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GABA transport and neuroinflammation are coupled in multiple sclerosis: Regulation of the GABA transporter-2 by ganaxolone
Neuroscience, pages 24 - 38
Interactions between neurotransmitters and the immune system represent new prospects for understanding neuroinflammation and associated neurological disease. GABA is the chief inhibitory neurotransmitter but its actions on immune pathways in the brain are unclear. In the present study, we investigated GABAergic transport in conjunction with neuroinflammation in models of multiple sclerosis (MS). Protein and mRNA levels of γ-amino butyric acid transporter 2 (GAT-2) were examined in cerebral white matter from MS and control (Non-MS) patients, in cultured human macrophages, microglia and astrocytes, and in spinal cords from mice with and without experimental autoimmune encephalomyelitis (EAE) using western blotting, immunocytochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). GABA levels were measured by HPLC. The GAT-2′s expression was increased in MS patients’ (n = 6) white matter, particularly in macrophage lineage cells, compared to Non-MS patients (n = 6) (p < 0.05). Interferon-γ (IFN-γ) stimulation of human macrophage lineage cells inducedGAT-2 expression and reduced extracellular GABA levels (p < 0.05) but soluble GABA treatment suppressedHLA-DRα,GAT-2 andXBP-1/s expression in stimulated macrophage lineage cells (p < 0.05). Similarly, the synthetic allopregnanolone analog, ganaxolone (GNX), repressed GAT-2, JAK-1 and STAT-1 expression in activated macrophage lineage cells (p < 0.05).In vivoGNX treatment reducedGat-2,Cd3ε,MhcII, andXbp-1/s expression in spinal cords following EAE induction (p < 0.05), which was correlated with improved neurobehavioral outcomes and reduced neuroinflammation, demyelination and axonal injury. These findings highlight altered GABAergic transport through GAT-2 induction during neuroinflammation. GABA transport and neuroinflammation are closely coupled but regulated by GNX, pointing to GABAergic pathways as therapeutic targets in neuroinflammatory diseases.
Abbreviations: BBB - blood–brain barrier, DMSO - dimethyl sulphoxide, EAE - experimental autoimmune encephalomyelitis, FBS - fetal bovine serum, GAT-2 - γ-amino butyric acid transporter 2, GNX - ganaxolone, HFAs - human fetal astrocytes, HFMs - human fetal microglia, HPLC - high-performance liquid chromatography, IFN-γ - interferon-γ, MBP - myelin basic protein, MDMs - monocyte-derived macrophages, MEM - minimal essential medium, MOG - myelin oligodendrocyte glycoprotein, MS - multiple sclerosis, PBLs - peripheral blood lymphocytes, PBMCs - peripheral blood mononuclear cells, PBS - phosphate-buffered saline.
Key words: multiple sclerosis, experimental autoimmune encephalitis, neuroinflammation, γ-amino butyric acid, γ-amino butyric acid transporter-2, ganaxolone.
There is a growing recognition that innate and adaptive immune processes are involved in neuroinflammation and subsequent neurodegeneration, which contribute to Alzheimer’s and Parkinson’s diseases, HIV-associated neurocognitive disorders and multiple sclerosis (MS) (Eikelenboom et al, 2002, Frohman et al, 2006, Noorbakhsh et al, 2006, and Tansey et al, 2008). MS is the prototype neuroinflammatory disease of the CNS defined by inflammatory demyelination and axonal loss ( Stadelmann, 2011 ). Defects in GABA signaling have been implicated in MS and other inflammatory neurodegenerative diseases (Demakova et al, 2003 and LeWitt et al, 2011). GABA has been reported also to regulate lymphoid and macrophage lineage cell activation (Stuckey et al, 2005 and Nigam et al, 2010). GABA’s reported effects on the immune system include the modulation of pro-inflammatory cytokine and chemokine production (Reyes-Garcia et al, 2007 and Tian et al, 2011) and diminished T cell proliferation (Shiratsuchi et al, 2009, Dionisio et al, 2011, and Soltani et al, 2011). GABA’s actions have been studied in the MS animal model, experimental autoimmune encephalitis (EAE) in which GABA-specific therapeutics displayed differential outcomes, highlighting the complexity of the GABAergic system ( Carmans et al., 2013 ).
The GABA (reuptake) transporters (GATs) are essential regulators of extra- and intra-cellular GABA levels ( Conti et al., 2004 ). Moreover, GATs’ expression is induced in macrophages and lymphocytes following immune activation, presumably leading to enhanced GABA cellular reuptake (Bhat et al, 2010 and Dionisio et al, 2011). Neuroactive steroids are produced within the nervous system ( Mellon et al., 2008 ) and bind to both intracellular steroid receptors and/or cell surface neurotransmitter receptors (Rupprecht et al, 1993, Robel and Baulieu, 1994, and Rupprecht, 1997). Several neuroactive steroids are positive allosteric modulators of the GABAAreceptor (GABA-A-R), including allopregnanolone, or they act to antagonize the GABA-A-R, such as DHEA-S (Rupprecht, 1997, Falkenstein et al, 2000, Hosie et al, 2006, and Mellon et al, 2008). Allopregnanolone is a cholesterol-derived neuroactive steroid, which is synthesized by neurons and glia (Rupprecht et al, 1993, Belelli and Lambert, 2005, and Houtchens, 2007). Importantly, allopregnanolone levels were decreased in cerebral white matter from MS patients and treatment with allopregnanolone reduced neuroinflammation and demyelination in EAE ( Noorbakhsh et al., 2011 ). Ganaxolone (GNX) is a synthetic analog of allopregnanolone ( Reddy and Woodward, 2004 ) and binds to a GABA-A-R site ( Carter et al., 1997 ). Studies in neurons indicate that this interaction enhances chloride (Cl−) permeability of the GABA-A ionophore receptor complex, which results in hyperpolarization of the post-synaptic membrane (Carter et al, 1997, Jorgensen, 2005, and Hosie et al, 2006). GNX has been investigated as an anti-epileptic drug in animal studies (Carter et al, 1997, Liptakova et al, 2000, and Reddy and Rogawski, 2010) and in human studies for epilepsy treatment ( Reddy, 2010 ) although its effects on the immune system remain unknown. Herein, we report the induction of γ-amino butyric acid transporter 2 (GAT-2) (SLC6A12) in the brains of MS patients, particularly on macrophage lineage cells, and also in animals with EAE. GNX suppressed GAT-2 expression in macrophage lineage cells leading to reduced neuroinflammatory gene expression with reduced disease severity in EAE.
