Telmisartan ameliorates lipopolysaccharide-induced innate immune response through peroxisome proliferator-activated receptor-γ activation in human monocytes

Abstract

Objective: Angiotensin II type 1 receptor (AT1) blockers (ARBs) reduce the bacterial endotoxin lipopolysaccharide (LPS)-induced innate immune response in human circulating monocytes expressing few AT1. To clarify the mechanisms of anti-inflammatory effects of ARBs with different peroxisome proliferator-activated receptor-γ (PPARγ)-activating potencies, we focused our study on telmisartan, an ARB with the highest PPARγ-stimulating activity.

Methods: Human circulating monocytes and monocytic THP-1 (human acute monocytic leukemia cell line) cells were exposed to 50 ng/ml LPS with or without pre-incubation with telmisartan. AT1 mRNA and protein expressions were determined by real-time PCR and membrane receptor binding assay, respectively. The expression of pro-inflammatory factors was determined by real-time PCR, western blot analysis and ELISA. PPARγ activation was measured by electrophoretic mobility shift assay and its role was determined by pharmacological inhibition and PPARγ gene silencing.

Results: In human monocytes, telmisartan significantly attenuated the LPS-induced expression of pro-inflammatory factors, the release of pro-inflammatory cytokines and prostaglandin E2, nuclear factor-κB activation and reactive oxygen species formation. In THP-1 cells, telmisartan significantly reduced LPS-induced tumor necrosis factor-α, inhibitor of κB-α, monocyte chemotactic protein-1 (MCP-1) and lectin-like oxidized low-density lipoprotein receptor-1 gene expression and MCP-1-directed migration. Telmisartan also stimulated the expression of the PPARγ target genes cluster of differentiation 36 and ATP-binding cassette subfamily G member 1 in monocytes. The anti-inflammatory effects of telmisartan were prevented by both PPARγ antagonism and PPARγ gene silencing. Anti-inflammatory effects of ARBs correlated with their PPARγ agonist potency.

Conclusion: Our observations demonstrate that in human monocytes, ARBs inhibit the LPS-induced pro-inflammatory response to a major extent through the PPARγ activation pathway and may be beneficial for the treatment of cardiovascular and metabolic disorders in which inflammation plays a major role.

Fig. 1

Telmisartan (Telm) inhibited lipopolysaccharide (LPS)-induced pro-inflammatory cytokine release and mRNA expression in human monocytes. Monocytes were incubated with 50 ng/ml LPS alone or after 2 h pre-incubation with different doses of Telm. (a) Cumulative release of pro-inflammatory cytokines 4 h after LPS addition as determined by ELISA. (b) Expression of cytokine mRNA after 2 h incubation with LPS as determined by real-time PCR. Results are means ± SEM from three independent experiments and are expressed as a percentage of LPS-induced response. IL, interleukin; TNF, tumor necrosis factor. ^#P<0.05; ^##P<0.01; ^###P<0.001 compared with LPS.

Fig. 2

Telmisartan (Telm) inhibited lipopolysaccharide (LPS)-induced pro-inflammatory responses in human monocytes. Human monocytes were pre-incubated for 2 h with vehicle (DMSO) or Telm and subsequently exposed to 50 ng/ml LPS. (a) Expression of pro-inflammatory marker mRNA 2 h after LPS addition. (b) Cyclooxygenase-2 (COX-2) protein expression and cumulative prostaglandin E2 (PGE₂) release in monocytes exposed to LPS for 24 h as determined by western blot and EIA, respectively. COX-2 protein expression was determined by western blot and quantified by densitometry. Values are presented as percentage of DMSO-treated group, as means ± SEM from at least three independent experiments. ***P<0.001 vs. DMSO; ^#P<0.05 vs. LPS. Picture shows a representative western blot. (c) Reactive oxygen species (ROS) production was determined by dichlorodihydrofluorescein diacetate probe oxidation in monocytes pretreated with vehicle or 10 μmol/l Telm and then exposed to LPS for 2 h. (d) Cell migration assay was performed on THP-1 monocytic cells pretreated with vehicle or 10 μmol/l Telm and subsequently stimulated with 100 ng/ml monocyte chemotactic protein-1 (MCP-1) for 4 h. Results are means ± SEM from at least three independent experiments. **P<0.01 vs. DMSO; ***P<0.001 vs. DMSO; ^#P<0.05 vs. LPS; ^##P<0.01 vs. LPS; ^###P<0.001 vs. LPS or MCP-1. ICAM-1, intercellular adhesion molecule 1; LOX-1, lectin-like oxidized low-density lipoprotein receptor-1.

