# Microglia and Astrocyte Activation by Toll-Like Receptor Ligands: Modulation by PPAR-γ Agonists

Authors: Catherine Gurley; Jessica Nichols; Shuliang Liu; Nirmal K. Phulwani; Nilufer Esen; Tammy Kielian
Journal: PPAR Research
 (2008)

---

## Abstract

Microglia and astrocytes express numerous members of the Toll-like receptor (TLR) family that are pivotal for recognizing conserved microbial motifs expressed by a wide array of pathogens. Despite the critical role for TLRs in pathogen recognition, when dysregulated these pathways can also exacerbate CNS tissue destruction. Therefore, a critical balance must be achieved to elicit sufficient immunity to combat CNS infectious insults and down-regulate these responses to avoid pathological tissue damage. We performed a comprehensive survey on the efficacy of various PPAR-γ agonists to modulate proinflammatory mediator release from primary microglia and astrocytes in response to numerous TLR ligands relevant to CNS infectious diseases. The results demonstrated differential abilities of select PPAR-γ agonists to modulate glial activation. For example, 15d-PGJ2 and pioglitazone were both effective at reducing IL-12 p40 release by TLR ligand-activated glia, whereas CXCL2 expression was either augmented or inhibited by 15d-PGJ2, effects that were dependent on the TLR ligand examined. Pioglitazone and troglitazone demonstrated opposing actions on microglial CCL2 production that were TLR ligand-dependent. Collectively, this information may be exploited to modulate the host immune response during CNS infections to maximize host immunity while minimizing inappropriate bystander tissue damage that is often characteristic of such diseases.


---

## Body

## 1. Introduction

Microglia and astrocytes participate in
the genesis of innate immune responses in the CNS parenchyma [1, 2]. Their strategic placement at
or near the
blood-brain barrier likely makes
both glial types sentinel cells for surveying pathogen entry in the CNS
parenchyma. Indeed, both microglia and astrocytes are capable of producing a
wide range of proinflammatory mediators in response to a diverse array of
microbial stimuli [3, 4]. Therefore, it is likely that
resident glia are pivotal for eliciting a local CNS inflammatory response
through the initial production of inflammatory mediators, which in turn, leads
to the recruitment of additional immune effector cells from the peripheral
circulation.Toll-like receptors (TLRs) are a group of
pattern recognition receptors (PRRs) responsible for recognizing conserved
motifs expressed on broad classes of microbes termed pathogen-associated
molecular patterns (PAMPs) [5, 6]. Typically, PAMPs represent
structural or nucleic acid motifs that are less likely to undergo mutation,
ensuring efficient pathogen recognition with a limited receptor arsenal [7]. A total of 13 TLRs have been
identified to date, each conferring recognition of conserved motifs from large
subclasses of bacteria, viruses, yeast, and fungi [5–7]. In addition, recent evidence
indicates that TLRs are also capable of sensing endogenous ligands produced
during stress or injury referred to as danger-associated molecular patterns
(DAMPs) that may participate in autoimmune induction [8–10]. Numerous TLRs have been
identified on microglia and astrocytes and both glial types are responsive to
numerous TLR ligands implicating their role in pathogen recognition (reviewed
in [11, 12]). In addition, recent
evidence links TLRs with the host response to CNS injury presumably via
recognition of endogenous “danger signals” since classical microbial TLR
ligands are not present (reviewed in [13–15]). Since TLRs have been
implicated in both infectious and noninfectious diseases of the CNS (reviewed
in [11, 13]), understanding their
potential to influence the course of neuroinflammation is paramount and under
certain conditions inappropriate TLR activation may contribute to excessive
tissue destruction. Therefore, modulating TLR activity to achieve optimal
benefit for the host may be a plausible strategy for minimizing tissue damage during
neuroinflammatory disorders.A group of
compounds with reported anti-inflammatory effects in several models of
inflammation, including the CNS, are ligands that interact with peroxisome
proliferator-activated receptor-gamma (PPAR-γ) [16–18]. PPAR-γ is a member
of the nuclear hormone receptor superfamily of ligand-activated transcription
factors that regulate the expression of genes involved in reproduction,
metabolism, development, and immune responses [17, 19]. A
wide array of both natural and synthetic agonists for PPAR-γ have been
identified including the naturally occurring prostaglandin metabolite
15d-PGJ2, thiazolidinediones (TZDs) a group of synthetic PPAR-γ agonists used for the treatment of
diabetes, polyunsaturated fatty acids, and certain high affinity tyrosine
derivatives. With regard to the CNS, several PPAR-γ agonists have been documented for their
ability to attenuate both microglial and astrocyte activation in response to a
diverse array of stimuli as well as impact the course of several
neurodegenerative diseases [16, 18, 20–25]. Although we and others have
demonstrated that select
PPAR-γ agonists are potent inhibitors of TLR2
and TLR4 activation (PGN and LPS, resp.) [23, 24, 26–29], a comprehensive examination
of the effects of PPAR-γ agonists on a wide array of TLR ligands
is lacking. In addition, although several studies describing the responses of
microglia and astrocytes to TLR ligands exist [30–34], no reports have
systematically investigated the ability of PPAR-γ ligands to modulate glial activation in
response to TLR signals. Therefore, the purpose of this study was to define the
actions of a panel of PPAR-γ agonists on TLR ligand-induced
activation of microglia and astrocytes. Although within the same class of compounds,
not all PPAR-γ agonists shared similar regulatory
properties in response to various TLR ligands. Indeed, in some cases,
inflammatory mediator production was enhanced following PPAR-γ agonist treatment. Collectively, these
results suggest selective actions of PPAR-γ agonists on glial responses to TLR
ligands that could be exploited for specific neuroinflammatory/infectious
conditions of the CNS.


## 2. Materials and Methods

### 2.1. TLR Ligands and PPAR-γ Agonists

The following TLR agonists were used in this study with  (see Table1)  the concentration of each and its TLR target identified in parenthesis:
Pam3CSK4 (TLR2, 1 μg/ml), polyinosine-polycytidylic acid
(polyI:C; TLR3, 25 μg/ml), lipopolysaccharide from E. coli O111:B4 (LPS; TLR4, 100 ng/ml),
flagellin from Salmonella typhimurium (TLR5, 10 μg/ml), single-stranded RNA (ssRNA; TLR7/8,
10 μg/ml), and synthetic unmethylated CpG oligonucleotide
(ODN; TLR9, 0.1 μM and 5 μM for microglia and astrocytes, resp.).
All TLR ligands were obtained from InvivoGen (San Diego,
Calif, USA).Table 1
Summary of PPAR-γ effects on PAMP-activated microglia
reported in this study.
TLR ligandPPAR-γ agonistPam3CyspolyI:CLPSFlagellinssRNAODN15d-PGJ2↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ TNF-α↓ TNF-α↓ TNF-α↓ TNF-αTNF-α ND*↓ TNF-α↓ CXCL2↑ CXCL2↑ CXCL2No effect CXCL2No effect CXCL2↑ CXCL2↓ CCL2↓ CCL2↓ CCL2↓ CCL2No effect CCL2↓ CCL2Rosiglitazone↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ TNF-α↓ TNF-αNo effect TNF-α↓ TNF-αTNF-α NDNo effect TNF-αPioglitazone↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↑ CXCL2↑ CXCL2*ND; not detectable.The natural PPAR-γ agonist 15d-PGJ2 and
synthetic TZDs ciglitazone, rosiglitazone, pioglitazone, and troglitazone were
purchased from Cayman Chemical (see Table 2; PPAR-γ Pak; Ann Arbor,
Mich, USA).
Dose-response studies were performed for all TZDs (10, 30, and 100 μM) and 15d- PGJ2 (5, 10, and
20 μM) in both TLR-activated microglia and
astrocytes.Table 2
Summary of PPAR-γ effects on PAMP-activated astrocytes
reported in this study.
TLR ligandPPAR-γ agonistPam3CyspolyI:CLPSFlagellinssRNAODN↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-1215d-PGJ2↑ CXCL2↓ NO↓ NO↑ CXCL2Ciglitazone↓ CXCL2↓ CXCL2↓ CXCL2↓ CXCL2NR*↓ CXCL2Pioglitazone↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↑ CXCL2↑ CXCL2↑ CXCL2↑ CCL2↑ CCL2Troglitazone↓ CCL2↓ CCL2NR↓ CCL2NR↓ CCL2*NR: not reported.


