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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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Schematic summary and hypothetical mechanisms of neuroinflammation and neurodegeneration. (Lower left) Chronic ethanol treatment potentiates poly I:C increases serum TNFα IL-1β, IL-6 and MCP-1 protein. These proteins in the blood enter the brain through transport systems or other mechanisms as described in the discussion (upper left). In brain these proinflammatory cytokines activate microglia. Ethanol can also directly activate NF-κB transcription. Activated microglia amplify the brain neuroinflammatory response through at least three potential mechanisms. Loop 1 represents microglial synthesis and release of cytokines that activate transcription factor NF-κB to synthesize and release more inflammatory cytokines, which further activates the microglia, producing more proinflammatory signals. Loop 2 involves activation of NADPH oxidase (NOX) in microglia that produces reactive oxygen species that activate transcription factor NF-κB to synthesize and release more inflammatory cytokines. Loop 3 involves HMGB1, a TLR activator, and TLR3 on microglia that stimulates NF-κB and microglial activation. Cytokine, glutamate and/or ethanol release of HMGB1 that can activate multiple TLR receptors on microglia. Our findings of ethanol increased HMGB1 and TLR3 expression in brain support a role for loop 3 in microglial activation. Together, these amplify proinflammatory responses that spread from microglia to neurons (upper right). Neuronal expression of NOX increases oxidative stress leading to neuronal death. Minocycline and naltrexone block microglial activation and blunt neuronal death. These studies suggest that blood proinflammatory signals contribute to neuroinflammation and neurodegeneration that can be prevented by blocking microglial proinflammatory activation
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Figure 13: Schematic summary and hypothetical mechanisms of neuroinflammation and neurodegeneration. (Lower left) Chronic ethanol treatment potentiates poly I:C increases serum TNFα IL-1β, IL-6 and MCP-1 protein. These proteins in the blood enter the brain through transport systems or other mechanisms as described in the discussion (upper left). In brain these proinflammatory cytokines activate microglia. Ethanol can also directly activate NF-κB transcription. Activated microglia amplify the brain neuroinflammatory response through at least three potential mechanisms. Loop 1 represents microglial synthesis and release of cytokines that activate transcription factor NF-κB to synthesize and release more inflammatory cytokines, which further activates the microglia, producing more proinflammatory signals. Loop 2 involves activation of NADPH oxidase (NOX) in microglia that produces reactive oxygen species that activate transcription factor NF-κB to synthesize and release more inflammatory cytokines. Loop 3 involves HMGB1, a TLR activator, and TLR3 on microglia that stimulates NF-κB and microglial activation. Cytokine, glutamate and/or ethanol release of HMGB1 that can activate multiple TLR receptors on microglia. Our findings of ethanol increased HMGB1 and TLR3 expression in brain support a role for loop 3 in microglial activation. Together, these amplify proinflammatory responses that spread from microglia to neurons (upper right). Neuronal expression of NOX increases oxidative stress leading to neuronal death. Minocycline and naltrexone block microglial activation and blunt neuronal death. These studies suggest that blood proinflammatory signals contribute to neuroinflammation and neurodegeneration that can be prevented by blocking microglial proinflammatory activation

