# PPAR-γ: Therapeutic Potential for Multiple Sclerosis

Authors: Paul D. Drew; Jihong Xu; Michael K. Racke
Journal: PPAR Research
 (2008)

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## Abstract

The role of peroxisome proliferator-activated receptors (PPARs) in altering lipid and glucose metabolism is well established. More recent studies indicate that PPARs also play critical roles in controlling immune responses. We and others have previously demonstrated that PPAR-γ agonists modulate the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). This review will discuss the cellular and molecular mechanisms by which these agonists are believed to modulate disease. The therapeutic potential of PPAR-γ agonists in the treatment of multiple sclerosis will also be considered.


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## Body

## 1. Introduction

Multiple sclerosis (MS) is the second
most common neurologic disorder of young adults, behind neurotrauma.
Approximately 350000–400000
individuals have physician diagnosed MS in the United States alone. MS is
commonly diagnosed around the third decade of life and many patients suffer the
devastating effects of the disease for much of their adult lives. The etiology
of MS is not completely understood but is believed to result from a combination
of genetic and environmental factors. The disease is characterized by
inflammation of the central nervous system (CNS), demyelination, and either
relapsing-remitting or progressive clinical presentations. Similarities to
experimental autoimmune encephalomyelitis (EAE), an established animal model of
MS which is elicited following generation and attack of autoreactive T cells
against brain tissues suggests an autoimmune origin for MS. In addition to
autoreactive T cells, other peripheral immune cells including B cells,
monocytes, and dendritic cells may play a role in the pathogenesis associated
with MS. In addition, resident CNS cells including chronically activated glial
cells are believed to play a role in disease pathogenesis [1].Nuclear receptors are a family of
transcription factors that regulate gene expression in response to ligand
binding. Nuclear receptor superfamily members include peroxisome
proliferator-activated receptors (PPARs) as well as androgen, estrogens,
progesterone, thyroid, and glucocorticoid receptors. Additional orphan nuclear
receptors exist for which ligands have not been identified.  The critical role of PPARs in modulating glucose
and lipid metabolism has been extensively documented  [2]. More recently, a role for PPARs in
altering immune responses has been established. A role for PPARs in modulation
of immune responses was suggested by the observation that indomethacin, a
nonsteroidal anti-inflammatory drug (NSAID) binds PPAR-γ. Furthermore, it was
documented that PPAR-γ is expressed by cells of the monocyte/macrophage
lineage. These observations led to seminal studies demonstrating that PPAR-γ agonists suppress the activation of monocyte/macrophages
[3, 4]. Three PPAR isoforms,
PPAR-α, -β/δ, and -γ, have been identified. These receptors exhibit distinct tissue
expression patterns and ligand specificities [2, 5]. Eicosanoids,
polyunsaturated fatty acids, and the cyclopentenone prostaglandin 15d-PGJ2 are naturally occurring PPAR-γ ligands. Synthetic PPAR-γ ligands include
thiazolidinediones which are used for the treatment of type II diabetes.As transcription factors, PPARs
primarily function to regulate the expression of specific genes. Similar to
other nuclear receptors, PPARs bind DNA and regulate gene expression as dimers.
PPARs form heterodimers with retinoid-X-receptors (RXRs), and bind DNA at
conservedperoxisome-proliferator
response elements (PPREs) present in the promoter of PPAR-responsive target
genes. Upon ligand binding, the PPAR/RXR heterodimer associates with
coactivator complexes, binds PPREs, and activates the transcription of
PPAR-responsive genes. In contrast, PPAR/RXR heterodimers not bound by ligand associate
with corepressor complexes resulting in suppression of gene transcription [2]. PPAR ligands principally activate transcription of genes encoding
proteins important in lipid and glucose metabolism by triggering PPAR/RXR
binding to PPREs present in the promoters of these genes. In contrast, PPAR
agonists generally suppress the expression of genes encoding proinflammatory
molecules through a mechanism not involving PPAR/RXR binding to
PPREs. This mechanism, termed receptor-dependent transrepression, is believed
to occur through physical interaction between PPAR/RXR and other transcription
factors which normally activate transcription of proinflammatory genes.
Physical interaction with PPAR/RXR inhibits binding of these transcription
factors to response elements present on genes encoding proinflammatory
molecules, thus suppressing the activation of these genes. Receptor-dependent
transrepression may also result from PPAR/RXR interaction with transcriptional
coactivator or corepressor molecules that are in limited supply, or PPAR/RXR interactions
with the basal transcription machinery [6, 7]. PPAR-γ agonists inhibit
transcription factors including NF-κB, AP-1, and STAT-1 from activating gene
expression through receptor-dependent transrepression [8]. The mechanisms resulting in
receptor-dependent transrepression have remained a mystery. However, recent
pioneering work by Glass et al. has begun to elucidate the molecular mechanisms
that control receptor-dependent transrepression of NF-κB responsive genes.
These studies demonstrate that in the presence of PPAR-γ ligands, PPAR-γ can
conjugate with small ubiquitin-like modifier-1 (SUMO1) resulting in the sumoylation
of PPAR-γ. Sumoylated PPAR-γ binds the corepressor molecule NCoR which maintains
the promoters of responsive genes in a repressed state, even in the presence of
NF-κB activating stimuli. The mechanisms by which NF-κB responsive genes are
believed to remain in a repressed state following sumoylation of PPAR-γ and
consequent association with NCoR are believed to involve inhibition of the recruitment of
ubiquitin conjugating enzymes to the corepressor complex following physical
association of sumoylated PPAR-γ with NCoR [9–11]. Interestingly, recent studies have
demonstrated that in addition to PPAR-γ, liver X receptor (LXR) mediated
transrepression involves sumoylation of receptor and association of NCoR [12]. This suggests the possibility of a
general mechanism of transrepression by PPARs and LXRs.As stated above, PPAR-γ agonists can
regulate gene expression in a receptor-dependent manner through receptor
binding to PPREs or through receptor-dependent transrepression. In addition,
PPAR-γ agonists including 15d-PGJ2 can regulate gene expression through
receptor-independent mechanisms. For example, 15d-PGJ2 blocks I-κB degradation
by inhibiting the activation of I-κB kinase resulting in the retention of NF-κB
in the cytoplasm [13, 14]. In addition, 15d-PGJ2 has been demonstrated to inhibit NF-κB binding to NF-κB DNA-response elements [15]. Thus, in summary, PPAR-γ agonists
can regulate gene expression through both receptor-dependent and
receptor-independent mechanisms.


