Immunological GABAergic interactions and therapeutic applications in autoimmune diseases

Autoimmunity Reviews 14 (2015) 1048–1056

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Review

Immunological GABAergic interactions and therapeutic applications in autoimmune diseases Gérald J. Prud'homme a,b,⁎, Yelena Glinka c, Qinghua Wang d,e,f,g a

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada Department of Laboratory Medicine and Keenan Research Centre for Biomedical Science, St. Michael's Hospital, 30 Bond Street, Toronto M5B1W8, Canada Keenan Research Centre for Biomedical Science, St. Michael's Hospital, 30 Bond Street, Toronto M5B1W8, Canada d Department of Endocrinology and Metabolism, Huashan Hospital, Medical College, Fudan University, Shanghai 200040, China e Department of Physiology, Faculty of Medicine, University of Toronto, Canada f Department of Medicine, Faculty of Medicine, University of Toronto, Canada g Division of Endocrinology and Metabolism, Keenan Research Centre for Biomedical Science of St. Michael's Hospital, 30 Bond Street, Toronto, Ontario M5B 1W8, Canada b c

a r t i c l e

i n f o

Article history: Received 6 July 2015 Accepted 17 July 2015 Available online 29 July 2015 Keywords: Diabetes EAE GABA GAD65 Immunotherapy Ion channel Inflammation

a b s t r a c t Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain. However, it is also produced in other sites; notably by pancreatic β cells and immune cells. The function of GABA in the immune system is at an early stage of study, but it exerts inhibitory effects that are relevant to autoimmune diseases. The study of GABAergic interactions in the immune system has centered on three main aspects: 1) the expression of GABA and the relevant GABAergic molecular machinery; 2) the in vitro response of immune cells; and 3) therapeutic applications in autoimmune diseases. T cells and macrophages can produce GABA, and express all the components necessary for a GABAergic response. There are two types of GABA receptors, but lymphocytes appear to express only type A (GABAAR); a ligand-gated chloride channel. Other immune cells may also express the type B receptor (GABABR); a G-protein coupled receptor. Activation of GABA receptors on T cells and macrophages inhibits responses such as production of inflammatory cytokines. In T cells, GABA blocks the activation-induced calcium signal, and it also inhibits NF-κB activation. In preclinical models, therapeutic application of GABA, or GABAergic (agonistic) drugs, protects against type 1 diabetes (T1D), experimental autoimmune encephalomyelitis (EAE), collagen-induced arthritis (CIA) and contact dermatitis. In addition, GABA exerts anti-apoptotic and proliferative effects on islet β cells, which may be applicable to islet transplantation. Autoimmunity against glutamic acid decarboxylase 65 (GAD65; synthesizes GABA) occurs in T1D. Antigen therapy of T1D with GAD65 or proinsulin in mice has protective effects, which are markedly enhanced by combined GABA therapy. Clinically, autoantibodies against GAD65 and/or GABA receptors play a pathogenic role in several neurological conditions, including stiff person syndrome (SPS), some forms of encephalitis, and autoimmune epilepsy. GABAergic drugs are widely used in medicine, and include benzodiazepines, barbiturates, anticonvulsants, and anesthetic drugs such as propofol. Native GABA can be administered orally to humans as a drug, and has few adverse effects. However, the immune effects of GABAergic drugs in patients are not well documented. GABAergic immunobiology is a recent area of research, which shows potential for the development of new therapies for autoimmune diseases. © 2015 Elsevier B.V. All rights reserved.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GABAergic molecular components and physiological responses . . . . . . . 2.1. GABA and its receptors . . . . . . . . . . . . . . . . . . . . . . 2.2. Physiological response . . . . . . . . . . . . . . . . . . . . . . Expression of GABA receptors by immune cells and GABA-induced responses .

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Abbreviations: CIA, collagen-induced arthritis; EAE, experimental autoimmune encephalomyelitis; GABA, gamma-aminobutyric acid; GABAAR, type A GABA receptor; GABABR, type B GABA receptor; GABA-T, GABA transaminase; GAD, glutamic acid decarboxylase; GAT, GABA transporter; MDSD, multiple low-dose streptozotocin-induced diabetes; NOD, nonobese diabetic mice; PBMC, peripheral blood mononuclear cells; T1D, type 1 diabetes; PMN, polymorphonuclear neutrophils; SPS, stiff person syndrome; VIAAT, vesicular inhibitory amino acid transporter. ⁎ Corresponding author at: Keenan Research Centre for Biomedical Science, 30 Bond Street, Room 418-LKSKI, St. Michael's Hospital, Toronto M5B1W8, Canada. E-mail addresses: [email protected] (G.J. Prud'homme), [email protected] (Y. Glinka), [email protected] (Q. Wang).

http://dx.doi.org/10.1016/j.autrev.2015.07.011 1568-9972/© 2015 Elsevier B.V. All rights reserved.

