P2X7R Signaling and Differential Regulation of Neuroinflammatory and Behavior Responses in Male and Female Mice During Chronic Ethanol Exposure

TL;DR

The study shows that chronic alcohol exposure causes brain inflammation and memory issues in male mice more than females, and blocking a specific receptor (P2X7R) can reduce these effects.

Contribution

The study reveals sex-specific differences in alcohol-induced neuroinflammation and the effectiveness of P2X7R inhibition in mitigating these effects.

Findings

Chronic ethanol exposure increased neuroinflammatory markers more in males than females.

P2X7R inhibition reduced cytokine levels and memory impairment in male mice.

Both sexes showed increased extracellular ATP and EVs, which were partially reduced by P2X7R blockade.

Abstract

Chronic alcohol exposure disrupts blood–brain barrier (BBB) integrity and promotes neuroinflammation, with P2X7 receptor (P2X7R) signaling playing a critical role. Our prior work in male mice linked P2X7R inhibition to reduced extracellular adenosine triphosphate (eATP) release, modulated extracellular vesicle (EV) cargo, and attenuated neuroinflammation in chronic intermittent ethanol (CIE)-exposed mice. However, sex-specific roles of P2X7R signaling and EV-mediated mechanisms in alcohol-induced neuroinflammation remain unclear. Male and female mice were exposed to ethanol vapor for three weeks and treated with Brilliant Blue G (BBG), a P2X7R inhibitor. Compared to their respective CIE-unexposed controls, brain gene expression of tumor necrosis factor–α (Tnf-α), interleukin-1 beta (Il-1b), interleukin-6 (Il-6), monocyte chemoattractant protein-1 (Mcp-1), and Fas ligand (Fasl) significantly increased in CIE-exposed males, while only Il-1b increased in females. P2X7R inhibition significantly reduced these cytokines. Pericyte immunostaining was decreased by CIE (indicating BBB injury) in male mice only and was restored by P2X7R inhibition with no difference between groups in females. Occludin staining (another BBB marker) did not differ between the treatment groups in male and female animals. Circulating cytokines (Macrophage inflammatory protein-1 alpha (MIP-1α), tumor necrosis factor–α (TNF-α), interleukin-1 beta (IL-1β), and interleukin-27 subunit p28/interleukin-30 (IL-27p28/IL-30) were significantly elevated in CIE-exposed males but not in females, with BBG treatment reducing cytokines in males. Circulating eATP, P2X7Rs, P-glycoprotein (P-gp), EVs, and EV-mtDNA, which we identified in our previous study, were increased in both sexes and partially decreased by P2X7R blockade. Spatial memory was impaired by CIE exposure in males but not females, and this deficit was reversed by BBG treatment. Our findings reveal sex differences in CIE-induced circulating cytokines, neuroinflammation, and memory impairment, with a stronger response in males. However, other markers of cell injury associated with CIE exposure were upregulated in both sexes; P2X7R inhibition effectively mitigated these effects, highlighting the functional relevance of targeting the P2X7R in alcohol-induced injury.

Figures

CIE increases serum levels of the P2X7R and P-gp. Serum P2X7R levels were higher in CIE-exposed (A) male and (B) female mice compared to their respective air control groups, and BBG treatment reduced these levels in CIE-exposed mice. CIE exposure elevated P-gp in males (C) and females (D) as compared with air controls. Statistical significance is indicated as for male mice (*p* = 0.0067) and * (*p* = 0.0361) and for female mice (*p* < 0.0001). Data represent the mean ± SEM from *n* = 3–5 mice per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test.

P2X7R inhibition reduces the CIE-induced increase in EV numbers and EV size in male and female mice. EV numbers were significantly increased in CIE male (A) and female mice (B) and were lowered in respective BBG-treated CIE-exposed mice. EV size was significantly increased by CIE and reduced in BBG-treated CIE-exposed (C) male and (D) female mice as compared to their respective CIE-exposed controls. Data are presented as the mean ± SEM (*n* = 4–7). One-way ANOVA followed by Tukey’s post hoc test was used to perform statistical analysis; * *p* ≤ 0.05, *p* ≤ 0.005 compared with the respective CI

Full text

1. Introduction

Chronic alcohol consumption remains a global health concern, contributing significantly to morbidity and mortality through its impact on multiple organs, particularly the central nervous system (CNS). Epidemiological data from the CDC report a 29% rise in alcohol-related deaths during the COVID-19 pandemic [1,2], while the WHO attributes over 3 million annual deaths to alcohol use globally [3]. Among its diverse pathological effects, alcohol-induced neurotoxicity is strongly associated with neuroinflammation and disruption of the blood–brain barrier (BBB), leading to cognitive impairment, mood disorders, and increased vulnerability to neurodegenerative diseases [4,5,6].

One of the key mechanisms underlying alcohol-induced brain injury is the compromise of BBB integrity, characterized by increased permeability and the downregulation of tight junction proteins [4,7]. BBB damage permits the infiltration of peripheral immune cells and the release of proinflammatory cytokines, creating a vicious cycle of neuroinflammation and neuronal damage [8,9,10]. The purinergic signaling pathway, particularly the P2X7 receptor (P2X7R), plays a central role in this process [11,12]. The P2X7R, an ATP-gated ion channel, gets activated in response to high extracellular adenosine triphosphate (eATP), a well-established danger-associated molecular pattern (DAMP) [13]. Activation of the P2X7R promotes calcium influx, inflammasome activation, and the release of inflammatory mediators, ultimately exacerbating BBB damage and CNS pathology [14,15,16].

Recent studies have highlighted the critical role of the P2X7R in regulating the release of extracellular vesicles (EVs), which serve as potent activators of intercellular communication during stress and injury [17,18,19]. These EVs can carry inflammatory cargo such as eATP, mitochondrial DNA (mtDNA), and cytokines, and have been implicated in propagating inflammation across the BBB [20,21]. The interplay between P2X7R signaling and EV dynamics represents a critical underexplored axis in alcohol-induced neuroinflammation. Further, the modulatory role of biological sex within this pathway remains largely uninvestigated.

