Previous research has described neuroinflammation associated with chronic alcohol consumption. We hypothesized that alcohol could induce macrophage infiltration into the brain and that this infiltration could drive the neuroinflammation observed after chronic alcohol. We therefore tested if blockade using a CCR2/5 dual inhibitor of the chemokine network associated with macrophage chemoattraction could reduce alcohol-induced neuroinflammation. Here, we show that chronic alcohol induces region-specific infiltration of IMs into the CNS that is associated with cytokine expression and microglial activation. Inhibition of CCR2/5 signaling using the small molecule inhibitor CVC abrogated the infiltration of macrophages, reduced cytokine expression, and partially normalized microglial morphology.
Although the CNS was long considered an immune-privileged compartment, an appreciation for peripheral immune infiltration in the setting of diseases has been accepted. Only recently has infiltration of peripheral macrophages been described in the setting of alcohol-induced neuroinflammation [11, 22]. Using a different alcohol model than previously studied in mice, we confirm this observation using flow cytometry of total immune cells in the brain, detecting an increase in CD11b+CD45hi macrophages. Peripheral macrophages have previously been distinguished from microglia (CD11b+CD45lo), and although expression of CD45 can shift during inflammation, gene expression studies suggest its expression level remains a distinguishing feature between the two cell types . Additionally, we created CX3CR1eGFP/+ CCR2RFP/+ mice to allow for visualization and localization of infiltrating CCR2+ macrophages. While we observed a trend toward increased macrophages in the cortex and cerebellum, the largest alcohol-induced increase observed was in the hippocampus, a region of significant alcohol-related inflammation in both rodents and humans [8, 26, 27]. These data suggest that alcohol-induced macrophage infiltration into the CNS may be region-specific and possibly linked to localized neural damage and immune signaling.
Blockade of CCR2/5 signaling with CVC successfully limited the alcohol-induced infiltration of peripheral macrophages into the CNS. Previously, CVC was shown to be effective at limiting macrophage chemotaxis to the liver in a model of fibrosis . While successful inhibition of peripheral immune cell infiltration is consistent with blockade of a chemokine receptor, blocking CCL2 signaling has additional advantages. In the CNS of CCL2 knockout mice, production of proinflammatory cytokines TNFα and IL-1β was significantly reduced after peripheral injection of bacterial endotoxin (lipopolysaccharide (LPS)) . Interestingly, these proinflammatory cytokines were expressed even before peripheral cell infiltration occurred, suggesting that, at least in this model, neuroinflammatory gene expression preceded the response of the peripheral immune system. This has important implications for our present study, as it suggests that continued proinflammatory signaling within the CNS depends on chemoattraction of peripheral immune cells such as monocytes, which is inhibited by the small molecule CVC. Reducing inflammatory signaling in the CNS, which in the case of LPS precedes immune cell infiltration, may contribute to the reduced expression of chemokines. Cell-specific knockout of CCR2 (such as using LysM-driven knockout in peripheral macrophages or CX3CR1-driven knockout in microglia, for example) could help to elucidate the importance of peripheral blockade of CCR2 vs central signaling.
Additionally, recent evidence suggests that, in the developing brain, mice deficient in either CCL2 or CCR2 are protected from alcohol-induced neuroinflammation (including proinflammatory cytokine expression and microglial activation) and neurotoxicity when treated with an acute alcohol exposure at postnatal day 4 . These results agree with the present data, which importantly is from adult mice with chronic alcohol consumption and provide further evidence for the importance of CCL2/CCR2 signaling in alcohol-induced neuroinflammation. Further experiments, including using CCL2 and CCR2 knockout adult mice under chronic alcohol conditions will also be informative.
Interestingly, we observed the most pronounced alterations in the surface activation markers between microglia of alcohol-fed mice and microglia from mice treated with CVC. It is important to note that for flow cytometric analysis for surface marker expression, we used total brain microglia and infiltrating macrophages, whereas other assays (such as histology and biochemical measurements) used more localized populations or tissues for analysis. Therefore, cell surface marker analysis may not fully reflect the micromilieu within subregions of the brain. A possible explanation for the alterations in microglial surface markers is that in the absence of IMs, the remaining microglia are forced to assume a more activated phenotype and respond to the damage induced by alcohol. Interestingly, while we observed surface protein activation markers in microglia, we also observed an activated morphologic phenotype with increased soma size and reduced microglial cell process length. For this analysis, we focused on microglia in the hippocampus, the site of significant peripheral macrophage infiltration (Fig. 2c) and an increase in protein and mRNA expression of proinflammatory cytokines (Fig. 4). Microglial morphology is one indicator of an inflammatory milieu, as these cells change their shape in response to immune activation, and our flow cytometry data suggested that microglia are significantly affected by alcohol and CVC administration (Fig. 5). CVC treatment partially rescued microglial morphology. Compared with alcohol-fed untreated mice, the CVC treatment groups had slightly larger cell bodies but retained the shortened cell process phenotype of alcohol-fed mice. This mixed microglial phenotype, despite the absence of IM infiltration, likely represents the innate response of microglia to alcohol which is characterized by activation and production of proinflammatory cytokines and reactive oxygen species [30,31,32]. The imaging in Fig. 5 suggests that microglial morphology was altered by chronic alcohol exposure although histologic examination of the complete microglial architecture is technically limited as some microglial processes may be disrupted by the histologic preparation. Additional studies have shown that alcohol consumption may only lead to partial microglia activation [33,34,35], similar to the findings we present here (Fig. 5a, b). Therefore, we examine the microglial morphologic changes in the context of other measures of a neuroinflammatory milieu including analysis of activation markers and proinflammatory gene expression.
