# The PPAR-γ Agonist 15-Deoxy-Δ12,14-Prostaglandin J2 Attenuates Microglial Production of IL-12 Family Cytokines: Potential Relevance to

Authors: Jihong Xu; Steven W. Barger; Paul D. Drew
Journal: PPAR Research
 (2008)

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## Abstract

Accumulation of amyloid-β peptide (Aβ) appears to contribute to the pathogenesis of Alzheimer's disease (AD). Therapeutic hope for the prevention or removal of
Aβ  deposits has been placed in strategies involving immunization against the Aβ peptide.  Initial Aβ immunization studies in animal models of AD showed great promise. However, when this strategy was attempted in human subjects with AD, an unacceptable degree of meningoencephalitis occurred. It is generally believed that this adverse outcome resulted from a T-cell response to Aβ. Specifically, CD4+ Th1 and Th17 cells may contribute to severe CNS inflammation and limit the utility of Aβ immunization in the treatment of AD.  Interleukin (IL)-12 and IL-23 play critical roles in the development of Th1 and Th17 cells, respectively. In the present study, Aβ1−42 synergistically elevated the expression of IL-12 and IL-23 triggered by inflammatory activation of microglia, and the peroxisome proliferator-activated receptor (PPAR)-γ agonist 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) effectively blocked the elevation of these proinflammatory cytokines. Furthermore, 15d-PGJ2 suppressed the Aβ-related synergistic induction of CD14, MyD88, and Toll-like receptor 2, molecules that play critical roles in neuroinflammatory conditions. Collectively, these studies suggest that PPAR-γ agonists may be effective in modulating the development of AD.


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## Body

## 1. Introduction

Alzheimer’s disease (AD) is a neurodegenerative disorder and the most common cause of dementia in the elderly. AD is characterized by
progressive memory deficits, changes in personality, and cognitive decline. It
is believed that abnormal accumulation of amyloid-β peptide (Aβ), either as a
soluble factor or as extracellular aggregates, contributes to the development
of AD [1–3]. Cleavage of amyloid precursor protein (APP) can produce amyloid-β
peptide 1-42 (Aβ1−42), the levels of which are correlated with
neurotoxicity and development of AD. The connection between Aβ and AD symptoms is further strengthened by
mouse models in which transgenic expression of the human Aβ precursor (APP)
results in accumulation of Aβ and deficits in memory tests [4]. Preclinical investigations of anti-Aβ therapies have come to rely on such mice as a
loose approximation of AD pathogenesis. The most successful anti-Aβ strategy demonstrated in these mice to date
involves recruiting the immune system through vaccination. APP-transgenic mice
that are immunized against Aβ at a young age never develop substantial Aβ deposits, and vaccination after deposition can
even reverse a significant degree of the Aβ accumulation [5]. Most importantly, behavioral deficits are alleviated by such
immunizations. These benefits correlate strongly with the titers of soluble
antibody generated against Aβ [6–8], and passive immunization by injection of anti-Aβ antibody alone is also effective [9, 10]. Unfortunately, the first attempt to translate this
vaccination approach to human AD patients generated iatrogenic
meningoencephalitis in about 6% of individuals [11]. Mice can be induced to undergo similar reactions when overexpressing
interferon (IFN)-γ [12], suggesting that immune responses tilted in favor of Th1 responses
foster cell-mediated and/or inflammatory reactions to the vaccination. There is
a considerable elaboration of inflammatory index in all AD brains [13, 14], including the activation of microglia; apparently, this neuroinflammation
is fostered by Aβ itself [15, 16].  It is possible
that these proinflammatory actions of Aβ create conditions unfavorable for the
development of humoral immune responses.IL-12 family cytokines are
heterodimeric proteins which include IL-12 and IL-23. IL-12 is composed of p40
and p35 subunits, and IL-23 is composed of the same p40 subunit together with a
unique p19 subunit [17]. IL-12 plays a critical role in the differentiation of CD4+ Th1 lymphocytes. These Th1 lymphocytes stimulate
cell-mediated immune responses important in clearing pathogens, including
viruses and bacteria. Th1 lymphocytes produce IFN-γ which activates
cells of the innate immune system and contributes to the clearance of these
pathogens. IL-23 stimulates the differentiation of a unique set of CD4+ T lymphocytes. These cells are characterized by the production of the cytokine
IL-17 and are termed as Th17 lymphocytes [18]. Recent studies indicated that mice genetically ablated of the p19
subunit of IL-23 are resistant to the development of experimental autoimmune
encephalomyelitis (EAE), whereas mice lacking the p35 subunit of IL-12 showed
similar or more severe EAE than that observed in
wild-type animals [19–21]. It thus appears that IL-12 and IL-23 each play important
yet distinct roles in the development of immune responses that tend towards
cell-mediated modalities which can include inflammation. Thus, suppressing the
production of these cytokines may be effective in the treatment of inflammatory
diseases.Peroxisome proliferator-activated receptors (PPARs) are
members of the nuclear hormone receptor family of transcriptional activators.
Three PPAR subtypes exist (PPAR-α, PPAR-γ, and PPAR-β/δ), each exhibiting
distinct patterns of tissue expression and ligand specificities [22]. The role of PPAR-γ in modulating adipogenesis and glucose metabolism
is well established. Thiazolidinediones are PPAR-γ agonists that are currently
used extensively in the treatment of type II diabetes. More recently, the role
of PPAR-γ agonists in modulating immune responses, including immune responses
in the CNS, has become appreciated. Nonsteroidal anti-inflammatory drugs
(NSAIDs) have been shown to reduce AD risk and ameliorate microglial reactivity
in AD brains [23]. Since NSAIDs bind to and activate PPAR-γ, resulting in reduced
expression of proinflammatory genes, this receptor may mediate the observed
anti-inflammatory effects of NSAIDs in AD brain. In addition, it has been
demonstrated that the PPAR-γ agonists, pioglitazone and
ibuprofen, reduced
glial inflammation and Aβ1−42 levels
in APPV717I transgenic mice [24]. Collectively, these studies suggest that PPAR-γ agonists may be
effective in the treatment of neurodegenerative diseases, including AD.Pattern recognition receptors termed as  Toll-like receptors (TLRs) play a critical role
in the innate immune response to pathogen-associated molecular patterns (PAMPs)
present in viruses, bacteria, and fungi [25]. They may also contribute to neuroinflammation triggered by endogenous
ligands [25] or simply overexpression of the receptors alone [26]. A series of eleven TLRs have been identified in mice and humans, each
capable of binding distinct PAMPs. The PAMP lipopolysaccharide (LPS) binds to
TLR4 in association with another pattern recognition receptor termed as CD14.
With the exception of TLR3, ligand binding to TLRs stimulates recruitment of
the adaptor molecule MyD88, activating a well-defined signal transduction
pathway that culminates in activation of the transcription factor NF-κB, which
elevates expression of a variety of proinflammatory genes [27]. TLR4 has been suggested to play a role in regulating the pathogenesis
of AD in humans [28, 29] and in animal models of AD [30]. This suggests that agents capable of altering MyD88-dependent TLR
signaling may modulate the development of AD.The current studies
indicate that the PPAR-γ agonist 15d-PGJ2 suppresses the production
of IL-12 and IL-23 by Aβ plus LPS-stimulated microglia. These cytokines play
critical roles in Th1 and Th17 cell differentiation.
These studies could have important implications concerning Aβ immunization as
therapy for AD. In addition, we demonstrate that 15d-PGJ2 inhibits
Aβ plus LPS stimulation of MyD88, CD14, and TLR2 expression by microglia,
suggesting that this cyclopentenone prostaglandin inhibits MyD88-dependent
signaling. This provides a potential mechanism by which the PPAR-γ agonist
15d-PGJ2 modulates the expression of proinflammatory cytokines.