Human brain tissue samples
Frontal brain white matter was collected at autopsy, with consent, from age and sex-matched MS subjects (n = 6; relapsing-remitting (RR-MS;n = 3), secondary progressive MS (n = 3)) and Non-MS patients (n = 6; sepsis, myocardial infarction, cancer, HIV/AIDS, all without neuropathological lesions), as previously reported ( Deslauriers et al., 2011 ). These studies were approved by the University of Alberta Ethics Committee (Pro00002291).
Human monocyte-derived macrophages (MDMs) and peripheral blood lymphocytes (PBLs) were prepared from blood collected from healthy donors as previously described (Tsutsui et al, 2004 and Acharjee et al, 2011). Briefly, human peripheral blood mononuclear cells (PBMCs) were purified from healthy blood with Histopaque (Sigma–Aldrich, Oakville, ON, Canada) ( Power et al., 1998 ). PBLs were isolated from PBMCs by removing adherent macrophages and maintained in RPMI 1640 medium with 15% fetal bovine serum (FBS) with phytohemagglutinin-P (PHA-P) (5 μg/ml) (Sigma–Aldrich, Oakville, ON, Canada) for 3 days. Cells were then harvested and cultured with anti-human anti-CD3 (5 μg/ml)-coated plates supplemented with recombinant human IL-2 (103 U/ml) (PeproTech, Rocky Hill, NJ, USA) for an additional 24 h with either GNX (100 μM) or dimethyl sulphoxide (DMSO) (Sigma–Aldrich, Oakville, ON, Canada) vehicle control in equal volume. MDMs were isolated from PBMCs, differentiated for 1 week ( Maingat et al., 2009 ). MDMs were pretreated with GNX (100 μM) or DMSO in minimal essential medium (MEM) containing 10% FBS (Life Technologies, Burlington, ON, Canada), 1% penicillin/streptomycin (Life Technologies, Burlington, ON, Canada) and 1%l-glutamine (Life Technologies, Burlington, ON, Canada), followed by recombinant human interferon-γ (IFN-γ) (400 U/mL) (PeproTech, Rocky Hill, NJ, USA) exposure for 24 h. Treated MDMs were lysed in either TRIzol® reagent (Life Technologies, Burlington, ON, Canada) followed by total RNA extraction or Laemmli buffer with 0.1% β-mercaptoethanol (Sigma–Aldrich, Oakville, ON, Canada) for protein isolation.
Human fetal astrocytes (HFAs) and microglia (HFMs) were prepared from 15–19-week-old fetal brains obtained, with consent (approved by the University of Alberta Ethics Committee, Pro00027660), as previously described (Zhu et al, 2009 and Vivithanaporn et al, 2010). Briefly, fetal brain tissues were dissected, meninges were removed and digested for 30 min with 2.5% trypsin (Life Technologies, Burlington, ON, Canada) and 2 mg/ml DNase I (Roche Diagnostics, Mannheim, Germany) and passed through a 70-μm cell strainer (BD Biosciences, Mississauga, ON, Canada). Cells were washed twice with centrifugation at 14,000 rpm for 10 min and plated in T-75 poly-l-ornithine-coated flasks (Sigma–Aldrich, Oakville, ON, Canada) at 6–8 × 107 cells/flask with media. Following a week of incubation (37 °C at 5% CO2), adherent cells (HFA) were separated from suspension cells (HFM) and re-plated for primary astrocytic or microglia differentiation (80% confluence). HFAs and HFMs were maintained in MEM containing 10% FBS (Life Technologies, Burlington, ON, Canada), 1% penicillin/streptomycin (Life Technologies, Burlington, ON, Canada), 1%l-glutamine (Life Technologies, Burlington, ON, Canada) and 10% dextrose (Life Technologies, Burlington, ON, Canada).
C57BL6 female mice were purchased from the Jackson Laboratory and maintained in the Health Sciences Laboratory Animal Services facility of the University of Alberta under conventional housing conditions. All experiments were approved by the University of Alberta Animal Care Committee (AUP00000317).
GNX (5α-pregnan-3β-methyl-3α-ol-20-one) (Steraloids Inc., Newport, RI, USA) was solubilized in DMSO and further diluted in 1× phosphate-buffered saline (PBS) (Life Technologies, Burlington, ON, Canada) and stored at 4 °C. Active therapeutic concentrations of GNX were derived from previous studies ( Ram et al., 2001 ). For intraperitoneal (i.p.) injections, GNX and DMSO were diluted in PBS to concentrations of low dose (15 mg/kg) and high dose (50 mg/kg), adjusted to physiological pH (7.4) and filter sterilized prior to administration. Forin vitrocell cultures, GNX and DMSO were further diluted in MDM or PBL MEM media (100 μM).
