Molecular Mechanism Of Arginine Regulating Intestinal Antiviral Innate Immune Function In Pigs

The body initiates signaling of the innate immune response through the TBK1-IRF3 signaling pathway,releasing IFN? to resist infection of external viruses.Nutritional stimulation may be a method,but it is unclear whether arginine regulates and how to regulate this pathway.Studies have shown that the key reasons of arginine regulates mammalian physiology are at least partly the ability of arginine to activate mTORC1,and then participates in many regulation,whereas CASTOR1 is a perceptive protein of arginine in the mTORC1 pathway.In this study,we investigated whether arginine-activated antiviral innate immune TBK1-IRF3 signaling passes through the Castor1-mTOR C1 signaling.The IPEC-J2 cell was used as a model for detecting the anti-viral innate immunity of arginine.The results of quantitative real-Time PCR showed that 0.4 mM,2 mM,4 mM arginine up-regulated the expression of IFN? and RIG-I,the antiviral experiment also demonstrated that 0.4 mM arginine inhibited replication of the VSV virus.Western-blotting results showed that arginine activated the TBK1-IRF3 signaling pathway to play antiviral effect.Here,we used TBK1 inhibitor and constructed IRF3 knockdown cell line to verify that the both inhibited significantly expression of IFN?.Since Castor1 is not only the receptor and perceptivator of arginine,it is also an inhibiter of rapamycin(mTOR)of the mammalian target.We constructed Castor1 knockdown cell line and also detected the expression of IFN?.Castor1 was found to inhibit its expression.Similarly,antiviral experiments are consistent with this,and Castor1 increases VSV viral replication.We also studied the mTORC1 signaling pathway,we used Western-blotting to detect phosphorylation of its downstream protein S6 K.Arginine did activate mTORC1 via Castor1.To demonstrate that Castor1-mTORC1 is involved in arginine-activated TBK1-IRF3 signaling to initiate antiviral innate immunity,we added mTORC1 inhibitor to IPEC-J2 cells to detect the expression of IFN? at mRNA level and the phosphorylation of IRF3 at protein level,both of which showed that the inhibitor did inhibit the TBK1-IRF3 signaling pathway,and the phosphorylation of IRF3 at the protein level was also detected using the Castor1 knockdown cell line.The results were consistent with the mTORC1 inhibitor,and Castor1 was also inhibited the phosphorylation of IRF3.In summary,this demonstrates that mTORC1 is critical for arginine to activate TBK1-IRF3 signaling.The experimental data of this study also lays a foundation for further research on the molecular mechanism of arginine and other nutrients in antiviral innate immune.