## 2. Materials and Methods

### 2.1. Reagents

15d-PGJ2 was obtained from Cayman Chemical Company (Ann Arbor, Mich, USA). Lipopolysaccharide and
lectin, Griffonia simplicifolia, were
obtained from Sigma (St. Louis,
Mo, USA).
Aβ1−42 was obtained from AnaSpec, Inc. (San Jose, Calif, USA). DMEM media, glutamine,
trypsin, and antibiotics used for tissue culture were obtained from BioWhittaker
(Walkersville, Md, USA).  OPI medium supplement was obtained from
Sigma.  Fetal bovine serum (FBS) was
obtained from Hyclone (Logan,
Utah, USA).  GM-CSF was obtained from BD Pharmingen (San Diego, Calif,
USA). N-2
supplement was obtained from Gibco Invitrogen Corporation (Carlsbad, Calif, USA). Glial fibrillary acidic
protein (GFAP) was obtained from Dako (Carpinteria,
Calif, USA).  C57BL/6 mice were obtained from Harlan (Indianapolis, Ind,
USA) and bred
in house.


### 2.2. Cell Culture

Primary
mouse microglia cultures were obtained through a modification of the McCarthy
and deVellis protocol [31].  Briefly, cerebral cortices from
1–3 day-old C57BL/6
mice were excised, meninges removed, and cortices minced into small
pieces.  Cells were separated by
trypsinization followed by trituration of cortical tissue.  The cell suspension was filtered through a 70μm cell strainer to remove debris.  Cells
were centrifuged at 153 × g for 5minutes at 4°C, resuspended in DMEM
medium containing 10% FBS, 1.4 mm L-glutamine, 100 U/mL
penicillin, 0.1mg/mL streptomycin, OPI medium supplement, and
0.5 ng/mL recombinant mouse GM-CSF, and plated into tissue culture
flasks.  Cells were allowed to grow to
confluency (7–10 days) at 37°C/5% CO2.  Flasks were then
shaken overnight (200 rpm at 37°C) in a temperature-controlled shaker to loosen
microglia and oligodendrocytes from the more adherent astrocytes.  These less adherent cells were plated for 2-3 hours and then
lightly shaken to separate oligodendrocytes from the more adherent microglia.
Microglia were seeded in 24-well plates or 6-well plates and incubated
overnight at 37°C/5% CO2. After overnight incubation, cells were treated with 15d-PGJ2 for
1 hour in the serum free medium with N-2 supplement, and then stimulated with
Aβ1−42 and/or LPS for 6 or 24 hours. Aβ1−42 peptides were
dissolved in DMSO to prepare a 5 mM stock solution, which was aliquoted and
stored at −80°C. Aβ1−42 stock solution was diluted with
culture medium to a concentration of 0.1 mM, and set at room temperature for 12–18 hours before
use. The final applied concentration of DMSO from Aβ1−42 was ≤0.2%. After the 24-hour stimulation, tissue
culture supernatants were collected for enzyme-linked immunosorbent assay
(ELISA), and cell viability was analyzed; 6 hours after stimulation, total RNA
was collected for real-time
quantitative RT-PCR (qRT-PCR) analysis. The purity of microglia cultures
was greater than 95% as determined by immunohistochemical staining with the
lectin, Griffonia simplicifolia.
Astrocyte contamination of the microglial cultures was assessed by
immunohistochemical staining with anti-GFAP.


