Effects of chronic and binge ethanol administration on mouse cerebellar and hippocampal neuroinflammation.

Background: Hippocampal and cerebellar neuropathology occurs in individuals with alcohol use disorders (AUD), resulting in impaired cognitive and motor function.Objectives: Evaluate the effects of ethanol on the expression of pro- and anti-inflammatory molecules, as well as the effects of the anti-inflammatory PPAR-γ agonist pioglitazone in suppressing ethanol-induced neuroinflammation.Methods: Adult male and female mice were treated chronically with ethanol for just under a month followed by a single acute binge dose of ethanol. Animals were provided liquid diet in the absence of ethanol (Control; n = 18, 9 M/9F), liquid diet containing ethanol (ethanol; n = 22, 11 M/11F), or liquid diet containing ethanol plus gavage administration of 30.0 mg/kg pioglitazone (ethanol + pioglitazone; n = 20, 10 M/10F). The hippocampus and cerebellum were isolated 24 h following the binge dose of ethanol, mRNA was isolated, and pro- and anti-inflammatory molecules were quantified by qRT-PCR.Results: Ethanol significantly (p < .05) increased the expression of pro-inflammatory molecules IL-1ß, TNF-α, CCL2, and COX2; increased the expression of inflammasome-related molecules NLRP3 and Casp1 but decreased IL-18; and altered the expression of anti-inflammatory molecules including TGFßR1 in the hippocampus and cerebellum, though some differences were observed between males and females and the two brain regions. The anti-inflammatory pioglitazone inhibited ethanol-induced alterations in the expression of most, but not all, inflammation-related molecules.Conclusion: Chronic plus binge administration of ethanol induced the expression of inflammatory molecules in adult mice and pioglitazone suppressed ethanol-induced neuroinflammation.

Ethanol modulation of cerebellar neuroinflammation in a postnatal mouse model of fetal alcohol spectrum disorders.

Fetal alcohol spectrum disorders (FASD) are alarmingly common, result in significant personal and societal loss, and there is no effective treatment for these disorders. Cerebellar neuropathology is common in FASD and causes aberrant cognitive and motor function. Ethanol-induced neuroinflammation is believed to contribute to neuropathological sequelae of FASD, and was previously demonstrated in the cerebellum in animal models of FASD. We now demonstrate neuroinflammation persists in the cerebellum several days following cessation of ethanol treatment in an early postnatal mouse model, with meaningful implications for timing of therapeutic intervention in FASD. We also demonstrate by Sholl analysis that ethanol decreases ramification of microglia cell processes in cells located near the Purkinje cell layer but not those near the external granule cell layer. Ethanol did not alter the expression of anti-inflammatory molecules or molecules that constitute NLRP1 and NLRP3 inflammasomes. Interestingly, ethanol decreased the expression of IL-23a (P19) and IL-12Rß1 suggesting that ethanol may suppress IL-12 and IL-23 signaling. Fractalkine-fractalkine receptor (CX3CL1-CX3CR1) signaling is believed to suppress microglial activation and our demonstration that ethanol decreases CX3CL1 expression suggests that ethanol modulation of CX3CL1-CX3CR1 signaling may contribute to cerebellar neuroinflammation and neuropathology. We demonstrate ethanol alters the expression of specific molecules in the cerebellum understudied in FASD, but crucial for immune responses. Ethanol increases the expression of NOX-2 and NGP and decreases the expression of RAG1, NOS1, CD59a, S1PR5, PTPN22, GPR37, and Serpinb1b. These molecules represent a new horizon as potential targets for development of FASD therapy.

Divergent and overlapping hippocampal and cerebellar transcriptome responses following developmental ethanol exposure during the secondary neurogenic period.