G.J. Prud'homme et al. / Autoimmunity Reviews 14 (2015) 1048–1056

3.1. Studies in rodents . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Studies in humans . . . . . . . . . . . . . . . . . . . . . . . . . 4. Immunotherapeutic effects of GABA in autoimmune diseases and inflammation 4.1. Type 1 diabetes (T1D) . . . . . . . . . . . . . . . . . . . . . . . 4.2. Experimental autoimmune encephalomyelitis (EAE) . . . . . . . . . 4.3. Collagen-induced arthritis (CIA) . . . . . . . . . . . . . . . . . . 4.4. Dermatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. GAD65 and GABA receptors as target antigens of autoimmunity . . . . . . . 5.1. T1D, stiff person syndrome (SPS), encephalitis and autoimmune epilepsy . 5.2. Therapeutic vaccination against GAD65 and insulin in T1D . . . . . . 6. Immunologic effects of clinical GABAergic drugs . . . . . . . . . . . . . . . 6.1. Anticonvulsant and anesthetic drugs . . . . . . . . . . . . . . . . 6.2. Administration of native GABA to humans . . . . . . . . . . . . . . 7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Take-home message . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Gamma-aminobutyric acid (GABA1) is the major inhibitory neurotransmitter in the brain. However, GABA is also produced in several sites outside the central nervous system (CNS) by non-neuronal cell types; notably the islet β cells of the pancreas [1] and immune cells [2]. Like neurons, immune cells [2–7] and pancreatic endocrine cells [8–10] express GABA receptors, which modulate their activity. These GABA-mediated effects are often inhibitory [2–9], as in the mature CNS; however, in islet β cells they are stimulatory [10–12]. Although the role of GABA in the CNS has been extensively studied and is well understood, this is not the case in non-neuronal sites. Indeed, its function is only beginning to be defined in the pancreas. Similarly, in the immune system investigations are still at an early stage, but GABA clearly exerts important inhibitory effects that are especially relevant to autoimmune diseases. The study of GABAergic interactions in the immune system has centered on three main aspects: 1) the expression of GABA, GABA receptors and the relevant GABAergic molecular machinery; 2) the in vitro response of immune cells to GABA or other agonists; and 3) therapeutic applications of GABA in autoimmune diseases. In this review, we will focus on these three aspects.

2. GABAergic molecular components and physiological responses 2.1. GABA and its receptors GABA is synthesized through the decarboxylation of L-glutamate by glutamic acid decarboxylase (GAD). There are two isoforms of this enzyme, GAD65 and GAD67, with considerable homology. In neurons GAD67 is distributed throughout the cytosol, whereas GAD65 is found in synaptic vesicles [13]. Human lymphocytes appear to express only GAD67 [6]. In pancreatic β cells, the main isoform in humans is GAD65, whereas in mice it is GAD67 [14,15]. In the brain, the concentration of GABA depends on the synthesis of GAD (GAD65 and GAD67 isoforms), inactivation of GABA by GABA transaminase (GABA-T), and the transfer of extracellular GABA into neurons by GABA transporters (GATs) [6,16]. In addition, the vesicular inhibitory amino acid transporter (VIAAT) is responsible for the loading of GABA into secretory vesicles [17]. There are two major types of GABA receptor, which differ markedly. The type A GABA receptors (GABAAR) are a heterogeneous group of pentameric cell-membrane receptors, with distinct physiological and pharmacologic properties. They form variably through the assembly of three types of subunits. Indeed, there are 19 GABAAR subunits, i.e., α1–6, β1–3, γ1–3, δ, ε, θ, π, and ρ1–3 [7,18–20]. Generally, at least

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in the CNS, a GABAAR consists of two α subunits, two β subunits, and one copy of another subunit. In this configuration, these receptors function as fast-acting chloride channels [18–20]. Muscimol is GABAAR agonist, whereas picrotoxin, bicuculline and SR-95531 are antagonists. In sharp contrast, there is a single type B receptor (GABABR), and it is a G-protein-coupled receptor (GPCR) [12,21]. It consists of two invariable subunits: B1 and B2. The GABABR is a slow acting receptor, which is linked to K+ channels. The activation of GABABR promotes the opening of K+ channels, causing hyperpolarization of the membrane and decreased adenylyl cyclase activity. This blocks the opening of sodium channels as well as voltage-gated Ca2+ channel (VGCC), to deliver an inhibitory signal. Baclofen is a GABABR agonist clinically used to treat spasticity [22], and saclofen is a useful antagonist for physiological studies. 2.2. Physiological response A detailed description of the CNS physiological aspects is beyond the scope of this review, but some key points will be mentioned. GABA exerts a fast inhibitory response characterized by neuronal membrane hyperpolarization, mainly through the activation of GABAAR [18–20, 23–25]. The opening of GABAAR Cl− channels on the membrane causes an influx of Cl− ions, resulting in hyperpolarization. In the synapse, these GABA receptors are of low affinity. However, GABA is also located at low concentrations outside the synapse, where it can bind to much higher affinity extrasynaptic GABAAR receptors [24,25]. These extrasynaptic receptors reduce neuronal excitability and are the target of numerous drugs, which include anxiolytics, anesthetics, anticonvulsants, alcohol and neurosteroids [18–20,23,25]. Similar high affinity extrasynaptic receptors have been described in non-neuronal cells, including lymphocytes and endocrine cells [4,10,12,25]. This is a particularly important feature, because GABA is only present at low (submicromolar) concentrations in the plasma and tissues outside the CNS [4]. 3. Expression of GABA receptors by immune cells and GABA-induced responses 3.1. Studies in rodents Investigators have examined the expression of GABAergic components in the lymphocytes and other immune cells of mice and rats. Tian et al. [26] reported the presence of GABAAR, at least at a functional level, in studies performed with mouse T lymphocytes. They showed that GABA inhibited the proliferative responses of T cells to CD3 antibody or cognate antigen in vitro. The proliferative response was reduced

