Anti-inflammatory actions of ß-funaltrexamine in a mouse model of lipopolysaccharide-induced inflammation.

Neuroinflammation is involved in a wide range of brain disorders, thus there is great interest in identifying novel anti-inflammatory agents to include in therapeutic strategies. Our previous in vitro studies revealed that beta-funaltrexamine (ß-FNA), a well-characterized selective mu-opioid receptor (MOR) antagonist, inhibits inflammatory signaling in human astroglial cells, albeit through an apparent MOR-independent mechanism. We also previously determined that lipopolysaccharide (LPS)-induced sickness behavior and neuroinflammation in mice are prevented by pretreatment with ß-FNA. Herein we investigated the temporal importance of ß-FNA treatment in this pre-clinical model of LPS-induced neuroinflammation. Adult, male C57BL/6J mice were administered an i.p. injection of LPS followed by treatment (i.p. injection) with ß-FNA immediately or 4 h post-LPS. Sickness behavior was assessed using an open-field test, followed by assessment of inflammatory signaling in the brain, spleen, and plasma. Levels of inflammatory chemokines/cytokines (interferon γ-induced protein, CXCL10; monocyte chemotactic protein 1, CCL2; and interleukin-6, IL-6) in tissues were measured using an enzyme-linked immunosorbent assay and nuclear factor-kappa B (NFκB), p38 mitogen activated kinase (p38 MAPK), and glial fibrillary acidic protein (GFAP) expression were measured by western blot. LPS-induced sickness behavior and chemokine expression were inhibited more effectively when ß-FNA treatment occurred immediately after LPS administration, as opposed to 4 h post-LPS; and ß-FNA-mediated effects were time-dependent as evidenced by inhibition at 24 h, but not at 8 h. The inhibitory effects of ß-FNA on chemokine expression were more evident in the brain versus the spleen or plasma. LPS-induced NFκB-p65 and p38 MAPK expression in the brain and spleen were inhibited at 8 and 24 h post-LPS. These findings extend our understanding of the anti-inflammatory effects of ß-FNA and warrant further investigation into its therapeutic potential.

Understanding depression: the hippocampus might hold the answer in a CREB-regulated transcription coactivator: An Editorial for 'AAV-mediated over-expression of CRTC1 in the hippocampal dentate gyrus ameliorates lipopolysaccharide-induced depression-like behavior in mice' on page 111.

The pathophysiology of major depressive disorders is not completely understood. In this issue of Journal of Neurochemistry, Ni and colleagues investigate the role of cyclic adenosine monophosphate response element-binding protein (CREB)-dependent signaling in the hippocampus on depressive-like behaviors. This editorial highlights the key findings reported by Ni et al., (2018) and how they demonstrated the importance of CREB-regulated transcription cofactor 1 in LPS-induced neuroinflammation and depressive-like behaviors.

Temporal and Site-Specific Changes in Central Neuroimmune Factors During Rapid Weight Gain After Ovariectomy in Rats.

Systemic inflammation is present in obesity and emerging evidence, primarily from studies using male rodents fed high-fat diets, suggests neuroimmune signaling also is involved. We investigated early changes in neuroimmune signaling during the weight gain that follows ovariectomy in rats. Ovariectomized (OVX) rats were given standard rat chow and terminated 5 days (baseline), 4 or 8 weeks after ovariectomy. Levels of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in plasma and periuterine adipose were not affected by ovariectomy. In contrast, compared to baseline levels, IL-6 expression in the arcuate nucleus (ARC) and dorsal vagal complex (DVC) decreased by 4 weeks after OVX, but was not affected in the paraventricular nucleus (PVN). MCP-1 expression decreased by 4 weeks in the ARC and by 8 weeks in the PVN, but was not affected in the DVC. Increased glial fibrillary acidic protein (GFAP) expression in the PVN indicated astrocyte activation; decreased toll-like receptor 4 (TLR4) expression in the ARC, but not other regions, suggested early effects on innate immune factors. Importantly, in reproductively intact rats, IL-6 and MCP-1 levels in plasma, periuterine adipose, and brain regions were not affected after 8 weeks. Unlike OVX rats, GFAP expression in the DVC of intact rats was decreased at 8 weeks, and TLR4 expression in the ARC was increased at 8 weeks. Taken together, these dynamic and selective changes in neuroimmune factors co-incident with post-ovariectomy weight gain provide insight into the role of neuroimmune signaling in obesity, particularly in females.