Sex differences in alcohol-related brain injury are increasingly recognized [22], with evidence suggesting that females metabolize alcohol less efficiently and exhibit heightened sensitivity to oxidative stress [23,24]. At the same time, males often show stronger innate immune responses [25,26]. Hormonal influences [27,28], mitochondrial resilience [29,30], and immune cell profiles [25,26] differ between sexes and likely contribute to differential neuroinflammatory outcomes. Despite its potential significance, the influence of sex on P2X7R activation and EV dynamics in alcohol-induced neuroinflammation remains largely unexplored—a gap this study aims to address.

In this study, we investigated sex-specific differences in P2X7R-driven neuroinflammatory responses and EV release in the context of chronic intermittent ethanol (CIE) exposure. By characterizing inflammatory markers, BBB disruption, and EV cargo profiles in male and female mice, we aim to elucidate novel sex-dependent mechanisms underlying alcohol-induced brain injury. Our findings may point to the development of targeted therapeutic strategies that account for sex as a biological variable in neuroinflammatory disorders.

2. Results

2.1. CIE Exposure Resulted in Comparable BEC Levels in Males and Females

Mice were exposed to ethanol vapors for 16 h per day, 4 days per week, throughout the experimental period to achieve and maintain pathophysiologically relevant blood ethanol concentrations (BECs). We observed that BEC levels of about 150–200 mg/dL were reached after 3 weeks of CIE exposure in both males and females (Figure 1). P2X7R blockade using BBG did not alter the blood ethanol concentration in both sexes.

2.2. Differences in Cytokine Gene Expression in the Brains of CIE-Exposed Male and Female Mice

Previous studies have shown that ethanol exposure activates neuroimmune signaling and increases proinflammatory cytokines [31,32,33,34,35]. Building on these findings, we investigated whether CIE induces similar neuroinflammatory responses in male and female mice. qPCR analysis of total brain tissue revealed sex-dependent changes in proinflammatory cytokines (Figure 2A,B). In males, CIE exposure significantly increased the expression of tumor necrosis factor–α (Tnf-α), interleukin-1 beta (Il-1b) monocyte chemoattractant protein-1 (Mcp-1), interleukin-6 (Il-6), and Fas ligand (Fasl) as compared to air control mice. BBG treatment in these mice reduced the gene expression of these cytokines. Females, however, exhibited a selective increase in Il-1b expression only following CIE exposure. This effect was significantly reduced by BBG treatment.

2.3. CIE Exposure Decreases Pericyte Coverage in Male Mice, Which Was Preserved upon P2X7R Inhibition

Pericyte loss and increased permeability due to disrupted tight junctions are early hallmarks of BBB damage. To determine the extent of BBB damage following CIE exposure, pericyte coverage and tight junction expression were determined using the intensity of CD13 and occludin immunostaining, respectively. Image analysis of immunohistochemical stains revealed a significant decrease in pericyte coverage in male mice exposed to CIE as compared to the air-exposed control group (Figure 3A,C). Inhibition of P2X7R activity using BBG preserved pericyte coverage in CIE-exposed male mice. In contrast, female mice did not show reductions in pericyte coverage following ethanol exposure (Figure 3B,D). Moreover, we did not observe any changes in occludin expression across the treatment groups in either male or female mice (Supplementary Figure S1).

2.4. Sex-Specific Modulation of Serum Cytokines by CIE Exposure

Clinical and preclinical studies have demonstrated elevated levels of circulating proinflammatory cytokines, such as tumor necrosis factor–α (TNF-α), interleukin-1 beta (IL-1β), and monocyte chemoattractant protein-1 (MCP-1), in alcohol-induced inflammation [8,36,37,38]. In the current study, we measured the levels of serum cytokines using multiplex MSD ELISA. CIE-exposed male mice showed increased levels of TNF-α, IL-1β, interleukin-27 subunit p28/interleukin-30 (IL-27p28/IL-30), and macrophage inflammatory protein-1 alpha (MIP-1) as compared to air-exposed control mice, and BBG treatment significantly reduced cytokine levels. While CIE exposure increased keratinocyte-derived chemokine (KC/GRO) and interferon-γ–induced protein 10 (IP-10) levels, P2X7R inhibition did not cause statistically significant reductions. In females, CIE exposure did not elevate any cytokine levels significantly, and BBG had no effect (Figure 4A,B). These results correlate with the whole-brain gene expression pattern in males and females for TNF-α and IL-1β. The effect of alcohol on TNF-α may not have been profound in females, possibly due to the already increased levels at baseline (TNF-α 19.96 ± 2.92 pg/mL in males vs. 52.26 ± 13.97 pg/mL in females). However, P2X7R blockade reduced IL-1β levels after CIE exposure in males and not females, given their comparable baseline values.

2.5. No Sex Difference in CIE-Induced Serum P2X7R Levels

Given the P2X7R’s central role in driving neuroinflammation through NLRP3 inflammasome activation and proinflammatory cytokine release, the P2X7R is a critical target for understanding and potentially mitigating alcohol-induced neuroinflammatory responses [39,40,41]. Our previous work demonstrated that CIE exposure in male mice elevated circulating P2X7R levels, reinforcing its contribution to alcohol-induced neuroinflammation. In the present study, we investigated whether similar P2X7R-mediated mechanisms occur and how P2X7R inhibition modulates these responses. We observed a trend toward increased serum P2X7R shedding levels in both male and female mice exposed to CIE compared to their respective air controls. Treatment with the selective P2X7R inhibitor BBG appeared to reduce these levels in CIE-exposed mice (Figure 5A,B); however, the differences among groups were not statistically significant.

2.6. P-Glycoprotein Levels Are Similarly Elevated in Male and Female CIE-Exposed Mice

To evaluate the impact of chronic ethanol exposure on BBB function, we measured serum P-gp levels [42]. Consistent with prior findings showing CIE-induced elevation of circulating P-glycoprotein (P-gp) in male mice [21], CIE exposure increased P-gp levels in both male and female mice compared to their respective air-controls (Figure 5C,D). P2X7R inhibition diminished P-gp levels in blood, but it was statistically significant only in females. These results suggest that CIE exposure caused BBB injury and increases in blood P-gp levels irrespective of sex. Figure 5CIE increases serum levels of the P2X7R and P-gp. Serum P2X7R levels were higher in CIE-exposed (A) male and (B) female mice compared to their respective air control groups, and BBG treatment reduced these levels in CIE-exposed mice. CIE exposure elevated P-gp in males (C) and females (D) as compared with air controls. Statistical significance is indicated as ** for male mice (p = 0.0067) and * (p = 0.0361) and **** for female mice (p < 0.0001). Data represent the mean ± SEM from n = 3–5 mice per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test.