### 2.2. Primary Microglia and Astrocyte Cultures

Primary microglia and astrocytes were isolated from C57BL/6
pups (2 to 4 days of age) as previously described [35, 36]. The purity of glial cultures
was evaluated by immunohistochemical staining using antibodies against CD11b
(BD Pharmingen) and glial fibrillary acidic protein (GFAP, Invitrogen, Carlsbad, Calif, USA) to
identify microglia and astrocytes, respectively. The purity of primary microglia
and astrocyte cultures was approximately 98% and 95%, respectively.Throughout this study, microglia and
astrocytes were seeded into 96-well plates at2×105 or 1×105 cells/well, respectively, and incubated overnight. The following day, glia were
pretreated with various PPAR-γ agonists for 1 hour prior to
stimulation with the TLR ligand panel for 24 hours. Cell-conditioned
supernatants were collected at 24 hours following TLR ligand treatment for
quantitation of proinflammatory mediator expression by ELISA.


### 2.3. Cell Viability Assays

The effects of PPAR-γ and TLR agonists on glial cell
viability were evaluated using a standard MTT assay based upon the
mitochondrial conversion of
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (MTT) into formazan crystals. Results are reported as the raw OD570 values (mean±SD).


### 2.4. Enzyme-Linked Immunosorbent Assays (ELISAs)

Protein
levels of TNF-α and CXCL2 (MIP-2) (Biosource) and IL-12 p40 and CCL2 (MCP-1, OptEIA, BD
Pharmingen, San Jose,
Calif, USA) were quantified in conditioned medium from PPAR-γ and TLR ligand-treated glia using ELISA
kits according to the manufacturer’s instructions (level of sensitivity = 15.6 pg/ml).


### 2.5. Nitrite Assay

Nitrite levels, a stable end product resulting from the reaction of
NO with molecular oxygen, were determined in astrocytes by adding 50μl of
cell-conditioned culture medium with 50 μl of Griess reagent (0.1%
naphtyletylenediamine dihydrochloride, 1% sulfanilamide, 2.5% phosphoric acid,
all from Sigma) in a 96-well plate. The absorbance at 550 nm was measured
on a plate reader (Spectra Max 190, Molecular Devices, Sunnyvale,
Calif, USA), and nitrite concentrations were calculated using a
standard curve with sodium nitrite (NaNO2, Sigma, level of
sensitivity, 0.4 μM). Based on our previous
findings where S. aureus-derived TLR
ligands were potent inducers of NO in astrocytes but not microglia [24, 33, 34], we only quantitated NO
levels in the former in the current study.


### 2.6. Statistics

Significant
differences between experimental groups were determined by the Student’st-test
at the 95% confidence interval using SigmaStat (SPSS Science, Chicago, Ill, USA).In this study, we performed a minimum of
two independent replicates of each experiment to confirm the results obtained. The
reporting of our results as representative of “x” number of independent
experiments was required since it is difficult to achieve identical levels of
proinflammatory mediator expression with distinct preparations of primary glia.
As a result, the absolute concentrations of the various proinflammatory
mediators differed between individual experiments; however, the trends were
consistent. This required us to report results from a single study where each
experimental treatment was represented by 3-4 individual
wells (i.e., biological replicates) and statistical analysis conducted.


## 2.1. TLR Ligands and PPAR-γ Agonists

The following TLR agonists were used in this study with  (see Table1)  the concentration of each and its TLR target identified in parenthesis:
Pam3CSK4 (TLR2, 1 μg/ml), polyinosine-polycytidylic acid
(polyI:C; TLR3, 25 μg/ml), lipopolysaccharide from E. coli O111:B4 (LPS; TLR4, 100 ng/ml),
flagellin from Salmonella typhimurium (TLR5, 10 μg/ml), single-stranded RNA (ssRNA; TLR7/8,
10 μg/ml), and synthetic unmethylated CpG oligonucleotide
(ODN; TLR9, 0.1 μM and 5 μM for microglia and astrocytes, resp.).
All TLR ligands were obtained from InvivoGen (San Diego,
Calif, USA).Table 1
Summary of PPAR-γ effects on PAMP-activated microglia
reported in this study.
TLR ligandPPAR-γ agonistPam3CyspolyI:CLPSFlagellinssRNAODN15d-PGJ2↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ TNF-α↓ TNF-α↓ TNF-α↓ TNF-αTNF-α ND*↓ TNF-α↓ CXCL2↑ CXCL2↑ CXCL2No effect CXCL2No effect CXCL2↑ CXCL2↓ CCL2↓ CCL2↓ CCL2↓ CCL2No effect CCL2↓ CCL2Rosiglitazone↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ TNF-α↓ TNF-αNo effect TNF-α↓ TNF-αTNF-α NDNo effect TNF-αPioglitazone↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↑ CXCL2↑ CXCL2*ND; not detectable.The natural PPAR-γ agonist 15d-PGJ2 and
synthetic TZDs ciglitazone, rosiglitazone, pioglitazone, and troglitazone were
purchased from Cayman Chemical (see Table 2; PPAR-γ Pak; Ann Arbor,
Mich, USA).
Dose-response studies were performed for all TZDs (10, 30, and 100 μM) and 15d- PGJ2 (5, 10, and
20 μM) in both TLR-activated microglia and
astrocytes.Table 2
Summary of PPAR-γ effects on PAMP-activated astrocytes
reported in this study.
TLR ligandPPAR-γ agonistPam3CyspolyI:CLPSFlagellinssRNAODN↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-1215d-PGJ2↑ CXCL2↓ NO↓ NO↑ CXCL2Ciglitazone↓ CXCL2↓ CXCL2↓ CXCL2↓ CXCL2NR*↓ CXCL2Pioglitazone↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↓ IL-12↑ CXCL2↑ CXCL2↑ CXCL2↑ CCL2↑ CCL2Troglitazone↓ CCL2↓ CCL2NR↓ CCL2NR↓ CCL2*NR: not reported.


## 2.2. Primary Microglia and Astrocyte Cultures

Primary microglia and astrocytes were isolated from C57BL/6
pups (2 to 4 days of age) as previously described [35, 36]. The purity of glial cultures
was evaluated by immunohistochemical staining using antibodies against CD11b
(BD Pharmingen) and glial fibrillary acidic protein (GFAP, Invitrogen, Carlsbad, Calif, USA) to
identify microglia and astrocytes, respectively. The purity of primary microglia
and astrocyte cultures was approximately 98% and 95%, respectively.Throughout this study, microglia and
astrocytes were seeded into 96-well plates at2×105 or 1×105 cells/well, respectively, and incubated overnight. The following day, glia were
pretreated with various PPAR-γ agonists for 1 hour prior to
stimulation with the TLR ligand panel for 24 hours. Cell-conditioned
supernatants were collected at 24 hours following TLR ligand treatment for
quantitation of proinflammatory mediator expression by ELISA.


## 2.3. Cell Viability Assays

The effects of PPAR-γ and TLR agonists on glial cell
viability were evaluated using a standard MTT assay based upon the
mitochondrial conversion of
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (MTT) into formazan crystals. Results are reported as the raw OD570 values (mean±SD).


## 2.4. Enzyme-Linked Immunosorbent Assays (ELISAs)

Protein
levels of TNF-α and CXCL2 (MIP-2) (Biosource) and IL-12 p40 and CCL2 (MCP-1, OptEIA, BD
Pharmingen, San Jose,
Calif, USA) were quantified in conditioned medium from PPAR-γ and TLR ligand-treated glia using ELISA
kits according to the manufacturer’s instructions (level of sensitivity = 15.6 pg/ml).


