Role Of MTOR In Regulating Pro-inflammatory Cytokines In TLR Signaling Pathway

Objectives：To investigate regulation of mTOR kinase in TLR signaling pathway in miceprimary cells, seeking for corresponding key target molecules, probing molecularregulatory mechanism. Providing targets for further investigation of TLR signalingpathway regulation and mTOR associated diseases therapy.Methods：Bone marrow derived dendtric cells, peritoneal macrophage and bone marrowderived macropgage were isolated and purificated from C57mice. Total RNAs wereextracted from BMDC upon LPS stimulated TLR4signaling with rapamycin pretreatmentfor30min, then multiple proinflammatory cytokines on mRNA level were screened andidentified mTOR targeted genes by qPCR. IL-10, IL-12, IL-23, CXCL1&TNFα weredetected in pMΦ and BMDM of mice according to former screened results in different timecourse. pMΦ was stimulated with LPS or R848alone and combined with R848respectively,then the protein levels of IL-10, IL-12&IL-23were detected by ELISA after inhibitingmTOR. Total proteins were extracted from pMΦ pretreated with rapamycin for30min，theninduced by LPS on1h,2h,4h,8h respectively, the activation of MAPKs families（ERK1/2、JNK1/2/3、p38）, IKKα/β, IκBα kinases and IRF3were detected by western bolt. Nucleartranslocation of transcription factors were detected from isolated nuclear proteins aftermTOR was blocked and phosphorylation of S6K1&4E-BP1were checked on total proteinlevel. The lentiviral pLKO.1-cMaf and pLKO.1-stat3vectors were constructed andtransiently transfected into293T along with packaging plasmids (pSPAX2, pMD2G) togenerate lentivirus, then were used to infect BMDM to knockdown cMaf and stat3, andcytokines productions of IL-10, IL-12&IL-23were detected by ELISA.Results：The mRNA levels of IL-12, IL-23, IL-10, CXCL1&TNFα and other moleculeswere detected after inhibited mTOR with rapamycin in TLR4signaling pathway. Theresults indicated that mTOR specifically promoted IL-10but inhibited IL-12and IL-23expression. While stimulated with LPS or R848alone or combined with LPS and R848, theresults shown that the mTOR regulated level of cytokines in combined stimulatory groupwere more significant than those of single group, moreover, regulation of mTOR shown dose dependent manner with rapamycin treatment. The protein levels shown that mTORdidn’t involve in activation of MAPK kinases (ERK, JNK, p38) and NF-κB signaling andits upstream kinases IKKα/β, IκBα, but also mTOR didn’t affect the activation of type interfon regulatory factors and its downstream genes IFNβ and CXCL10expression.Detection of nuclear proteins suggested that mTOR exerted less affection on nucleartranslocation of ATF2, β-catenin and other transcription factors but significantly affectedcMaf and stat3. Furtherly checked total protein levels of cMaf and stat3shown that cMafprotein synthesis and stat3phosphorylation were greatly impaired after inhibited mTOR,but didn’t affect protein synthesis of stat3. The cytokines expression level were measured incMaf knockdown BMDM triggered with LPS combined with R848suggested thatIL-12p40and IL-12p70productions were significantly enhanced while mTOR inhibition,moreover, IL-10expression was impaired but not IL-23p19. In stat3knockdown BMDM,productions of IL-10, IL-12p40, p70as well as IL-23p19were enhanced compared to wildtypes BMDM.Conclutions：1. mTOR specifically promoted proinflammatory cytokines IL-10but inhibited IL-12、IL-23expression in TLR4&TLR7/8signaling pathway.2. mTOR didn’t involve in activation of MAPK kinases (ERK、JNK、p38) and IKK kinasessignaling in TLR4signaling pathway.3. mTOR promoted IL-10but restrained IL-12expression through controlling translation oftranscription fator cMaf，inhibited IL-10, IL-12&IL-23expression by contributing to stat3phosphorylation in TLR4&TLR7/8signaling pathway.