MTORC1 Mediated IRF3 Translocation To Lysosome Is Required For IRF3 S396 Phosphorylation In TLR3/4 Signaling Pathway

TLR3/4 is indispensable in host defense against pathogens.TLR4 recruits Mal-Myd88 and TRAM-TRIF to promote inflammatory cytokines and IFNβproduction,while TLR3 signals TRIF to promote IFNβproduction.Once activated,TRIF interacts with TRAF3 to active TBK1 and downstream molecule IRF3.TRIF also interacts with TRAF6-RIP1 to active TAK1 and downstream molecule NFкB.IRF3 can be phosphorylated directly by TBK1 at Ser396.The phosphorylated IRF3 will dimerize and translocate into nucleus to promote IFNβtranscription.mTOR is a highly conserved Serine/Threonine kinase.It is a pivotal and common component of mTORC1 and mTORC2.By phosphorylating different substrates,mTORC1 functions as a master regulator in promoting anabolic metabolism and inhibiting autophagy.mTORC2 is insensitive to nutrient treatment and involves in actin cytoskeleton reorganization and cell survival.mTORC1 activation relies on mTOR translocation from cytoplasm to lysosomes.The process is regulated by amino acid sensing signaling pathways,which are orchestrated and locate on lysosome.Given that TRIF is activated on endo-lysosome in TLR3/4 signaling pathway as well,whether and how mTORC1 and related proteins involve in TLR3/4-TRIF signaling pathway remains an open question.Here,we show that TLR3/4 induced IRF3 activation is impaired in mTOR^-/-BMDMs,whereas TBK1 activation is normal.The phenotype has signaling pathway specificity because both of c GAS-STING or MDA5-MAVS induced TBK1 and IRF3 activation are not affected when mTOR is deficient.The phenotype is also verified in rapamycin pre-treated wt BMDMs,PF-4708671 pre-treated wt BMDMs,Raptor^-/-BMDMs,Rictor^-/-BMDMs,respectively.We find that mTORC1,rather than mTORC2,plays a role in TLR3/4 induced IRF3 activation.Mechanistically,mTOR interacts with IRF3 in a TLR3/4 signaling dependent manner.After stimulation,we can detect colocalization between IRF3 and lyso-tracker in mTOR^+/+BMDMs,but the colocalization is abolished in mTOR^-/-BMDMs.This gives a potent indication that mTOR confers IRF3 localization on the lysosome after stimulation.Further,different mutations which anchor IRF3 to lysosome,mitochondrion or cell membrene are constructed.Stable expression of wt IRF3 or mutations are established in Raw264.7 cell lines.By comparing,TLR3/4 induced IRF3 phosphorylation on Ser396 arise in cells stably expressing wt IRF3 or mutations which target to lysosome.Other mutations failed to be phosphorylated on Ser396 after stimulation.These data indicate that subcellular localization on lysosome is crucial for TLR3/4 induced IRF3 activation.In addition,we use amino acids depriviated cell culture medium to dissociate mTOR from lysosome before stimulation.In this instance,BMDMs is responseless to poly（I:C）because IRF3 phosphorylation on Ser396is almostly undetectable.The phosphorylation can be rescued when essential amino acids mixture are added.Accordingly,Lys M-Cre/mTOR^fl/fl mice are more susceptible to E.coli infection in peritoneal cavity than mTOR^fl/fl mice because of decreased cytokines production and increased bacteria burden.Taken together,we conclude that,there are two things which are needed for IRF3 activation in TLR3/4 signaling pathway.One is IRF3 translocation from cytoplasm to lysosome.It is mediated by mTORC1.The other is IRF3 phosphorylation by TBK1 at Ser396.Our data helps increase understanding of the molecular mechanisms between metabolism and immune responses.