## 2.5. Nitrite Assay

Nitrite levels, a stable end product resulting from the reaction of
NO with molecular oxygen, were determined in astrocytes by adding 50μl of
cell-conditioned culture medium with 50 μl of Griess reagent (0.1%
naphtyletylenediamine dihydrochloride, 1% sulfanilamide, 2.5% phosphoric acid,
all from Sigma) in a 96-well plate. The absorbance at 550 nm was measured
on a plate reader (Spectra Max 190, Molecular Devices, Sunnyvale,
Calif, USA), and nitrite concentrations were calculated using a
standard curve with sodium nitrite (NaNO2, Sigma, level of
sensitivity, 0.4 μM). Based on our previous
findings where S. aureus-derived TLR
ligands were potent inducers of NO in astrocytes but not microglia [24, 33, 34], we only quantitated NO
levels in the former in the current study.


## 2.6. Statistics

Significant
differences between experimental groups were determined by the Student’st-test
at the 95% confidence interval using SigmaStat (SPSS Science, Chicago, Ill, USA).In this study, we performed a minimum of
two independent replicates of each experiment to confirm the results obtained. The
reporting of our results as representative of “x” number of independent
experiments was required since it is difficult to achieve identical levels of
proinflammatory mediator expression with distinct preparations of primary glia.
As a result, the absolute concentrations of the various proinflammatory
mediators differed between individual experiments; however, the trends were
consistent. This required us to report results from a single study where each
experimental treatment was represented by 3-4 individual
wells (i.e., biological replicates) and statistical analysis conducted.


## 3. Results

### 3.1. Ability of PPAR-γ
Agonists to Modulate Microglial Cytokine Production in Response to Diverse TLR Ligands

Microglia represent the main innate immune effector in the
CNS parenchyma as evident by their expression of numerous TLRs [32, 37, 38]. Although much emphasis has
been placed on the neurodestructive properties of activated microglia, recent
studies have revealed that in the correct context microglia can also facilitate
CNS repair [39–41]. Therefore, striking the
correct balance between regulated and inappropriate microglial activation may
lead to optimal outcomes for a wide range of CNS neuroinflammatory conditions.
To determine whether PPAR-γ agonists could serve to modulate
microglial activation in the context of CNS infection, the effects of these
compounds on microglial cytokine production in response to diverse TLR ligands
were examined. The natural PPAR-γ agonist 15d-PGJ2 was
uniformly found to inhibit IL-12 p40 release in response to all TLR ligands
tested including Pam3Cys4, polyI:C, LPS, flagellin, ssRNA, and ODN (Figure 1). Fairly
comprehensive reductions in IL-12 p40 production were also observed with all
TLR ligands tested in response to the synthetic PPAR-γ agonists rosiglitazone (Figure 2) and
pioglitazone (Figure 3) although the extent of inhibition was dramatically less
compared to 15d-PGJ2. Rosiglitazone exhibited significant toxicity
to primary microglia at the highest dose tested (i.e., 100 μM), hence it was not included in the
final analysis. In contrast, ciglitazone did not dramatically affect IL-12 p40
production in response to the majority of TLR ligands examined (data not
shown). Troglitazone exhibited microglial toxicity at the two highest doses of
agonist tested (i.e., 100 and 30 μM); therefore, the results of this PPAR-γ agonist on microglial mediator
production in response to TLR ligands are not presented.The natural PPAR-γ agonist 15d-PGJ2 is a potent
inhibitor of microglial IL-12 p40 production in response to a vast array of TLR
ligands. Primary microglia were plated at 2×105 cells/well in
96-well plates and incubated overnight. The following day, cells were
pretreated for 1 hour with the indicated concentrations of 15d-PGJ2 prior to the addition of TLR ligands. Cell-conditioned supernatants were
collected at 24 hours following TLR ligand exposure, whereupon IL-12 p40 levels
were determined by ELISA (a). The effects of 15d-PGJ2 on microglial
viability were assessed using an MTT assay (b). Significant differences between
microglia treated with TLR ligands alone versus TLR ligands + 15d-PGJ2 are noted with asterisks (**P<.001). Results presented are
representative of two independent experiments.
(a)(b)The synthetic PPAR-γ agonist rosiglitazone attenuates IL-12
p40 production in response to TLR ligands in microglia. Primary microglia
were plated at 2×105 cells/well in 96-well plates and incubated
overnight. The following day, cells were pretreated for 1 hour with the
indicated concentrations of rosiglitazone prior to the addition of TLR ligands.
Cell-conditioned supernatants were collected at 24 hours following TLR ligand
exposure, whereupon IL-12 p40 levels were determined by ELISA (a). The effects
of rosiglitazone on microglial viability were assessed using an MTT assay (b).
Significant differences between microglia treated with TLR ligands alone versus
TLR ligands + rosiglitazone are noted with asterisks (*P<.05). Results presented are representative of two independent experiments.
(a)(b)The TZD pioglitazone inhibits microglial IL-12 p40 expression
in response to diverse TLR ligands. Primary microglia were plated at 2×105 cells/well in 96-well plates and incubated overnight. The following day, cells
were pretreated for 1 hour with the indicated concentrations of pioglitazone
prior to the addition of TLR ligands. Cell-conditioned supernatants were
collected at 24 hours following TLR ligand exposure, whereupon IL-12 p40 levels
were determined by ELISA (a). The effects of pioglitazone on microglial
viability were assessed using an MTT assay (b). Significant differences between
microglia treated with TLR ligands alone versus TLR ligands + pioglitazone are
noted with asterisks (*P<.05;**P<.001). Results presented are representative of two
independent experiments.
(a)(b)Another proinflammatory cytokine with
potent effects on the blood-brain barrier as well as glial activation is TNF-α [42]. This cytokine is expressed
at high levels in numerous CNS infectious diseases including bacterial
meningitis, brain abscess, as well as viral infections [43–46]. In some cases, excessive
TNF-α production during these infectious
diseases has been implicated in contributing to bystander damage to surrounding
host tissue [45, 46]. Therefore, strategies aimed
at achieving optimized cytokine expression may prove beneficial for favorable
disease outcomes. Of the PPAR-γ agonists tested, 15d-PGJ2 was found to exert the most global inhibition of TNF-α production in response to the battery
of TLR ligands tested (Figure 4). Specifically, TNF-α release by microglia in response to
Pam3Cys4, polyI:C, flagellin, and ODN was significantly attenuated by 15d-PGJ2,
whereas cytokine production following LPS treatment was not as dramatically
affected. Single-stranded RNA (ssRNA) was a poor inducer of TNF-α by primary microglia (Figure 4).
Similar to results with IL-12 p40, rosiglitazone was the next more global
inhibitor of TNF-α production in response to the TLR
ligands tested (Figure 5), whereas the other PPAR-γ agonists (i.e., ciglitazone and
pioglitazone) were largely without effect (data not shown). Collectively, these
results indicate that not all PPAR-γ agonists are equally effective at
modulating proinflammatory cytokine release from primary microglia and suggest
that tailored responses to specific pathogen motifs may be achieved through the
use of distinct PPAR-γ agonists.Figure 4
The potency of 15d-PGJ2 to attenuate TNF-α production varies according to the TLR
ligand examined. Primary microglia were plated at 2×105 cells/well in 96-well plates and incubated overnight. The following day, cells
were pretreated for 1 hour with the indicated concentrations of 15d-PGJ2 prior to the addition of TLR ligands. Cell-conditioned supernatants were
collected at 24 hours following TLR ligand exposure, whereupon TNF-α levels were determined by ELISA.
Significant differences between microglia treated with TLR ligands alone versus
TLR ligands + 15d-PGJ2 are noted with asterisks (*P<.05;**P<.001). Results
presented are representative of two independent experiments.Figure 5
The synthetic PPAR-γ agonist rosiglitazone selectively
inhibits microglial TNF-α expression in response to TLR ligands. Primary microglia were plated at 2×105 cells/well in 96-well
plates and incubated overnight. The following day, cells were pretreated for 1 hour with the indicated concentrations of rosiglitazone prior to the addition
of TLR ligands. Cell-conditioned supernatants were collected at 24 hours
following TLR ligand exposure, whereupon TNF-α levels were determined by ELISA.
Significant differences between microglia treated with TLR ligands alone versus
TLR ligands + rosiglitazone are noted with asterisks (*P<.05). Results presented are representative of two independent experiments.