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by approximately 40–50% at GABA concentrations of 0.3 to 1 mM. These and subsequent findings by other investigators [6,11] revealed that this inhibitory effects is partial, and is only modestly increased at higher concentrations (N 1 mM) [26]. This has not been fully explained, but perhaps only a subpopulation of T cells expresses GABA receptors. In vitro, the suppressive effect was duplicated by a GABAAR agonist (muscimol), but not a GABABR agonist (baclofen) [26]. It was abrogated by a GABAAR antagonist (bicuculline). This suggested that GABA was acting only through GABAAR. Tian et al. [26] also observed that, in vivo, GABA treatment partially inhibited delayed-type hypersensitivity (DTH) responses. In a subsequent study, Tian et al. [3] identified specific subunits of GABAAR in T cells by RT-PCR. They reported that the α1, α2, β1, β2, and δ subunits were expressed by naïve and activated CD4+ T cells of NOD mice. γ3 was also expressed in activated T cells. This study revealed that the subunits required to form a functional receptor were present. Other T-cell subpopulations were not examined. Bjurstöm et al. [4] also reported the presence of GABAAR subunits, but with some differences, possibly because they were examining a different mouse strain or population of T cells. They detected α1, α4, β2, β3, δ and γ1 transcripts in encephalitogenic CD4+ T-cell lines derived from B10.RIII mice. Interestingly, they observed inhibition or T-cell responses by GABA at concentrations as low as 100 nM, which was considerably lower than in previous reports. This concentration (100 nM) corresponds to normal GABA levels in plasma [27], and suggests potential activation of these receptors under normal physiological conditions. Importantly, these authors [4] recorded GABAinduced currents in T cells, using patch-clamp configurations. The GABA-activated channels in T cells generated currents similar to those produced by extra-synaptic channels in neurons. Mendu et al. [7] examined the expression of all known GABAAR subunits, and identified eight subunits in mouse CD4+ and CD8+ T cells, with no difference between the two cell types. They reported differences in the number and type of subunits between mouse, rat and human T cells. By immunoblotting and immunocytochemistry they showed abundant expression of GABAAR in T cells of all three species. Fluorescent staining revealed a punctate pattern on the membrane of the cells. Moreover, using a patch-clamp technique they found that these channels were activated by GABA, and generated whole-cell transient and tonic currents. Bhat et al. [2] observed GABAAR subunits in mouse macrophages, but surprisingly in view of other reports could not identify any in T cells. They identified other GABAergic components in both macrophages and T cells, including GAD65, GABA (which was secreted), GABA-T and GAT-2 (one of the four GATs). Dendritic cells (DCs) expressed GAD65 and secreted GABA. Activation of T cells or macrophages increased the levels of the

detected components. In addition, electrophysiological studies (wholecell voltage clamp) revealed GABA-induced inward currents in macrophages, as in control hippocampal neurons although in macrophages they were smaller in amplitude and slower to rise and decay. Other investigators have also identified GABAAR subunits in macrophages. For instance, Reyes-Garcia et al. [28] detected GABAAR subunits in mouse peritoneal macrophages, and showed that the application of GABA inhibited LPS-induced cytokine production (IL-6 and IL-12). In rats T cells, several GABAAR subunits have been identified, including α, β, γ, δ, θ, π and ρ components, but as in mice have differed partially between studies and strains [7,29,30]. As recently reviewed [30], GABAergic signaling has also been observed in mouse and human myeloid DCs. These cells have been found to have GABAAR, which is activated by GABA as confirmed in electrophysiological studies [31]. DCs infected with toxoplasma were found to secrete large amounts of GABA [31]. In contrast to inhibitory immune effects noted previously in T cells and macrophage, GABA appears to have stimulatory effects on DCs, at least on chemotactic responses, motility and transmigration [30,31], although antigen presentation and other functions are suppressed as mentioned in other sections. 3.2. Studies in humans As in rodents, several GABAergic components have been identified in human immune cells (Table 1). Alam et al. [5] examined GABAAR subunit expression in human peripheral blood mononuclear cells (PBMCs) and Jurkat cells (a human CD4+ T-cell line). They detected mRNA expression of α1–4, β2, β3, γ2, δ and ε subunits in PBMCs, and the same as well as additional types in the Jurkat T-cell line. They also examined specific subpopulation of cells, and found that T cells, B cells and monocytes were all positive for some α and β chain components. Immunoblotting studies with an anti-α1 antibody revealed expression of this chain in PBMC, T cells and B cells. Dionisio et al. [6] also examined the expression of GABAAR subunits, as well as other GABAergic components. Furthermore, they studied the response of human T cells to GABA in a number of assays. These experiments demonstrated that human lymphocytes possess all the components for a functional GABAergic system. This includes the necessary GABAAR receptor subunits, GAT transporters (GAT-1 and GAT-2), GABA-T, VIAAT, and GAD67 (but not GAD65). The level of these components was higher in activated T cells. To demonstrate that there are functional GABAAR channels, they performed whole-cell recordings, and identified GABA-induced currents in 10–15% of activated lymphocytes. They also reported partial inhibition of T-cell proliferation by GABA.

Table 1 GABAergic components of the human immune system.

GABA GAD GABA-T GAT-1,2 VIAAT GABAAR α Subunits

PBMC

L

+

+ + (GAD67) + + + +

+ α1–4

CD4+

CD8+

M

DCs

B cell

PMN

+ + (GAD65/67)

+ α1,3

+ α1

+ α1

α1,5 β2

α1,5 β2

−

β1

β1

+

+ α1,3

+ −

β2

−

α1,3,6 β Subunits

β2,3 β3

β2 3rd subunit

γ2,δ,ε γ2,δ,ρ2 π,ρ2

GABABR

+

π,ρ2 mo

+

Ref [25,63] [6,33] [6] [6] [6] [5–7,25,30–33,63] [5] [6] [7] [5] [6] [7] [32] [5] [6] [7] [33,62,65]

Abbreviations: +, mRNA, protein, or mediator expression have been detected in one or more studies; DCs, dendritic cells; GABAAR, type A GABA receptor; GABABR, type B GABA receptor; GABA-T, GABA transaminase; GAD, glutamic acid decarboxylase; GAT, GABA transporter; L, lymphocytes (unfractionated); M, monocytes or macrophages; mo, reported in mice only; PMN, polymorphonuclear neutrophils; VIAAT, vesicular inhibitory amino acid transporter.

