Many genes differentially expressed in brain tissue from human alcoholics and animals that have consumed large amounts of alcohol are components of the innate immune toll-like receptor (TLR) pathway. TLRs initiate inflammatory responses via two branches: (1) MyD88-dependent or (2) TRIF-dependent. All TLRs signal through MyD88 except TLR3. Prior work demonstrated a direct role for MyD88-dependent signaling in regulation of alcohol consumption. However, the role of TLR3 as a potential regulator of excessive alcohol drinking has not previously been investigated. To test the possibility TLR3 activation regulates alcohol consumption, we injected mice with the TLR3 agonist polyinosinic:polycytidylic acid (poly(I:C)) and tested alcohol consumption in an every-other-day two-bottle choice test. Poly(I:C) produced a persistent increase in alcohol intake that developed over several days. Repeated poly(I:C) and ethanol exposure altered innate immune transcript abundance; increased levels of TRIF-dependent pathway components correlated with increased alcohol consumption. Administration of poly(I:C) before exposure to alcohol did not alter alcohol intake, suggesting that poly(I:C) and ethanol must be present together to change drinking behavior. To determine which branch of TLR signaling mediates poly(I:C)-induced changes in drinking behavior, we tested either mice lacking MyD88 or mice administered a TLR3/ dsRNA complex inhibitor. MyD88 null mutants showed poly(I:C)-induced increases in alcohol intake. In contrast, mice pretreated with a TLR3/dsRNA complex inhibitor reduced their alcohol intake, suggesting poly(I:C)-induced escalations in alcohol intake function are, at least partially, dependent on TLR3. Together, these results strongly suggest that TLR3-dependent signaling drives excessive alcohol drinking behavior.

Although there are sex differences in the effects of alcohol on immune responses, it is unclear if sex differences in immune response can influence drinking behavior. Activation of toll-like receptor 3 (TLR3) by polyinosinic:polycytidylic acid (poly(I:C)) produced a rapid proinflammatory response in males that increased alcohol intake over time (Warden et al., 2019). Poly(I:C) produced a delayed and prolonged innate immune response in females. We hypothesized that the timecourse of innate immune activation could regulate drinking behavior in females. Therefore, we chose to test the effect of two time points in the innate immune activation timecourse on every-other-day two-bottle-choice drinking: (1) peak activation; (2) descending limb of activation. Poly(I:C) reduced ethanol consumption when alcohol access occurred during peak activation. Poly(I:C) did not change ethanol consumption when alcohol access occurred on the descending limb of activation. Decreased levels of MyD88-dependent pathway correlated with decreased alcohol intake and increased levels of TRIF-dependent pathway correlated with increased alcohol intake in females. To validate the effects of poly(I:C) were mediated through MyD88, we tested female mice lacking Myd88. Poly(I:C) did not change alcohol intake in Myd88 *

Overactivation of neuroimmune signaling has been linked to excessive ethanol consumption. Tolllike receptors (TLRs) are a major component of innate immune signaling and initiate anti-and pro-inflammatory responses via intracellular signal transduction cascades. TLR7 is upregulated in post-mortem brain tissue from humans with alcohol use disorder (AUD) and animals with prior exposure to ethanol. Despite this evidence, the role of TLR7 in the regulation of voluntary ethanol consumption has not been studied. We test the hypothesis that TLR7 activation regulates voluntary ethanol drinking behavior by administering a TLR7 agonist (R848) during an intermittent access drinking procedure in mice. Acute activation of TLR7 reduced ethanol intake, preference, and total fluid intake due, at least in part, to an acute sickness response. However, chronic pre-treatment with R848 resulted in tolerance to the adverse effects of the drug and a subsequent increase in ethanol consumption. To determine the molecular machinery that mediates these behavioral changes, we evaluated gene expression after acute and chronic TLR7 activation. We found that acute TLR7 activation produces brain region specific changes in expression of immune pathway genes, whereas chronic TLR7 activation causes downregulation of TLRs and blunted cytokine induction, suggesting molecular tolerance. Our results demonstrate a novel role for TLR7 signaling in regulating voluntary ethanol consumption. Taken together, our findings suggest TLR7 may be a viable target for development of therapies to treat AUD.

Astrocytes are fundamental building blocks of the central nervous system. Their dysfunction has been implicated in many psychiatric disorders, including alcohol use disorder, yet our understanding of their functional role in ethanol intoxication and consumption is very limited. Astrocytes regulate behavior through multiple intracellular signaling pathways, including G-protein coupled-receptor (GPCR)-mediated calcium signals. To test the hypothesis that GPCR-induced calcium signaling is also involved in the behavioral effects of ethanol, we expressed astrocyte-specific excitatory DREADDs in the prefrontal cortex (PFC) of mice. Activating Gq-GPCR signaling in PFC astrocytes increased drinking in ethanol-naïve mice, but not in mice with a history of ethanol drinking. In contrast, reducing calcium signaling with an astrocyte-specific calcium extruder reduced ethanol intake. Cortical astrocyte calcium signaling also altered the acute stimulatory and sedative-hypnotic effects of ethanol. Astrocyte-specific Gq-DREADD activation increased both the locomotor-activating effects of low dose ethanol and the sedative-hypnotic effects of a high dose, while reduced astrocyte calcium signaling diminished sensitivity to the hypnotic effects. In addition, we found that adenosine A1 receptors were required for astrocyte calcium activation to increase ethanol sedation. These results support integral roles for PFC astrocytes in the behavioral actions of ethanol that are due, at least in part, to adenosine receptor activation.