2.7. Increased Release of Serum ATP (eATP) Is Similar in Male and Female CIE-Exposed Mice

The P2X7R plays a pivotal role in regulating eATP levels [43], thereby linking ATP signaling to neuroinflammation [44,45] and CNS pathologies [46]. To explore this, we assessed serum ATP levels and found that CIE exposure increased ATP concentrations in both sexes. There was a trend in the reduction of ATP levels in CIE-exposed male and female mice after BBG treatment, which did not reach statistical significance (Figure 6A,B).

2.8. No Sex Bias in CIE-Induced EV Release and P2X7R Inhibition Effect

P2X7R activation promotes EV release in inflammatory and pathological conditions. In a previous study using male mice, we demonstrated that ethanol exposure increases [19,20,47] EV numbers, suggesting alterations in vesicle biogenesis [21]. Here, we examined the effects of CIE exposure and P2X7R inhibition on EV release and assessed sex differences in EV numbers between male and female mice. CIE exposure increased circulatory EVs in both sexes. EV counts were reduced in BBG-treated CIE-exposed male and female mice when compared to their respective CIE-exposed controls (Figure 7A,B).

Next, we evaluated whether CIE exposure influences EV size and whether P2X7R inhibition modulates these changes in both sexes. CIE exposure increased EV size in male and female mice. BBG-dependent P2X7R inhibition markedly decreased EV size in both BBG-treated CIE-exposed male and female mice as compared to respective CIE-exposed controls (Figure 7C,D). Figure 7P2X7R inhibition reduces the CIE-induced increase in EV numbers and EV size in male and female mice. EV numbers were significantly increased in CIE male (A) and female mice (B) and were lowered in respective BBG-treated CIE-exposed mice. EV size was significantly increased by CIE and reduced in BBG-treated CIE-exposed (C) male and (D) female mice as compared to their respective CIE-exposed controls. Data are presented as the mean ± SEM (n = 4–7). One-way ANOVA followed by Tukey’s post hoc test was used to perform statistical analysis; * p ≤ 0.05, ** p ≤ 0.005 compared with the respective CIE-exposed control mice.

2.9. CIE Exposure Induces Similar EV mtDNA Signatures in Male and Female Mice and Similar Changes After P2X7R Blockade

Studies from our lab and others have shown that EVs isolated from ethanol-exposed cells contain increased levels of mtDNA [20,21], which act as DAMPs and lead to the activation of autocrine and paracrine signaling [48]. We employed digital PCR to quantify the copy numbers of three mitochondrial genes: mt-Nd2, mt-Atp8, and mt-Cox2. CIE exposure increased the levels of all three mtDNA genes in EVs from CIE-exposed groups compared to air-exposed controls. The baseline copy numbers for mt-Nd2 and mt-Atp8 were much higher in male mice compared to female mice. These baseline sex differences may reflect higher mitochondrial density or metabolic activity in males, potentially influencing EV biogenesis, although this was not directly tested in the present study. While baseline levels of mt-Cox2 were similar in male and female mice, the increase in CIE expression was approximately 5-fold higher in males than in females. Notably, P2X7R inhibition with BBG effectively counteracted this increase, substantially reducing mtDNA levels (Figure 8A,B).

2.10. Sex Bias in the Spatial Memory Performance of CIE-Exposed Mice

Spatial memory performance after CIE exposure in male and female mice was assessed using the object placement test (OPT). Significant group differences were observed in the discrimination index (DI) and exploration ratio (ER) (Kruskal–Wallis test, p < 0.05). While control males and females showed no difference, CIE exposure significantly impaired ER in males (p < 0.05), but not in females (Figure 9A,B). Similar effects were observed for the DI (Figure 9C,D). Notably, P2X7R inhibition with BBG restored the ER and DI in CIE-exposed male mice (p < 0.05 vs. untreated CIE males). CIE did not affect cognition in females, and BBG treatment showed no significant effect in females. This indicates a sex-specific effect of P2X7R signaling on spatial memory.

3. Discussion

The results of the current study reveal differences in the inflammatory responses to CIE exposure in male and female mice and the importance of the P2X7R in the regulation of these effects. Although male and female mice had similar BEC levels after CIE exposure, their brain inflammatory profiles diverged considerably. These results reinforce the notion that sex is a critical biological variable in neuroimmune research and suggest that P2X7R-targeted therapies may have different effects in males versus females.

We observed significant upregulation of Il-1b, Tnf-α, Il-6, Ccl2 (MCP-1), and Fasl transcripts in the whole-brain tissue of male mice after CIE exposure, indicating a strong proinflammatory and pro-apoptotic transcriptional response. CIE exposure induces a transcriptional program in the brain consistent with widespread neuroinflammatory activation. Multiple in vivo studies, including ours, have reported ethanol-dependent upregulation of Il-1b, Tnf-α, and Ccl2/Mcp1 in isolated brain microvessels and cortical, hippocampal, and cerebellar regions [9,21,37,49,50,51]. Parallel increases in Il-6 expression have been documented in chronic ethanol vapor and liquid diet models [36,52,53]. Similarly, an increase in Fasl transcription after ethanol exposure has been linked to inflammatory cytokine induction and apoptotic mechanisms that underlie ethanol-associated neuronal loss and degeneration [54,55]. Our findings align with those of Niedzwiedz-Massey et al. [56], who reported that chronic plus binge ethanol exposure increases Il-1b, Tnf-α, and Ccl2 expression in the hippocampus and cerebellum of adult mice. Interestingly, we observed an increase only in Il-1b levels in females, underscoring the sex-specific nature of ethanol-induced neuroinflammation. Similar sex-specific responses have been reported previously; for example, Anton et al. [33] found that intermittent binge ethanol exposure in aged female mice significantly elevated hippocampal Il-1b, Tnf-α, and Ccl2 expression. In contrast, despite prior reports suggesting heightened ethanol neurotoxicity in females, our results indicate a relatively modest neuroinflammatory response in CIE-exposed females, characterized by an isolated elevation in Il-1b. Several reports demonstrate sex-dependent neuroimmune transcriptional differences and highlight the IL-1β pathway as being particularly sensitive in females [22,26,57,58]. This suggests that sex-specific outcomes may reflect differences in hormonal status, immune priming, glial reactivity, and regional vulnerability to injury. P2X7R inhibition attenuated CIE-induced Il-1b gene upregulation in female mice, mirroring the results in male mice, underscoring the P2X7R’s role, likely via NLRP3 inflammasome activation, in mediating ethanol’s effects [59,60].