### 2.3. Cell Viability Assay

Cell
viability was determined by MTT reduction assay as described previously [32].  Optical densities were
determined using a Spectromax 190 microplate reader (Molecular Devices, Sunnyvale, Calif,
USA) at 570 nm.  Results were reported as percent
viability relative to untreated cultures.


### 2.4. Enzyme-Linked Immunosorbent Assay (ELISA)

Cytokine
(IL-12p40, IL-12p70, and IL-1β) levels in tissue culture media were determined
by ELISA as described by the manufacturer (OptEIA Sets, Pharmingen, San Diego, Calif,
USA). Cytokine
IL-23 (p19/p40) levels in tissue culture media were determined by ELISA as
described by the manufacturer (eBioscience, San Diego, Calif, USA). Optical densities were
determined using a Spectromax 190 microplate reader (Molecular Devices, Sunnyvale, Calif,
USA) at 450 nm.
Cytokine concentrations in media were determined from standards containing
known concentrations of the proteins.


### 2.5. RNA Isolation and cDNA Synthesis

Total RNA was isolated from microglia using the RNeasy Mini Kit (Qiagen Sciences,
Md, USA).
RNA samples were treated with DNAse1 (Invitrogen, Carlsbad, Calif, USA) to remove any traces of
contaminating DNA. The reverse transcription (RT) reactions were carried out
using an iScript cDNA synthesis kit (Bio-Rad, Hercules, Calif, USA) according to the
manufacturer's instructions.


### 2.6. Real-Time Quantitative RT-PCR Assay

IL-12p40, IL-12p35, IL-23p19, IL-1β, CD14,
MyD88, TLR2, and TLR4 mRNAs were quantified by real-time PCR using an iCycler IQ multicolor real-time PCR
detection system (Bio-Rad). All primers and TaqMan MGB probes (FAMdye-labeled) were
designed and synthesized by Applied Biosystems (Foster City, Calif, USA). The real-time PCR reactions
were performed in a total volume of 25μL using an iCycler kit (Bio-Rad).
The levels of  IL-12p40, IL-12p35, IL-23p19, IL-1β, CD14,
MyD88, TLR2, and TLR4 mRNA expression in primary microglia were
calculated after normalizing cycle thresholds against the “housekeeping” gene
GAPDH, and are presented as the fold induction value (2−ΔΔCt)
relative to LPS-stimulated microglia.


### 2.7. Statistics

Data were
analyzed by one-way ANOVA followed by a Bonferroni posthoc test to determine
the significance of difference.


## 2.1. Reagents

15d-PGJ2 was obtained from Cayman Chemical Company (Ann Arbor, Mich, USA). Lipopolysaccharide and
lectin, Griffonia simplicifolia, were
obtained from Sigma (St. Louis,
Mo, USA).
Aβ1−42 was obtained from AnaSpec, Inc. (San Jose, Calif, USA). DMEM media, glutamine,
trypsin, and antibiotics used for tissue culture were obtained from BioWhittaker
(Walkersville, Md, USA).  OPI medium supplement was obtained from
Sigma.  Fetal bovine serum (FBS) was
obtained from Hyclone (Logan,
Utah, USA).  GM-CSF was obtained from BD Pharmingen (San Diego, Calif,
USA). N-2
supplement was obtained from Gibco Invitrogen Corporation (Carlsbad, Calif, USA). Glial fibrillary acidic
protein (GFAP) was obtained from Dako (Carpinteria,
Calif, USA).  C57BL/6 mice were obtained from Harlan (Indianapolis, Ind,
USA) and bred
in house.


## 2.2. Cell Culture

Primary
mouse microglia cultures were obtained through a modification of the McCarthy
and deVellis protocol [31].  Briefly, cerebral cortices from
1–3 day-old C57BL/6
mice were excised, meninges removed, and cortices minced into small
pieces.  Cells were separated by
trypsinization followed by trituration of cortical tissue.  The cell suspension was filtered through a 70μm cell strainer to remove debris.  Cells
were centrifuged at 153 × g for 5minutes at 4°C, resuspended in DMEM
medium containing 10% FBS, 1.4 mm L-glutamine, 100 U/mL
penicillin, 0.1mg/mL streptomycin, OPI medium supplement, and
0.5 ng/mL recombinant mouse GM-CSF, and plated into tissue culture
flasks.  Cells were allowed to grow to
confluency (7–10 days) at 37°C/5% CO2.  Flasks were then
shaken overnight (200 rpm at 37°C) in a temperature-controlled shaker to loosen
microglia and oligodendrocytes from the more adherent astrocytes.  These less adherent cells were plated for 2-3 hours and then
lightly shaken to separate oligodendrocytes from the more adherent microglia.
Microglia were seeded in 24-well plates or 6-well plates and incubated
overnight at 37°C/5% CO2. After overnight incubation, cells were treated with 15d-PGJ2 for
1 hour in the serum free medium with N-2 supplement, and then stimulated with
Aβ1−42 and/or LPS for 6 or 24 hours. Aβ1−42 peptides were
dissolved in DMSO to prepare a 5 mM stock solution, which was aliquoted and
stored at −80°C. Aβ1−42 stock solution was diluted with
culture medium to a concentration of 0.1 mM, and set at room temperature for 12–18 hours before
use. The final applied concentration of DMSO from Aβ1−42 was ≤0.2%. After the 24-hour stimulation, tissue
culture supernatants were collected for enzyme-linked immunosorbent assay
(ELISA), and cell viability was analyzed; 6 hours after stimulation, total RNA
was collected for real-time
quantitative RT-PCR (qRT-PCR) analysis. The purity of microglia cultures
was greater than 95% as determined by immunohistochemical staining with the
lectin, Griffonia simplicifolia.
Astrocyte contamination of the microglial cultures was assessed by
immunohistochemical staining with anti-GFAP.


