The Role of CHRFAM7A in the Impairment of Blood-Brain Barrier Via Facilitating Brain Microvascular Endothelial Pyroptosis By Cryptococcus Neoformans

Background:

Cryptococcal meningitis is a AIDS-defining disease and the common invasive fungal infection in the world. In less developed areas, cryptococcal meningitis is the main cause of death among adults living with HIV. The alpha7 nicotinic acetylcholine receptor (α7nAChR) played an important role in immune regulation, the occurrence and regulation of inflammation. CHRFAM7A is a human specific gene whose gene product is a negative regulator of α7nAChR. It is associated with a variety of inflammatory diseases and is crucial in maintaining body homeostasis. The cleavage of GSDMD by Caspase-1 leading to the release of its N-terminal domain (GSDMD-NT) and the subsequent formation of pores in the cell membrane is a potential mechanism of pyroptosis. Pathogenic microorganisms cause activation of NLRP3 and release of the pro-inflammatory cytokines IL-1β and IL-18, and induce GSDMD-mediated pyroptosis, leading to pro-inflammatory cell death. Increased permeability of cerebral microvascular endothelial cells is an important index to evaluate blood-brain barrier (BBB) integrity. In the previous stage, our group has confirmed that Cryptococcus neoformans (Cn) can cause pyroptosis of cerebral microvascular endothelial cells, the main component of BBB and this study intends to explore the effect of CHRFAM7A on Cn impairs blood-brain barrier via facilitating brain endothelial pyroptosis.

Methods:

pcDNA3.3-mCherry (Addgene plasmid # 26823 ) / pcDNA3.1-CHRFAM7A-mCherry (Addgene plasmid # 62635) plasmid was transfected into bEnd.3 Mouse Brain Capillary Endothelial Cell Line. The transfected cells were cultured with 10^7/ml Cryptococcus neoformans(B-4500F02) at 37℃ for 3 hours. After three times of PBS cleaning, the absorbance under wavelength OD450 was measured after 3.5 hours with 10% Cell Counting Kit-8 (CCK8) reagent is added, and the inhibition rate was calculated. The total soluble protein of bEnd.3 in each group was extracted with RIPA lysis buffer for western blot analysis. Image J and SPSS (IBM SPSS Statistics 26) software were used for grayscale and statistical analysis respectively. One-way ANOVA was used for data analysis.

Results:

The cell activity rate and transfection rate of the cells transfected with two plasmids were detected by cell activity assay and fluorescence detection, and the results showed that there was no statistical difference between the two groups (P>0.05). In cell activity assay test, the survival rate of pcDNA3.1-CHRFAM7A-mCherry transfection group was higher than that of pcDNA3.3-mCherry transfection group (P<0.001). In Western Blotting experiment, the expression levels of NLRP3, Caspase 1, GSDMD-NT and IL-1β in pcDNA3.1-CHRFAM7A-mCherry transfection group were down-regulated compared with that in pcDNA3.3-mCherry transfection group (P<0.05). The expression levels of IL-18 in pcDNA3.1-CHRFAM7A-mCherry transfection group were significantly down-regulated compared with that in pcDNA3.3-mCherry transfection group (P<0.01).

Conclusion:

In this study, bEnd.3 without CHRFAM7A were introduced with related gene plasmids and verified at the cell level and protein molecular level, both of which were shown CHRFAM7A gene product can reduce the occurrence of Cn induced BBB pyroptosis via NLRP3, Caspase 1 and GSDMD-NT signaling pathways. And the CHRFAM7A gene product inhibits the release of cytokines IL-1β and IL-18 from cells injured with Cn, reducing inflammation and immune response. Synthesize above, CHRFAM7A gene may be a target for the treatment of Cn impairs blood-brain barrier via facilitating brain endothelial pyroptosis. (Acknowledgements:This study was supported by Grant from School of Public Health of Southern Medical University, China, Grant No. GW202431 to H. Cao; Corresponding author: Hong Cao, gzhcao@smu.edu.cn)

No relevant conflicts of interest to declare.