α7 Nicotinic acetylcholine receptor potentiation downregulates chemotherapy-induced inflammatory overactivation by overlapping intracellular mechanisms.

We studied, using a combination of animal and cellular models, the glial mechanisms underlying the anti-neuropathic and anti-inflammatory properties of PAM-2 [(E)-3-furan-2-yl-N-p-tolyl-acrylamide], a positive allosteric modulator of α7 nicotinic acetylcholine receptors (nAChRs). In mice, PAM-2 decreased the inflammatory process induced by the combination of oxaliplatin (OXA), a chemotherapeutic agent, and interleukin-1ß (IL-1ß), a pro-inflammatory molecule. In the brain and spinal cord of treated animals, PAM-2 reduced pro-inflammatory cytokines/chemokines by mechanisms involving mRNA downregulation of factors in the toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB pathway, and increased the precursor of brain-derived neurotrophic factor (proBDNF). To determine the molecular mechanisms underlying the anti-inflammatory activity of PAM-2, both human C20 microglia and normal human astrocytes (NHA) were used. The results showed that PAM-2-induced potentiation of glial α7 nAChRs decreases OXA/IL-1ß-induced overexpression of inflammatory molecules by different mechanisms, including mRNA downregulation of factors in the NF-κB pathway (in microglia and astrocyte) and ERK (only in microglia). The OXA/IL-1ß-mediated reduction in proBDNF was prevented by PAM-2 in microglia, but not in astrocytes. Our findings also indicate that OXA/IL-1ß-induced organic cation transporter 1 (OCT1) expression is decreased by PAM-2, suggesting that decreased OXA influx may be involved in the protective effects of PAM-2. The α7-selective antagonist methyllycaconitine blocked the most important effects mediated by PAM-2 at both animal and cellular levels, supporting a mechanism involving α7 nAChRs. In conclusion, glial α7 nAChR stimulation/potentiation downregulates neuroinflammatory targets, and thereby remains a promising therapeutic option for cancer chemotherapy-induced neuroinflammation and neuropathic pain.

Anti-inflammatory effects of ß-FNA are sex-dependent in a pre-clinical model of LPS-induced inflammation.

BACKGROUND: Inflammation is present in neurological and peripheral disorders. Thus, targeting inflammation has emerged as a viable option for treating these disorders. Previous work indicated pretreatment with beta-funaltrexamine (ß-FNA), a selective mu-opioid receptor (MOR) antagonist, inhibited inflammatory signaling in vitro in human astroglial cells, as well as lipopolysaccharide (LPS)-induced neuroinflammation and sickness-like-behavior in mice. This study explores the protective effects of ß-FNA when treatment occurs 10 h after LPS administration and is the first-ever investigation of the sex-dependent effects of ß-FNA on LPS-induced inflammation in the brain and peripheral tissues, including the intestines. RESULTS: Male and female C57BL/6J mice were administered LPS followed by treatment with ß-FNA-immediately or 10 h post-LPS. Sickness- and anxiety-like behavior were assessed using an open-field test and an elevated-plus-maze test, followed by the collection of whole brain, hippocampus, prefrontal cortex, cerebellum/brain stem, plasma, spleen, liver, large intestine (colon), proximal small intestine, and distal small intestine. Levels of inflammatory chemokines/cytokines (interferon γ-induced-protein, IP-10 (CXCL10); monocyte-chemotactic-protein 1, MCP-1 (CCL2); interleukin-6, IL-6; interleukin-1ß, IL-1ß; and tumor necrosis factor-alpha, TNF-α) in tissues were measured using an enzyme-linked immunosorbent assay. Western blot analysis was used to assess nuclear factor-kappa B (NF-κB) expression. There were sex-dependent differences in LPS-induced inflammation across brain regions and peripheral tissues. Overall, LPS-induced CXCL10, CCL2, TNF-α, and NF-κB were most effectively downregulated by ß-FNA; and ß-FNA effects differed across brain regions, peripheral tissues, timing of the dose, and in some instances, in a sex-dependent manner. ß-FNA reduced LPS-induced anxiety-like behavior most effectively in female mice. CONCLUSION: These findings provide novel insights into the sex-dependent anti-inflammatory effects of ß-FNA and advance this agent as a potential therapeutic option for reducing both neuroinflammation an intestinal inflammation.

