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Despite advancements in critical care and resuscitation, traumatic injuries are one of the leading causes of death around the world and can bring about long-term disabilities in survivors. One of the primary causes of death for trauma patients are secondary phase complications that can develop weeks or months after the initial insult. These secondary complications typically occur due to systemic immune dysfunction that develops in response to injury which can lead to immunosuppression, coagulopathy, multiple organ failure, unregulated inflammation, and potentially sepsis in patients. Recently, extracellular vesicles (EVs) have been identified as mediators of these processes since their levels are increased in circulation after traumatic injury and they encapsulate cargo that can aggravate secondary complications. In this review, we will discuss the role of EVs in the post-trauma pathologies that arise after burn injuries, trauma to the central nervous system, and infection. Additionally, we will examine the use of EVs as biomarkers for predicting late-stage trauma outcomes and as therapeutics for reversing the pathological processes that develop post-trauma. Overall, EVs have emerged as critical mediators of trauma-associated pathology and their use as a therapeutic agent represents an exciting new field of biomedicine.

Alcohol misuse contributes to the dysregulation of immune responses and multiorgan dysfunction across various tissues, which are associated with higher risk of morbidity and mortality in people with alcohol use disorders. Organ-specific immune cells, including microglia in the brain, alveolar macrophages in the lungs, and Kupffer cells in the liver, play vital functions in host immune defense through tissue repair and maintenance of homeostasis. However, binge drinking and chronic alcohol misuse impair these immune cells' abilities to regulate inflammatory signaling and metabolism, thus contributing to multiorgan dysfunction. Further complicating these delicate systems, immune cell dysfunction associated with alcohol misuse is exacerbated by aging and gut barrier leakage. This critical review describes recent advances in elucidating the potential mechanisms by which alcohol misuse leads to derangements in host immunity and highlights current gaps in knowledge that may be the focus of future investigations.

Plasma-derived extracellular vesicles (EV) can serve as markers of cell damage/disease but can also have therapeutic utility depending on the nature of their cargo, such as miRNA. Currently, there are challenges and lack of innovations regarding early diagnosis and therapeutic options within different aspects of management of patients suffering from chronic pancreatitis (CP). Use of EV as biomarkers for pancreatic health and/or as adjuvant therapy would make a difference in management of these patients. The aim of this study was to characterize the miRNA cargo of EV purified from the plasma of CP patients and compared to those of healthy participants. EVs were isolated from plasma of 15 CP patients and 10 healthy controls. Nanoparticle tracking analysis was used to determine frequency and size, while NanoString technology was used to characterize the miRNA cargo. Relevant clinical parameters were correlated with EV miRNA cargo. ~ 30 miRNA species were identified to have significantly (p

Alcohol 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 causes the secretion of pro‐inflammatory microvesicles (MVs) from brain tissue. Ethanol‐induced MVs reproduce the pro‐inflammatory gene induction and microglial activation seen with ethanol alone, and inhibition of their secretion blocks ethanol‐induced neuroinflammation. Microglia are required for the generation of pro‐inflammatory MVs, with MV secretion involving activation of microglial PI3K.

Adolescence is a critical neurodevelopmental window for maturation of brain structure, neurocircuitry, and glia. This development is sculpted by an individual's unique experiences and genetic background to establish adult level cognitive function and behavioral makeup. Alcohol abuse during adolescence is associated with an increased lifetime risk for developing an alcohol use disorder (AUD). Adolescents participate in heavy, episodic binge drinking that causes persistent changes in neurocircuitry and behavior. These changes may underlie the increased risk for AUD and might also promote cognitive deficits later in life. In this chapter, we have examined research on the persistent effects of adolescent binge-drinking both in humans and in rodent models. These studies implicate roles for neuroimmune signaling as well as epigenetic reprogramming of neurons and glia, which create a vulnerable neuroenvironment. Some of these changes are reversible, giving hope for future treatments to prevent many of the long-term consequences of adolescent alcohol abuse.