## 2. Effects of PPAR-γ on Immune Cell Function

### 2.1. CNS Resident Cells

Microglia are bone marrow-derived cells
that migrate to the CNS during embryonic development. Normally, these cells
exist in a quiescent state in the CNS. Likewise, astrocytes, resident CNS cells
that protect neurons through production of neurotrophic factors as well as uptake
of glutamate and other neurotoxic molecules, commonly are quiescent in the CNS.
However, these glial cells may become activated in response to insults including
stress, trauma, and pathogens, and under these conditions may initiate protective
immune responses. Upon activation, microglia and astrocytes produce proinflammatory
molecules including nitric oxide (NO), cytokines, and chemokines. These proinflammatory molecules play critical
roles in removing pathogens and debris from the infected or injured CNS. In
contrast, chronically activated glia are believed to contribute to CNS damage
characteristic of neuroinflammatory and neurodegenerative disorders including
MS.Van Eldik et al. were the first to evaluate the effects of PPAR-γ agonists on immune
function in glial cells. They demonstrated that the PPAR-γ agonist 15d-PGJ2 inhibited LPS induction of NO and iNOS expression in the murine BV-2 microglial
cell line. However, troglitazone which is a PPAR-γ agonist and thiazolidinedione
did not suppress LPS induction of these molecules. These results were
interpreted to indicate that 15d-PGJ2 functioned through a
receptor-independent mechanism. Using the same BV-2 microglial cell system, the
Van Eldik laboratory also demonstrated that 15d-PGJ2 suppressed LPS
induction of TNF-α, IL-1β, and COX-2 [16]. However, 15d-PGJ2 increased
intracellular glutathione levels as well as expression of heme oxygenase-1, an
enzyme known to stimulate antioxidant production [17]. Minghetti et al. were the first to
investigate the effects of PPAR-γ agonists on immune function in primary
microglia. These studies indicated that 15d-PGJ2 as well as the thiazolidinedione
ciglitazone suppressed LPS induction of iNOS and TNF-α expression by primary rat microglia. These
PPAR-γ agonists also suppressed IFN-γ induction of major histocompatibility
class II in these cells. Because 15d-PGJ2 and ciglitazone effects on
microglial immune cell function were similar in these studies, it was
interpreted that 15d-PGJ2 functioned through a receptor-dependent mechanism
[18].More
recently, we compared the effects of a series of thiazolidinediones or 15d-PGJ2 on the production of proinflammatory molecules by primary microglia and
astrocytes. These studies demonstrated that both thiazolidinediones and 15d-PGJ2 inhibited the production of NO, the proinflammatory cytokines TNF-α, IL-1β, and
IL-6, and the chemokine MCP-1 by these glial cells [19]. However, even though 15d-PGJ2 binds PPAR-γ with less affinity than each of the thiazolidinediones, this cyclopentenone
prostaglandin more strongly inhibited production of these proinflammatory
molecules by the glial cells, suggesting that 15d-PGJ2 acts at least
in part through receptor-independent mechanisms. A receptor-dependent effect of
15d-PGJ2 in regulating glial cell immune function is supported by
studies demonstrating that microglial cell activation is suppressed in a
cooperative manner by this PPAR-γ ligand in combination with 9-cis retinoic
acid, the ligand for the retinoic acid receptor RXR [20]. This supports the hypothesis that glial cell activation is maximally
suppressed in the presence of ligands following formation of PPAR-γ/RXR heterodimers. Our observation that
monocyte/microglia and astrocytes express increased levels of PPAR-γ during
active EAE suggests that this receptor may modulate disease, perhaps through
effects on glial cell activation [21]. Luna-Medina et al. [22] demonstrated that thiazolidinediones
inhibited LPS induction of proinflammatory molecules by microglia and
astrocytes. In addition, thiazolidinedione treatment of glial cultures
suppressed the production of neurotoxic molecules. The effects of
thiazolidinediones on glia in these studies were abrogated by PPAR-γ antagonists
suggesting a receptor-dependent mechanism [22]. Minghetti et al. demonstrated that two
flurbiprofen derivatives demonstrated to release NO suppressed glial cell
activation through activation of PPAR-γ [23, 24]. Interestingly, one of these compounds,
NXC 2216, initially activated the receptor, but later stimulated nitration and
inactivation of PPAR-γ [24]. This suggested that these flurbiprofen
derivatives could differentially activate or suppress glial immune cell
function depending on length of treatment. Interestingly, PGA2 potently suppressed microglia and astrocyte production of proinflammatory
molecules [25]. Structurally, PGA2 is a
cyclopentenone prostaglandin like 15d-PGJ2. However, PGA2 is not
believed to bind PPAR-γ, suggesting that the cyclopentenone ring structure
itself may modulate glial cell activation.Astrocytes,
like microglia, react to pathogens through a series of pattern recognition
receptors which stimulate toll-like receptor signaling [26, 27]. Kielian et al. demonstrated that both
15d-PGJ2 and the thiazolidinedione ciglitazone suppressed
Staphylococcus aureus induction of NO and IL-1β by primary astrocytes. Suppression
of these proinflammatory molecules by 15d-PGJ2 and ciglitazone
occurred in both wild-type and PPAR-γ deficient astrocytes, suggesting that
these compounds mediated their effects in a receptor-independent manner [28].PPAR-γ agonists have been demonstrated to modulate a variety of signaling pathways.
Singh et al. showed that 15d-PGJ2 inhibited LPS induction of NO and
proinflammatory molecules in primary astrocytes. Transfection of wild-type
PPAR-γ, dominant-negative PPAR-γ, or treatment of cells with a PPAR-γ