BACKGROUND: The developing hippocampus and cerebellum, unique among brain regions, exhibit a secondary surge in neurogenesis during the third trimester of pregnancy. Ethanol (EtOH) exposure during this period is results in a loss of tissue volume and associated neurobehavioral deficits. However, mechanisms that link EtOH exposure to teratology in these regions are not well understood. We therefore analyzed transcriptomic adaptations to EtOH exposure to identify mechanistic linkages. METHODS: Hippocampi and cerebella were microdissected at postnatal day (P)10, from control C57BL/6J mouse pups, and pups treated with 4 g/kg of EtOH from P4 to P9. RNA was isolated and RNA-seq analysis was performed. We compared gene expression in EtOH- and vehicle-treated control neonates and performed biological pathway-overrepresentation analysis. RESULTS: While EtOH exposure resulted in the general induction of genes associated with the S-phase of mitosis in both cerebellum and hippocampus, overall there was little overlap in differentially regulated genes and associated biological pathways between these regions. In cerebellum, EtOH additionally induced gene expression associated with the G2/M-phases of the cell cycle and sonic hedgehog signaling, while in hippocampus, EtOH-induced the pathways for ribosome biogenesis and protein translation. Moreover, EtOH inhibited the transcriptomic identities associated with inhibitory interneuron subpopulations in the hippocampus, while in the cerebellum there was a more pronounced inhibition of transcripts across multiple oligodendrocyte maturation stages. CONCLUSIONS: These data indicate that during the delayed neurogenic period, EtOH may stimulate the cell cycle, but it otherwise results in widely divergent molecular effects in the hippocampus and cerebellum. Moreover, these data provide evidence for region- and cell-type-specific vulnerability, which may contribute to the pathogenic effects of developmental EtOH exposure.

Protection against alcohol-induced neuronal and cognitive damage by the PPARγ receptor agonist pioglitazone.

Binge alcohol drinking has emerged as a typical phenomenon in young people. This pattern of drinking, repeatedly leading to extremely high blood and brain alcohol levels and intoxication is associated with severe risks of neurodegeneration and cognitive damage. Mechanisms involved in excitotoxicity and neuroinflammation are pivotal elements in alcohol-induced neurotoxicity. Evidence has demonstrated that PPARγ receptor activation shows anti-inflammatory and neuroprotective properties. Here we examine whether treatment with the PPARγ agonist pioglitazone is beneficial in counteracting neurodegeneration, neuroinflammation and cognitive damage produced by binge alcohol intoxication. Adult Wistar rats were subjected to a 4-day binge intoxication procedure, which is commonly used to model excessive alcohol consumption in humans. Across the 4-day period, pioglitazone (0, 30, 60mg/kg) was administered orally twice daily at 12-h intervals. Degenerative cells were detected by fluoro-jade B (FJ-B) immunostaining in brain regions where expression of pro-inflammatory cytokines was also determined. The effects of pioglitazone on cognitive function were assessed in an operant reversal learning task and the Morris water maze task. Binge alcohol exposure produced selective neuronal degeneration in the hippocampal dentate gyrus and the adjacent entorhinal cortex. Pioglitazone reduced FJ-B positive cells in both regions and prevented alcohol-induced expression of pro-inflammatory cytokines. Pioglitazone also rescued alcohol-impaired reversal learning in the operant task and spatial learning deficits in the Morris water maze. These findings demonstrate that activation of PPARγ protects against neuronal and cognitive degeneration elicited by binge alcohol exposure. The protective effect of PPARγ agonist appears to be linked to inhibition of pro-inflammatory cytokines.

Pioglitazone blocks ethanol induction of microglial activation and immune responses in the hippocampus, cerebellum, and cerebral cortex in a mouse model of fetal alcohol spectrum disorders.

