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Chronic ethanol increases systemic TLR3 agonist-induced neuroinflammation and neurodegeneration.

Qin L, Crews FT - J Neuroinflammation (2012)

Bottom Line: Here, we investigate the effects of chronic ethanol on neuroinflammation and neurodegeneration triggered by toll-like receptor 3 (TLR3) agonist poly I:C.Escalating blood and brain proinflammatory responses were found with ethanol, poly I:C, and ethanol-poly I:C treatment.Ethanol potentiation of poly I:C was associated with ethanol-increased expression of TLR3 and endogenous agonist HMGB1 in the brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bowles Center for Alcohol Studies, School of Medicine, University of North Carolina at Chapel Hill, NC 27599, USA.

ABSTRACT

Background: Increasing evidence links systemic inflammation to neuroinflammation and neurodegeneration. We previously found that systemic endotoxin, a TLR4 agonist or TNFα, increased blood TNFα that entered the brain activating microglia and persistent neuroinflammation. Further, we found that models of ethanol binge drinking sensitized blood and brain proinflammatory responses. We hypothesized that blood cytokines contribute to the magnitude of neuroinflammation and that ethanol primes proinflammatory responses. Here, we investigate the effects of chronic ethanol on neuroinflammation and neurodegeneration triggered by toll-like receptor 3 (TLR3) agonist poly I:C.

Methods: Polyinosine-polycytidylic acid (poly I:C) was used to induce inflammatory responses when sensitized with D-galactosamine (D-GalN). Male C57BL/6 mice were treated with water or ethanol (5 g/kg/day, i.g., 10 days) or poly I:C (250 μg/kg, i.p.) alone or sequentially 24 hours after ethanol exposure. Cytokines, chemokines, microglial morphology, NADPH oxidase (NOX), reactive oxygen species (ROS), high-mobility group box 1 (HMGB1), TLR3 and cell death markers were examined using real-time PCR, ELISA, immunohistochemistry and hydroethidine histochemistry.

Results: Poly I:C increased blood and brain TNFα that peaked at three hours. Blood levels returned within one day, whereas brain levels remained elevated for at least three days. Escalating blood and brain proinflammatory responses were found with ethanol, poly I:C, and ethanol-poly I:C treatment. Ethanol pretreatment potentiated poly I:C-induced brain TNFα (345%), IL-1β (331%), IL-6 (255%), and MCP-1(190%). Increased levels of brain cytokines coincided with increased microglial activation, NOX gp91phox, superoxide and markers of neurodegeneration (activated caspase-3 and Fluoro-Jade B). Ethanol potentiation of poly I:C was associated with ethanol-increased expression of TLR3 and endogenous agonist HMGB1 in the brain. Minocycline and naltrexone blocked microglial activation and neurodegeneration.

Conclusions: Chronic ethanol potentiates poly I:C blood and brain proinflammatory responses. Poly I:C neuroinflammation persists after systemic responses subside. Increases in blood TNFα, IL-1β, IL-6, and MCP-1 parallel brain responses consistent with blood cytokines contributing to the magnitude of neuroinflammation. Ethanol potentiation of TLR3 agonist responses is consistent with priming microglia-monocytes and increased NOX, ROS, HMGB1-TLR3 and markers of neurodegeneration. These studies indicate that TLR3 agonists increase blood cytokines that contribute to neurodegeneration and that ethanol binge drinking potentiates these responses.

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Ethanol increases TLR3 and HMGB1 expression. Chronic ethanol treatment of C57BL/6 mice (5 g/kg, i.g., daily for 10 days) increased mRNA and protein expression (+IR) of brain TLR3 and HMGB1. (A) Quantitation of TLR3 mRNA and TLR3 + IR. (A-a) Level of brain TLR3 mRNA 27 hours following the last dose of ethanol treatment was measured using real-time PCR as described in the methods. Ethanol exposure significantly increased brain TLR3 mRNA. (A-b) TLR3 + IR cells were counted in mouse cortex after TLR3 immunostaining. Ethanol significantly increased the number of TLR3 + IR cells. (A-c) Representative images of immunohistochemical staining for TLR3 in the cortex of control and ethanol-treated mice. (B) Quantitation of HMGB1 mRNA and HMGB1 + IR. (B-a) HMGB1 mRNA was measured by real-time PCR in which ethanol increased by about 2 fold. (B-b) Quantitative evaluation of HMGB1 + IR. The number of HMGB1 + IR cells was increased about 2 fold. (B-c) The representative images of immunohistochemical staining for HMGB1 in the cortex of control and ethanol-treated mice. *P <0.05, **P <0.01, compared with water control group. Scale bar, 50 μm.
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Figure 8: Ethanol increases TLR3 and HMGB1 expression. Chronic ethanol treatment of C57BL/6 mice (5 g/kg, i.g., daily for 10 days) increased mRNA and protein expression (+IR) of brain TLR3 and HMGB1. (A) Quantitation of TLR3 mRNA and TLR3 + IR. (A-a) Level of brain TLR3 mRNA 27 hours following the last dose of ethanol treatment was measured using real-time PCR as described in the methods. Ethanol exposure significantly increased brain TLR3 mRNA. (A-b) TLR3 + IR cells were counted in mouse cortex after TLR3 immunostaining. Ethanol significantly increased the number of TLR3 + IR cells. (A-c) Representative images of immunohistochemical staining for TLR3 in the cortex of control and ethanol-treated mice. (B) Quantitation of HMGB1 mRNA and HMGB1 + IR. (B-a) HMGB1 mRNA was measured by real-time PCR in which ethanol increased by about 2 fold. (B-b) Quantitative evaluation of HMGB1 + IR. The number of HMGB1 + IR cells was increased about 2 fold. (B-c) The representative images of immunohistochemical staining for HMGB1 in the cortex of control and ethanol-treated mice. *P <0.05, **P <0.01, compared with water control group. Scale bar, 50 μm.