Chronic alcohol exposure induces a complex, multi-organ response with activation of a variety of immune responses and inflammatory expression. Previous research has sought to characterize the gene expression changes caused by chronic alcohol in various parts of the brain. Gene expression analyses in humans [36,37,38,39] and rodents  reveal alterations in genes related to neurons and neurogenesis, axonal growth, myelin regulation, intracellular signaling, protein trafficking, and other critical cell processes. Interestingly, neuroimmune genes were significantly increased in the frontal cortex of human patients with alcohol use disorder , while PET studies using markers of glial density have revealed that alcohol reduces or alters the behavior of CNS glia, likely representing changes in the local neuroimmune milieu [41,42,43]. Using a targeted screen, we observed alterations in inflammation-related transcripts in the CNS after chronic alcohol in mice and expanded our focus to investigate the hippocampus, cortex, and cerebellum. We observed the upregulation of multiple proinflammatory genes similar to previous descriptions, and some of these were altered by the treatment of CVC to block CCR2/5 signaling. As noted previously, some cytokine expression is increased prior to the infiltration of peripheral immune cells in other neuroinflammation models , which may provide an explanation for why CVC treatment only altered the expression of some inflammatory genes in the brain regions investigated.
Our data suggest that infiltrating macrophages specifically target the hippocampus as a site of increased infiltration. The hippocampus is a critical center for learning and memory and has been implicated in the pathology associated with AUD for decades, and this increased peripheral immune cell infiltration may, in part, underline this regional vulnerability. Hippocampal volume loss is associated with alcohol consumption in a dose-dependent manner. In a recently published study, researchers followed individuals for over three decades and show that increasing amounts of alcohol consumption are associated with a greater risk of hippocampal atrophy . Alcohol also influences key processes in memory formation and learning by suppressing long-term potentiation (LTP) of synaptic connections [44,45,46] and altering proper maturation and maintenance of dendritic spines and synaptic connections within the hippocampus [47, 48].
Microglia play a critical role in the synaptic pruning process , and alterations in their normal function have been shown to disrupt proper developmental synaptic pruning . In our study, alcohol consumption led to morphologic changes in microglia and changes in surface marker expression (Fig. 5). Also, the lysosomal protein CD68 was downregulated in the hippocampal microglia. Thus, the effect of alcohol on microglia is significant and may lead to altered microglial phagocytic function. An interesting future direction would be to assess synaptic pruning in this model. There is also evidence that proinflammatory cytokines may have an influence on synapse function, much like complement involvement in the regulation of synapse development. For example, TNFα has been shown to modulate glutamate receptors and decrease synaptic strength [50,51,52]. Interestingly, while the resident source is likely from the microglia, peripheral macrophages also express TNFα and may be involved in influencing synapses in addition to their possible role promoting neuroinflammation [52, 53]. Additionally, we noted increased cytokine protein expression in the hippocampus (Fig. 4), and cytokines have been implicated in synapse dysregulation in hippocampus neurons in vitro . Alcohol consumption may therefore be inducing microglial activation and changes in cytokine production that could affect the synapse structure function as well as the responsiveness of microglia to CNS immune surveillance and pathogen defense.
Previously, we have used various inhibitors of the proinflammatory NLRP3 inflammasome to show that reduction in inflammation via small molecule inhibitors may affect alcohol consumption and preference. Using therapeutic treatments to inhibit NLRP3, caspase-1, and IL-1β, we found a reduction in ethanol consumption and preference in a two-bottle choice test . Thus, in the treatment of alcohol-related pathology such as alcoholic liver disease, some therapeutics may operate via a mechanism that includes reduced alcohol consumption. Using the two-bottle choice paradigm, we found that CVC treatment did not influence ethanol consumption or preference. Additionally, using the Lieber-DeCarli chronic alcohol consumption model, we observed no difference in alcohol consumption between control and CVC-treated mice. Therefore, we can conclude that the data presented here, which included a reduction of both peripheral macrophage infiltration to the CNS and markers of inflammation within the CNS parenchyma, is likely due to the direct effects of CVC blockade on the CCR2/5 receptors and is not related to a change in the amount of alcohol consumed and/or a preference for alcohol. This is consistent with our previous findings that CVC both prevented and ameliorated alcoholic liver disease in mice .
Interestingly, previous behavioral studies by Blednov et al. have shown that CCR2-knockout mice have reduced preference and consumption of ethanol, while CCR5-knockout mice increase their consumption and preference compared with wild-type mice . CVC acts as a dual inhibitor blocking both the CCR2 and CCR5 receptors. Therefore, our observation of no change in consumption or preference in CVC-treated mice may be explained by the previous observations that CCR2 and CCR5 genetic deficiencies have opposite effects on alcohol use or preference. Using a dual inhibitor, we may have negated any effect on ethanol-seeking behavior and thus isolated a direct biologic effect of the CVC molecule on the underlying biologic pathology associated with alcohol use rather than influencing behavior and ethanol exposure.
Inflammatory processes in disease states generally occur as an evolutionary beneficial host response. However, chronic dysregulated inflammation often contributes to organ pathology and disease and is a key feature of chronic alcohol exposure. Infiltrating macrophages to the CNS in the setting of chronic alcohol may be protective or detrimental to the overall CNS milieu. Whereas we have previously shown that CVC reduces features of alcoholic liver disease, the hallmark pathologic features of chronic alcohol on the CNS are continuing to be defined, especially within the realm of inflammation, and this present study stimulates many interesting areas for future research including on synapse structures and functions, functional connections between glia and neurons, viability of neurons and glial cells, integrity of the blood-brain barrier, global neuronal network connections, and more.