Extracellular vesicles (EVs) have emerged as key regulators of immune function across multiple diseases. Severe burn injury is a devastating trauma with significant immune dysfunction that results in an ∼12% mortality rate due to sepsis-induced organ failure, pneumonia, and other infections. Severe burn causes a biphasic immune response: an early (0–72 h) hyper-inflammatory state, with release of damage-associated molecular pattern molecules, such as high-mobility group protein 1 (HMGB1), and proinflammatory cytokines (e.g., IL-1β), followed by an immunosuppressive state (1–2+ wk post injury), associated with increased susceptibility to life-threatening infections. We have reported that early after severe burn injury HMGB1 and IL-1β are enriched in plasma EVs. Here we tested the impact of EVs isolated after burn injury on phenotypic and functional consequences in vivo and in vitro using adoptive transfers of EV. EVs isolated early from mice that underwent a 20% total body surface area burn injury (burn EVs) caused similar hallmark cytokine responses in naïve mice to those seen in burned mice. Burn EVs transferred to RAW264.7 macrophages caused similar functional (i.e., cytokine secretion) and immune gene expression changes seen with their associated phase of post-burn immune dysfunction. Burn EVs isolated early (24 h) induced MCP-1, IL-12p70, and IFNγ, whereas EVs isolated later blunted RAW proinflammatory responses to bacterial endotoxin (LPS). We also describe significantly increased HMGB1 cargo in burn EVs purified days 1 to 7 after injury. Thus, burn EVs cause immune outcomes in naïve mice and macrophages similar to findings after severe burn injury, suggesting EVs promote post-burn immune dysfunction.

BackgroundMicroglia 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.

BACKGROUND: Many brain disorders, including alcohol use disorder (AUD), are associated with induction of multiple proinflammatory genes. One aspect of proinflammatory signaling is progressive increases in expression across cells and induction of other innate immune genes. High-mobility group box 1 (HMGB1) heteromers contribute to amplification by potentiating multiple proinflammatory responses, including Toll-like receptors (TLRs). TLR signaling recruits coupling proteins linked to nuclear transcription factors that induce proinflammatory cytokines and chemokines as well as their respective receptors. We tested the hypothesis that AUD induction of TLR expression increases levels of proinflammatory genes as well as cellular signaling cascades in association with neurodegeneration in the orbitofrontal cortex (OFC). METHODS: Post-mortem human OFC tissue samples (n = 10) from male individuals diagnosed with AUD were compared to age-matched moderate drinking controls (CON). Neuroimmune signaling molecules were assessed using immunohistochemistry for protein and reverse transcription polymerase chain reaction for messenger RNA (mRNA). RESULTS: In the AUD OFC, we report induction of the endogenous TLR agonist HMGB1 as well as all TLRs assessed (i.e., TLR2-TLR9) except TLR1. This was accompanied by increased expression of the TLR adaptor protein myeloid differentiation primary response 88 (MyD88), activation of the proinflammatory nuclear transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and downstream induction of proinflammatory cytokines, chemokines, and their corresponding receptors. Expression of several of these proinflammatory signaling markers is observed in glia and neurons. Induction of HMGB1-TLR-MyD88-NFκB proinflammatory signaling pathways correlates with neurodegeneration (i.e., Fluoro-Jade B) as well as lifetime alcohol consumption and age of drinking onset. CONCLUSION: These data implicate induction of HMGB1-TLR-MyD88-NFκB cascades through coordinated glial and neuronal signaling as contributing to neurodegeneration in the post-mortem human OFC of individuals with AUD.

Although the cause of progressive neurodegeneration is often unclear, neuronal death can occur through several mechanisms. In conditions such as Alzheimer’s or alcohol use disorder (AUD), Toll-like receptor (TLR) induction is observed with neurodegeneration. However, links between TLR activation and neurodegeneration are lacking. We report a role of apoptotic neuronal death in AUD through TLR7-mediated induction of death receptor signaling. In postmortem human cortex, a two-fold increase in apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in neurons was found in AUD versus controls. This occurred with the increased expression of TLR7 and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors. Binge ethanol treatment in C57BL/6 mice increased TLR7 and induced neuronal apoptosis in cortical regions that was blocked by TLR7 antagonism. Mechanistic studies in primary organotypic brain slice culture (OBSC) found that the inhibition of TLR7 and its endogenous ligand let-7b blocked ethanol-induced neuronal cell death. Both IMQ and ethanol induced the expression of TRAIL and its death receptor. In addition, TRAIL-neutralizing monoclonal antibodies blocked both imiquimod (IMQ) and ethanol induced neuronal death. These findings implicate TRAIL as a mediator of neuronal apoptosis downstream of TLR7 activation. TLR7 and neuronal apoptosis are implicated in other neurodegenerative diseases, including Alzheimer’s disease. Therefore, TRAIL may represent a therapeutic target to slow neurodegeneration in multiple diseases.

