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. 2007 Apr 23;150(8):3457–3464. doi: 10.1210/en.2008-1757

Pioglitazone Inhibits Toll-Like Receptor Expression and Activity in Human Monocytes and db/db Mice

Mohan R Dasu 1, Samuel Park 1, Sridevi Devaraj 1, Ishwarlal Jialal 1
PMCID: PMC2717888  PMID: 19389833

Abstract

Toll-like receptors (TLRs) are key innate immune sensors of endogenous damage signals and play an important role in inflammatory diseases like diabetes and atherosclerosis. Pioglitazone (PIO), a peroxisome proliferator-activated receptor (PPAR)-γ agonist, has been reported to be an antiinflammatory agent. Thus, in the present study, we examined the antiinflammatory effects of PIO on TLR2 and TLR4 expression in human monocytes exposed to Pam3CSK4 (Pam; TLR2 ligand) and purified lipopolysaccharide (LPS; TLR4 ligand) using flow cytometry and real-time RT-PCR. Monocytes were isolated from healthy human volunteers and pretreated with PIO (1 μm) followed by Pam (170 ng/ml) and LPS (160 ng/ml) challenge. PIO significantly decreased Pam- and LPS-induced TLR2 (−56%) and TLR4 (−78%) expression (P < 0.05). In addition, PIO decreased TLR ligand-induced nuclear factor-κB activity (−63%), IL-1β (−50%), IL-6 (−52%), monocyte chemoattractant protein-1(−83%), and TNF-α (−87%) compared with control. Next, PIO-treated db/db mice (n = 6/group) showed decreased TLR2 (−60%) and TLR4 (−45%) expression in peritoneal macrophages compared with vehicle control mice (P < 0.001) with associated decrease in MyD88-dependent signaling and nuclear factor-κB activation. Data suggest that Pam- and LPS-induced TLR2 and TLR4 expression are inhibited by PIO in human monocytes and db/db mice. Thus, we define a novel pathway by which PIO could induce antiinflammatory effects.

Pioglitazone treatment of human monocytes and db/db mice ameliorates ligand-induced TLR2 and TLR4 expression and functional activation and may impact CVD associated with T2D.

Toll-like receptors (TLRS) are highly conserved pattern recognition receptors, expressed on a variety of immune cells including monocytes. TLRs play a key role in the activation and regulation of the innate immune system and inflammation (1). Each member of the TLR family recognizes specific components of pathogens, which on activation trigger a signaling cascade leading to cytokine production and adaptive immune response (2). TLRs are increased in a broad range of inflammatory diseases including sepsis syndrome, asthma, rheumatoid arthritis, atherosclerosis, systemic lupus erythematosus, and recently diabetes mellitus (1,2,3). TLR2 and TLR4 have been shown to play a role in the inflammatory processes in the pathogenesis of insulin resistance, diabetes, and atherosclerosis in both clinical and experimental conditions (3,4,5,6,7,8).

Ligands for peroxisome proliferator-activated receptor (PPAR)-γ, such as thiazolidinediones (TZDs), increases insulin sensitivity by selectively binding to PPARs (9). Among the TZDs, pioglitazone (PIO) is considered safe because of reduced risk of myocardial ischemic events (10). PIO is a widely used TZD for treating type 2 diabetes patients, and it is known that PIO ameliorates insulin resistance via the activation of PPAR-γ (11). In addition to increasing insulin sensitivity, PIO is known to exert its antiinflammatory effects by decreasing inflammation and endothelial dysfunction (12), reduction in plasminogen activator inhibitor-1 (13), increased adiponectin (14), and reduced blood pressure (15); however, the mechanism of these effects are not fully understood. Recent studies have shown that PPAR-γ activation by PIO decreased the expression of inflammatory genes such as ILs and TNF-α (16,17,18) and suppressing the nuclear factor-κ B (NF-κB) pathways (19).

Given the antiinflammatory effects of PIO and the expression of TLRs in inflammatory conditions such as diabetes, in this study we examined the effects of PIO on TLR2 and TLR4 expression in human monocytes in vitro and in db/db mice in vivo.

Materials and Methods

Monocyte isolation

Blood was collected from healthy, fasting, human adult volunteers with informed consent and study approval from the Institutional Review Board of University of California, Davis, Medical Center. Three volunteers were pooled for each experiment to minimize variations in the data collected. Peripheral blood mononuclear cells were isolated from heparinized blood via Ficoll-Hypaque gradient (20). Monocytes were subsequently isolated from the peripheral blood mononuclear cells magnetically by depletion technique (Miltenyi Biotech, Auburn, CA). Eighty-eight percent of these cells were CD14 positive by flow cytometry (20) confirming its characterization as monocytes.

Endotoxin levels were measured in media and reagents (RPMI 1640, PBS, BSA, etc.) to verify levels were less than 100 EU/ml using limulus amoebocyte lysate assays (catalog no. 50-6474; Lonza, Walkersville, MD), ensuring accurate TLR expression measurements free from endotoxin interference. All experiments were performed on four occasions.

Fluorescence-activated cell sorter analysis of TLR2 and TLR4 in human monocytes

Expression of TLR2 and TLR4 was measured in human monocytes pretreated with PIO (0.1, 1, 5 μm) for 2 h followed by synthetic lipoprotein Pam3Cys-Ser-(Lys)4 [TLR2 ligand; Pam3CSK4 (Pam), 170 ng/ml; Invivogen, San Diego, CA] and lipopolysaccharide (LPS; TLR4 ligand; Escherichia coli 026:B6; Sigma, St. Louis, MO) challenge overnight (12 h), with suitable vehicular controls. After treatment, cells were washed twice with cold PBS-BSA (0.1%), incubated with antihuman TLR2 (catalog no. 17-922; eBioscience, San Diego, CA), antihuman TLR4 (catalog no. 12-9912; eBioscience), or isotype-matched IgG controls (mouse IgG2a, K catalog no. 14-4724), depending on the cell treatment; 5,000–10,000 events were analyzed with the BD fluorescence-activated cell sorter array bioanalyzer (BD Biosciences, San Jose, CA) as described previously (8). Viability and integrity of monocytes after Pam/LPS and PIO treatments was confirmed by trypan blue exclusion and morphology examination using microscopy. Results are expressed as mean fluorescence intensity (MFI)/105 cells. Intra- and interassay coefficients of variation (CVs) were determined to be less than 10%.