### 3.2. PPAR-γ Agonists Differentially Affect Chemokine Release by Microglia Following TL Rligand Treatment

Chemokines are small molecular weight (8–14 kDa)
chemotactic cytokines that are produced locally at sites of inflammation and
establish a concentration gradient resulting in the active recruitment of
target cell populations [47]. Chemokines are a
structurally and functionally related family of proteins subdivided into four
groups based on the relative position of conserved N-terminal cysteine residues
[47–49]. In general, the chemokine
subfamilies show similar, often overlapping specificity with regards to the
movements of the target cell populations they orchestrate. One key chemokine
involved in the recruitment of neutrophils into areas of inflammation,
including the CNS, is CXCL2 (MIP-2) [50–52]. The effects of 15d-PGJ2 on microglial CXCL2 expression were complex and varied with each TLR ligand.
Specifically, CXCL2 release was either enhanced (polyI:C, LPS, and ODN),
reduced (Pam3Cys4), or remained unchanged (flagellin and ssRNA) (Figure 6). Increases
in CXCL2 production were also observed following pioglitazone treatment in
Pam3Cys- and ODN-stimulated microglia (Figure 7). The overall stimulatory
activity of 15d-PGJ2 on CXCL2 production is in agreement with reports
from other groups [53, 54].Figure 6
15d-PGJ2 demonstrates differential effects on
CXCL2 production by microglia, which are TLR ligand-dependent. Primary
microglia were plated at 2×105 cells/well in 96-well plates and
incubated overnight. The following day, cells were pretreated for 1 hour with
the indicated concentrations of 15d-PGJ2 prior to the addition of
TLR ligands. Cell-conditioned supernatants were collected at 24 hours following
TLR ligand exposure, whereupon CXCL2 levels were determined by ELISA.
Significant differences between microglia treated with TLR ligands alone versus
TLR ligands + 15d-PGJ2 are noted with asterisks (*P<.05;**P<.001). Results
presented are representative of two independent experiments.Figure 7
Pioglitazone augments microglial CXCL2 production in response
to Pam3Cys4 and ODN. Primary microglia were plated at 2×105 cells/well in 96-well plates and incubated overnight. The following day, cells
were pretreated for 1 hour with the indicated concentrations of pioglitazone
prior to the addition of Pam3Cys4 or ODN. Cell-conditioned supernatants were
collected at 24 hours following TLR ligand exposure, whereupon CXCL2 levels
were determined by ELISA. Significant differences between microglia treated
with TLR ligands alone versus TLR ligands + pioglitazone are noted with
asterisks (*P<.05;**P<.001). Results presented are representative of two independent experiments.Another chemokine that is associated with
mononuclear cell infiltration during various CNS infections is CCL2 (MCP-1),
which targets monocyte and lymphocyte entry [52, 55, 56]. Unlike CXCL2, which was
differentially regulated by 15d-PGJ2 in response to diverse TLR
ligands, CCL2 production was uniformly and potently attenuated by this PPAR-γ agonist in response to the full array
of TLR ligands tested (Figure 8). Similar to IL-12 p40 production, the
synthetic TZDs demonstrated differential effects on CCL2 release from TLR
ligand activated microglia. Specifically, rosiglitazone was fairly comprehensive
in its ability to attenuate CCL2 production with significant reductions
observed in response to Pam3Cys4, polyI:C, LPS, flagellin, and ODN, whereas the
other TZDs tested (ciglitazone and pioglitazone) did not have much effect on CCL2
release in response to the majority of TLR ligands tested (data not shown). In
summary, these results reveal that PPAR-γ agonists display a wide range of
effects on chemokine production by microglia elicited by TLR ligands.Figure 8
15d-PGJ2 is a potent inhibitor of CCL2 release by
microglia in response to a wide range of TLR ligands. Primary microglia
were plated at 2×105 cells/well in 96-well plates and incubated
overnight. The following day, cells were pretreated for 1 hour with the
indicated concentrations of 15d-PGJ2 prior to the addition of TLR
ligands. Cell-conditioned supernatants were collected at 24 hours following TLR
ligand exposure, whereupon CCL2 levels were determined by ELISA. Significant
differences between microglia treated with TLR ligands alone versus TLR ligands
+ 15d-PGJ2 are noted with asterisks (*P<.05;**P<.001). Results presented are
representative of two independent experiments.


### 3.3. Effects of PPAR-γ
Agonists on Astrocytic Proinflammatory Mediator Production in Response to TLR Ligands

Astrocytes participate in CNS innate immune responses as
evident by their ability to produce a wide array of inflammatory mediators in
response to diverse stimuli [1, 4]. As already mentioned, these
molecules can have dramatic consequences on CNS infection and tissue damage
with net effects dictated by factors such as timing and duration of release. To
determine the effects of PPAR-γ agonists on astrocyte responses to TLR
ligands, we examined the production of two proinflammatory mediators produced
by activated astrocytes, namely, NO and IL-12 p40. Both polyI:C and LPS were
capable of reproducibly inducing NO expression in astrocytes as previously
described [31, 57], which was attenuated by 15d-PGJ2 in a dose-dependent manner (Figure 9). Similar inhibitory effects on astrocytic
NO release in response to polyI:C and LPS were observed with troglitazone and
ciglitazone, whereas rosiglitazone and pioglitazone did not modulate NO
production (data not shown). Of note was the fact that unlike microglia, which
exhibited significant cell death in response to the highest dose of 15d-PGJ2 tested (i.e., 20 μM), astrocyte viability was not adversely
affected by 15d-PGJ2 at any of the concentrations examined. This
finding is in agreement with previous reports demonstrating that, in general,
primary astrocytes are more refractory to the toxic effects of PPAR-γ agonists compared to primary microglia [23, 24, 27].15d-PGJ2 attenuates NO production by astrocytes in
response to polyI:C and LPS stimulation. Primary astrocytes were plated at 1×105 cells/well in 96-well plates and incubated overnight. The
following day, cells were pretreated for 1 hour with the indicated
concentrations of 15d-PGJ2 prior to the addition of polyI:C or LPS.
Cell-conditioned supernatants were collected at 24 hours following TLR ligand
exposure, whereupon NO levels were determined by the Griess reagent (a). The
effects of 15d-PGJ2 on astrocyte viability were assessed using an
MTT assay (b). Significant differences between astrocytes treated with TLR
ligands alone versus TLR ligands + 15d-PGJ2 are noted with asterisks (*P<.05;**P<.001). Results presented are representative of two independent experiments.
(a)(b)Similar to the findings obtained with microglia,
15d-PGJ2 was a universal and potent inhibitor of IL-12 p40
production by astrocytes in response to all TLR ligands tested (Figure 10). In
addition, both pioglitazone and troglitazone were capable of attenuating IL-12
p40 expression in response to the full array of TLR ligands examined (Figure 11
and data not shown), whereas the effects of ciglitazone were variable.15d-PGJ2 is a global inhibitor of astrocytic
IL-12 p40 release following TLR ligand exposure. Primary astrocytes were
plated at 1×105 cells/well in 96-well plates and incubated
overnight. The following day, cells were pretreated for 1 hour with the
indicated concentrations of 15d-PGJ2 prior to the addition of TLR
ligands. Cell-conditioned supernatants were collected at 24 hours following TLR
ligand exposure, whereupon IL-12 p40 levels were determined by ELISA (a) and (b).
Significant differences between astrocytes treated with TLR ligands alone
versus TLR ligands + 15d-PGJ2 are noted with asterisks (*P<.05;**P<.001). Results
presented are representative of two independent experiments.
(a)(b)The TZD pioglitazone inhibits astrocytic IL-12 p40
expression in response to diverse TLR ligands. Primary astrocytes were
plated at 1×105 cells/well in 96-well plates and incubated
overnight. The following day, cells were pretreated for 1 hour with the
indicated concentrations of pioglitazone prior to the addition of TLR ligands.
Cell-conditioned supernatants were collected at 24 hours following TLR ligand
exposure, whereupon IL-12 p40 levels were determined by ELISA (a). The effects
of pioglitazone on astrocyte viability were assessed using an MTT assay (b).
Significant differences between astrocytes treated with TLR ligands alone
versus TLR ligands + pioglitazone are noted with asterisks (*P<.05;**P<.001). Results
presented are representative of two independent experiments.
(a)(b)


