• Energy Research

Innate immune signaling is an important feature in the pathology of alcohol use disorders. Alcohol abuse causes persistent innate immune activation in the brain. This is seen in postmortem human alcoholic brain specimens, as well as in primate and rodent models of alcohol consumption. Further, in vitro models of alcohol exposure in neurons and glia also demonstrate innate immune activation. The activation of the innate immune system seems to be important in the development of alcohol use pathology, as anti-immune therapies reduce pathology and ethanol self-administration in rodent models. Further, innate immune activation has been identified in each of the stages of addiction: binge/intoxication, withdrawal/negative affect, and preoccupation/craving. This suggests that innate immune activation may play a role both in the development and maintenance of alcoholic pathology. In this chapter, we discuss the known contributions of innate immune signaling in the pathology of alcohol use disorders, and present potential therapeutic interventions that may be beneficial for alcohol use disorders.

Abstract Background Toll-like receptor (TLR) signaling is emerging as an important component of neurodegeneration. TLR7 senses viral RNA and certain endogenous miRNAs to initiate innate immune responses leading to neurodegeneration. Alcoholism is associated with hippocampal degeneration, with preclinical studies linking ethanol-induced neurodegeneration with central innate immune induction and TLR activation. The endogenous miRNA let-7b binds TLR7 to cause neurodegeneration. Methods TLR7 and other immune markers were assessed in postmortem human hippocampal tissue that was obtained from the New South Wales Tissue Bank. Rat hippocampal-entorhinal cortex (HEC) slice culture was used to assess specific effects of ethanol on TLR7, let-7b, and microvesicles. Results We report here that hippocampal tissue from postmortem human alcoholic brains shows increased expression of TLR7 and increased microglial activation. Using HEC slice culture, we found that ethanol induces TLR7 and let-7b expression. Ethanol caused TLR7-associated neuroimmune gene induction and initiated the release let-7b in microvesicles (MVs), enhancing TLR7-mediated neurotoxicity. Further, ethanol increased let-7b binding to the danger signaling molecule high mobility group box-1 (HMGB1) in MVs, while reducing let-7 binding to classical chaperone protein argonaute (Ago2). Flow cytometric analysis of MVs from HEC media and analysis of MVs from brain cell culture lines found that microglia were the primary source of let-7b and HMGB1-containing MVs. Conclusions Our results identify that ethanol induces neuroimmune pathology involving the release of let-7b/HMGB1 complexes in microglia-derived microvesicles. This contributes to hippocampal neurodegeneration and may play a role in the pathology of alcoholism.

AbstractAlcohol use disorder (AUD) pathology features pro‐inflammatory gene induction and microglial activation. The underlying cellular processes that promote this activation remain unclear. Previously considered cellular debris, extracellular vesicles (EVs) have emerged as mediators of inflammatory signaling in several disease states. We investigated the role of microvesicles (MVs, 50 nm–100 µm diameter EVs) in pro‐inflammatory and microglial functional gene expression using primary organotypic brain slice culture (OBSC). Ethanol caused a unique immune gene signature that featured: temporal induction of pro‐inflammatory TNF‐α and IL‐1β, reduction of homeostatic microglia state gene Tmem119, progressive increases in purinergic receptor P2RY12 and the microglial inhibitory fractalkine receptor CX3CR1, an increase in the microglial presynaptic gene C1q, and a reduction in the phagocytic gene TREM2. MV signaling was implicated in this response as reduction of MV secretion by imipramine blocked pro‐inflammatory TNF‐α and IL‐1β induction by ethanol, and ethanol‐conditioned MVs (EtOH‐MVs) reproduced the ethanol‐associated immune gene signature in naïve OBSC slices. Depletion of microglia prior to ethanol treatment prevented pro‐inflammatory activity of EtOH‐MVs, as did incubation of EtOH‐MVs with the HMGB1 inhibitor glycyrrhizin. Ethanol caused HMGB1 secretion from cultured BV2 microglia in MVs through activation of PI3 kinase. In summary, these studies find MVs modulate pro‐inflammatory gene induction and microglial activation changes associated with ethanol. Thus, MVs may represent a novel therapeutic target to reduce neuroinflammation in the setting of alcohol abuse or other diseases that feature a neuroimmune component. [Correction added on 5 April 2021, after first online publication: The copyright line was changed.]