Ten to twelve- week-old C57BL6 female mice were injected subcutaneously in each hind leg with 50 μg of myelin oligodendrocyte glycoprotein peptide (MOG35–55peptide) (Peptide Synthesis Facility, University of Alberta, Edmonton, AB, Canada) emulsified in complete Freund’s adjuvant (Sigma–Aldrich, Oakville, ON, Canada) supplemented with 5 mg/mol heat-killed mycobacteria H37 RA (Difco Laboratories, Detroit, MI, USA) (1:1) ( Ellestad et al., 2009 ). Animals received 0.3-μg i.p. injections of pertussis toxin (List Biological Laboratories, Campbell, CA, USA) on the day of induction with MOG35–55and were then boosted 48 h later. Control animals were immunized with complete Freund’s adjuvant and 1× PBS (1:1) and boosted with pertussis toxin, as above. Animals were assessed daily for EAE severity (behavioral score) for 30 days using a 0–14 rating scale ( Ellestad et al., 2009 ). EAE mice received i.p. injections of GNX (15 mg/kg and 50 mg/kg) daily beginning on the day of EAE induction (Day 0). Control animals received daily i.p. injections of DMSO in matched volumes to those used in the GNX-treated animals.
High-performance liquid chromatography (HPLC)
Fluorometric HPLC was adapted from a previously described protocol (Grant et al, 2006 and Maingat et al, 2009). Cell culture supernatants were mixed to a 60× final concentration with 100% methanol then centrifuged at 12,000gfor 4 min at 4 °C. A 1:1 (5 μl) aliquot of the supernatant was mixed with derivatizing agent (2 mg ofN-isobutyryl-l-cysteine and 1 mg o-phthaldialdehyde dissolved in 100 μl of methanol, followed by addition of 900 μl of 0.1 M sodium borate buffer), and then was placed into a sample management system (Waters Alliance 2690XE; Waters Corporation, Milford, MA, USA). HPLC separation was achieved on a Symmetry C18 column (4.6 × 150 mm; 3.5 μm particle diameter) coupled with a guard column of the same stationary phase (Waters Corporation, Milford, MA, USA). The column heater was set to 30 °C and the sample cooler was held at 4 °C. To separate the derivatized amino acids of interest, a gradient was established from equal parts of solvents A (850 ml of 0.04 M sodium phosphate buffer and 150 ml of methanol, pH 6.2) and B (670 ml of 0.04 M sodium phosphate buffer, 555 ml of methanol, and 30 ml of tetrahydrofuran, pH 6.2) to only solvent B by ∼45 min, with a flow rate of 500 μl/minute. The run time was 60 min for column washout and equilibrium, and 30 min to elute all compounds. A Waters 2475 fluorescence detector (Waters Corporation, Milford, MA, USA) was used to quantify the eluted derivatized GABA (excitation, 344 nm; emission, 433 nm).
Human MDMs were plated in 12 well plates until 80% confluent, pre-treated with IFN-γ (400U/ml) for 1 h, followed by GNX (100 μM) or DMSO (comparable volume) for 24 h. THP-1 cells were plated at 2.0 × 105 cells/well in 12 well plates and treated under the same conditions. Cells were then lysed with Laemmli buffer with 0.1% β-mercaptoethanol and boiled at 95 °C for 10 min. Proteins from whole-cell lysates were separated using polyacrylamide gel electrophoresis and protein fractions were transferred to a nitrocellulose membrane overnight (Bio-Rad, Mississauga, ON, Canada) ( Zhu et al., 2009 ). The membrane was blocked with 5% milk for 1 h and labeled with monoclonal mouse anti-STAT-1 (p86/p90) (1:1000, Cell Signaling Technology, Boston, MA, USA), monoclonal mouse anti-p-STAT-1 (Tyr701) (1:500, Abcam, Eugene, OR, USA), monoclonal mouse anti-JAK-1 (1:1000, Cell Signaling Technology, Boston, MA, USA) and polyclonal rabbit anti-GAT-2 (1:1000, GeneTex, Inc., Irvine, CA, USA) overnight at 4°. The immunolabeled membrane was then probed with secondary peroxidase-conjugated goat anti-rabbit IgG (1:500, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 2 h. β-Actin-HRP was used as a loading control (1:1000, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Membranes were developed with Pierce ECL Western blotting substrate (Fisher Scientific, Ottawa, ON, Canada) and exposed on film (Canon Canada, Inc., Mississauga, ON, Canada).