antagonist did not alter 15d-PGJ2 effects on astrocytes in these
studies suggesting that the agonist functioned through a receptor-independent
mechanism. The PPAR-γ agonist decreased NF-κB activity in these studies, presumably
by inhibiting I-κB kinase. Singh et al. also demonstrated that 15d-PGJ2 inhibited the phosphatidylinositol 3-kinase-Akt signaling pathway, suggesting
an additional mechanism by which the agonist inhibited production of
proinflammatory molecules in astrocytes [29]. Additional
studies indicated that both the thiazolidinedione ciglitazone and 15d-PGJ2 stimulated MAP kinase pathways in astrocytes through receptor-independent
mechanisms involving production of reactive oxygen species [30]. The PPAR-γ agonists rosiglitazone and 15d-PGJ2 also were demonstrated to modulate the
JAK/STAT pathway by inhibiting the phosphorylation of specific JAK and STAT
molecules following induced expression of suppressor of cytokine signaling
(SOCS) 1 and 3 proteins in astrocytes and microglia [31].Collectively,
the studies discussed above suggest that PPAR-γ agonists may regulate immune function
in glia through receptor-dependent or alternatively through
receptor-independent mechanisms. Factors that may determine if
receptor-dependent or receptor-independent mechanisms are employed may include
the specific PPAR-γ agonist studied, the concentration of the agonist used, and
the cell type studied. For example,
responses are likely to differ between primary and transformed cells and may
vary depending on the developmental state of the tissue from which the glial
cells are derived.The
function and phenotype of T cells can be dramatically altered by glia. For
example, the IL-12 family of cytokines which includes IL-12, IL-23, and IL-27 is
believed to alter T cell phenotype and modulate the development of EAE and MS [32]. Specifically, the IL-12 family of
cytokines modulates the differentiation of Th1 cells which are
believed to contribute to the development of EAE. In addition, IL-23
contributes to the production of Th17 cells which have recently
been demonstrated to play a critical role in autoimmunity. IL-12 family members
are heterodimeric. IL-12 consists of a dimer of p40 and p35 subunits. IL-23
consists of the same p40 subunit in association with p19. IL-27 exists as a dimer
of p28 in association with EBV-induced molecule 3 (EBI3). Previously, we
demonstrated that the PPAR-γ agonist 15d-PGJ2 potently inhibited LPS
induction of IL-12 p40 secretion by N9 mouse microglial cells and primary rat
microglia [33]. More recently, we demonstrated that 15d-PGJ2 and the thiazolidinedione rosiglitazone inhibited LPS induction of IL-12 p40,
IL-12 p70, IL-23, and IL-27 p28 in primary microglia. In addition, 15d-PGJ2 inhibited IL-12 p40, IL-23, and IL-27 p28, while rosiglitazone inhibited IL-23,
and IL-27 p28, but not IL-12 p40 in primary astrocytes. LPS did not stimulate
the production of IL-12 p70 in astrocytes [34]. These studies suggest that PPAR-γ
agonists may modulate the development of EAE in part by modulating IL-12 family
cytokine production by glia, which may alter T cell phenotype. Costimulatory
molecules may be expressed by antigen presenting cells (APCs) including CNS microglia. Interaction of costimulatory molecules including
CD40, CD80, and CD86 on APCs with their cognate receptors present of CD4+ T cells is important in the activation and differentiation of these T cells,
which likewise modulate the development of EAE and possibly MS. Our previous
studies indicated that 15d-PGJ2 inhibited microglial expression of CD40,
but had no effect on the expression of CD80 and CD86 costimulatory molecules.
Therefore, through modulation of costimulatory molecule expression by microglia,
PPAR-γ agonists may  alter the
pathogenesis of EAE [21, 35].PPAR-γ
agonists can alter the viability of neurons and oligodendrocytes, which are CNS
cells compromised in MS. These agonists may alter the viability of these cells
directly or indirectly by suppressing the production of cytotoxic molecules by
activated microglia and astrocytes. Combs et al. [36] demonstrated that treatment of glial
cultures with a variety of PPAR-γ agonists suppressed β-amyloid mediated
toxicity of cortical neurons. Similarly, PPAR-γ agonists including
thiazolidinediones and 15d-PGJ2 inhibited LPS induction of neuronal cell
death in studies utilizing a rat cortical neuron-glial coculture paradigm [37]. Furthermore, more recent studies
indicated that thiazolidinediones treatment of cortical neuron-mixed glia
cocultures resulted in protection of neurons. Neuron protection was abrogated
in these studies by a PPAR-γ antagonist suggesting that neuron protection
occurred by a receptor-dependent mechanism [22]. In addition to protecting neurons
through suppression of glial activation, PPAR-γ agonists can also directly
protect neurons. For example, PPAR-γ agonists have been demonstrated to protect
neurons from a variety of neurotoxic agents including NMDA [38] and apolipoprotein E4 [39]. Interestingly, neurons express PPAR-γ
and several studies suggest that neuron cell viability may be mediated through PPAR-γ
activation [40, 41]. However, the mechanisms by which PPAR-γ
regulates neuron cell viability have not been fully elucidated. Recent studies
suggest that one mechanism by which PPAR-γ agonists may modulate neuron cell
viability is through modulation of the antiapoptotic factor Bcl-2 [42]. Interestingly, PPAR-γ also regulates
neural stem cell proliferation and differentiation [43]. Less is known concerning the role of
PPAR-γ in modulating oligodendrocyte cell viability and differentiation. However,
studies suggest that PPAR-γ protects oligodendrocyte progenitors [44] and modulates oligodendrocyte differentiation
[45].