Plasma-derived extracellular vesicles (EV) can serve as markers of cell damage/disease but can also have therapeutic utility depending on the nature of their cargo, such as miRNA. Currently, there are challenges and lack of innovations regarding early diagnosis and therapeutic options within different aspects of management of patients suffering from chronic pancreatitis (CP). Use of EV as biomarkers for pancreatic health and/or as adjuvant therapy would make a difference in management of these patients. The aim of this study was to characterize the miRNA cargo of EV purified from the plasma of CP patients and compared to those of healthy participants. EVs were isolated from plasma of 15 CP patients and 10 healthy controls. Nanoparticle tracking analysis was used to determine frequency and size, while NanoString technology was used to characterize the miRNA cargo. Relevant clinical parameters were correlated with EV miRNA cargo. ~ 30 miRNA species were identified to have significantly (p 0.05) different expression in EV from individuals with CP compared to healthy individuals; ~ 40 miRNA were differentially expressed in EV from pre-diabetic versus non-diabetic CP patients. miR-579-3p, while exhibiting significantly lower (~ 16-fold) expression in CP compared to healthy and lower (~ 24-fold) in CP narcotic users compared to the non-users, is actually enriched (~ 32-fold) within EV in pre-diabetic CP patients compared to non-diabetic CP patients. A unique pattern was identified in female CP patients. These data support the prospect of using a plasma-derived EV cargo to assess pancreatic health and its therapeutic potential in CP patients.

On November 15, 2019, the 24th annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held as a satellite conference during the annual Society for Leukocyte Biology meeting in Boston, Massachusetts. The 2019 meeting focused on alcohol, immunity, and organ damage, and included two plenary sessions. The first session highlighted new research exploring the mechanisms of alcohol-induced inflammation and liver disease, including effects on lipidomics and lipophagy, regulatory T cells, epigenetics, epithelial cells, and age-related changes in the gut. The second session covered alcohol-induced injury of other organs, encompassing diverse areas of research ranging from neurodegeneration, to lung barrier function, to colon carcinogenesis, to effects on viral infection. The discussions also highlighted current laboratory and clinical research used to identify biomarkers of alcohol use and disease.

Severe burn injury is a devastating form of trauma that results in persistent immune dysfunction with associated morbidity and mortality. The underlying drivers of this immune dysfunction remain elusive, and there are no prognostic markers to identify at-risk patients. Extracellular vesicles (EVs) are emerging as drivers of immune dysfunction as well as biomarkers. We investigated if EVs after burn injury promote macrophage activation and assessed if EV contents can predict length of hospital stay. EVs isolated early from mice that received a 20% total body surface area (TBSA) burn promoted proinflammatory responses in cultured splenic macrophages. Unbiased LC-MS/MS proteomic analysis of early EVs (&lt, 72 h post-injury) from mice and humans showed some similarities including enrichment of acute phase response proteins such as CRP and SAA1. Semi-unbiased assessment of early human burn patient EVs found alterations consistent with increased proinflammatory signaling and loss of inhibition of CRP expression. In a sample of 50 patients with large burn injury, EV SAA1 and CRP were correlated with TBSA injury in both sexes and were correlated with length of hospital stay in women. These findings suggest that EVs are drivers of immune responses after burn injury and their content may predict hospital course.