Mentions: We report here that intraperitoneal poly I:C when sensitized with d-GalN increased cytokines (TNFα, IL-1β, IL-6) and the cytokine-chemokine (MCP-1) in both blood and brain. Either poly I:C or d-GalN alone did not elevate serum and brain TNFα levels (data not shown), which is consistent with previous studies [4,46]. The three-day time course indicated poly I:C-induced brain and blood TNFα peaked at approximately three hours. Previously we found that intraperitoneal LPS, a toll-like receptor 4 (TLR4) agonist, increases liver, brain and blood TNFα that peaked one hour after treatment [1]. Increases in blood TNFα by TNFα injection or induced through LPS treatment required blood–brain barrier TNFα receptor-mediated transport to fully activate brain neuroinflammatory responses [1]. Many tissues may release cytokines into blood, spreading proinflammatory responses to other tissues. The liver and gut have large numbers of monocyte-like cells making it likely they release cytokines into the blood. Although poly I:C-stimulated brain and blood TNFαpeaked at the same time, blood levels returned to near zero by one day, whereas brain TNFα levels remained elevated for three days, the longest time point studied. We found a single LPS injection induced a blood response of less than 24 hours, whereas the brain neuroinflammatory response lasted for more than 10 months [1], consistent with the hypothesis that increases in blood proinflammatory cytokines trigger a persistent increase in neuroinflammation. A delayed increase in liver anti-inflammatory IL-10 may contribute to loss of systemic responses, whereas brain shows a delayed decrease in IL-10, possibly contributing to persistent brain neuroinflammation [27]. In this study, we investigated TNFα, IL-1β, IL-6 and MCP-1 in both blood and brain across four treatment groups that provided graded responses increasing in magnitude, for example, low controls, small ethanol alone responses, significant poly I:C responses and the largest response from ethanol-poly I:C treatment. For example, serum MCP-1 and brain MCP-1 mRNA and protein increase in parallel from controls that are a fewfold less than ethanol alone, with poly I:C alone manyfold larger and sequential ethanol-poly I:C treatment being significantly more than any other treatments. We also found that microglia, the innate immune cells of brain, showed morphological activation that paralleled the level of proinflammatory gene induction across control, ethanol, poly I:C and ethanol-poly I:C groups consistent with microglia responding to blood proinflammatory signals and amplifying the responses. We show here that TNFα, IL-1β, IL-6, and MCP-1 each shows graded increases in blood that resemble graded increases in brain mRNA and protein as well as stages of microglial activation across treatment groups. Proinflammatory cytokines have a blood-to-brain saturable transport system that carries cytokines and chemokines across the blood–brain barrier into brain [41]. Increased mRNA indicates brain protein increases are likely both synthesis and transport. Microglia are the innate immune cells of brain that express cytokine and TLR receptors that respond to many immune signals including cytokines and endogenous TLR agonists, such as HMGB1, a ubiquitous protein and TLR receptor agonist [42,47,48]. Microglia are uniquely sensitive to the brain environment and are thought to initiate neuroinflammatory responses [6]. This is consistent with our finding of increasing morphological activation of microglia coinciding with induction of brain TNFα, IL-1β, IL-6, and MCP-1 mRNA and blood protein levels of these cytokines and chemokines (Figure 13). However, endothelial cells in brain form the blood–brain barrier and both transport and increase synthesis and secretion of cytokines into brain [49]. Our findings are consistent with increases in blood proinflammatory cytokines contributing to activation of brain microglia, endothelial cells and neuroinflammatory gene induction in multiple types of brain cells (See Figure 13).


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

Qin L, Crews FT - J Neuroinflammation (2012)