Luciferase reporter gene assays for TLR2 and TLR4

Assays were performed as described earlier (20). Briefly, 293T cells were cotransfected with TLR4 and TLR2 expression plasmids, a luciferase plasmid containing NF-κB (two times)-binding site and β-galactosidase plasmid and corresponding empty vectors as controls using SuperFect transfection reagent (catalog no. 301305; QIAGEN, Valencia, CA), following manufacturer’s instructions. Transfected cells were pretreated with PIO (1 μm) for 2 h followed by high-glucose or synthetic TLR ligands for 15 h before lysis. Luciferase and β-galactosidase enzyme activities were determined using the luciferase assay system and β-galactosidase enzyme system (catalog no. E2000; Promega, Madison, WI). Luciferase activity was normalized by β-galactosidase activity to correct the transfection efficiency.

Animal model

To determine the in vivo effects of pioglitazone, male db/db mice on C57BL/6J background (8–10 wk of age, stock no. 000697; Jackson Labs, Bar Harbor, ME) were placed into two groups of six, one of which received a 10-d course of PIO injections dissolved in dimethylsulfoxide-PBS (10 mg/kg body weight, ip) and the control group received a suitable vehicular control. All the animals had ad libitum access to food and water. After 10 d the animals were euthanized and peritoneal macrophages were isolated for TLR2 and TLR4 analysis. All procedures performed on animals were approved by the Animal Care and Use Committee of University of California, Davis (Davis, CA). PIO was purchased from Cayman Chemical (catalog no. 71745; Ann Arbor, MI) and was dissolved according to vendor protocol in a 1:1 solution of dimethylsulfoxide-PBS (pH 7.2). The dose of PIO was chosen based on a dose response study (2.5, 5, 10, 20 mg/kg body wt for 10 d ip) performed in mice (n = 3 mice/group) and measured effects on TLR2 and TLR4 expression.

Metabolic measurements

Serum samples were saved for measurement of glucose, insulin, triglyceride, and cholesterol. Glucose was measured using a glucose HK assay from Sigma (catalog no. GAHK20), triglyceride (catalog no. 10010303) and total cholesterol (catalog no. 10067640) were measured by assays from Cayman Chemical, and insulin was measured using an assay from ALPCO Diagnostics (Salem, NH; catalog no. 80-INSMS-E01). These values were used to calculate homeostasis model assessment of insulin resistance (HOMA-IR) as described earlier (21).

Fluorescence-activated cell sorter analysis of TLR2 and TLR4 in peritoneal macrophages of db/db mice

TLR expression in db/db mice peritoneal macrophages was measured after PIO treatment. Analysis of TLR expression was performed using antimouse TLR2 (catalog no. 51-9021; eBioscience) and antimouse TLR4 (catalog no. 12-9041; eBioscience) with flow cytometry as described earlier (22). Eighty to 90% of the cells isolated were CD68 positive.

ELISA and multiplex cytokine assay

Adiponectin (catalog no. 44-ADPMS-E01) and serum amyloid A (catalog no. 41-SAAMS-E01) levels were measured in sera using immunoassays (ALPCO Diagnostics). The release of cytokines, IL-1β, IL-6, monocyte chemoattractant protein (MCP)-1, and TNF-α were measured in mice serum and PIO/ligand treated and control supernatants of human monocytes using a multiplex assay (mouse: MPXMCYTO-70K and human: MPXHCYTO-60K; Millipore, St. Charles, MO). Intra- and interassay CVs were determined to be less than 14%.

Real-time RT-PCR

RNA was extracted from the cells using TRI reagent (Invitrogen, Carlsbad, CA) reagent. The first-strand of cDNA was synthesized using total RNA (1 μg/reaction). cDNA (50–100 ng) was amplified using primer probe sets for TLR2, TLR4, and 18s (SA Biosciences, Gaithersburg, MD) following manufacturer’s cycling parameters. Data are calculated using the 2 −ΔΔCT method and are presented as ratio of transcripts for TLR gene normalized to 18s (20,22).

Nuclear/cytoplasmic extracts and transcription factor assays

Nuclear and cytoplasmic extracts were prepared from isolated macrophages and monocytes as described previously (20,22). Nuclear extracts were used to perform PPAR-γ (catalog no. 40196) and NF-κB (catalog no. 40096) transcription factors activation assays (Active Motif, Carlsbad, CA) to verify activation of PPAR-γ and down-regulation of NF-κB in the presence of PIO, indicating increased insulin sensitivity and decreased inflammation, respectively. Both assays were performed in accordance to manufacturer’s protocols. Intra- and interassay CV for these assays was less than 7%.

Western blotting

Western blot analysis was used to examine the downstream signaling events. Twenty micrograms of total protein were resolved, transferred, and probed with antibodies for IL-1 receptor-associated kinase (IRAK)-1, Myeloid differentiation factor 88 (MyD88), p38, ERK1/2, p65 (Santa Cruz Biotechnology, Santa Cruz, CA), and Toll/IL-1 receptor (TIR) domain-containing adapter-inducing interferon-β (TRIF; Abcam, Cambridge, MA). Stripped membranes were further incubated with β-actin. Representative blots from each group of mice were presented. Band intensities were determined using Image Quant software (GE Healthcare Biosciences, Piscataway, NJ) as described previously (20) and normalized to β-actin and presented as a ratio.