BACKGROUND: Fetal alcohol spectrum disorders (FASD) result from fetal exposure to alcohol and are the leading cause of mental retardation in the United States. There is currently no effective treatment that targets the causes of these disorders. Thus, novel therapies are critically needed to limit the neurodevelopmental and neurodegenerative pathologies associated with FASD. METHODS: A neonatal mouse FASD model was used to examine the role of the neuroimmune system in ethanol (EtOH)-induced neuropathology. Neonatal C57BL/6 mice were treated with EtOH, with or without pioglitazone, on postnatal days 4 through 9, and tissue was harvested 1 day post treatment. Pioglitazone is a peroxisome proliferator-activated receptor (PPAR)-γ agonist that exhibits anti-inflammatory activity and is neuroprotective. We compared the effects of EtOH with or without pioglitazone on cytokine and chemokine expression and microglial morphology in the hippocampus, cerebellum, and cerebral cortex. RESULTS: In EtOH-treated animals compared with controls, cytokines interleukin-1ß and tumor necrosis factor-α mRNA levels were increased significantly in the hippocampus, cerebellum, and cerebral cortex. Chemokine CCL2 mRNA was increased significantly in the hippocampus and cerebellum. Pioglitazone effectively blocked the EtOH-induced increase in the cytokines and chemokine in all tissues to the level expressed in handled-only and vehicle-treated control animals. EtOH also produced a change in microglial morphology in all brain regions that was indicative of microglial activation, and pioglitazone blocked this EtOH-induced morphological change. CONCLUSIONS: These studies indicate that EtOH activates microglia to a pro-inflammatory stage and also increases the expression of neuroinflammatory cytokines and chemokines in diverse regions of the developing brain. Further, the anti-inflammatory and neuroprotective PPAR-γ agonist pioglitazone blocked these effects. It is proposed that microglial activation and inflammatory molecules expressed as a result of EtOH treatment during brain development contribute to the sequelae associated with FASD. Thus, pioglitazone and anti-inflammatory pharmaceuticals more broadly have potential as novel therapeutics for FASD.

Effects of ethanol on immune response in the brain: region-specific changes in adolescent versus adult mice.

BACKGROUND: Alcohol use occurs across the life span beginning in adolescence and continuing through adulthood. Ethanol (EtOH)-induced pathology varies with age and includes changes in neurogenesis, neurodegeneration, and glial cell activation. EtOH-induced changes in glial activation and immune activity are believed to contribute to EtOH-induced neuropathology. Recent studies indicate an emerging role of glial-derived neuroimmune molecules in alcohol abuse and addiction. METHODS: Adolescent and adult C57BL/6 mice were treated via gavage with 6 g/kg EtOH for 10 days, and tissue was harvested 1 day post treatment. We compared the effects of EtOH on chemokine and cytokine expression and astrocyte glial fibrillary acidic protein (GFAP) immunostaining and morphology in the hippocampus, cerebellum, and cerebral cortex. RESULTS: EtOH increased mRNA levels of the chemokine CCL2/MCP-1 in all 3 regions of adult mice relative to controls. The cytokine interleukin-6 (IL-6) was selectively increased only in the adult cerebellum. EtOH did not affect mRNA levels of the cytokine tumor necrosis factor-alpha (TNF-α) in any of these brain regions in adult animals. Interestingly, CCL2, IL-6, and TNF-α mRNA levels were not increased in the hippocampus, cerebellum, or cortex of adolescent mice. EtOH treatment of adult and adolescent mice resulted in increased GFAP immunostaining. CONCLUSIONS: Collectively, these data indicate an age- and region-specific susceptibility to EtOH regulation of neuroinflammatory and addiction-related molecules as well as astrocyte phenotype. These studies may have important implications concerning differential alcohol-induced neuropathology and alcohol addiction across the life span.

Developmental Ethanol Exposure Impacts Purkinje Cells but Not Microglia in the Young Adult Cerebellum.

Fetal alcohol spectrum disorders (FASD) caused by developmental ethanol exposure lead to cerebellar impairments, including motor problems, decreased cerebellar weight, and cell death. Alterations in the sole output of the cerebellar cortex, Purkinje cells, and central nervous system immune cells, microglia, have been reported in animal models of FASD. To determine how developmental ethanol exposure affects adult cerebellar microglia and Purkinje cells, we used a human third-trimester binge exposure model in which mice received ethanol or saline from postnatal (P) days 4-9. In adolescence, cerebellar cranial windows were implanted and mice were aged to young adulthood for examination of microglia and Purkinje cells in vivo with two-photon imaging or in fixed tissue. Ethanol had no effect on microglia density, morphology, dynamics, or injury response. However, Purkinje cell linear frequency was reduced by ethanol. Microglia-Purkinje cell interactions in the Purkinje Cell Layer were altered in females compared to males. Overall, developmental ethanol exposure had few effects on cerebellar microglia in young adulthood and Purkinje cells appeared to be more susceptible to its effects.

