Neurochemistry & Neuropharmacology Immunomodulation of Glatiramer Acetate in Multiple Sclerosis

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease in central nervous system (CNS) characterized by demyelination as well as axonal and neuronal degeneration. Glatiramer acetate is a mixture of synthetic polypeptides comprising four amino acids resembling the myelin basic protein (MBP), and approved as an immunoregulatory drug for the treatment of relapsing-remitting MS. The mechanism of action of GA in MS patients and the animal model experimental autoimmune encephalomyelitis (EAE) were extensively investigated over years. The cumulative findings indicate GA exerts its therapeutic activity by immunomodulating various levels of the immune response. This includes the blockade of major histocompatibility complex (MHC) molecules, T cell receptor antagonist, induction of GA-specific suppressor Th2 cells, an increase in frequency and function of CD4+CD25+FoxP3+ regulatory T cells, the down-regulation of Th1 and Th17 differentiation; the development of type II antigen presenting cells (APCs). In the review, we aim to provide a comprehensive overview of the immunoregulatory properties of GA in adaptive and innate immune response, in particular on the CD4+ effector T cells.


Immunopathogenesis of multiple sclerosis
The evidence from the animal model of MS, experimental autoimmune encephalomyelitis (EAE) and clinical data from MS patients support the notion that MS occurs as a consequence of the activation of autoreactive myelin-specific T helper (Th) cells. It is likely that exposure to an unknown microbial antigen, which contains protein sequences cross-reactive with self-myelin antigens, results in the activation of myelin-specific T cells. Activated T cells subsequently released pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and Interleukin-1 (IL-1) in the periphery. These activated cells undergo adhesion to the endothelia barrier, attraction, and active invasion into CNS. In the CNS, myelin specific T cells can be further activated by local and infiltrating antigen presenting cells. Reactivation of these cells results in increased secretion of pro-inflammatory cytokines and chemokines, which in turn recruit and activate macrophages and other inflammatory cells. Activated T cells may directly attack the oligodendrocytes and destroy the myelin. Activated macrophages/microglia can secrete toxic molecules (e.g., nitric oxide) to further enhance myelin destruction.

The Pathogenic Role of CD4 T Helper Cell Subsets in MS
CD4+ T helper (Th) cells are essential regulators of immune responses and inflammatory diseases. Based on their cytokine secretion and transcription factor expression, CD4 + Th cells can be divided into several subsets: Th1, Th2 and Th17. Th1 cells secrete IFN-γ and promote cell-mediated immunity against intracellular pathogens; Th2 cells secrete IL-4 and IL-10 and mediate humoral immunity and defend against extracellar pathogens and parasites; Th17 cells that producing IL-17 participate in the autoimmunity and tissue inflammation, protect the host against certain pathogens.
In the past years, Th1 cells are thought originally to be the main pathogenic T cells in MS and its animal model EAE, while Th2 cells are thought to be protective. Th1 cells secreting IFN-γ are closely associated with the clinical severity of EAE and could independently induce EAE when adoptively transferred into naïve mice [5]. Recently, accumulating evidence indicates that a new-identified Th subset: Th17 cells play an important role in the pathogenesis of MS and EAE. Th17 cells were found in the brain lesions of patients with MS; IL-17 expression was elevated in the serum and cerebrospinal fluid (CSF) of patients with MS [6]. IL-17 knockout mice show a significant, but not complete, reduction in severity of EAE. Administration of neutralizing anti-IL-17 antibody in vivo reduced the severity of EAE [7,8]. Further, adoptive transfer of Th17 cells directly induced severe EAE in mice. The evidence supported that Th17 and Th1 cells both attributes to the pathogenesis of MS and EAE. CD4+CD25+Foxp3+ regulatory T cells (Treg) are an important subclass of regulatory cells that maintain immune tolerance by suppressing self-reactive Th cells. Forkhead transcription factor Foxp3 is the key transcription factor in the physiological development of Treg [9]. There is evidence to suggest that the function of Treg cells in MS patients is impaired. Their inhibitory effect on antigen-specific