Hormonal and immune regulation likely underlie the different responses exhibited in males and females. For example, estrogens can modulate immune function, suppressing proinflammatory cytokine production and enhancing glucocorticoid anti-inflammatory effects [25]. Numerous studies have demonstrated that alcohol-induced neuroinflammation is characterized by the increased expression of proinflammatory cytokines, such as TNF-α, IL-1β, and MCP-1, primarily driven by the activation of innate immune receptors [49,61,62]. In our study, CIE exposure markedly elevated blood cytokines (TNF-α, IL-1β, KC/GRO, IP-10, IL-27p28/IL-30, and MIP-1) in male mice, and BBG treatment significantly reduced these levels, reinforcing the P2X7R’s role in peripheral inflammation. These results align with previous studies implicating P2X7R in amplifying inflammatory responses in both the CNS and peripheral tissues during alcohol exposure [21,63,64,65]. Interestingly, a sex-specific pattern emerged where female mice showed no significant serum cytokine changes with CIE exposure or BBG treatment, in contrast to the robust inflammatory response observed in male mice. This observation is consistent with earlier reports indicating attenuated or delayed immune responses in females following ethanol exposure, likely due to differences in sex hormones [25,66], immune signaling pathways [25,67], and ethanol metabolism [25]. Females metabolize alcohol faster than males, which may contribute to their blunted peripheral inflammatory profile [66,67].

Sex differences have also been found in the pericyte coverage of brain microvessels, with significant diminution of CD13 in CIE males but no difference in females. Pericytes play an important role in supporting the functions of the brain endothelium, and their loss coincides with a compromised BBB, enhanced neuroinflammation, and cognitive decline [68]. P2X7R inhibition normalized CD13 expression in CIE-exposed male animals, highlighting the important contribution of receptor activation to pericyte changes in alcohol exposure, similar to the pattern of cytokine gene expression in the brain. Importantly, image analysis of occludin immunostaining performed in the same brain samples/areas demonstrated no changes in all treatment groups, regardless of sex. Occludin, which connects brain endothelial cells, assures BBB tightness and has been shown to be downregulated after alcohol exposure in human brain endothelial cells in vitro [69]. One potential explanation could be that CIE exposure led to the phosphorylation/modification of occludin without changing its content [69], or inherent differences between in vivo and in vitro experiments.

Changes in brain cytokines paralleled the systemic inflammatory profile, where serum cytokine levels were significantly increased in ethanol-exposed males but remained relatively unchanged in females. This supports the “two-hit” hypothesis of ethanol-induced neuroimmune damage proposed by Crews and colleagues, in which peripheral cytokines first compromise BBB integrity, facilitating secondary neuroinflammatory insults [70,71,72]. BBG treatment reversed the elevation of peripheral cytokines in males, further strengthening the mechanistic link between P2X7R signaling and systemic inflammation [73]. This receptor, predominantly expressed on immune and endothelial cells, is activated by high eATP and is known to promote cytokine release, immune cell activation, and vascular damage [14].

Quantitative analysis revealed a significant elevation in circulating ATP levels following ethanol exposure in both male and female mice, an effect that was partially, albeit non-significantly, attenuated by P2X7R inhibition. The lack of statistical significance may reflect cohort-specific variability, a modest effect size, or limited statistical power rather than the absence of a biological effect. Consistent with previous reports by Di Virgilio et al., P2X7R-mediated ATP release is recognized as a pivotal event in the initiation and amplification of inflammatory signaling cascades within both the CNS and peripheral compartments [74]. eATP functions as a key DAMP that activates the P2X7R, subsequently triggering the NLRP3 inflammasome-mediated release of IL-1β and IL-18 [14,59]. The observed elevation in ATP likely both contributed to and resulted from inflammatory processes, establishing a self-perpetuating feed-forward loop of P2X7R activation [21,39].

Consistent with our previous findings in the male cohort [21], CIE exposure significantly increased circulating P-gp levels compared to air controls in both sexes. BBG treatment reduced P-gp levels, but statistical significance was observed only in females in this cohort. These data suggest that BBG mitigates CIE-induced brain endothelial injury irrespective of sex, though larger, repeated, and time-course studies are needed to determine if the sex-specific significance reflects true biology or cohort variability. P-gp (ABCB1), a key efflux transporter at the BBB, is transcriptionally regulated by inflammatory cytokines, such as TNF-α and endothelin-1 [75,76], and its expression often parallels systemic or CNS inflammation. Elevated eATP levels observed in our study may contribute to this upregulation, since purinergic receptor activation, particularly through the P2X7R, can amplify the cytokine release and signaling cascades that modulate P-gp expression [77]. Previous reports also indicate that chronic ethanol and other drugs of abuse alter ABC transporter expression and function at the BBB [78]. Taken together, these results bridge our eATP data with subsequent findings on P2X7R activity, suggesting that P-gp elevation may represent an adaptive BBB response secondary to P2X7R-mediated neuroinflammatory signaling rather than a sex-dependent process.