Innate immune signaling molecules, such as Toll-like receptors (TLRs), cytokines and transcription factor NF&kappa, B, are increased in post-mortem human alcoholic brain and may play roles in alcohol dependence and neurodegeneration. Innate immune signaling involves microglia -neuronal signaling which while poorly understood, may impact learning and memory. To investigate mechanisms of ethanol induction of innate immune signaling within and between brain cells, we studied immortalized BV2 microglia and SH-SY5Y human neuroblastoma to model microglial and neuronal signaling. Cells were treated alone or in co-culture using a Transwell system, which allows transfer of soluble mediators. We determined immune signaling mRNA using real-time polymerase chain reaction. Ethanol induced innate immune genes in both BV2 and SH-SY5Y cultured alone, with co-culture altering gene expression at baseline and following ethanol exposure. Co-culture blunted ethanol-induced high mobility group box protein 1 (HMGB1)-TLR responses, corresponding with reduced ethanol induction of several proinflammatory NF&kappa, B target genes. In contrast, co-culture resulted in ethanol upregulation of cytokines IL-4 and IL-13 in BV2 and corresponding receptors, that is, IL-4 and IL-13 receptors, in SH-SY5Y, suggesting induction of a novel signaling pathway. Co-culture reduction in HMGB1-TLR levels occurs in parallel with reduced proinflammatory gene induction and increased IL-4 and IL-13 ligands and receptors. Findings from these immortalized and tumor-derived cell lines could provide insight into microglial-neuronal interactions via release of soluble mediators in vivo.

Rationale Alcohol use disorder (AUD) involves dysregulation of innate immune signaling in brain. Toll-like receptor 3 (TLR3), an innate immune receptor that is upregulated in post-mortem human alcoholics, leads to induction of interferon (IFN) signaling. IFNs have been linked to depressive-like symptoms and therefore may play a role in addiction pathology. Astrocyte-neuronal signaling may contribute to maladaptation of neuronal circuits. Objectives In this manuscript, we examine ethanol (EtOH) induction of IFN signaling in neuronal, astrocyte, and microglial cell lines and assess astrocyte-neuronal interactions. Methods U373 astrocytes, SH-SY5Y neurons, and BV2 microglia were treated with EtOH and analyzed for autocrine/paracrine IFN signaling. Results EtOH induced TLR3, IFNβ, and IFNγ in SH-SY5Y neurons and U373 astrocytes, but not in BV2 microglia. The IFN response gene TRAIL was also strongly upregulated by TLR3 agonist Poly(I:C) and EtOH in U373 astrocytes. TRAIL blockage via neutralizing antibody prevented induction of IFNs in SH-SY5Y neurons but not in U373 astrocytes. Blocking TRAIL in conditioned media from EtOH-treated astrocytes prevented induction of IFNs in SH-SY5Y neurons. Finally, an in vivo model of chronic 10-day binge EtOH exposure in C57BL6/J mice, as well as single acute treatment with Poly(I:C), showed increased TRAIL +IR cells in both orbitofrontal and entorhinal cortex. Conclusions This study establishes a role of astrocyte to neuron TRAIL release in EtOH-induced IFN responses. This may contribute to alcohol associated negative affect and suggest potential therapeutic benefit of TRAIL inhibition in AUD.

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.

Modulating immune responses to sepsis and trauma remain one of the most difficult challenges in modern medicine. Large burn injuries (LBI) are a severe form of trauma associated with sepsis, immune impairment, and mortality. Immune dysfunction after LBI is complex, involving both enhanced and impaired immune activation. The release of Damage-Associated Molecular Patterns (DAMPs), such as HMGB1, and cytokines (e.g. IL-1β) creates an environment of immune dysfunction often leading to end organ failure and death. Both HMGB1 and IL-1β have been found to play critical roles in sepsis and post-burn immune dysfunction. HMGB1 and IL-1β have been shown previously to form potent complexes in vitro. We recently identified the presence of HMGB1/IL-1β heterocomplexes in human tissue. We now find HMGB1/IL-1β complexes in human and mouse plasma, and identify a synergistic role of HMGB1/IL-1β complexes in post-burn immune dysfunction. In both humans and mice, we found that HMGB1 was enriched in plasma microvesicles (MVs) after LBI. HMGB1 was found form complexes with IL-1β. Using flow cytometry of mouse plasma MVs, we identified an increase in an HMGB1+/IL-1β+ MVs. Using co-IP, HMGB1 was found to bind the pro-form of IL-1β in mouse and human plasma. Pro-IL-1β, which is traditionally considered inactive, became active when complexed with HMGB1. Human THP-1 monocytes treated with HMGB1-pro-IL-1β complexes showed increased transcription of LBI associated cytokines IL-6 and IFNβ along with suppression of iNOS, mimicking findings associated with LBI. These findings identify that HMGB1/IL-1β complexes released after burn injuries can modulate immune responses, and microvesicles are identified as a novel reservoir for these immune mediators. These complexes might serve as novel immune targets for the treatment of systemic immune responses due to LBI or other causes of sepsis.