## 2.3. Cell Viability Assay

Cell
viability was determined by MTT reduction assay as described previously [32].  Optical densities were
determined using a Spectromax 190 microplate reader (Molecular Devices, Sunnyvale, Calif,
USA) at 570 nm.  Results were reported as percent
viability relative to untreated cultures.


## 2.4. Enzyme-Linked Immunosorbent Assay (ELISA)

Cytokine
(IL-12p40, IL-12p70, and IL-1β) levels in tissue culture media were determined
by ELISA as described by the manufacturer (OptEIA Sets, Pharmingen, San Diego, Calif,
USA). Cytokine
IL-23 (p19/p40) levels in tissue culture media were determined by ELISA as
described by the manufacturer (eBioscience, San Diego, Calif, USA). Optical densities were
determined using a Spectromax 190 microplate reader (Molecular Devices, Sunnyvale, Calif,
USA) at 450 nm.
Cytokine concentrations in media were determined from standards containing
known concentrations of the proteins.


## 2.5. RNA Isolation and cDNA Synthesis

Total RNA was isolated from microglia using the RNeasy Mini Kit (Qiagen Sciences,
Md, USA).
RNA samples were treated with DNAse1 (Invitrogen, Carlsbad, Calif, USA) to remove any traces of
contaminating DNA. The reverse transcription (RT) reactions were carried out
using an iScript cDNA synthesis kit (Bio-Rad, Hercules, Calif, USA) according to the
manufacturer's instructions.


## 2.6. Real-Time Quantitative RT-PCR Assay

IL-12p40, IL-12p35, IL-23p19, IL-1β, CD14,
MyD88, TLR2, and TLR4 mRNAs were quantified by real-time PCR using an iCycler IQ multicolor real-time PCR
detection system (Bio-Rad). All primers and TaqMan MGB probes (FAMdye-labeled) were
designed and synthesized by Applied Biosystems (Foster City, Calif, USA). The real-time PCR reactions
were performed in a total volume of 25μL using an iCycler kit (Bio-Rad).
The levels of  IL-12p40, IL-12p35, IL-23p19, IL-1β, CD14,
MyD88, TLR2, and TLR4 mRNA expression in primary microglia were
calculated after normalizing cycle thresholds against the “housekeeping” gene
GAPDH, and are presented as the fold induction value (2−ΔΔCt)
relative to LPS-stimulated microglia.


## 2.7. Statistics

Data were
analyzed by one-way ANOVA followed by a Bonferroni posthoc test to determine
the significance of difference.


## 3. Results

### 3.1. Effects of 15d-PGJ2 on IL-1β production by
β-Amyloid Plus LPS-Stimulated Microglia

A variety
of studies suggest that the inflammatory cytokine IL-1β plays a significant
role in modulating the pathogenesis of AD [33]. In the present study, we investigated whether Aβ1−42 plus a
low dose of LPS could induce IL-1β production by primary mouse microglial
cells. Our results showed that Aβ1−42 alone did not induce microglia
production of IL-1β protein (Figure 1(a)) and IL-1β mRNA (Figure 1(b)). LPS
(10 ng/mL) alone stimulated microglial production of IL-1β protein and mRNA,
while a combination of Aβ1−42 and LPS synergistically induced the
expression of IL-1β protein and mRNA. Interestingly, the PPAR-γ agonist 15d-PGJ2 strongly suppressed induction of IL-1β in Aβ1−42 plus LPS-stimulated
primary microglial cells. The PPAR-γ agonist did not decrease the viability of
these microglial cells compared to cells treated with Aβ1−42 plus
LPS as determined by MTT analysis (data not shown). Therefore, the effects of
15d-PGJ2 on the production of IL-1β were not due to effects on cell
viability. These studies suggest that 15d-PGJ2 may suppress the
production of IL-1β, an inflammation-related cytokine associated with the
pathogenesis of AD.15d-PGJ2 inhibits IL-1β expression by Aβ1−42
plus LPS-activated microglia. (a) Primary mouse microglial
cells were pretreated for 1 hour with 15d-PGJ2 (2.5μM). Aβ1−42 (5μM), LPS (10 ng/mL),
or Aβ1−42 (5μM) plus LPS (10 ng/mL) was added as indicated, and 24
hours later, the concentration of IL-1β in the culture medium was determined.
Values represent the mean ± s.e.m for a representative experiment run in
triplicate. At least three independent experiments were conducted.  (b) Cells were pretreated for 1 hour with 15d-PGJ2 (2.5μM). Aβ1−42 (10μM), LPS (5 ng/mL), or Aβ1−42 (10μM) plus LPS (5 ng/mL) was added as
indicated, and 6 hours later, total RNA was isolated. IL-1β mRNA levels were determined by real-time quantitative RT-PCR. Results
are expressed as fold inductions in GAPDH normalized mRNA values versus levels
in LPS-treated cells. Values
are mean ± s.e.m of six samples derived from three independent
experiments, with each experiment performed in duplicate.  P**<.01 and P***<.001
versus Aβ1−42 + LPS-treated cultures.
(a)(b)


