 3315. documented in a variety of CNS injuries that are often followed by 23. Luo, J., A. H. Lin, E. Masliah, and T. Wyss-Coray. 2006. Bioluminescence imaging of Smad signaling in living mice shows correlation with excitotoxic seizures (53), therapeutic strategies to knock down IL-6 expres- . Proc. Natl. Acad. Sci. USA 103: 18326–18331. sion and/or signaling in the CNS may prove beneficial. 24. Tesseur, I., K. Zou, L. Esposito, F. Bard, E. Berber, J. V. Can, A. H. Lin, L. Crews, P. Tremblay, P. Mathews, et al. 2006. Deficiency in neuronal TGF-b signaling promotes neurodegeneration and Alzheimer’s pathology. J. Clin. In- Acknowledgments vest. 116: 3060–3069. 25. Town, T., Y. Laouar, C. Pittenger, T. Mori, C. A. Szekely, J. Tan, R. S. Duman, We thank Dr. Ram Gal for valuable editorial comments. and R. A. Flavell. 2008. Blocking TGF-b-Smad2/3 innate immune signaling mitigates Alzheimer-like pathology. Nat. Med. 14: 681–687. 26. Fleidervish, I. A., L. Libman, E. Katz, and M. J. Gutnick. 2008. Endogenous Disclosures polyamines regulate cortical neuronal excitability by blocking voltage-gated Na+ The authors have no financial conflicts of interest. channels. Proc. Natl. Acad. Sci. USA 105: 18994–18999. 1722 TGF-b SIGNALING–INDUCED EPILEPTOGENESIS

27. Weissberg, I., A. Reichert, U. Heinemann, and A. Friedman. 2011. Blood-brain 41. Spooren, A., K. Kolmus, G. Laureys, R. Clinckers, J. De Keyser, G. Haegeman, barrier dysfunction in epileptogenesis of the . Epilepsy Res. Treat. and S. Gerlo. 2011. Interleukin-6, a mental cytokine. Brain Res. Brain Res. Rev. 2011: 143908. 67: 157–183. 28. Morikawa, M., D. Koinuma, K. Miyazono, and C. H. Heldin. 2013. Genome- 42. Grabenstatter, H. L., Y. C. Del Angel, J. Carlsen, M. F. Wempe, A. M. White, wide mechanisms of Smad binding. Oncogene 32: 1609–1615. M. Cogswell, S. J. Russek, and A. R. Brooks-Kayal. 2014. The effect of STAT3 29. McLin, J. P., and O. Steward. 2006. Comparison of seizure phenotype and inhibition on and subsequent spontaneous seizures in the pi- neurodegeneration induced by systemic kainic acid in inbred, outbred, and hy- locarpine model of acquired epilepsy. Neurobiol. Dis. 62: 73–85. brid mouse strains. Eur. J. Neurosci. 24: 2191–2202. 43. Bankstahl, M., C. J. Muller,€ E. Wilk, K. Schughart, and W. Lo¨scher. 2012. 30. Schauwecker, P. E. 2012. Strain differences in seizure-induced cell death fol- Generation and characterization of pilocarpine-sensitive C57BL/6 mice as lowing pilocarpine-induced status epilepticus. Neurobiol. Dis. 45: 297–304. a model of . Behav. Brain Res. 230: 182–191. 31. Zhu, Y., G. Y. Yang, B. Ahlemeyer, L. Pang, X. M. Che, C. Culmsee, S. Klumpp, 44. Yang, J., B. Houk, J. Shah, K. F. Hauser, Y. Luo, G. Smith, E. Schauwecker, and and J. Krieglstein. 2002. Transforming growth factor-b1 increases bad phos- G. N. Barnes. 2005. Genetic background regulates semaphorin gene expression phorylation and protects neurons against damage. J. Neurosci. 22: 3898–-3909. and epileptogenesis in mouse brain after kainic acid status epilepticus. Neuro- 32. Qian, L., S. J. Wei, D. Zhang, X. Hu, Z. Xu, B. Wilson, J. El-Benna, J. S. Hong, science 131: 853–869. and P. M. Flood. 2008. Potent anti-inflammatory and neuroprotective effects of 45. Borges, K., M. Gearing, D. L. McDermott, A. B. Smith, A. G. Almonte, TGF-b1 are mediated through the inhibition of ERK and p47phox-Ser345 phos- B. H. Wainer, and R. Dingledine. 