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.

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.

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.

Adolescents binge drink more than any other age group, increasing risk of disrupting the development of the frontal cortex. We hypothesized that adolescent binge drinking would lead to persistent alterations in adulthood. In this study, we modeled adolescent weekend underage binge-drinking, using adolescent mice (post-natal days [P] 28-37). The adolescent intermittent binge ethanol (AIE) treatment includes 6 binge intragastric doses of ethanol in an intermittent pattern across adolescence. Assessments were conducted in adulthood following extended abstinence to determine if there were persistent changes in adults. Reversal learning, open field and other behavioral assessments as well as brain structure using magnetic imaging and immunohistochemistry were determined. We found that AIE did not impact adult Barnes Maze learning. However, AIE did cause reversal learning deficits in adults. AIE also caused structural changes in the adult brain. AIE was associated with adulthood volume enlargements in specific brain regions without changes in total brain volume. Enlarged regions included the orbitofrontal cortex (OFC, 4%), cerebellum (4.5%), thalamus (2%), internal capsule (10%) and genu of the corpus callosum (7%). The enlarged OFC volume in adults after AIE is consistent with previous imaging studies in human adolescents. AIE treatment was associated with significant increases in the expression of several extracellular matrix (ECM) proteins in the adult OFC including WFA (55%), Brevican (32%), Neurocan (105%), Tenacin-C (25%), and HABP (5%). These findings are consistent with AIE causing persistent changes in brain structure that could contribute to a lack of behavioral flexibility.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12974-017-0799-4) contains supplementary material, which is available to authorized users.

Alcohol consumption and stress increase brain levels of known innate immune signaling molecules. Microglia, the innate immune cells of the brain, and neurons respond to alcohol, signaling through Toll-like receptors (TLRs), high-mobility group box 1 (HMGB1), miRNAs, pro-inflammatory cytokines and their associated receptors involved in signaling between microglia, other glia and neurons. Repeated cycles of alcohol and stress cause a progressive, persistent induction of HMGB1, miRNA and TLR receptors in brain that appear to underlie the progressive and persistent loss of behavioral control, increased impulsivity and anxiety, as well as craving, coupled with increasing ventral striatal responses that promote reward seeking behavior and increase risk of developing alcohol use disorders. Studies employing anti-oxidant, anti-inflammatory, anti-depressant, and innate immune antagonists further link innate immune gene expression to addiction-like behaviors. Innate immune molecules are novel targets for addiction and affective disorders therapies. This article is part of the Special Issue entitled "Alcoholism".

Background: 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. Methods: To determine whether adolescent ethanol (AE) binge drinking alters the adult brain, male C57BL/6 mice were treated with either water or ethanol during adolescence (5 g/kg/d, i.g., postnatal days P28 to P37) and assessed during adulthood (P60 to P88). An array of neurotransmitter-specific genes, behavioral tests (i.e., reversal learning, prepulse inhibition, and open field), and postmortem brain structure using magnetic resonance imaging (MRI) and immunohistochemistry, were employed to assess persistent alterations in adult brain. Results: At P38, 24 hours after AE binge, many neurotransmitter genes, particularly cholinergic and dopaminergic, were reduced by ethanol treatment. Interestingly, dopamine receptor type 4 mRNA was reduced and confirmed using immunohistochemistry. Normal control maturation (P38 to P88) resulted in decreased neurotransmitter mRNA, e.g., an average decrease of 56%. Following AE treatment, adults showed greater gene expression reductions than controls, averaging 73%. Adult spatial learning assessed in the Morris water maze was not changed by AE treatment, but reversal learning experiments revealed deficits. Assessment of adult brain region volumes using MRI indicated that the olfactory bulb and basal forebrain were smaller in adults following AE. Immunohistochemical analyses found reduced basal forebrain area and fewer basal forebrain cholinergic neurons. Conclusions: Adolescent binge ethanol treatment reduces adult neurotransmitter gene expression, particularly cholinergic genes, reduces basal forebrain and olfactory bulb volumes, and causes a reduction in the density of basal forebrain acetylcholine neurons. Loss of cholinergic neurons and forebrain structure could underlie adult reversal learning deficits following adolescent binge drinking. Language: en