### 3.2. Effects of 15d-PGJ2 on IL-12 Family Cytokines
by β-Amyloid Plus LPS-Stimulated Microglia

IL-12
family cytokines are believed to contribute to the differentiation of Th1 and Th17 cells. Aβ1−42 alone had little or no effect
on the production of IL-12 family cytokines by microglia. LPS (10 ng/mL)
stimulated microglia to secrete IL-12 family cytokines including IL-12p40
(Figure 2(a)), IL-12p70 (Figure 2(b)), and IL-23 (Figure 2(c)). In the context
of this inflammatory priming, Aβ1−42 further increased microglial
production of each of these IL-12 family proteins significantly. Furthermore, the PPAR-γ agonist 15d-PGJ2 significantly suppressed the expression of these
IL-12 family proteins.15d-PGJ2 inhibits IL-12 family
cytokines production by Aβ1−42
plus
LPS-activated microglia. Cells were pretreated for 1 hour
with 15d-PGJ2 (2.5μM). Aβ1−42 (5μM), LPS (10 ng/mL), or Aβ1−42 (5μM) plus LPS (10 ng/mL) was added as indicated,
and 24 hours later, the concentration of IL-12p40 (a), IL-12p70 (p35/p40) (b),
and IL-23 (p19/p40) (c) in the culture medium was determined. Values represent
the mean ± s.e.m for a representative experiment run in triplicate. At least
three independent experiments were conducted. P**<.01 and P***<.001
versus Aβ1−42 + LPS-treated cultures.
(a)(b)(c)


### 3.3. Effects of 15d-PGJ2 on Expression of IL-12
Family Cytokine Subunit mRNAs by β-Amyloid Plus LPS-Stimulated Microglia

Aβ1−42 alone had little or no effect on stimulating the expression of IL-12 family
cytokine subunit mRNAs including IL-12p35 (Figure 3(a)), IL-12p40 (Figure 3(b)),
and IL-23p19 (Figure 3(c)). Low doses of LPS (5 ng/mL) alone slightly induced
the expression of these mRNAs. However, Aβ1−42 in combination with
LPS elicited significantly higher levels of IL-12 family subunit mRNAs compared
to microglia stimulated with LPS alone. Pretreatment with 15d-PGJ2 significantly suppressed the expression of IL-12 family subunit mRNAs. Thus,
15d-PGJ2 inhibits the expression of IL-12 family cytokines and the
mRNAs that encode these proteins. IL-12 and IL-23 play critical roles in the
differentiation of Th1 and Th17 cells, which may
contribute to the inflammatory events that resulted in cessation of clinical
trials involving immunization of Aβ in the treatment of AD. Thus, cotreatment
with 15d-PGJ2 may increase the utility of Aβ immunotherapy for AD
patients.15d-PGJ2 inhibits microglial mRNA expression of IL-12p40,
IL-12p35, and IL-23p19 induced by Aβ1−42
plus LPS. Cells were
pretreated for 1 hour with 15d-PGJ2 (2.5μM). Aβ1−42 (10μM), LPS (5 ng/mL), or Aβ1−42 (10μM) plus LPS (5 ng/mL)  was added as indicated, and 6 hours later,
total RNA was isolated. IL-12p35 (a), IL-12p40 (b), and IL-23p19 (c) mRNA
levels were determined by real-time quantitative RT-PCR. Results are
expressed as fold inductions in GAPDH normalized mRNA values versus levels in
LPS-treated cells. Values
are mean ± s.e.m of six samples derived from three independent
experiments, with each experiment performed in duplicate. P*<.05,
P**<.01, and P***<.001 versus Aβ1−42 + LPS-treated
cultures.
(a)(b)(c)


### 3.4. Effects of 15d-PGJ2 on Expression of  Toll-Like Receptor Signaling

The
MyD88-dependent TLR signaling pathway plays a critical role in modulating the
response to PAMPs including LPS. We demonstrate that a combination of Aβ1−42 plus LPS significantly induced the expression of CD14 relative to microglia
treated with LPS alone (Figure 4(a)). In addition, Aβ1−42 plus LPS
also trended towards inducing MyD88 expression relative to each stimulus alone
(Figure 4(b)). CD14 and MyD88 are critical intermediates in MyD88-dependent
signaling. As we have demonstrated previously, LPS does not significantly
induce the expression of TLR4, but does induce the expression of TLR2 [34]. Similarly, Aβ1−42 in combination with LPS did not induce
microglial expression of TLR4 (Figure 4(c)), but did induce the expression of
TLR2 (Figure 4(d)). Interestingly, 15d-PGJ2 inhibited Aβ1−42 plus LPS induction of MyD88, CD14, and TLR2 mRNA expression in microglia. These
studies suggest that 15d-PGJ2 may suppress inflammatory responses stimulated by
Aβ1−42 plus LPS by inhibiting MyD88-dependent TLR signaling.The effects of 15d-PGJ2 on microglial mRNA expression of CD14,
MyD88, TLR4, and TLR2 induced by Aβ1−42
plus LPS. Cells were
pretreated for 1 hour with 15d-PGJ2 (2.5μM). Aβ1−42 (10μM), LPS (5 ng/mL), or Aβ1−42 (10μM) plus LPS (5 ng/mL) was added as
indicated, and 6 hours later, total RNA was isolated. CD14 (a), MyD88 (b), TLR4 (c), and TLR2 (d) mRNA levels were determined by real-time
quantitative RT-PCR. Results are expressed as fold inductions in GAPDH
normalized mRNA values versus levels in LPS-treated cells. Values are
mean ± s.e.m of six samples derived from three independent
experiments, with each experiment performed in duplicate. P*<.05,
P**<.01, and P***<.001 versus Aβ1−42 + LPS-treated
cultures.
(a)(b)(c)(d)


