Effect Of Endosome Acidifying Maturation Dysfunction On LPS-Activated Macrophages And Its Mechanism Study

Sepsis is systemic inflammatory response syndrome (SIRS) with an infectious origin.Sepsis may progress into septic shock and multiple organ dysfunction syndrome (MODS)which ultimately threaten the pathients’ lives. Lipopolysaccharides (LPS), one componentof the cell walls of Gram-negative bacteria, are regarded as the major pathogen associatedmolecule pattern (PAMP) which leads to sepsis. LPS is transferred to TLR4which is thepattern recognition receptors (PRR) for LPS, then LPS-TLR4complex activate theMyD88-dependent and TRIF/TRAM-dependent inflammatory signal transduction pathway,then the expression of inflammatory cytokines was upregulated in innate immunecells.Over expression of inflammatory cytokines could cause sepsis.Internalization is the cell process that extracellular material transport into cell throughendocytosis.After internalization, LPS-TLR4complex located in early endosome,accompanying acidifying maturation of endosome, LPS-TLR4complex then translocated tolysosome for degradation. Recent researches show that internalization of LPS-TLR4has avery tight relationship with the activation of monocytes/macrophages. Our recentresearches also show that when internalization was inhibited, the inflammatory responsewas depressed, but the mechanism is unclear. We also observed that with CQ pretreatment,LPS-TLR4complex dislocated after internalization. This is contrary with the previouscognitive, the relationship between this phenomenon and LPS internalization and activationof macrophages is not known yet.So we took this study to investigate the Effect of endosome acidifying maturationdysfunction on LPS-activated Macrophages and its Mechanism.ObjectUse Chloroquine as an inhibitor of endosome acidifying maturation, to investigate theeffect of endosome acidifying maturation dysfunction on LPS-activated Macrophages andits Mechanism.Methods 1. Investigate the effect of endosome acidifying maturation dysfunction onLPS-activated Macrophages.(1)RAW264.7cells were incubated in the presence or absence of CQ(20μg/ml) for1hour, then stimulated with LPS (100ng/ml) for24h, then TNF-α and IL-6secretion in thecell-free culture supernatants was evaluated using ELISA.(2) Using siRNA interference technology to knockdown MyD88and TRAM inRAW264.7cells, then stimulated with LPS (100ng/ml) for24h, then TNF-α and IL-6secretion in the cell-free culture supernatants was evaluated using ELISA.(3)Cytokine secretion in the cell-free culture supernatants was evaluated usingCytokine antibody array, in the MyD88and TRAM dysfunction model cells andCQ(20μg/ml) incubated cells stimulated by LPS (100ng/ml) for24h. And then draw ancomparative analysis.2. Investigate the mechanism of endosome acidifying maturation dysfunction onLPS-activated Macrophages.(1) Investigate the internalized LPS-TLR4complex dislocation phenomenon inducedby CQ in RAW264.7cells.①Using CQ(20μg/ml) and NH4CL (500μg/ml) to inhibit endosome acidifyingmaturation of RAW264.7cells, then stimulated by FITC-LPS (200ng/ml) for1h, observethe colocation of FITC-LPS and Cy3-TLR4after internalization by confocal laser scaningmicroscopy.②Using CQ(20μg/ml) to inhibit endosome acidifying maturation of RAW264.7cells,then stimulated by6FAM-CpG DNA (100ng/ml) for30min, observe the colocation of6FAM-CpG DNA and Cy5-TLR9by confocal laser scaning microscopy.③Observe LPS-TLR4colocation and dislocation on live.Transfect TLR4::YFP plasmid to HEK293cell, and observe the colocation ofFITC-LPS and TLR4YFPby confocal laser scaning microscopy on live. Observe LPS-TLR4colocation and dislocation induced by CQ in different time piont.④Investigate the effect of endosome acidifying maturation dysfunction on theexpression of endosome cycling regulate moleculars.Using CQ(20μg/ml) to inhibit endosome acidifying maturation of RAW264.7cells,then incubation with LPS (100ng/ml) for4h, the total RNA were extracted and the Real time-PCR assay was applied to check the mRNA expression of Rab7b、Rab10and Rab11a.