We first tested the ability of homotaurine to limit disease progression in the chronic model of EAE that was induced by immunizing C57BL/6 mice with a myelin oligodendrocyte glycoprotein peptide (MOG 35–55 )35. Eleven to thirteen days after receiving the encephalitic regimen, all of the mice began to develop EAE. When an animal reached an EAE severity score of 1 it was randomly assigned to receive plain drinking water, or water containing homotaurine (0.25 mg/ml) for the rest of the observation period. This homotaurine dose was chosen based on our dosing studies of homotaurine’s ability to reverse disease in newly diabetic NOD mice (manuscript in preparation). Control and homotaurine-treated groups of mice drank similar amounts of water each day (≈4 ml/mouse/day). While the control group of mice quickly progressed to more severe EAE, the homotaurine treatment group of mice remained at low clinical score of EAE (Fig. 1A).

Figure 1 Homotaurine inhibits EAE progression after the onset of symptoms in both monophasic and relapsing-remitting mouse models of MS. (A) C57BL/6 mice were immunized with MOG 35–55 and monitored daily for clinical signs of EAE as described in Methods. Eleven to thirteen days post-immunization all of the mice developed EAE. When the mice reached an EAE score of 1 they were randomized to receive plain water or water containing homotaurine (0.25 mg/ml). Graph shows mean EAE scores ± SEM for mice that received plain water (circles) and homotaurine (triangles) after EAE onset. N = 5 mice/group, overall p = 0.01, by Kruskal-Wallis analysis. (B) SJL mice were immunized with PLP 139–151 and ten to twelve days later all of the mice develeoped clinical symptoms. When the mice reached an EAE score of 1 they were randomized to receive plain water or water containing homotaurine. Graph shows mean EAE scores ± SEM for mice that received plain water (circles) or homotaurine (triangles) continuously after EAE onset. N = 8 mice/group, *p < 0.05, overall p < 0.01. (C) Spinal cord (left panel) and cerebellum (right panel) in control and homotaurine-treated PLP 139–151 immunized SJL mice. Twenty-seven days after treatment, adjacent sections were stained with hematoxylin and eosin (H &E, top row) and luxol fast blue or black gold (bottom row). Arrow in H&E stained sections indicates infiltrates. Inserts in black gold stained cerebellum images show boxed areas magnified 1.5X revealing greater myelination and fine myelin fibers in homotaurine-treated mice (arrows). Left panel scale bar = 1 mm, right panel scale bar = 250 µm. Full size image

We next tested homotaurine treatment in a relapsing-remitting model of EAE that was induced by immunizing SJL mice with a proteolipid protein peptide (PLP 139–151 )36. Ten to 12 days after receiving the encephalitic regimen, all of the mice began to develop EAE. When an animal reached an EAE severity score of 1 it was randomly assigned to receive plain drinking water, or water containing homotaurine (0.25 mg/ml) for the rest of the study. Control mice displayed classical relapsing-remitting EAE. Homotaurine-treated mice exhibited a reduced mean EAE severity throughout the observation period compared to those given plain water, and eventually displayed almost complete remission (Fig. 1B). Histological analysis of their brains and spinal cords revealed reduced mononuclear cell infiltration and areas of myelin loss in the cerebellum and spinal cords of homotaurine-treated mice (Fig. 1C). Thus, treatment with homotaurine after the clinical onset of EAE inhibited disease progression in both monophasic and relapsing-remitting mouse models of MS. Previous studies using GABA to inhibit T1D and RA in mouse models could not distinguish the contributions of GABA A -Rs versus GABA B -Rs to those observations. Our results using homotaurine point directly to GABA A -R-mediated pathways as major components of GABA’s anti-inflammatory effects.

To begin to understand the basis for homotaurine’s therapeutic effects, we next examined homotaurine’s effects on lymphocytes since T cells are the major mediators of EAE and MS. SJL mice were immunized with PLP 139–151 and 10 days later (when most mice had an EAE score of about 1), the mice were given plain water or water containing homotaurine (0.25 mg.ml) continuously for five days after which their splenic T cells were analyzed by ELISPOT. Consistent with previous studies of GABA treatment2,5,8,10,11, we observed that homotaurine treatment significantly decreased the frequency of splenic IFNγ (Th1) responses, but increased IL-10-secreting responses to PLP 139–151 (Fig. 2). In addition, we show for the first time, that GABA A -R activation significantly reduces the frequency of PLP 139–151- reactive IL-17A (Th17) spot-forming colonies (SFC) (Fig. 2). These IL-17A SFC represent antigen-specific activated/memory CD4+ T cells since peptide 13mers do not stimulate CD8+ T cells effectively, and it has been demonstrated in the PLP 139–151 /SJL model that IL-17 secreting SFC arise from CD4+ cells and not other cell types37. Homotaurine had no effect on Th2 frequencies (data not shown).

