Inhibitory Effect Of CCT-ODN On Ligand-induced Intracellular Trafficking Of TLR7/9and Its Possible Mechanism

Toll-like receptor (TLRs) detect infections by highly conserved components ofpathogens that are either not present in our own cells or are normally sequestered incellular compartments that are inaccessible to the TLRs. Most TLRs are expressed onthe cell surface, where they have been shown to detect pathogen-expressed molecules.A subset of TLRs, including TLR3,7,8, and9, are expressed intracellularly withinone or more endosomal compartments and detect nucleic acids. Amulti-transmembrane protein found in the ER at steady state, Unc93B1, controlstrafficking of all endosomal TLRs, TLR3,7,8, and9. Because pathogen and hostnucleic acids have very similar structures, these endosomal TLRs may face an extrachallenge to induce anti-pathogen immune responses while avoiding the induction ofautoimmune diseases.B cells and plasmacytoid DCs express TLR7and TLR9in human. At steady state,the majority of TLR7and TLR9reside in the ER in cells expressing WT Unc93B1.Upon TLR stimulation, TLR9is preferentially transported through the earlyendosome to the acidified endosomes by Unc93B1, whereby it is cleaved to becomecompetent for signaling. TLR7and TLR9are active only once they are cleaved in theacidified ‘‘signaling endosomes’’ by endosomal proteases. Once within the signalingendosomal compartment, TLR9and TLR7recruit the adaptor protein MyD88andtrigger signals leading to inflammatory cytokine expression through NF-κB activation.In contrast, D34A mutant Unc93B1transports TLR7, but not TLR9, to the signalingendosome even in the absence of external stimulation.As we know, CCT-ODN with the sequence of human microsatellite DNAcontained CCT repeats can suppress TLR9more powerfully than TLR7, but it cannotinhibit TLR3and TLR4. Therefore, we can forward a hypothesis that CCT-ODN maysuppress the activity of pDCs by targeting Unc93B1resulting in the trafficking ofTLR7and TLR9. The CCT-ODN mediated inhibition on induced TLR7/9trafficking possibly through trapping Unc93B1and making it unable to carry TLR7of TLR9tomove.1. Effect of CCT-ODN on intracellular trafficking of TLR7and TLR9In present work, to understand the molecular mechanism about how theCCT-ODN down regulates TLR7and TLR9activation, we contrast stained forTLR7/9and endoplasmic reticulum (ER) through indirect immunofluorescencetechnique and studied the effect of CCT-ODN on the intracellular trafficking of TLR7and TLR9in human pDCs. The result showed that CCT-ODN could inhibit theintracellular trafficking of TLR7and TLR9induced by ligands. Then, mousesplenocytes were used to repeat the experiment mentioned above. The result revealedthat CCT-ODN could also effectively inhibit TLR9trafficking in the mousesplenocytes when activated by2006, which was similar with that in CAL-1cells.2. Effect of CCT-ODN on intracellular trafficking of Unc93B1Unc93B1, an ER protein with12membrane-spanning domains, could selectivelyassociate with and transport TLR7and TLR9from ER to endolysosomes uponTLR7/9activation. To explain the possible mechanism of CCT-ODN for inhibitingTLR7/9trafficking, CAL-1cells were cultured with TLR9ligand (CpG2006) orTLR7ligand (Imiquimod) in the absence or presence of CCT-ODN, respectively, for2hours and then fixed on slides. After staining with anti-Unc93B1and Calnexinantibodies followed by fluorescence labeled secondary antibodies, the slides weredetected under confocal microscope. The result demonstrated that both TLR9ligandand TLR7ligand could induce Unc93B1transport away from ER. Compared theeffect of2006and Imiquimod on inducing Unc93B1movement,2006displayed morestrong role than Imiquimod. CCT-ODN could hold Unc93B1in ER during TLR7/9activation. Thus, SAT05f could inhibit TLR7/9trafficking possibly through trappingUnc93B1.3. CCT-ODN was unable to bind to ER and Unc93B1To find whether CCT-ODN was capable of targeting ER or Unc93B1, we labeledSAT05f at3’-end with Cy3and conducted up-taking assay in CAL-1cells usingSAT05f-Cy3. CAL-1cells were cultured with CCT-ODN-Cy3at37°C for60minutesat dose of8ug/ml, and then stained with either ER-Tracker Green or Alexa Fluor488 labeled anti-Unc93B1for ER or Unc93B1. MS19was as a negative control ODN. Weexamined the intracellular localization of CCT-ODN-Cy3and Unc93B1by confocalmicroscopy. The result showed that CCT-ODN-Cy3and Unc93B1couldn’t belocalized in the same site in ER, indicating that CCT-ODN inhibited TLR7/9trafficking by targeting Unc93B1indirectly.4. CCT-ODN up-regulated the TLR7and TLR9mRNA expression in CAL-1cellsTo find whether CCT-ODN could influence the mRNA expression levels ofhuman TLR7and TLR9in CAL-1cells, CAL-1cells were cultured in the absence orpresence of agonists of TLR7and TLR9for2and24hours, the total RNA of CAL-1cells was extracted after lysis of cells. Human TLR7and TLR9mRNA expressionwas measured using quantitative real-time RT-PCR, and the expression levels ofhuman TLR7and TLR9were normalized to those