### 3.4. PPAR-γ
Agonists Modulate Chemokine Production by Astrocytes Following TLR Ligand Exposure

Although astrocytes are capable of releasing cytokines in response to diverse antigens, they are often considered the major
source of chemokines during CNS inflammation [1, 4]. Similar to our recent
report, 15d-PGJ2 slightly augmented CXCL2 production by astrocytes
in response to several TLR ligands examined, namely Pam3Cys4, PGN, and LPS (Figure 12 and data not shown) [24]. Similar increases in CXCL2
release following PPAR-γ agonist exposure have also been
reported by others [53, 54]. In contrast, each synthetic
TZD appeared to differentially regulate CXCL2 release. For example, ciglitazone
inhibited CXCL2 expression in response to the entire battery of TLR ligands
examined (Figure 13). Conversely, pioglitazone significantly augmented CXCL2
expression in response to polyI:C, flagellin, and ssRNA particularly at the
highest dose examined (i.e., 100 μM; Figure 14), whereas rosiglitazone did
not dramatically alter CXCL2 levels response to any of the TLR ligands tested
in astrocytes (data not shown).CXCL2 release is augmented in astrocytes by 15d-PGJ2 in response to distinct TLR ligands. Primary astrocytes were plated at 1×105 cells/well in 96-well plates and incubated overnight. The
following day, cells were pretreated for 1 hour with the indicated
concentrations of 15d-PGJ2 prior to the addition of Pam3Cys4 or LPS.
Cell-conditioned supernatants were collected at 24 hours following TLR ligand
exposure, whereupon CXCL2 levels were determined by ELISA (a). The effects of
15d-PGJ2 on astrocyte viability were assessed using an MTT assay
(b). Significant differences between astrocytes treated with TLR ligands alone
versus TLR ligands + 15d-PGJ2 are noted with asterisks (*P<.05;**P<.001). Results
presented are representative of two independent experiments.
(a)(b)Ciglitazone attenuates astrocytic CXCL2 expression in
response to several TLR ligands. Primary astrocytes were plated at 1×105 cells/well in 96-well plates and incubated overnight. The following day, cells
were pretreated for 1 hour with the indicated concentrations of ciglitazone
prior to the addition of TLR ligands. Cell-conditioned supernatants were
collected at 24 hours following TLR ligand exposure, whereupon CXCL2 levels
were determined by ELISA (a). The effects of ciglitazone on astrocyte viability
were assessed using an MTT assay (b). Significant differences between
astrocytes treated with TLR ligands alone versus TLR ligands + ciglitazone are
noted with asterisks (*P<.05;**P<.001). Results presented are representative of two
independent experiments.
(a)(b)Figure 14
Pioglitazone enhances CXCL2 release by astrocytes following
TLR ligand exposure. Primary astrocytes were plated at 1×105 cells/well in 96-well plates and incubated overnight. The following day, cells
were pretreated for 1 hour with the indicated concentrations of pioglitazone
prior to the addition of polyI:C, flagellin, or ssRNA. Cell-conditioned supernatants
were collected at 24 hours following TLR ligand exposure, whereupon CXCL2
levels were determined by ELISA. Significant differences between astrocytes
treated with TLR ligands alone versus TLR ligands + pioglitazone are noted with
asterisks (*P<.05;**P<.001). Results presented are representative of two independent experiments.Similar to CXCL2, pioglitazone treatment
increased CCL2 production in response to flagellin, and ssRNA primarily at the
highest dose tested, whereas troglitazone led to significant reductions in CCL2
release following Pam3Cys4, polyI:C, flagellin, and ODN treatment (Figure15).
In general, ciglitazone and rosiglitazone had little effect on CCL2 production
by astrocytes in response to the majority of TLR ligands tested (data not
shown). These results indicate that despite their inclusion within the same
family, specific PPAR-γ agonists differentially target
proinflammatory genes in distinct manners.TZDs exert differential effects on astrocytic CCL2
production following TLR ligand treatment. Primary astrocytes were plated
at 1×105 cells/well in 96-well plates and incubated overnight. The
following day, cells were pretreated for 1 hour with the indicated
concentrations of pioglitazone (a) or troglitazone, (b) prior to the addition
of TLR ligands. Cell-conditioned supernatants were collected at 24 hours
following TLR ligand exposure, whereupon CCL2 levels were determined by ELISA
(a) and (b). Significant differences between astrocytes treated with TLR ligands
alone versus TLR ligands + TZDs are noted with asterisks (*P<.05;**P<.001). Results
presented are representative of two independent experiments.
(a)(b)


