Role of interleukin-10 (IL-10) in regulation of GABAergic transmission and acute response to ethanol

Highlights

• The anti-inflammatory cytokine IL-10 rapidly regulates GABAergic transmission.

• PI3K is involved in postsynaptic regulation of GABA_A receptors by IL-10.

• Brain IL-10 content is increased 1 h after a single intoxicating dose of EtOH.

• IL-10 regulates EtOH sensitivity by reducing acute EtOH-induced hypnosis.

• Early increases in IL-10 may contribute to disrupted sleep following EtOH exposure.

Abstract

Mounting evidence indicates that ethanol (EtOH) exposure activates neuroimmune signaling. Alterations in pro-inflammatory cytokines after acute and chronic EtOH exposure have been heavily investigated. In contrast, little is known about the regulation of neurotransmission and/or modulation by anti-inflammatory cytokines in the brain after an acute EtOH exposure. Recent evidence suggests that interleukin-10 (IL-10), an anti-inflammatory cytokine, is upregulated during withdrawal from chronic EtOH exposure. In the present study, we show that IL-10 is increased early (1 h) after a single intoxicating dose of EtOH (5 g/kg, intragastric) in Sprague Dawley rats. We also show that IL-10 rapidly regulates GABAergic transmission in dentate gyrus neurons. In brain slice recordings, IL-10 application dose-dependently decreases miniature inhibitory postsynaptic current (mIPSC) area and frequency, and decreases the magnitude of the picrotoxin sensitive tonic current (I_tonic), indicating both pre- and postsynaptic mechanisms. A PI3K inhibitor LY294002 (but not the negative control LY303511) ablated the inhibitory effects of IL-10 on mIPSC area and I_tonic, but not on mIPSC frequency, indicating the involvement of PI3K in postsynaptic effects of IL-10 on GABAergic transmission. Lastly, we also identify a novel neurobehavioral regulation of EtOH sensitivity by IL-10, whereby IL-10 attenuates acute EtOH-induced hypnosis. These results suggest that EtOH causes an early release of IL-10 in the brain, which may contribute to neuronal hyperexcitability as well as disturbed sleep seen after binge exposure to EtOH. These results also identify IL-10 signaling as a potential therapeutic target in alcohol-use disorders and other CNS disorders where GABAergic transmission is altered.

Introduction

A growing body of evidence from behavioral, molecular, genomic and electrophysiological studies indicates that ethanol (EtOH) exposure activates neuroimmune signaling (Crews et al., 2011, Mayfield et al., 2013, Bajo et al., 2015a). A number of hypotheses exist as to how EtOH causes increased activation of pro-inflammatory cytokines. For example, EtOH-induced liposaccharide (LPS) release into the systemic circulation and increased high-mobility group box 1 (HMGB1) protein release from neurons are implicated, both of which increase toll-like receptor 4 activity (Wang et al., 2010, Zou and Crews, 2014). This activity causes microglial activation, leading to further expression of pro-inflammatory genes, that are associated with increased alcohol consumption (Mayfield et al., 2013). Thus, mice treated with a high EtOH dose show an increase in systemic levels of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) and monocyte chemoattractant protein-1 (MCP-1), accompanied with a persistent increase in pro-inflammatory cytokines in the brain (Norkina et al., 2007, Qin et al., 2008). EtOH preference and amount of EtOH consumed are significantly reduced in mice lacking interleukin-1 receptor antagonist (IL-1ra) and in interleukin-6 (IL-6) knockout mice (Blednov et al., 2012).

In addition to neuroimmune modulation after EtOH exposure, cytokines and their receptors regulate neurotransmission and synaptic plasticity (Vezzani and Viviani, 2015), including ligand-gated ion channel trafficking and function via kinase-dependent mechanisms (Viviani et al., 2003, Yang et al., 2005). TNFα acts on neuronal TNFR1 by a phosphatidylinositol-3-kinase (PI3K) mechanism to exocytose AMPA receptors (AMPAR), thereby increasing excitatory neurotransmission in hippocampal neurons (Stellwagen et al., 2005). Conversely, TNFα causes endocytosis of GABA_A receptors (GABA_ARs) (Stellwagen et al., 2005). Another pro-inflammatory cytokine, interleukin-1β (IL-1β), reduces the frequency of AMPAR-dependent excitatory synaptic currents, but enhances NMDAR-mediated currents by activation of tyrosine kinases (Viviani et al., 2003, Yang et al., 2005). In addition, the IL-1 receptor 1 (IL-1R1) and NMDA receptor families interact with each other (Gardoni et al., 2011). IL-1β also increases recruitment of GABA_ARs to cell-surface via IL-1R1-dependent PI3K activation, causing a delayed enhancement of GABA currents (Serantes et al., 2006). Overall, investigations of GABA_AR indicate that the two pro-inflammatory cytokines TNFα and IL-1β diminish and augment inhibitory synaptic transmission, respectively.

In contrast to pro-inflammatory cytokines, less is known about the regulation of neurotransmission and/or modulation of anti-inflammatory cytokines in the brain after acute EtOH exposure. Indeed, recent investigations show that IL-1ra, an endogenous anti-inflammatory factor, regulates GABAergic transmission in central nucleus of amygdala and EtOH effects on synaptic activity (Bajo et al., 2015b). Also, mice lacking IL-1ra show reduced EtOH consumption (Blednov et al., 2015). In this study, we focused on interleukin-10 (IL-10), an anti-inflammatory cytokine. The −592C > A IL-10 gene polymorphism is associated with alcoholism in Spanish subjects (Marcos et al., 2008). A recent human study showed that after a single binge EtOH exposure, LPS-induced pro-inflammatory state is followed by an anti-inflammatory state in blood samples collected 2–5 h post-binge (Afshar et al., 2015). A single 24 h incubation with EtOH (25 mM) also increases IL-10 production by human monocytes (Norkina et al., 2007). In a 4-day binge EtOH model, hippocampal IL-10 levels were upregulated 7 days after withdrawal (Marshall et al., 2013). Interestingly, no blood-brain-barrier (BBB) disruption or upregulation of hippocampal pro-inflammatory cytokines occurred at this time point. Despite increases peripherally from acute exposure and centrally from chronic exposure, much remains unknown about alterations in anti-inflammatory cytokines in the brain after a single binge EtOH exposure and their possible modulation of GABAergic neurotransmission.

In the current study, we examine the effects of a single intoxicating dose of EtOH on rat hippocampal IL-10 levels. We also examine IL-10 effects on inhibitory tonic and phasic GABAergic currents in hippocampal dentate gyrus (DG) neurons and the involvement of PI3-kinase in these effects. Finally, we examine the behavioral contributions of IL-10 to EtOH's hypnotic responses, which may reflect neuroimmune modulation of sleep. Overall, these results suggest that early increases in brain IL-10 after a single intoxicating dose of EtOH may contribute to disrupted sleep following EtOH exposure.