of human GAPDH. The resultsshowed that CCT-ODN up-regulated TLR7and TLR9mRNA expression in CAL-1cells after stimulating2or24hours. In addition, TLR7/9activation is an extremelycomplex balance between the production of inflammatory and anti-inflammatorycytokine. To examine whether CCT-ODN inhibited activation of TLR7/9byup-regulating inhibitory cytokine containing IL-10, IL-10mRNA expression wasmeasured using quantitative real-time RT-PCR. The results showed that SAT05fdisplayed no obvious effect of IL-10in CAL-1cells after stimulating24h. The dataindicated that CCT-ODN suppressed the activation of TLR7and TLR9not byup-regulating of IL-10.5. Effect of CCT-ODN on the expression of TLR9on CAL-1cell surfaceCCT-ODN could inhibit intracellular trafficking of TLR9in human pDCs, andcould up-regulate the expression level of TLR9. The inhibition of TLR9intracellulartrafficking causes the suppression of downstream signaling pathway. TLR9have notbeen consumed, may be not need to initiate upstream transcriptional activation tosupplement TLR9. But the result showed CCT-ODN could up-regulate TLR9expression. Based on the up-regulation, we proposed new expression TLR9shouldnot be accumulated in ER and select other pathways. Related reports indicated that TLR9could be detected on cell surface. We uncertain whether CCT-ODN can induceTLR9to transfer to cell surface different from the pathway of CpG ODN trafficking.In order to explain the possibility, we detected the effect of CCT-ODN on theexpression of TLR9on CAL-1cell surface using Flow Cytometry. The result showedTLR9could be not detected on CAL-1cell surface, and CCT-ODN could not induceTLR9to transfer to CAL-1cell surface.6. Effect of CCT-ODN on the mRNA expression level of TLR7/9-mediatedsignaling pathway related moleculesTo investigate the effect of CCT-ODN on TLR7/9-mediated innate immunitysignaling activation, the research has detected influences of CCT-ODN onTLR7/9-mediated signaling pathway related molecules. The results indicatedCCT-ODN could up-regulate classical signaling pathway transcription factors, such asNF-κB, IRF-7and IFN-mRNA expression level, but no significance significantlyinfluence on TNF-. Additionally, we have detected the effect of CCT-ODN onmRNA expression of upstream related molecules of IRF-7signaling pathway,including STING-dependent DNA sensors: DAI, Lrrfip1, IFI16and Trex1, othersabout STING-independent DNA sensors: AIM2and RNA poly III. The resultsindicated CCT-ODN could act on cytosolic DNA sensors.7. Structure-activity relationship of CCT-ODN on CpG2006-induced TLR9traffickingTo explore the structure-activity relationship of CCT-ODN, CCT-ODN and itsderivatives were tested for their inhibitory effect on TLR9trafficking induced byCpG2006in CAL-1cells. Besides CCT-ODN with8CCT motifs, based on thebackbone structure of CCT-ODN, the number of CCT motif in (CCT)2,(CCT)6,(CCT)10and (CCT)12are changed to2,6,10and12, respectively;3’ end CCT motifin (CCT)6-(AAG)2and (CCT)4-(AAG)4are substituted with two or four AAG,respectively;(AAG)2-(CCT)6and (CCT)3-(AAG)2-(CCT)3by substituting two CCTmotifs with AAG at5’ end or in the middle of (CCT)8; and (AGG)2-(CCT)6and(CCT)6-(AGG)2by substituting CCTCCT at5’-end or3’-end, respectively, withAGGAGG to form hairpin. The result showed that (CCT)6-(AAG)2and (CCT)3-(AAG)2-(CCT)3could induce TLR9trafficking, whereas (AGG)2-(CCT)6and(CCT)6-(AGG)2displayed the strongest effect on inhibiting TLR9trafficking inducedby CpG2006among CCT-ODN and its derivatives.8. Effect of the derivatives of CCT-ODN on STING-dependent and–independentpathway related molecules expression levelDue to there are different effects of the derivatives of CCT-ODN on TLR9intracellular trafficking, the characteristics of the allosteric CCT-ODNs are different.Subsequently, we will confirm the effects of the allosteric CCT-ODNs on TLR7/9gene expression level. The derivatives were tested for their roles on TLR7/9expression level by real time PCR. The results showed that the tendency of thederivatives represented accordance on TLR7/9expression level, but there aredifferences in inhibitory intensity. Accordingly, we detected the effect of the ODNs onSTING-dependent and-independent pathway related molecules expression level. Theresults indicated (CCT)2,(CCT)10,(CCT)4-(AAG)4,(AAG)2-(CCT)6,(CCT)3-(AAG)2-(CCT)3,(CCT)6-(AGG)2and (AGG)2-(CCT)6could activateSTING-dependent signaling pathway, but (CCT)2,(CCT)12and (CCT)4-(AAG)4couldalternatively activate AIM2-mediated STING-dependent signaling pathway.Thus, the inhibitory activity of CCT-ODN may be by blocking the movement ofUnc93B1, resulting in TLR7/9lacking in endolysosomes. To overcome the lacking,the pDCs adoptively reacts in two ways. One is to express more TLR7/9mRNAs totry to fill up the TLR7/9lacking in endolysosomes. Another is to initiate analternative STING-dependent signaling pathway through IRF-7that is activated byup-stream signaling DNA sensors capable of recognizing and binding cytosolic DNAmolecules.