Effects of ethanol on immune response in the brain: region-specific changes in aged mice.

BACKGROUND: Alcohol abuse has dramatic effects on the health of the elderly. Recent studies indicate that ethanol increases immune activity in younger animals and that some of these proinflammatory molecules alter alcohol consumption and addiction. However, the effects of alcohol on immune activation in aged animals have not been thoroughly investigated. FINDINGS: We compared the effects of ethanol on chemokine and cytokine expression in the hippocampus, cerebellum, and cerebral cortex of aged C57BL/6 mice. Mice were treated via gavage with 6 g/kg ethanol for 10 days and tissue was harvested 1 day post-treatment. Ethanol selectively increased mRNA levels of the chemokine (C-C motif) ligand 2/monocyte chemotactic protein-1 in the hippocampus and cerebellum, but not in the cortex of aged mice relative to control animals. In this paradigm, ethanol did not affect mRNA levels of the cytokines IL-6 or TNF-α in any of these brain regions in aged animals. CONCLUSIONS: Collectively, these data indicate a region-specific susceptibility to ethanol regulation of neuroinflammatory and addiction-related molecules in aged mice. These studies could have important implications concerning alcohol-induced neuropathology and alcohol addiction in the elderly.

Ethanol Induces Neuroinflammation in a Chronic Plus Binge Mouse Model of Alcohol Use Disorder via TLR4 and MyD88-Dependent Signaling.

Ethanol induces neuroinflammation, which is believed to contribute to the pathogenesis of alcohol use disorder (AUD). Toll-like receptors (TLRs) are a group of pattern recognition receptors (PRRs) expressed on both immune cells, including microglia and astrocytes, and non-immune cells in the central nervous system (CNS). Studies have shown that alcohol activates TLR4 signaling, resulting in the induction of pro-inflammatory cytokines and chemokines in the CNS. However, the effect of alcohol on signaling pathways downstream of TLR4, such as MyD88 and TRIF (TICAM) signaling, has not been evaluated extensively. In the current study, we treated male wild-type, TLR4-, MyD88-, and TRIF-deficient mice using a chronic plus binge mouse model of AUD. Evaluation of mRNA expression by qRT-PCR revealed that ethanol increased IL-1ß, TNF-α, CCL2, COX2, FosB, and JunB in the cerebellum in wild-type and TRIF-deficient mice, while ethanol generally did not increase the expression of these molecules in TLR4- and MyD88-deficient mice. Furthermore, IRF3, IRF7, and IFN-ß1, which are associated with the TRIF-dependent signaling cascade, were largely unaffected by alcohol. Collectively, these results suggest that the TLR4 and downstream MyD88-dependent signaling pathways are essential in ethanol-induced neuroinflammation in this mouse model of AUD.

Ethanol-induced cerebellar transcriptomic changes in a postnatal model of fetal alcohol spectrum disorders: Focus on disease onset.

