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. 2017 Dec 12:10:409.
doi: 10.3389/fnmol.2017.00409. eCollection 2017.

Toll-Like Receptor 4 Mediates Methamphetamine-Induced Neuroinflammation through Caspase-11 Signaling Pathway in Astrocytes

Si-Hao Du  1 , Dong-Fang Qiao  1 , Chuan-Xiang Chen  1 , Si Chen  1 , Chao Liu  2 , Zhoumeng Lin  3 , Huijun Wang  1 , Wei-Bing Xie  1
Affiliations

Toll-Like Receptor 4 Mediates Methamphetamine-Induced Neuroinflammation through Caspase-11 Signaling Pathway in Astrocytes

Si-Hao Du et al. Front Mol Neurosci. .

Abstract

Methamphetamine (METH) is an amphetamine-typed stimulant drug that is increasingly being abused worldwide. Previous studies have shown that METH toxicity is systemic, especially targeting dopaminergic neurons in the central nervous system (CNS). However, the role of neuroinflammation in METH neurotoxicity remains unclear. We hypothesized that Toll-like receptor 4 (TLR4) and Caspase-11 are involved in METH-induced astrocyte-related neuroinflammation. We tested our hypothesis by examining the changes of TLR4 and Caspase-11 protein expression in primary cultured C57BL/6 mouse astrocytes and in the midbrain and striatum of mice exposed to METH with western blot and double immunofluorescence labeling. We also determined the effects of blocking Caspase-11 expression with wedelolactone (a specific inhibitor of Caspase-11) or siRNA on METH-induced neuroinflammation in astrocytes. Furthermore, we determined the effects of blocking TLR4 expression with TAK-242 (a specific inhibitor of TLR4) or siRNA on METH-induced neuroinflammation in astrocytes. METH exposure increased Caspase-11 and TLR4 expression both in vitro and in vivo, with the effects in vitro being dose-dependent. Inhibition of Caspase-11 expression with either wedelolactone or siRNAs reduced the expression of inflammasome NLRP3 and pro-inflammatory cytokines. In addition, blocking TLR4 expression inhibited METH-induced activation of NF-κB and Caspase-11 in vitro and in vivo, suggesting that TLR4-Caspase-11 pathway is involved in METH-induced neuroinflammation. These results indicate that Caspase-11 and TLR4 play an important role in METH-induced neuroinflammation and may be potential gene targets for therapeutics in METH-caused neurotoxicity.

Keywords: Caspase-11; Toll-like receptor 4 (TLR4); astrocyte; inflammasome; methamphetamine; neuroinflammation.