Relationship Between Circulating Metabolic Hormones and Their Central Receptors During Ovariectomy-Induced Weight Gain in Rats.

Although increasing research focuses on the phenomenon of body weight gain in women after menopause, the complexity of body weight regulation and the array of models used to investigate it has proven to be challenging. Here, we used ovariectomized (OVX) rats, which rapidly gain weight, to determine if receptors for ghrelin, insulin, or leptin in the dorsal vagal complex (DVC), arcuate nucleus (ARC), or paraventricular nucleus (PVN) change during post-ovariectomy weight gain. Female Sprague-Dawley rats with ad libitum access to standard laboratory chow were bilaterally OVX or sham OVX. Subgroups were weighed and then terminated on day 5, 33, or 54 post-operatively; blood and brains were collected. ELISA kits were used to measure receptors for ghrelin, insulin, and leptin in the DVC, ARC, and PVN, as well as plasma ghrelin, insulin, and leptin. As expected, body weight increased rapidly after ovariectomy. However, ghrelin receptors did not change in any of the areas for either group, nor did circulating ghrelin. Thus, the receptor:hormone ratio indicated comparable ghrelin signaling in these CNS areas for both groups. Insulin receptors in the DVC and PVN decreased in the OVX group over time, increased in the PVN of the Sham group, and were unchanged in the ARC. These changes were accompanied by elevated circulating insulin in the OVX group. Thus, the receptor:hormone ratio indicated reduced insulin signaling in the DVC and PVN of OVX rats. Leptin receptors were unchanged in the DVC and ARC, but increased over time in the PVN of the Sham group. These changes were accompanied by elevated circulating leptin in both groups that was more pronounced in the OVX group. Thus, the receptor:hormone ratio indicated reduced leptin signaling in the DVC and PVN of both groups, but only in the OVX group for the ARC. Together, these data suggest that weight gain that occurs after removal of ovarian hormones by ovariectomy is associated with selective changes in metabolic hormone signaling in the CNS. While these changes may reflect behavioral or physiological alterations, it remains to be determined whether they cause post-ovariectomy weight gain or result from it.

Proinflammatory cytokines and HIV-1 synergistically enhance CXCL10 expression in human astrocytes.

HIV encephalitis (HIVE), the pathologic correlate of HIV-associated dementia (HAD) is characterized by astrogliosis, cytokine/chemokine dysregulation, and neuronal degeneration. Increasing evidence suggests that inflammation is actively involved in the pathogenesis of HAD. In fact, the severity of HAD/HIVE correlates more closely with the presence of activated glial cells than with the presence and amount of HIV-infected cells in the brain. Astrocytes, the most numerous cell type within the brain, provide an important reservoir for the generation of inflammatory mediators, including interferon-gamma inducible peptide-10 (CXCL10), a neurotoxin and a chemoattractant, implicated in the pathophysiology of HAD. Additionally, the proinflammatory cytokines, IFN-gamma and TNF-alpha, are also markedly increased in CNS tissues during HIV-1 infection. In this study, we hypothesized that the interplay of host cytokines and HIV-1 could lead to enhanced expression of the toxic chemokine, CXCL10. Our findings demonstrate a synergistic induction of CXCL10 mRNA and protein in human astrocytes exposed to HIV-1 and the proinflammatory cytokines. Signaling molecules, including JAK, STATs, MAPK (via activation of Erk1/2, AKT, and p38), and NF-kappaB were identified as instrumental in the synergistic induction of CXCL10. Understanding the mechanisms involved in HIV-1 and cytokine-mediated up-regulation of CXCL10 could aid in the development of therapeutic modalities for HAD.

The opioid antagonist, beta-funaltrexamine, inhibits chemokine expression in human astroglial cells.