Figure 2 Homotaurine treatment at time of clinical EAE onset reduces the frequency of autoreactive IL-17A- and IFNγ-secreting T cell responses, while increasing IL-10-secreting responses. Mice were PLP 139–151 immunized and after reaching an EAE score of 1, they received plain water or homotaurine for five days. Splenic T cells were isolated from individual mice and the percentages of IL-17A, IFNγ and IL-10 secreting T cells responding to PLP 139–151 were measured by ELISPOT. Data shown are the mean SFC ± SEM of each group of mice (n = 5 per group) from two separate experiments. IL-4 responses to PLP 139–151 were at background levels (data not shown). Wells containing cells from control or homotaurine-treated mice that were incubated with medium alone had 0–5 SFC. Pairwise comparisons were performed by 2-tailed Student’s t test. **p < 0.01 vs. the controls. Full size image

GABA treatment can promote CD4+ Treg responses in mice8,12. It is unknown whether GABA A -R activation might also modulate CD8+CD122+PD-1+ and CD8+CD122+PD-1− regulatory T cells or IL-10-secreting Bregs which can play crucial roles in inhibiting inflammation and T cell autoimmunity. We found that the percentages of splenic CD4+Foxp3+ Tregs (Fig. 3A) and CD8+CD122+PD-1+, but not CD8+CD122+PD-1−, Tregs (Fig. 3B,C) in the homotaurine-treated mice were significantly higher than in the controls. There was no significant difference in the frequency of splenic CD19+IL-10+ Bregs (data not shown).

Figure 3 Homotaurine treatment enhances CD4+ and CD8+ Treg responses in mice. SJL mice were immunized with PLP 139–151 in 50% CFA and when the mice developed EAE with a score of 1, they were randomized and provided with plain water and water containing 0.25 mg/ml homotaurine for five days. The percentages of splenic (A) CD4+Foxp3+, (B,C) CD8α+CD122+ and CD8α+CD122+PD-1+ Tregs were determined by flow cytometry. The cells were gated first on living lymphocytes and CD4+ or CD8+ and the percentages of CD4+Foxp3+, CD8α+CD122+PD-1− and CD8α+CD122+PD-1+ Tregs were analyzed. Data are representative flow cytometry charts or expressed as the mean ± SEM of each group of mice (n = 5 per group) from two separate experiments. *p < 0.05, **p < 0.01 vs. the controls. (D) Splenic CD8+CD122+PD-1+ and CD8+CD122+PD-1− Tregs were sorted by flow cytometry and mixed with lymph node mononuclear cells from PLP 139–151 -immunized SJL mice at the indicated ratios and assessed for PLP 139–151 -stimulated T cell proliferation. Data are expressed as mean ± SD of each group of cells. The cultures with responders in medium alone served the negative controls (with CPM of 850–940) and peptide-stimulated cultures without any inhibitor provided positive controls. Additional positive control cells were stimulated with PPD or anti-CD3 (with CPM of 20821–26759). *p < 0.05, **p < 0.01 vs. the peptide-stimulated positive controls. Full size image

To test the inhibitory function of CD8+CD122+PD-1+ and CD8+CD122+PD-1− Tregs, SJL mice were immunized with PLP 139–151 and 10 days later, they were randomized to receive homotaurine (0.25 mg/ml) through their drinking water, or plain water for the next six days. Their splenic CD8+CD122+PD-1+ and CD8+CD122+PD-1− Tregs were sorted by flow cytometry and mixed with lymph node mononuclear cells from PLP 139–151 -immunized SJL mice for proliferation assays. Co-culture with CD8+CD122+PD-1+ Tregs, but not CD8+CD122+PD-1− Tregs, from either homotaurine-treated or control (plain water)-treated mice significantly inhibited the proliferation of responders in a dose-dependent manner (Fig. 3D). The inhibitory effect of CD8+CD122+PD-1+ cells from homotaurine-treated mice was comparable to those from control mice, indicating that homotaurine treatment did not significantly alter their suppressive activity. These data, together with the increased frequency of CD4+Foxp3+ and CD8+CD122+PD-1+ Tregs in the homotaurine-treated mice indicate that CD4+Foxp3+ and CD8+CD122+PD-1+ Tregs play an important role in inhibiting the pathogenic T cell autoimmunity, contributing to the decreased severity of EAE.

Very few of the many drugs that can inhibit EAE successfully translated to the clinic. Factors that contribute to this failure include; (1) inherent differences between EAE and MS, (2) toxicity or side-effects in humans, and (3) supra-physiological doses were used in the EAE studies. In regard to those issues, human T cells express GABA A -Rs whose activation can limit PBMC inflammatory responses3,4,5,38, homotaurine appears to be safe for long-term human use, and using FDA guidelines (www.fda.gov/downloads/Drugs/…/Guidances/UCM078932.pdf) for scaling mouse doses to the equivalent human dosage, the homotaurine dose we used to inhibit EAE was 6–10-fold lower than that used in the Alzheimer’s disease clinical trials.

Murine and human T cells express relatively high levels of transcripts encoding the δ and ρ2 subunits of GABA A -Rs2,3,4,5,38. The δ and ρ2 subunits are of particular interest because thay are found in “extrasynaptic” GABA A -Rs that can be orders of magnitude more sensitive to GABA than the typical GABA A -Rs subtypes that are clustered in neuronal synapses39,40,41,42,43,44. These δ and /or ρ2 subunits may confer T cells with sensitivity to low levels of GABA and contribute to CNS “immunological privilege”. In this scenario, endogenous CNS GABA may help regulate the low-frequency CNS-reactive T cells that spontaneously activate under normal conditions, but it is insufficient to regulate the large number of autoreactive T cells that are experimentally activated in EAE. As we show here, administration of a BBB-permeable high-affinity GABA A -R agonist is capable of limiting CNS autoreactivity under those conditions. Additionally, homotaurine may have acted on antigen presenting cells (APC) such as macrophages, dendritic cells, and microglia that contribute to EAE pathogenesis21,45,46. These APC also express GABA A -Rs and GAB A A-R agonists have been shown to inhibit their inflammatory activity11,21,47,48,49.