Jorge Montesinos

Ethanol induces TLR4/TLR2 association, triggering an inflammatory response in microglial cells.

Alcohol consumption can induce brain damage, demyelination, and neuronal death, although the mechanisms are poorly understood. Toll‐like receptors are sensors of the innate immune system and their activation induces inflammatory processes. We have reported that ethanol activates and recruits Toll‐like receptor (TLR)4 receptors within the lipid rafts of glial cells, triggering the production of inflammatory mediators and causing neuroinflammation. Since TLR2 can also participate in the glial response and in the neuroinflammation, we investigate the effects of ethanol on TLR4/TLR2 responses. Here, we demonstrate that ethanol up‐regulates TLR4 and TLR2 expression in microglial cells, inducing the production of inflammatory mediators which triggers reactive oxygen species generation and neuronal apoptosis. Ethanol also promotes TLR4/TLR2 recruitment into lipid rafts‐caveolae, mimicking their activation by their ligands, lipopolysaccharide, and lipoteichoic acid (LTA). Immunoprecipitation and confocal microscopy studies reveal that ethanol induces a physical association between TLR2 and TLR4 receptors, suggesting the formation of heterodimers. Using microglia from either TLR2 or TLR4 knockout mice, we show that TLR2 potentiates the effects of ethanol on the TLR4 response reflected by the activation of MAPKs and inducible NO synthase. In summary, we provide evidence for a mechanism by which ethanol triggers TLR4/TLR2 association contributing to the neuroinflammation and neurodegeneration associated with alcohol abuse.

Impact of the Innate Immune Response in the Actions of Ethanol on the Central Nervous System.

The innate immune response in the central nervous system (CNS) participates in both synaptic plasticity and neural damage. Emerging evidence from human and animal studies supports the role of the neuroimmune system response in many actions of ethanol (EtOH) on the CNS. Research studies have shown that alcohol stimulates brain immune cells, microglia, and astrocytes, by activating innate immune receptors Toll-like receptors (TLRs) and NOD-like receptors (inflammasome NLRs) triggering signaling pathways, which culminate in the production of pro-inflammatory cytokines and chemokines that lead to neuroinflammation. This review focuses on evidence that indicates the participation of TLRs and the inflammasome NLRs signaling response in many effects of EtOH on the CNS, such as neuroinflammation associated with brain damage, cognitive and behavioral dysfunction, and adolescent brain development alterations. It also reviews findings that indicate the role of TLR4-dependent signaling immune molecules in alcohol consumption, reward, and addiction. The research data suggest that overactivation of TLR4 or NLRs increases pro-inflammatory cytokines and mediators to cause neural damage in the cerebral cortex and hippocampus, while modest TLR4 activation, along with the generation of certain cytokines and chemokines in specific brain areas (e.g., amygdala, ventral tegmental area), modulate neurotransmission, alcohol drinking, and alcohol rewards. Elimination of TLR4 and NLRP3 abolishes many neuroimmune effects of EtOH. Despite much progress being made in this area, there are some research gaps and unanswered questions that this review discusses. Finally, potential therapies that target neuroimmune pathways to treat neuropathological and behavioral consequences of alcohol abuse are also evaluated.

Involvement of TLR4 in the long-term epigenetic changes, rewarding and anxiety effects induced by intermittent ethanol treatment in adolescence

Studies in humans and experimental animals have demonstrated the vulnerability of the adolescent brain to actions of ethanol and the long-term consequences of binge drinking, including the behavioral and cognitive deficits that result from alcohol neurotoxicity, and increased risk to alcohol abuse and dependence. Although the mechanisms that participate in these effects are largely unknown, we have shown that ethanol by activating innate immune receptors, toll-like receptor 4 (TLR4), induces neuroinflammation, impairs myelin proteins and causes cognitive dysfunctions in adolescent mice. Since neuroimmune signaling is also involved in alcohol abuse, the aim of this study was to assess whether ethanol treatment in adolescence promotes the long-term synaptic and molecular events associated with alcohol abuse and addiction. Using wild-type (WT) and TLR4-deficient (TLR4-KO) adolescent mice treated intermittently with ethanol (3g/kg) for 2 weeks, we showed that binge-like ethanol treatment in adolescent mice promotes short- and long-term alterations in synaptic plasticity and epigenetic changes in the promoter region of bdnf and fosb, which increased their expression in the mPFC of young adult animals. These molecular events were associated with long-term rewarding and anxiogenic-related behavioral effects, along with increased alcohol preference. Our results further showed the participation of neuroimmune system activation and the TLR4 signaling response since deficient mice in TLR4 (TLR4-KO) are protected against molecular and behavioral alterations of ethanol in the adolescent brain. Our results highlight a new role of the neuroimmune function and open up new avenues to develop pharmacological treatments that can normalize the immune signaling responsible for long-term effects in adolescence, including alcohol abuse and related disorders.

