Neuroinflammatory Responses and Blood-Brain Barrier Injury in Chronic Alcohol Exposure: Role of Purinergic P2X7 Receptor Signaling

Abstract

Alcohol consumption leads to neuroinflammation and blood-brain barrier (BBB) damage, resulting in neurological impairment. We previously demonstrated that ethanol-induced disruption of barrier function in human brain endothelial cells was associated with mitochondrial injury, increased ATP and extracellular vesicle (EV) release, and purinergic receptor P2X7R activation. Therefore, we aimed to evaluate the effect of P2X7r blockade on peripheral and neuro-inflammation in EtOH-exposed mice. In a chronic intermittent ethanol (CIE)-exposed mouse model, P2X7R was inhibited by two different methods: Brilliant Blue G (BBG) or gene knockout. We assessed blood ethanol concentration (BEC), plasma P2X7R and P-gp, number of extra-cellular vesicles (EV), serum ATP and EV-ATP levels. Brain microvessel gene expression and EV mtDNA copy numbers were measured by RT2 PCR array and digital PCR, respectively. A RT2 PCR array of brain microvessels revealed significant upregulation of proinflammatory genes involved in apoptosis, vasodilation, and platelet activation in CIE-exposed animals, which were decreased 15-50-fold in BBG-treated CIE-exposed animals. Plasma P-gp levels and serum P2X7R shedding were significantly increased in CIE-exposed animals. Pharmacological or genetic suppression of P2X7R decreased P2X7R shedding to levels equivalent to those in control group. The increase in EV number and EV-ATP content in the CIE-exposed mice was significantly reduced by P2X7R inhibition. CIE mice showed augmented EV-mtDNA copy numbers which were reduced in EVs after P2X7R inhibition or receptor knockout. These observations suggested that P2X7R signaling plays a critical role in ethanol-induced brain injury. Increased eATP, EV-ATP, EV numbers, and EV-mtDNA copy numbers highlight a new mechanism of brain injury during alcohol exposure via P2X7R and biomarkers of such damage. In this study, for the first time, we report the in vivo involvement of P2X7R signaling in CIE-induced brain injury.

Keywords: ATP; Blood-brain barrier; CIE; Extracellular vesicles; P2X7R.

Figure 1. Schematic of the experiment and blood ethanol concentrations (BECs).

(A) CIE exposure paradigm. Created with BioRender.com (B and C) BECs were assessed to ensure that pathophysiologically relevant ethanol levels were obtained at the end of the experiment. The mean BECs were 154.98 ± 10.70 mg/dl, 161 ± 10.19 mg/dL, 223.59 ± 20.15 mg/dL, and 259.73 ± 13.73 mg/dL in the CIE, BBG-CIE, CIE and CIE-P2X7r^−/− groups, respectively.

Figure 2. P2X7R inhibition downregulated the increase in the expression of genes involved in inflammation, apoptosis, vasodilation, and platelet activation in brain microvessels and serum cytokine levels in BBG-treated CIE-exposed animals.

(A) Heatmap shows the upregulation of genes involved in inflammation, apoptosis, vasodilation, and platelet activation in the brain microvessels of CIE animals. BBG treatment led to significant downregulation of these genes. (B) Cytokine levels after CIE exposure were analyzed by MSD ELISA. The levels of the proinflammatory cytokines TNF-a, KC/GRO, and IL-2 were significantly lower in BBG-treated CIE-exposed animals than in CIE-exposed animals only. A two-tailed t test was used for the statistical analyses. The values are presented as the mean ± SEM;n = 3–7; *p <0.05, and **** p < 0.0001 compared with CIE-exposed mice.

Figure 3. Serum levels of P2X7R upregulated by CIE were suppressed by receptor inhibition.

A) BBG treatment and B) P2X7R^−/− resulted in significantly less P2X7R shedding in the serum than that in the serum of the CIE-exposed mice. One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analyses; ** p ≤ 0.01, ***p ≤ 0.001, **** p < 0.0001 compared with CIE-exposed mice as controls. (n = 3–7, mean ± SEM).

Figure 4. CIE exposure resulted in increased blood levels of P-glycoprotein (P-gp).

The levels of P-gp after CIE exposure were analyzed by ELISA. Compared with those in air-control mice,plasma P-gp levels in CIE-exposed mice were significantly greater. Treatment with BBG had no effect on P-gp levels in BBG-treated CIE-exposed animals. One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analyses. n = 5–7, mean ± SEM and ****p < 0.0001.

Figure 5. Serum ATP levels in CIE exposed animals were normalized by pharmacologic or genetic P2X7R inhibition.

Serum ATP levels were lower in (A) BBG-treated CIE-exposed and B) P2X7R−/− CIE-exposed mice than in CIE-exposed mice. One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analyses. N = 5–7, mean ± SEM and *p ≤ 0.05, **p < 0.01, and ***p < 0.0001; ns= nonsignificant. A two-tailed t test was used for the statistical analyses.

Figure 6. EV ATP content increased in CIE animals were diminished by pharmacologic or genetic P2X7R inhibition.

The serum EV-ATP levels upregulated 7–10-fold by CIE were reduced in (A) BBG-treated CIE-exposed and (B) P2X7R^−/− CIE-exposed mice. One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analyses. n = 5–7, mean ± SEM and *p £ 0.05, **p < 0.01 and ***p < 0.001, ns= nonsignificant; two-tailed t test was used for the statistical analyses.

Figure 7. Genetic and pharmacologic P2X7R inhibition reduced EV numbers in CIE exposed mice.

EV numbers were significantly lower in (A) BBG+CIE-exposed and (B) P2X7R^−/− CIE-exposed mice than in the respective CIE-exposed mice. One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analyses; ***p < 0.001, **** p < 0.0001, and ns= nonsignificant (n = 5–7, mean ± SEM).

Figure 8. Genetic and pharmacologic P2X7R inhibition downregulated mitochondrial gene expression enhanced by CIE.

Digital PCR was used to quantify the copy numbers of mtDNA present in EVs, and bar graphs represent the copy number of genes per microliter of DNA in the experimental groups. (A) P2X7R inhibition or (B) P2X7R^−/− knockout reduces mtDNA copy numbers in the EVs isolated from BBG-treated CIE exposed animals and CIE-exposed P2X7R^−/− compared to that of EVs from CIE-exposed animals. One-way ANOVA followed by Tukey’s post hoc test were used for the statistical analyses. mean ± SEM, * p ≤ 0.01, **p ≤ 0.001, **** p < 0.0001, and ns= nonsignificant. (n = 3–8)