2003. Neuronal and glial pathological changes Exp. Neurol. phorylation and translocation in microglia. J. Immunol. 181: 660–668. during epileptogenesis in the mouse pilocarpine model. 182: 21–34. 33. Makwana, M., L. L. Jones, D. Cuthill, H. Heuer, M. Bohatschek, M. Hristova, 46. Schauwecker, P. E., and O. Steward. 1997. Genetic determinants of susceptibility to excitotoxic cell death: implications for gene targeting approaches. Proc. Natl. S. Friedrichsen, I. Ormsby, D. Bueringer, A. Koppius, et al. 2007. Endogenous Acad. Sci. USA 94: 4103–4108. transforming growth factor b1 suppresses inflammation and promotes survival in 47. Becker, A. J., J. Pitsch, D. Sochivko, T. Opitz, M. Staniek, C. C. Chen, adult CNS. J. Neurosci. 27: 11201–11213. K. P. Campbell, S. Schoch, Y. Yaari, and H. Beck. 2008. Transcriptional up- 34. Naert, G., and S. Rivest. 2011. CC chemokine receptor 2 deficiency aggravates regulation of Cav3.2 mediates epileptogenesis in the pilocarpine model of epi- cognitive impairments and amyloid pathology in a transgenic mouse model of lepsy. J. Neurosci. 28: 13341–13353. Alzheimer’s disease. J. Neurosci. 31: 6208–6220. 48. Li, H., K. D. Graber, S. Jin, W. McDonald, B. A. Barres, and D. A. Prince. 2012. 35. Wyss-Coray, T., P. Borrow, M. J. Brooker, and L. Mucke. 1997. Astroglial Gabapentin decreases epileptiform discharges in a chronic model of neocortical overproduction of TGF-b1 enhances inflammatory dis- trauma. Neurobiol. Dis. 48: 429–438. Downloaded from ease in transgenic mice. J. Neuroimmunol. 77: 45–50. 49. Carlen, P. L. 2012. Curious and contradictory roles of glial connexins and 36. Wyss-Coray, T., J. D. Loike, T. C. Brionne, E. Lu, R. Anankov, F. Yan, pannexins in epilepsy. Brain Res. 1487: 54–60. S. C. Silverstein, and J. Husemann. 2003. Adult mouse astrocytes degrade 50. Steinha¨user, C., and D. Boison. 2012. Epilepsy: crucial role for astrocytes. Glia amyloid-b in vitro and in situ. Nat. Med. 9: 453–457. 60: 1191. 37. Feng, X. H., and R. Derynck. 2005. Specificity and versatility in TGF-b sig- 51. Anderova´, M., T. Antonova, D. Petrı´k, H. Neprasova´, A. Chva´tal, and E. Sykova´. naling through Smads. Annu. Rev. Cell Dev. Biol. 21: 659–693. 2004. Voltage-dependent potassium currents in hypertrophied rat astrocytes after 38. Mullen, A. C., D. A. Orlando, J. J. Newman, J. Love´n, R. M. Kumar, a cortical stab wound. Glia 48: 311–326.

S. Bilodeau, J. Reddy, M. G. Guenther, R. P. DeKoter, and R. A. Young. 2011. 52. D’Ambrosio, R., D. O. Maris, M. S. Grady, H. R. Winn, and D. Janigro. 1999. http://www.jimmunol.org/ Master transcription factors determine cell-type-specific responses to TGF-b Impaired K+ homeostasis and altered electrophysiological properties of post- signaling. Cell 147: 565–576. traumatic hippocampal glia. J. Neurosci. 19: 8152–8162. 39. Zhang, Y., X. H. Feng, and R. Derynck. 1998. Smad3 and Smad4 cooperate with 53. Vossel, K. A., A. J. Beagle, G. D. Rabinovici, H. Shu, S. E. Lee, G. Naasan, c-Jun/c-Fos to mediate TGF-b-induced transcription. Nature 394: 909–913. M. Hegde, S. B. Cornes, M. L. Henry, A. B. Nelson, et al. 2013. Seizures and 40. Erta, M., A. Quintana, and J. Hidalgo. 2012. Interleukin-6, a major cytokine in epileptiform activity in the early stages of Alzheimer disease. JAMA Neurol. 70: the central nervous system. Int. J. Biol. Sci. 8: 1254–1266. 1158–1166. by guest on October 1, 2021