We observed systemic markers consistent with P2X7R-mediated neuroinflammatory mechanisms. Serum P2X7R levels showed a trend toward elevation in both male and female mice following CIE exposure, reflecting the ethanol-induced upregulation of P2X7R observed in microglia in prior studies [79,80]. Notably, treatment with the P2X7R antagonist resulted in a reduction of serum P2X7R levels in CIE-exposed mice. This observation, albeit modest, supports the therapeutic potential of P2X7R inhibition in mitigating alcohol-related neuroimmune activation. We and others showed in preclinical studies that P2X7R antagonists attenuate ethanol-induced neuroinflammatory markers and cognitive deficits [21,81,82]. The trend toward reduction, even without statistical significance, suggests that circulating P2X7R—likely shed from immune, endothelial, or glial cells—could serve as a surrogate biomarker for neuroimmune activation following ethanol exposure. Non-significant changes may reflect transient shedding [39], cohort variability, or subtle sex-dependent differences in P2X7R signaling [83,84,85]. Although serum P2X7R levels did not differ significantly between sexes, the existing literature suggests that subtle differences in P2X7R signaling could still have functional implications for neuroimmune outcomes. Several studies have shown that microglial P2 receptor expression, including that of the P2X7R, exhibits pronounced sex- and age-dependent variation, with male microglia displaying a more reactive, proinflammatory transcriptional and proteomic profile, whereas female microglia tend to adopt reparative or neuroprotective states [28,85,86]. These intrinsic differences in purinergic responsiveness suggest that equivalent circulating P2X7R levels may yield distinct downstream effects in males and females. Moreover, estrogen signaling can attenuate P2X7R-mediated inflammatory outputs in CNS cells, which may help explain the reduced functional consequences of P2X7R activation in females [87]. P2X7R activation induces membrane blebbing and EV formation through the acid sphingomyelinase, p38 MAPK/ROCK, and inflammasome-linked pathways, and has been directly implicated in the export of mitochondrial material in EVs—mechanisms that connect receptor activation to EV abundance and mtDNA cargo [88,89].

In the context of CIE exposure, in vivo studies by our group and others have shown increased eATP, EVs, and EV-mtDNA, whereas pharmacologic or genetic inhibition of the P2X7R markedly reduces these responses [21,89,90]. Consistent with these reports, we found increased EV numbers and sizes in both sexes, and BBG reversed these effects with no sex bias. EVs carry bioactive cargo and mediate immune communication [91,92,93,94]. Alcohol induces EV release from liver and immune cells, propagating inflammatory signals to distant tissues, including the brain [20,95,96,97,98,99]. Notably, the CIE-induced shift toward larger EVs indicates cellular activation or injury [100,101]. EVs isolated from CIE-exposed animals likely reflect ethanol-induced mitochondrial damage coupled with the active release of mtDNA-containing vesicles [102,103,104]. mt-Nd2, mt-Atp8, and mt-Cox2 encode core subunits of respiratory chain complexes I, V, and IV, respectively, and these loci (and other mitochondrial targets) are commonly used as gene-specific probes to quantify EV-associated mtDNA [105,106]. EV-encapsulated mtDNA is not an inert cargo: it can be transferred to recipient cells and engage innate nucleic acid sensors such as TLR9 and the cGAS–STING axis, thereby promoting inflammatory cytokine production [103,104,107]. Alcohol exposure models (including chronic-plus-binge paradigms) have demonstrated the increased production of mtDNA-enriched EVs that potentiate neutrophilia and proinflammatory signaling, supporting the designation of EV-mtDNA as a biologically active DAMP cargo in ethanol injury [102,108]. Defects in mitochondrial function and stress-activated kinase pathways have been implicated in mtDNA release and subsequent packaging into EVs, providing a plausible connection between ethanol-induced mitochondrial dysfunction and altered EV biogenesis [103,107]. The P2X7R plays a role in regulating EV release via cytoskeletal and membrane effects [109,110], explaining BBG’s EV-normalizing effect.

An important finding was the increase in mtDNA cargo within EVs (EV-mtDNA) after ethanol exposure in both sexes, which was markedly blunted by P2X7R inhibition. Elevated EV-associated mtDNA has been linked to autoimmune and neurodegenerative diseases [111], yet its role in alcohol-induced neuroinflammation remains underexplored [112]. In our study, EV-mtDNA levels increased along with ATP and P-gp levels and EV numbers in both sexes, suggesting that EV-mtDNA may serve as a sex-independent biomarker of ethanol-induced inflammation. The suppression of EV-mtDNA by BBG highlights the P2X7R’s role in dictating EV cargo. The P2X7R not only governs EV release but also indirectly enhances their inflammatory potential by promoting conditions that favor mitochondrial stress and mtDNA incorporation into vesicles. This process likely occurs secondary to P2X7R-induced ionic flux, ROS generation, and inflammasome activation, which together destabilize mitochondria and facilitate mtDNA leakage into the cytosol and EVs [19,89,113,114]. Prior reports, including our recent study, suggest that P2X7R-dependent EV-mtDNA release may act as an intermediary between systemic mitochondrial dysfunction and CNS immune activation during CIE exposure [21,102,114,115], although the direct contribution of this pathway is yet to be fully elucidated.

Baseline sex-related differences in mtDNA copy number may reflect differences in mitochondrial content per cell, inherent metabolic differences, or variation in mtDNA packaging into EVs. However, the present data do not distinguish between these possibilities. Future studies incorporating direct measures of mitochondrial mass [116] and citrate synthase activity [117], as well as a detailed analysis of EV cargo composition, will be required to clarify these mechanisms.

Sex differences in the neuroimmune response to CIE exposure have emerged as critical determinants of cognitive outcomes, with mounting evidence implicating inflammasome activation and neuroinflammatory signaling pathways in mediating these effects [26,118,119]. Our results indicate that CIE exposure impairs spatial memory in males, which was effectively restored by BBG treatment. Females did not exhibit any impairment in special memory after CIE exposure. These behavioral patterns are consistent with previous molecular evidence demonstrating sex-dependent differences in neuroinflammatory and neurotoxic responses to ethanol exposure [118]. CIE produced a male-specific deficit in object placement discrimination that co-occurred with a broad proinflammatory/pro-apoptotic whole-brain transcriptional profile (↑ Tnf-α, ↑ Mcp-1/Ccl2, ↑ Il-6, ↑ Il-1b, and ↑ Fasl), whereas females showed only a modest Il-1b increase and preserved spatial memory. An isolated Il-1b rise alone does not inevitably produce cognitive impairment; rather, male vulnerability appears to reflect escalation to a multi-node inflammatory and death pathway program that is more likely to disrupt hippocampal circuits supporting spatial memory [120,121,122]. Several male-specific mediators plausibly drive cognitive impairment. Dysregulated TNF-α and IL-1β are known to suppress hippocampal long-term potentiation and impair memory [123,124], while sustained MCP-1/CCL2 recruits and activates microglia to sustain inflammation and worsen cognition [125,126]. Concurrent FASL upregulation implicates death receptor signaling that can promote neuronal apoptosis or maladaptive synaptic pruning, compounding cytokine-mediated deficits and helping explain persistent DI impairment in males [28,54,55].