Immunofluorescence and immunohistochemistry
MDM or THP-1 cultures were stimulated with IFN-γ (400 U/ml) for 1 h followed by GNX (100 μM) or DMSO for 24 h. Cells were fixed with 2% paraformaldehyde (Fisher Scientific, Ottawa, ON, Canada) for 30 min, washed 3× with PBS + 2% FBS, blocked with Odessey blocking buffer (Li-cor, Guelph, ON, Canada) for 2 h and incubated overnight at 4° with polyclonal rabbit anti-GAT-2 (1:1000, Abcam, Eugene, OR, USA) and monoclonal mouse anti-HLA-DR (1:100) (Abcam, Eugene, OR, USA) primary antibodies. Following overnight incubation, fluorescent conjugated secondary antibodies (Alexa488 goat anti-mouse (Invitrogen, Eugene, ON, Canada) and Alexa Fluor 680 goat anti-rabbit (Invitrogen, Eugene, ON, Canada) were incubated for 2 h and washed 3× with PBS + 2% FBS. Cells were washed and mounted with gelvatol on microscope slides and imaged on an upright fluorescent microscope (Axioskop2; Carl Zeiss MicroImaging Inc., Thornwood, NY, USA) Spinal cords isolated from untreated and treated animals (n = 4) were formalin-fixed (4%) and paraffin-embedded. The subsequent sections were de-paraffinized, hydrated, and boiled in 0.01 M trisodium citrate buffer (pH 6) for 10 min for antigen retrieval. Sections were incubated in 0.3% hydrogen peroxide for 20 min. Sections were blocked with Odyssey blocking buffer (Li-cor, Guelph, ON, Canada) for 2 h at room temperature then probed with polyclonal rabbit anti-CD3 (1:100, Abcam, Eugene, OR, USA), monoclonal mouse anti-MHCII (1:50, eBioscience, San Diego, CA, USA), monoclonal mouse anti-STAT-1 (1:1000, Cell Signaling Technology, Boston, MA, USA), polyclonal rabbit anti-GAT-2 (1:1000, Abcam, Eugene, OR, USA), polyclonal rabbit anti-GFAP (1:500, BD Biosciences, Mississauga, ON, Canada) and monoclonal mouse myelin basic protein (MBP) (1:500, Abcam, Eugene, OR, USA) antibodies were used to detect lymphocyte infiltration, MHC Class II activation, GAT-2 and MBP abundance. Bielschowsky’s silver staining was performed for axonal identification, as described previously ( Tsutsui et al., 2004 ). Formalin-fixed (4%), paraffin-embedded autopsy white matter from MS and Non-MS samples were prepared as above. Immunolabeling with polyclonal rabbit anti-GAT-2 (1:1000, Abcam, Eugene, OR, USA) antibodies and monoclonal mouse anti-HLA-DRα (1:100, Abcam, Eugene, OR, USA) were used to detect co-localization of GAT-2 expression in MHC Class II+cells conjugated to DAB or alkaline peroxidase, anti-rabbit or anti-mouse secondary antibodies, respectively (Life Technologies, Burlington, ON, Canada). DAB peroxidase substrate kit (Vector Laboratories, Burlington, ON, Canada) or alkaline phosphatase substrate kit (Vector Laboratories, Burlington, ON, Canada). Sections were dehydrated with xylene, mounted with acrytol (Leica Biosystems, Buffalo Grove, IL, USA) and viewed under a light microscope (Axioskop2; Carl Zeiss MicroImaging Inc., Thornwood, NY, USA). Immunofluorescence images were quantified as mean pixel intensity using ImageJ software ( Schneider et al., 2012 ).
Human MDMs were prepared as above with surface phenotype labeling of activated MDMs with anti-HLA-DR and GAT-2 and gating was assessed by total percentage (%) of the HLA-DR+/GAT-2+population compared to secondary only controls. Primary antibodies included PE-conjugated mouse anti-HLA (1:100, eBioscience, San Diego, CA, USA), polyclonal rabbit anti-GAT-2 (1:200, Abcam, Eugene, OR, USA; immunofluorescence), polyclonal rabbit anti-GAT-2 (1:100, GeneTex, Irvine CA; Western blot) and PE-conjugated anti-rabbit secondary IgG (1:500, eBioscience, San Diego, CA, USA) antibodies. Mouse splenocytes were harvested, permeabilized with permeabilization buffer (eBioscience, San Diego, CA, USA) and stained with efluor450-conjugated mouse anti-T-bet (1:100, eBioscience, San Diego, CA, USA) or PerCPCy5.5-conjugated mouse anti-GATA3 (1:100, eBioscience, San Diego, CA, USA). Cells were fixed, washed with PBS + 2% FBS and subsequently stained with PerCP-conjugated mouse anti-CD3ε (1:1000, eBioscience, San Diego, CA, USA) and APC-conjugated mouse anti-CD4 (1:1000, eBioscience, San Diego, CA, USA). CellTrace CFSE™ cell proliferation kit (Molecular Probes, Eugene, OR, USA) was used for proliferation assays, according to the manufacturer’s protocol. In all experimental procedures, anti-CD3ε, anti-CD4, anti-GAT-2, anti-HLA, anti-Tbet, and anti-GATA3 antibodies were compensated with compbeads (BD Biosciences, Mississauga, ON, Canada) for single color compensation controls. The immunolabeled cells were analyzed on BD FACSCanto flow cytometer (BD Biosciences, Mississauga, ON, Canada) using FACSDiva software (BD Biosciences, Mississauga, ON, Canada) for acquisition and FlowJo for analysis (Tree Star Inc., Ashland, OR, USA).
Cell proliferation assay
Splenocytes were isolated from EAE induced C57BL/6 mice at day 30 post induction and stained with Cell Trace CFSE cell proliferation kit (Invitrogen, Eugene, OR, USA) (1 μM), according to the manufacturer’s protocol ( Ellestad et al., 2009 ). Briefly, total splenocytes were seeded at 2 × 105 cells/well in a 96-well round-bottomed plate with or without MOG35–55(20 μg/ml) peptide for 5 days, followed by subsequent fixation and flow cytometric analysis, as above. Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with 10% FBS (Life Technologies, Burlington, ON, Canada), 1% penicillin/streptomycin (Life Technologies, Burlington, ON, Canada), 2 mMl-glutamine (Life Technologies, Burlington, ON, Canada) and 1% nonessential amino acids (Life Technologies, Burlington, ON, Canada) were used for cell culture.
Quantitative real-time RT-PCR
Human white matter (normal appearing white matter), HFAs, HFMs, hMDMs and murine lumbar spinal cords were homogenized in TRIzol® reagent (Life Technologies, Burlington, ON, Canada) and total RNA was isolated and purified using RNeasy mini columns (Qiagen, Alameda, CA, USA) (Noorbakhsh et al, 2006, Antony et al, 2007, and Ellestad et al, 2009). Complementary (cDNA) was prepared by oligo(dT)-primed reverse transcription of mRNA using either Superscript II or Superscript III reverse transcriptase (Invitrogen, Eugene, ON, Canada) according to the manufacturer’s recommended protocols. Quantitative real-time PCR (qRT-PCR) was performed using Bio-Rad iQ 2× SYBRgreen supermix (Bio-Rad, Mississauga, ON, CA) on the iQ5 cycler (Bio-Rad, Mississauga, ON, CA) according to the manufacturer’s recommended protocols. All data were analyzed using the ΔΔCt method and described as relative fold change (RFC). Specific oligonucleotide primer sequences are provided in Table 1 .
|mMhc II I-Eα||ACACACCATCATCCAGGCAGAGTT||TCTGGAGTGTTGGAACGCTCT|
All statistical tests were performed using GraphPad InStat version 3.0 (GraphPad Software, La Jolla, CA, USA) with both parametric and nonparametric comparisons.pvalues of <0.05 were considered significant (∗).