Fetal alcohol spectrum disorders (FASD) are a group of neurodevelopmental disorders caused by ethanol exposure in utero, which can result in neurocognitive and behavioral impairments, growth defects, and craniofacial anomalies. FASD affects up to 1-5% of school-aged children in the United States, and there is currently no cure. The underlying mechanisms involved in ethanol teratogenesis remain elusive and need greater understanding to develop and implement effective therapies. Using a third trimester human equivalent postnatal mouse model of FASD, we evaluate the transcriptomic changes induced by ethanol exposure in the cerebellum on P5 and P6, after only 1 or 2 days of ethanol exposure, with the goal of shedding light on the transcriptomic changes induced early during the onset and development of FASD. We have highlighted key pathways and cellular functions altered by ethanol exposure, which include pathways related to immune function and cytokine signaling as well as the cell cycle. Additionally, we found that ethanol exposure resulted in an increase in transcripts associated with a neurodegenerative microglia phenotype, and acute- and pan-injury reactive astrocyte phenotypes. Mixed effects on oligodendrocyte lineage cell associated transcripts and cell cycle associated transcripts were observed. These studies help to elucidate the underlying mechanisms that may be involved with the onset of FASD and provide further insights that may aid in identifying novel targets for interventions and therapeutics.

Cerebellar Transcriptomic Analysis in a Chronic plus Binge Mouse Model of Alcohol Use Disorder Demonstrates Ethanol-Induced Neuroinflammation and Altered Glial Gene Expression.

Alcohol use disorder (AUD) is one of the most common preventable mental health disorders and can result in pathology within the CNS, including the cerebellum. Cerebellar alcohol exposure during adulthood has been associated with disruptions in proper cerebellar function. However, the mechanisms regulating ethanol-induced cerebellar neuropathology are not well understood. High-throughput next generation sequencing was performed to compare control versus ethanol-treated adult C57BL/6J mice in a chronic plus binge model of AUD. Mice were euthanized, cerebella were microdissected, and RNA was isolated and submitted for RNA-sequencing. Down-stream transcriptomic analyses revealed significant changes in gene expression and global biological pathways in control versus ethanol-treated mice that included pathogen-influenced signaling pathways and cellular immune response pathways. Microglial-associated genes showed a decrease in homeostasis-associated transcripts and an increase in transcripts associated with chronic neurodegenerative diseases, while astrocyte-associated genes showed an increase in transcripts associated with acute injury. Oligodendrocyte lineage cell genes showed a decrease in transcripts associated with both immature progenitors as well as myelinating oligodendrocytes. These data provide new insight into the mechanisms by which ethanol induces cerebellar neuropathology and alterations to the immune response in AUD.

Developmental ethanol exposure has minimal impact on cerebellar microglial dynamics, morphology, and interactions with Purkinje cells during adolescence.

Introduction: Fetal alcohol spectrum disorders (FASD) are the most common cause of non-heritable, preventable mental disability, occurring in almost 5% of births in the United States. FASD lead to physical, behavioral, and cognitive impairments, including deficits related to the cerebellum. There is no known cure for FASD and their mechanisms remain poorly understood. To better understand these mechanisms, we examined the cerebellum on a cellular level by studying microglia, the principal immune cells of the central nervous system, and Purkinje cells, the sole output of the cerebellum. Both cell types have been shown to be affected in models of FASD, with increased cell death, immune activation of microglia, and altered firing in Purkinje cells. While ethanol administered in adulthood can acutely depress the dynamics of the microglial process arbor, it is unknown how developmental ethanol exposure impacts microglia dynamics and their interactions with Purkinje cells in the long term. Methods: To address this question, we used a mouse model of human 3rd trimester exposure, whereby L7cre/Ai9+/-/Cx3cr1G/+ mice (with fluorescently labeled microglia and Purkinje cells) of both sexes were subcutaneously treated with a binge-level dose of ethanol (5.0 g/kg/day) or saline from postnatal days 4-9. Cranial windows were implanted in adolescent mice above the cerebellum to examine the long-term effects of developmental ethanol exposure on cerebellar microglia and Purkinje cell interactions using in vivo two-photon imaging. Results: We found that cerebellar microglia dynamics and morphology were not affected after developmental ethanol exposure. Microglia dynamics were also largely unaltered with respect to how they interact with Purkinje cells, although subtle changes in these interactions were observed in females in the molecular layer of the cerebellum. Discussion: This work suggests that there are limited in vivo long-term effects of ethanol exposure on microglia morphology, dynamics, and neuronal interactions, so other avenues of research may be important in elucidating the mechanisms of FASD.