## 3.1. Ability of PPAR-γ
Agonists to Modulate Microglial Cytokine Production in Response to Diverse TLR Ligands

Microglia represent the main innate immune effector in the
CNS parenchyma as evident by their expression of numerous TLRs [32, 37, 38]. Although much emphasis has
been placed on the neurodestructive properties of activated microglia, recent
studies have revealed that in the correct context microglia can also facilitate
CNS repair [39–41]. Therefore, striking the
correct balance between regulated and inappropriate microglial activation may
lead to optimal outcomes for a wide range of CNS neuroinflammatory conditions.
To determine whether PPAR-γ agonists could serve to modulate
microglial activation in the context of CNS infection, the effects of these
compounds on microglial cytokine production in response to diverse TLR ligands
were examined. The natural PPAR-γ agonist 15d-PGJ2 was
uniformly found to inhibit IL-12 p40 release in response to all TLR ligands
tested including Pam3Cys4, polyI:C, LPS, flagellin, ssRNA, and ODN (Figure 1). Fairly
comprehensive reductions in IL-12 p40 production were also observed with all
TLR ligands tested in response to the synthetic PPAR-γ agonists rosiglitazone (Figure 2) and
pioglitazone (Figure 3) although the extent of inhibition was dramatically less
compared to 15d-PGJ2. Rosiglitazone exhibited significant toxicity
to primary microglia at the highest dose tested (i.e., 100 μM), hence it was not included in the
final analysis. In contrast, ciglitazone did not dramatically affect IL-12 p40
production in response to the majority of TLR ligands examined (data not
shown). Troglitazone exhibited microglial toxicity at the two highest doses of
agonist tested (i.e., 100 and 30 μM); therefore, the results of this PPAR-γ agonist on microglial mediator
production in response to TLR ligands are not presented.The natural PPAR-γ agonist 15d-PGJ2 is a potent
inhibitor of microglial IL-12 p40 production in response to a vast array of TLR
ligands. Primary microglia were plated at 2×105 cells/well in
96-well plates and incubated overnight. The following day, cells were
pretreated for 1 hour with the indicated concentrations of 15d-PGJ2 prior to the addition of TLR ligands. Cell-conditioned supernatants were
collected at 24 hours following TLR ligand exposure, whereupon IL-12 p40 levels
were determined by ELISA (a). The effects of 15d-PGJ2 on microglial
viability were assessed using an MTT assay (b). Significant differences between
microglia treated with TLR ligands alone versus TLR ligands + 15d-PGJ2 are noted with asterisks (**P<.001). Results presented are
representative of two independent experiments.
(a)(b)The synthetic PPAR-γ agonist rosiglitazone attenuates IL-12
p40 production in response to TLR ligands in microglia. Primary microglia
were plated at 2×105 cells/well in 96-well plates and incubated
overnight. The following day, cells were pretreated for 1 hour with the
indicated concentrations of rosiglitazone prior to the addition of TLR ligands.
Cell-conditioned supernatants were collected at 24 hours following TLR ligand
exposure, whereupon IL-12 p40 levels were determined by ELISA (a). The effects
of rosiglitazone on microglial viability were assessed using an MTT assay (b).
Significant differences between microglia treated with TLR ligands alone versus
TLR ligands + rosiglitazone are noted with asterisks (*P<.05). Results presented are representative of two independent experiments.
(a)(b)The TZD pioglitazone inhibits microglial IL-12 p40 expression
in response to diverse TLR ligands. Primary microglia were plated at 2×105 cells/well in 96-well plates and incubated overnight. The following day, cells
were pretreated for 1 hour with the indicated concentrations of pioglitazone
prior to the addition of TLR ligands. Cell-conditioned supernatants were
collected at 24 hours following TLR ligand exposure, whereupon IL-12 p40 levels
were determined by ELISA (a). The effects of pioglitazone on microglial
viability were assessed using an MTT assay (b). Significant differences between
microglia treated with TLR ligands alone versus TLR ligands + pioglitazone are
noted with asterisks (*P<.05;**P<.001). Results presented are representative of two
independent experiments.
(a)(b)Another proinflammatory cytokine with
potent effects on the blood-brain barrier as well as glial activation is TNF-α [42]. This cytokine is expressed
at high levels in numerous CNS infectious diseases including bacterial
meningitis, brain abscess, as well as viral infections [43–46]. In some cases, excessive
TNF-α production during these infectious
diseases has been implicated in contributing to bystander damage to surrounding
host tissue [45, 46]. Therefore, strategies aimed
at achieving optimized cytokine expression may prove beneficial for favorable
disease outcomes. Of the PPAR-γ agonists tested, 15d-PGJ2 was found to exert the most global inhibition of TNF-α production in response to the battery
of TLR ligands tested (Figure 4). Specifically, TNF-α release by microglia in response to
Pam3Cys4, polyI:C, flagellin, and ODN was significantly attenuated by 15d-PGJ2,
whereas cytokine production following LPS treatment was not as dramatically
affected. Single-stranded RNA (ssRNA) was a poor inducer of TNF-α by primary microglia (Figure 4).
Similar to results with IL-12 p40, rosiglitazone was the next more global
inhibitor of TNF-α production in response to the TLR
ligands tested (Figure 5), whereas the other PPAR-γ agonists (i.e., ciglitazone and
pioglitazone) were largely without effect (data not shown). Collectively, these
results indicate that not all PPAR-γ agonists are equally effective at
modulating proinflammatory cytokine release from primary microglia and suggest
that tailored responses to specific pathogen motifs may be achieved through the
use of distinct PPAR-γ agonists.Figure 4
The potency of 15d-PGJ2 to attenuate TNF-α production varies according to the TLR
ligand examined. Primary microglia were plated at 2×105 cells/well in 96-well plates and incubated overnight. The following day, cells
were pretreated for 1 hour with the indicated concentrations of 15d-PGJ2 prior to the addition of TLR ligands. Cell-conditioned supernatants were
collected at 24 hours following TLR ligand exposure, whereupon TNF-α levels were determined by ELISA.
Significant differences between microglia treated with TLR ligands alone versus
TLR ligands + 15d-PGJ2 are noted with asterisks (*P<.05;**P<.001). Results
presented are representative of two independent experiments.Figure 5
The synthetic PPAR-γ agonist rosiglitazone selectively
inhibits microglial TNF-α expression in response to TLR ligands. Primary microglia were plated at 2×105 cells/well in 96-well
plates and incubated overnight. The following day, cells were pretreated for 1 hour with the indicated concentrations of rosiglitazone prior to the addition
of TLR ligands. Cell-conditioned supernatants were collected at 24 hours
following TLR ligand exposure, whereupon TNF-α levels were determined by ELISA.
Significant differences between microglia treated with TLR ligands alone versus
TLR ligands + rosiglitazone are noted with asterisks (*P<.05). Results presented are representative of two independent experiments.


## 3.2. PPAR-γ Agonists Differentially Affect Chemokine Release by Microglia Following TL Rligand Treatment

Chemokines are small molecular weight (8–14 kDa)
chemotactic cytokines that are produced locally at sites of inflammation and
establish a concentration gradient resulting in the active recruitment of
target cell populations [47]. Chemokines are a
structurally and functionally related family of proteins subdivided into four
groups based on the relative position of conserved N-terminal cysteine residues
[47–49]. In general, the chemokine
subfamilies show similar, often overlapping specificity with regards to the
movements of the target cell populations they orchestrate. One key chemokine
involved in the recruitment of neutrophils into areas of inflammation,
including the CNS, is CXCL2 (MIP-2) [50–52]. The effects of 15d-PGJ2 on microglial CXCL2 expression were complex and varied with each TLR ligand.
Specifically, CXCL2 release was either enhanced (polyI:C, LPS, and ODN),
reduced (Pam3Cys4), or remained unchanged (flagellin and ssRNA) (Figure 6). Increases
in CXCL2 production were also observed following pioglitazone treatment in
Pam3Cys- and ODN-stimulated microglia (Figure 7). The overall stimulatory
activity of 15d-PGJ2 on CXCL2 production is in agreement with reports
from other groups [53, 54].Figure 6
15d-PGJ2 demonstrates differential effects on
CXCL2 production by microglia, which are TLR ligand-dependent. Primary
microglia were plated at 2×105 cells/well in 96-well plates and
incubated overnight. The following day, cells were pretreated for 1 hour with
the indicated concentrations of 15d-PGJ2 prior to the addition of
TLR ligands. Cell-conditioned supernatants were collected at 24 hours following
TLR ligand exposure, whereupon CXCL2 levels were determined by ELISA.
Significant differences between microglia treated with TLR ligands alone versus
TLR ligands + 15d-PGJ2 are noted with asterisks (*P<.05;**P<.001). Results
presented are representative of two independent experiments.Figure 7
Pioglitazone augments microglial CXCL2 production in response
to Pam3Cys4 and ODN. Primary microglia were plated at 2×105 cells/well in 96-well plates and incubated overnight. The following day, cells
were pretreated for 1 hour with the indicated concentrations of pioglitazone
prior to the addition of Pam3Cys4 or ODN. Cell-conditioned supernatants were
collected at 24 hours following TLR ligand exposure, whereupon CXCL2 levels
were determined by ELISA. Significant differences between microglia treated
with TLR ligands alone versus TLR ligands + pioglitazone are noted with
asterisks (*P<.05;**P<.001). Results presented are representative of two independent experiments.Another chemokine that is associated with
mononuclear cell infiltration during various CNS infections is CCL2 (MCP-1),
which targets monocyte and lymphocyte entry [52, 55, 56]. Unlike CXCL2, which was
differentially regulated by 15d-PGJ2 in response to diverse TLR
ligands, CCL2 production was uniformly and potently attenuated by this PPAR-γ agonist in response to the full array
of TLR ligands tested (Figure 8). Similar to IL-12 p40 production, the
synthetic TZDs demonstrated differential effects on CCL2 release from TLR
ligand activated microglia. Specifically, rosiglitazone was fairly comprehensive
in its ability to attenuate CCL2 production with significant reductions
observed in response to Pam3Cys4, polyI:C, LPS, flagellin, and ODN, whereas the
other TZDs tested (ciglitazone and pioglitazone) did not have much effect on CCL2
release in response to the majority of TLR ligands tested (data not shown). In
summary, these results reveal that PPAR-γ agonists display a wide range of
effects on chemokine production by microglia elicited by TLR ligands.Figure 8
15d-PGJ2 is a potent inhibitor of CCL2 release by
microglia in response to a wide range of TLR ligands. Primary microglia
were plated at 2×105 cells/well in 96-well plates and incubated
overnight. The following day, cells were pretreated for 1 hour with the
indicated concentrations of 15d-PGJ2 prior to the addition of TLR
ligands. Cell-conditioned supernatants were collected at 24 hours following TLR
ligand exposure, whereupon CCL2 levels were determined by ELISA. Significant
differences between microglia treated with TLR ligands alone versus TLR ligands
+ 15d-PGJ2 are noted with asterisks (*P<.05;**P<.001). Results presented are
representative of two independent experiments.