Chunhua Chen*
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA

Abstract
Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease in central nervous system (CNS) characterized by demyelination as well as axonal and neuronal degeneration. Glatiramer acetate is a mixture of synthetic polypeptides comprising four amino acids resembling the myelin basic protein (MBP), and approved as an immunoregulatory drug for the treatment of relapsing-remitting MS. The mechanism of action of GA in MS patients and the animal model experimental autoimmune encephalomyelitis (EAE) were extensively investigated over years. The cumulative findings indicate GA exerts its therapeutic activity by immunomodulating various levels of the immune response. This includes the blockade of major histocompatibility complex (MHC) molecules, T cell receptor antagonist, induction of GA-specific suppressor Th2 cells, an increase in frequency and function of CD4+CD25+FoxP3+ regulatory T cells, the down-regulation of Th1 and Th17 differentiation; the development of type II antigen presenting cells (APCs). In the review, we aim to provide a comprehensive overview of the immunoregulatory properties of GA in adaptive and innate immune response, in particular on the CD4+ effector T cells.
T cell proliferation was significantly reduced compared to healthy individuals [10].

Development of Glatiramer Acetate
Glatiramer acetate (also known as GA, Copaxone) is a synthetic random copolymer composed of the four amino acids L-alanine, L-lysine, L-glutamate and L-tyrosine in a defined molar ratio of 4.2:3.4:1.4:1.0 with a length of 40-100 residues. It was first synthesized to mimic the encephalitogenic properties of myelin basic protein (MBP), one of the major myelin autoantigen involved in the induction of EAE, the animal model of MS. Instead of inducing EAE disease, it has been shown to be effective in preventing and suppressing EAE by various encephalitogens in a variety of species [11,12]. GA has also been shown to have beneficial effects on the clinical course and MRIdefined brain lesions of patients with multiple sclerosis. In 1996, GA is approved as an immunoregulatory drug by the United States Food and Drug Administration (FDA) for the treatment of relapsing-remitting MS that slows the progression of disability and reduces relapse rate [13].

Mechanism of action of GA in MS
The mechanism by which GA induces its beneficial effect in EAE animals and MS patients was largely investigated for many years. These studies demonstrate that GA has unique immune regulatory properties on adaptive and innate immune system. We compile the current knowledge on the immunomodulatory mechanism of GA in the treatment of MS and EAE.

Competition with MBP for binding to MHC molecules
It was demonstrated that GA exhibits a high-affinity and promiscuous binding to a variety of major histocompatibility complex (MHC) class II molecules on antigen presenting cells (APC). GA does not undergo the processing before binding to MHC molecules. GA may compete with myelin-basic protein (MBP) for binding to MHC II molecules on the surface of APC cells, and even displaced the MBP antigen when already bound. This competition for binding to the MHC can consequently lead to the inhibition of myelin-reactive T cell response [14,15]. Aharoni et al. reported that GA was shown to inhibit the response to the immunodominant epitope of MBP peptide 82-100 by acting as a T cell receptor (TCR) antagonist. The immunodominant determinant 82-100 of MBP, a major target of T cells in brain lesions of MS patients. In contrast to the broad specificity of the MHC blocking induced by GA, its TCR antagonistic activity was restricted to the 82-100 determinant of MBP [16].

Induction of GA-specific suppressor Th2 cells
In untreated MS patients and healthy subjects, the majority of GA-reactive CD4+ T cells belong to the Th1 subset. Th1 cells characteristically produce a spectrum of pro-inflammatory cytokines such as IFN-γ, IL-2 and IL-12 [17]. During treatment with GA, the cytokine profile of the GA-reactive T cells shifts towards the Th2 type [18]. GA-reactive Th2 cells produce anti-inflammatory cytokines, including IL-4, IL-5, IL-6 and IL-13 [19,20]. These cells migrate into the CNS, and reactivate locally by cross-reactivity with myelin antigen. After local re-stimulation, GA-reactive Th2 cells secrete antiinflammatory cytokines and inhibit the action of encephalitogenic T cells, which dampen the local inflammation process. The process is termed "bystander supression" [21,22]. These GA-specific Th2 cells can produce neurotrophic factors BDNF, which might favor remyelination and axon protection [23][24][25].