Plasma profile of pro-inflammatory cytokines and chemokines in cocaine users under outpatient treatment: influence of cocaine symptom severity and psychiatric co-morbidity.

The treatment for cocaine use constitutes a clinical challenge because of the lack of appropriate therapies and the high rate of relapse. Recent evidence indicates that the immune system might be involved in the pathogenesis of cocaine addiction and its co‐morbid psychiatric disorders. This work examined the plasma pro‐inflammatory cytokine and chemokine profile in abstinent cocaine users (n = 82) who sought outpatient cocaine treatment and age/sex/body mass‐matched controls (n = 65). Participants were assessed with the diagnostic interview Psychiatric Research Interview for Substance and Mental Diseases according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM‐IV‐TR). Tumor necrosis factor‐alpha, chemokine (C‐C motif) ligand 2/monocyte chemotactic protein‐1 and chemokine (C‐X‐C motif) ligand 12 (CXCL12)/stromal cell‐derived factor‐1 (SDF‐1) were decreased in cocaine users, although all cytokines were identified as predictors of a lifetime pathological use of cocaine. Interleukin‐1 beta (IL‐1β), chemokine (C‐X3‐C motif) ligand 1 (CX3CL1)/fractalkine and CXCL12/SDF‐1 positively correlated with the cocaine symptom severity when using the DSM‐IV‐TR criteria for cocaine abuse/dependence. These cytokines allowed the categorization of the outpatients into subgroups according to severity, identifying a subgroup of severe cocaine users (9–11 criteria) with increased prevalence of co‐morbid psychiatric disorders [mood (54%), anxiety (32%), psychotic (30%) and personality (60%) disorders]. IL‐1β was observed to be increased in users with such psychiatric disorders relative to those users with no diagnosis. In addition to these clinical data, studies in mice demonstrated that plasma IL‐1β, CX3CL1 and CXCL12 were also affected after acute and chronic cocaine administration, providing a preclinical model for further research. In conclusion, cocaine exposure modifies the circulating levels of pro‐inflammatory mediators. Plasma cytokine/chemokine monitoring could improve the stratification of cocaine consumers seeking treatment and thus facilitate the application of appropriate interventions, including management of heightened risk of psychiatric co‐morbidity. Further research is necessary to elucidate the role of the immune system in the etiology of cocaine addiction.

LPS or ethanol triggers clathrin‐ and rafts/caveolae‐dependent endocytosis of TLR4 in cortical astrocytes

Toll‐like receptor 4 (TLR4) activation and signalling in glial cells play critical roles in neurological disorders and in alcohol‐induced brain damage. TLR4 endocytosis upon lipopolysaccharide (LPS) stimulation regulates which signalling pathway is activated, the MyD88‐dependent or the TIR‐domain‐containing adapter‐inducing interferon‐β (TRIF)‐dependent pathway. However, it remains elusive whether ethanol‐induced TLR4 signalling is associated with receptor internalization and trafficking, and which endocytic pathway(s) are used in cortical astrocytes. Using the adenoviral over‐expression of TLR4GFP, confocal microscopy and the imagestream technique, we show that upon ethanol or LPS stimulation, TLR4 co‐localizes with markers of the clathrin and caveolin endocytic pathways, and that this endocytosis is dependent on dynamin. Using chlorpromazin and filipin as inhibitors of the clathrin and rafts/caveolae endocytic pathways, respectively, we demostrate that TRIF‐dependent signalling relies on an intact clathrin pathway, whereas disruption of rafts/caveolae inhibits the MyD88‐ and TRIF‐dependent signalling pathways. Immunofluorescence studies also suggest that lipid rafts and clathrin cooperate for appropriate TLR4 internalization. We also show that ethanol can trigger similar endocytic pathways as LPS does, although ethanol delays clathrin internalization and alters TLR4 vesicular trafficking. Our results provide new insights into the effects of ethanol or LPS on TLR4 signalling in cortical astrocytes, events that may underlie neuroinflammation and brain damage.

