The Role And Mechanism Of Rosmarinic Acid In Regulating Interferon Response Induced By Viral Infection

As an important barrier against the infection of pathogenic microorganisms,the innate immune system plays a crucial role in the identification and elimination of pathogenic microorganisms.The host recognizes pathogen-associated molecular patterns（PAMPs）and damaged-associated molecular patterns（DAMPs）mainly through pattern recognition receptors（PRRs）on the cell membrane or inside the cell to activate innate immune response.The nucleic acid of pathogenic microorganism is a typical class of pathogen-associated molecular patterns,which can be recognized by nucleic acid sensors and induce innate immune response.Many nucleic acid sensors have been identified at the moment,mainly including DNA sensors and RNA sensors.DNA sensors mainly include cytoplasmic DNA sensor c GAS（Cyclic GMP-AMP synthase）,TLR9（Toll-like receptor 9）located on endosomes,AIM2（absent in melanoma 2）and so on.RNA sensors consist of RLRs（Retinoic acid-inducible gene I-like receptors）family,TLR3,TLR7,TLR8 located on endosomes,PKR（double-stranded RNA-activated protein kinase R）and so on.Since the discovery of cytoplasmic DNA sensor c GAS by Professor Zhijian Chen in 2013,related research continues to emerge.The activation of c GAS signaling pathway is a key mechanism of cytoplasmic DNA mediated innate immune response.When exogenous pathogens invade,DNA released by the pathogens binds to c GAS and activates the enzymatic activity of c GAS,which catalyzes the cyclization of ATP（Adenosine triphosphate）and GTP（Guanosine triphosphate）to synthesize c GAMP（2’3’cyclic GMP-AMP）.c GAMP binds to and activates STING（Stimulator of interferon genes）localized on the endoplasmic reticulum,which translocates from the endoplasmic reticulum to the Golgi apparatus,where it further recruits and activates TBK1（TANK-binding kinase 1）.Activated TBK1 phosphorylates STING and interferon regulatory factor 3（IRF3）.After phosphorylation,IRF3 undergoes dimerization and induces type I interferon expression in the nucleus.For pattern recognition of RNA molecules,the RLRs family is an important RNA sensor in the cytoplasm.Its main members include RIG-I（Retinoic acid-inducible gene1）,MDA5（melanoma differentiation-associated protein 5）and LGP2（Laboratory of genetics and physiology 2）.RIG-I and MDA5 play an important role in monitoring RNA virus invasion as cytoplasmic RNA sensors.During the invasion of RNA virus,RNA released by the virus is rapidly recognized by RIG-I or MDA5 and MAVS（mitochondrial antiviral signaling protein）is recruited,which oligomerizes and activates TBK1.The activated TBK1 recruits and activates IRF3,and phosphorylated IRF3 enters the nucleus to induce the expression of type I interferon.Sensing viral nucleic acid by the innate immune system is an important process in response to viral infection.When the body is infected with the virus,the viral nucleic acid is recognized by the nucleic acid sensors,and the downstream signaling pathway is activated to induce the production of type I interferon.At the same time,the production of interferon is also a"double-edged sword".When the virus cannot be completely eliminated for a while,the antiviral immune response produced by the natural immune system will be over-activated,which will aggravate the production of interferon and inflammatory factors,and eventually cause severe inflammation of tissues and organs,multiple organ failure and even death.Therefore,the storm of inflammatory factors caused by viruses induces immune damage and even failure of vital organs of the body,which is one of the main causes of viral lethality.Therefore,finding drugs that can inhibit the excessive activation of immune response induced by cytoplasmic nucleic acid is of great significance for antiviral research and has potential clinical application value.Interferon-stimulated response elements（ISRE）fluorescent reporter system can indicate the activation of interferon signaling pathway by fluorescence.We constructed TM3 cells stably expressing the ISRE reporter system and screened a variety of nonsteroidal anti-inflammatory compounds using this cell model.Based on the screening results,we excluded the compounds with high cytotoxicity and selected the eleven compounds with the best inhibitory effect for further validation.Based on the verification results,we selected five compounds with the best inhibitory effect on interferon production and analyzed the half cytotoxicity concentration(CC₅₀)and the half inhibition concentration(IC₅₀).By comparison of CC₅₀ and IC₅₀,we found that Rosmarinic acid（RA）had the highest drug selection index(CC₅₀/IC₅₀).Therefore,we selected RA for in-depth study.Further studies showed that RA inhibited cytoplasmic DNA-and cytoplasmic RNA-induced type I interferon production,which suggested that RA had a potential inhibitory effect on the activation of c GAS-STING and RIG-I-MAVS pathways.To further determine the inhibitory effect of RA,we stimulated cells using HT-DNA,HSV-1,Poly（I:C）and VSV.We found that RA effectively inhibited the production of interferon induced by the above four stimuli and significantly inhibited the phosphorylation of TBK1 and IRF3 induced by HT-DNA and Poly（I:C）.Thus,RA could inhibit cytoplasmic DNA mediated the activation of c GAS-STING signaling pathway as well as cytoplasmic RNA mediated the activation of RIG-I-MAVS signaling pathway.We next investigated the mechanism by which RA inhibits c GAS-STING and RIG-I-MAVS pathways.We found that RA inhibited cytoplasmic DNA induced phosphorylation of TBK1,STING,IRF3 and the generation of c GAMP,but did not affect c GAMP-induced signaling pathway activation.Similarly,RA inhibited cytoplasmic RNA induced phosphorylation of TBK1 and IRF3 in the RNA signaling pathway.Therefore,RA acts upstream of c GAMP and TBK1.RA may target c GAS and RIG-I to exert its effect.In order to verify this conjecture,we examined the interaction between RA and c GAS or RA and RIG-I through pull-down assay,and the results showed that RA could bind to c GAS but not RIG-I.The above results suggested that RA might target the key regulators of both c GAS-STING signaling pathway and RIG-I-MAVS signaling pathway.Our previous studies had shown that G3BP1 could act as a platform molecule to regulate the activation of c GAS-STING signaling pathway and RIG-I-MAVS signaling pathway.Thus,we examined the binding of RA to G3BP1,and the results showed that RA could bind to G3BP1,which suggested that RA might regulate cytoplasmic nucleic acid mediated immune response by targeting G3BP1.To further clarify the mechanism of RA,we examined the effect of RA on the interaction between c GAS and G3BP1 as well as RIG-I and G3BP1 by co-immunoprecipitation assay.The results showed that RA was able to inhibit the interaction between c GAS and G3BP1 as well as RIG-I and G3BP1.In addition,RA did not inhibit the production of interferon induced by cytoplasmic nucleic acid in G3BP1 deficient cells,showing that the inhibitory effect of RA was achieved through G3BP1.The results of above experiments indicated that RA inhibited cytoplasmic nucleic acid induced immune response by targeting G3BP1.In summary,our study identified RA as a nonsteroidal anti-inflammatory compound could target G3BP1 to inhibit cytoplasmic DNA as well as cytoplasmic RNA induced over-immune response,which also provided potential therapeutic strategies and theoretical basis for the over-activation of immune response induced by viral infection.