Protection of neurons and microglia against ethanol in a mouse model of fetal alcohol spectrum disorders by peroxisome proliferator-activated receptor-γ agonists.

Fetal alcohol spectrum disorders (FASD) result from ethanol exposure to the developing fetus and are the most common cause of mental retardation in the United States. These disorders are characterized by a variety of neurodevelopmental and neurodegenerative anomalies which result in significant lifetime disabilities. Thus, novel therapies are required to limit the devastating consequences of FASD. Neuropathology associated with FASD can occur throughout the central nervous system (CNS), but is particularly well characterized in the developing cerebellum. Rodent models of FASD have previously demonstrated that both Purkinje cells and granule cells, which are the two major types of neurons in the cerebellum, are highly susceptible to the toxic effects of ethanol. The current studies demonstrate that ethanol decreases the viability of cultured cerebellar granule cells and microglial cells. Interestingly, microglia have dual functionality in the CNS. They provide trophic and protective support to neurons. However, they may also become pathologically activated and produce inflammatory molecules toxic to parenchymal cells including neurons. The findings in this study demonstrate that the peroxisome proliferator-activated receptor-γ agonists 15-deoxy-Δ12,15 prostaglandin J2 and pioglitazone protect cultured granule cells and microglia from the toxic effects of ethanol. Furthermore, investigations using a newly developed mouse model of FASD and stereological cell counting methods in the cerebellum elucidate that ethanol administration to neonates is toxic to both Purkinje cell neurons as well as microglia, and that in vivo administration of PPAR-γ agonists protects these cells. In composite, these studies suggest that PPAR-γ agonists may be effective in limiting ethanol-induced toxicity to the developing CNS.

Ethanol effects on cerebellar myelination in a postnatal mouse model of fetal alcohol spectrum disorders.

Fetal alcohol spectrum disorders (FASD) are alarmingly common, result in significant personal and societal loss, and there are no effective treatments for these disorders. Cerebellar neuropathology is common in FASD and can cause impaired cognitive and motor function. The current study evaluates the effects of ethanol on oligodendrocyte-lineage cells, as well as molecules that modulate oligodendrocyte differentiation and function in the cerebellum in a postnatal mouse model of FASD. Neonatal mice were treated with ethanol from P4-P9 (postnatal day), the cerebellum was isolated at P10, and mRNAs encoding oligodendrocyte-associated molecules were quantitated by qRT-PCR. Our studies demonstrated that ethanol significantly reduced the expression of markers for multiple stages of oligodendrocyte maturation, including oligodendrocyte precursor cells, pre-myelinating oligodendrocytes, and mature myelinating oligodendrocytes. Additionally, we determined that ethanol significantly decreased the expression of molecules that play critical roles in oligodendrocyte differentiation. Interestingly, we also observed that ethanol significantly reduced the expression of myelin-associated inhibitors, which may act as a compensatory mechanism to ethanol toxicity. Furthermore, we demonstrate that ethanol alters the expression of a variety of molecules important in oligodendrocyte function and myelination. Collectively, our studies increase our understanding of specific mechanisms by which ethanol modulates myelination in the developing cerebellum, and potentially identify novel targets for FASD therapy.

Ethanol modulation of hippocampal neuroinflammation, myelination, and neurodevelopment in a postnatal mouse model of fetal alcohol spectrum disorders.