## 3.3. Effects of PPAR-γ
Agonists on Astrocytic Proinflammatory Mediator Production in Response to TLR Ligands

Astrocytes participate in CNS innate immune responses as
evident by their ability to produce a wide array of inflammatory mediators in
response to diverse stimuli [1, 4]. As already mentioned, these
molecules can have dramatic consequences on CNS infection and tissue damage
with net effects dictated by factors such as timing and duration of release. To
determine the effects of PPAR-γ agonists on astrocyte responses to TLR
ligands, we examined the production of two proinflammatory mediators produced
by activated astrocytes, namely, NO and IL-12 p40. Both polyI:C and LPS were
capable of reproducibly inducing NO expression in astrocytes as previously
described [31, 57], which was attenuated by 15d-PGJ2 in a dose-dependent manner (Figure 9). Similar inhibitory effects on astrocytic
NO release in response to polyI:C and LPS were observed with troglitazone and
ciglitazone, whereas rosiglitazone and pioglitazone did not modulate NO
production (data not shown). Of note was the fact that unlike microglia, which
exhibited significant cell death in response to the highest dose of 15d-PGJ2 tested (i.e., 20 μM), astrocyte viability was not adversely
affected by 15d-PGJ2 at any of the concentrations examined. This
finding is in agreement with previous reports demonstrating that, in general,
primary astrocytes are more refractory to the toxic effects of PPAR-γ agonists compared to primary microglia [23, 24, 27].15d-PGJ2 attenuates NO production by astrocytes in
response to polyI:C and LPS stimulation. Primary astrocytes were plated at 1×105 cells/well in 96-well plates and incubated overnight. The
following day, cells were pretreated for 1 hour with the indicated
concentrations of 15d-PGJ2 prior to the addition of polyI:C or LPS.
Cell-conditioned supernatants were collected at 24 hours following TLR ligand
exposure, whereupon NO levels were determined by the Griess reagent (a). The
effects of 15d-PGJ2 on astrocyte viability were assessed using an
MTT assay (b). Significant differences between astrocytes treated with TLR
ligands alone versus TLR ligands + 15d-PGJ2 are noted with asterisks (*P<.05;**P<.001). Results presented are representative of two independent experiments.
(a)(b)Similar to the findings obtained with microglia,
15d-PGJ2 was a universal and potent inhibitor of IL-12 p40
production by astrocytes in response to all TLR ligands tested (Figure 10). In
addition, both pioglitazone and troglitazone were capable of attenuating IL-12
p40 expression in response to the full array of TLR ligands examined (Figure 11
and data not shown), whereas the effects of ciglitazone were variable.15d-PGJ2 is a global inhibitor of astrocytic
IL-12 p40 release following TLR ligand exposure. Primary astrocytes were
plated at 1×105 cells/well in 96-well plates and incubated
overnight. The following day, cells were pretreated for 1 hour with the
indicated concentrations of 15d-PGJ2 prior to the addition of TLR
ligands. Cell-conditioned supernatants were collected at 24 hours following TLR
ligand exposure, whereupon IL-12 p40 levels were determined by ELISA (a) and (b).
Significant differences between astrocytes treated with TLR ligands alone
versus TLR ligands + 15d-PGJ2 are noted with asterisks (*P<.05;**P<.001). Results
presented are representative of two independent experiments.
(a)(b)The TZD pioglitazone inhibits astrocytic IL-12 p40
expression in response to diverse TLR ligands. Primary astrocytes were
plated at 1×105 cells/well in 96-well plates and incubated
overnight. The following day, cells were pretreated for 1 hour with the
indicated concentrations of pioglitazone prior to the addition of TLR ligands.
Cell-conditioned supernatants were collected at 24 hours following TLR ligand
exposure, whereupon IL-12 p40 levels were determined by ELISA (a). The effects
of pioglitazone on astrocyte viability were assessed using an MTT assay (b).
Significant differences between astrocytes treated with TLR ligands alone
versus TLR ligands + pioglitazone are noted with asterisks (*P<.05;**P<.001). Results
presented are representative of two independent experiments.
(a)(b)


## 3.4. PPAR-γ
Agonists Modulate Chemokine Production by Astrocytes Following TLR Ligand Exposure

Although astrocytes are capable of releasing cytokines in response to diverse antigens, they are often considered the major
source of chemokines during CNS inflammation [1, 4]. Similar to our recent
report, 15d-PGJ2 slightly augmented CXCL2 production by astrocytes
in response to several TLR ligands examined, namely Pam3Cys4, PGN, and LPS (Figure 12 and data not shown) [24]. Similar increases in CXCL2
release following PPAR-γ agonist exposure have also been
reported by others [53, 54]. In contrast, each synthetic
TZD appeared to differentially regulate CXCL2 release. For example, ciglitazone
inhibited CXCL2 expression in response to the entire battery of TLR ligands
examined (Figure 13). Conversely, pioglitazone significantly augmented CXCL2
expression in response to polyI:C, flagellin, and ssRNA particularly at the
highest dose examined (i.e., 100 μM; Figure 14), whereas rosiglitazone did
not dramatically alter CXCL2 levels response to any of the TLR ligands tested
in astrocytes (data not shown).CXCL2 release is augmented in astrocytes by 15d-PGJ2 in response to distinct TLR ligands. Primary astrocytes were plated at 1×105 cells/well in 96-well plates and incubated overnight. The
following day, cells were pretreated for 1 hour with the indicated
concentrations of 15d-PGJ2 prior to the addition of Pam3Cys4 or LPS.
Cell-conditioned supernatants were collected at 24 hours following TLR ligand
exposure, whereupon CXCL2 levels were determined by ELISA (a). The effects of
15d-PGJ2 on astrocyte viability were assessed using an MTT assay
(b). Significant differences between astrocytes treated with TLR ligands alone
versus TLR ligands + 15d-PGJ2 are noted with asterisks (*P<.05;**P<.001). Results
presented are representative of two independent experiments.
(a)(b)Ciglitazone attenuates astrocytic CXCL2 expression in
response to several TLR ligands. Primary astrocytes were plated at 1×105 cells/well in 96-well plates and incubated overnight. The following day, cells
were pretreated for 1 hour with the indicated concentrations of ciglitazone
prior to the addition of TLR ligands. Cell-conditioned supernatants were
collected at 24 hours following TLR ligand exposure, whereupon CXCL2 levels
were determined by ELISA (a). The effects of ciglitazone on astrocyte viability
were assessed using an MTT assay (b). Significant differences between
astrocytes treated with TLR ligands alone versus TLR ligands + ciglitazone are
noted with asterisks (*P<.05;**P<.001). Results presented are representative of two
independent experiments.
(a)(b)Figure 14
Pioglitazone enhances CXCL2 release by astrocytes following
TLR ligand exposure. Primary astrocytes were plated at 1×105 cells/well in 96-well plates and incubated overnight. The following day, cells
were pretreated for 1 hour with the indicated concentrations of pioglitazone
prior to the addition of polyI:C, flagellin, or ssRNA. Cell-conditioned supernatants
were collected at 24 hours following TLR ligand exposure, whereupon CXCL2
levels were determined by ELISA. Significant differences between astrocytes
treated with TLR ligands alone versus TLR ligands + pioglitazone are noted with
asterisks (*P<.05;**P<.001). Results presented are representative of two independent experiments.Similar to CXCL2, pioglitazone treatment
increased CCL2 production in response to flagellin, and ssRNA primarily at the
highest dose tested, whereas troglitazone led to significant reductions in CCL2
release following Pam3Cys4, polyI:C, flagellin, and ODN treatment (Figure15).
In general, ciglitazone and rosiglitazone had little effect on CCL2 production
by astrocytes in response to the majority of TLR ligands tested (data not
shown). These results indicate that despite their inclusion within the same
family, specific PPAR-γ agonists differentially target
proinflammatory genes in distinct manners.TZDs exert differential effects on astrocytic CCL2
production following TLR ligand treatment. Primary astrocytes were plated
at 1×105 cells/well in 96-well plates and incubated overnight. The
following day, cells were pretreated for 1 hour with the indicated
concentrations of pioglitazone (a) or troglitazone, (b) prior to the addition
of TLR ligands. Cell-conditioned supernatants were collected at 24 hours
following TLR ligand exposure, whereupon CCL2 levels were determined by ELISA
(a) and (b). Significant differences between astrocytes treated with TLR ligands
alone versus TLR ligands + TZDs are noted with asterisks (*P<.05;**P<.001). Results
presented are representative of two independent experiments.
(a)(b)