The rescue of male behavior deficits by P2X7R antagonism supports an upstream ATP → P2X7 → inflammasome axis as an amplifier of male neuroinflammation. P2X7R activation drives IL-1β release and downstream cytokine cascades; BBG reduces IL-1β/TNF transcription and improves cognition in multiple preclinical paradigms, consistent with our molecular and behavioral normalization in males [114,127].

Our data indicate that P2X7R inhibition is most effective when ethanol exposure triggers an inflammatory response (as in males). Key limitations of our study include the whole-brain transcriptomic readout (no cell-type or sub-regional resolution) and lack of protein/time-course or causal manipulations. Overall, CIE exposure elicits a male-predominant, multi-node inflammatory/apoptotic program that coincides with spatial memory loss, while females show a restricted Il-1b response and behavioral resilience; P2X7R inhibition reverses molecular and behavioral pathology in males, highlighting the P2X7R–inflammasome axis as a sex-dependent therapeutic hub.

Although our findings demonstrate that P2X7R drives sex-specific neuroinflammation with increased proinflammatory transcript and circulatory cytokine levels and cognitive impairment following CIE exposure, several limitations should be acknowledged. While our study focused on the effects of CIE and the P2X7R, we were unable to explore the molecular mediators linking sex hormones with P2X7R signaling. Previous work suggests that estrogen and testosterone distinctly modulate immune signaling and ethanol metabolism, potentially contributing to the observed sex-dependent inflammatory responses [25,28]. Despite comparable BECs between sexes, females are known to metabolize alcohol more rapidly, which could explain their relatively blunted cytokine activation profile [66,67]. Our gene expression analyses were performed on whole-brain tissue, which may mask region- and cell-type-specific responses. Moreover, while this study identified key associations between mitochondrial EVs, mtDNA, and P2X7R signaling, causal relationships remain to be established.

A limitation of this study is that the estrous stage was not monitored in female mice, and fluctuations in estrogen, FSH, and LH can influence inflammatory signaling. Consequently, variability in cycle stage may have increased within-group variability and potentially masked phase-specific responses. Future studies will be helpful to explore how sex hormones modulate the P2X7R and downstream signaling cascades in brain cell types, including microglia, astrocytes, and endothelial cells. High-resolution single-cell or spatial transcriptomics could clarify cell-specific inflammatory signatures, while functional assays using P2X7R-deficient or hormone receptor knockout models could help uncover mechanistic crosstalk. Further, defining the bioactive cargo of EVs, particularly mtDNA, may clarify how peripheral and central inflammation intersect during alcohol exposure [10,102,128]. Lastly, given the promising protective effects of P2X7R inhibition, therapeutic targeting of purinergic signaling or EV-mediated mitochondrial pathways could offer novel sex-specific strategies to prevent or treat alcohol-related neuroinflammation [114].

In summary, our findings support a model in which ethanol-induced ATP release and P2X7R activation drive a systemic inflammatory cascade (cytokines and EVs with mtDNA) that damages the BBB and triggers neuroinflammation. Consistent with the “two-hit” hypothesis, peripheral cytokines (from P2X7R signaling) can prime the brain for injury. In male mice, CIE induced a pronounced increase in systemic ATP and soluble P2X7Rs, which could further activate P2X7Rs and the inflammasome, thereby creating a feed-forward loop of inflammation. Concurrently, CIE increased EV number, size, and mtDNA content in circulation, which were all reversed by BBG, indicating that the P2X7R drives these peripheral signals. Together, elevated cytokines, EVs, and ATP in males likely contribute to signaling processes associated with neuroinflammation. In contrast, females showed similar upstream signals but exhibited a much weaker downstream inflammatory response, which may reflect hormonal modulation of purinergic and inflammasome pathways. This sex-specific immune trajectory may explain why males suffer greater ethanol-induced neural injury and cognitive decline while females remain relatively protected. Our results thus highlight the P2X7R as a key mediator of ethanol-induced neuroinflammation and suggest that P2X7R antagonists may offer neuroprotection, particularly in males (Figure 10). Future studies should explore combined or alternative strategies, such as the use of hormone modulators, to optimize therapeutic efficacy across sexes in alcohol-related neuroimmune injury.

In males (top panel), CIE exposure increases P2X7R activation and eATP signaling, resulting in mitochondrial dysfunction and the release of mtDNA-containing EVs. These changes are associated with the elevated expression of tumor necrosis factor–α (Tnf-α), interleukin-1 beta (Il-1b), interleukin-6 (Il-6), monocyte chemoattractant protein-1 (Mcp-1), and Fas ligand (Fasl), as well as BBB impairment and cognitive deficits. P2X7R inactivation significantly attenuated these molecular and functional changes in males.

In females (bottom panel), although gene expression and cognitive outcomes were largely unchanged, CIE exposure altered EV number, mtDNA content, and EV size, indicating subtle effects on extracellular vesicle dynamics.

Symbols denote P2X7R activation/inactivation, ATP/eATP, mitochondrial DNA, extracellular vesicles, and inflammatory gene expression. The dashed line indicates the observed sex difference in downstream molecular and functional outcomes (Figure created using Biorender.com).

4. Materials and Methods

4.1. Animals and Experimental Groups

C57BL/6 wild-type male and female mice were obtained from Jackson Laboratories. Mice were grouped into eight experimental groups: four for each sex (male and female). Animals were further selected based on an average body weight of 30–35 gm to ensure comparable baseline characteristics across groups. Each sex included four groups: air control, BBG-treated CIE-unexposed (BBG), CIE-exposed (CIE), and BBG-treated CIE-exposed (BBG-CIE). Group sizes ranged from 5 to 15 mice to ensure statistical power: air control (n = 5), BBG (n = 5), CIE (n = 10), and BBG-CIE (n = 10) per sex.

