New Microglial Mechanisms Revealed in Alcohol Use Disorder: How Does that Translate?

Alcohol use disorder (AUD) is a complex brain disorder characterized by an impaired ability to cease or moderate drinking despite adverse consequences to the individual. Myriad studies in recent years have pointed to neuroinflammation as being inextricably linked to alcohol and other substance use disorders (AUD/SUD). Under normal conditions, microglia, the resident macrophages and phagocytic cells of the brain, play an important role in maintaining homeostasis in the brain and serve as the first line of defense against pathogens. However, under conditions such as in aging or disease, the immune system can become dysregulated, resulting in chronic inflammation. This chronic pro-inflammatory state can lead to compromised blood-brain barrier (BBB) integrity, invasion of peripheral immune cells, mitochondrial dysfunction and dampened neurogenesis.

Changes in microglial and neuroimmune-related gene expression have been identified in murine and human brains after chronic alcohol consumption (1). Other studies have stressed the importance of disease and microglial phenotype. One study in particular found the presence of both M1 (pro-inflammatory) and M2 (resolution and repair) microglia in the brain in a male rat model of four days binge ethanol exposure, suggesting that anti-inflammatory microglia are present to mitigate the pro-inflammatory response to ethanol (2). Furthermore, pharmacologic depletion of microglia by the colony stimulating factor 1 receptor inhibitor, PLX5622, inhibited expression of pro-inflammatory genes and enhanced expression of anti-inflammatory genes in a mouse model of acute binge ethanol withdrawal (3).

While it is clear that microglia play a role in AUD, the nature of that role has remained elusive. Are activated microglia a key contributor to disease pathogenesis or merely a consequence of exposure to chronic alcohol consumption?

An article published in this issue of Biological Psychiatry helps to address these questions and makes significant strides in establishing a causal link between microglial activation and AUD (4). In order to test the hypothesis that microglial inhibition dampens the neuroinflammatory cascade following binge ethanol withdrawal, this study by Warden et al. used PLX5622, to inhibit microglia in the well-established chronic intermittent ethanol vapor two-bottle choice (CIE-2BC) mouse model. Following dietary administration of PLX5622 to male alcohol-dependent and non-dependent mice for four weeks, authors assessed several endpoints including: withdrawal-associated anxiety behavior, inflammatory gene expression, gamma-aminobutyric acid (GABA)ergic and glutamatergic gene expression and activity in brain regions important in AUD, the central nucleus of the amygdala (CeA) and the medial prefrontal cortex (mPFC).

There were several key findings in the study. Depletion of microglia prevented increased drinking after dependence was established. The authors noted that microglial inhibition prevented increased alcohol consumption in dependent mice without altering drinking in non-dependent mice. This is consistent with another recent study showing that microglial depletion does not change non-dependent drinking, but does prevent immune (toll-like receptor 4, tlr4)-enhanced drinking (5). Warden et al. also found that ethanol exposure increased microglial proliferation in the mPFC, as determined by histological analysis of expression of the microglial marker ionized calcium-binding adapter molecule 1 (Iba1) as well as transmembrane protein 119 (TMEM119) which distinguishes microglia from resident and infiltrating macrophages. This observation was not consistent with another recent translational work that showed an alcohol-induced reduction in Iba1 staining (6). However, a direct comparison cannot be made between the preclinical alcohol dependence models used in these two studies, as they used different ethanol drinking procedures and two different rodent species: the CIE-2BC procedure in C57BL/6L mice (4) and the 30 day access to two-drinking bottles in the Marchigian-Sardinian alcohol-preferring (msP) rats (6).

Warden et al. additionally discovered that microglial inhibition resulted in decreased anxiety-like behavior associated with withdrawal, as determined by the novelty suppressed feeding test. Inhibition of microglia also resulted in lower expression of genes associated with immunity and reversed changes in GABAergic and glutamatergic gene expression. In fact, many of the genes found to be associated with both microglial inhibition and alcohol dependence, such as interleukin-6 (IL-6), beta-2 microglobulin (B2m), and cathepsin S (Ctss) are pro-inflammatory and are known from the literature to be involved in alcohol consumption (7). Microglial inhibition was also shown to decrease GABAergic and glutamatergic synaptic transmission in the CeA. Overall, these data help to support the hypothesis that microglia play a role in the shift from voluntary alcohol consumption to alcohol dependence in mice, thus establishing a possible causal role and specific mechanisms, for microglia in AUD pathogenesis.