## 4. Discussion

In order for the CNS to respond to
infectious insults, a rapid and efficient host immune response must be
initiated and directed to expedite pathogen elimination. One means to achieve
this goal is through the triggering of TLRs expressed on resident glia that
signal proinflammatory mediator release in an attempt to quell infection [11, 12]. However, recent evidence
also suggests that these normally protective immune responses can become dysregulated,
culminating in the destruction of surrounding normal CNS parenchyma [13]. Therefore, fine tuning the
resultant immune response to achieve maximal pathogen clearance concomitant
with minimal tissue damage would represent a best case scenario for the
management of a wide array of CNS infectious diseases including bacterial
meningitis, HIVE, brain abscess, and other viral infections. The purpose of
this study was to perform a comprehensive analysis of the ability of several
PPAR-γ agonists to regulate glial activation
in response to a panel of TLR ligands that may be encountered during native CNS
infections. It is envisioned that this information may be exploited as a first
step towards the derivation of specific treatment strategies that could be
implemented with existing therapies for CNS infections to maximize benefit to
patients.One obvious distinction between the
various PPAR-γ agonists tested to modulate
TLR-dependent glial activation was the finding that 15d-PGJ2 consistently led to more dramatic and widespread decreases in inflammatory
mediator production compared to the synthetic TZDs. This relationship was
observed with both primary microglia and astrocytes and is in agreement with
previous reports by us and other laboratories where 15d-PGJ2 exerted
potent inhibitory effects at lower effective concentrations compared to TZDs,
despite the fact that the former exhibits a lower binding affinity to PPAR-γ [26, 27, 58, 59]. In addition, differences
were observed between the immune modulatory effects within the group
of TZDs tested. This was somewhat surprising; however, Storer et al. also reported
that ciglitazone and pioglitazone had no effect on TNF-α or CCL2 production by microglia in
response to LPS [27], similar to our findings in
the present study. However, a few discrepancies between these reports also
exist, which might be explained by the fact that only a single TLR ligand was
examined (i.e., LPS) and the concentrations of several TZDs exceeded the
maximal dose tested in the current study. In general, only one TZD,
rosiglitazone, exerted rather global effects on the inflammatory mediators
examined here, whereas the other compounds (i.e., ciglitazone, troglitazone,
and pioglitazone) demonstrated differential effects that were dependent on the
TLR ligand as well as the proinflammatory mediator measured. The reason
responsible for these differences is not clear but distinct chemical structures
of the various TZDs have been suggested to contribute to their unique
properties [60]. The widespread effects of
rosiglitazone on glial inflammatory mediator production are in agreement with
the fact that this TZD has been reported to impact numerous PPAR-γ isoforms including PPAR-γ1 and PPAR-γ2, whereas other TZDs have been reported
to selectively target a single PPAR-γ subtype [61, 62]. Other studies have revealed
distinct differences in the effectiveness of TZD members in regulating changes
in glucose metabolism in astrocytes [63] and mitochondrial function [64]. In addition, the EC50 of individual TZD compounds for PPAR-γ does vary within this group of
molecules depending on the experimental readout examined, which may contribute to their
differential effects [61, 62]. Alternatively, studies have
shown that the coactivator proteins interacting with PPAR-γ differ in a ligand-dependent manner [65]. Unfortunately, there are few
reports where comprehensive side-by-side comparisons have been made with regard
to the effects of various TZD compounds on neuroinflammation either in vitro or in vivo. Making direct comparisons between various TZDs in
disparate models of neuroinflammation should be viewed cautiously since
differences in disease models, inflammatory stimuli, and species examined all
have the potential to influence the results obtained.Our findings demonstrating the inhibitory
effects of 15d-PGJ2 on TLR ligand-induced glial activation are
similar to those observed following LPS stimulation. Specifically, 15d-PGJ2 has been shown to inhibit LPS-induced NO [27–29, 66], TNF-α [27, 28, 66], and IL-12 family member
expression [26] in microglia and/or
astrocytes. Importantly, this report has provided a more comprehensive analysis
of the effects of PPAR-γ agonists on glial activation with the inclusion
of a battery of TLR ligands that would be encountered during various CNS
infectious diseases. Earlier studies have primarily focused on the immune
modulatory effects of PPAR-γ agonists in response to the TLR4 ligand
LPS, and therefore, this study is also novel from the perspective that numerous
TLR ligands were evaluated.Interestingly, both 15d-PGJ2 and pioglitazone were found to augment CXCL2 release by microglia and
astrocytes in response to specific TLR ligands. In particular, 15d-PGJ2 enhanced CXCL2 production in response to LPS stimulation in both microglia and
astrocytes, in agreement with previous reports [53, 54]. In addition, recent results
from our laboratory have revealed that CXCL2 release was slightly increased by
15d-PGJ2 and downregulated by ciglitazone in response to the
gram-positive pathogen S. aureus in
primary astrocytes [24], in corroboration with the current report.Elevated levels of endogenous 15d-PGJ2 have been associated with the resolution of inflammation in vivo, suggesting
that it functions as negative feedback regulator of inflammatory responses [67, 68]. This study demonstrates that
15d-PGJ2 is an effective and selective inhibitor of glial activation
in response to TLR ligands, suggesting that it may be capable of modulating
chronic microglial and astrocyte responses during the course of CNS infectious
diseases. Evidence to support this concept is provided by our findings that
15d-PGJ2 effectively inhibited IL-12 p40 and CCL2 expression in
microglia and astrocytes, two molecules with important functions in the
differentiation of CD4+ Th1 cells and monocyte and T cell influx
into the infected CNS [52, 55, 56, 69, 70]. It is possible that the
downregulation of these mediators in microglia and astrocytes following 15d-PGJ2 treatment results in the failure to recruit and/or stimulate Ag-specific T
cells in the CNS parenchyma. Therefore, preventing chronic microglial
activation by 15d-PGJ2 or synthetic PPAR-γ agonists may help to resolve
inflammation earlier, resulting in less damage to surrounding normal brain
parenchyma.In summary, these studies demonstrate
that not all PPAR-γ agonists are created equal in terms of
their ability to modulate proinflammatory mediator release by activated
microglia and astrocytes. Specifically, differences were specific to the type
of TLR ligand examined. When considering the potential utility of PPAR-γ agonists for modulating pathological
inflammation typical of several CNS infectious diseases, critical issues such
as the timing and length of PPAR-γ administration and doses of compound
must be considered. For example, it appears likely that compounds should be
delivered at periods at or nearing pathogen clearance since a significant
attenuation of the host immune response would be counterproductive to infection
resolution. Upon pathogen elimination, PPAR-γ agonists may minimize damage to
surrounding tissue by downregulating exaggerated CNS immune responses that
could be perpetuated by microbial debris (i.e., cell wall fragments such as LPS
and PGN or pathogen nucleic acids) via continued engagement of TLRs. Indeed,
recent studies by our laboratory have revealed that the PPAR-γ agonist ciglitazone demonstrates
beneficial effects with delayed administration in an experimental brain abscess
model as revealed by accelerated abscess encapsulation and reduction in
bacterial burdens [71]. In addition, recent studies by other groups have revealed
beneficial effects of PPAR-γ agonists in other infectious disease
paradigms [72–76]. Therefore, the current study
can be used as a guide to facilitate the selection of PPAR-γ agonists as candidates for intervention
during CNS infectious diseases.



---

*Source: 453120-2008-06-19.xml*