Study Of Tolerogenic Spleen Dendritic Cells And Its Negative Immunoregulation In Multiple Organs Dysfunction Syndrome Mice

Objective: (1) To explore the changes of spleen dendritic cell function:immunoactivation or immunetolerogenity in MODS. (2) To confirm the effect of tolerogenicdendritic cell formation on the immune organ damage and immunosuppression.in late stage ofMODS; (3) To explore the mechanism and pathway of immunotolerance formation inducedby tolerogenic dendritic cell and the improvement of the body immune status with anti-PD-L1antibody use.Method: MODS mice model were established by intraperitoneal injection of zymosan.three hundred and twenty C57BL/6 mice were randomly divided into control(n=10 ),zymosan challenge 6 hours(n= 15), 12 hours(n=15) ,24 hours(n=15), 48 hours(n=30 ), 5day(n=30 )and 12 day(n=50 ). Serum and spleen tissue samples were harvested and frozen untilrelate experiment was done. Spleen dendritic cell and pan T cells were separated by MACSmicrobeads. The expression of CD11c, MHC-â…¡, costimulatory molecule CD86, PD-L1,PD-1 on dendritc cell and surface makers on T cell were detected by flow cytometry,immunochistochemistry method, immunoflurence labeling and RT-PCR techniques. Thecytokines in serum, spleen tissue and cell culture supernate were detected by ELISA. Theeffect of dentricit cell activating T cell was got by mixed lymphocyte reaction. Theproliferation of T cell was examined by the MTT method. Apoptotic cells of spleen weredetected by means of TUNEL and flow cytometry. Pathological changes of spleen wereobserved by light microscope.Result: The expression of MCH-â…¡and CD86 on splenic dentritic cell were increased atearly stage and acute stage in MODS. Contrast, the expression of MCH-â…¡and CD86 onsplenic dentritic cell were decreased at remission stage and late stage in MODS. Theexpression of PL-L1 on splenic dentritic cell was increased transiently at acute stage inMODS, while the expression of PL-L1 on splenic dentritic cell was increased constantly atlate stage in MODS. There was no expression of PD-1 in normal control group, but there wasexpression of PD-1 at all stages in MODS group. The positive expression rate of PD-1 increased from 8% at early stage to 40% at late stage. There was a small amout of expressionPD-1 on CD4 ~+T cell in spleen at acute stage in MODS, but the expression of PD-1 wasincreased siginificantly at late stage in MODS. The expression of tolerogenic PIR-B ondendritic cell was increased siginificantly at remission stage until to late stage in MODS. Thelevel of IL-10 and IL-12p40 secreated by splenic dendritic cell were increased and the level ofIL-12p70 decreased at late stage in MODS. After acute stage, the level of HMGB1 increasedconconstantly and reached the peak at late stage in MODS. The function of DC inducing theproliferation of T cell decreased at late stage in MODS, while dendritic cell secreated lessIL-2 and the function of T cell decreased. The number of CD4~+foxp3~+Treg increasedsiginificantly at late stage in MODS, which was related with the expression of PD-L1~+dendritic cell. Spleen white pulp took an atrophy changes, meanwhile the numbers oflymphocyte decreased significantly and lymphocyte appeared apoptosis at late stage inMODS. The T cell proliferation index and IL-12 level increased, while the number of Tregdecreased and the white pulp restored to the normal in the anti PD-L1 antibody treated group.Conclusion: 1. After analysing the splenic dendritic cell immunophenotype at late stagein MODS, the expression of MHC- and costimulatory moleculeâ…¡CD86 on spleen dendriticcell begin to decrease at remission stage and reached the lowest at late stage of MODS. Theexpression of coinhibitor PD-L1 and tolerogenic molecular PIR-B began to increase atremission stage until to late stage in MODS. Though the number of dendritic cells was morecompared with the normal control group, the function of dendritic cell changed fromexcessive activation to low activity and it turned into tolerogenic dendritic cell. It suggestedtolerogenic dendritic cell was predominantly, which was the major reason forimmunosuppression.2 .There was a high expression of PD-L1 on splenic dendritic cell, high level of IL-12p40and IL-10, high expression of PD-1 on CD4~+T, decreased number of T cell, increased numberof Treg and increased number of apoptotic cell at late stage in MODS. It suggestedtolerogenic dendritic cell may prime the PD-1/PD-L1 pathway to secret immunoinhibitor andto induce T cell angery. Tolerogenic dendritic cell played negative immunoregulatory andleaded to immunosuppression and multiple organs dysfunction at late stage in MODS.3. It was first time that the PD-1was detected on spleen dendritic cells in MODS by flowcytometry and immunfluorescence technique, and its expression was related with the functionof dendritic cell activation and tolerance formation. Normally, mouse spleen dendritic cellsdoes not express PD-1, we are the 1st to find the expression of PD-1 on spleen dendritic cell inMODS. It is speculated that the expression of PD-1 receptor on spleen dendritic cells in MODSisrelatedtothetolerogenityandthePD-L1/PD-1pathway.4. After blcoking the PD-L1 / PD-1pathway with anti-PD-L1 antibody, the contains ofIL-12p40 secreted by dendritic cell decreased and the contains of IL-12p70 by dendritic cellincreased,promotedthe proliferation of Tcell and IL-2increasing. BlockingthePD-L1/PD-1pathwaymaybeatargetforimprovingtheimmuneatlatestageinMODS.