Emerging evidence indicates that neuroinflammatory responses in astroglia, including chemokine expression, are altered by opioids. Astroglial chemokines, such as CXCL10, are instrumental in response to many neuropathological insults. Opioid mediated disruption of astroglial CXCL10 expression may be detrimental in opioid abusers or patients receiving acute opioid therapy. We have characterized the in vitro effects of opioids on CXCL10 protein expression in human astroglial (A172) cells. The proinflammatory cytokine, tumor necrosis factor (TNF)alpha induced CXCL10 expression in A172 cells. Using MG-132, helenalin and SN50 [inhibitors of the transcription factor, nuclear factor (NF)-kappaB], we determined that NF-kappaB activation is instrumental in TNFalpha-induced CXCL10 expression in A172 astroglia. Morphine exposure during the 24 h TNFalpha stimulation period did not alter CXCL10 expression. However, fentanyl, a more potent mu-opioid receptor (MOR) agonist, inhibited TNFalpha-induced CXCL10 expression. Interestingly, neither the non-selective opioid receptor antagonist, naltrexone nor beta-funaltrexamine (beta-FNA), a highly selective MOR antagonist, blocked fentanyl mediated inhibition of TNFalpha-induced CXCL10 expression. Rather, beta-FNA dose-dependently inhibited TNFalpha-induced CXCL10 expression with a greater potency than that observed for fentanyl. Immunoblot analysis indicated that morphine, fentanyl and beta-FNA each reduced TNFalpha-induced nuclear translocation of NF-kappaB p65. These data show that beta-FNA and fentanyl inhibit TNFalpha-induced CXCL10 expression via a MOR-independent mechanism. Data also suggest that inhibition of TNFalpha-induced CXCL10 expression by fentanyl and beta-FNA is not directly related to a reduction in NF-kappaB p65 nuclear translocation. Further investigation is necessary in order to fully elucidate the mechanism through which these two opioid compounds inhibit CXCL10 expression. Understanding the mechanism by which chemokine expression is suppressed, particularly by the opioid antagonist, beta-FNA, may provide insights into the development of safe and effective treatments for neuroinflammation.

The Oct DNA motif participates in the alcohol inhibition of the inducible nitric oxide synthase gene promoter in rat C6 glioma cells.

Induction of nitric oxide synthase-2 (iNOS) by cytokines and bacterial products is associated with protein binding at the proximal promoter and in an upstream enhancer region of the Nos2 gene. To clarify how ethanol suppresses rat iNOS activity, we constructed several deletion mutants of the Nos2 promoter fused to the luciferase gene and transfected the constructs into C6 glial cells. Acute ethanol exposure of stably transfected cells for 24 h inhibits induced activity of Nos2 promoter constructs containing deletions in the 5' flanking region, including a 94 bp promoter that lacks any known NF-kappaB site but which carries a C/EBPbeta and overlapping gamma-IRE, GAS and Oct motifs. Ethanol failed to inhibit the endogenous activity of a smaller, 78 bp promoter that lacks the C/EBPbeta and overlapping, gamma-IRE and GAS motifs and showed no inducible activity. As another approach, in vivo DNA footprinting was used and identified protein protections at five regions of the proximal Nos2 promoter in induced cells. Exposure to acute ethanol diminished protein occupation in the five promoter regions including the gamma-IRE/NF-kappaB and the overlapping gamma-IRE/GAS/Oct sites. Site-directed mutagenesis in the octamer domain of the gamma-IRE/GAS/Oct motifs was studied in a 1002 bp promoter to examine its role in ethanol inhibition of cytokine and lipopolysaccharide induced activity. The data indicate that ethanol failed to inhibit promoter activity when the Oct motif is missing. Electrophoretic mobility shift assays performed using a 22-mer probe containing the overlapping gamma-IRE/GAS/Oct sites showed three complexes with one of the complexes being competed by an octamer-1 antibody. These observations demonstrate the role of protein-DNA binding at the core promoter, and the likely involvement of the octamer motif, in ethanol modulation of cytokine and lipopolysaccharide induced iNOS expression.

The opioid antagonist, ß-funaltrexamine, inhibits lipopolysaccharide-induced neuroinflammation and reduces sickness behavior in mice.

