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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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Effect of chronic ethanol treatment on poly I:C-induced blood and brain TNFα and IL-1β. As described in the methods, male C57BL/6 mice were treated intragastrically with ethanol (5 g/kg, i.g. daily for 10 days) and 24 hours after the last dose of ethanol treatment injected intraperitoneally with poly I:C (250 μg/kg) plus D-GalN (20 mg/kg). Brains were collected three hours after poly I:C injection for all groups, that is, ethanol alone is 27 hours after the last dose of ethanol. The levels of serum TNFα and IL-1β protein and brain TNFα and IL-1β mRNA and protein were measured by real-time PCR and ELISA. (A) Poly I:C treatment increased serum TNFα protein and brain TNFα mRNA and protein. Ethanol treatment did not alter serum TNFα protein, but increased brain TNFα mRNA and protein. Ethanol exposure potentiated poly I:C-induced serum TNFα protein as well as brain TNFα mRNA and protein. (B) Poly I:C treatment increased serum IL-1β protein and brain IL-1β mRNA and protein. Ethanol alone had no significant effect. Ethanol pretreatment potentiated poly I:C-induced serum IL-1β protein and brain IL-1β gene expression and protein synthesis. The results are the means ± SEM in two independent experiments with seven animals per group. *P <0.05, **P <0.01, compared with the vehicle control group. #P <0.05, compared with the corresponding poly I:C treated group.
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Figure 2: Effect of chronic ethanol treatment on poly I:C-induced blood and brain TNFα and IL-1β. As described in the methods, male C57BL/6 mice were treated intragastrically with ethanol (5 g/kg, i.g. daily for 10 days) and 24 hours after the last dose of ethanol treatment injected intraperitoneally with poly I:C (250 μg/kg) plus D-GalN (20 mg/kg). Brains were collected three hours after poly I:C injection for all groups, that is, ethanol alone is 27 hours after the last dose of ethanol. The levels of serum TNFα and IL-1β protein and brain TNFα and IL-1β mRNA and protein were measured by real-time PCR and ELISA. (A) Poly I:C treatment increased serum TNFα protein and brain TNFα mRNA and protein. Ethanol treatment did not alter serum TNFα protein, but increased brain TNFα mRNA and protein. Ethanol exposure potentiated poly I:C-induced serum TNFα protein as well as brain TNFα mRNA and protein. (B) Poly I:C treatment increased serum IL-1β protein and brain IL-1β mRNA and protein. Ethanol alone had no significant effect. Ethanol pretreatment potentiated poly I:C-induced serum IL-1β protein and brain IL-1β gene expression and protein synthesis. The results are the means ± SEM in two independent experiments with seven animals per group. *P <0.05, **P <0.01, compared with the vehicle control group. #P <0.05, compared with the corresponding poly I:C treated group.

Mentions: To investigate chronic ethanol proinflammatory responses and poly I:C TLR3 agonist responses across multiple proinflammatory agents, we determined proinflammatory responses in ethanol alone, poly I:C alone or sequential ethanol-poly I:C administration in C57BL/6 mice. We compared induction of cytokines, TNFα, IL-1β, IL-6 and the chemokine, MCP-1, that we previously found increased in postmortem human alcoholic brain [25]. Brains of mice treated for 10 days with a binge-drinking dose of ethanol followed by 27 hours of abstinence showed a significant increase in both TNFα mRNA and protein, although TNFα did not show an elevation in serum after chronic ethanol (Figure 2). Brains of ethanol-treated mice also showed increased IL-6 and MCP-1 mRNA and protein. Serum of ethanol-treated mice showed a 4 fold increase in MCP-1 and a 50% increase in IL-6, although values remained relatively low compared to those found with poly I:C treatment (Figure 3). Poly I:C treatment increased serum levels of TNFα, IL-1β, IL-6, and MCP-1 manyfold over vehicle control basal levels. Similarly brain mRNA and protein for TNFα, IL-1β, IL-6, and MCP-1 were increased manyfold by poly I:C treatment (Figures 2 and 3). Interestingly, ethanol pretreatment potentiated poly I:C responses increasing levels of proinflammatory cytokines in both blood and brain. The blood IL-1β level increased more than 10 fold in ethanol-poly I:C-treated mice (Figure 2). In brain, ethanol pretreatment potentiated poly I:C induction of TNFα mRNA from 4 to 10 fold, IL-1β mRNA from 4 to 8 fold, IL-6 mRNA from 6 to 12 fold, and MCP-1 mRNA from 28 to 49 fold (Figures 2 and 3). Ethanol pretreatment also increased poly I:C induction of TNFα, IL-1β, IL-6, and MCP-1 protein levels in brain (Figures 2 and 3). These results indicate that acute serum proinflammatory responses mimic brain proinflammatory responses. Both blood and brain show a modest ethanol response, marked poly I:C TLR3 agonist response and sequential ethanol-poly I:C amplification of proinflammatory gene induction.