Increased GAT-2 in MS white matter
Previous studies have implicated GABA’s actions on leukocytes in the regulation of neuroinflammation (GAT-2Bhat et al, 2010 and Carmans et al, 2013), prompting the investigation of GABA transporter expression in cerebral white matter.GAT-2transcript levels were significantly increased in white matter specimens from MS compared to Non-MS patients whileGAT-1andGAT-3transcript levels were similar between groups ( Fig. 1 A). Western blotting showed increased GAT-2 immunoreactivity in MS (n = 2) compared to Non-MS patients’ (n = 2) white matter lysates ( Fig. 1 B). Immunohistochemical detection of MHC Class II (HLA-DRα) in cerebral white matter sections from Non-MS, MS (normal appearing white matter, MS-NAWM) and MS (demyelinating lesion, MS-Lesion) patients ( Fig. 1 C, top panels) disclosed minimal immunoreactivity in Non-MS white matter, occasional microglial cells in MS-NAWM and numerous immunopositive cells in the MS-Lesion white matter. Similarly, GAT-2 immunoreactivity was minimal in Non-MS white matter, but detected on rare cells and as background in MS-NAWM, but apparent in cells and interstitial tissue in MS-Lesion white matter; GAT-2 (brown) was co-localized with MHC Class II (blue) immunoreactivity in MS-Lesion ( Fig. 1 C inset (i), bottom panels). These data suggested that GAT-2 was up-regulated in MS white matter, particularly brain macrophage lineage cells.
IFN-γ dependent induction of GAT-2 in human macrophages
As GAT-2 induction was most evident on brain macrophage lineage cells expressing MHC Class II in MS lesions, we investigated cultured human microglia, MDMs and astrocytes, all of which are capable of expressing MHC Class II. Following IFN-γ stimulation, increasedMHC-IIexpression was observed in all cell types ( Fig. 2 A).GAT-2was also induced in stimulated MDMs but not in astrocyte or microglial cultures ( Fig. 2 A). To verify these transcriptional changes, MHC Class II and GAT-2 immunoreactivity was examined in human MDMs revealing their expression was minimal in unstimulated MDMs ( Fig. 2 B, D) but IFN-γ stimulation significantly increased the immunoreactivity for both proteins in MDMs ( Fig. 2 C, D). In addition to increased expression of GAT-2, MDMs exposed to IFN-γ also showed decreased extracellular GABA levels in culture supernatants compared to unexposed MDMs’ supernatants ( Fig. 2 E). To explore the direct cellular effects of GABA, the human macrophage lineage (THP-1) cell line was stimulated with recombinant human IFN-γ and then treated with GABA (at physiologically relevant concentrations). These studies disclosed that GABA (300 ng/ml) exposure significantly reduced the expression of bothHLA-DRα( Fig. 2 F) andGAT-2( Fig. 2 G) in stimulated MDMs. Moreover, the endoplasmic reticulum stress marker,XBP-1/s, showed reduced expression in stimulated MDMs exposed to GABA ( Fig. 2 H). Thus, GAT-2 was induced in MDMs by IFN-γ, which was associated with reduced extracellular GABA levels. Moreover, increased extracellular GABA prevented IFN-γ-mediated induction of MHC Class II, GAT-2 and the ER stress indicator, XBP-1s.
GNX suppresses inflammatory gene expression in human macrophage lineage cells
Since GABA was found to inhibit IFN-γ dependent responses in macrophages, we examined the effects of the positive allosteric GABA-A-R modulator, GNX in human MDMs. IFN-γ stimulated MDMs receiving GNX treatment displayed a reduction inHLA-DRα( Fig. 3 A) andGAT-2( Fig. 3 B) transcript levels, relative to DMSO (vehicle)-treated MDMs. Flow cytometric analyses confirmed that IFN-γ stimulated HLA-DRα+expression in human MDMs (treated with DMSO) with increased GAT-2 expression but GAT-2 was suppressed with GNX treatment (GAT-2+/HLA-DRα+%) ( Fig. 3 C). Likewise confocal imaging of MDMs showed that IFN-γ stimulated cells exhibited robust GAT-2 and HLA-DRα expression ( Fig. 3 D, F), which was significantly reduced by GNX treatment ( Fig. 3 E, F). Given that IFN-γ acts through the JAK-1/STAT-1 pathway, the expression ofSTAT-1was examined in IFN-γ-stimulated MDMs and THP-1 cells, revealing that GNX treatment suppressedSTAT-1transcript levels in both cell types ( Fig. 3 G). In addition, JAK-1 and STAT-1 immunoreactivity was increased in IFN-γ-stimulated THP-1 cells but GNX treatment suppressed JAK-1 and STAT-1 immunoreactivity, compared to DMSO-treated controls ( Fig. 3 H). Thus, GNX treatment reduced HLA-DRα and GAT-2 expression together with diminished JAK-1 and STAT-1 abundance in human macrophage lineage cells stimulated with IFN-γ.
