Transcriptional therapy with the histone deacetylase inhibitor trichostatin A ameliorates experimental autoimmune encephalomyelitis

Abstract

We demonstrate that the histone deacetylase (HDAC) inhibitor drug trichostatin A (TSA) reduces spinal cord inflammation, demyelination, neuronal and axonal loss and ameliorates disability in the relapsing phase of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). TSA up-regulates antioxidant, anti-excitotoxicity and pro-neuronal growth and differentiation mRNAs. TSA also inhibits caspase activation and down-regulates gene targets of the pro-apoptotic E2F transcription factor pathway. In splenocytes, TSA reduces chemotactic, pro-Th1 and pro-proliferative mRNAs. A transcriptional imbalance in MS may contribute to immune dysregulation and neurodegeneration, and we identify HDAC inhibition as a transcriptional intervention to ameliorate this imbalance.

Introduction

Multiple sclerosis (MS) is a Th1 cytokine-driven inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS) (Peterson et al., 2001). Neuronal or axonal injury may account for disability in MS and can occur in the absence of overt inflammation (Trapp et al., 1999). Mechanisms that have been implicated in MS pathogenesis include oxidative stress, excitotoxicity, autoimmunity and hormonal imbalance. Therapies that target these distinct pathophysiologic events have been shown to be effective in experimental autoimmune encephalomyelitis (EAE), a Th1 cytokine-driven model of MS. Based on these findings, some are being tested in humans (Youssef et al., 2002). However, drugs with preferential effects on the chronic relapsing (i.e., post-inflammation peak) phase of EAE, in which axonal loss correlates with disability (Wujek et al., 2002), have yet to be identified (Steinman, 1999).

Histone deacetylase (HDAC) enzymes repress genes via condensation of the nucleosome and interactions with transcription factors. For instance, HDACs bind repressor element 1 silencing transcription factor (REST), which is known to counteract neuronal differentiation traits (Ballas et al., 2001), and Sp1, another transcription factor that mediates neuronal antioxidant pathways (Ryu et al., 2003a, Ryu et al., 2003b). Compounds that inhibit HDAC enzymes range from nonspecific agents such as the short-chain fatty acid sodium phenylbutyrate (SPB), to highly specific hydroxamic acids such as trichostatin A (TSA). These structurally diverse agents induce histone hyperacetylation with concomitant up-regulation of neuronal maturation and antioxidant gene expression, indicating REST (Ballas et al., 2001, Hakimi et al., 2002) and Sp1 (Ryu et al., 2003a, Ryu et al., 2003b) transcription factor derepression.

E2F1 is a transcription factor that promotes immune cell proliferation (Wu et al., 2001). E2F1 activity is triggered by activation of T cells via the IL-2 receptor (IL-2R), and thus holds special relevance for MS and EAE, diseases driven by activated Th1 T cells. In fact, we have shown that peripheral blood mononuclear cells (PBMCs) from MS patients exhibit an activated E2F pathway signature (Iglesias et al., 2004), suggesting E2F as a potential therapeutic target in this disease. Accordingly, E2f1-deficient mice have less severe EAE and their splenocytes have decreased γ-IFN and elevated IL-4 responses to antigenic stimulation in vitro (Iglesias et al., 2004), suggesting a contribution by E2F1 to Th1 differentiation. E2F1 overexpression, in addition, contributes to neuronal apoptosis (Hou et al., 2000), further supporting our rationale for targeting the E2F pathway in MS.

We have previously shown that HDAC mRNAs are elevated in activated immune cells (Dangond et al., 1998), suggesting that they may serve as markers of activation and potential targets of treatment. We have also shown that TSA blocks immune cell proliferation and suppresses the pro-Th1 factor γ-IFN (Dangond and Gullans, 1998), in accordance with a growing literature suggesting that HDAC inhibitors cause a shift to a Th2 phenotype (Saemann et al., 2000). For instance, HDAC inhibitors down-regulate the CD28 costimulatory molecule (Moreira et al., 2003), the IL-2 receptor (Moreira et al., 2003) and IL-12 (p35 and p40) (Saemann et al., 2000) and up-regulate IFN-α and -β (Shuttleworth et al., 1983). Since MS is associated with transcriptional dysregulation (Iglesias et al., 2004), we tested whether the transcriptional drug TSA is clinically beneficial for EAE and whether it has neuroimmunoprotective effects.