### 2.2. Peripheral Immune Cells

As mentioned previously, autoreactive
T cells are believed to contribute to MS pathogenesis. Clark et al. initially
demonstrated that T cells express PPAR-γ and that 15d-PGJ2 and ciglitazone
inhibited T cell secretion of IL-2 and altered T cell proliferation [46]. PPAR-γ agonists have been
demonstrated to induce apoptosis of T cells [47]. However, others studies indicate
that PPAR-γ agonists promote the survival of T cells [48]. The exact reason for the
discrepancy between these studies is not clear, but may involve differences in
the concentration of PPAR-γ agonists used in the studies. Regulatory T cells
play a critical role in suppressing the development of autoimmune diseases.
Interestingly, recent studies indicate that PPAR-γ agonists enhance the
generation and function of regulatory T cells [49, 50]. It is now clear that B cells have a
significant role in modulating EAE and MS. Phipps et al. demonstrated that B
cells express PPAR-γ and that PPAR-γ agonists stimulate the apoptosis of these
cells [51, 52]. Collectively, these studies suggest
that PPAR-γ may modulate MS in part by altering the
viability and function of lymphocytes.As stated previously, macrophages
express PPAR-γ and PPAR-γ agonists regulate the function of these cells [53]. Like macrophages, dendritic cells
are also of monocytic origin and function as professional antigen presenting
cells. Dendritic cells also play a significant role in MS [54, 55]. Interestingly, PPAR-γ agonists have
been shown to alter the viability and function of dendritic cells [56]. For example, cyclopentenone
prostaglandins induced the apoptosis of dendritic cells, although apoptosis
occurred through a receptor-independent mechanism [57]. PPAR-γ agonists were also shown to
inhibit the migration of dendritic cells [58]. Furthermore, PPAR-γ agonists
inhibited toll-like receptor mediated activation of dendritic cells by
suppressing MAP kinase and NF-κB signaling pathways [59]. PPAR-γ effects on dendritic cell
function have also been demonstrated to contribute to the development of CD4+ T cell anergy [60]. Collectively, these studies
indicate that PPAR-γ agonists may modulate MS in part through effects on
monocytic cells.Activated peripheral immune cells
including antigen specific T cells and macrophages are capable of entering the
CNS and contributing to MS pathology. Extravasation of peripheral immune cells
is mediated by a variety of factors including chemokines and adhesion molecule
expression on the cerebral vascular endothelium. Chemokines are synthesized
under inflammatory conditions and generate a concentration gradient to which
cells with the appropriate chemokine receptors migrate. The expression of
specific chemokines is increased in EAE and MS [61, 62]. PPAR-γ agonists decrease the expression of MCP-1
which is a chemoattractant for monocytes and microglia [63, 64] as well as the T cell
chemoattractants IP-10 (CXCL3), Mig (CXCL3), and I-TAC (CXCL3) [65]. Adhesion molecules present on the
cerebral vascular endothelium facilitate extravasation of peripheral immune
cells into the CNS. PPAR-γ agonists modulate the expression of various specific
adhesion molecules suggesting an additional mechanism controlling immune cell
extravasation into the CNS [66–68]. Future studies will be important in
determining more detailed mechanisms by which PPAR-γ agonists modulate immune
cell movement into the CNS.


