Effect And Mechanism Research Of Serotonin On Antiviral Innate Immunity

When a variety of DNA viruses,such as HSV-1 and herpes zoster virus,invade the body,they elicit a strong immune response that induces the onset and progression of inflammation.For example,when such viruses invade the brain,microglia residing in the brain initiate antiviral immune pathways such as the c GAS-STING pathway to produce type I interferons to resist virus invasion.However,overactivated microglia can induce viral encephalitis,which can be life-threatening in severe cases.Therefore,how to precisely regulate the antiviral immune process and balance the inflammatory damage induced by DNA viruses is of great significance for elucidating the mechanism of innate immune regulation and the development of antiviral drugs.5-Hydroxytryptamine(5-HT)is an inhibitory neurotransmitter that was first found in serum and is also known as serotonin.It is widely expressed in mammalian tissues,especially in the cerebral cortex and neural synapses.In recent years,it has been reported that serotonin can affect the immune system.For example,serotonin regulates the activation,proliferation,and differentiation of T-lymphocytes.Physiological concentrations of serotonin inhibit IFN-γ-induced expression of MHC class II molecules in macrophages,thereby regulating the production of chemokines and cytokines from various immune cells.In conclusion,serotonin is involved in many steps in the control of immune and inflammatory responses,but the role of serotonin in the field of innate antiviral immunity has not been reported.Our laboratory previously found that serotonin levels in the blood were significantly elevated after HSV-1 infection in mice,suggesting that serotonin may play a key role in the process of HSV-1 infection.Infection of RAW 264.7 cells with HSV-1resulted in a significant increase in the expression of the membrane protein selective serotonin reuptake inhibitor(SSRI,also called 5-HT transporter,5-HTT),which is responsible for transporting serotonin into the cells,and downregulation of the expression of ALDH2,a key enzyme in serotonin metabolism,suggesting that serotonin may accumulate and play a regulatory role in the cells.Having established that serotonin might be associated with immunity to viral infection,we first tested the effect of serotonin on innate immunity against viral infection in vitro.We found that serotonin pretreatment significantly reduced the type I interferon response induced by the DNA virus HSV-1 and exogenous DNA in RAW264.7 cells and BV2 cells,promoted viral replication,and inhibited the activation of the downstream interferon-related genes CXCL10 and MX1,indicating that serotonin could regulate the intracellular immune process against DNA viruses in vitro.To further explore the mechanism by which serotonin affects antiviral innate immunity,we first used receptor antagonists to inhibit the activity of the serotonin receptor.After exogenous DNA transfection,we found that those receptor antagonists could not restore the inhibitory effect of serotonin on type I interferon production.However,pretreatment with 5-HTP,a precursor of serotonin,reduced DNA-mediated type I interferon production,indicating that serotonin regulated intracellular innate antiviral immunity,instead,it does not depend on its classic cell surface receptor signaling pathway.Furthermore,the serotonin transporter inhibitor fluoxetine could effectively reverse the serotonin-mediated immunosuppression phenotype,indicating that serotonin needs to enter cells via the serotonin transporter to exert its effects.Next,we investigated the effects of serotonin on interferon-related signaling pathways and found that serotonin could significantly inhibit the phosphorylation of STING and IRF3,key proteins in the c GAS-STING pathway,and then inhibit the production of downstream type I interferon.These results indicated that serotonin affected the downstream immune response of interferon by inhibiting the c GAS-STING pathway and then regulated the antiviral immune process of the body.In addition to mechanistic research,we further explored the effect of serotonin on antiviral immunity in vivo.After intraperitoneal injection of serotonin and infection with the HSV-1 virus,we found a significant decrease in the level of type I interferon in the peripheral blood of mice and an increase in viral replication in the spleen after serotonin treatment.These results indicated that the innate immune response in mice was suppressed after pretreatment with serotonin,and virus replication in mice was elevated.Since serotonin is an endogenous small molecule,5-HT precursor 5-HTP administration is an effective way to elevate 5-HT levels in vivo.After intraperitoneal injection of5-HTP,mice were infected with HSV-1.It was observed that type I interferon levels were reduced in the blood of the mice after treatment with 5-HTP,which was consistent with the effect of exogenous injection of serotonin,indicating that increasing the content of endogenous serotonin could inhibit the HSV-1-induced innate immune response.Chronic administration of fluoxetine in drinking water was used to inhibit serotonin function in vivo.Mice were infected by HSV-1 after three weeks of treatment.As expected,the level of type I interferon in blood was elevated,and the level of virus replication in the kidney was suppressed significantly.These results indicated that chronic reduction of serotonin by fluoxetine can effectively promote the development of inflammation and inhibit the proliferation of the virus in vivo,which demonstrates that serotonin plays a negative regulatory role in the immune process of DNA virus infection.In addition,this study demonstrated that fluoxetine,which has been used as an antidepressant in clinical practice,has good antiviral activity and can be used as a basis for the design of related antiviral drugs in the future.In summary,our study shows that serotonin,a classical neurotransmitter,can regulate antiviral innate immune inflammation.Serotonin can enter the cell through serotonin transporters to inhibit the phosphorylation of key proteins in the c GAS-STING pathway,specifically reduce the level of type I interferon induced by DNA viruses or exogenous DNA,downregulate the expression of related downstream genes of interferon,inhibit the innate immune process and prevent the occurrence and development of viral inflammation.At the animal level,we found that exogenous or endogenous elevation of serotonin content could effectively inhibit the antiviral innate immune response,while the serotonin reuptake inhibitor fluoxetine could enhance the immune response and achieve stronger virus clearance.In the next step,we will explore the function of serotonin in DNA-related autoimmune diseases such as sickle cell anemia and arthritis,in an effort to generate novel diagnostic and therapeutic approaches.