Statistical analysis

All in vitro experiments were performed four times in duplicates with the results being reported as the means ± sd. Differences were analyzed by ANOVA with appropriate post hoc analyses. P < 0.05 was considered significant. Correlation analysis was performed using Pearson’s equations. All statistical analyses were performed using GraphPad Prism software (San Diego, CA).

Results

PIO decreases the expression of TLR2 and TLR4 protein and mRNA expression in human monocytes

Pam and LPS are known inducers of TLR2 and TLR4 protein expression, respectively (8,20). We first examined the effect of PIO treatment on Pam- and LPS-induced TLR2 and TLR4 expression by flow cytometric analysis. Half-maximum doses of Pam and LPS used in the present study were determined in pilot dose-response experiments (data not shown). Stimulation of human monocytes with Pam (170 ng/ml) or LPS (160 ng/ml) led to significant increase (36 ± 4 and 25 ± 4 MFI/105 cells) in TLR2 and TLR4 surface expression compared with control (6 ± 2 and 7 ± 2 MFI/105 cells; P < 0.01, n = 4), and pretreatment with PIO significantly reduced the expression of TLR2 and TLR4 protein in a concentration-dependent manner with a minimum significant reduction at 1 μm PIO (Fig. 1A). Furthermore, to determine whether the decrease in monocyte TLR2 and TLR4 expression by PIO resulted from reduced mRNA expression, we investigated the TLR2 and TLR4 mRNA levels by real-time RT-PCR. Treatment with PIO (1 μm) significantly reduced the Pam- or LPS-induced TLR2 and TLR4 mRNA expression (Fig. 1B). Because 1 μm PIO significantly inhibited both TLR2 and TLR4, this dose was used in all subsequent experiments.

Figure 1.

Figure 1

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A, TLR2 and TLR4 (black bars) protein expression was measured in human monocytes after Pam or LPS challenge in the presence of PIO (0.1–5 μm) by flow cytometry as described in Materials and Methods. Values are expressed as MFI/105 cells. *, P < 0.05 vs. control; **, P < 0.05 vs. Pam or LPS; n = 4 experiments. B, TLR2 and TLR4 (black bars) mRNA expression ratios in human monocytes after Pam or LPS challenge in the presence of PIO (1 μm) by real-time RT-PCR as described in Materials and Methods. Values are expressed as mean ratio ± sd. *, P < 0.001 vs. control; **, P < 0.05 vs. Pam or LPS; n = 4 experiments. C, The DNA binding activity of nuclear NF-κB p65 in human monocytes after Pam or LPS (black bars) exposure with or without PIO was assessed by ELISA as detailed in Materials and Methods. Values are normalized to mg nuclear protein and expressed as mean ± sd. *, P < 0.05 vs. control; **, P < 0.05 vs. Pam/LPS; n = 4 experiments in duplicate.

PIO-mediated reduction of TLR2 and TLR4 attenuates proinflammatory effects in human monocytes

In human cell culture studies, activation of TLR2 and TLR4 by their ligands has previously been shown to up-regulate expression of proinflammatory mediators such as IL-1β, IL-6, MCP-1, and TNF-α (8,20,22). To assess the functional relevance of reduced monocyte TLR2 and TLR4 expression by PIO, human monocytes were challenged with Pam (170 ng/ml) or LPS (160 ng/ml) in the presence or absence of PIO (1 μm) before measuring IL-1β, IL-6, MCP-1, and TNF-α protein concentration in the cell supernatants. Pretreatment of cells with PIO significantly reduced Pam-induced IL-1β (−51%), IL-6 (−52%), MCP-1 (−77%), and TNF-α (−87%) compared with control (P < 0.05, n = 4). Also, PIO significantly decreased LPS-induced IL-1β (−67%), IL-6 (−52%), MCP-1 (−77%), and TNF-α (−78%) compared with control (P < 0.05, n = 4, Table 1). In addition, PIO significantly reduced Pam- or LPS-induced activation of NF-κB p65 activity in human monocytes compared with controls (Fig. 1C). PIO treatment did not show significant inhibition in basal NF-κB p65 activity in control cells (0.8 ± 0.3 vs. 0.63 ± 0.2 ng/mg protein).

Table 1.

Effect of PIO on IL-1β, IL-6, MCP-1, and TNF-α secretion in human monocytes

Supernatant (pg/mg protein) Control Pam (170 ng/ml) Pam+PIO (1 μm) LPS (160 ng/ml) LPS+PIO (1 μm)
IL-1β 116 ± 17 443 ± 22a 218 ± 51b 845 ± 120a 274 ± 85b
IL-6 1.6 ± 0.6 3 ± 0.4a 1.6 ± 0.3b 3.4 ± 1a 2 ± 0.1b
MCP-1 17 ± 4 109 ± 11a 24 ± 6b 74 ± 13a 17 ± 6b
TNF-α 5 ± 1.4 20 ± 4a 3 ± 0.5b 37 ± 6a 8 ± 2b
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Values are expressed as mean ± sd. 

a

P < 0.05 vs. control; 

b

P < 0.01 vs. Pam/LPS (n = 5 experiments). 

PIO inhibits TLR2 and TLR4 expression in transfected 293T cells

We ectopically expressed TLR2/4 in HEK293T cells to further confirm the inhibitory effects of PIO using NF-κB reporter based cotransfection assays (20). We recently showed that high glucose (HG) significantly induces TLR2 and TLR4 expression in human monocytes (20). In line with our findings, PIO reduced the HG- (15 mm) or Pam-induced NF-κB transactivation in TLR2 cotransfected 293T cells (PIO+HG 66 ± 4; PIO+ Pam 100 ± 11 compared with HG: 198 ± 9 or Pam: 200 ± 10 relative luciferase activity, P < 0.05). In addition, PIO significantly inhibited HG- (15 mm) or LPS-induced NF-κB transactivation in TLR4 cotransfected 293T cells (PIO+HG 95 ± 3; PIO+LPS 110 ± 9 compared with HG: 288 ± 9 or LPS: 245 ± 10 relative luciferase activity, P < 0.05). These results demonstrate that PIO inhibits HG- and ligand-induced TLR2 and TLR4 receptor activity and resulting NF-κB activation further confirming our data. Furthermore, LPS had no effect on TLR2 transfected cells and Pam had no effect on TLR4 cotransfected cells due to their absence in HEK293T cells.