G.J. Prud'homme et al. / Autoimmunity Reviews 14 (2015) 1048–1056

As noted above, Mendu et al. [7] also identified GABAAR in human T cells. They detected only five GABAAR subunits (α1, α5, β1, π, and ρ2) in human CD4+ and CD8+ T cells, which were comparably expressed in the two cell types. They reported that human T cells, unlike mouse T cells, did not express the γ2 chain. This is medically relevant because this chain is required for responsiveness to benzodiazepines. As previously reviewed by other authors [25,30], there have been marked differences in GABAAR subunit expression identified in T cells, both inside and between species. The significance of this is not clear, and merits further investigation. It is noteworthy that subsets of CD4+ T cells, such as Th1, Th2, Th17 and regulatory T cells (Tregs), have not been examined. Thus, it is unknown whether all these T-cell subsets express GABA receptors and, if they do, whether they express different subtypes of the receptors. Other cells of the human immune system also express GABA receptors. In accord with previous work [5], Wheeler et al. [32] found that human monocytes express functional GABAAR, and this appears to be relevant to their inhibition by some anesthetic agents. As noted above, the GABAergic responses of human DCs are similar to those of mice [31]. Neutrophils express GABAAR and GABABR receptors, and it appears that type B receptors can stimulate neutrophilic chemotaxis [33]. The mechanisms by which GABA inhibits immune responses are not fully elucidated. Alam et al. [5] reported that the stimulation of PBMC with fMLP or LPS induced an increase in intracellular Ca2+, as detected by flow cytometry, which was partially inhibited by GABA. This inhibitory effect was mimicked by muscimol, and it was blocked by bicuculline, whereas baclofen had no effect, suggesting it was a GABAAR-dependent response. However, T cells were not directly activated in these experiments. We examined human T cells simulated with anti-CD3 mAb or concanavalin A [34], and found that GABA had inhibitory effects similar to those previously reported in mouse cells. For instance, GABA inhibited CD3-induced T-cell proliferation, in a GABAARdependent way. We further examined potential mechanisms of T-cell suppression, and observed that GABA inhibited the increase in intracellular Ca2+ that is an early signal for T-cell activation [34]. The mechanism of calcium signaling blockade has not been established. However, lymphocytes have relatively high intracellular Cl− levels, and it has been proposed that the opening of GABAAR channels results in an efflux of Cl−, which causes membrane depolarization and inhibits Ca2+ entry into the cell [3]. Indeed, a strongly negative membrane potential favors Ca2+ entry [35], and this will be counteracted by Cl− efflux. In this respect, it is of interest that lectin stimulation of Jurkat T cells increases intracellular Cl− levels [36], and this might be required to maintain a negative membrane potential sufficient to promote Ca2+ influx [37]. Recently, we demonstrated that GABA also inhibits NF-κB activation in both human T lymphocytes and pancreatic islet cells. We also found, at least in islet cells, that some of GABA's effects are mediated by sirtuin

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1 (SIRT1) [38]. SIRT1 is an NAD+-dependent deacetylase that can counteract inflammatory signals. GABA increased SIRT1 and NAD+ (an essential cofactor), resulting in increased enzymatic activity that was associated with deacetylation of the p65 component of NF-κB. This type of deacetylation has been shown to block the activation NF-κB [39]. Because this pathway plays a key role in both innate and adaptive immunity, its inhibition may represent a major mode of action of GABA. 4. Immunotherapeutic effects of GABA in autoimmune diseases and inflammation 4.1. Type 1 diabetes (T1D) GABA, administered to mice orally or intra-peritoneally, has been found to protect against T1D and other autoimmune diseases (Table 2). Tian et al. [3] were the first to demonstrate the protective effects of GABA in non-obese diabetic (NOD) mouse T1D. They reported that GABA (100 μM, in vitro) suppressed activated T-cell responses against islet. Furthermore, they showed activity of GABA in vivo as an immunoprotective drug. Indeed, GABA inhibited the adoptive transfer of T1D, consistent with the suppression of diabetogenic effector T cells. Moreover, GABA markedly protected against the development of T1D when the treatment was initiated in prediabetic mice. Their findings suggested that the protection against disease was primarily due to a reduction in Th1-mediated autoimmunity. Interestingly, they observed that GABA blocked T-cell cycling in the G0/G1 phase. This was not associated with activated T-cell apoptosis, and they suggested that the persistence of GABA was required to maintain its immunosuppressive activity. We observed similar protection against T1D in our studies [11]; however, we found that GABA had a major direct action on islet β cells, which likely contributed to the beneficial therapeutic effect. We have recently reviewed the positive effects of GABA on islet β cells, which consist of anti-apoptotic, proliferative and regenerative effects [12], and here we will focus mainly on immunologic findings. In vivo, the suppressive actions of GABA are not limited to Th1 cells, since it prevented disease induced by diabetogenic CD8+ cytotoxic T lymphocytes (CTLs) in the transgenic TCR-8.3 NOD model [11]. In vitro, we observed that GABA inhibits the proliferation and/or cytokine production of activated CD4+ and CD8+ T cells as well as macrophages. For instance, it markedly inhibited IFN-γ production by T cells and IL-12 production by macrophages. In contrast, GABA augmented T-cell secretion of TGF-β1 [11], which is a major suppressive and immunoprotective cytokine. T1D is a T-cell dependent disease, but inflammatory mechanisms also appear to play a key pathogenic role [40,41]. Local (in the islets) and systemic inflammation is most evident in multiple low-dose streptozotocin (STZ)-induced diabetes (MDSD) [42], but is also present

Table 2 Immunotherapeutic effects of GABA and other GABAergic drugs. Disease

Strain/model

Therapy

Immune effects

Disease course

Ref.

