Telmisartan Protects a Microglia Cell Line from LPS Injury Beyond AT1 Receptor Blockade or PPARγ Activation

Abstract

The Angiotensin II Receptor Blocker (ARB) Telmisartan reduces inflammation through Angiotensin II AT1 receptor blockade and peroxisome proliferator-activated receptor gamma (PPARγ) activation. However, in a mouse microglia-like BV2 cell line, imitating primary microglia responses with high fidelity and devoid of AT1 receptor gene expression or PPARγ activation, Telmisartan reduced gene expression of pro-injury factors, enhanced that of anti-inflammatory genes, and prevented LPS-induced increase in inflammatory markers. Using global gene expression profiling and pathways analysis, we revealed that Telmisartan normalized the expression of hundreds of genes upregulated by LPS and linked with inflammation, apoptosis and neurodegenerative disorders, while downregulating the expression of genes associated with oncological, neurodegenerative and viral diseases. The PPARγ full agonist Pioglitazone had no neuroprotective effects. Surprisingly, the PPARγ antagonists GW9662 and T0070907 were neuroprotective and enhanced Telmisartan effects. GW9226 alone significantly reduced LPS toxic effects and enhanced Telmisartan neuroprotection, including downregulation of pro-inflammatory TLR2 gene expression. Telmisartan and GW9662 effects on LPS injury negatively correlated with pro-inflammatory factors and upstream regulators, including TLR2, and positively with known neuroprotective factors and upstream regulators. Gene Set Enrichment Analysis (GSEA) of the Telmisartan and GW9662 data revealed negative correlations with sets of genes associated with neurodegenerative and metabolic disorders and toxic treatments in cultured systems, while demonstrating positive correlations with gene sets associated with neuroprotection and kinase inhibition. Our results strongly suggest that novel neuroprotective effects of Telmisartan and GW9662, beyond AT1 receptor blockade or PPARγ activation, include downregulation of the TLR2 signaling pathway, findings that may have translational relevance.

Keywords: Angiotensin receptor blockers; Inflammation; Neuroprotection, microglia; PPARγ; TLR2.

Fig. 1

Expression of AT1 and PPARγ genes in mouse cortex, BV2 cells treated with DMSO and BV2 cells treated with DMSO + LPS. In contrast to the mouse frontal cortex (CTX) BV2 cells do not express the AT1 receptor gene, whether treated with LPS or not (A). PPARg gene expression is extremely low in BV2 cells when compared to mouse frontal cortex and does not change whether treated with LPS or not. Results are means of three samples analyzed independently. Data are expressed as fold-change relative to CTX after correction for GAPDH expression and were analyzed by one way ANOVA followed by Duncan test. ***p < 0.0001 vs all others

Fig. 2

The PPARγ agonist Pioglitazone does not reduce LPS-induced increase in IL-1β and IL-6 gene expression. a Pretreatment with the PPARγ full agonist Pioglitazone (Pio) 10 μM for 2 h does not decrease the enhanced IL-1β gene expression produced after 1 h of exposure to LPS (100 ng/ml). ANOVA F (3, 16) = 18.07, p < 0.0001. b Pretreatment with the PPARγ full agonist Pioglitazone (Pio) 10 μM for 2 h does not decrease the enhanced IL-6 gene expression produced after 1 h of exposure to LPS (100 ng/ml). ANOVA F (3, 16) = 9.578, p = 0.0007. Results are means ± SEM for three to five groups analyzed independently. Data were analyzed by one-way ANOVA with Newman-Keuls to correct for multiple comparisons. ****p < 0.0001, **p < 0.01, *p<0.05 compared to DMSO; ^####p < 0.0001, ^###p < 0.001, ^##p<0.01 compared to Pio; ns (not significant)