The in vitro oxidation of low-density lipoprotein (LDL) by hypochlorous acid produces a modified form (HOCl-LDL) capable of stimulating platelet function. We now report that HOCl-LDL is highly effective at inducing platelet function, causing stable aggregation and α-granule secretion. Such stimulation depended on the presence of low levels of primary agonists such as adenosine diphosphate (ADP) and thrombin, or others like epinephrine (EPI) and macrophage-derived chemokine (MDC, CCL22). Agonist levels, which by themselves induced little or reversible aggregation, caused strong stable aggregation when combined with low levels of HOCl-LDL. Platelet activation by HOCl-LDL and ADP (1 μM) caused P-selectin (CD62P) exposure, without serotonin or adenosine triphosphate (ATP) secretion. Intracellular calcium levels rose slowly (from 100 to 200 nM) in response to HOCl-LDL alone and rapidly when combined with ADP to about 300 nM. p38 mitogen-activated protein kinase (MAPK) became phosphorylated in response to HOCl-LDL alone. This phosphorylation was not blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide, which reduced the extent of aggregation and calcium increase. However, the p38 MAPK inhibitor SB203580 blocked platelet aggregation and phosphorylation of p38 MAPK. These findings suggest that HOCl-LDL exposed during atherosclerotic plaque rupture, coupled with low levels of primary agonists, can rapidly induce extensive and stable thrombus formation. (Blood. 2004;104:380-389)

The prefrontal cortex (PFC) is associated with higher cognitive functions including attention and working memory and has been implicated in the regulation of impulsivity as well as the pathology of complex mental illnesses. N-methyl D-aspartate (NMDA) antagonist treatment with dizocilpine induces cell death which is greatest in the frontal cortex on post-natal day seven (P7), however the long-term structural and behavioral effects of this treatment are unknown. This study investigates both the acute neurotoxicity of P7 dizocilpine and the persistent effects of this treatment on pyramidal cells and parvalbumin interneurons in the adult PFC, a brain region involved in the regulation of impulsivity. Dizocilpine treatment on P7 increased cleaved caspase-3 immunoreactivity (IR) in the PFC on P8. In adult mice (P82), P7 dizocilpine treatment resulted in 50% fewer parvalbumin-positive interneurons (p

Adolescent Alcohol abuse is common and alters brain development leading to persistent changes in adult brain and behavior. Adolescent intermittent ethanol (AIE) treatment (5 gm/kg, i.g.; 2 days on/2 days off; P25-P55) of wistar rats was used to model adolescent drinking and assessed in adulthood. AIE increased …

Background Athina Markou and her colleagues discovered persistent changes in adult behavior following adolescent exposure to ethanol or nicotine consistent with increased risk for developing addiction. Building on Dr. Markou’s important work and that of others in the field, researchers at the Bowles Center for Alcohol Studies have found that persistent changes in behavior following adolescent stress or alcohol exposure may be linked to induction of immune signaling in brain. Aim This study aims to illuminate the critical interrelationship of the innate immune system (e.g., toll-like receptors [TLRs], high-mobility group box 1 [HMGB1]) in the neurobiology of addiction. Method This study reviews the relevant research regarding the relationship between the innate immune system and addiction. Conclusion Emerging evidence indicates that TLRs in brain, particularly those on microglia, respond to endogenous innate immune agonists such as HMGB1 and microRNAs (miRNAs). Multiple TLRs, HMGB1, and miRNAs are induced in the brain by stress, alcohol, and other drugs of abuse and are increased in the postmortem human alcoholic brain. Enhanced TLR-innate immune signaling in brain leads to epigenetic modifications, alterations in synaptic plasticity, and loss of neuronal cell populations, which contribute to cognitive and emotive dysfunctions. Addiction involves progressive stages of drug binges and intoxication, withdrawal-negative affect, and ultimately compulsive drug use and abuse. Toll-like receptor signaling within cortical-limbic circuits is modified by alcohol and stress in a manner consistent with promoting progression through the stages of addiction.