PIO treatment decreases TLR2 and TLR4 expression in peritoneal macrophages of db/db mice

With the novel in vitro observations in human monocytes, we tested the TLR-mediated antiinflammatory effects of PIO in db/db mice, in vivo. We initially examined the various biochemical parameters in PIO-treated male db/db mice. Table 2 shows the various results for each group. Treatment of db/db mice with PIO for 10 d had no significant effect on body weight. Plasma glucose (−33%), insulin (−51%), HOMA-IR (−76%), and triglycerides (−74%) were markedly decreased in the PIO group compared with the vehicle control diabetic group. Furthermore, PIO treatment significantly reduced serum amyloid A (−68%) and increased adiponectin levels (66%) compared with vehicle control (P < 0.05). No changes in the above biochemical parameters and TLR2 (13 ± 1 vs. 8 ± 2 untreated MFI/105 cells) and TLR4 (14 ± 3 vs. 10 ± 2 vs. untreated MFI/105 cells) expression were observed in the control C57BL6/J mice treated with PIO for 10 d (10 mg/kg body weight).

Table 2.

Biochemical parameters in db/db mice

Vehicle control PIO (10 mg/kg body weight) Change, %
Body weight (g) 45 ± 5 42 ± 6 −6
Glucose (mg/dl) 343 ± 80 227 ± 35a −33
Insulin (μU/ml) 9.4 ± 0.4 4.6 ± 0.2a −51
HOMA-IR index 8.6 ± 3 2.6 ± 0.8a −76
Total cholesterol (mg/dl) 321 ± 70 317 ± 41 −1.2
Triglycerides (mg/dl) 35.5 ± 2.3 9.18 ± 0.4a −74
Serum amyloid A (ng/ml) 1895 ± 376 604 ± 148a −68
Adiponectin (ng/ml) 9 ± 0.6 15 ± 0.16a 66
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Values are expressed as mean ± sd. −, Decrease. 

a

P < 0.05 vs. control (n = 6 mice/group). 

We then examined TLR2 and TLR4 expression in PIO-treated db/db mice peritoneal macrophages. PIO treatment significantly reduced TLR2 (−59%) and TLR4 (−45%) in db/db mice macrophages compared with vehicle control diabetic mice (Fig. 2).

Figure 2.

Figure 2

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PIO treatment (10 mg/kg body weight, 10 d) inhibits TLR2 and TLR4 expression in peritoneal macrophages of db/db mice (black bars; n = 6/group) compared with vehicle controls. Values are expressed as MFI/105 cells. *, P < 0.001 vs. db/db.

In addition, there was a significant correlation between HOMA-IR and TLR2 expression (r = 0.94; P < 0.001) and TLR4 expression (r = 0.73; P < 0.01). Furthermore, glucose levels significantly correlated with TLR2 expression (r = 0.4; P < 0.05) and TLR4 expression (r = 0.9; P < 0.05), respectively in PIO-treated db/db mice. Increased adiponectin levels in PIO-treated db/db mice correlate with decreased TLR2 (r = 0.5; P < 0.05) and TLR4 (r =0.6; P < 0.01), whereas PPAR-γ levels correlate with both TLR2 and TLR4 (r = 0.3; P < 0.02).

PIO inhibits TLR-mediated signaling in db/db mice

We determined the effect of PIO on TLR mediated MyD88-dependent signaling pathway using Western blot technique. TLR2 and TLR4 both engage MyD88 and activate NF-κB, which are common downstream signaling components for all TLRs except TLR3. Therefore, activation of MyD88-dependent pathway and MAPK was used as a readout to determine the inhibitory effects of PIO on the activation of TLR2 and TLR4.

PIO significantly inhibited phosphorylation of IRAK-1, p38, and ERK1/2; MyD88 and TRIF protein expression in cytoplasmic extract; and p65 protein levels in nuclear extracts of peritoneal macrophages compared with untreated db/db mice, with no change in total protein and β-actin levels (Fig. 3, A–D), suggesting inhibition of TLR-mediated signaling cascade (both MyD88 dependent and independent) including MAPKs by PIO in db/db mice. Densitometric ratios further corroborate the data (Fig. 3, A–D).

Figure 3.

Figure 3

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Representative Western blot depicting the effect of PIO (10 mg/kg body weight) on MyD88, TRIF, p65 (A), IRAK-1 (B), p38 (C), and ERK1/2 phosphorylation (D) in peritoneal macrophages of db/db mice. After treatment for 10 d, peritoneal macrophages were lysed, and 20 μg of protein were blotted for the indicated total, phosphoproteins, and β-actin as described in Materials and Methods (n = 6/group). Densitometric values are expressed as ratio ± sd (n = 4/group). *, P < 0.05 vs. db/db.

PIO treatment increases PPAR-γ and decreases NF-κB p65 activation in peritoneal macrophages of db/db mice

To further investigate TLR2 and TLR4-mediated inflammation and determine the antiinflammatory effects of PIO, we measured PPAR-γ and NF-κB activities in the nuclear extracts of peritoneal macrophages of treated and control db/db mice. PIO treatment for 10 d significantly increased PPAR-γ activity (69%, P < 0.001) and decreased NF-κB p65-dependent DNA binding activity (−62%, P < 0.01) (Fig. 4, A and B).

Figure 4.

