2022, Vol. 26 ›› Issue (24): 3826-3832

CD1d-mediated inhibition of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammatory factor expression via signal regulatory protein-alpha

Xiang Mingzhi1, 2, Yuan Zilin2, 3, Wang Gang2, 3, Cheng Jie2, 4, Diao Bo2, 3, Liu Yueping2, 3   

Abstract: BACKGROUND: Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammatory factors have been proven to be associated with a variety of inflammatory diseases, and regulating their expression will provide some insights into the treatment of related diseases.
OBJECTIVE: To explore whether CD1d regulates the expression of NLRP3 inflammatory factors so as to verify the underlying mechanism that CD1d regulates the NLRP3 inflammasomes expression via signal regulatory protein-α (SIRPα).
METHODS: (1) 293T cells were transfected with Flag-CD1d and HA-SIRPα lentivirus overexpression vectors for co-immunoprecipitation assay to verify the interaction between CD1d and SIRPα. (2) RAW264.7 was transfected with the Flag-CD1d lentiviral overexpression vector. RT-qPCR was used to detect the expression of NLRP3 and other genes. (3) RAW264.7 was transfected with the overexpression vector of HA-SIRPα lentivirus. The gene and protein expression levels of NLRP3   were detected by RT-qPCR and western blot assay, respectively. (4) SIRPα lentivirus RNAi vector was used to transfect RAW264.7. After lipopolysaccharide stimulation, gene and protein expression levels of NLRP3 were detected by RT-qPCR and western blot assay.
RESULTS AND CONCLUSION: (1) Co-immunoprecipitation assay confirmed the interactions between CD1d and SIRPα. (2) After overexpression of CD1d gene, the expression levels of NLRP3, pro-IL-1β and pro-IL-18 gene in the experimental group were significantly lower than those in the normal group (P < 0.05). (3) The expression of NLRP3 in the experimental group was significantly lower than that in the normal group after lipopolysaccharide stimulation (P < 0.05), but there were no differences in the expression of pro-IL-1β and pro-IL-18 between the experimental group and the control group (P > 0.05). The protein expression of p38, P-P38, NLRP3, pro-IL-1β and pro-IL-18 in the experimental group was significantly lower than that in the SIRPα interference group (P < 0.01). (4) The expression of NLRP3, pro-IL-1β, and pro-IL-18 gene in the experimental group was significantly higher than that in the normal group after lipopolysaccharide stimulation (P < 0.05). The protein expression of p38, p-p38, NLRP3, pro-IL-1β, and pro-IL-18 in the experimental group was significantly higher than that in the overexpression SIRPα group (P < 0.01). (5) It is concluded that CD1d forms a complex with SIRPα on the cell membrane, then inhibits the downstream p38MAPK/NF-κB pathway via SIRPα, and finally inhibits the expression of NLRP3 inflammatory factors.
Key words: CD1d, signal regulatory protein-α, NLRP3, inflammatory factor, p38MAPK, NF-κB, pathway