Regulation of GA on CD4+ Th cells
Th1 and Th17 are pathogenic T cells in the development of MS and EAE. Kantengwa et al. reported that GA inhibited Th1 differentiation of CD4+ T cells at various T cell maturation stages and in an antigen-independent manner [26]. In vivo GA treatment biased differentiation of CD4+ T cells from the detrimental Th1 phenotype towards the anti-inflammatory Th2 phenotype [27]. Recently, our study has demonstrated GA inhibited Th17 differentiation through down-regulation of STAT3 phosphorylation and transcription factors RORγt and RORα expression in vivo. In vitro human and mouse Th17 differentiation system, GA inhibited the differentiation of Th17 in a dose-dependence manner. In Th1 differentiation system, GA also suppressed Th1 differentiation in vitro. Further, we investigated which T h subset (Th17 or Th1) was chiefly responsible for the treatment effect of GA. Our data indicated that the treatment effect of GA in EAE was mainly attributable to its regulatory property on Th17 differentiation [28].
CD4+CD25+Foxp3+ regulatory T cells have the beneficial effect in the development of MS by suppression of pathogenic T cells. Viglietta et al. reported that the effector function and the frequency of Treg is significantly decreased in the peripheral blood of patients with MS [29]. Several studies provided the evidence that GA have the beneficial effect on induction of CD4+CD25+ Treg cells. In vitro human and animal system, GA induced the conversion of peripheral CD4+CD25to CD4+CD25+ regulatory T cells through the activation of transcript factor Foxp3. GA treatment led to a significant increase in Foxp3 expression in CD4+ T cells in MS patients whose Foxp3 expression was reduced at baseline [30].

Regulation of GA on APC cells
In the past, it is considered that T cells are primary target of GA. Early studies focused on its influence on the adaptive immune system. APCs including monocytes and dendritic cells (DCs) play the central role in the initial and development of immune response. The interaction between APCs and T cells is fundamental for any adaptive T cell immune response. More recent studies indicate that GA may affect the properties of APCs. Vieira et al. reported that in vitro DCs exposure to GA have an impaired capacity to secrete Th1 polarizing factor IL-12p70, therefore preferentially induce Th2 cells and enhanced levels of the anti-inflammatory cytokine IL-10 [31,32]. Further, Kim et al. reported that mococytes from GA-treated patients produced significantly higher amounts of IL-10 and lower amounts of IL-12. GA therapy leads to the generation of type II monocyte, which contributes to Th2 deviation both in the periphery and CNS of MS patients [33]. Weber et al. also reported that lipopolysaccharide (LPS)induced activation marker CD150/SLAM expression and TNF-α production were significantly reduced in monocytes from GA-treated patients compared with controls [34]. These studies clearly indicated that GA treatment promotes Th2 cells differentiation by modifying the phenotype of APCs. Our study has demonstrated that GA inhibited Th17 differentiation by the reduction of IL-6 in treated monocytes. GA primarily interacts with monocytes and inhibits the production of IL-6, critically required for Th17 differentiation through STAT3 activity in T cells [28]. These findings provide the possibility that GA treatment may compromise innate immune responses in GA-treated MS patients.

Conclusion
Glatiramer acetate is a random polymer of four amino acids enriched in myelin basic protein, and approved as an immunomodulatory drug for the treatment of relapsing MS. It has unique immune regulatory properties on adaptive and innate immune system. GA regulates the immune response at different levels, including binding to MHC II molecules as MHC blocker and TCR antagonist; preferential Th2 deviation in CD4 T cells; the down-regulation of Th1 and Th17 differentiation; restoration of frequency and function of Treg cells; biasing dendritic cells and monocytes toward to anti-inflammatory phenotype. The comprehensive understanding of the mechanism of action of GA may provide potential therapy target and useful insight for the development of new efficient drugs in the future.