The Function And Molecular Mechanisms Of Sirt3 In Antiviral Innate Immune Responses

Viral infection is one of the leading causes inducing body injuries and infectious diseases.Upon viral infection,the innate immune system recognizes pathogen-associated molecular patterns（PAMPs）by pattern-recognition receptors（PRRs）,which then triggers the antiviral innate immune responses and induces the production of interferon and inflammatory factors to resist the virus invasion.Since viruses cannot survive independently,they rely on the host cells to obtain energy and materials for their replication.In the meantime,they manipulate the metabolism of the hosts to improve their survival.Glucose metabolism is one of the most important metabolic pathways,providing primary carbon and energy for all creatures.Viruses manipulate and change the glucose metabolism by promoting the glycolysis when a rapid requirement of energy is demanded.The changes in the glycolytic machinery induced by viruses may also contribute to the pathogenesis of the infections.Accumulating evidence have revealed the modulation function of glucose metabolism in the antiviral innate responses.However,the interaction between them and the potent mechanism still needs to be investigated.Sirt3,the nicotinamide adenine dinucleotide（NAD）-dependent deacetylase,belongs to the silent mating type information regulation 2 homolog（Sirtuins）family.Sirt3 is involved in a variety of biological activities including metabolism,apoptosis,cellular signal transductions and so on.Sirt3 also participates in glucose metabolism by targeting key enzymes and regulating them,which further functions in innate immunity such as inflammatory responses and autophagy.However,the role of Sirt3 in antiviral innate immune responses has not been reported yet.To find out the function of Sirt3 in innate immunity during viral infections,the functional screening was conducted by knocking down the expression of Sirt1-7 in peritoneal macrophages using si RNA.It was found that Sirt3 knockdown significantly increased the replication of VSV in infected macrophages and decreased the m RNA expression of interferon-β（IFN-β）,indicating that Sirt3 might function as a positive role during viral infection.Moreover,we found that the productions of lactate and succinate were also increased in Sirt3-knockdown peritoneal macrophages upon VSV infection.To further confirm the influence of Sirt3 on glucose metabolism,we conducted extracellular flux assays to measure the oxygen consumption rate（OCR）and extracellular acidification rate（ECAR）and found that Sirt3-knockdown significantly promoted glycolysis while inhibited tricarboxylic acid cycle（TCA）cycle upon viral infection.These results demonstrated that Sirt3 functioned as a positive regulator in antiviral immune responses,which was related with glycolysis and TCA cycle.To further investigate the function of Sirt3 in viral infections,we infected peritoneal macrophages with VSV,Se V and HSV and found that the production of IFN-βwas significantly decreased in Sirt3 knockdown peritoneal macrophages upon different viral infections.Moreover,the lactate and succinate productions were also significantly increased,which was correlated with the production of IFN-βproduction.Then,to further confirm the function of Sirt3 in promoting antiviral responses,as well as inhibiting glycolysis and increasing TCA cycle during viral infections,macrophage cell strain Raw264.7 cells were stably over-expressed Sirt3 and infected with VSV.It was found that Sirt3 overexpression significantly decreased VSV replication and increased IFN-βproduction,with less production of lactate and succinate at the same time.Data above suggested that Sirt3 could promote antiviral innate immunity by decreasing glycolysis and increasing TCA cycle and IFN-βproduction,which was required for hosts to limit virus entry at early stage and inhibit virus replication at late stage of viral infection.To further confirm these results,we next investigate the function of Sirt3 on viral infection in vivo and in vitro.To further investigate the function of Sirt3 in vivo,the littermates of Sirt3^+/-and Sirt3^-/-mice were infected with VSV.We found that the overall survival rate of Sirt3^-/-mice was significantly lower than Sirt3^+/-mice upon VSV infection.Moreover,the production of IFN-βinduced by viral infection in the serum of Sirt3^-/-mice was significantly lower than that in Sirt3^+/-mice.In addition,inflammatory responses induced by viral infection were more severe in Sirt3^-/-mice,with more infiltration of inflammatory cells in lung and more production of proinflammatory cytokines,including tumor necrosis factor-α（TNF-α）and interleukin-6（IL-6）,in targeted organs.These results revealed that Sirt3^-/-mice were more susceptible with lower antiviral innate immune responses and more severe organ damages upon VSV infection.We also found that lactate and succinate,which were important metabolic intermediates downstream of glucose,were significantly upregulated in the serum of Sirt3^-/-mice compared with that of Sirt3^+/-mice.These results revealed that Sirt3promoted antiviral immune responses by inhibiting glycolysis and promoting TCA cycle in vivo.We next explored the positive function of Sirt3 in antiviral immunity in