## 2.1. CNS Resident Cells

Microglia are bone marrow-derived cells
that migrate to the CNS during embryonic development. Normally, these cells
exist in a quiescent state in the CNS. Likewise, astrocytes, resident CNS cells
that protect neurons through production of neurotrophic factors as well as uptake
of glutamate and other neurotoxic molecules, commonly are quiescent in the CNS.
However, these glial cells may become activated in response to insults including
stress, trauma, and pathogens, and under these conditions may initiate protective
immune responses. Upon activation, microglia and astrocytes produce proinflammatory
molecules including nitric oxide (NO), cytokines, and chemokines. These proinflammatory molecules play critical
roles in removing pathogens and debris from the infected or injured CNS. In
contrast, chronically activated glia are believed to contribute to CNS damage
characteristic of neuroinflammatory and neurodegenerative disorders including
MS.Van Eldik et al. were the first to evaluate the effects of PPAR-γ agonists on immune
function in glial cells. They demonstrated that the PPAR-γ agonist 15d-PGJ2 inhibited LPS induction of NO and iNOS expression in the murine BV-2 microglial
cell line. However, troglitazone which is a PPAR-γ agonist and thiazolidinedione
did not suppress LPS induction of these molecules. These results were
interpreted to indicate that 15d-PGJ2 functioned through a
receptor-independent mechanism. Using the same BV-2 microglial cell system, the
Van Eldik laboratory also demonstrated that 15d-PGJ2 suppressed LPS
induction of TNF-α, IL-1β, and COX-2 [16]. However, 15d-PGJ2 increased
intracellular glutathione levels as well as expression of heme oxygenase-1, an
enzyme known to stimulate antioxidant production [17]. Minghetti et al. were the first to
investigate the effects of PPAR-γ agonists on immune function in primary
microglia. These studies indicated that 15d-PGJ2 as well as the thiazolidinedione
ciglitazone suppressed LPS induction of iNOS and TNF-α expression by primary rat microglia. These
PPAR-γ agonists also suppressed IFN-γ induction of major histocompatibility
class II in these cells. Because 15d-PGJ2 and ciglitazone effects on
microglial immune cell function were similar in these studies, it was
interpreted that 15d-PGJ2 functioned through a receptor-dependent mechanism
[18].More
recently, we compared the effects of a series of thiazolidinediones or 15d-PGJ2 on the production of proinflammatory molecules by primary microglia and
astrocytes. These studies demonstrated that both thiazolidinediones and 15d-PGJ2 inhibited the production of NO, the proinflammatory cytokines TNF-α, IL-1β, and
IL-6, and the chemokine MCP-1 by these glial cells [19]. However, even though 15d-PGJ2 binds PPAR-γ with less affinity than each of the thiazolidinediones, this cyclopentenone
prostaglandin more strongly inhibited production of these proinflammatory
molecules by the glial cells, suggesting that 15d-PGJ2 acts at least
in part through receptor-independent mechanisms. A receptor-dependent effect of
15d-PGJ2 in regulating glial cell immune function is supported by
studies demonstrating that microglial cell activation is suppressed in a
cooperative manner by this PPAR-γ ligand in combination with 9-cis retinoic
acid, the ligand for the retinoic acid receptor RXR [20]. This supports the hypothesis that glial cell activation is maximally
suppressed in the presence of ligands following formation of PPAR-γ/RXR heterodimers. Our observation that
monocyte/microglia and astrocytes express increased levels of PPAR-γ during
active EAE suggests that this receptor may modulate disease, perhaps through
effects on glial cell activation [21]. Luna-Medina et al. [22] demonstrated that thiazolidinediones
inhibited LPS induction of proinflammatory molecules by microglia and
astrocytes. In addition, thiazolidinedione treatment of glial cultures
suppressed the production of neurotoxic molecules. The effects of
thiazolidinediones on glia in these studies were abrogated by PPAR-γ antagonists
suggesting a receptor-dependent mechanism [22]. Minghetti et al. demonstrated that two
flurbiprofen derivatives demonstrated to release NO suppressed glial cell
activation through activation of PPAR-γ [23, 24]. Interestingly, one of these compounds,
NXC 2216, initially activated the receptor, but later stimulated nitration and
inactivation of PPAR-γ [24]. This suggested that these flurbiprofen
derivatives could differentially activate or suppress glial immune cell
function depending on length of treatment. Interestingly, PGA2 potently suppressed microglia and astrocyte production of proinflammatory
molecules [25]. Structurally, PGA2 is a
cyclopentenone prostaglandin like 15d-PGJ2. However, PGA2 is not
believed to bind PPAR-γ, suggesting that the cyclopentenone ring structure
itself may modulate glial cell activation.Astrocytes,
like microglia, react to pathogens through a series of pattern recognition
receptors which stimulate toll-like receptor signaling [26, 27]. Kielian et al. demonstrated that both
15d-PGJ2 and the thiazolidinedione ciglitazone suppressed
Staphylococcus aureus induction of NO and IL-1β by primary astrocytes. Suppression
of these proinflammatory molecules by 15d-PGJ2 and ciglitazone
occurred in both wild-type and PPAR-γ deficient astrocytes, suggesting that
these compounds mediated their effects in a receptor-independent manner [28].PPAR-γ agonists have been demonstrated to modulate a variety of signaling pathways.
Singh et al. showed that 15d-PGJ2 inhibited LPS induction of NO and
proinflammatory molecules in primary astrocytes. Transfection of wild-type
PPAR-γ, dominant-negative PPAR-γ, or treatment of cells with a PPAR-γ
antagonist did not alter 15d-PGJ2 effects on astrocytes in these
studies suggesting that the agonist functioned through a receptor-independent
mechanism. The PPAR-γ agonist decreased NF-κB activity in these studies, presumably
by inhibiting I-κB kinase. Singh et al. also demonstrated that 15d-PGJ2 inhibited the phosphatidylinositol 3-kinase-Akt signaling pathway, suggesting
an additional mechanism by which the agonist inhibited production of
proinflammatory molecules in astrocytes [29]. Additional
studies indicated that both the thiazolidinedione ciglitazone and 15d-PGJ2 stimulated MAP kinase pathways in astrocytes through receptor-independent
mechanisms involving production of reactive oxygen species [30]. The PPAR-γ agonists rosiglitazone and 15d-PGJ2 also were demonstrated to modulate the
JAK/STAT pathway by inhibiting the phosphorylation of specific JAK and STAT
molecules following induced expression of suppressor of cytokine signaling
(SOCS) 1 and 3 proteins in astrocytes and microglia [31].Collectively,
the studies discussed above suggest that PPAR-γ agonists may regulate immune function
in glia through receptor-dependent or alternatively through
receptor-independent mechanisms. Factors that may determine if
receptor-dependent or receptor-independent mechanisms are employed may include
the specific PPAR-γ agonist studied, the concentration of the agonist used, and
the cell type studied. For example,
responses are likely to differ between primary and transformed cells and may
vary depending on the developmental state of the tissue from which the glial
cells are derived.The
function and phenotype of T cells can be dramatically altered by glia. For
example, the IL-12 family of cytokines which includes IL-12, IL-23, and IL-27 is
believed to alter T cell phenotype and modulate the development of EAE and MS [32]. Specifically, the IL-12 family of
cytokines modulates the differentiation of Th1 cells which are
believed to contribute to the development of EAE. In addition, IL-23
contributes to the production of Th17 cells which have recently
been demonstrated to play a critical role in autoimmunity. IL-12 family members
are heterodimeric. IL-12 consists of a dimer of p40 and p35 subunits. IL-23
consists of the same p40 subunit in association with p19. IL-27 exists as a dimer
of p28 in association with EBV-induced molecule 3 (EBI3). Previously, we
demonstrated that the PPAR-γ agonist 15d-PGJ2 potently inhibited LPS
induction of IL-12 p40 secretion by N9 mouse microglial cells and primary rat
microglia [33]. More recently, we demonstrated that 15d-PGJ2 and the thiazolidinedione rosiglitazone inhibited LPS induction of IL-12 p40,
IL-12 p70, IL-23, and IL-27 p28 in primary microglia. In addition, 15d-PGJ2 inhibited IL-12 p40, IL-23, and IL-27 p28, while rosiglitazone inhibited IL-23,
and IL-27 p28, but not IL-12 p40 in primary astrocytes. LPS did not stimulate
the production of IL-12 p70 in astrocytes [34]. These studies suggest that PPAR-γ
agonists may modulate the development of EAE in part by modulating IL-12 family
cytokine production by glia, which may alter T cell phenotype. Costimulatory
molecules may be expressed by antigen presenting cells (APCs) including CNS microglia. Interaction of costimulatory molecules including
CD40, CD80, and CD86 on APCs with their cognate receptors present of CD4+ T cells is important in the activation and differentiation of these T cells,
which likewise modulate the development of EAE and possibly MS. Our previous
studies indicated that 15d-PGJ2 inhibited microglial expression of CD40,
but had no effect on the expression of CD80 and CD86 costimulatory molecules.
Therefore, through modulation of costimulatory molecule expression by microglia,
PPAR-γ agonists may  alter the
pathogenesis of EAE [21, 35].PPAR-γ
agonists can alter the viability of neurons and oligodendrocytes, which are CNS
cells compromised in MS. These agonists may alter the viability of these cells
directly or indirectly by suppressing the production of cytotoxic molecules by
activated microglia and astrocytes. Combs et al. [36] demonstrated that treatment of glial
cultures with a variety of PPAR-γ agonists suppressed β-amyloid mediated
toxicity of cortical neurons. Similarly, PPAR-γ agonists including
thiazolidinediones and 15d-PGJ2 inhibited LPS induction of neuronal cell
death in studies utilizing a rat cortical neuron-glial coculture paradigm [37]. Furthermore, more recent studies
indicated that thiazolidinediones treatment of cortical neuron-mixed glia
cocultures resulted in protection of neurons. Neuron protection was abrogated
in these studies by a PPAR-γ antagonist suggesting that neuron protection
occurred by a receptor-dependent mechanism [22]. In addition to protecting neurons
through suppression of glial activation, PPAR-γ agonists can also directly
protect neurons. For example, PPAR-γ agonists have been demonstrated to protect
neurons from a variety of neurotoxic agents including NMDA [38] and apolipoprotein E4 [39]. Interestingly, neurons express PPAR-γ
and several studies suggest that neuron cell viability may be mediated through PPAR-γ
activation [40, 41]. However, the mechanisms by which PPAR-γ
regulates neuron cell viability have not been fully elucidated. Recent studies
suggest that one mechanism by which PPAR-γ agonists may modulate neuron cell
viability is through modulation of the antiapoptotic factor Bcl-2 [42]. Interestingly, PPAR-γ also regulates
neural stem cell proliferation and differentiation [43]. Less is known concerning the role of
PPAR-γ in modulating oligodendrocyte cell viability and differentiation. However,
studies suggest that PPAR-γ protects oligodendrocyte progenitors [44] and modulates oligodendrocyte differentiation
[45].