T1D

NOD (prediabetic) NOD (diabetic) NOD-TCR8.3 (prediabetic) CD1 mice (MDSD, diabetic) NOD (islet Tx) NOD (diabetic) SJL/J (PLPp Ag) C57BL/6J (MOGp Ag)

GABA GABA GABA GABA GAD-Vax + GABA ProIns-Vax + GABA Vigabatrin, topiramate Vigabatrin GABA GABA Baclofen Baclofen

Th1 sup, IFN-γ↓, IL-12↓, Treg↑

Prevented (insulitis↓) Reversed transiently Prevented (insulitis↓) Reversed (insulitis↓, β-cell regeneration) Prolonged islet survival Long-term reversal (insulitis↓, β-cell replication) Ameliorated or reversed Ameliorated Aggravated Ameliorated Ameliorated Ameliorated

[3,11] [11,108] [11] [11] [51] [108] [2] [58] [58] [61] [62] [65]

EAE

CIA HCD

DBA/1 DBA/1 C57BL/6 (DNFB)

CTL response↓ IL-1↓, TNF-α↓ IL-12↓, IFN-γ↓ TGF-β↑ IFN-γ↓,IL-10↑, Treg↑ IL-1β↓, IL-6↓, APCs sup TNF-α↓, IL-6↑, IFN-γ↑ T-cell proliferation↓ APCs sup, AutoAb↓ Th17↓, DCs sup, IL-6↓ Immune-cell infiltrate↓

Abbreviations: ↑, increased; ↓, decreased; Ag, antigen; DNFB, dermatitis induced by 2,4-dinitrofluzorobenzene; AutoAb, autoantibody; CIA, collagen-induced arthritis; EAE, experimental autoimmune encephalomyelitis; GAD-Vax; GAD65/alum vaccination; HCD, hypersensitivity contact dermatitis; MDSD, multiple low-dose streptozotocin-induced diabetes; MOGp, myelin oligodendrocyte protein peptide; NOD, nonobese diabetic mice; NOD-TCR8.3, transgenic NOD mice bearing TCR8.3+ CTLs; PBMC, peripheral blood mononuclear cells; PLPp, proteolipid protein peptide; ProIns-Vax, vaccination with proinsulin/alum; sup, functionally suppressed; T1D, type 1 diabetes; Tx transplantation.

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in NOD mice [41]. Consistent with this, anti-inflammatory therapy is protective in the MDSD [42] and autoimmune NOD-mouse form of diabetes [41,43,44]. Supporting an anti-inflammatory action, GABA therapy in the MDSD model prevented insulitis and reduced serum levels of inflammatory cytokines (IL-1β, IL-12, TNF-α and IFN-γ) [11]. The anti-inflammatory effect might be due to the suppression of the NF-κB pathway. Indeed, as noted above, GABA inhibits NF-κB activation in both lymphocytes and islet cells. This is a key pathway that promotes inflammation, acting at several levels, and it has been shown to contribute to islet-cell apoptosis [45–47]. Of importance, GABA increased Tregs of the Foxp3+/neuropilin-1+ phenotype [11], which have been shown to be mainly of the thymusderived type (tTreg) [48,49]. We hypothesize that an increase in the number of Tregs is at least partially responsible for the immunosuppressive effects of GABA, but additional work is required to confirm this. In addition to these findings in T1D, GABA also appears to be protective in T2D. Tian, et al. [50], examined obesity and hyperglycemia in high fat diet-fed mice. In these mice obesity-related inflammation appears to be a major pathogenic factor. Oral GABA treatment inhibited inflammation, and ameliorated glucose tolerance and insulin sensitivity. As in our studies, Treg numbers were increased, which they postulate contributed to the therapeutic effect. GABA treatment shows promise for the amelioration of islet transplantation. It improved transplanted islet survival in NOD mice, especially when combined with antigen-based therapy [51], as described in another section. A major limitation of clinical islet transplantation is that the majority of islets die soon after transplantation due to a combination of factors; most notably hypoxia and an acute inflammatory response [52–54]. In studies with human cells, we demonstrated that GABA markedly reduced islet-cell apoptosis in culture, and stimulated insulin production [34,55]. Islet cells die spontaneously in culture, and commonly used immunosuppressive drugs further impair islet-cell survival [34,56]. GABA considerably improved islet-cell survival in vitro. Moreover, it protected islet cells against apoptosis induced by cytokines [55], as well as rapamycin, tacrolimus, and mycophenolate mofetil [34]. As noted previously, these protective effects appear dependent on increased SIRT1 [38]. Importantly, GABA did not interfere with the immunosuppressive effects of rapamycin, but actually collaborated with that drug to suppress T lymphocytes. These findings suggest that GABA might be used to improve islet-cell survival before and after transplantation. Furthermore, in patients chronically treated with immunosuppressive drugs for other reasons, GABA might improve islet-cell survival and function. 4.2. Experimental autoimmune encephalomyelitis (EAE) EAE has long been considered an animal model of multiple sclerosis (MS). It is mediated by autoaggressive CD4+ effector T cells (Th1 and Th17), responding to CNS antigens such as myelin basic protein (MBP), which cause inflammatory and demyelination lesions in the CNS. This disease can be induced in mice and other species, and although not a perfect model of MS it has nevertheless been useful in designing therapies [57]. Bjurstöm et al. [4] demonstrated that low physiological concentrations of GABA could suppress the proliferation of mouse encephalitogenic T cells responding to MBP peptides in vitro. Bhat et al. [2] analyzed GABAergic treatment in vitro and in vivo. GABA does not cross the blood brain barrier (BBB) to any significant extent, and they treated the mice with either topiramate or vigabatrin (GABAergic drugs in clinical use). Topiramate is a GABAAR agonist, whereas vigabatrin inhibits GABA-T and this results in increased GABA levels. For in vitro studies, they used mice expressing a transgenic TCR specific for myelin oligodendroglial protein (MOG). Splenocytes of these mice responded to a MOG peptide by proliferating and secreting T-cell (e.g., IFN-γ and IL-17) and antigen-presenting cell (APC) cytokines (e.g., IL-1β, TNF-α, and IL6). Proliferation and cytokine production were inhibited by GABAAR agonistic drugs and vigabatrin, and this was reversed by a GABAAR antagonist. However, in contrast to other studies, they reported that the