GNX treatment prevents EAE-associated neurobehavioral deficits
To extend the above findings to anin vivomodel, EAE was induced in C57BL6 mice with MOG35–55and treated daily by intraperitoneal (i.p.) injections with a vehicle control (DMSO) or GNX. Controls included PBS with complete Freund’s adjuvant and without MOG35–55, and with daily GNX or DMSO treatments. Among the EAE groups, neurological deficits were evident by day 11 post-induction and peaked by day 17 post-induction ( Fig. 4 A). Treatment of EAE mice with GNX at low (15 mg/kg) or high (50 mg/kg) doses reduced the EAE-associated neurological deficits compared to the DMSO-treated EAE group, although the therapeutic benefit was greater with the higher dose of GNX ( Fig. 4 A). Animals receiving either GNX concentration also showed significant reductions in the maximum severity score. Again, the therapeutic effect was greatest in the high-dose-treated group ( Fig. 4 B). Thus, GNX exerted beneficial effects on EAE severity in a concentration-dependent manner.
Reduced inflammatory gene expression in the spinal cord during EAE with GNX treatment
The corresponding molecular changes were assessed in lumbar spinal cords from control and EAE animals receiving GNX treatment revealing thatMhc-II( Fig. 5 A),Gat-2( Fig. 5 B),Cd3ε( Fig. 5 C) andXbp-1/s( Fig. 5 D) andF4/80( Fig. 5 E) transcript expression was induced in EAE animals compared to healthy control animals but these RNA levels were significantly decreased in EAE/GNX-treated animals at day 16 post-induction of EAE (peak severity). Conversely,Gfapwas minimally activated in EAE animals at day 16 with negligible effects mediated by GNX treatment ( Fig. 5 F). Indeed, these GNX-mediated effects persisted for all genes at day 30 post-induction ( Fig. 6 A–D).
Neurobehavioral deficits also showed significant (positive) correlations with several host transcript levels includingMhc-II( Fig. 5 G),Gat-2( Fig. 5 H),Cd3( Fig. 5 I) when comparing their severity scores with the expression levels at day 16 post-EAE induction. These data indicated thatGat-2was increased in EAE in conjunction with several immune genes that were correlated with neurobehavioral deficits, recapitulating immunomolecular features of MS. However, these effects were ameliorated in EAE by GNX treatment.
Since GNX is assumed to function by way of binding to the GABA-A-R, the expression levels of the principalGABA-A-Rsubunitsα1andβ1were examined, showing a decrease in the expression of theGABA-A-R α1andβ1subunits in EAE mice at day 16 after induction while GNX exerted no effects on the expression level of these subunits at either day 16 or day 30 ( Fig. 7 A, B). BothIl-1β( Fig. 7 C) andIl-6( Fig. 7 E) exhibited increased expression in EAE animals’ spinal cords at day 16, but surprisingly GNX treatment enhanced the activation of both genes at this time point, although GNX had no apparent effects at day 30 ( Fig. 7 D, F).
T-bet+ T cells are suppressed by GNX treatment in EAE
As GNX treatment suppressed the inflammatory environment in spinal cords of EAE mice, it was imperative to define the impact of GNX on lymphocyte activation outside of CNS in EAE animals. EAE/GNX-treated mice showed a reduction in the T-bet+CD4+population (3.3%) derived from splenocytes isolated at day 16, compared to the EAE/DMSO-treated group (24.3%) ( Fig. 8 A). Furthermore, CTL/GNX-treated animals showed a reduced abundance of the T-bet+CD4+population (1.60%) compared to the CTL/DMSO-treated animals (5.96%) ( Fig. 8 A). Since total CD3ε transcripts were reduced in the CNS of GNX-treated EAE animals ( Fig. 5 C), we also examined total T-bet/GAPDH transcript abundance in the CNS, observing a reduction of T-bet transcripts at day 16, but not at day 30 of EAE/GNX-treated animals ( Fig. 8 B). Since GNX appeared to reduce T-bet+CD4+populations in the periphery ( Fig. 8 A), we next determined if GNX treatment reduced T cell proliferation in response to an antigen-specific challenge (MOG). Splenocytes isolated at day 30 post-induction that were stimulated with the MOG35–55peptide showed similar CFSElowpercentages of CD4+T cell populations from both EAE-induced mice treated with DMSO or GNX ( Fig. 8 C). Moreover, both GATA3+CD4+(Th2) and Tbet+CD4+(Th1) subsets exposed to MOG35–55showed CFSElowproliferation profiles that were similar in both EAE groups ( Fig. 8 D, E) suggesting GNX did not affect MOG-specific clonal expansion of Th1- or Th2-polarized cells. Thus, GNX’s effects appeared limited to T-bet expression (transcript and protein) at peak severity, which regulates transcription of pro-inflammatory genes, such as IFN-γ, as well as, potentially reducing total Th1 cell numbers during peak disease, both within the CNS and in the periphery.