## 2.2. Peripheral Immune Cells

As mentioned previously, autoreactive
T cells are believed to contribute to MS pathogenesis. Clark et al. initially
demonstrated that T cells express PPAR-γ and that 15d-PGJ2 and ciglitazone
inhibited T cell secretion of IL-2 and altered T cell proliferation [46]. PPAR-γ agonists have been
demonstrated to induce apoptosis of T cells [47]. However, others studies indicate
that PPAR-γ agonists promote the survival of T cells [48]. The exact reason for the
discrepancy between these studies is not clear, but may involve differences in
the concentration of PPAR-γ agonists used in the studies. Regulatory T cells
play a critical role in suppressing the development of autoimmune diseases.
Interestingly, recent studies indicate that PPAR-γ agonists enhance the
generation and function of regulatory T cells [49, 50]. It is now clear that B cells have a
significant role in modulating EAE and MS. Phipps et al. demonstrated that B
cells express PPAR-γ and that PPAR-γ agonists stimulate the apoptosis of these
cells [51, 52]. Collectively, these studies suggest
that PPAR-γ may modulate MS in part by altering the
viability and function of lymphocytes.As stated previously, macrophages
express PPAR-γ and PPAR-γ agonists regulate the function of these cells [53]. Like macrophages, dendritic cells
are also of monocytic origin and function as professional antigen presenting
cells. Dendritic cells also play a significant role in MS [54, 55]. Interestingly, PPAR-γ agonists have
been shown to alter the viability and function of dendritic cells [56]. For example, cyclopentenone
prostaglandins induced the apoptosis of dendritic cells, although apoptosis
occurred through a receptor-independent mechanism [57]. PPAR-γ agonists were also shown to
inhibit the migration of dendritic cells [58]. Furthermore, PPAR-γ agonists
inhibited toll-like receptor mediated activation of dendritic cells by
suppressing MAP kinase and NF-κB signaling pathways [59]. PPAR-γ effects on dendritic cell
function have also been demonstrated to contribute to the development of CD4+ T cell anergy [60]. Collectively, these studies
indicate that PPAR-γ agonists may modulate MS in part through effects on
monocytic cells.Activated peripheral immune cells
including antigen specific T cells and macrophages are capable of entering the
CNS and contributing to MS pathology. Extravasation of peripheral immune cells
is mediated by a variety of factors including chemokines and adhesion molecule
expression on the cerebral vascular endothelium. Chemokines are synthesized
under inflammatory conditions and generate a concentration gradient to which
cells with the appropriate chemokine receptors migrate. The expression of
specific chemokines is increased in EAE and MS [61, 62]. PPAR-γ agonists decrease the expression of MCP-1
which is a chemoattractant for monocytes and microglia [63, 64] as well as the T cell
chemoattractants IP-10 (CXCL3), Mig (CXCL3), and I-TAC (CXCL3) [65]. Adhesion molecules present on the
cerebral vascular endothelium facilitate extravasation of peripheral immune
cells into the CNS. PPAR-γ agonists modulate the expression of various specific
adhesion molecules suggesting an additional mechanism controlling immune cell
extravasation into the CNS [66–68]. Future studies will be important in
determining more detailed mechanisms by which PPAR-γ agonists modulate immune
cell movement into the CNS.