TLR4 elimination prevents synaptic and myelin alterations and long-term cognitive dysfunctions in adolescent mice with intermittent ethanol treatment

The adolescent brain undergoes important dynamic and plastic cell changes, including overproduction of axons and synapses, followed by rapid pruning along with ongoing axon myelination. These developmental changes make the adolescent brain particularly vulnerable to neurotoxic and behavioral effects of alcohol. Although the mechanisms of these effects are largely unknown, we demonstrated that ethanol by activating innate immune receptors toll-like receptor 4 (TLR4), induces neuroinflammation and brain damage in adult mice. The present study aims to evaluate whether intermittent ethanol treatment in adolescence promotes TLR4-dependent pro-inflammatory processes, leading to myelin and synaptic dysfunctions, and long-term cognitive impairments. Using wild-type (WT) and TLR4-deficient (TLR4-KO) adolescent mice treated intermittently with ethanol (3.0g/kg) for 2weeks, we show that binge-like ethanol treatment activates TLR4 signaling pathways (MAPK, NFκB) leading to the up-regulation of cytokines and pro-inflammatory mediators (COX-2, iNOS, HMGB1), impairing synaptic and myelin protein levels and causing ultrastructural alterations. These changes were associated with long-lasting cognitive dysfunctions in young adult mice, as demonstrated with the object recognition, passive avoidance and olfactory behavior tests. Notably, elimination of TLR4 receptors prevented neuroinflammation along with synaptic and myelin derangements, as well as long-term cognitive alterations. These results support the role of the neuroimmune response and TLR4 signaling in the neurotoxic and behavioral effects of ethanol in adolescence.

Gender differences in the inflammatory cytokine and chemokine profiles induced by binge ethanol drinking in adolescence

Heavy binge drinking in adolescence can cause long‐term cognitive and behavioral dysfunctions. Recent experimental evidence indicates the participation of immune system activation in the effects of ethanol in the adolescent brain and suggests gender differences. The present study aims to assess plasma cytokine and chemokine levels in male and female adolescents and young adults during acute alcohol intoxication and to correlate these results with the toll‐like receptor 4 (TLR4) response. The potential role of the TLR4 signaling response was also assessed in plasma and prefrontal cortex (PFC) of adolescent wild‐type and TLR4‐knockout male and female mice with binge ethanol treatment. The results showed that alcohol intoxication increased the plasma levels of several cytokine and chemokine [interferon‐γ, interleukin (IL)‐10, IL‐17A, IL‐1β, IL‐2, IL‐4, IL‐6, IL‐8, fractalkine, monocyte chemoattractant protein 1 (MCP‐1) and macrophage inflammatory protein 1α (MIP‐1α)] and the upregulation of TLR4 mRNA levels occurred in intoxicated females, while elevation of colony‐stimulating factor was only observed in the plasma of males. In wild‐type female adolescent mice, intermittent ethanol treatment increased the levels of several cytokines (IL‐17A and IL‐1β) and chemokines (MCP‐1, MIP‐1α and fractalkine) in PFC and in serum (IL‐17A, MCP‐1 and MIP‐1α), but significant differences in the fractalkine levels in PFC were observed only in male mice. No changes in serum or prefrontal cortex cytokine and chemokine levels were noted in ethanol‐treated male or female TLR4‐knockout mice. Our findings revealed that females are more vulnerable than males to inflammatory effects of binge ethanol drinking and suggested that TLR4 is an important target of ethanol‐induced inflammation and neuroinflammation in adolescence.