Brain pathologies such as neurodegenerative diseases, infection, traumatic brain injury, and mood disorders produce enormous personal and economic burdens. It is well established that neuroinflammation plays an important role in the etiology and/or manifestation of such disorders. Previously, we discovered that beta-funaltrexamine (ß-FNA) inhibits inflammatory signaling in human astrocytes in vitro, resulting in reduced expression of proinflammatory cytokines/chemokines. The present study examines the effects of peripherally administered ß-FNA on lipopolysaccharide (LPS)-induced neuroinflammation and sickness behavior in vivo. Adult male C57BL/6J mice were administered ß-FNA and were then immediately administered bacterial lipopolysaccharide (LPS). At 24h post-injections, sickness behavior was assessed in an open-field test. Following behavioral analysis plasma and brains were collected. Levels of interleukin-6 (IL-6), interferon-γ inducible protein-10 (CXCL10), and monocyte chemoattractant protein-1 (CCL2) were determined by enzyme-linked immunosorbant assay (ELISA). At 24h post-LPS injection, IL-6, CCL2 and CXCL10 were increased in the plasma, whereas, only CCL2 and CXCL10 were elevated in the brain. ß-FNA significantly inhibited LPS-induced CXCL10 and CCL2 expression in brain, but minimally or not at all in the plasma. LPS-induced sickness behavior, as indicated by a reduction in distance moved, was prevented by ß-FNA. Overall, CXCL10 expression in the brain was most positively and significantly correlated with sickness behavior; whereas, anxiety-like behavior was most positively and significantly correlated with IL-6 and CCL2 levels in the plasma and levels of CXCL10 and CCL2 in the brain. The reduction in sickness behavior may be in part due to decreased chemokine expression in the brain; further examination of the anti-inflammatory and neuroprotective effects of ß-FNA is warranted.

Soil characteristics and phosphorus level effect on phosphorus loss in runoff.

The loss of phosphorus (P) in runoff from agricultural soils may accelerate eutrophication in lakes and streams as well as degrade surface water quality. Limited soil specific data exist on the relationship between runoff P and soil P. This study investigated the relationship between runoff dissolved reactive phosphorus (DRP) and soil P for three Oklahoma benchmark soils: Richfield (fine, smectitic, mesic Aridic Argiustoll), Dennis (fine, mixed, active, thermic Aquic Argiudoll), and Kirkland (fine, mixed, superactive, thermic Udertic Paleustoll) series. These soils were selected to represent the most important agricultural soils in Oklahoma across three major land resource areas. Surface soil (0-15 cm) was collected from three designated locations, treated with diammonium phosphate (18-46-0) to establish a wide range of water-soluble phosphorus (WSP) (3.15-230 mg kg(-1)) and Mehlich-3 phosphorus (M3P) (27.8-925 mg kg(-1)). Amended soils were allowed to reach a steady state 210 d before simulated rainfall (75 mm h(-1)). Runoff was collected for 30 min from bare soil boxes (1.0 x 0.42 m and 5% slope) and analyzed for DRP and total P. Soil samples collected immediately before rainfall simulation were analyzed for the following: M3P, WSP, ammonium oxalate P saturation index (PSI(ox)), water-soluble phosphorus saturation index (PSI(WSP)), and phosphorus saturation index calculated from M3P and phosphorus sorption maxima (P(sat)). The DRP in runoff was highly related (p < 0.001) to M3P for individual soil series (r2 > 0.92). Highly significant relationships (p < 0.001) were found between runoff DRP and soil WSP for the individual soil series (r2 > 0.88). Highly significant relationships (p < 0.001) existed between DRP and different P saturation indexes. Significant differences (p < 0.05) among the slopes of the regressions for the DRP-M3P, DRP-WSP, DRP-PSI(ox), DRP-PSI(WSP), and DRP-P(sat) relationships indicate that the relationships are soil specific and phosphorus management decisions should consider soil characteristics.

The opioid antagonist, ß-funaltrexamine, inhibits NF-κB signaling and chemokine expression in human astrocytes and in mice.