Ethanol treatment on postnatal day seven (P7) causes robust brain cell death and is a model of late gestational alcohol exposure (Ikonomidou et al., 2000). To investigate the long-term effects of P7 ethanol treatment on adult brain, mice received either two doses of saline or ethanol on P7 (2.5g/kg, s.c., 2 hours apart) and were assessed as adults (P82) for brain volume (using postmortem MRI) and cellular architecture (using immunohistochemistry). Adult mice that received P7 ethanol had reduced MRI total brain volume (4%) with multiple brain regions being reduced in both males and females. Immunohistochemistry indicated reduced frontal cortical parvalbumin immunoreactive (PV+IR) interneurons (18-33%) and reduced Cux1+IR layer II pyramidal neurons (15%) in both sexes. Interestingly, markers of adult hippocampal neurogenesis differed between sexes, with only ethanol treated males showing increased doublecortin and Ki67 expression (52 and 57% respectively) in the dentate gyrus, consistent with increased neurogenesis compared to controls. These findings suggest that P7 ethanol treatment causes persistent reductions in adult brain volume and frontal cortical neurons in both males and females. Increased adult neurogenesis in males, but not females, is consistent with differential adaptive responses to P7 ethanol toxicity between the sexes. One day of ethanol exposure, e.g. P7, causes persistent adult brain dysmorphology.

Binge-drinking is common in human adolescents. The adolescent brain is undergoing structural maturation and has a unique sensitivity to alcohol neurotoxicity. Therefore, adolescent binge ethanol may have long-term effects on the adult brain that alter brain structure and behaviors that are relevant to alcohol use disorders.

AbstractBackgroundMicroglia are critical mediators of neuroimmune pathology across multiple neurologic disorders. Microglia can be persistently activated or “primed” by Toll-like receptor (TLR) activation, ethanol, stress, and other insults. Thus, strategies to prevent or reverse microglial priming may be beneficial for conditions that involve progressively increasing microglial activation. Microglial depletion with repopulation is emerging as a potential therapy to normalize chronic immune activation. Primary organotypic hippocampal slice culture (OHSC) allows for the study of neuroimmune activation as well as microglial depletion and repopulation without involvement of peripheral immune activation. OHSC undergoes functional maturation and retains cytoarchitecture similar toin vivo.MethodsOHSC underwent microglial depletion with the CSF1R antagonist PLX3397 with or without repopulation after removal of PLX3397. Immune, trophic, and synaptic gene changes in response to agonists of TLRs 2, 3, 4, 7, and 9 as well as ethanol were assessed in the settings of microglial depletion and repopulation. Gi-DREADD inhibition of microglia was used to confirm select findings seen with depletion. The ability of microglial repopulation to prevent progressive proinflammatory gene induction by chronic ethanol was also investigated.ResultsMicroglia were depleted (> 90%) by PLX3397 in OHSC. Microglial depletion blunted proinflammatory responses to several TLR agonists as well as ethanol, which was mimicked by Gi-DREADD inhibition of OHSC microglia. Removal of PLX3397 was followed by complete repopulation of microglia. OHSCs with repopulated microglia showed increased baseline expression of anti-inflammatory cytokines (e.g., IL-10), microglial inhibitory signals (e.g., CX3CL1), and growth factors (e.g., BDNF). This was associated with blunted induction (~ 50%) of TNFα and IL-1β in response to agonists to TLR4 and TLR7. Further, chronic cycled ethanol from 4 days in vitro (DIV) to 16DIV caused immediate 2-fold inductions of TNFα and IL-1β that grew to ~4-fold of age-matched control slices by 40DIV. This persistent inflammatory gene expression was completely reversed by microglial depletion and repopulation after chronic ethanol.ConclusionsMicroglia in OHSCs mediate proinflammatory responses to TLR agonists and ethanol. Microglial repopulation promoted an anti-inflammatory, trophic neuroenvironment and normalized proinflammatory gene expression. This supports the possibility of microglial depletion with repopulation as a strategy to reverse chronic neuroimmune activation.

Neuroimmune activation is a key feature of the pathologies of numerous psychiatric disorders including alcoholism, depression, and anxiety. Both HMGB1 and IL-1β have been implicated in brain disorders. Previous studies find HMGB1 andIL-1β form heterocomplexes in vitro with enhanced immune responses, lead to our hypothesis that HMGB1 and IL-1β heterocomplexes formed in vivo to contribute to the pathology of alcoholism. HMGB1/IL-1β heterocomplexes were prepared in vitro and found to potentiate IL-1β receptor proinflammatory gene induction compared to IL-1β alone in hippocampal brain slice culture. These HMGB1/IL-1β complexes were found to be increased in post-mortem human alcoholic hippocampus by co-immunoprecipiation. In mice, acute binge ethanol induced both HMGB1 and IL-1β in the brain and plasma. HMGB1 and IL-1β complexes were found only in mouse brain, with confocal microscopy revealing an ethanol-induced HMGB1 and IL-1β cytoplasmic co-localization. Surprisingly, IL-1β was found primarily in neurons. Studies in hippocampal brain slice culture found ethanol increased HMGB1/IL-1β complexes in the media. These studies suggest a novel neuroimmune mechanism in the pathology of alcoholism. Immunogenic HMGB1/IL-1β complexes represent a novel target for immune modulatory therapy in alcohol use disorders, and should be investigated in other psychiatric diseases that involve a neuroimmune component.