Diminished neuropathology in EAE with GNX treatment
Spinal cords from healthy (CTL/DMSO or CTL/GNX) and EAE mice with DMSO or GNX treatments showed that MHC Class II-immunopositive cells were not detected in the CTL/DMSO- and CTL/GNX-treated animals’ spinal cords while EAE/DMSO animals exhibited abundant Class II immune-labeled cells within the parenchyma and perivascular regions, compared to EAE/GNX animals that showed substantially less MHC Class II immunoreactivity ( Fig. 9 A). Total STAT-1 was evident in EAE/DMSO-treated animals’ spinal cords but displayed diminished in expression in EAE/GNX-treated animals ( Fig. 9 A, insets (i and ii)). GAT-2 immunoreactivity was apparent in perivascular cells among CTL/DMSO and CTL/GNX animals’ spinal cords although GAT-2 immunoreactivity was markedly enhanced in the spinal cords of the EAE/DMSO animals in contrast to the EAE/GNX-treated animals (arrows), which displayed similar GAT-2 immunoreactivity to that observed in the control groups ( Fig. 9 B). GAT-2 (brown) and MHC Class II (blue) co-localized immunoreactivity was present in cells resembling macrophages ( Fig. 9 B, insets (i and ii)) in both EAE/DMSO and EAE/GNX spinal cords. Bielchowsky (silver) staining to evaluate axonal viability revealed that CTL/DMSO and CTL/GNX animals showed abundant axonal staining within the spinal cords ( Fig. 9 C). Among EAE/DMSO mice, there was a striking paucity of axons while EAE/GNX-treated animals’ spinal cords showed numerous axons ( Fig. 9 C). CD3+cells were not evident in CTL/DMSO and CTL/GNX animals but numerous CD3+cells were detected in the dorsal column white matter of EAE/DMSO animals with reduced CD3+lymphocytes observed in EAE/GNX-treated animals ( Fig. 9 D). Similarly, MBP immunoreactivity was readily observed in CTL/DMSO and CTL/GNX animals’ spinal cords but EAE/DMSO-treated animals showed reduced MBP immunoreactivity in spinal cord white matter (dorsal column, arrow), which was comparatively preserved in EAE/GNX-treated animals ( Fig. 9 E). Astrocyte and microglia activation was assessed by GFAP and Iba-1 immunoreactivity, respectively, showing increased immunolabeling of both cell types in the EAE/DMSO-treated animals’ spinal cords, which was minimally diminished with GNX treatment ( Fig. 9 F, G, respectively). Nonetheless, these morphological findings were congruent with the molecular and neurobehavioral data above, in that among EAE animals exhibiting neurological deficits there was increased GAT-2 expression with concurrent inflammatory changes, which were reduced by GNX treatment.
The present study represents the first report of increased GAT-2 levels in MS white matter, which were chiefly localized in activated brain macrophage lineage cells expressing MHC Class II, in demyelinating lesions. Complementing these observations, exposure of human macrophages to IFNγ, induced GAT-2 expression with a concurrent reduction in extracellular GABA levels. Supplementing extracellular GABA levels suppressed the expression of GAT-2 and several immune genes in IFNγ-stimulated macrophage lineage cells. Likewise, the positive allosteric modulator of the GABA-A-R, GNX, suppressed GAT-2 expression in IFNγ-activated macrophage lineage cells in conjunction with repressing both the JAK-1/STAT-1 signaling pathway and downstream pro-inflammatory gene expression that was apparent in bothex vivoandin vivomodels. These findings highlight GABA’s ability to regulate GAT-2 levels as well as immune activation, which was also reflected by GNX’s capacity to inhibit inflammation, likely acting via intracellular steroid receptors and perhaps the GABA-A-R. Thus, the previously recognized immune effects of GABA are affected by GAT-2 abundance, which is regulated by a synthetic neuroactive steroid with downstream effects on pro-inflammatory mechanisms implicated in MS.
GABA is in constant efflux/influx equilibrium that is regulated by action potentials, electrochemical concentration gradients, and its re-uptake transporters (GATs) (Ruiz-Tachiquin et al, 2002, Cordeiro et al, 2003, and Jorgensen, 2005). In the present study, we focused on the mechanism involving the GABA re-uptake transporter, GAT-2, because its expression was increased in white matter from persons with MS and furthermore, GAT-2 was localized in HLA-DRα+cell populations that were confined to regions in which active demyelination was observed. GAT-2 is a transmembrane spanning receptor that contains an intracellular carboxy-terminus containing two putative phosphorylation sites (Goncalves et al, 1999 and Soudijn and van Wijngaarden, 2000). Possible adaptor protein interaction(s) with GAT-2 might mediate GAT-2’s signaling pathways and downstream transcriptional involvement with genes involved in neuroinflammation.
GAT-2 is the least abundant GAT in the human nervous system despite being constitutively expressed on neurons and astrocytes (Conti et al, 1999 and Christiansen et al, 2007). It has also been immunodetected in meninges and endothelial cells of the CNS, as well as, outside the CNS in immune cells (Conti et al, 2004 and Bhat et al, 2010). It is noteworthy that, although not statistically significant, IFN-γ stimulation induced an increase in GAT-2 expression within astrocytes; thus astrocytes, the chief cell type within the CNS, might also participate in the regulation of GABA-mediated regulation of inflammation. To extend our findings of GAT-2 induction in MS brain tissue, GAT-2 was induced in IFN-γ stimulated macrophages with ensuing high levels of MHC Class II expression; indeed, the increase of GAT-2 in MDMs was associated with decreased GABA levels in supernatant from these cells. Thus, a loss or reduction of extracellular GABA through GAT-2 induction might augment inflammation within the brain at sites of active demyelination. To confirm the importance of GABA in inhibiting active inflammation we treated macrophage lineage cells with soluble GABA at physiological concentrations ( Ferkany et al., 1978 ) while also stimulating the same cells with IFNγ. Soluble GABA was able to reduce both MHC Class II and GAT-2 expression, supporting our hypothesis that the regulation of GABAergic signaling is important in controlling inflammation.
Assuming GABA homeostasis is dysregulated in MS, GABA replacement would maintain homeostasis and restrict inflammation. However, soluble GABA cannot cross the blood–brain barrier (BBB) but GNX is BBB-permeable. To investigate GNX’s impact on inflammation, we pre-treated IFN-γ-activated MDMs with GNX and found that indeed both GAT-2 and MHC Class II subunits are suppressed at both the transcript and the protein levels. To determine the mechanism of action by which GNX exerted its effects, human macrophage lineage cells were pre-treated with GNX or DMSO with subsequent IFN-γ stimulation, revealing a reduction in total JAK-1/STAT-1 abundance; thus GNX pre-treatment suppressed IFN-γ stimulation of macrophage lineage cells. STAT-1 can activate transcription of the Class II Transactivator (CIITA), which regulates MHC Class II subunit expression (Linhoff et al, 2001, Nickerson et al, 2001, and Barbaro Ade et al, 2002). Not surprisingly, diminished STAT-1 was observed in IFN-γ-stimulated THP-1 cells that were treated with GNX accompanied by reduced (downstream) MHC Class II expression. Furthermore, thein vivoEAE studies showed that both STAT-1 protein and downstream MHC Class II (I-Eα) subunit expression were both suppressed with GNX treatment, suggesting an overall suppression of the JAK-1/STAT-1 signaling pathway. The outcome of suppressing MHC Class II+cells would result in reduced peptide presentation and lymphocyte engagement, thereby preventing the formation of MOG-specific, auto-inflammatory helper T cells, which play integral roles in MS-defined pathogenesis.