Figure 4

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A, The DNA binding activity of PPAR-γ in peritoneal macrophages of db/db mice after PIO treatment (black bars) was assessed by ELISA as detailed in Materials and Methods. Values are normalized to milligrams nuclear protein and expressed as mean ± sd. *, P < 0.05 vs. control; n = 6 mice/group. B, The DNA binding activity of nuclear NF-κB p65 in peritoneal macrophages of db/db mice after PIO treatment (black bars) was assessed by ELISA as detailed in Materials and Methods. Values are normalized to milligrams nuclear protein and expressed as mean ± sd. *, P < 0.001 vs. control; n = 6 mice/group.

PIO-mediated reduction of TLR2 and TLR4 expression decreases proinflammatory effects in db/db mice

Next, we measured IL-1β, IL-6, MCP-1, and TNF-α serum concentration in PIO-treated db/db mice as a functional readout of NF-κB activation. There was a significant reduction in IL-1β (47%), IL-6 (84%), MCP-1(65%), and TNF-α (57%) concentration in PIO-treated db/db mice compared with controls (Table 3).

Table 3.

Effect of PIO on IL-1β, IL-6, MCP-1, and TNF-α secretion in db/db mice

Serum (pg/ml) Vehicle control PIO (10 mg/kg body weight)
IL-1β 128 ± 10 68 ± 4a
IL-6 38 ± 6 6 ± 2a
MCP-1 131 ± 14 46 ± 9a
TNF-α 15 ± 2 6 ± 0.7a
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Values are expressed as mean ± sd. 

a

P < 0.05 vs. vehicle control (n = 6 mice/group). 

Discussion

Determining the pathophysiology initiating the development of inflammation should expand our capacity to identify novel therapeutic targets for the prevention and treatment of type 2 diabetes (T2D) (23,24). This process remains incompletely understood in part due to the complexity of the interaction of multiple cells and organ systems plus the multiplicity of intracellular perturbations within these systems that mediate the development of T2D. The major cells implicated in inflammation include monocytes, macrophages, T cells, adipocytes, and endothelial cells (25). Increasing our understanding of this biology will require the combination of studying cross talking signaling networks and the pleiotropic effects of known therapeutic drugs (26).

The use of the TZD class of insulin sensitizers like PIO for the treatment of T2D has proven to be effective in restoring normal glycemic control in patients primarily through lowering glucose levels and reducing cardiovascular events (10,11,12,13,14). Whether these effects are operational in the immune system are less certain and, if present, whether these changes are linked to inflammation is not clear. Therefore, in this study, to understand the mechanisms of antiinflammatory effects of PIO, we determined the altered expression of TLRs, key receptors of innate immune system known to be induced by glucose, and further delineated the TLR-mediated signaling and inflammation in both human monocytes and db/db mice after PIO treatment. We chose PIO for this study due to its better safety profile associated with myocardial events in T2D patients (10).

Recently several studies have shown that the TLRs recognize molecular patterns and direct several aspects of innate immunity, acquired immunity, and inflammation, including cytokine gene expression (27). More than 10 TLRs have been identified, and all of them have an ectodomain of leucine-rich repeats involved in ligand binding and a cytoplasmic TIR domain that interacts with TIR domain-containing adaptor molecules. Upon specific ligand binding, TLRs use two individual pathways: the MyD88-dependent and -independent pathways. In the MyD88-dependent pathway, the signal from TLR is transduced via MyD88 and IRAK, finally activating NF-κB (28,29). The activated NF-κB moves to the nucleus and induces the expression of inflammatory cytokines such as IL-1, IL-6, and MCP-1 (20,29,30). MyD88-independent pathway is executed in TLR3 activation, leading to interferon regulatory factor-3 instead of NF-κB and induces interferon-inducible genes (30). TLR2 and TLR4 expression is increased in the human atherosclerotic plaque, animals models of atherosclerosis, and monocytes of type 1 diabetes patients (3,4,5,6,7,8,31). Interestingly, total loss of TLR4 gene is associated with reduction in lesion size, lipid content, and macrophage infiltration in hypercholesterolemic apoE−/− mice (32). In addition, double-knockout mice models of TLR2−/−/low-density lipoprotein receptor−/− and TLR2−/−/apoE−/− showed reduced development of atherosclerosis (33,34). Accordingly, in this study we focused on the TLR2 and TLR4 interaction and their role in inflammatory processes, and we showed here that Pam and LPS increased the TLR2 and TLR4 expression in protein and mRNA levels in human monocytes along with activation of NF-κB and consequent increase in inflammation. Furthermore, in this study, we demonstrate that the insulin sensitizer, PIO, suppresses the Pam- and LPS-induced TLR2 and TLR4 expression with corresponding decrease in NF-κB activation, inflammatory cytokine production, and increase in PPAR-γ activation and insulin sensitivity. In addition, results from 293T cells transfected with TLR2 and TLR4 NF-κB-luciferase reporter plasmids, suggest that PIO treatment abolished HG- and ligand-induced TLR2 and TLR4 protein expression. These results indicate that the altered expression levels of TLR2 and TLR4 in monocytes may modulate inflammation in conditions associated with insulin resistance like obesity, metabolic syndrome, and T2D and that TLR2 and TLR4 are target genes, whose expression is modified by PIO.