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Figures

Figure 1
Figure 1
Astrocytes are activated after Methamphetamine (METH) exposure in vitro and in vivo. Male C57BL/6 mice were divided randomly into control and experiment groups (n = 5). Animals were injected intraperitoneally with saline or METH (15 mg/kg/injection, 8 injections, at 12 h intervals). (A) Immunohistochemical staining of GFAP in the striatum and midbrain. Scale bar, 50 μm. Western blot (B,C) and quantitative analyses (B1–C2) were performed to determine IL-1β and IL-18 protein expression in the striatum and midbrain. Primary cultured astrocytes were exposed to 0.5, 1.0, 1.5, 2.0 and 2.5 mM METH for 24 h. (D) Morphological changes were observed. Western blot (E) and quantitative analyses (E1) were performed to determine IL-1β and IL-18 protein expression. β-actin was used as a loading control. Fold induction relative to vehicle-treated group is shown. *p < 0.05 vs. vehicle-treated group. Data were analyzed with Student’s t-test or one-way analyses of variance (ANOVA) followed by LSD post hoc analysis. Data are expressed as mean ± standard deviation (SD).
Figure 2
Figure 2
Caspase-11 mediates IL-1β and IL-18 expression in METH-exposed astrocytes. (A,A1) Primary cultured astrocytes were exposed to 0.5, 1.0, 1.5, 2.0 and 2.5 mM METH for 24 h. (B–B3) Primary cultured astrocytes were transfected with siRNAs targeting Caspase-11 or control siRNA for 24 h followed by METH (2 mM) treatment for 24 h. (C–C3) Primary cultured astrocytes were exposed to Wed (30 μM) for 2 h prior to METH (2 mM) treatment as indicated. Western blot (A–C) and quantitative analyses were performed to determine Caspase-11, IL-1β and IL-18 protein expression. All the experiments were repeated three times. Data are expressed as mean ± SD. *p < 0.05 vs. non-METH-treated group. #p < 0.05 vs. the scrambled + METH treated group. Data in (A) were analyzed with one-way ANOVA followed by LSD post hoc analyses; data in (B) were analyzed with two-way ANOVA followed by LSD post hoc analyses; data in (C) was analyzed with 2 × 2 factorial ANOVA followed by LSD post hoc analyses.
Figure 3
Figure 3
Inflammasomes are involved in Caspase-11-mediated IL-1β and IL-18 protein expression caused by METH in astrocytes. (A,A1) Primary cultured astrocytes were exposed to 0.5, 1.0, 1.5, 2.0 and 2.5 mM METH for 24 h. (B–B2) Cells were transfected with siRNAs targeting Caspase-11 or control siRNA for 24 h followed by METH (2 mM) treatment for 24 h. (C–C2) Cells were exposed to Wed (30 μM) for 2 h prior to METH (2 mM) treatment as indicated. (D–D3) Cells were transfected with siRNAs targeting apoptosis-associated speck-like protein containing CARD (ASC) or control siRNA for 24 h followed by METH (2 mM) treatment for 24 h. Western blot (A–D) and quantitative analyses were performed to determine Caspase-11, NLRP3, ASC, Caspase-1, IL-1β and IL-18 protein expression. All the experiments were repeated three times. Data are expressed as mean ± SD. *p < 0.05 vs. non-METH-treated group. #p < 0.05 vs. the scrambled + METH treated group. Data in (A) were analyzed with one-way ANOVA followed by LSD post hoc analyses; data in (B,D) were analyzed with two-way ANOVA followed by LSD post hoc analyses; data in (C) were analyzed with 2 × 2 factorial ANOVA followed by LSD post hoc analyses.
Figure 4
Figure 4
Toll-like receptor 4 (TLR4) is necessary for METH-induced IL-1β and IL-18 expression in astrocytes. (A,A1) Primary cultured astrocytes were exposed to 0.5, 1.0, 1.5, 2.0 and 2.5 mM METH for 24 h. (B–B3) Cells were transfected with siRNAs targeting TLR4 or control siRNA for 24 h followed by METH (2 mM) treatment for 24 h. (C–C3,D) Cells were exposed to TAK-242 (100 nM) for 2 h prior to METH (2 mM) treatment. Western blot (A–C) and quantitative analyses were performed to determine TLR4, IL-1β and IL-18 protein expression. Immunolabeling and confocal imaging analysis (D) showed elevated TLR4 expression in the cells treated with METH compared with controls. (E) Male C57BL/6 mice were divided randomly into control, METH, TAK-242 and METH + TAK-242 groups (n = 3/group). TAK-242 was injected intraperitoneally with DMSO and saline (3 mg/kg/injection, five injections, at 24 h intervals). At the 2nd day, animals were injected intraperitoneally with saline or METH (15 mg/kg/injection, eight injections, at 12 h intervals). Midbrain tissues were harvested at 2 h after the last dosing. Immunolabeling and confocal imaging analysis showed elevated TLR4 expression in the midbrain of METH-exposed mice compared with controls. Yellow arrow refers to TLR4. Cell experiments were repeated three times. Data are expressed as mean ± SD. *p < 0.05 vs. non-METH-treated group. #p < 0.05 vs. the scrambled + METH treated group. Data in (A) were analyzed with one-way ANOVA followed by LSD post hoc analyses; data in (B,C) were analyzed with 2 × 2 factorial ANOVA followed by LSD post hoc analyses.