All mice were housed in groups of five per cage in an uncrowded, quiet animal facility room on a 12-h light/dark cycle, with free access to lab chow and water. All in vivo procedures were conducted per the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and the Animal Research: Reporting in Vivo Experiments (ARRIVE) guidelines (www.nc3rs.org.uk/arrive-guidelines; accessed on 14 April 2025). Experimental protocols were approved by the Temple University Institutional Animal Care and Use Committee (IACUC).

4.2. CIE Exposure

A CIE exposure protocol was employed to induce alcohol-related neuroinflammation in mice based on previously established methods from our prior published work [21]. Mice assigned to the CIE and BBG-CIE groups were exposed to ethanol vapor for 16 h daily, followed by 8 h in ambient air four days per week and three weeks [21]. Mice in the BBG and BBG-CIE groups were administered BBG (45 mg/kg in 100 µL of 0.9% saline; Abcam, ab120389, Cambridge, UK) intraperitoneally prior to ethanol exposure, following protocols established in earlier studies [21,129]. BBG was chosen for its well-established in vivo use as a P2X7R antagonist and its documented neuroprotective effects in multiple neuroinflammatory models, making it suitable for this translational study [130,131,132].

4.3. BEC Determination

BECs were assessed at the end of the exposure protocol to confirm systemic ethanol levels [21]. Blood was collected via submandibular vein puncture into tubes containing 0.5 M EDTA (pH 8) immediately after the mice were removed from the ethanol vapor chamber. The isolated plasma was analyzed using a colorimetric enzymatic assay (ECET-100™ Ethanol Assay Kit; BioAssay Systems, San Francisco, CA, USA), following the manufacturer’s instructions.

4.4. qPCR Assay

Total RNA was extracted from whole brain tissue using the Trizol extraction method (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. RNA purity and concentration were assessed using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). DNA was eliminated from the total nucleic acid preparation using the DNA-free™ DNA Removal Kit (Catalog number AM1906, Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s protocol.

cDNA was synthesized from isolated RNA using a High-Capacity cDNA Reverse Transcription Kit (Catalog number: 4368814; Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. Reverse transcription cycling parameters were as follows: Step 1—25 °C for 10 min to allow primer annealing; Step 2—37 °C for 120 min for cDNA synthesis; Step 3—85 °C for 5 min to inactivate reverse transcriptase; Step 4—4 °C hold until retrieval.

For qPCR analysis, commercially available TaqMan probe-based assays were used (Table 1), and the corresponding assay IDs are provided below.

qPCR was performed using the QuantStudio™ 3 System (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Cycling conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 60 s, with data acquisition at 60 °C.

4.5. Immunohistochemistry and Image Analysis

Frozen tissue sections (10 µm) from the right cerebral hemisphere were cut using a cryostat (Leica CM1850, Wetzlar, Germany). Sections were fixed in methanol/acetone (50:50, v/v), followed by permeabilization with 0.05% Triton X-100 for 20 min at room temperature. After permeabilization, sections were washed with 1× TBS. Non-specific antibody binding was blocked using 5% BSA in PBS prior to primary antibody incubation. Tissue sections were incubated with primary antibodies overnight at 4 °C in a humidified chamber. After primary antibody incubation, sections were washed with 1× TBS and incubated with appropriate secondary antibodies for immunofluorescence visualization. Double immunostaining was performed using antibodies against the pericyte marker CD13 (goat polyclonal, 1:50; R&D Systems, #AF2335, Minneapolis, MN, USA) and the tight junction protein occludin (rabbit polyclonal, 1:200; Novus Biologicals, #NBP1-87402, Centennial, CO, USA). Secondary antibodies included Alexa Fluor 488 donkey anti-goat (Invitrogen, #A11055, Carlsbad, CA, USA) and Alexa Fluor 594 donkey anti-rabbit (Invitrogen, #A21207). Fluorescent images were acquired under a 20× objective lens using an upright microscope (i80 Eclipse, Nikon, Tokyo, Japan) configured with an IRIS 9 camera (Teledyne Photometrics, Tucson, AZ, USA). From each group, 3 mice were utilized for microscopy analysis. Ten different random regions within the anterior right brain were selected based on the localized presence of both endothelial (occludin) and pericyte (CD13) markers. Fluorescent images were captured using FITC and TRITC channels corresponding to CD13 and occludin staining. Exposure time and camera gain settings were kept consistent for each channel across all analyzed tissues. Then, all images were analyzed using Nikon’s NIS-Elements software (Version 5.21.03).

To determine pericyte coverage, CD13-positive fluorescence signals associated with occludin-positive fluorescence signals were manually marked as regions of interest (ROIs) in each image. Using an automated measurement tool, the green (CD13) and red (occludin) intensities of each ROI were noted. For each mouse, the arithmetic mean intensity of CD13-positive ROIs was calculated and plotted as the pericyte coverage per mouse.

4.6. Multiplex Detection of Serum Proinflammatory Markers

Serum proinflammatory markers were quantified using the V-PLEX Mouse Cytokine 19-Plex Kit (MSD) (Cat No: K15255D; Meso Scale Discovery, Rockville, MD, USA), following the manufacturer’s instructions. Data from the V-PLEX Meso Scale assays were analyzed using standard curves within the respective assay programs using MSD Discovery Workbench software (DISCOVERY WORKBENCH version 4.0.13, Meso Scale Discovery).

4.7. EV Isolation and Nanoparticle Tracking Analysis

EVs were isolated from plasma samples using a kit-based protocol (cat. no. 4484450; Invitrogen, Carlsbad, CA, USA) [20,133]. Nanoparticle tracking analysis (NTA) of the isolated EVs was performed using the NanoSight NS300 system, equipped with a 488 nm laser (Malvern Technologies, Malvern, UK). In brief, the EV samples were diluted (1:500) in 1 mL of particle-free Milli-Q water (MilliporeSigma, Burlington, VT, USA) and introduced into the NanoSight chamber with a 1 mL BD slip-tip syringe (Cat. No. 309659, Becton Dickinson (BD), Franklin Lakes, NJ, USA). Before analysis, the system was calibrated using 100 nm latex beads (Malvern Instruments Inc., Malvern, UK, Cat. No. NTA4088). The resulting data were processed using NTA 3.3.104 software [20,21].