## 3. Effects of PPAR-γ Agonists on EAE and Multiple Sclerosis

### 3.1. EAE

EAE is a well-established
animal model of MS. The disease is induced following immunization of CNS
antigens, is mediated by myelin-specific T cells, and is characterized by CNS
inflammation, demyelination, and remittent paralysis [1]. The blood-brain-barrier is believed compromised in
EAE. However, the relative bioavailability of PPAR-γ agonists into the CNS
varies, and it is important to consider this variable when interpreting studies
designed to evaluate the effects on these agonists on EAE [69, 70]. The effects of PPAR-γ agonists in modulating EAE were
first investigated by Niino et al. who demonstrated that the thiazolidinedione troglitazone
inhibited the development of EAE elicited by MOG35–55 immunization
of C57BL/6 mice. Troglitazone did not alter T cell proliferation or T cell
production of IFN-γ in vitro in these studies [71]. We demonstrated that 15d-PGJ2 inhibited
the proliferation of splenic MBPAc1-11 transgenic T cells and
inhibited IL-4 and IFN-γ production by these cells in vitro [21]. In vitro treatment of these transgenic T cells
with 15d-PGJ2 decreased the encephalitogenicity of these cells
following adoptive transfer into naïve mice. This PPAR-γ agonist also inhibited
the development of EAE when administered prior to or following onset of disease
in an active model of disease involving immunization of B10.PL mice with MBPAc1-11 [21]. These studies suggest that PPAR-γ
agonists may be effective in the treatment of established MS. Feinstein et al. demonstrated
that monophasic EAE was inhibited by thiazolidinediones including pioglitazone.
Although pioglitazone had no effect on the initial phase of relapsing-remitting
EAE, disease severity was reduced upon subsequent relapses. In addition, these
studies demonstrated that pioglitazone protected against axonal demyelination [72]. Bright et
al. demonstrated that the PPAR-γ agonists 15d-PGJ2 and ciglitazone decreased
IL-12 expression and differentiation of Th1 cells which was associated
with decreased severity of active and passive EAE [73]. This team of investigators later showed that heterozygous
PPAR-γ deficient mice demonstrated more severe EAE than wild-type mice [74]. They also demonstrated that more
severe EAE developed following treatment with PPAR-γ antagonists [75]. We showed that a combination of
the PPAR-γ agonist 15d-PGJ2 and the RXR agonist 9-cis retinoic acid
cooperatively inhibited the development of EAE [20]. Collectively, these studies
support a role for PPAR-γ in modulating EAE. Homozygous PPAR-γ mutations are
lethal thus complicating additional studies designed to evaluate the role of
this receptor in modulating disease. Development of conditional PPAR-γ knockout
mice as well as highly specific PPAR-γ antagonists will help define the role of
PPAR-γ in modulation of EAE.


### 3.2. MS

Heneka et al. investigated the effects of
thiazolidinediones pioglitazone and ciglitazone and the nonthiazolidinedione
PPAR-γ agonist GW347845 on the function of peripheral blood mononuclear cells
(PBMCs) from MS patients and healthy donors. These studies demonstrated that
all of these PPAR-γ agonists decreased phytohemagglutinin (PHA) induced T cell
proliferation and production of the cytokines TNF-α and IFN-γ by PBMCs.
Interestingly, proliferation and cytokine secretion were further suppressed
following pretreatment of cells with PPAR-γ agonists. These studies also
demonstrated that the PPAR-γ agonists decreased bcl-2 expression and induced
apoptosis of activated T cells [76]. Additional studies by the same
group indicated that PPAR-γ expression was reduced in PBMCs from MS patients
relative to healthy donors, which correlated with decreased anti-inflammatory
effects of pioglitazone on patient-derived PBMCs [77]. PHA stimulation of PBMCs from
healthy donors resulted in decreased PPAR-γ expression, which was overcame by pretreatment of these cells with PPAR-γ agonist.
Long-term treatment of diabetes patients with pioglitazone also overcame the
decrease in PPAR-γ expression in PHA treated PBMCs from these patients. These
studies indicate that pioglitazone treatment in humans can protect against loss
of PPAR-γ expression resulting from inflammation. These studies also indicated
that preincubation of PBMCs with pioglitazone resulted in increased PPAR-γ and
decreased NF-κB DNA-binding activity [77]. Collectively, these studies suggest that sustained
activation of PPAR-γ may prevent inflammation induced reduction of the
expression of this receptor. These studies may have important implications
concerning the use of PPAR-γ agonists in the treatment of MS.A small clinical
study supports the idea that PPAR-γ agonists may be effective in the treatment of
MS [78]. Larger scale clinical trials are currently
underway to further assess the therapeutic potential of PPAR-γ agonists for the
treatment of MS.


