HIV-1 gp120 Impairs Blood-Brain Barrier Via Inducing Brain Endothelial Pyroptosis

Background:

Brain microvascular endothelial cells (BMECs) are a key component of the blood-brain barrier (BBB) which is critical for maintaining brain homeostasis as an important line of defense for brain tissue against pathogen invasion. It has been shown that free envelope protein gp120 (Gp120) released into blood circulation by damaged cells infected with HIV-1 is a vital factor affecting the integrity of BBB. Further studies found that Gp120 reduces the expression levels of tight junction-associated proteins ZO-1, ZO-2, and Occludin, which are associated with the induction of protein degradation via the proteasome, thereby decreasing transepithelial electrical resistance and increasing the permeability of BBB. Notably, BMECs also express CD4 and chemokine receptors which are essential to the infection of HIV-1, implying that Gp120 might function directly on BMECs to injury the BBB. However, it is still largely elusive about the cellular and molecular mechanism of the abrasive interaction between Gp120 and BMECs. Here, we focus on the pathogenesis of direct impairment to the BMECs by Gp120.

Methods:

The immortalized human BMECs (HCMEC/D3) were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin, and maintained at 37 °C in a 5% CO₂ incubator. The cells were incubated with HIV-1 gp120 protein in different final concentrations (20 nM, 40 nM, 80 nM, 160 nM) for 24 hours at 37 ℃ for LDH release experiment. The control group was treated with isovolumic vehicle (phosphate buffered saline, PBS). Then, total soluble proteins of HCMEC/D3 treated with 20 nM HIV-1 gp120 or isovolumic vehicle were extracted by RIPA lysis buffer for western blotting to compare the expression level change of pyroptosis associated proteins. In addition, confocal fluorescence microscopy was performed to detect the expression level of NLRP3 in endothelial cells. ImageJ 1.53t and GraphPad Prism 8.3 software were used for optical density and statistical analysis respectively. The data are expressed as mean ± SD. For comparisons between two groups, unpaired t-test was performed; for comparisons among multiple groups, one-way analysis of variance (ANOVA) test was performed with Tukey's post-hoc test. P < 0.05 was considered statistically significant.

Results:

We found that the relative LDH release was elevated in Gp120-treated endothelial cells with the increase of concentration gradient (P < 0.05). Immunoblotting results showed that the expression levels of pyroptosis-related protein, including NLRP3, ASC, CASP 1, N-terminal of GSDMD especially, were significantly upregulated in HCMEC/D3 treated with 20 nM Gp120 compared to control group (P < 0.05), in spite of the fact that there was no statistically significant difference in the expression of full length GSDMD (P > 0.05). To further visually validate the effects of NLRP3 inflammasome activation induced by Gp120, immunofluorescence experiments of NLRP3 were performed in HCMEC/D3. The results showed that the expression level of NLRP3 was remarkably increased as well (P < 0.05).

Conclusion:

In this study, we preliminarily showed that HIV-1 gp120 induces human brain endothelial pyroptosis leading to BBB breakdown. More intensive investigations are still required to elucidate the underlying mechanism how pyroptosis is triggered and transmitted in the interaction between HIV-1 gp120 and BMECs. (Acknowledgements:This study was supported by National Natural Science Foundation of China, No. 82172259 to H. Cao and Grant from School of Public Health of Southern Medical University, China, Grant No. GW202329 to H. Cao and Undergraduate Training Program for Innovation and Entrepreneurship of SMU, No. 202412121357; Corresponding author: Hong Cao, gzhcao@smu.edu.cn)

No relevant conflicts of interest to declare.