vitro.Upon VSV infection,Sirt3^-/-peritoneal macrophages produced significantly less IFN-βwith more replication of VSV.To investigate whether Sirt3 deficiency regulates glycolysis,the OCR and ECAR were assayed.In consistent with extracellular flux assay that Sirt3 deficiency increased glycolysis and blocked TCA cycle in macrophages,the concentrations of lactate and succinate were significantly upregulated in Sirt3^-/-macrophages upon VSV infection.Then we analyzed the signaling transduction of antiviral responses and found that Sirt3deficiency decreased the activation of tank binding kinase 1（TBK1）and interferon-regulatory factor 3（IRF3）.Moreover,the activation of mitogen-activated protein kinase（MAPK）and nuclear factor-κB（NF-κB）signaling was also decreased in the Sirt3^-/-macrophages.These results indicated that Sirt3 promoted the antiviral signaling transduction and responses by regulating glucose metabolism reprogram.To further find out the mechanism how Sirt3 promoted antiviral responses and how it affected the metabolites of glucose metabolism,we evaluated the interaction between pyruvate kinase M2（PKM2）and Sirt3.Findings in immunoprecipitation assays and Western blot showed that Sirt3 and PKM2 was interacted with each other,and the interaction between them was enhanced upon VSV infection.Immunofluorescence also showed that Sirt3 co-localized with PKM2,and the co-localization was enhanced upon VSV infection.We further explored the mechanism of the interaction between Sirt3 and PKM2 during viral infection.We found that the acetylation of PKM2 was increased upon VSV infection,which was stronger in the Sirt3^-/-peritoneal macrophages.However,the phosphorylation of PKM2 decreased upon VSV infection only in the Sirt3^-/-macrophages,which indicated that the Sirt3-mediated deacetylation promoted the phosphorylation of PKM2.Given that Sirt3was the deacetylase,we tested whether the interaction between Sirt3 and PKM2 in antiviral immune responses was enzyme activity dependent.We overexpressed wild-type Sirt3 or catalytic mutant Sirt3（Sirt3-H248Y）and found that the acetylation of PKM2 was decreased potently by the co-overexpression of Sirt3 upon VSV infection,but not by the overexpression of Sirt3-H248Y.The phosphorylation was also increased intensively by Sirt3overexpression,while not by Sirt3-H248Y overexpression,which further proved that Sirt3-mediated deacetylation prompted the phosphorylation of PKM2.Then we confirmed the phosphorylation and acetylation of PKM2 caused by its agonists or antagonist.It was found that the agonists decreased the phosphorylation of PKM2 but increased the acetylation of it,while the antagonist had opposite effects.However,the changes of phosphorylation induced by PKM2 antagonist and agonists disappeared with the overexpression of Sirt3.These data revealed that Sirt3 could decrease the acetylation of PKM2 and increase the phosphorylation of it upon viral infection,the process of which was enzyme-dependent.In consideration that Sirt3 deficiency increased glycolysis and decreased TCA cycle,Sirt3 might be required for efficiently inducing antiviral responses by inhibiting glycolysis and promoting TCA cycle via deacetylating PKM2.Lactate has been reported to be a natural suppressor of retinoic acid-inducible-1-like receptors（RLRs）signaling by targeting mitochondrial antiviral-signaling protein（MAVS）,participating the cross-regulation between antiviral signaling and energy metabolism.To further confirm the function of PKM2 on antiviral immunity via regulating glycolysis and TCA cycle,we employed PKM2 antagonist to evaluate the effect.It was found that the inhibition of PKM2 significantly increased ECAR and decreased OCR in macrophages upon viral infection,with more production of lactate.The inhibition of PKM2 also led to more VSV replication and less IFN-βproduction.The activation of PKM2 had opposite effects.Moreover,the pretreatment of PKM2 agonist could also significantly decrease viral replication and increase IFN-βexpression in both lung and spleen of mice.Above results indicated that PKM2 promoted antiviral innate immunity by inhibiting glycolysisTo further explore the function of the extracellular lactate in antiviral responses,we next investigated its function in vitro and in vivo by the extra addition of lactate.We found that extracellular lactate could increase virus entry and replication before sufficient IFN-βtranscription both in vivo and in vitro.These findings collectively illustrated that PKM2-reprogramed glucose metabolism was required for reducing viral entry and fully inducing antiviral responses via decreasing glycolysis.In summary,our research reveals that host cells reprogram their metabolism and initiate antiviral innate immunity to fight against virus.Upon viral infection,Sirt3 activates PKM2by deacetylating it,which inhibits glycolysis while promotes TCA cycle and reduces the production of lactate,functioning as a protective role in antiviral innate immune responses.These findings demonstrate the interaction between glucose metabolism and antiviral innate immunity,helping us to better understand the mechanism of it,which also outline that controlling glucose intake or targeting glycolysis may be a promising supplementary therapy strategy for viral infection.