Mentions: To investigate the mechanisms of ethanol proinflammatory responses we measured the expression of TLR3 and HMGB1, a ubiquitous TLR3 co-agonist. Ethanol treatment increased brain TLR3 mRNA (Figure 8A-a). Toll-like receptor positive immunoreactivity (+IR) provides insight into protein levels and ethanol increased TLR3 + IR cells by at least 2 fold in cortex (Figure 8 A-b). Cells with upregulated TLR3 expression appear to be neurons, which are consistent with previous findings by others that TLR3 was expressed in many cell types of the brain in mice [18], including neurons [41], microglia [9] and astrocytes [23]. These measurements were assessed 24 hours after the last ethanol dose indicating that TLR increases persist after blood ethanol concentrations return to zero and during the poly I:C responses. HMGB1 can bind to and activate multiple TLR receptors [42] being required for TLR3 receptor activation [22]. Ethanol treatment increased both brain HMGB1 mRNA (Figure 8B-a) and HMGB1 + IR (Figure 8B-b) by about 2 fold in cortex. Thus, ethanol treatment increased both HMGB1 and TLR3 receptors in brain (Figure 8).


Chronic ethanol increases systemic TLR3 agonist-induced neuroinflammation and neurodegeneration.

Qin L, Crews FT - J Neuroinflammation (2012)

Ethanol increases TLR3 and HMGB1 expression. Chronic ethanol treatment of C57BL/6 mice (5 g/kg, i.g., daily for 10 days) increased mRNA and protein expression (+IR) of brain TLR3 and HMGB1. (A) Quantitation of TLR3 mRNA and TLR3 + IR. (A-a) Level of brain TLR3 mRNA 27 hours following the last dose of ethanol treatment was measured using real-time PCR as described in the methods. Ethanol exposure significantly increased brain TLR3 mRNA. (A-b) TLR3 + IR cells were counted in mouse cortex after TLR3 immunostaining. Ethanol significantly increased the number of TLR3 + IR cells. (A-c) Representative images of immunohistochemical staining for TLR3 in the cortex of control and ethanol-treated mice. (B) Quantitation of HMGB1 mRNA and HMGB1 + IR. (B-a) HMGB1 mRNA was measured by real-time PCR in which ethanol increased by about 2 fold. (B-b) Quantitative evaluation of HMGB1 + IR. The number of HMGB1 + IR cells was increased about 2 fold. (B-c) The representative images of immunohistochemical staining for HMGB1 in the cortex of control and ethanol-treated mice. *P <0.05, **P <0.01, compared with water control group. Scale bar, 50 μm.
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Figure 8: Ethanol increases TLR3 and HMGB1 expression. Chronic ethanol treatment of C57BL/6 mice (5 g/kg, i.g., daily for 10 days) increased mRNA and protein expression (+IR) of brain TLR3 and HMGB1. (A) Quantitation of TLR3 mRNA and TLR3 + IR. (A-a) Level of brain TLR3 mRNA 27 hours following the last dose of ethanol treatment was measured using real-time PCR as described in the methods. Ethanol exposure significantly increased brain TLR3 mRNA. (A-b) TLR3 + IR cells were counted in mouse cortex after TLR3 immunostaining. Ethanol significantly increased the number of TLR3 + IR cells. (A-c) Representative images of immunohistochemical staining for TLR3 in the cortex of control and ethanol-treated mice. (B) Quantitation of HMGB1 mRNA and HMGB1 + IR. (B-a) HMGB1 mRNA was measured by real-time PCR in which ethanol increased by about 2 fold. (B-b) Quantitative evaluation of HMGB1 + IR. The number of HMGB1 + IR cells was increased about 2 fold. (B-c) The representative images of immunohistochemical staining for HMGB1 in the cortex of control and ethanol-treated mice. *P <0.05, **P <0.01, compared with water control group. Scale bar, 50 μm.
Mentions: To investigate the mechanisms of ethanol proinflammatory responses we measured the expression of TLR3 and HMGB1, a ubiquitous TLR3 co-agonist. Ethanol treatment increased brain TLR3 mRNA (Figure 8A-a). Toll-like receptor positive immunoreactivity (+IR) provides insight into protein levels and ethanol increased TLR3 + IR cells by at least 2 fold in cortex (Figure 8 A-b). Cells with upregulated TLR3 expression appear to be neurons, which are consistent with previous findings by others that TLR3 was expressed in many cell types of the brain in mice [18], including neurons [41], microglia [9] and astrocytes [23]. These measurements were assessed 24 hours after the last ethanol dose indicating that TLR increases persist after blood ethanol concentrations return to zero and during the poly I:C responses. HMGB1 can bind to and activate multiple TLR receptors [42] being required for TLR3 receptor activation [22]. Ethanol treatment increased both brain HMGB1 mRNA (Figure 8B-a) and HMGB1 + IR (Figure 8B-b) by about 2 fold in cortex. Thus, ethanol treatment increased both HMGB1 and TLR3 receptors in brain (Figure 8).