## 3.1. EAE

EAE is a well-established
animal model of MS. The disease is induced following immunization of CNS
antigens, is mediated by myelin-specific T cells, and is characterized by CNS
inflammation, demyelination, and remittent paralysis [1]. The blood-brain-barrier is believed compromised in
EAE. However, the relative bioavailability of PPAR-γ agonists into the CNS
varies, and it is important to consider this variable when interpreting studies
designed to evaluate the effects on these agonists on EAE [69, 70]. The effects of PPAR-γ agonists in modulating EAE were
first investigated by Niino et al. who demonstrated that the thiazolidinedione troglitazone
inhibited the development of EAE elicited by MOG35–55 immunization
of C57BL/6 mice. Troglitazone did not alter T cell proliferation or T cell
production of IFN-γ in vitro in these studies [71]. We demonstrated that 15d-PGJ2 inhibited
the proliferation of splenic MBPAc1-11 transgenic T cells and
inhibited IL-4 and IFN-γ production by these cells in vitro [21]. In vitro treatment of these transgenic T cells
with 15d-PGJ2 decreased the encephalitogenicity of these cells
following adoptive transfer into naïve mice. This PPAR-γ agonist also inhibited
the development of EAE when administered prior to or following onset of disease
in an active model of disease involving immunization of B10.PL mice with MBPAc1-11 [21]. These studies suggest that PPAR-γ
agonists may be effective in the treatment of established MS. Feinstein et al. demonstrated
that monophasic EAE was inhibited by thiazolidinediones including pioglitazone.
Although pioglitazone had no effect on the initial phase of relapsing-remitting
EAE, disease severity was reduced upon subsequent relapses. In addition, these
studies demonstrated that pioglitazone protected against axonal demyelination [72]. Bright et
al. demonstrated that the PPAR-γ agonists 15d-PGJ2 and ciglitazone decreased
IL-12 expression and differentiation of Th1 cells which was associated
with decreased severity of active and passive EAE [73]. This team of investigators later showed that heterozygous
PPAR-γ deficient mice demonstrated more severe EAE than wild-type mice [74]. They also demonstrated that more
severe EAE developed following treatment with PPAR-γ antagonists [75]. We showed that a combination of
the PPAR-γ agonist 15d-PGJ2 and the RXR agonist 9-cis retinoic acid
cooperatively inhibited the development of EAE [20]. Collectively, these studies
support a role for PPAR-γ in modulating EAE. Homozygous PPAR-γ mutations are
lethal thus complicating additional studies designed to evaluate the role of
this receptor in modulating disease. Development of conditional PPAR-γ knockout
mice as well as highly specific PPAR-γ antagonists will help define the role of
PPAR-γ in modulation of EAE.


## 3.2. MS

Heneka et al. investigated the effects of
thiazolidinediones pioglitazone and ciglitazone and the nonthiazolidinedione
PPAR-γ agonist GW347845 on the function of peripheral blood mononuclear cells
(PBMCs) from MS patients and healthy donors. These studies demonstrated that
all of these PPAR-γ agonists decreased phytohemagglutinin (PHA) induced T cell
proliferation and production of the cytokines TNF-α and IFN-γ by PBMCs.
Interestingly, proliferation and cytokine secretion were further suppressed
following pretreatment of cells with PPAR-γ agonists. These studies also
demonstrated that the PPAR-γ agonists decreased bcl-2 expression and induced
apoptosis of activated T cells [76]. Additional studies by the same
group indicated that PPAR-γ expression was reduced in PBMCs from MS patients
relative to healthy donors, which correlated with decreased anti-inflammatory
effects of pioglitazone on patient-derived PBMCs [77]. PHA stimulation of PBMCs from
healthy donors resulted in decreased PPAR-γ expression, which was overcame by pretreatment of these cells with PPAR-γ agonist.
Long-term treatment of diabetes patients with pioglitazone also overcame the
decrease in PPAR-γ expression in PHA treated PBMCs from these patients. These
studies indicate that pioglitazone treatment in humans can protect against loss
of PPAR-γ expression resulting from inflammation. These studies also indicated
that preincubation of PBMCs with pioglitazone resulted in increased PPAR-γ and
decreased NF-κB DNA-binding activity [77]. Collectively, these studies suggest that sustained
activation of PPAR-γ may prevent inflammation induced reduction of the
expression of this receptor. These studies may have important implications
concerning the use of PPAR-γ agonists in the treatment of MS.A small clinical
study supports the idea that PPAR-γ agonists may be effective in the treatment of
MS [78]. Larger scale clinical trials are currently
underway to further assess the therapeutic potential of PPAR-γ agonists for the
treatment of MS.


## 4. Conclusions

PPAR-γ agonists have been demonstrated to limit pathology in animal models of human
neuroinflammatory and neurodegenerative disorders. These studies suggest that
these agonists may be effective in the treatment of human diseases including
MS. Type II diabetes is commonly treated with PPAR-γ agonists termed thiazolidinediones
which include pioglitazone and rosiglitazone. These medications have an
excellent safety profile which should facilitate future clinical trials designed
to evaluate the efficacy of these PPAR-γ agonists in the treatment of human
disorders of the CNS. However, basic research designed to better understand the
cellular and molecular mechanisms by which PPAR-γ agonists regulate CNS
inflammation will be critical in developing more effective treatment strategies
for neuroinflammatory disorders including MS.Figure 1
Effects of PPAR-γ on immune cell function. Glial cells including microglia and astrocytes may become activated in response to
inflammatory stimuli including stress, trauma, and pathogens. Upon activation,
microglia, and astrocytes produce a wide range of cytokines as well as NO. These
molecules may be toxic to CNS cells, including myelin-producing
oligodendrocytes and neurons, which are compromised in the course of MS. PPAR-γ
agonists block the activation of glial cells resulting in repression of
production of cytokines and NO, and protect oligodendrocytes and neurons from
the toxic effects of these molecules. PPAR-γ agonists can also directly protect
neurons from a variety of neurotoxic agents including NMDA and apolipoprotein
E4. Chemokines secreted by activated glia cells establish a concentration
gradient to which target cell populations migrate, and play important roles in
recruiting cells into inflammatory sites in the CNS. PPAR-γ agonists may regulate the extravasation of peripheral
immune cells into the CNS by suppressing chemokine expression. In addition, activated microglia serve as the major
antigen-presenting cells (APCs) in the CNS, and dendritic cells and macrophages
serve as APCs in the periphery. These APCs are capable of secreting IL-12
family cytokines upon activation. IL-12 and IL-23 play a critical role
in the development of Th1 and Th17 cells. PPAR-γ agonists
may inhibit Th1 and Th17 cell
production by repressing IL-12 family cytokine secretion by these APCs, which
is believed to protect against EAE/MS.



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*Source: 627463-2008-06-29.xml*