A Preliminary Study Related To The Gene Regulation Mechanism Of Lentiviral-mediated TREM1 And LBP Gene Silencing On LPS-induced Buffalo Mononuclear Macrophages

Gram-negative bacteria,such as E.coli,dysentery bacillus and brucellosis,can cause inflammatory diseases of livestock which has brought great losses to the livestock industry,such as sepsis,mastitis,or reproductive system infections.The Endotoxin,also known as lipopolysaccharide(LPS),is a major virulence factor in the outer wall of Gram-negative bacteria.It can activate a variety of receptors such as TLRs on transmembrane and intracellular signaling,releasing inflammatory mediators and inducing organism inflammation injury.Myeloid cell trigger receptor 1(TREM1)is a cell membrane pattern recognition receptor which amplifies LPS inflammatory response through the TREM1/DAP12 signaling pathway.Lipopolysaccharide binding protein(LBP)has high affinity of LPS and belongs to the upstream genes in TLR4 signaling pathway.TREM1 and LBP can expand the inflammation,which is essential for LPS signaling.Studies have shown that silencing or knockout of TREM1 or LBP can significantly reduce the expression of inflammatory mediators and control over-inflammation,and there is synergistic interaction between TREM1 and TLR4 signaling pathway.In order to understand the role of buffalo TREM1 and LBP genes in LPS-induced inflammatory response and the mechanism of inflammation-induced signal transduction,we used lentiviral vector-mediated RNA interference and transcriptase technology as the main means in vitro to study the effect of TREM1/LBP on the expression of LPS-induced gene expression in buffalo peripheral blood mononuclear macrophages,and to analyze the molecular regulation mechanism of TREM1/LBP gene silencing in LPS-induced gene expression in order to lay the foundation for improving disease resistance.The main results of this study are as follows:1.Isolation and culture of mononuclear macrophages from peripheral blood of buffaloFor the purpose of obtainning the buffalo mononuclear macrophages with good growth status,the primary mononuclear macrophages of the buffalo were isolated and purified.Purified buffalo mononuclear macrophages were identified by cell phagocytosis,cell surface specific antigen analysis and qRT-PCR.The results showed that compared with the 5%and 7.5%autologous serum groups,the 2.5%autologous serum culture group was better for the cell adherence after inoculation,and the purified cells could specifically express TREM1 and CD 14 genes,and the positive rate of CD14 and MHC-? was 98.50%.2.Effects of inhibition of TREM1 gene expression on expression of inflammation related genes in buffalo monocyte/macrophages induced by LPSIn order to investigate the effect of inhibition of TREM1 gene expression on LPS-induced expression of macrophage inflammatory gene in buffalo mononuclear macrophages,TREM1-shRNA294 recombinant plasmid was used to package the virus,and the suitable conditions for target cell infection were explored.Followed by qRT-PCR and ELISA methods,we detected the inflammation-related genes in virus-infected cells.The results showed that the infection rate was more than 50%under the condition of the virus titer was>4×108 IU/mL,the lentivirus infection index(MOI)was 300,and 7 days.Compared with LPS control group,the expression of TREM1 gene was decreased by 69%,and the expression of DAP 12,TLR4,MYD88,TNF-?,IL-1? were significantly lower than those of LPS group(P<0.05).The level of cytokine TNF-? in the TREM1-sh group was significantly lower than LPS control group(604.92 pg/ml,P<0.05),and the cytokine IL-1? content(230.65 pg/ml)was significantly lower than the LPS control group(387.12 pg/ml,P<0.05).3.Effects of LBP gene expression on LPS-induced expression of inflammation-associated gene in buffalo monocyte/macrophagesIn order to investigate knockdown of LBP gene expression in LPS-induced buffalo mononuclear/macrophages,the lentiviral packaging,conditions for target cell infection and gene expression analysis were performed in the same way as above.The results showed that the virus titer was>5×108 IU/mL.Compared with LPS control group,the expression of LBP,CD 14,TLR4,TNF-?,IL-1?,IL-8 were decreased significantly(P<0.05).The content of cytokine TNF-?,IL-1? and soluble LBP(sLBP)protein was significantly lower(P<0.05).For different proportions of LV infection,boeh the mRNA level and content of TNF-? and IL-1? were significantly decreased than those of LPS group(P<0.05),and there was no significant difference in groups(P>0.05).When the ratio of TREM1-shRNA and LBP-shRNA was 2:1,the expression of Bcl-2 gene was significantly decreased while Caspase-3 increased(P<0.05).4.Transcriptomics analysi in groups s of silencing TREM1 and LBP in buffalo mononuclear/macrophagesIn order to fully analyze the molecular regulation mechanism of TREM1/LBP gene silencing on LPS-induced signal transduction,3 samples named TREM1-sh,LBP-sh and NC(LPS stimulated alone)were analyzed by RNA-seq,analyzed by bioinformatics method,and the DEGs were verified by qRT-PCR.The analysis showed that compared with NC group,there were 1355 genes in the TREM1-sh group while 897 genes in the LBP-sh group(P<0.001).Compared with LBP-sh group,TREM1-sh had 2536 DEGs(P<0.001).The GO enrichment analysis found that the main enrichment of the biological processes were mainly concentrated in the cellular immune,immune process,antigen presentation and so on.The KEGG analysis showed TREM1-sh and LBP-sh groups were significantly enriched in NF-kB signaling pathway,cytokine pathway,whlie TREM1-sh group in JAK/STAT and antigen presentation.A total of 182 common genes were found in all 3 comparision groups.Maybe 9 genes including NFKBIB,TNFRSF13B,TNFRSF17,IL-26,C5AR1,HIF1A,IL2RA,IL-8 and MMP-9,were likely to be co-regulated by TREM1 and LBP gene silencing about immune-inflammatory responses,mediated by HIF-1A/C5AR1/cytokines interaction,STAT/TNFSF/NF-KB/IL-8/MMP-9,LBP/TREM1 interaction or other signal transduction pathways.The above results suggest that inhibition of TREM1/LBP gene expression can effectively reduce the expression of LPS-induced inflammation-related genes of monocyte/macrophage,and regulate the LPS-induced inflammatory response.The TREM1 pathway interacts with LBP signal transmission.