(2) Investigate the effect of endosome acidifying maturation dysfunction on theexpression of inflammation negative regulate moleculars of RAW264.7cells.Using CQ(20μg/ml) to inhibit endosome acidifying maturation of RAW264.7cells,then incubation with LPS (100ng/ml) for4h, the total RNA were extracted and the Realtime-PCR assay was applied to check the mRNA expression of A20、MyD88s and Tollip.Results1. Investigate the effect of endosome acidifying maturation dysfunction onLPS-activated Macrophages.(1)ELISA data indicated that pretreatment of RAW264.7cells with CQ, the expressionof IL-6and TNF-α sitimulated by LPS in protein level significantly decreased (P<0.01).(2) ELISA data indicated that after siRNA knockdown MyD88and TRAM expressionin RAW264.7cells, the expression of IL-6and TNF-α sitimulated by LPS in protein levelsignificantly decreased (P<0.01). Especially, when MyD88was knocked down, theexpression of TNF-α decreased more significant than IL-6, and when TRAM was knockeddown, the expression of IL-6decreased more significant than TNF-α.(3) Results of cytokines arrays showed that pretreatment with CQ could inhibited theexpression of all cytokines or chemokines of RAW264.7cells induced by LPS. After siRNAknockdown MyD88and TRAM expression in RAW264.7cells, the expression of cytokinesor chemokines induced by LPS decreased almost the same way that pretreatment with CQ.2. Investigate the mechanism of endosome acidifying maturation dysfunction onLPS-activated Macrophages.(1) Investigate the internalized LPS-TLR4complex dislocation phenomenon inducedby CQ in RAW264.7cells.①After endosome acidifying maturation was inhibited by CQ or NH4CL, thedislocation of FITC-LPS and Cy3-TLR4after internalization was observed by confocallaser scaning microscopy.②After endosome acidifying maturation was inhibited by CQ, the intracellulardislocation of6FAM-CpG DNA and Cy5-TLR9was observed by confocal laser scaningmicroscopy.③Observe LPS-TLR4colocation and dislocation on live. The transfection of TLR4::YFP plasmid to HEK293cells was failed.Observe LPS-TLR4colocation and dislocation induced by CQ every10min in1hour,the colocation phenomenon reduced gradually, and disappeared after40min.④Investigate the effect of endosome acidifying maturation dysfunction on theexpression of endosome cycling regulate moleculars.Real time-PCR data shows that after LPS sitimulation, the mRNA expression ofRab7b increased significantly(P<0.05), and the mRNA expression of Rab10and Rab11adecreased significantly(P<0.05). When pretreatment with CQ, the mRNA expression ofRab7b decreased significantly(P<0.05), and the mRNA expression of Rab10and Rab11aincreased significantly(P<0.05) sitimulated by LPS.(2) Investigate the effect of endosome acidifying maturation dysfunction on theexpression of inflammation negative regulate moleculars.Real time-PCR data shows that after LPS sitimulation, the mRNA expression of A20，MyD88s and Tollip increased significantly(P<0.05), When pretreatment with CQ, themRNA expression of A20，MyD88s and Tollip increased more significantly(P<0.05)compared with non-CQ pretreatment.Conclusions(1) Endosome acidifying maturation dysfunction could inhibit MyD88-dependent andTRIF/TRAM-dependent signaling transduction pathway activited by LPS-TLR4.(2)Endosome acidifying maturation dysfunction could lead to LPS-TLR4complexdislocated and scattered in RAW264.7cells. Moreover, related inflammationnegative-regulate moleculars’ expression upregulate, which could be tightly related with theinhibition of MyD88-dependent and TRIF/TRAM-dependent signaling transductionpathway when endosome acidifying maturation is blocked.(3) The mechanism that endosome acidifying maturation dysfunction leads toLPS-TLR4complex dislocated may be: Endosome acidifying maturation could not be theonly factor for receptors dislocated from ligands, there may have some other factors workedtogether. Endosome acidifying maturation dysfunction could influence the whole endosomematuration program and the normal activity of receptors and ligands in endosomes. Becauseof Rab7b decreased, the fusion of endosome and lysosome was blocked, LPS-TLR4complex could not degradate normally after separate, also due to Rab10and Rab11a increased, the transport between early-endosome and golgi and the transport fromrecycle-endosome to early-endosome are inhanced, TLR4couldn’t transport back to cellmembrane to continue its work normally, TLR4circulation blocked, finally TLR4andLPS accumulated, scattered and dislocated as we observed. The molecular mechanismneeds a lot of work to do.