inhibitory effect was apparent only on APCs, and not T cells. In vivo, they induced EAE by immunizing SJL/J mice with a myelin proteolipid protein peptide, and treated the mice with either topiramate or vigabatrin. Both GABAergic agents ameliorated EAE when treatment was begun at the same time as the immunization, or after induction of the disease. Therapy resulted in reduced inflammatory lesions in the CNS, and decreased generation of effector T cells in the spleen and lymph nodes. In accord with previous findings [2], Carmans et al. [58] found that vigabatrin considerably ameliorates EAE. In contrast, they reported that daily GABA treatment was not beneficial, and actually aggravated the disease. GABA-treated mice displayed increased immunity against the encephalitogenic peptide (MOG35–55). The reason for the enhancement of some immune responses by GABA is unclear, and discrepant with other reports in the literature. In terms of therapeutic effectiveness, unmodified GABA is not well suited for CNS diseases due to its minimal ability to penetrate the BBB. This might explain why only vigabatrin ameliorated disease. The expression of GABA transporters by lymphocytes appears to be of importance. These transporters (GATs) increase intracellular GABA in neurons, but their role in lymphocytes is not as well defined. Wang et al. [59] reported that the GABA transporter GAT-1 negatively regulates T cells, and protects against EAE. GAT-1 knockout mice had much more severe disease and higher T-cell responses against antigen. Furthermore, GAT-1 deficiency was associated with greater activation of the NF-κB pathway. The GAT-1-related effects were limited to T cells, and not macrophages or B cells. In another study, Wang et al. [60] reported that GAT-1 reduces T-cell activation and survival through protein kinase C (PKC) signaling pathways. In T cells, lack of GAT-1 resulted in increased cell cycle entry and reduced apoptosis. Moreover, the phosphorylation of PKCθ was promoted, and there was downregulation of cyclin-dependent kinase inhibitor p27kip1, an increase in antiapoptotic proteins, and activation NF-κB. 4.3. Collagen-induced arthritis (CIA) CIA is an autoimmune form of arthritis, usually induced in mice, with some features of rheumatoid arthritis (RA). Tian et al. [61] showed that activation of peripheral GABA receptors ameliorates this disease in mice. The T cells of mice treated with GABA displayed reduced proliferative responses to collagen, and their APCs reduced stimulatory capacity. Furthermore, the treated mice had lower levels of collagen-reactive IgG2a, but not IgG1 antibodies, suggesting suppressed Th1 responses. Autoantibody levels correlated with disease scores. Thus, in these experiments, GABA suppressed both cellular and humoral immune responses against collagen. Recently, Huang et al. [62] demonstrated that baclofen (GABABR agonist) alleviates CIA. Oral administration of baclofen decreased Th17 cell numbers, and suppressed IL-6 production by DCs. The priming of Th17 cells by DCs was diminished. Antagonistic and genetic blockade of GABABRs in DCs reduced the effects of baclofen, supporting the conclusion that these effects in DCs are mediated by the type B receptor. 4.4. Dermatitis GABAergic therapy might be beneficial in some forms of dermatitis. GABAAR-positive immune cells have been identified in the skin of psoriatic patients [63]. This included macrophages, lymphocytes and neutrophils. Furthermore, treatment of these patients with the GABA analogues gabapentin and pregabalin had beneficial effects [64]. GABABR expression also appears relevant [65]. The GABABR agonist baclofen was found to reduce the chemotaxis of human PBMCs towards several chemokines [65]. Furthermore, baclofen protected against hypersensitivity contact dermatitis (HCD) in a preclinical model. It reduced inflammation, and impaired recruitment of immune cells (neutrophils, monocytes and lymphocytes) into the skin.

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5. GAD65 and GABA receptors as target antigens of autoimmunity 5.1. T1D, stiff person syndrome (SPS), encephalitis and autoimmune epilepsy Autoimmunity against GABAergic molecular components occurs in several neurological diseases, as well as T1D [66–76]. In the case of T1D, GAD65 autoantibodies have been regarded mostly as an early marker of disease, and are not thought to be pathogenic (see below) [76]. In neurological disorders, antibodies are directed against GAD65, GABA receptors, and in some cases other neuronal receptors as well. Unlike T1D, there is evidence that GAD65 and/or GABA receptor autoantibodies are pathogenic, although the mechanisms of injury are not completely elucidated [66–74]. The range of clinical disorders includes SPS, various forms of encephalitis, autoimmune epilepsy and psychosis. These conditions can be severely debilitating or fatal. Indeed, as recently reviewed [74], anti-GAD and related antibodies define a newly recognized group of syndromes denoted hyperexcitability disorders. In this group linked to anti-GAD65 antibodies, the best studied disease is SPS [66–74]. In this case, 60–80% of patients have anti-GAD antibodies (mostly against GAD65), but it should be noted that antibodies against GABAAR-associated protein are also common [66,77]. The latter antibodies inhibit the expression of GABAAR on the neuronal membrane, and possibly contribute to the pathogenesis. T1D occurs in approximately 30% o of SPS patients, but in contrast very few T1D patients have SPS (1 in 10 000 patients) [78]. Of interest, there are distinguishing features between GAD65 antibodies in the two diseases [66,79,80]. Thus, in SPS the antibodies are present in both the cerebrospinal fluid (CSF) and the circulation, whereas in T1D they are found only in the blood. Furthermore, the antibodies in the two diseases bind to different portions of GAD65 (although there is overlap), and appear much more likely to block enzymatic activity in SPS than in T1D. Nevertheless, it remains unclear how an antibody against an intracellular antigen causes disease. Interestingly, GAD65-containing immune complexes have been identified in the serum of patients, suggesting GAD65 is somehow accessible to antibodies [69]. Moreover, the disease can be adoptively transferred to rats with the serum of patients with high titer anti-GAD65 [73]. The possibility that effector T cells are involved also has to be considered [81]. In some cases, the onset of SPS has followed a viral infection or neoplasia [66]. Indeed, in approximately 10% of cases SPS is a paraneoplastic manifestation of either breast or other types of cancer, but these patients may only have low titers of GAD65 antibodies (or none), and high titers of other antibodies such as anti-amphiphysin [82]. The treatment of SPS is problematic, but GABAergic drugs acting on either GABAAR or GABABR are of benefit [83,84]. Furthermore, measures frequently used to treat other autoimmune diseases are indicated, such as immunosuppressive drugs, IVIG, plasmapheresis, and B-cell depletion with rituximab [66,83–85]. It is increasingly recognized that autoantibodies against GAD and GABA receptors are a feature of some forms of autoimmune encephalitis and/or epilepsy [72,74,86–90]. However, these patients often have autoantibodies to other neuronal target molecules, and the relative pathogenic role of each antibody is not always clear. For instance, in autoimmune epilepsy (including autoimmune status epilepticus), antibodies have been identified against GAD65, GABAAR, GABABR, Nmethyl-D-aspartate receptor (NMDAR), voltage-gated potassium channel complex and other targets [89], and their presence serves as helpful markers for diagnosis. This disease is recalcitrant to anticonvulsant treatment. As in SPS, these conditions can be improved by therapy targeting the autoimmune manifestations [88–90]. As well, a search for a tumor (often malignant) is always indicated, due to the high risk of a paraneoplastic factor (as in SPS).