Figure 5
Figure 5
Caspase-11 is involved in TLR4-mediated IL-1β and IL-18 expression in METH-exposed astrocytes. Primary cultured astrocytes transfected with siRNAs targeting Caspase-11 (A,A1), TLR4 (C–C3) or control siRNA for 24 h followed by METH (2 mM) treatment for 24 h. Cells were exposed to Wed (30 μM) (B,B1) or TAK-242 (100 nM) (D–D3) for 2 h prior to METH (2 mM) treatment as indicated. Western blot (A–D) and quantitative analyses were performed to determine TLR4, Caspase-11, ASC and Caspase-1 protein expression. All the experiments were repeated three times. Data are expressed as mean ± SD. *p < 0.05 vs. non-METH-treated group. #p < 0.05 vs. the scrambled + METH treated group. Data in (A) were analyzed with two-way ANOVA followed by LSD post hoc analyses; data in (B–D) were analyzed with 2 × 2 factorial ANOVA followed by LSD post hoc analyses.
Figure 6
Figure 6
TLR4 induces translocation of NF-κB into the nucleus, leading to an increase of Caspase-11 transcription in METH-exposed astrocytes. (A–A2) Primary cultured astrocytes were transfected with siRNAs targeting NF-κB or control siRNA for 24 h followed by METH (2 mM) treatment for 24 h. (B–D) Cells were exposed to TAK-242 (100 nM) for 2 h prior to METH (2 mM) treatment as indicated, nucleus proteins (C,C1) and cytoplasm (B,B1) proteins were extracted separately. Western blot (A–C) and quantitative analyses were performed to determine NF-κB, and Caspase-11 protein expression. H3 was used as the loading control of nucleus proteins. Immunolabeling and confocal imaging analysis (D) showed elevated NF-κB expression in the METH-treated cells compared with controls. (E) The animal exposure paradigms were the same as described in Figure 4. Immunolabeling and confocal imaging analysis showed elevated NF-κB expression in the midbrain of METH-exposed mice compared with controls. White arrow refers to NF-κB. Cell experiments were repeated three times. Data are expressed as mean ± SD. *p < 0.05 vs. non-METH-treated group. #p < 0.05 vs. the scrambled + METH treated group. Data were analyzed with 2 × 2 factorial ANOVA followed by LSD post hoc analyses.
Figure 7
Figure 7
TLR4 mediates METH-induced IL-1β and IL-18 expression through both Myd88-dependent and Myd88-independent signaling pathways. Primary cultured astrocytes transfected with siRNAs targeting TLR4 (A–A3), TRIF (C–C6), Myd88 (D–D6) or control siRNA for 24 h followed by METH (2 mM) treatment for 24 h. Cells were exposed to TAK-242 (100 nM) (B–B3) for 2 h prior to METH (2 mM) treatment as indicated. Western blot (A–D) and quantitative analyses were performed to determine TLR4, TRIF, TIRAP, Myd88, NF-κB, Caspase-11 and/or IL-1β protein expression. All the experiments were repeated three times. Data are expressed as mean ± SD. *p < 0.05 vs. non-METH-treated group. #p < 0.05 vs. the scrambled + METH treated group. Data in (A,B) were analyzed with 2 × 2 factorial ANOVA followed by LSD post hoc analyses; data in (C,D) were analyzed with two-way ANOVA followed by LSD post hoc analyses.
Figure 8
Figure 8
Silencing of TLR4 expression reduces METH-induced IL-1β and IL-18 expression in vivo. Mice were exposed to saline vehicle, METH, TAK-242, or METH + TAK-242 as described in the methods section and in Figure 4 legend. Striatum and midbrain tissues were harvested at 24 h after the last dosing. Western blot (A,B) and quantitative analyses (A1–A6,B1–B6) were performed to determine TLR4, NF-κB, Caspase-11, ASC, IL-18 and IL-1β protein expression. Data are expressed as mean ± SD. *p < 0.05 vs. non-METH-treated group. #p < 0.05 vs. METH-treated group. Data were analyzed with 2 × 2 factorial ANOVA followed by LSD post hoc analyses.
Figure 9
Figure 9
A schematic depicting the role of TLR4-NFkB-Caspase-11 signaling pathway in METH-induced astrocyte-related neuroinflammation. Briefly, TLR4 expression is increased following METH treatment. Increased TLR4 upregulates the expression of Myd88 and TRIF. The Myd88-dependent and Myd88-independent pathways upregulate the expression of transcription factor NF-κB. Then NF-κB elevates Caspase-11 expression, which mediates the inflammasome NLRP3 pathway by upregulating the expression of Caspase-1 and ASC. The increased NLRP3 inflammasomes induce the expression of pro-inflammatory cytokines IL-1β and IL-18.
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