## 3.1. Effects of 15d-PGJ2 on IL-1β production by
β-Amyloid Plus LPS-Stimulated Microglia

A variety
of studies suggest that the inflammatory cytokine IL-1β plays a significant
role in modulating the pathogenesis of AD [33]. In the present study, we investigated whether Aβ1−42 plus a
low dose of LPS could induce IL-1β production by primary mouse microglial
cells. Our results showed that Aβ1−42 alone did not induce microglia
production of IL-1β protein (Figure 1(a)) and IL-1β mRNA (Figure 1(b)). LPS
(10 ng/mL) alone stimulated microglial production of IL-1β protein and mRNA,
while a combination of Aβ1−42 and LPS synergistically induced the
expression of IL-1β protein and mRNA. Interestingly, the PPAR-γ agonist 15d-PGJ2 strongly suppressed induction of IL-1β in Aβ1−42 plus LPS-stimulated
primary microglial cells. The PPAR-γ agonist did not decrease the viability of
these microglial cells compared to cells treated with Aβ1−42 plus
LPS as determined by MTT analysis (data not shown). Therefore, the effects of
15d-PGJ2 on the production of IL-1β were not due to effects on cell
viability. These studies suggest that 15d-PGJ2 may suppress the
production of IL-1β, an inflammation-related cytokine associated with the
pathogenesis of AD.15d-PGJ2 inhibits IL-1β expression by Aβ1−42
plus LPS-activated microglia. (a) Primary mouse microglial
cells were pretreated for 1 hour with 15d-PGJ2 (2.5μM). Aβ1−42 (5μM), LPS (10 ng/mL),
or Aβ1−42 (5μM) plus LPS (10 ng/mL) was added as indicated, and 24
hours later, the concentration of IL-1β in the culture medium was determined.
Values represent the mean ± s.e.m for a representative experiment run in
triplicate. At least three independent experiments were conducted.  (b) Cells were pretreated for 1 hour with 15d-PGJ2 (2.5μM). Aβ1−42 (10μM), LPS (5 ng/mL), or Aβ1−42 (10μM) plus LPS (5 ng/mL) was added as
indicated, and 6 hours later, total RNA was isolated. IL-1β mRNA levels were determined by real-time quantitative RT-PCR. Results
are expressed as fold inductions in GAPDH normalized mRNA values versus levels
in LPS-treated cells. Values
are mean ± s.e.m of six samples derived from three independent
experiments, with each experiment performed in duplicate.  P**<.01 and P***<.001
versus Aβ1−42 + LPS-treated cultures.
(a)(b)


## 3.2. Effects of 15d-PGJ2 on IL-12 Family Cytokines
by β-Amyloid Plus LPS-Stimulated Microglia

IL-12
family cytokines are believed to contribute to the differentiation of Th1 and Th17 cells. Aβ1−42 alone had little or no effect
on the production of IL-12 family cytokines by microglia. LPS (10 ng/mL)
stimulated microglia to secrete IL-12 family cytokines including IL-12p40
(Figure 2(a)), IL-12p70 (Figure 2(b)), and IL-23 (Figure 2(c)). In the context
of this inflammatory priming, Aβ1−42 further increased microglial
production of each of these IL-12 family proteins significantly. Furthermore, the PPAR-γ agonist 15d-PGJ2 significantly suppressed the expression of these
IL-12 family proteins.15d-PGJ2 inhibits IL-12 family
cytokines production by Aβ1−42
plus
LPS-activated microglia. Cells were pretreated for 1 hour
with 15d-PGJ2 (2.5μM). Aβ1−42 (5μM), LPS (10 ng/mL), or Aβ1−42 (5μM) plus LPS (10 ng/mL) was added as indicated,
and 24 hours later, the concentration of IL-12p40 (a), IL-12p70 (p35/p40) (b),
and IL-23 (p19/p40) (c) in the culture medium was determined. Values represent
the mean ± s.e.m for a representative experiment run in triplicate. At least
three independent experiments were conducted. P**<.01 and P***<.001
versus Aβ1−42 + LPS-treated cultures.
(a)(b)(c)


## 3.3. Effects of 15d-PGJ2 on Expression of IL-12
Family Cytokine Subunit mRNAs by β-Amyloid Plus LPS-Stimulated Microglia