Fig. 3

Anti-inflammatory effects of telmisartan (Telm) in human monocytes involved inhibition of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) activation. Human monocytes were pre-incubated for 2 h with vehicle (DMSO) or 10 μmol/l Telm and subsequently exposed to lipopolysaccharide (LPS) (50 ng/ml). (a) Representative western blot image showing inhibitory effect of Telm on LPS-induced phosphorylation of p38 MAPK and extracellular signal-regulated kinases 1/2 (ERK1/2) in monocytes incubated for 30 min with LPS. Quantitative densitometric values are presented as percentage of DMSO-treated group, and as means ± SEM from at least three independent experiments. **P<0.01 vs. DMSO; ***P<0.001 vs. DMSO; ^##P<0.01 vs. LPS; ^###P<0.001 vs. LPS. (b) Telm dose dependently inhibited inhibitor of κB-α (IκB-α) mRNA expression induced by 2 h incubation with LPS, and prevented LPS-induced IκB-α protein degradation time dependently. IκB-α protein levels are presented as means ± SEM from three independent experiments. *P<0.05 vs. control (0 h); ^#P<0.05 vs. LPS with Telm group. Pictures are representative western blots. (c) Nuclear proteins extracted from human monocytes treated with LPS with or without Telm were used to measure NF-κB p65 protein expression by western blot. Representative western blot is shown. NF-κB p65 protein values are presented as means ± SEM from three independent experiments. ***P<0.001 vs. DMSO; ^###P<0.001 vs. LPS.

Fig. 4

Telmisartan (Telm) activated peroxisome proliferator-activated receptor-γ (PPARγ) and increased PPARγ target gene expression in human monocytes and THP-1 cells. Primary human monocytes or THP-1 cells were incubated for 4 h with vehicle (DMSO) or 10 μmol/l Telm. PPARγ activation was analyzed by electrophoretic mobility shift assay. Representative pictures show PPARγ–DNA binding in human monocytes (a) and THP-1 cells (b), and the intensity of each band was measured by densitometry for quantitative analysis. Anti-PPARγ antibody (2 μg) and a 100-fold excess of cold probe were used to determine the specificity of the shift. Pre-incubation of nuclear extracts from Telm-treated cells with an anti-PPARγ antibody led to a profound decrease in PPARγ–DNA binding activity, demonstrating the reaction specificity. Expression of mRNA of PPARγ and PPARγ target genes cluster of differentiation 36 (CD36) and ATP-binding cassette subfamily G member 1 (ABCG1) in monocytes (c) and THP-1 cells (d) was determined by real-time PCR. Results are means ± SEM of three independent experiments. *P<0.05 vs. DMSO; ***P<0.001 vs. DMSO.

Fig. 5

Peroxisome proliferator-activated receptor-γ (PPARγ) gene silencing eliminated the effect of telmisartan (Telm) on lipopolysaccharide (LPS)-induced gene expression in THP-1 cells. Human PPARγ-specific or nontargeting scrambled control small interfering RNA (siRNA) were transfected into THP-1 cells. PPARγ protein levels were measured at 48 and 72 h posttransfection by western blot (a). PPARγ mRNA expression (b) and cluster of differentiation 36 (CD36) and ATP-binding cassette subfamily G member 1 (ABCG1) mRNA expression (c) were measured at 72 h of posttransfection by real-time PCR. ***P<0.001 vs. scrambled siRNA or DMSO; ^###P<0.001 vs. Telm. The inflammatory markers tumor necrosis factor-α (TNFα) and inhibitor of κB-α (IκB-α) were measured in cells treated for 2 h with LPS (50 ng/ml) alone or in a combination with 10 μmol/l Telm with and without PPARγ siRNA transfection (d). Results are means ± SEM from three independent experiments. ^###P<0.001 vs. LPS; ***P<0.001 vs. LPS with Telm.

Fig. 6

Peroxisome proliferator-activated receptor-γ (PPARγ) antagonist prevents telmisartan (Telm) amelioration of lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNFα) release. Monocytes pretreated for 1 h with 1 μmol/l PPARγ antagonist T0070907 (T007) were incubated for 1 h with 10 μmol/l Telm or 10 μmol/l PPARγ agonist troglitazone (Tgz) followed by 4 h incubation in the presence of 50 ng/ml LPS. Cumulative TNFα release was determined in culture medium by ELISA. Results are means ± SEM of three independent experiments and are expressed as a percentage of LPS-induced response. ^#P<0.05 vs. LPS; ^###P<0.001 vs. LPS; *P<0.05 vs. LPS with Telm or LPS with Tgz.