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

Qin L, Crews FT - J Neuroinflammation (2012)

Effect of chronic ethanol treatment on poly I:C-induced blood and brain TNFα and IL-1β. As described in the methods, male C57BL/6 mice were treated intragastrically with ethanol (5 g/kg, i.g. daily for 10 days) and 24 hours after the last dose of ethanol treatment injected intraperitoneally with poly I:C (250 μg/kg) plus D-GalN (20 mg/kg). Brains were collected three hours after poly I:C injection for all groups, that is, ethanol alone is 27 hours after the last dose of ethanol. The levels of serum TNFα and IL-1β protein and brain TNFα and IL-1β mRNA and protein were measured by real-time PCR and ELISA. (A) Poly I:C treatment increased serum TNFα protein and brain TNFα mRNA and protein. Ethanol treatment did not alter serum TNFα protein, but increased brain TNFα mRNA and protein. Ethanol exposure potentiated poly I:C-induced serum TNFα protein as well as brain TNFα mRNA and protein. (B) Poly I:C treatment increased serum IL-1β protein and brain IL-1β mRNA and protein. Ethanol alone had no significant effect. Ethanol pretreatment potentiated poly I:C-induced serum IL-1β protein and brain IL-1β gene expression and protein synthesis. The results are the means ± SEM in two independent experiments with seven animals per group. *P <0.05, **P <0.01, compared with the vehicle control group. #P <0.05, compared with the corresponding poly I:C treated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Effect of chronic ethanol treatment on poly I:C-induced blood and brain TNFα and IL-1β. As described in the methods, male C57BL/6 mice were treated intragastrically with ethanol (5 g/kg, i.g. daily for 10 days) and 24 hours after the last dose of ethanol treatment injected intraperitoneally with poly I:C (250 μg/kg) plus D-GalN (20 mg/kg). Brains were collected three hours after poly I:C injection for all groups, that is, ethanol alone is 27 hours after the last dose of ethanol. The levels of serum TNFα and IL-1β protein and brain TNFα and IL-1β mRNA and protein were measured by real-time PCR and ELISA. (A) Poly I:C treatment increased serum TNFα protein and brain TNFα mRNA and protein. Ethanol treatment did not alter serum TNFα protein, but increased brain TNFα mRNA and protein. Ethanol exposure potentiated poly I:C-induced serum TNFα protein as well as brain TNFα mRNA and protein. (B) Poly I:C treatment increased serum IL-1β protein and brain IL-1β mRNA and protein. Ethanol alone had no significant effect. Ethanol pretreatment potentiated poly I:C-induced serum IL-1β protein and brain IL-1β gene expression and protein synthesis. The results are the means ± SEM in two independent experiments with seven animals per group. *P <0.05, **P <0.01, compared with the vehicle control group. #P <0.05, compared with the corresponding poly I:C treated group.
Mentions: To investigate chronic ethanol proinflammatory responses and poly I:C TLR3 agonist responses across multiple proinflammatory agents, we determined proinflammatory responses in ethanol alone, poly I:C alone or sequential ethanol-poly I:C administration in C57BL/6 mice. We compared induction of cytokines, TNFα, IL-1β, IL-6 and the chemokine, MCP-1, that we previously found increased in postmortem human alcoholic brain [25]. Brains of mice treated for 10 days with a binge-drinking dose of ethanol followed by 27 hours of abstinence showed a significant increase in both TNFα mRNA and protein, although TNFα did not show an elevation in serum after chronic ethanol (Figure 2). Brains of ethanol-treated mice also showed increased IL-6 and MCP-1 mRNA and protein. Serum of ethanol-treated mice showed a 4 fold increase in MCP-1 and a 50% increase in IL-6, although values remained relatively low compared to those found with poly I:C treatment (Figure 3). Poly I:C treatment increased serum levels of TNFα, IL-1β, IL-6, and MCP-1 manyfold over vehicle control basal levels. Similarly brain mRNA and protein for TNFα, IL-1β, IL-6, and MCP-1 were increased manyfold by poly I:C treatment (Figures 2 and 3). Interestingly, ethanol pretreatment potentiated poly I:C responses increasing levels of proinflammatory cytokines in both blood and brain. The blood IL-1β level increased more than 10 fold in ethanol-poly I:C-treated mice (Figure 2). In brain, ethanol pretreatment potentiated poly I:C induction of TNFα mRNA from 4 to 10 fold, IL-1β mRNA from 4 to 8 fold, IL-6 mRNA from 6 to 12 fold, and MCP-1 mRNA from 28 to 49 fold (Figures 2 and 3). Ethanol pretreatment also increased poly I:C induction of TNFα, IL-1β, IL-6, and MCP-1 protein levels in brain (Figures 2 and 3). These results indicate that acute serum proinflammatory responses mimic brain proinflammatory responses. Both blood and brain show a modest ethanol response, marked poly I:C TLR3 agonist response and sequential ethanol-poly I:C amplification of proinflammatory gene induction.

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