Also, we have shown in vivo that TLR2 and TLR4 expression in peritoneal macrophages of db/db mice is increased compared with control mice and that treatment with PIO inhibits TLR2/TLR4 expression. These results are very important when we understand how TLR2/TLR4 expression mediates inflammation in diabetic conditions. We recently showed that hyperglycemia induces and activates both TLR2 and TLR4, leading to MyD88-dependent signaling in the human monocytes culminating in NF-κB activation and inflammatory cytokine production (20). As shown in our results, enhanced TLR expression and signaling is suppressed by PIO by increased insulin sensitivity, increased PPAR-γ activation, and adiponectin production in line with our recent findings. Next, we have shown that there is a significant correlation between TLR expression and insulin sensitivity (HOMA-IR index) and inflammation, indicating that TLRs play a pivotal role in the inflammation seen in db/db mice and experimental diabetes. Future studies are needed to confirm these findings in T2D patients. TZDs were reported to reduce inflammation in neonatal rat cardiac myocytes, reduce myocardial hypertrophy (35), and have antiarteriosclerotic effects by preventing the N-omega-nitro-l-arginine methyl ester-induced coronary inflammation and arteriosclerosis (36). PPAR-γ is inhibited and TLRs are activated in db/db mice macrophages, and PIO treatment attenuates the TLR response and enhances PPAR-γ. Thus, activation of PPAR-γ and inhibition of TLRs in macrophages may reduce cytokine production, limiting the local inflammatory response, hence arresting atherogenesis in T2D.

Recent clinical studies using TZD therapy in T2D have shown to reduce serum levels of matrix metalloproteinase-9 and C-reactive protein (37,38), suggesting direct antiinflammatory effects (39). T2D patients treated with PIO for 6 months’ duration had significantly reduced carotid intima thickness compared with placebo group, further indicating the inhibitory potential of TZD therapy in T2D and atherosclerosis (40). PIO and rosiglitazone were shown to lower angiotensin II-induced hypertension, decrease markers of inflammation, rectify structural abnormalities in the vessel wall, and improve endothelial function in a rat model of hypertension (41). However, there is a paucity of data examining the effects of TZDs on TLR expression and activity. In the present report, we show that PIO decreases TLR2 and TLR4 protein and mRNA expression and reduces NF-κB p65-dependent activation with concomitant reduction in key inflammatory mediator production in vitro. Furthermore, administration of PIO to db/db mice resulted in significant reduction in TLR2 and TLR4 protein and mRNA expression compared with vehicle control mice. In addition, PIO treatment attenuated TLR mediated signaling (MyD88, IRAK-1, TRIF, and p65 protein expression) in db/db mice.

Our findings are consistent with those of a number of studies exploring the function of the PPAR-γ system. Although, so far, very little is known about the molecular mechanisms by which cooperated stimulation of these MAPKs via TLR2, TLR4, and PPAR-γ modulate inflammation, observations from the current study point toward a common inflammatory output on their activation. Bai et al. (42) have shown that rosiglitazone inhibited the in vitro activity of p38 and NF-κB, with a concomitant reduction in proinflammatory gene expression. Similar findings were reported with troglitazone on ERK-1/2 phosphorylation (43). Several in vitro reports strengthen our data that TLR-induced MyD88-dependent signaling culminates in NF-κB activation via p38 and ERK1/2 (44,45). Furthermore, activation of PPAR in adipocytes inhibits the inflammatory response to LPS by decreasing the NF-κB activity (46). In addition, we confirm our in vitro findings in db/db mice. In future studies we will investigate this in T2D patients. Thus, we suggest that the novel mechanisms of pioglitazone are attributable to decreasing inflammation by inhibiting TLR2 and TLR4 expression and also indicate that this reduction may represent a alternate strategy to limit TLR mediated inflammatory processes. By improving insulin sensitivity, along with the additional pleiotropic effects, specifically the antiinflammatory effects via TLR2 and TLR4, PIO may have a significant impact on the cardiovascular disease burden associated with T2D.

Footnotes

This work was supported by American Diabetes Association Grant 7-07-JF-16 (to M.R.D.), National Institutes of Health Grants K24 AT00596 and JDRF-1-2007-585 (to I.J.).

Disclosure Summary: The authors have nothing to disclose.

First Published Online April 23, 2007

Abbreviations: CV, Coefficient of variation; HG, high glucose; HOMA-IR, homeostasis model assessment of insulin resistance; IRAK, IL-1 receptor-associated kinase; LPS, lipopolysaccharide; MCP, monocyte chemoattractant protein; MFI, mean fluorescence intensity; MyD88, myeloid differentiation factor 88; NF-κB, nuclear factor-κ B; Pam, Pam3CSK4; PIO, pioglitazone; PPAR, peroxisome proliferator-activated receptor; T2D, type 2 diabetes; TIR, Toll/IL-1 receptor; TLR, Toll-like receptor; TRIF, TIR domain-containing adapter-inducing interferon-β; TZD, thiazolidinedione.