Increased localization of APP-C99 in mitochondria-associated ER membranes causes mitochondrial dysfunction in Alzheimer disease

In the amyloidogenic pathway associated with Alzheimer disease (AD), the amyloid precursor protein (APP) is cleaved by β‐secretase to generate a 99‐aa C‐terminal fragment (C99) that is then cleaved by γ‐secretase to generate the β‐amyloid (Aβ) found in senile plaques. In previous reports, we and others have shown that γ‐secretase activity is enriched in mitochondria‐associated endoplasmic reticulum (ER) membranes (MAM) and that ER–mitochondrial connectivity and MAM function are upregulated in AD. We now show that C99, in addition to its localization in endosomes, can also be found in MAM, where it is normally processed rapidly by γ‐secretase. In cell models of AD, however, the concentration of unprocessed C99 increases in MAM regions, resulting in elevated sphingolipid turnover and an altered lipid composition of both MAM and mitochondrial membranes. In turn, this change in mitochondrial membrane composition interferes with the proper assembly and activity of mitochondrial respiratory supercomplexes, thereby likely contributing to the bioenergetic defects characteristic of AD.

Role of the innate immune system in the neuropathological consequences induced by adolescent binge drinking

Adolescence is a critical stage of brain maturation in which important plastic and dynamic processes take place in different brain regions, leading to development of the adult brain. Ethanol drinking in adolescence disrupts brain plasticity and causes structural and functional changes in immature brain areas (prefrontal cortex, limbic system) that result in cognitive and behavioral deficits. These changes, along with secretion of sexual and stress‐related hormones in adolescence, may impact self‐control, decision making, and risk‐taking behaviors that contribute to anxiety and initiation of alcohol consumption. New data support the participation of the neuroimmune system in the effects of ethanol on the developing and adult brain. This article reviews the potential pathological bases that underlie the effects of alcohol on the adolescent brain, such as the contribution of genetic background, the perturbation of epigenetic programming, and the influence of the neuroimmune response. Special emphasis is given to the actions of ethanol in the innate immune receptor toll‐like receptor 4 (TLR4), since recent studies have demonstrated that by activating the inflammatory TLR4/NFκB signaling response in glial cells, binge drinking of ethanol triggers the release of cytokines/chemokines and free radicals, which exacerbate the immune response that causes neuroinflammation/neural damage as well as short‐ and long‐term neurophysiological, cognitive, and behavioral dysfunction. Finally, potential treatments that target the neuroimmune response to treat the neuropathological and behavioral consequences of adolescent alcohol abuse are discussed.

Binge-like ethanol treatment in adolescence impairs autophagy and hinders synaptic maturation: Role of TLR4

Adolescence is a developmental period of brain maturation in which remodeling and changes in synaptic plasticity and neural connectivity take place in some brain regions. A different mechanism participates in adolescent brain maturation, including autophagy processes that play a role in synaptic development and plasticity. Alcohol is a neurotoxic compound whose abuse in adolescence causes TLR4 response activation by triggering neuroinflammation, neural damage and behavioral alterations. However, the potential participation of autophagy in long-term neurochemical and cognitive dysfunctions induced by binge ethanol drinking in adolescence is uncertain. We therefore evaluated whether binge ethanol drinking alters autophagy pathways by contributing to adolescent synaptic dysfunctions, and if the immune receptor TLR4 response participates in these events. With wild-type (WT) and TLR4-deficient (TLR4-KO) adolescent mice treated intermittently with ethanol (3.0 g/kg) for 2 weeks, we show that binge-like ethanol exposure in adolescence impairs autophagy machinery by increasing autophagy inhibitor mTOR by lowering LC3-II levels and accumulating p62. Inhibition of mTOR, by rapamycin, restores the levels of excitatory scaffolding synaptic proteins (PSD-95 or SHANK3), p62, and partly reestablishes the LC3-II levels in the prefrontal cortices of ethanol-treated WT mice. Elimination of the TLR4 receptors using TLR4-KO mice prevents autophagy dysfunctions and reduces the number or size of the synaptic connections induced by ethanol. These results suggest the role of autophagy dysfunctions in the structural synaptic plasticity alterations induced by binge alcohol in adolescence, and support the participation of the TLR4 response in these events.