Opioid-immune crosstalk occurs when opioid drugs alter the activity of the immune system. In this study, the opioid antagonist ß-funaltrexamine (ß-FNA) decreases the expression and release of an inflammatory chemokine, interferon-γ inducible protein-10 (CXCL10) from normal human astrocytes stimulated by interleukin 1ß (IL-1ß). ß-FNA decreased CXCL10 by an unknown action that did not involve the mu opioid receptor (MOR). As IL-1ß acts through its receptor to activate NF-κB/MAPK signaling pathways which leads to CXCL10 expression and release, key steps in the IL-1ß signaling pathways were examined following ß-FNA treatment. IL-1ß-induced activation of p38 mitogen-activated protein kinases (p38 MAPK) was inhibited by ß-FNA as shown by decreased p38 MAPK phosphorylation in treated cells. ß-FNA also decreased the levels of activated subunits of NF-κB (p50 and p65) in treated astrocytes. The impact of ß-FNA was also observed in proteins that act to negatively regulate NF-κB signaling. IL-1ß upregulated the expression of A20, a ubiquitin (Ub)-editing enzyme that dampens NF-κB signaling by altering ubiquination patterns on IL-1 receptor second messengers, and the increase in A20 was significantly inhibited by ß-FNA treatment. Inhibition of the Ub-activating enzyme E1 by the inhibitor PYR41 also decreased CXCL10 release, like ß-FNA, and concurrent treatment with both PYR41 and ß-FNA inhibited CXCL10 more than did either agent alone. In mice, lipopolysaccharide-induced CXCL10 expression in the brain was inhibited by treatment with ß-FNA. These findings suggest that ß-FNA exerts an anti-inflammatory action in vitro and in vivo that is MOR-independent and possibly due to the alkylating ability of ß-FNA.

Ethanol increases nuclear factor-kappa B activity in human astroglial cells.

Alcohol abuse adversely affects essentially all the organs of the body, either directly or indirectly. Ethanol may contribute to brain damage via inflammation. Ethanol may also alter CNS immunocompetence and further the progression of certain CNS infections. Nuclear factor (NF)-kappa B helps regulate inflammatory gene expression in glia. It is possible that ethanol effects on CNS pathology are partly a consequence of ethanol modulation of NF-kappa B-associated pathways in glia. We have assessed the effects of 0.5-6 h ethanol exposure on cytokine (5 ng/ml interleukin-1 beta + 100 ng/ml interferon gamma + 30 ng/ml tumor necrosis factor-alpha)-induced NF-kappa B activation in human A172 astroglial cells. Immunoblot analysis indicated that NF-kappa B p65 nuclear translocation occurred within 0.5 h after cytokine stimulation. Stimulation in the presence of ethanol resulted in increased nuclear p65 levels at 3 h, with 200 mM causing a greater increase than 50 mM ethanol. Gel shift assay data suggested that cytokine-induced NF-kappa B binding activity was greatest in cells exposed to 50 mM ethanol, followed by 200 and 0 mM ethanol exposed cells, respectively. Thus, in cytokine-stimulated cells, 200 mM ethanol resulted in greater nuclear p65 levels, yet, 50 mM ethanol exposure resulted in more pronounced DNA binding by NF-kappa B. These findings demonstrate that acute ethanol enhances p65 activity in human astroglia and further support the hypothesis that ethanol-mediated brain pathology involves modulation of NF-kappa B pathways. A better understanding of the mechanistic events involved in ethanol-induced CNS pathology should provide for therapeutic strategies to combat detrimental effects of alcohol on the CNS.

Chronic ethanol inhibits CXC chemokine ligand 10 production in human A172 astroglia and astroglial-mediated leukocyte chemotaxis.

Astroglia are the most prevalent cell type in the human central nervous system (CNS) and perform important roles in normal tissue homeostasis, during pathological events and following trauma. Astroglial-derived chemokines have important neurotrophic effects and are important to CNS immunocompetence and response to injury, in part, due to their direct role in leukocyte and microglial cell recruitment. However, while ethanol is known to induce CNS pathologies and to be peripherally immunosuppressive, ethanol effects on chemokine expression in human astroglia are essentially unknown. We have demonstrated that chemotaxis of human U937 leukocytic cells, across a 0.5 microm pore polycarbonate transmembrane insert, is induced in response to culture media collected from 10 microg/ml lipopolysaccharide (LPS) + 10 ng/ml interleukin (IL)-1beta-stimulated A172 human astroglia cells. The involvement of the chemokine CXCL10 (also known as interferon-gamma inducible protein or IP-10) in astroglial-induced chemotaxis of U937 cells has been indicated, as chemotaxis can be reduced by an anti-CXCL10 neutralizing antibody. Interestingly, chemotaxis of U937 cells, in response to astroglial-exposed media, is reduced when astroglia are chronically (9 days) exposed to 50 mM ethanol before stimulation with LPS + IL-1beta. Furthermore, we observed that LPS + IL-1beta-stimulated CXCL10 production is inhibited in human A172 astroglia exposed to chronic 50 mM ethanol. Thus, alterations in astroglial CXCL10 expression may disrupt CNS immunocompetence and play an important role in ethanol-induced CNS pathologies.