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or related peptides, have been administered by a variety of routes (subcutaneous, oral or nasal) and adjuvant formulations [76,91–98]. Genetic (DNA) vaccination has also been investigated [99–102]. The major goal has been to generate tolerance against the target antigen by either inactivating reactive T cells (clonal anergy or deletion), biasing the response to a nonpathogenic type (Th1 to Th2 shift) or, perhaps more commonly, generating regulatory T cells (Treg, Tr1 and other) [91]. The major target antigens in human T1D are proinsulin/insulin, GAD65, islet antigen 2 (IA-2) and zinc transporter protein 8 (ZnT8) [76,91]. Several other islet antigens have also been recognized, mostly in preclinical investigations. However, insulin and GAD65 stand out as the best studied, and the major ones administered in clinical trials. Despite many encouraging preclinical studies, the clinical benefits of antigen therapy in T1D have been limited, and much below expectations. It is beyond the scope of this manuscript to discuss these studies in detail, but they have been recently reviewed [76,92,98]. Almost all immunological studies of GAD65 in T1D have focused on its role as an antigen, with rarely any attention directed at its function. It was initially identified as an antigen by precipitation with antibodies in the serum of diabetic patients [103]. It was subsequently extensively characterized as a β-cell target antigen of islet cells in NOD mice. T-cell responsiveness against it was clearly demonstrated [104]. However, its true importance as an antigen has always been somewhat suspect [105]. For instance, GAD65 knockout mice still develop diabetes at the same rate [106], and GAD65 is scant in mouse islets, as compared to human islets [14,15]. Indeed, mouse β cells express mostly GAD67. Nevertheless, the induction of protective tolerance by immunization with GAD65 or specific GAD peptides has been frequently demonstrated, by a number of approaches [91]. GAD-reactive T-cell clones with regulatory function have been generated and, indeed, some authors have labeled GAD65 the regulatory antigen of T1D [91]. To our knowledge, only one study examined GABA production, although it was intended as an antigen study [107]. Transgenic mice expressing high levels of human GAD65 in their islets, and locally producing high levels of GABA, had a lower incidence of diabetes. We speculate that in this model high levels of GABA act on both islet cells and reactive T cells to improve the outcome. Importantly, immunization of NOD mice with a GAD65/alum preparation was protective [96], although contradictory results subsequently appeared in murine models of T1D [105]. Based on encouraging preclinical data, clinical trials were initiated in T1D patients by administration of a recombinant human GAD65/alum (GAD/alum) vaccine [96]. Phase I and II results were encouraging but, unfortunately, the phase III trial did not show improvement in clinical outcomes [98]. In view of the limitations of antigen therapy, as outlined above, some investigators have examined combining it with GABA treatment. Tian et al. [51] found that combining a GAD65/alum vaccine with oral GABA treatment markedly improved the survival of transplanted syngeneic islets in diabetic NOD mice. GAD65/alum alone and GABA alone improved the outcome transiently, but the combination of the two was much superior. Tian et al. [108] also treated diabetic NOD mice with a proinsulin/ alum vaccine combined with oral GABA therapy. The vaccine failed to improve the disease, whereas GABA alone had a transient effect. Remarkably, however, combined therapy reversed the disease with long-term or permanent remissions. Combined therapy reduced Th1 responses to autoantigens, and enhanced Treg and IL-10 responses. Furthermore, in accord with our previous work, GABA induced β-cell replication to restore normoglycemia. Thus, this therapy exerted beneficial immunoregulatory and β-cell regenerative effects. 6. Immunologic effects of clinical GABAergic drugs 6.1. Anticonvulsant and anesthetic drugs

5.2. Therapeutic vaccination against GAD65 and insulin in T1D Antigen therapy for the prevention or treatment of autoimmune diseases, particularly T1D, had been investigated for many years. Antigens,

Several drugs mediate their pharmacological effects at least in part by promoting the activation of the GABAAR [109,110]. This includes benzodiazepines, barbiturates, general anesthetics (e.g., thiopental and