Fetal alcohol spectrum disorders (FASD) are alarmingly common and result in significant personal and societal loss. Neuropathology of the hippocampus is common in FASD leading to aberrant cognitive function. In the current study, we evaluated the effects of ethanol on the expression of a targeted set of molecules involved in neuroinflammation, myelination, neurotransmission, and neuron function in the developing hippocampus in a postnatal model of FASD. Mice were treated with ethanol from P4-P9, hippocampi were isolated 24 h after the final treatment at P10, and mRNA levels were quantitated by qRT-PCR. We evaluated the effects of ethanol on both pro-inflammatory and anti-inflammatory molecules in the hippocampus and identified novel mechanisms by which ethanol induces neuroinflammation. We further demonstrated that ethanol decreased expression of molecules associated with mature oligodendrocytes and greatly diminished expression of a lacZ reporter driven by the first half of the myelin proteolipid protein (PLP) gene (PLP1). In addition, ethanol caused a decrease in genes expressed in oligodendrocyte progenitor cells (OPCs). Together, these studies suggest ethanol may modulate pathogenesis in the developing hippocampus through effects on cells of the oligodendrocyte lineage, resulting in altered oligodendrogenesis and myelination. We also observed differential expression of molecules important in synaptic plasticity, neurogenesis, and neurotransmission. Collectively, the molecules evaluated in these studies may play a role in ethanol-induced pathology in the developing hippocampus and contribute to cognitive impairment associated with FASD. A better understanding of these molecules and their effects on the developing hippocampus may lead to novel treatment strategies for FASD.

ß-Lapachone ameliorization of experimental autoimmune encephalomyelitis.

ß-Lapachone is a naturally occurring quinine, originally isolated from the bark of the lapacho tree (Tabebuia avellanedae) which is currently being evaluated in clinical trials for the treatment of cancer. In addition, recent investigations suggest its potential application for treatment of inflammatory diseases. Multiple sclerosis (MS) is an autoimmune disorder characterized by CNS inflammation and demyelination. Reactive T cells including IL-17 and IFN-γ-secreting T cells are believed to initiate MS and the associated animal model system experimental autoimmune encephalomyelitis (EAE). IL-12 family cytokines secreted by peripheral dendritic cells (DCs) and CNS microglia are capable of modulating T-cell phenotypes. The present studies demonstrated that ß-lapachone selectively inhibited the expression of IL-12 family cytokines including IL-12 and IL-23 by DCs and microglia, and reduced IL-17 production by CD4(+) T-cells indirectly through suppressing IL-23 expression by microglia. Importantly, our studies also demonstrated that ß-lapachone ameliorated the development on EAE. ß-Lapachone suppression of EAE was associated with decreased expression of mRNAs encoding IL-12 family cytokines, IL-23R and IL-17RA, and molecules important in Toll-like receptor signaling. Collectively, these studies suggest mechanisms by which ß-lapachone suppresses EAE and suggest that ß-lapachone may be effective in the treatment of inflammatory diseases such as MS.

Liver X receptor agonist regulation of Th17 lymphocyte function in autoimmunity.

CD4+ Th17 cells are believed to play an important role in the development of a variety of autoimmune diseases including EAE, an animal model of MS. Previously, we and others demonstrated that LXR agonists suppressed the activation of primary glial cells and blocked the development of EAE. The present studies demonstrated that the LXR agonist T0901317 suppressed IL-17A expression from splenocytes derived from Valpha2.3/Vbeta8.2 TCR transgenic mice and from MOG(35-55)-immunized C57BL/6 mice. Furthermore, in vitro treatment with IL-23 alone or in combination with MOG(35-55) induced IL-17A expression from splenocytes derived from MOG(35-55)-immunized mice, and T0901317 blocked this induction. In vitro treatment with the LXR agonist suppressed IL-23R expression by splenocytes. In addition, in vivo treatment with the LXR agonist suppressed IL-17A and IL-23R mRNA and protein expression in EAE mice. These studies suggest that LXR agonists suppress EAE, at least in part by suppressing IL-23 signaling. Recent studies indicate that the cytokines IL-21 and IL-22 are produced by Th17 cells and modulate immune responses. Our studies demonstrate that the LXR agonist T0901317 suppressed MOG(35-55)-induced expression of IL-21 and IL-22 mRNA in splenocytes derived from MOG(35-55)-immunized mice. Finally, we demonstrate that the LXR agonist T0901317 suppressed the development of EAE in an experimental paradigm involving treatment of established EAE. Collectively, these studies suggest that LXR agonists may be effective in the treatment of MS.