Acute ethanol exposure modulates expression of inducible nitric-oxide synthase in human astroglia: evidence for a transcriptional mechanism.

Astroglia are important in immunocompetence and response to injury within the CNS. Activated astroglia respond, in part, by expressing inducible nitric-oxide synthase (iNOS) and subsequent catalytic production of nitric oxide. Results from a previous study in our laboratory, in the human A172 astroglial cell line, revealed that induction of iNOS activity by tumor necrosis factor-alpha + interferon-gamma + interleukin-1 beta was inhibited by 24-h exposure to a high ethanol concentration (200 mM), but enhanced by 50 mM ethanol. In the work reported in this article, we tested the working hypothesis that ethanol acts transcriptionally to modulate cytokine-induced expression of the iNOS gene, NOS2A, in human astroglia. Ethanol, 50 or 200 mM, did not directly alter in vitro catalytic activity of the iNOS enzyme, indicating that ethanol does not affect the enzyme directly. Likewise, ethanol exposure after a 12-h cytokine-stimulation period had no effect on in vivo iNOS activity. However, when cells were simultaneously exposed to ethanol and cytokines for 12 h, in vivo iNOS activity was altered. That ethanol must be present during cytokine stimulation to influence iNOS activity is consistent with a transcriptional mechanism of action. In addition, steady-state expression of iNOS protein and NOS2A mRNA levels were modulated in a biphasic manner by ethanol similar to that noted previously for iNOS activity. These findings strongly support the suggestion that ethanol modulates cytokine-induced iNOS expression in A172 cells at a pretranslational site. These findings should be instrumental in the identification of the critical ethanol-sensitive elements involved in the regulation of NOS2A in human astroglia.

Β-funaltrexamine inhibits chemokine (CXCL10) expression in normal human astrocytes.

Neuroinflammation is an integral component of neurodegenerative disorders, CNS infection and trauma. Astroglial chemokines, such as CXCL10, are instrumental in neuroinflammatory signaling as well as neurotoxicity. We have utilized proinflammatory-induced CXCL10 expression in normal human astrocytes (NHA) as a model in which to assess the anti-inflammatory actions of the selective, mu-opioid receptor (MOR) antagonist, ß-funaltrexamine (ß-FNA). Interferon (IFN)γ+HIV-1 Tat-induced CXCL10 expression (secreted protein and mRNA) was inhibited by co-treatment with ß-FNA. Neither the MOR-selective antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2 (CTAP) nor the nonselective opioid receptor antagonist, naltrexone inhibited IFNγ+HIV-1 Tat-induced CXCL10 expression. Furthermore, co-treatment with excess CTAP or naltrexone did not prevent ß-FNA mediated inhibition of IFNγ+HIV-1 Tat-induced CXCL10 expression. Additionally, we utilized an inhibitor of NF-κB activation (SN50) to demonstrate that IFNγ+HIV-1 Tat-induced CXCL10 expression is NF-κB-dependent in NHA. Subsequent experiments revealed that ß-FNA did not significantly affect NF-κB activation. Interestingly, we discovered that ß-FNA inhibited p38 activation as indicated by decreased expression of phospho-p38. Together, these findings suggest that the inhibitory actions of ß-FNA are MOR-independent and mediated, in part, via a transcriptional mechanism. These findings add to our understanding of the mechanism by which chemokine expression is inhibited by ß-FNA. In conjunction with future investigations, these novel findings are expected to provide insights into the development of safe and effective treatments for neuroinflammation.

Evaluation of neurobehavioral and neuroinflammatory end-points in the post-exposure period in rats sub-acutely exposed to manganese.

Manganese (Mn) can cause manganism, a neurological disorder similar to Parkinson' Disease (PD). The neurobehavioral and neuroinflammatory end-points in the Mn post exposure period have not been studied yet. Rats were injected on alternate days with 8 doses of MnCl2 (25mg/kg) or saline, then euthanized 1, 10, 30 or 70 days following the last dose. Whole-blood (WB) (p<0.05), urine (p<0.05) and brain cortical (p<0.0001) Mn levels were significantly increased 24h after the last dose. Decreases in the rats' ambulation were noted 1, 10 and 30 days after the last Mn dose (p<0.001; p<0.05; p<0.001, respectively) and also in the rearing activity at the four time-points (p<0.05). Cortical glial fibrillary acid protein immunoreactivity (GFAP-ir) was significantly increased at 1, 10, 30 (p<0.0001) and 70 (p<0.001) days after the last Mn dose, as well as tumor necrosis α (TNF-α) levels (p<0.05) but just on day 1. Taken together, the results show that, during the 70-day clearance phase of Mn, the recovery is not immediate as behavioral alterations and neuroinflammation persist long after Mn is cleared from the cortical brain compartment.