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propofol), and some antiepileptic drugs (e.g., loreclezole, ganaxolone and topiramate). Vigabatrin increases GABA levels by irreversibly blocking the GABA-T enzyme. Gabapentin, valproic acid and tiagabine are other drugs that promote GABAergic activity, although their mechanisms of action in epileptic disorders are not completely elucidated. Baclofen, in contrast, activates the GABABR, and is used as an anti-spasmatic agent, to treat SPS for example. Despite the common clinical use of these drugs, the literature on their immunologic effects is scant and inconsistent [109,110], and the contribution of GABAergic mechanisms is largely unknown. As a confounding factor, most of these drugs also have other (non-GABA related) molecular targets. For instance, benzodiazepines bind to a peripheral receptor that appears to produce immunological effects, but it is unrelated to GABA receptors [111,112]. Nevertheless, as mentioned previously, gabapentin ameliorates psoriasis [64] and this might be related to a GABAergic effect, but this requires further confirmation. The best documented immunological effects have been obtained with anesthetics. The GABAAR agonistic drug propofol is a powerful general anesthetic administered intravenously [110]. It that has been linked to immune impairment and increased postoperative infections [110, 112,113], although there are many concurrent factors in that setting. Propofol impairs neutrophilic and macrophage function; reducing reactive oxygen species and cytokine production [112,113]. However, it was not determined whether GABAergic signaling contributed to any of these findings. Recently, Wheeler et al. [32] reported that propofol and thiopental (both GABAAR agonists), depressed human monocyte function in chemotaxis and phagocytosis assays, and this was reversed by GABAAR antagonists. They also found that monocyte receptors were insensitive to a benzodiazepine, which suggests that these cells lack the GABAAR subunit required for that response. 6.2. Administration of native GABA to humans To our knowledge there have been no controlled studies of the effects of GABA administration on human immune function. It has been known for over 50 years that GABA is absorbed following oral administration. Indeed, in the late 1950s, orally administered GABA was studied as a potential therapy for epilepsy but this was unsuccessful, likely due to a failure to cross the BBB. Large doses (several grams per day) did not cause major adverse effects, and elevated serum levels of GABA were detected [114]. Subsequently, small trials showed that it could increase circulating insulin levels, but did not alter glucose levels in normal subjects [115,116]. These findings suggest that the oral administration of GABA is feasible, and we are currently studying its pharmacological properties. The fact that orally administered GABA has minimal effects on the brain is an advantage to treat non-neurological diseases, because this is expected to markedly reduce adverse effects. GABA is present in the normal diet, but in relatively low amounts (milligrams) in vegetables such as potatoes and tomatoes, and in larger amounts in green tea leaves [see http://www.fda.gov/ucm/groups/fdagov-public/@ fdagov-foods-gen/documents/document/ucm264254.pdf]. It is also added to many dietary supplement products. Of interest, GABA is available without prescription and advocated for the relief of sleep disorders, anxiety, hypertension, and to boost GH release and increase muscle mass. There is only minimal scientific evidence to justify these uses, although a significant increase in GH has been reported [117,118]. 7. Conclusion In this review we have outlined current knowledge about the immunological effects of GABA. These studies confirm that immune cells possess all the molecular components of a GABAergic system. Indeed, they produce GABA and respond to this mediator in several assays, usually in an inhibitory way, although some stimulatory effects may occur especially in DCs. Mouse, rat and human T cells express GABAAR subunits but the receptor composition has varied between studies,

and a detailed description of receptor expression in major lymphocytes subsets (Th1, Th2, Th17, Treg and others) is lacking. Activation of the receptor in T lymphocytes blocks the early calcium signal, which may be a key element of the inhibitory effect. Furthermore, GABA clearly has an anti-inflammatory action, which is associated with inhibition of NF-κB activation. NF-κB activation is also blocked in pancreatic β cells, which may be of considerable therapeutic importance because this pathway induces apoptosis in these cells. In preclinical models, GABA has ameliorated autoimmune diseases, including T1D, EAE and CIA. In T1D, GABA has protective and stimulatory effects on β cells, but suppressive effects on the autoimmune response. Indeed, in this context, it is a rare agent that acts beneficially on both the endocrine and immune systems. We describe autoimmune diseases where autoantibodies are directed at GABAergic components (GAD65 and GABA receptors). In the case of T1D, anti-GAD65 antibodies occur but do not appear pathogenic. However, pathogenic antibodies against these GABAergic components have been described in several neurological conditions, including SPS, some forms of encephalitis, as well as autoimmune epilepsy. These diseases can be ameliorated by administration of GABAergic agonists, and/ or therapies that attenuate the autoimmune responses such as immunosuppressive drugs, IVIG, plasmapheresis and B-cell depletion. Antigen therapy with GAD65 or insulin has been beneficial in NODmouse T1D, but minimally effective in clinical trials. Recent results show that combining a vaccine preparation of these antigens with oral GABA therapy markedly improves the outcome in mice. To our knowledge this has not been examined in humans. Finally, the question arises whether existing GABAergic drugs will be useful for the treatment of autoimmune diseases. Although the results are impressive in animals, there is only a very limited clinical literature on the immune effects of these drugs, and this is an important area for future investigation. A major limitation is that most GABAergic drugs were selected for their ability to cross the BBB, to treat neurological diseases, but this may not be desirable for other diseases. Native GABA can be administered orally and is well tolerated. It also has the advantage that it activates both types of GABA receptors, unlike most GABAergic drugs. Prospective clinical trials are required to examine the immunological effects of therapy with GABA or mimetic drugs in humans, but this represents a promising area for future drug development. Take-home message • Lymphocytes and macrophages produce GABA and possess the GABAergic machinery required to respond to this mediator. • GABA inhibits immune cells in vitro and in vivo, blocks NF-κB activation and reduces inflammatory cytokine production. • In preclinical models, GABA therapy protects against T1D, EAE, CIA and hypersensitivity dermatitis. • GABA treatment markedly improves antigen therapy of T1D with GAD65 or proinsulin. • GABA protects islet cells against apoptosis induced by cytokines, conventional immunosuppressive drugs and other stresses, and this may be relevant to islet transplantation. • Autoimmunity against GAD65 is a feature of T1D, but its pathogenic role is unclear. • Autoantibodies reactive to GAD65 and GABA receptors are a feature of SPS, encephalitis and autoimmune epilepsy. • GABAergic drugs and native GABA can be administered to humans, but their effects on the immune system requires further investigation.

Acknowledgments Our studies were supported by Juvenile Diabetes Research Foundation (JDRF), Canadian Institute for Health Research (CIHR), Canadian

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Diabetes Association (CDA) and National Science Foundation China (NSFC). These funding sources had no input in the study design; collection, analysis and interpretation of the data; writing the report; or the decision to publish the article.

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