Aβ1−42 alone had little or no effect on stimulating the expression of IL-12 family
cytokine subunit mRNAs including IL-12p35 (Figure 3(a)), IL-12p40 (Figure 3(b)),
and IL-23p19 (Figure 3(c)). Low doses of LPS (5 ng/mL) alone slightly induced
the expression of these mRNAs. However, Aβ1−42 in combination with
LPS elicited significantly higher levels of IL-12 family subunit mRNAs compared
to microglia stimulated with LPS alone. Pretreatment with 15d-PGJ2 significantly suppressed the expression of IL-12 family subunit mRNAs. Thus,
15d-PGJ2 inhibits the expression of IL-12 family cytokines and the
mRNAs that encode these proteins. IL-12 and IL-23 play critical roles in the
differentiation of Th1 and Th17 cells, which may
contribute to the inflammatory events that resulted in cessation of clinical
trials involving immunization of Aβ in the treatment of AD. Thus, cotreatment
with 15d-PGJ2 may increase the utility of Aβ immunotherapy for AD
patients.15d-PGJ2 inhibits microglial mRNA expression of IL-12p40,
IL-12p35, and IL-23p19 induced by Aβ1−42
plus LPS. Cells were
pretreated for 1 hour with 15d-PGJ2 (2.5μM). Aβ1−42 (10μM), LPS (5 ng/mL), or Aβ1−42 (10μM) plus LPS (5 ng/mL)  was added as indicated, and 6 hours later,
total RNA was isolated. IL-12p35 (a), IL-12p40 (b), and IL-23p19 (c) mRNA
levels were determined by real-time quantitative RT-PCR. Results are
expressed as fold inductions in GAPDH normalized mRNA values versus levels in
LPS-treated cells. Values
are mean ± s.e.m of six samples derived from three independent
experiments, with each experiment performed in duplicate. P*<.05,
P**<.01, and P***<.001 versus Aβ1−42 + LPS-treated
cultures.
(a)(b)(c)


## 3.4. Effects of 15d-PGJ2 on Expression of  Toll-Like Receptor Signaling

The
MyD88-dependent TLR signaling pathway plays a critical role in modulating the
response to PAMPs including LPS. We demonstrate that a combination of Aβ1−42 plus LPS significantly induced the expression of CD14 relative to microglia
treated with LPS alone (Figure 4(a)). In addition, Aβ1−42 plus LPS
also trended towards inducing MyD88 expression relative to each stimulus alone
(Figure 4(b)). CD14 and MyD88 are critical intermediates in MyD88-dependent
signaling. As we have demonstrated previously, LPS does not significantly
induce the expression of TLR4, but does induce the expression of TLR2 [34]. Similarly, Aβ1−42 in combination with LPS did not induce
microglial expression of TLR4 (Figure 4(c)), but did induce the expression of
TLR2 (Figure 4(d)). Interestingly, 15d-PGJ2 inhibited Aβ1−42 plus LPS induction of MyD88, CD14, and TLR2 mRNA expression in microglia. These
studies suggest that 15d-PGJ2 may suppress inflammatory responses stimulated by
Aβ1−42 plus LPS by inhibiting MyD88-dependent TLR signaling.The effects of 15d-PGJ2 on microglial mRNA expression of CD14,
MyD88, TLR4, and TLR2 induced by Aβ1−42
plus LPS. Cells were
pretreated for 1 hour with 15d-PGJ2 (2.5μM). Aβ1−42 (10μM), LPS (5 ng/mL), or Aβ1−42 (10μM) plus LPS (5 ng/mL) was added as
indicated, and 6 hours later, total RNA was isolated. CD14 (a), MyD88 (b), TLR4 (c), and TLR2 (d) mRNA levels were determined by real-time
quantitative RT-PCR. Results are expressed as fold inductions in GAPDH
normalized mRNA values versus levels in LPS-treated cells. Values are
mean ± s.e.m of six samples derived from three independent
experiments, with each experiment performed in duplicate. P*<.05,
P**<.01, and P***<.001 versus Aβ1−42 + LPS-treated
cultures.
(a)(b)(c)(d)