References

  1. Verstak B, Hertzog P, Mansell A 2007 Toll-like receptor signaling and the clinical benefits that lie within. Inflamm Res 56:1–10 [DOI] [PubMed] [Google Scholar]
  2. O'Neill LA 2003 Therapeutic targeting of Toll-like receptors for inflammatory and infectious diseases. Curr Opin Pharmacol 3:396–403 [DOI] [PubMed] [Google Scholar]
  3. Devaraj S, Dasu MR, Rockwood J, Winter W, Griffen SC, Jialal I 2008 Increased toll-like receptor (TLR)2 and TLR4 expression in monocytes from patients with type 1 diabetes: further evidence of a proinflammatory state. J Clin Endocrinol Metab 93:578–583 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Xu XH, Shah PK, Faure E, Equils O, Thomas L, Fishbein MC, Luthringer D, Xu XP, Rajavashisth TB, Yano J, Kaul S, Arditi M 2001 Toll-like receptor-4 is expressed by macrophages in murine and human lipid-rich atherosclerotic plaques and upregulated by oxidized LDL. Circulation 104:3103–3108 [DOI] [PubMed] [Google Scholar]
  5. Shi H, Kokoeva MV, Inouye K, Tzameli I, Yin H, Flier JS 2006 TLR4 links innate immunity and fatty acid-induced insulin resistance. J Clin Invest 116:3015–3025 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Li H, Sun B 2007 Toll-like receptor 4 in atherosclerosis. J Cell Mol Med 11:88–95 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Shinohara M, Hirata K, Yamashita T, Takaya T, Sasaki N, Shiraki R, Ueyama T, Emoto N, Inoue N, Yokoyama M, Kawashima S 2007 Local overexpression of Toll-like receptors at the vessel wall induces atherosclerotic lesion formation. Synergism of TLR2 and TLR4. Arterioscler Thromb Vasc Biol 27:2384–2391 [DOI] [PubMed] [Google Scholar]
  8. Edfeldt K, Swedenborg J, Hansson GK, Yan ZQ 2002 Expression of toll-like receptors in human atherosclerotic lesions: a possible pathway for plaque activation. Circulation 105:1158–1161 [PubMed] [Google Scholar]
  9. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA 1995 An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator activated receptor γ. J Biol Chem 270:12953–12956 [DOI] [PubMed] [Google Scholar]
  10. Dormandy JA, Charbonnel B, Eckland DJ, Erdmann E, Massi-Benedetti M, Moules IK, Skene AM, Tan MH, Lefèbvre PJ, Murray GD, Standl E, Wilcox RG, Wilhelmsen L, Betteridge J, Birkeland K, Golay A, Heine RJ, Korányi L, Laakso M, Mokán M, Norkus A, Pirags V, Podar T, Scheen A, Scherbaum W,et al. 2005 Secondary prevention of macrovascular events in patients with type 2 diabetes in the PROactive Study (PROspective pioglitAzone Clinical Trial In macroVascular Events): a randomised controlled trial. Lancet 366:1279–1289 [Google Scholar]
  11. Mazzone T 2007 Prevention of macrovascular disease in patients with diabetes mellitus: opportunities for intervention. Am J Med 120(9 Suppl 2):S26–S32 [DOI] [PubMed] [Google Scholar]
  12. Sjöholm A, Nyström T 2005 Endothelial inflammation in insulin resistance. Lancet 365:610–612 [DOI] [PubMed] [Google Scholar]
  13. Igarashi M, Hirata A, Yamaguchi H, Jimbu Y, Tominaga M 2008 Pioglitazone reduces atherogenic outcomes in type 2 diabetic patients. J Atheroscler Thromb 15:34–40 [DOI] [PubMed] [Google Scholar]
  14. Bajaj M, Suraamornkul S, Piper P, Hardies LJ, Glass L, Cersosimo E, Pratipanawatr T, Miyazaki Y, DeFronzo RA 2004 Decreased plasma adiponectin concentrations are closely related to hepatic fat content and hepatic insulin resistance in pioglitazone-treated type 2 diabetic patients. J Clin Endocrinol Metab 89:200–206 [DOI] [PubMed] [Google Scholar]
  15. Konrad T, Lübben G, Franzen C 2005 Pioglitazone lowers blood pressure in hypertensive patients with type 2 diabetes mellitus: an open, multicentre, observational study. Clin Drug Investig 25:337–340 [DOI] [PubMed] [Google Scholar]
  16. Zhang WY, Schwartz EA, Permana PA, Reaven PD 2008 Pioglitazone inhibits the expression of inflammatory cytokines from both monocytes and lymphocytes in patients with impaired glucose tolerance. Arterioscler Thromb Vasc Biol 28:2312–2318 [DOI] [PubMed] [Google Scholar]
  17. Pitocco D, Giubilato S, Zaccardi F, Di Stasio E, Buffon A, Biasucci LM, Liuzzo G, Crea F, Ghirlanda G 2009 Pioglitazone reduces monocyte activation in type 2 diabetes. Acta Diabetol 46:75–77 [DOI] [PubMed] [Google Scholar]
  18. Chen J, Li D, Zhang X, Mehta JL 2004 Tumor necrosis factor-α-induced apoptosis of human coronary artery endothelial cells: modulation by the peroxisome proliferator-activated receptor-γ ligand pioglitazone. J Cardiovasc Pharmacol Ther 9:35–41 [DOI] [PubMed] [Google Scholar]
  19. Mehta JL, Hu B, Chen J, Li D 2003 Pioglitazone inhibits LOX-1 expression in human coronary artery endothelial cells by reducing intracellular superoxide radical generation. Arterioscler Thromb Vasc Biol 23:2203–2208 [DOI] [PubMed] [Google Scholar]
  20. Dasu MR, Devaraj S, Zhao L, Hwang DH, Jialal I 2008 High glucose induces toll-like receptor expression in human monocytes: mechanism of activation. Diabetes 57:3090–3098 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Devaraj S, Siegel D, Jialal I 2007 Simvastatin (40 mg/day), adiponectin levels, insulin sensitivity, in subjects with the metabolic syndrome. Am J Cardiol 100:1397–1399 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Dasu MR, Riosvelasco AC, Jialal I 2009 Candesartan inhibits Toll-like receptor expression and activity both in vitro and in vivo. Atherosclerosis 202:76–83 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Garber AJ 1998 Vascular disease and lipids in diabetes. Med Clin North Am 82:931–948 [DOI] [PubMed] [Google Scholar]