This work provides significant mechanistic insight into the role of microglia and the neuroimmune response in AUD. The significant attenuation of drinking and anxiety-like behavior in dependent mice in direct response to pharmacologic inhibition of microglia compared to non-dependent mice alone is strong data supportive of a causal role of microglia in AUD. The mechanistic data with regard to gene expression and synaptic transmission also support the hypothesis and are useful to the field. The study introduces follow-up questions that will serve to translate the findings from the bench to human laboratory studies.

The authors themselves reported that one limitation in their work was the exclusion of female mice from the study. Sex differences are an important topic in research and these differences are readily apparent in both animal models of AUD as well as humans. While more men have previously been diagnosed with AUD, this gap is narrowing in recent years, with more women being diagnosed than ever before. It is increasingly important that researchers focus on the translational aspects of their work, including sex differences. Additionally, sex differences have been noted in microglia in murine studies, with more neuroprotection observed in adult female mouse brains compared to their male counterparts following ischemic insult by middle cerebral artery occlusion (MCAo). Additionally, female and male microglia transplanted into the brains of the opposite sex maintained their sex-specific phenotypic characteristics, further supporting that microglia have sex-specific features (8). A study in rats that sought to explain why females require two to three times more morphine than males, demonstrated key sex differences in microglia in the periaqueductal gray, with females showing an increased pro-inflammatory “activated” microglial phenotype that opposes the analgesic effects of morphine (9). Future studies can expound upon the findings in the article by Warden et al. by addressing any sex differences in drinking behavior and anxiety outcomes related to microglial activation states.

The study focused on two specific brain regions, the CeA and the mPFC. Future studies should expand their search to other areas of the brain implicated in AUD, including other parts of the limbic system and the cerebellum. It would be particularly important to address any inflammation and microglial activation in the hippocampus, as hippocampal neurogenesis has been shown to be negatively impacted by alcohol consumption.

Another way to increase the translational applicability of this work is to focus more on activation states or microglial phenotype. That is, future studies should look to methods for shifting microglia from a pro-inflammatory M1 to an anti-inflammatory M2 status. While the current preclinical study achieved the goal of supporting the hypothesis that activated microglia contribute to alcohol dependence in mice, it would not be feasible to completely inhibit microglia in human studies. Future studies should use the findings from Warden et al. study as impetus to use clinically relevant methods to alleviate neuroinflammation.

To that end, increased attention has been given to the effect of the gut microbiome on a number of brain disorders, including alcohol-induced inflammation in the brain. One recent study found that alcohol consumption induced neuroinflammation and microglial activation in mice, whereas reduction of gut bacteria with antibiotic treatment reduced pro-inflammatory cytokine expression and abrogated microglial activation (10). An appealing element of studying the effect of the gut microbiome on neuroinflammation in AUD in humans is that it’s non-invasive and any results would be highly relevant clinically.

In addition, pharmacotherapeutic research for AUD has also focused on therapeutic targets related to inflammation. Currently, disulfiram, naltrexone, and acamprosate are the only FDA-approved medications for treating AUD. However, research into the use of medications currently used for other conditions as well as medication development are always underway. Using the microglial-specific genetic information gleaned from the current Biological Psychiatry study, future pharmacotherapy could use these genes as potential therapeutic targets.

The study by Warden et al. breaks new ground in establishing a causal link between microglia and alcohol dependence in mice, moving the field forward in its understanding of the role of neuroinflammation in AUD. The detailed mechanistic findings therein can be translated from the bench to clinical studies in effort to shift the alcohol-induced pro-inflammatory status to an anti-inflammatory state that promotes recovery and brain repair using a clinically relevant intervention. There are a number of clinical trials in process that address the neuroimmune response in AUD and hopefully these new insights will add to the growing number of studies in this area.