The JAK-1/STAT-1 pathways are not limited to macrophages and thus, other possible targets for GNX-suppressive effects are the circulating MOG-specific T helper cells of the T-bet lineage (Th1), as STAT-1 is involved in the transcriptional regulation of T-bet ( Oestreich and Weinmann, 2012 ). The Th1 (T-bet+CD3+) population in EAE has been directly correlated with MS disease severity ( Yeh et al., 2011 ). GNX treatment showed a marked reduction in T-bet+CD4+cells in spleens isolated from GNX-treated EAE animals, compared to EAE/DMSO controls. Furthermore, animals not immunized with the MOG35–35peptide, which did not develop disease (CTL/DMSO or CTL/GNX), also showed a reduced percentage of the T-bet+CD4+population with GNX treatment compared to DMSO-treated CTL animals. Furthermore, the associated Th1 (T-bet+) population was increased in EAE/DMSO lumbar spinal cords, with a reduction of this marker in EAE/GNX-treated animals at day 16. These results confirm that the systemic T-bet+Th1 immune response is dampened with GNX treatment in the presence or absence of the auto-antigen in both the CNS and the periphery at peak disease.
To investigate the antigen-specific, adaptive immune response to MOG35–55in GNX-treated EAE animals, anex vivoproliferation assay was performed using CFSE as an indicator of cell proliferation. Since GNX treatment resulted in reduced T-bet+Th1 pro-inflammatory lymphocyte profiles at day 16, it was important to determine if GNX was functionally interfering with the clonal expansion of Th1 cells.Ex vivoproliferation in response to the cognate (MOG35–55) antigen confirmed that antigen-specific responses to MOG35–55displayed no differences between the total CD4+population in spleens of mice treated with DMSO or GNX isolated at day 30. These latter observations suggested that lymphocyte expansion in response to the MOG35–55antigen was similar in both EAE/DMSO and EAE/GNX-treated animals. Further investigation within the individual lymphocyte profiles revealed no differences between CFSElowproliferating T-bet+CD4+(Th1) and GATA3+CD4+(Th2) populations compared to DMSO-treated EAE animals, indicating that there was no difference in the adaptive immune profiles expressed between EAE/GNX-treated animals and EAE/DMSO animals. Daily GNX treatment delayed the progression of Th1 polarization in EAE animals at day 16, measured by reduced T-bet+CD4+lymphocyte profiles but this effect was not apparent at day 30 post-induction, measured by theex vivoproliferation assay. Despite similar responsiveness to the MOG35–55peptide, measured byex vivocell division, EAE/GNX-treated animals at day 30 were generally healthier than EAE/DMSO animals. A potential explanation for this finding might be that GNX was able to directly repress the transcription of T-bet in CD4+cells in doing so, reduced the proportion of Th1 cells detected in the CNS of EAE/GNX-treated animals compared to EAE/DMSO-treated animals. Nonetheless, GNX-treatment conferred protection in mice with EAE as it limited the proportion of T-bet+cells expressed in the CNS at peak severity. Of relevance, the roles of other T-bet expressing cell types such as, CD8+ T lymphocytes, were not studied but might also be affected by GNX.
Like soluble GABA-treated macrophage lineage cells, GNX-treatmentin vivoalso exerted direct effects on immune gene transcription. For example, the biomarker for endoplasmic reticulum stress,XBP-1/s, which is increased in MS brains and associated with inflammation, was repressed. These findings highlight the capacity for GNX to act through several mechanisms including, positive allosteric modulation of the GABA-A-R and selective transcriptional repression. Moreover, GNX’s natural analog, allopregnanolone, has been linked to promoting myelination as it is one of the principal neuroactive steroids secreted from oligodendrocytes in the CNS and Schwann cells in the PNS (Robel and Baulieu, 1994, Rupprecht, 1997, and Belelli and Lambert, 2005). This raises other possible therapeutic targets for GNX, as disease severity in EAE/GNX-treated animals was abrogated with preservation of myelin and axonal integrity, confirmed by immunohistological analyses of spinal cord sections by silver staining and MBP immunoreactivity. These results suggest that GNX treatment prevented demyelination and given that demyelination is the pivotal event leading to the clinical manifestations of MS, GNX treatment might represent a valuable therapeutic approach to prevention and possible reversal of this neuroinflammatory-driven disease process.
The authors thank Dr. Kenneth Warren for helpful discussions and Gail Rauw for preparation and analyses of all HPLC samples and the University of Alberta Animal Services for excellent care of laboratory animals. JGW holds an Alberta Innovates-Health Solutions (AI-HS) Fellowship. CP holds a Canada Research Chair (CRC) (Tier 1) in Neurological Infection and Immunity. These studies were supported by the Multiple Sclerosis Society of Canada (CP). None of the authors have commercial interests or activities related to the contents of the present manuscript.
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a Department of Medicine, University of Alberta, Edmonton, AB, Canada
b Department of Laboratory Medicine & Pathology, University of Alberta, Edmonton, AB, Canada
c Department of Psychiatry, University of Alberta, Edmonton, AB, Canada
© 2014 IBRO, Published by Elsevier B.V.