Fig. 3

The PPARγ antagonist GW9662 enhances the Telmisartan-induced reduction of LPS-induced increase in IL-1β gene expression and eliminates the LPS-induced increase in TNFα gene expression. A Exposure to the PPARγ antagonist GW9662 (GW) 10 μM of for 2 h potentiates the effect of Telmisartan to reduce the increase in IL-1β gene expression produced after 1 h of exposure to LPS (100 ng/ml). ANOVAF (6, 14) = 27.95, p < 0.0001. B Exposure to the PPARγ antagonist GW9662 (GW) 10 μM or Telmisartan (Telm) 10 μM alone for 2 h eliminates the increase in TNFα gene expression produced after 1 h of exposure to LPS (100 ng/ml). ANOVA F (5, 16) = 13.19, p < 0.0001 Results are means ± SEM for three to five groups analyzed independently. Data were analyzed by one-way ANOVA with Newman- Keuls to correct for multiple comparisons. ****p < 0.0001, ***p < 0.001, *p < 0.05 compared to DMSO; ^####p < 0.0001, ^###p < 0.001, ^#p < 0.05 compared to Telm; ⁺⁺⁺⁺p < 0.0001, ⁺⁺⁺p<0.001, +p < 0.05 compared to GW; ^$$$p < 0.001, ^$$p<0.01 compared to LPS; ^%%p<0.01 compared to LPS + Telm; ^&&&p < 0.001 compared to GW+ LPS

Fig. 4

Telmisartan significantly reduces LPS-induced IL-β, IL-6, TNFα and Iκβα gene expression. Pretreatment with Telmisartan 10 μM (Telm) for 2 h significantly reduces the increase in a IL-1β gene expression produced after 1 h of exposure to LPS (100 ng/ml). ANOVA F (3, 14) = 37.9 p < 0.0001. b IL-6 gene expression produced after 1 h of exposure to LPS (100 ng/ml). ANOVA F (3, 10) = 52.9 p < 0.0001. c TNFα gene expression produced after 1 h of exposure to LPS (100 ng/ml). ANOVA F (3, 8) = 196.4 p<0.0001. d Iκβα gene expression produced after 1 h of exposure to LPS (100 ng/ml) F (3, 8) = 6.114 p = 0.0182. Results are means ± SEM for three to five groups analyzed independently. Data are expressed as fold-change relative to DMSO and were analyzed by one-way ANOVA followed by Newman-Keuls test for multiple comparisons. ****p < 0.0001, *p < 0.05 compared to DMSO; ^####p < 0.0001, ^###p < 0.001, ^#p < 0.05 compared to Telm; ⁺⁺p < 0.01, ⁺p < 0.05 compared to LPS

Fig. 5

Gene Set Enrichment Analysis (GSEA) of BV2 cells treated with Telmisartan + LPS versus Telmisartan + LPS + GW9662. Gene signatures (vertical bars) from GEO (NCBI) and MSIG (Broad Institute) were overlaid on the ranked list of genes from our microarray data (red and blue bar). In mouse astrocytes, genes activated by FGF2 and up regulated by U0126 correlate with genes upregulated by GW9662 (Fig. 4a) [114]. Genes up regulated by IL-4 in murine microglia are negatively correlated with genes up-regulated by GW9662 (Fig. 4b, GSE49329, [146]). Genes upregulated by glutamate and down regulated by Candesartan in rat cerebellar cortical neurons correlate with genes downregulated by GW9662 (Fig. 4c, GSE67036 [14]). In the striatum of dyskinetic rats treated with L-DOPA, genes down regulated by the anti-dyskinesia MEK inhibitor (PD98059) are also down-regulated by GW9662 (Fig. 4d, GSE93695 [147]). Genes up-regulated by PDGF in the neuroblastoma cell line SH-SY5Y pre-treated by the MEK inhibitor U0126 are negatively correlated with genes up-regulated by GW9662 (Fig. 4e [117, 144]). In the MCF7 breast cancer cell line stably overexpressing ligand-activable EGFR, genes down-regulated by EGFR are also down-regulated by GW9662 (Fig. 4f [148]). In the MCF7 breast cancer cell line stably overexpressing constitutively active MAP2K1 (MEK), genes down-regulated by MEK are also down-regulated by GW9662 (Fig. 4g [148]). In the MCF7 breast cancer cell line stably overexpressing constitutively active RAF1, genes upregulated by RAF1 are downregulated by GW9662 (Fig. 4h GSE3542 [148])