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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.

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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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TLR3 agonist poly I:C induction of TNFα in mouse serum and brain. Levels of proinflammatory cytokine TNFα were determined following a single poly I:C (250 μg/kg, i.p.) and d-galactosamine (D-GalN, 20 mg/kg, i.p.) injection into C57BL/6 mice. At the time points indicated, mice were sacrificed and brain extracts and sera prepared as described in methods. Note both brain and serum TNFα peaked at three hours. Interestingly, blood (serum) TNFα declined to control level by 24 hours whereas brain TNFα level remained elevated at about half the peak level for at least 72 hours. The results shown are the means ± SEM of two experiments performed with seven mice per time point. *P <0.05, **P <0.01, compared to the corresponding vehicle controls.
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Figure 1: TLR3 agonist poly I:C induction of TNFα in mouse serum and brain. Levels of proinflammatory cytokine TNFα were determined following a single poly I:C (250 μg/kg, i.p.) and d-galactosamine (D-GalN, 20 mg/kg, i.p.) injection into C57BL/6 mice. At the time points indicated, mice were sacrificed and brain extracts and sera prepared as described in methods. Note both brain and serum TNFα peaked at three hours. Interestingly, blood (serum) TNFα declined to control level by 24 hours whereas brain TNFα level remained elevated at about half the peak level for at least 72 hours. The results shown are the means ± SEM of two experiments performed with seven mice per time point. *P <0.05, **P <0.01, compared to the corresponding vehicle controls.

Mentions: We have previously found that induction of brain TNFα following intraperitoneal injections of LPS, a toll-like receptor 4 agonist, is related to blood TNFα that is transported into the brain inducing a response that lasted at least 10 months [1]. We hypothesized that poly I:C, a TLR3 agonist known to activate systemic and brain innate immune responses, would induce parallel systemic and brain proinflammatory responses that cause persistent brain activation. Poly I:C treatment of mice increased TNFα serum levels that peak around three hours at more than tenfold basal levels returning to near zero by 24 hours (Figure 1). Poly I:C treatment increased brain levels of TNFα that peaked at three hours after poly I:C at about 6 fold basal levels and remained significantly elevated for at least three days. These findings are consistent with acute systemic proinflammatory activation contributing to persistent brain neuroinflammatory responses.


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

Qin L, Crews FT - J Neuroinflammation (2012)

TLR3 agonist poly I:C induction of TNFα in mouse serum and brain. Levels of proinflammatory cytokine TNFα were determined following a single poly I:C (250 μg/kg, i.p.) and d-galactosamine (D-GalN, 20 mg/kg, i.p.) injection into C57BL/6 mice. At the time points indicated, mice were sacrificed and brain extracts and sera prepared as described in methods. Note both brain and serum TNFα peaked at three hours. Interestingly, blood (serum) TNFα declined to control level by 24 hours whereas brain TNFα level remained elevated at about half the peak level for at least 72 hours. The results shown are the means ± SEM of two experiments performed with seven mice per time point. *P <0.05, **P <0.01, compared to the corresponding vehicle controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3412752&req=5

Figure 1: TLR3 agonist poly I:C induction of TNFα in mouse serum and brain. Levels of proinflammatory cytokine TNFα were determined following a single poly I:C (250 μg/kg, i.p.) and d-galactosamine (D-GalN, 20 mg/kg, i.p.) injection into C57BL/6 mice. At the time points indicated, mice were sacrificed and brain extracts and sera prepared as described in methods. Note both brain and serum TNFα peaked at three hours. Interestingly, blood (serum) TNFα declined to control level by 24 hours whereas brain TNFα level remained elevated at about half the peak level for at least 72 hours. The results shown are the means ± SEM of two experiments performed with seven mice per time point. *P <0.05, **P <0.01, compared to the corresponding vehicle controls.
Mentions: We have previously found that induction of brain TNFα following intraperitoneal injections of LPS, a toll-like receptor 4 agonist, is related to blood TNFα that is transported into the brain inducing a response that lasted at least 10 months [1]. We hypothesized that poly I:C, a TLR3 agonist known to activate systemic and brain innate immune responses, would induce parallel systemic and brain proinflammatory responses that cause persistent brain activation. Poly I:C treatment of mice increased TNFα serum levels that peak around three hours at more than tenfold basal levels returning to near zero by 24 hours (Figure 1). Poly I:C treatment increased brain levels of TNFα that peaked at three hours after poly I:C at about 6 fold basal levels and remained significantly elevated for at least three days. These findings are consistent with acute systemic proinflammatory activation contributing to persistent brain neuroinflammatory responses.

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