Mechanism Of BLV Inhibition Of Lactoferrin Synthesis In Bovine Mammary Epithelial Cells

Bovine leukemia virus(BLV)is a retrovirus that is widely circulating worldwide and causes persistent infection in cows through the invasion of B lymphocytes and is latent in host cells as a provirus that suppresses immune function in dairy cows.BLV can infect the mammary tissue of cows via the bloodstream,causing damage to bovine mammary epithelial cells(BMECs),resulting in decreased lactation.Bovine lactoferrin(b LF)is mainly synthesized and secreted by BMECs and neutrophils,and has broad-spectrum anti-infection,antioxidant and immunomodulatory functions,which are important for the mammary gland health of cows.Does BLV infection affect the b LF content in bovine milk and through which pathways does it affect b LF synthesis? Therefore,an in-depth study on the effect of BLV infection of BMECs on b LF expression and the pathway of action is important to enhance the b LF content of bovine milk and improve the nutritional quality of milk.To clarify the effect of BLV infection on the b LF content in the blood and milk of dairy cows,blood and milk were collected from healthy cows(n=17),low BLV-loaded cows(n=18),and high BLV-loaded cows(n=17)in this study.Serum,whey,and neutrophil samples were isolated and analyzed for b LF content by ELISA,high-performance liquid chromatography,and q RT-PCR.The results showed that compared with negative cattle,there were no significant changes in serum b LF content and peripheral blood neutrophil b LF m RNA expression,and there was no correlation between serum b LF and BLV load.The results of whey ELISA showed that compared with negative cattle,the b LF content of positive cattle decreased by an average of 30.29%,and the b LF content in the milk of low-load cattle and high-load cattle decreased by an average of 25.5% and35.4%.The results of High-performance liquid chromatography showed that compared with negative cattle,the b LF content in milk with low BLV load and high BLV load was significantly decreased(P < 0.05).The above results showed that BLV load was correlated with b LF in milk.The higher the BLV load,the lower the b LF content in milk,while the BLV load was not correlated with the b LF content in bovine blood.The primary cultured BMECs were subsequently treated with BLV to establish an in vitro infection model,The effect of BLV infection was verified by immunofluorescence,q RT-PCR,and Western Blot to detected BLV-related genes and protein,and the expression level of b LF after BLV treatment of BMECs was examined.The m RNA and protein results showed that the primary cultured cells expressed the epithelial cell marker protein CK18.The m RNA and protein expression levels of b LF were detected in the 5th,6th and 7th passages cells without significant changes,indicating that the cultured cells were epithelial cells and stably expressed b LF.The results of BLV-treated cells showed that the m RNA of three structural genes of BLV,env,gag,and pol,and the core protein p24,were expressed in BMECs,and with the increase of BLV treatment dose,the expression level was significantly increased(P < 0.01).The results of the CCK8 assay showed that the cell viability level was significantly reduced when 5 MOI BLV infected cells(P < 0.05).These results demonstrated that BLV infected BMECs.Because 5 MOI BLV affected epithelial cells proliferation,the maximum dose of BLV used in subsequent experiments was 4 MOI.The results of b LF assay showed that BLV-treated BMECs significantly reduced the relative expression levels of b LF m RNA and protein(P < 0.05),and intracytoplasmic localization was reduced.The above results indicated that BLV inhibited b LF synthesis in BMECs.To investigate whether BLV inhibits with b LF transcription by affecting b LF-associated transcriptional regulators,BMECs were infected in vitro with different MOI BLV,and transcription factors with binding sites on the b LF promoter,such as STAT3,were examined by q RT-PCR,Western Blot,Dual luciferase reporter gene assay,Chromatin immunoprecipitation,and Cellular immunofluorescence methods.The results of m RNA and protein showed that BLV infection significantly upregulated the expression of p-STAT3(P < 0.01)and significantly downregulated the expression of NF-κB p-p65,p-STAT5,p-c-Jun and SP1(P < 0.01).Dual luciferase reporter gene assay showed that BLV repressed b LF promoter activity,suggesting that BLV may inhibit b LF transcription by affecting the expression of b LF-related transcription factors.The results of chromatin immunoprecipitation showed that the enrichment of c-Jun and SP1 in the b LF promoter region was significantly reduced after BLV infection of cells(P < 0.01).Therefore,we speculated that BLV could reduce b LF transcription activity and inhibit b LF expression by inhibiting c-Jun phosphorylation or SP1 expression.To further determine the role of c-Jun and SP1 in the reduction of b LF synthesis resulting from BLV treatment of BMECs,cells were pretreated with c-Jun inhibitor and activator,SP1 si RNA and overexpression vector,followed by treatment of BMECs with BLV.The transcriptional synthesis of b LF was detected by q RT-PCR,Western Blot,Chromatin coimmunoprecipitation,and Dual luciferase reporter gene assay.The results showed that the enrichment of both in the b LF promoter region was reduced after inhibition of c-Jun phosphorylation or SP1 expression alone,but the b LF promoter activity and protein expression were not affected.Promotion of c-Jun phosphorylation or SP1 expression alone similarly failed to affect b LF promoter activity and protein expression,whereas simultaneous inhibition of c-Jun phosphorylation and SP1 expression decreased b LF promoter activity and downregulated protein expression significantly(P < 0.01).After simultaneous promotion of both expressions,the promoter activity was enhanced,and the protein expression was upregulated significantly(P <0.05).These results suggested that c-Jun and SP1 co-regulated b LF transcription.After simultaneous overexpression of c-Jun and SP1 and reinfection with BLV,compared with the BLVinfected group,the b LF promoter activity was restored,and b LF expression was no longer inhibited.The above results showed that BLV reduced b LF promoter activity and inhibited b LF synthesis by simultaneously inhibiting c-Jun phosphorylation and SP1 expression.Activation of c-Jun phosphorylation while overexpressing SP1 attenuated the inhibitory effect of BLV on b LF transcription.In summary,BLV infection reduced the b LF content in bovine milk,and the higher the BLV load,the lower the b LF content;In BMECs,BLV reduces the binding activity of c-Jun and SP1 to the b LF promoter region transcriptional regulatory site by inhibiting c-Jun phosphorylation and SP1 expression and ultimately inhibits b LF transcription;Promotion of c-Jun phosphorylation and SP1 expression antagonizes the inhibitory effect of BLV on b LF transcription.The above results provide a theoretical basis for the study of the mechanism of b LF synthesis.They also provide a reference for studies related to the scientific enhancement of b LF content in milk.