165 THE EFFECT OF shRNA TARGETING CLUSTER OF DIFFERENTIATION ANTIGEN 14 ON GENE EXPRESSION OF TNF-α, TLR4, AND IL-6 IN LIPOPOLYSACCHARIDE-INDUCED BUFFALO PERIPHERAL BLOOD MONOCYTE/MACROPHAGE

Abstract

Cluster of differentiation antigen 14 (CD14) plays a crucial role in the inflammatory response to lipopolysaccharide (LPS), which interacts with TLR4 and MD-2 to enable cell activation, leading to inflammation. Several studies have proved that upstream inhibition of bacterial LPS/toll-like receptor 4 (TLR4)/CD14-mediated inflammation pathway is an effective therapeutic approach for attenuating damaging immune activation. In this study, to explore the effect of CD14 down-regulation on TLR4 signal conductive-related genes expression after stimulation by LPS, five CD14 shRNA (319/421/755/970/1041) sequences and a negative control sequence (NC-1864) were synthesised and used to construct lentiviral recombinant plasmid pSicoR-GFP-shRNA. Lentiviral recombinant plasmids of pSicoR-GFP-shRNA and fusion expression vector of pDsRed-N1-buffalo CD14 were co-transfected into HEK293 using liposome. At 72 h after transfection, the expression of exogenous buffalo CD14 mRNA was reduced at different level for all shRNA plasmids, in which shRNA-1041 had the highest interfering efficiency by RT-qPCR and fluorescence-activated cell sorting analysis. Then, buffalo peripheral blood monocyte/macrophage was purified and infected by the CD14 shRNA lentivirus. After 7 days of infection, the cells were stimulated by 1 µg mL^–1 LPS for 3 h, then the mRNA expression level of CD14, TLR4, IL-6, and TNF-α transcripts in the cells were detected by the RT-qPCR method. After stimulation by LPS, the expression of endogenous CD14 was significantly reduced by CD14 shRNA-1041, the mRNA expression level of TLR4, IL-6, and TNF-α genes was also significantly down-regulated in comparison with control group (P ≤ 0.01). In conclusion, the selected CD14 shRNA-1041 cannot only inhibit the expression of endogenous CD14 mRNA in buffalo peripheral blood monocyte/macrophage, but also downregulate the mRNA expression of CD14, TLR4, IL-6, and TNF-α. The above results demonstrate that knockdown of endogenous CD14 has obvious coordination effects on the signal conductive function of TLR4 after stimulating by LPS, and shRNA technology will provide a new way to prevent endotoxin-related diseases in livestock.

This work was supported by the National Transgenic Project (2009ZX08007-009B), Guangxi natural science funding (2012GXNSFCB053002), and funding of State Key Laboratory of Subtropical Bioresource Conservation and Utilisation (KSL-CUSAb-2012-02).