  24. 1999 A desktop guide to type 2 diabetes mellitus. European Diabetes Policy group. Diabetes Med 16:716–730 [PubMed] [Google Scholar]
  25. Luster AD, Alon R, von Andrian UH 2005 Immune cell migration in inflammation: present and future therapeutic targets. Nat Immunol 6:1182–1190 [DOI] [PubMed] [Google Scholar]
  26. Khanderia U, Pop-Busui R, Eagle KA 2008 Thiazolidinediones in type 2 diabetes: a cardiology perspective. Ann Pharmacother 42:1466–1474 [DOI] [PubMed] [Google Scholar]
  27. Takeda K, Kaisho T, Akira S 2003 Toll-like receptors. Annu Rev Immunol 21:335–376 [DOI] [PubMed] [Google Scholar]
  28. Uematsu S, Akira S 2006 Toll-like receptors and innate immunity. J Mol Med 84:712–725 [DOI] [PubMed] [Google Scholar]
  29. Kawai T, Akira S 2006 TLR signaling. Cell Death Differ 13:816–825 [DOI] [PubMed] [Google Scholar]
  30. Kawai T, Adachi O, Ogawa T, Takeda K, Akira S 1999 Unresponsiveness of MyD88 deficient mice to endotoxin. Immunity 11:115–122 [DOI] [PubMed] [Google Scholar]
  31. Stoll LL, Denning GM, Weintraub NL 2006 Endotoxin, TLR4 signaling and vascular inflammation: potential therapeutic targets in cardiovascular disease. Curr Pharm Des 12:4229–4245 [DOI] [PubMed] [Google Scholar]
  32. Mullick AE, Tobias PS, Curtiss LK 2006 Toll-like receptors and atherosclerosis: key contributors in disease and health? Immunol Res 34:193–209 [DOI] [PubMed] [Google Scholar]
  33. Michelsen KS, Wong MH, Shah PK, Zhang W, Yano J, Doherty TM, Akira S, Rajavashisth TB, Arditi M 2004 Lack of Toll-like receptor 4 or myeloid differentiation factor 88 reduces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotein E. Proc Natl Acad Sci USA 101:10679–10684 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Liu X, Ukai T, Yumoto H, Davey M, Goswami S, Gibson 3rd FC, Genco CA 2008 Toll-like receptor 2 plays a critical role in the progression of atherosclerosis that is independent of dietary lipids. Atherosclerosis 196:146–154 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ye P, Yang W, Wu SM, Sheng L 2006 Effect of pioglitazone on the expression of inflammatory cytokines in attenuating rat cardiomyocyte hypertrophy. Methods Find Exp Clin Pharmacol 28:691–696 [DOI] [PubMed] [Google Scholar]
  36. Ishibashi M, Egashira K, Hiasa K, Inoue S, Ni W, Zhao Q, Usui M, Kitamoto S, Ichiki T, Takeshita A 2002 Antiinflammatory and antiarteriosclerotic effects of pioglitazone. Hypertension 40:687–693 [DOI] [PubMed] [Google Scholar]
  37. Haffner SM, Greenberg AS, Weston WM, Chen H, Williams K, Freed MI 2002 Effect of rosiglitazone treatment on nontraditional markers of cardiovascular disease in patients with type 2 diabetes mellitus. Circulation 106:679–684 [DOI] [PubMed] [Google Scholar]
  38. Marx N, Froehlich J, Siam L, Ittner J, Wierse G, Schmidt A, Scharnagl H, Hombach V, Koenig W 2003 Antidiabetic PPARγ-activator rosiglitazone reduces MMP-9 serum levels in type 2 diabetic patients with coronary artery disease. Arterioscler Thromb Vasc Biol 23:283–288 [DOI] [PubMed] [Google Scholar]
  39. Iwata M, Haruta T, Usui I, Takata Y, Takano A, Uno T, Kawahara J, Ueno E, Sasaoka T, Ishibashi O, Kobayashi M 2001 Pioglitazone ameliorates tumor necrosis factor-α-induced insulin resistance by a mechanism independent of adipogenic activity of peroxisome proliferator-activated receptor-γ. Diabetes 50:1083–1092 [DOI] [PubMed] [Google Scholar]
  40. Koshiyama H, Shimono D, Kuwamura N, Minamikawa J, Nakamura Y 2001 Rapid communication: inhibitory effect of pioglitazone on carotid arterial wall thickness in type 2 diabetes. J Clin Endocrinol Metab 86:3452–3456 [DOI] [PubMed] [Google Scholar]
  41. Llorens S, Mendizabal Y, Nava E 2007 Effects of pioglitazone and rosiglitazone on aortic vascular function in rat genetic hypertension. Eur J Pharmacol 575:105–112 [DOI] [PubMed] [Google Scholar]
  42. Bai YP, Liu YH, Chen J, Song T, You Y, Tang ZY, Li YJ, Zhang GG 2007 Rosiglitazone attenuates NF-κB-dependent ICAM-1 and TNF-α production caused by homocysteine via inhibiting ERK1/2/p38MAPK activation. Biochem Biophys Res Commun 360:20–26 [DOI] [PubMed] [Google Scholar]
  43. Bae MA, Song BJ 2003 Critical role of c-Jun N-terminal protein kinase activation in troglitazone-induced apoptosis of human HepG2 hepatoma cells. Mol Pharmacol 63:401–408 [DOI] [PubMed] [Google Scholar]
  44. Vabulas RM, Ahmad-Nejad P, da Costa C, Miethke T, Kirschning CJ, Häcker H, Wagner H 2001 Endocytosed HSP60s use toll-like receptor 2 (TLR2) and TLR4 to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells. J Biol Chem 276:31332–31339 [DOI] [PubMed] [Google Scholar]
  45. Thobe BM, Frink M, Hildebrand F, Schwacha MG, Hubbard WJ, Choudhry MA, Chaudry IH 2007 The role of MAPK in Kupffer cell toll-like receptor (TLR) 2-, TLR4-, and TLR9-mediated signaling following trauma-hemorrhage. J Cell Physiol 210:667–675 [DOI] [PubMed] [Google Scholar]
  46. Rodríguez-Calvo R, Serrano L, Coll T, Moullan N, Sánchez RM, Merlos M, Palomer X, Laguna JC, Michalik L, Wahli W, Vázquez-Carrera M 2008 Activation of peroxisome proliferator-activated receptor β/Δ inhibits lipopolysaccharide-induced cytokine production in adipocytes by lowering nuclear factor-κB activity via extracellular signal-related kinase 1/2. Diabetes 57:2149–2157 [DOI] [PMC free article] [PubMed] [Google Scholar]

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