## 4. Discussion

AD currently affects over 200
million people worldwide. Disease incidence is expected to increase as the
population ages, and the socioeconomic impact of AD is staggering. The disease
is characterized in part by the presence of neuritic plaques which contain
accumulations of insoluble Aβ. Vaccination with Aβ synthetic peptides in animal
models of AD suggested that such immunizations may be effective in the
treatment of AD in humans. For example, Aβ immunization of APP transgenic mice decreases
the density and number of Aβ deposits in the brains of these mice. Decreased Aβ
deposits in these mice are associated with decreased neuritic dystrophy and
gliosis [7]. Intranasal administration of Aβ engenders humoral responses that
include immunoglobulin isotypes consistent with a Th2 response, and this is
associated with increased clearance of amyloid [35]. Significantly, active immunization against Aβ in APP transgenic mice
decreases memory deficits in these mice [6, 35, 36]. Interestingly, passive administration of monoclonal
antibodies specific for Aβ peptides is also effective in clearing Aβ and
improving memory deficits in APP transgenic mice [37, 38]. This suggests that Aβ-specific antibodies produced
following immunization are the critical factor mediating AD-like pathology in
these animal models of AD. Three potential mechanisms have been suggested that
may determine how anti-Aβ antibodies reduce Aβ deposits in the brains of APP
transgenic mice. Aβ antibodies (1) may directly dissolve Aβ deposits, (2) may
stimulate Fc-receptor-mediated phagocytosis of Aβ by microglia, and/or (3) may
stimulate Aβ efflux from the brain to the plasma [3].Animal studies indicating that Aβ
immunization of APP transgenic mice reduced plaque burden in mice stimulated
human clinical trials designed to evaluate the clinical efficacy of Aβ
immunization in the treatment of AD. Small-scale phase I trials indicated
apparent safety of Aβ immunization, and demonstrated that the majority of mild
to moderate AD patients immunized in these studies produced anti-Aβ antibodies [39, 40]. However, subsequent larger-scale phase II clinical trials
were halted when approximately 6% of Aβ immunized patients developed
meningoencephalitis [11]. Postmortem evaluation indicated that Aβ immunization resulted in
decreased plaque burden in the cortex of treated patients [41–43], and these brain regions were associated with abundant Aβ
immunoreactive microglia, suggesting that these cells were involved in the
removal of Aβ [43]. Interestingly, although anti-Aβ antibodies are believed to contribute
to the reduction in Aβ plaques in AD patients, antibodies titers did not
correlate with the development of meningoencephalitis [11, 40]. Several studies suggest that T cell responses to Aβ may
have stimulated the development of meningoencephalitis in immunized AD patients
[44–46]. Furthermore, a higher T cell reactivity to Aβ has been
observed in some elderly and AD patients not immunized with Aβ. This suggests
that the elderly population and AD patients may exhibit increased
susceptibility to the development of meningoencephalitis following Aβ
vaccination [46]. Cases of meningoencephalitis were associated with increased
infiltration of both CD4+ and CD8+ T cells [42]. However, it is generally believed that CD4+ Th1 cells triggered the development of meningoencephalitis following Aβ
immunization [3]. The potential role of the recently described CD4+Th17 cells in the production of meningoencephalitis has not been evaluated. However,
studies indicating that these cells play a critical role in the development of
MS and other autoimmune disorders suggest that these cells may also play a role
in the development of meningoencephalitis in Aβ immunized AD patients.Our current studies indicate that
the PPAR-γ agonist 15d-PGJ2 inhibits microglial production of IL-12
and IL-23, which play critical roles in the differentiation of Th1 and Th17 cells, respectively. This suggests that 15d-PGJ2 could potentially increase the efficacy and safety of Aβ immunization of AD
patients by decreasing or abolishing the development of meningoencephalitis in
these patients. Epidemiological studies indicated that nonsteroidal anti-inflammatory
drugs (NSAIDs) reduced the risk of AD. Some NSAIDs are capable of activating
PPAR-γ, suggesting that these drugs may modulate development of AD through
activation of this receptor [47, 48]. The role of PPAR-γ in modulating AD is supported by
studies indicating that ibuprofen reduced Aβ1−42 levels in APP
transgenic mouse models of AD, while low levels of the thiazolidinedione
pioglitazone stimulated a slight yet statistically insignificant reduction of
Aβ1−42 levels in these mice [49]. In a later study, higher levels of pioglitazone decreased astrocyte
and microglial activation and Aβ plaque burden in APP transgenic mice [24]. Similarly, the thiazolidinedione rosiglitazone also decreased Aβ1−42 levels in animal models of AD [50]. Collectively, these studies support a role for PPAR-γ in modulating AD
pathology. Studies indicate that PPAR-γ activation suppresses expression of β-site
of APP cleaving enzyme (BACE)-1, suggesting that PPAR-γ agonists may modulate
AD pathogenesis at least in part by altering Aβ homeostasis [51]. Importantly, recent clinical studies demonstrated that rosiglitazone
was effective in improving cognition in a subset of AD patients [52, 53].  The fact that
rosiglitazone exhibits poor blood-brain barrier penetration suggests that this
PPAR-γ agonist may act in the periphery and not directly in the CNS.We and others have previously
demonstrated that PPAR-γ agonists are capable of suppressing the activation of
NF-κB, which is a potent transcriptional activator of a variety of genes
encoding proinflammatory molecules. MyD88-dependent signaling results in the
activation of NF-κB. In the current studies, we demonstrate that the PPAR-γ
agonist 15d-PGJ2 suppressed microglial expression of MyD88 and CD14
which are critical intermediates in MyD88-dependent TLR signaling. In addition,
we demonstrate that 15d-PGJ2 inhibits microglial expression of
IL-1β, a cytokine believed to contribute to AD pathogenesis [33]. Thus, PPAR-γ agonists may act as general suppressors of classical
activation of microglia. Since classically activated microglia produce
neurotoxic molecules, suppression of microglial activation may protect against
AD. However, it should also be noted that some form of microglial activation
may help remove Aβ plaques from AD brains through phagocytosis. In addition,
TLR and CD14 molecules have been suggested to contribute to—or alternatively
protect against—the development
of AD [30, 54, 55]. It clearly appears that microglia and microglial products
modulate AD through a series of complex and potentially conflicting mechanisms.In summary, we have demonstrated
that the PPAR-γ agonist 15d-PGJ2 inhibits production of IL-12 and
IL-23 by Aβ plus LPS-activated microglia. These cytokines regulate the
differentiation of Th1 and Th17 cells, which may limit
the efficacy of Aβ immunotherapy for the treatment of AD. Furthermore, we
demonstrate that 15d-PGJ2 inhibits the production of IL-1β by
microglia, a cytokine known to play a role in AD pathogenesis. Finally, we
demonstrate that 15d-PGJ2 inhibits the expression of MyD88-dependent
signaling intermediates, suggesting a mechanism by which this PPAR-γ agonist
may suppress inflammation. Collectively, these studies contribute to the body
of evidence indicating that PPAR-γ agonists may be effective in the treatment
of AD.



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*Source: 349185-2008-07-02.xml*