Background The inbred mouse strain C57BL/6 is widely used in both models of addiction and immunological disease. However, there are pronounced phenotypic differences in ethanol (EtOH) consumption and innate immune response between C57BL/6 substrains. The focus of this study was to examine the effects of substrain on innate immune response and neuroimmune‐induced escalation of voluntary EtOH consumption. The main goal was to identify whether substrain differences in immune response can account for differences in EtOH behavior. Methods We compared acute innate immune response with a viral dsRNA mimic, polyinosinic:polycytidylic acid (poly(I:C)), in brain using qRT‐PCR in both C57BL/6N and C57BL/6J mice. Next, we used a neuroimmune model of escalation using poly(I:C) to compare drinking behavior between substrains. Finally, we compared brain neuroimmune response with both EtOH and repeated poly(I:C) in both substrains as a way to account for differences in EtOH behavior. Results We found that C57BL/6 substrains have differing immune response and drinking behaviors. C57BL/6N mice have a shorter but more robust inflammatory response to acute poly(I:C). In contrast, C57BL/6J mice have a smaller but longer‐lasting acute immune response to poly(I:C). In our neuroimmune‐induced escalation model, C57BL/6J mice but not C57BL/6N mice escalate EtOH intake after poly(I:C). Finally, only C57BL/6J mice show enhanced proinflammatory transcript abundance after poly(I:C) and EtOH, suggesting that longer‐lasting immune responses are critical to neuroimmune drinking phenotypes. Conclusions Altogether, this work has elucidated additional influences that substrain has on both innate immune response and drinking phenotypes. Our observations highlight the importance of considering and reporting the source and background used for production of transgenic and knockout mice. These data provide further evidence that genetic background must be carefully considered when investigating the role of neuroimmune signaling in EtOH abuse.

Alcohol abuse induces changes in microglia morphology and immune function, but whether microglia initiate or simply amplify the harmful effects of alcohol exposure is still a matter of debate. Here, we determine microglia function in acute and voluntary drinking behaviors using a colony‐stimulating factor 1 receptor inhibitor (PLX5622). We show that microglia depletion does not alter the sedative or hypnotic effects of acute intoxication. Microglia depletion also does not change the escalation or maintenance of chronic voluntary alcohol consumption. Transcriptomic analysis revealed that although many immune genes have been implicated in alcohol abuse, downregulation of microglia genes does not necessitate changes in alcohol intake. Instead, microglia depletion and chronic alcohol result in compensatory upregulation of alcohol‐responsive, reactive astrocyte genes, indicating astrocytes may play a role in regulation of these alcohol behaviors. Taken together, our behavioral and transcriptional data indicate that microglia are not the primary effector cell responsible for regulation of acute and voluntary alcohol behaviors. Because microglia depletion did not regulate acute or voluntary alcohol behaviors, we hypothesized that these doses were insufficient to activate microglia and recruit them to an effector phenotype. Therefore, we used a model of repeated immune activation using polyinosinic:polycytidylic acid (poly(I:C)) to activate microglia. Microglia depletion blocked poly(I:C)‐induced escalations in alcohol intake, indicating microglia regulate drinking behaviors with sufficient immune activation. By testing the functional role of microglia in alcohol behaviors, we provide insight into when microglia are causal and when they are consequential for the transition from alcohol use to dependence.

INTRODUCTION: Rapid Point of Care Tests for infection (POCT) do not consistently improve antibiotic stewardship (ASP) of suspected ICU infection. We measured 1) the effect of a negative PCR-POCT on antibiotic stop decisions, and 2) clinico-behavioural factors that prevent stopping.METHODS: Vignettes of antibiotic treated respiratory infection, with 4 distinct trajectories were presented to ICU clinicians: overall improvement, clinical improvement/biological worsening, clinical worsening/biological improvement, overall worsening. Initial and post PCR-POCT antibiotic decisions (stop or continue) /confidence levels were recorded. The PCR-POCT offer was voluntary but always presented and negative. Linear regression determined association of their final decision with influencing factors.RESULTS: Seventy clinicians responded. A negative PCR-POCT increased stop decisions in all scenarios (p<0.001) except improvement (already high); especially in discordant clin worse(49% pre-POCT vs 74% post-POCT). Inclination to stop was reduced by an ambiguous/worsening trajectory(p=0.015), initial confidence to continue(p<0.001), and involuntary receipt of POCT(p<0.001), not clinician experience or risk averseness. CONCLUSIONS: Negative PCR-POCT increases the inclination to stop antibiotics, particularly in ambiguous/worsening trajectories of ICU infection. Clinician intuition to continue and disinterest in POCT reduce its influence to stop. Highlighting and quantifying the predictive impact of behavioural-trajectorial factors can improve antibiotic stewardship and study design in ICU related infection.

MotivationDetecting novel functional modules in molecular networks is an important step in biological research. In the absence of gold standard functional modules, functional annotations are often used to verify whether detected modules/communities have biological meaning. However, as we show, the uneven distribution of functional annotations means that such evaluation methods favor communities of well-studied proteins.ResultsWe propose a novel framework for the evaluation of communities as functional modules. Our proposed framework, CommWalker, takes communities as inputs and evaluates them in their local network environment by performing short random walks. We test CommWalker’s ability to overcome annotation bias using input communities from four community detection methods on two protein interaction networks. We find that modules accepted by CommWalker are similarly co-expressed as those accepted by current methods. Crucially, CommWalker performs well not only in well-annotated regions, but also in regions otherwise obscured by poor annotation. CommWalker community prioritization both faithfully captures well-validated communities and identifies functional modules that may correspond to more novel biology.Availability and implementationThe CommWalker algorithm is freely available at opig.stats.ox.ac.uk/resources or as a docker image on the Docker Hub at hub.docker.com/r/lueckenmd/commwalker/.Supplementary information Supplementary data are available at Bioinformatics online.