Schematic summary and hypothetical mechanisms of neuroinflammation and neurodegeneration. (Lower left) Chronic ethanol treatment potentiates poly I:C increases serum TNFα IL-1β, IL-6 and MCP-1 protein. These proteins in the blood enter the brain through transport systems or other mechanisms as described in the discussion (upper left). In brain these proinflammatory cytokines activate microglia. Ethanol can also directly activate NF-κB transcription. Activated microglia amplify the brain neuroinflammatory response through at least three potential mechanisms. Loop 1 represents microglial synthesis and release of cytokines that activate transcription factor NF-κB to synthesize and release more inflammatory cytokines, which further activates the microglia, producing more proinflammatory signals. Loop 2 involves activation of NADPH oxidase (NOX) in microglia that produces reactive oxygen species that activate transcription factor NF-κB to synthesize and release more inflammatory cytokines. Loop 3 involves HMGB1, a TLR activator, and TLR3 on microglia that stimulates NF-κB and microglial activation. Cytokine, glutamate and/or ethanol release of HMGB1 that can activate multiple TLR receptors on microglia. Our findings of ethanol increased HMGB1 and TLR3 expression in brain support a role for loop 3 in microglial activation. Together, these amplify proinflammatory responses that spread from microglia to neurons (upper right). Neuronal expression of NOX increases oxidative stress leading to neuronal death. Minocycline and naltrexone block microglial activation and blunt neuronal death. These studies suggest that blood proinflammatory signals contribute to neuroinflammation and neurodegeneration that can be prevented by blocking microglial proinflammatory activation
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 13: Schematic summary and hypothetical mechanisms of neuroinflammation and neurodegeneration. (Lower left) Chronic ethanol treatment potentiates poly I:C increases serum TNFα IL-1β, IL-6 and MCP-1 protein. These proteins in the blood enter the brain through transport systems or other mechanisms as described in the discussion (upper left). In brain these proinflammatory cytokines activate microglia. Ethanol can also directly activate NF-κB transcription. Activated microglia amplify the brain neuroinflammatory response through at least three potential mechanisms. Loop 1 represents microglial synthesis and release of cytokines that activate transcription factor NF-κB to synthesize and release more inflammatory cytokines, which further activates the microglia, producing more proinflammatory signals. Loop 2 involves activation of NADPH oxidase (NOX) in microglia that produces reactive oxygen species that activate transcription factor NF-κB to synthesize and release more inflammatory cytokines. Loop 3 involves HMGB1, a TLR activator, and TLR3 on microglia that stimulates NF-κB and microglial activation. Cytokine, glutamate and/or ethanol release of HMGB1 that can activate multiple TLR receptors on microglia. Our findings of ethanol increased HMGB1 and TLR3 expression in brain support a role for loop 3 in microglial activation. Together, these amplify proinflammatory responses that spread from microglia to neurons (upper right). Neuronal expression of NOX increases oxidative stress leading to neuronal death. Minocycline and naltrexone block microglial activation and blunt neuronal death. These studies suggest that blood proinflammatory signals contribute to neuroinflammation and neurodegeneration that can be prevented by blocking microglial proinflammatory activation
Mentions: We report here that intraperitoneal poly I:C when sensitized with d-GalN increased cytokines (TNFα, IL-1β, IL-6) and the cytokine-chemokine (MCP-1) in both blood and brain. Either poly I:C or d-GalN alone did not elevate serum and brain TNFα levels (data not shown), which is consistent with previous studies [4,46]. The three-day time course indicated poly I:C-induced brain and blood TNFα peaked at approximately three hours. Previously we found that intraperitoneal LPS, a toll-like receptor 4 (TLR4) agonist, increases liver, brain and blood TNFα that peaked one hour after treatment [1]. Increases in blood TNFα by TNFα injection or induced through LPS treatment required blood–brain barrier TNFα receptor-mediated transport to fully activate brain neuroinflammatory responses [1]. Many tissues may release cytokines into blood, spreading proinflammatory responses to other tissues. The liver and gut have large numbers of monocyte-like cells making it likely they release cytokines into the blood. Although poly I:C-stimulated brain and blood TNFαpeaked at the same time, blood levels returned to near zero by one day, whereas brain TNFα levels remained elevated for three days, the longest time point studied. We found a single LPS injection induced a blood response of less than 24 hours, whereas the brain neuroinflammatory response lasted for more than 10 months [1], consistent with the hypothesis that increases in blood proinflammatory cytokines trigger a persistent increase in neuroinflammation. A delayed increase in liver anti-inflammatory IL-10 may contribute to loss of systemic responses, whereas brain shows a delayed decrease in IL-10, possibly contributing to persistent brain neuroinflammation [27]. In this study, we investigated TNFα, IL-1β, IL-6 and MCP-1 in both blood and brain across four treatment groups that provided graded responses increasing in magnitude, for example, low controls, small ethanol alone responses, significant poly I:C responses and the largest response from ethanol-poly I:C treatment. For example, serum MCP-1 and brain MCP-1 mRNA and protein increase in parallel from controls that are a fewfold less than ethanol alone, with poly I:C alone manyfold larger and sequential ethanol-poly I:C treatment being significantly more than any other treatments. We also found that microglia, the innate immune cells of brain, showed morphological activation that paralleled the level of proinflammatory gene induction across control, ethanol, poly I:C and ethanol-poly I:C groups consistent with microglia responding to blood proinflammatory signals and amplifying the responses. We show here that TNFα, IL-1β, IL-6, and MCP-1 each shows graded increases in blood that resemble graded increases in brain mRNA and protein as well as stages of microglial activation across treatment groups. Proinflammatory cytokines have a blood-to-brain saturable transport system that carries cytokines and chemokines across the blood–brain barrier into brain [41]. Increased mRNA indicates brain protein increases are likely both synthesis and transport. Microglia are the innate immune cells of brain that express cytokine and TLR receptors that respond to many immune signals including cytokines and endogenous TLR agonists, such as HMGB1, a ubiquitous protein and TLR receptor agonist [42,47,48]. Microglia are uniquely sensitive to the brain environment and are thought to initiate neuroinflammatory responses [6]. This is consistent with our finding of increasing morphological activation of microglia coinciding with induction of brain TNFα, IL-1β, IL-6, and MCP-1 mRNA and blood protein levels of these cytokines and chemokines (Figure 13). However, endothelial cells in brain form the blood–brain barrier and both transport and increase synthesis and secretion of cytokines into brain [49]. Our findings are consistent with increases in blood proinflammatory cytokines contributing to activation of brain microglia, endothelial cells and neuroinflammatory gene induction in multiple types of brain cells (See Figure 13).

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