Inhibitory role for GABA in autoimmune inflammation

Abstract

GABA, the principal inhibitory neurotransmitter in the adult brain, has a parallel inhibitory role in the immune system. We demonstrate that immune cells synthesize GABA and have the machinery for GABA catabolism. Antigen-presenting cells (APCs) express functional GABA receptors and respond electrophysiologically to GABA. Thus, the immune system harbors all of the necessary constituents for GABA signaling, and GABA itself may function as a paracrine or autocrine factor. These observations led us to ask further whether manipulation of the GABA pathway influences an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Increasing GABAergic activity ameliorates ongoing paralysis in EAE via inhibition of inflammation. GABAergic agents act directly on APCs, decreasing MAPK signals and diminishing subsequent adaptive inflammatory responses to myelin proteins.

Fig. 1.

A GABAergic system is present in the immune system. (A) Macrophages and DCs were purified and stimulated with LPS, and CD4⁺ T cells were stimulated with α-CD3 and α-CD28 for 24–48 h. GAD enzyme was detected by immunoblotting in resting (MR or DR or TR) or stimulated (MS or DS or TS) peritoneal macrophages, dendritic cells, and T cells, respectively. Astrocytes (Ast) and brain extract (Br) were used as positive controls. (B) GABA secreted into conditioned media supernatant over purified immune cells, stimulated as above, was measured by dot blot. Controls are commercial pure GABA (2 μM) and same volume growth media used without cells: serum-free RPMI used for growth of DCs and macrophages (Con1) or X-Vivo 20 used for growing T cells (Con2). (C) Representative trace of a voltage-clamp recording showing functional GABA receptors in peritoneal macrophages during the first 10 s of recording (Top) and showing lack of response to GABA application 20 min after the initial responses (Middle). Bottom: Hippocampal neurons cultured 14 days in vitro are used as control. Arrowheads point to currents induced by focal application of 100 μM GABA for 1-s duration (marked by solid bars) in peritoneal macrophages. Arrows point to GABA currents induced by focal application of 100 μM GABA for 0.5-s duration in hippocampal neurons. In addition, neurons show spontaneous IPSCs, some denoted by asterisks. N = 11, n = 7 for macrophages and N = 3, n = 3 for neurons, where N is the total number of cells and n is the number of cells showing responses to GABA application. (D–F) mRNA was measured by RT-PCR in immune cells stimulated as in A. GABAT is the enzyme that degrades GABA, and GAT-2, a GABA transporter. Brain (Br), liver (Liv), and β-actin are used as controls.

Fig. 2.

GABAergic agents act directly on APCs through the GABA-A-R and MAPK to suppress inflammation. (A) Naive splenocytes from C57BL/6 mice transgenic for the MOG TCR were activated in vitro with 0–10 μg/mL MOG in the presence of vehicle or various concentrations of topiramate: 2 μm, 20 μM, or 200 μM. Data are shown as mean ± SD cytokine secretion (pg/mL) of duplicate cultures measured by ELISA and are representative of the range of concentrations tested (*P < 0.05; **P < 0.005). (B and C) Purified T cell cultures were activated with 0.1–1 μg/mL plate-bound α-CD3 and α-CD28. Proliferation rates (B) and cytokine production (pg/mL) (C) were measured in the presence of vehicle, 200 μM topiramate, 100 μM gabaculine, or 100 μM muscimol. Data are shown as mean ± SD of triplicates and are representative of the range of concentrations tested. (D) Peritoneal macrophages were purified from mice treated with vehicle, vigabatrin (400 mg/kg per day), or topiramate (100 mg/kg per day) for 1 week and activated in vitro with LPS. (E) Peritoneal macrophages were stimulated with 0–800 ng/mL LPS in the presence of the GABAergic agents in vitro, 20–200 μM topiramate, 500 μM vigabatrin, 10–100 μM muscimol, or 50–500 μM gabaculine, and for each GABAergic agent with 10–100 μM picrotoxin, the GABA-A channel blocker. For D and E, data represent IL-1β production (pg/mL), mean ± SD of duplicate cultures (*P < 0.05; **P < 0.005) and are shown for a representative concentration. (F) Macrophages or T cells were purified separately from MOG TCR transgenic mice treated with vehicle (−) or topiramate (+) (100 mg/kg per day) orally for 1 week. Macrophages and T cells were mixed reciprocally and the culture stimulated in vitro with 0–20 μg/mL MOG. Cytokine production then measured shown as mean ± SD (pg/mL) of duplicate cultures is representative of the range of concentration of MOG. *P < 0.05. (G) Peritoneal macrophages purified from mice treated as in D were stimulated in vitro with LPS for various time points, as shown. Western blotting for the phosphorylated p44/42 MAPK and p38/ERK proteins was performed and compared with nonphosphorylated forms and β-actin control.

Fig. 3.

GABAergic agents ameliorate EAE. (A–C) SJL/J mice were immunized with 100 μg of PLP 139-151 in CFA. Graphs represent prevention of EAE (A) by oral treatment with topiramate (100 mg/kg per day) or vigabatrin (400 mg/kg per day) starting at the time of immunization, as indicated by the arrow; and treatment of established EAE (B and C) by oral topiramate (100 mg/kg per day) or vigabatrin (400 mg/kg per day) starting at the peak of disease, as indicated by the arrow. Data represent clinical scores, as described in Materials and Methods, mean ± SEM, representative of two independent experiments, n = 10 per group. *P < 0.05, Mann-Whitney analysis. In B and C, mice were treated daily except during days 32–42, as indicated by the solid bar, when they were treated every 3 days. (D–F) H&E-stained transverse sections of spinal cord of mice killed at the end of the experiment (day 37; A). Representative control (D), topiramate- (E), and vigabatrin-treated animals (F) are shown. *Parenchymal inflammatory foci; ⁺meningeal inflammatory foci. 125× magnification.

Fig. 4.

GABAergic agents ameliorate EAE through an effect on the immune system. (A and B) SJL/J mice were immunized with PLP 139-151 (PLP) and treated with vehicle (PBS), topiramate (100 mg/kg per day), or vigabatrin (400 mg/kg per day) for 10 days after immunization. Splenocytes were then taken and restimulated ex vivo with 0–25 μg/mL PLP. Cytokine responses (A) and proliferation rates (B) of cells to rechallenge with 10 μg/mL of PLP are shown as mean ± SD of triplicate culture wells. *P < 0.05; **P < 0.005. Results are representative of three independent experiments and the range of PLP concentration. (C) Splenocytes were taken from PLP-immunized SJL/J donor mice treated with topiramate, vigabatrin, or vehicle for 10 days as in A and B and adoptively transferred into untreated naïve recipient SJL/J mice. EAE induced adoptively in recipients is shown as mean clinical scores ± SEM, n = 7–10 per group. *P < 0.05, Mann-Whitney analysis.