4.8. Quantification of Serum P2X7R Levels

P2X7R levels in serum were measured using a mouse purinergic P2X7R ELISA kit (Cat. No. E12339m, American Research Products, Waltham, MA, USA) following the manufacturer’s instructions with minor modifications, as described previously [21]. Briefly, serum samples collected at the time of organ harvest were subjected to ELISA, and absorbance was recorded at 450 nm using a SpectraMax^®^ M5 microplate reader (Molecular Devices, San Jose, CA, USA).

4.9. Serum P-gp Measurement

Serum samples were analyzed using a commercially available kit (Cat. No. MBS450526; MyBioSource, San Diego, CA, USA) to assess circulatory P-gp levels, as described previously [21]. Briefly, serum was incubated in pre-coated wells, followed by the sequential addition of detection antibodies and the substrate solution. The final colorimetric reaction was quantified at 450 nm using a SpectraMax^®^ M5 microplate reader, and P-gp concentrations were calculated against a standard curve generated from known concentrations provided with the kit.

4.10. ATP Quantification in Serum

ATP levels in serum samples were measured using the ATP Determination Kit (Cat. no. A22066, Thermo Fisher Scientific; Waltham, MA, USA) with slight modifications to the manufacturer’s protocol [134]. Briefly, serially diluted ATP standards (1 nM–1 µM) and 20 µL of serum samples were added to a Corning^®^ black transparent-bottom 96-well plate (Cat #3603, Corning Inc., Corning, NY, USA) containing a reaction mix of Tricine buffer, MgSO_4_, EDTA, DTT, D-luciferin, and luciferase. Luminescence was recorded immediately using an Infinite^®^ 200 M PRO plate reader (Tecan Austria GmbH, Grödig, Austria), and ATP concentrations were calculated from the standard curve after background subtraction.

4.11. EV-DNA Quantification and Digital PCR Analysis

The DNA attached to the EV surface was eliminated by incubating 100 µL of the EV suspension with 10 U of DNase (LGC Biosearch Technologies, Cat. No. DB0715K, Hoddesdon, UK) for 20 min at 37 °C. The reaction was stopped by adding 10 µL of 10X DNase stop solution. Afterward, the suspension was diluted with 100 µL of nuclease-free water (NFW), and EV lysis was achieved by adding 20 µL of proteinase K (Cat. no. 4485229, Thermo Fisher Scientific, Waltham, MA, USA) at room temperature. DNA was then isolated from the lysed EV suspension using the DNeasy^®^ Blood & Tissue Kit (Qiagen, Cat. no. 69506, Hilden, Germany) [20,21].

The concentration of isolated EV-DNA was measured using a NanoDrop UV–Vis ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Based on these measurements, the DNA was diluted with nuclease-free water to a working concentration of 2 ng/µL for downstream digital PCR analysis. Mitochondrial gene-specific Taqman™ probes for ATP8 [mt-Atp8] (Cat. no. 4331182 Mm04225236_g1), NADH dehydrogenase 2 [mt-Nd2] (Cat. no. 4331182 Mm04225288_s1), and cytochrome c oxidase subunit II [mt-Cox2] (Cat. no. 4331182 Mm03294838_g1) were used in this experiment (Thermo Fisher Scientific; Waltham, MA, USA) [108]. PCRs were performed using 2 µL of the 5× Absolute Q™ DNA Digital PCR Master Mix (Cat. no. A52490, Thermofisher, Waltham, MA, USA), 2 µL of the EV-DNA template (2 ng), 0.5 µL of the FAM-Taqman™ probe, and 5.5 µL of NFW. First, 9 µL of the above reaction mixtures were loaded onto the QuantStudioTMMAP16 Digital PCR plate (Cat. no. 10246917, ThermoFisher, Waltham, MA, USA). Following the addition of 15 µL of QuantStudio^TM^ Absolute QTM Isolation Buffer (Cat. no. A52730, ThermoFisher, Waltham, MA, USA) to each sample, the wells were sealed using gaskets provided with the dPCR plates. The PCR conditions for mtDNA dPCR were 10 min at 96 °C, followed by 40 cycles of 5 s at 96 °C and 15 s at 60 °C. The QuantStudio™ Absolute Q Digital PCR System and QuantStudio dPCR software (Version 6.3) were used for DNA amplification, and the number of microchambers with successful mtDNA amplification was counted.

4.12. OPT

We performed the OPT to evaluate hippocampus-dependent spatial memory in mice, with minor modifications from established methods [135]. After the end of the CIE protocol, animals were acclimated to the testing room for 30 min and then individually placed into empty testing chambers for 30 min to habituate, with chambers cleaned using quatricide between each testing. The next day, after a 30-min acclimation, mice underwent a 10-min habituation in the chamber without objects, followed by a 5-min break in holding cages while two identical objects were positioned parallel to each other, about 4–6 cm from the chamber edges. Mice were then returned for a 10-min familiarization phase, with their behavior recorded. After at least 30 min, during which one object was moved diagonally to a new location, mice were reintroduced to the chamber for a 10-min testing phase, and exploration was recorded. Exploration was defined as the animal’s nose being within 2 cm of an object. Behavioral scoring and analysis were performed by an investigator blinded to treatment group and sex. Animals not meeting minimum exploration criteria were excluded. Exploration times for each object were recorded to calculate the exploration ratio (moved object exploration time/total exploration time) and discrimination index ([moved − unmoved object time]/total exploration time), where a ratio ≥ 0.5 or a positive discrimination index indicated intact spatial memory. This protocol was adapted from established methods [136,137].

4.13. Statistical Analysis

Data were analyzed using Prism Version 10.6.1 (799) software (GraphPad Software Inc., La Jolla, CA, USA). A p-value of ≤0.05 was considered statistically significant. Results are presented as the mean ± SEM. ANOVA with Tukey’s post hoc test was used for comparisons between multiple groups. For behavioral outcome, we performed the OPT test, and the differences in groups were assessed using the Kruskal–Wallis test. For microscopic analysis, multiple group comparisons were performed using one-way analysis of variance (Brown-Forsythe and Welch ANOVA test) with Dunnett’s T3 post hoc test.