Bottom Line: Here, we investigate the effects of chronic ethanol on neuroinflammation and neurodegeneration triggered by toll-like receptor 3 (TLR3) agonist poly I:C.Escalating blood and brain proinflammatory responses were found with ethanol, poly I:C, and ethanol-poly I:C treatment.Ethanol potentiation of poly I:C was associated with ethanol-increased expression of TLR3 and endogenous agonist HMGB1 in the brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bowles Center for Alcohol Studies, School of Medicine, University of North Carolina at Chapel Hill, NC 27599, USA.

ABSTRACT

Background: Increasing evidence links systemic inflammation to neuroinflammation and neurodegeneration. We previously found that systemic endotoxin, a TLR4 agonist or TNFα, increased blood TNFα that entered the brain activating microglia and persistent neuroinflammation. Further, we found that models of ethanol binge drinking sensitized blood and brain proinflammatory responses. We hypothesized that blood cytokines contribute to the magnitude of neuroinflammation and that ethanol primes proinflammatory responses. Here, we investigate the effects of chronic ethanol on neuroinflammation and neurodegeneration triggered by toll-like receptor 3 (TLR3) agonist poly I:C.

Methods: Polyinosine-polycytidylic acid (poly I:C) was used to induce inflammatory responses when sensitized with D-galactosamine (D-GalN). Male C57BL/6 mice were treated with water or ethanol (5 g/kg/day, i.g., 10 days) or poly I:C (250 μg/kg, i.p.) alone or sequentially 24 hours after ethanol exposure. Cytokines, chemokines, microglial morphology, NADPH oxidase (NOX), reactive oxygen species (ROS), high-mobility group box 1 (HMGB1), TLR3 and cell death markers were examined using real-time PCR, ELISA, immunohistochemistry and hydroethidine histochemistry.

Results: Poly I:C increased blood and brain TNFα that peaked at three hours. Blood levels returned within one day, whereas brain levels remained elevated for at least three days. Escalating blood and brain proinflammatory responses were found with ethanol, poly I:C, and ethanol-poly I:C treatment. Ethanol pretreatment potentiated poly I:C-induced brain TNFα (345%), IL-1β (331%), IL-6 (255%), and MCP-1(190%). Increased levels of brain cytokines coincided with increased microglial activation, NOX gp91phox, superoxide and markers of neurodegeneration (activated caspase-3 and Fluoro-Jade B). Ethanol potentiation of poly I:C was associated with ethanol-increased expression of TLR3 and endogenous agonist HMGB1 in the brain. Minocycline and naltrexone blocked microglial activation and neurodegeneration.

Conclusions: Chronic ethanol potentiates poly I:C blood and brain proinflammatory responses. Poly I:C neuroinflammation persists after systemic responses subside. Increases in blood TNFα, IL-1β, IL-6, and MCP-1 parallel brain responses consistent with blood cytokines contributing to the magnitude of neuroinflammation. Ethanol potentiation of TLR3 agonist responses is consistent with priming microglia-monocytes and increased NOX, ROS, HMGB1-TLR3 and markers of neurodegeneration. These studies indicate that TLR3 agonists increase blood cytokines that contribute to neurodegeneration and that ethanol binge drinking potentiates these responses.

Show MeSH
Related in: MedlinePlus