α-Synuclein potentiates interleukin-1ß-induced CXCL10 expression in human A172 astrocytoma cells.

Neuroinflammation and neuronal degeneration observed in Parkinson's disease (PD) has been attributed in part to glial-mediated events. Increased expression of proinflammatory cytokines and abnormal accumulation of the neuronal protein, α-synuclein in the brain are also characteristic of PD. While increasing evidence suggests that astrocytes contribute to neuroinflammation and dopaminergic neuronal degeneration associated with PD, there remains much to learn about these astroglial-mediated events. Therefore, we investigated the in vitro effects of interleukin-1ß (IL-1ß) and α-synuclein on astroglial expression of interferon-γ inducible protein-10 (CXCL10), a proinflammatory and neurotoxic chemokine. IL-1ß-induced CXCL10 protein expression was potentiated by co-exposure to α-synuclein. α-Synuclein did not significantly affect IL-1ß-induced CXCL10 mRNA expression, but did mediate increased CXCL10 mRNA stability, which may explain, in part, the increased levels of secreted CXCL10 protein. Future investigations are warranted to more fully define the mechanism by which α-synuclein enhances IL-1ß-induced astroglial CXCL10 expression. These findings highlight the importance of α-synuclein in modulating inflammatory events in astroglia. These events may be particularly relevant to the pathology of CNS disorders involving α-synuclein accumulation, including PD and HIV-1 associated dementia.

Chronic inorganic mercury exposure induces sex-specific changes in central TNFα expression: importance in autism?

Mercury is neurotoxic and increasing evidence suggests that environmental exposure to mercury may contribute to neuropathologies including Alzheimer's disease and autism spectrum disorders. Mercury is known to disrupt immunocompetence in the periphery, however, little is known about the effects of mercury on neuroimmune signaling. Mercury-induced effects on central immune function are potentially very important given that mercury exposure and neuroinflammation both are implicated in certain neuropathologies (i.e., autism). Furthermore, mounting evidence points to the involvement of glial activation in autism. Therefore, we utilized an in vivo model to assess the effects of mercury exposure on neuroimmune signaling. In prairie voles, 10 week mercury exposure (60ppm HgCl(2) in drinking water) resulted in a male-specific increase in TNFα protein expression in the cerebellum and hippocampus. These findings are consistent with our previously reported male-specific mercury-induced deficits in social behavior and further support a role for heavy metals exposure in neuropathologies such as autism. Subsequent studies should further evaluate the mechanism of action and biological consequences of heavy metals exposure. Additionally, these observations highlight the potential of neuroimmune markers in male voles as biomarkers of environmental mercury toxicity.

Neuromelanin inhibits CXCL10 expression in human astroglial cells.

Increasing evidence indicates neuroinflammation is instrumental in the pathogenesis of Parkinson's disease (PD). In PD, there is selective degeneration of neuromelanin (NM)-containing dopamine neurons. Neuromelanin is predominantly cytoprotective within dopaminergic neurons, whereas, NM released from damaged neurons activates microglia. However, the effects of NM on astroglial cells remain largely unknown. Astroglia are essential to neuronal homeostasis and responsive to injury, in part, through secretion of chemokines, including interferon γ inducible protein-10 (CXCL10). Thus, we used an in vitro approach to identify the effects of NM on TNFα-induced CXCL10 expression in human astroglial cells. TNFα-induced CXCL10 expression was inhibited in NM exposed cells. Additionally, TNFα-induced NF-кB activation was inhibited by NM. Given that CXCL10 expression is NF-кB-dependent in human astroglial cells, these findings suggest that NM may inhibit CXCL10 expression, in part, through an NF-кB-dependent mechanism. While the in vivo consequences of NM mediated effects on astroglial CXCL10 expression remain to be fully elucidated, insights obtained in this study further our understanding of the effects of NM on inflammatory signaling in human astroglial cells.