Study Of The Regulatory Role And Mechanism Of Spleen Dendritic Cells And Its Function In Multiple Organs Dysfunction Syndrome By Ubiquitination

Objective:To study the changes of spleen dendritic cells(DC) and the ubiquitin-dependent effects of MHC-Ⅱ protein in the course of multiple organ dysfunctionsyndrome(MODS). To confirm the influences of MHC-Ⅱ and spleen DC in the courseof MODS by the ubiquitin-proteasome pathway. To explore the ubiquitinationregulatory mechanisms of MHC-Ⅱ in spleen DC during the first strike period ofMODS by the E3ubiquitin ligase enzyme MARCH1. To observe the influences ofsplenic DC immune function during the first strike period of MODS by theintervention of MARCH1, and to find a new target for the immune regulation andprevention of MODS.Methods: MODS mice model were established by intraperitoneal injection ofzymosan.120mice were randomly divided into normal control group and the MODS1d,3d,5d,7d and10d groups. Spleen and thymus tissue samples of each group wereisolated, part of the spleen tissue samples were used to detect the changes of thenumber of CD11c+DC by immunofluorescence method, and the rest tissue sampleswere used to separate Spleen DC and T cells for cytological experiments in vitro byMiniMACS microbeads. The expression of CD86, MHC-Ⅱ, ubiquitin and E3ubiquitin ligase (MARCH1-8) on DC, the levels of IL-12and TNF-α secreted by DC,the proliferative activity of T cells and the levels of IL-2and IFN-γ secreted by T cellswere detected by flow cytometry, Real-Time PCR, Western blot and MTT method.Then the ubiquitination levels of MHC-Ⅱ proteins in DC of the normal control groupand MODS1d group were detected by co-immunoprecipitation and western Blot.After treating the two groups with MG132respectively for6h, we detected theapoptosis rate of DC, the expression of MHC-Ⅱ protein on DC, the proliferative activity of T cells and the levels of IL-2, IFN-γ and TNF-α secreted by T cells by PIstaining, Real-Time PCR, Western blot and MTT method. Cellular cDNA wasextracted from the tail of mouse as a template. The MARCH1cDNA was synthesizedby PCR with the specific primers. After restriction endonuclease EcoRⅠand BamHⅠdouble digested it,the MARCH1cDNA was cloned in pMB-HA vector to constructrecombinant vectors. The result of recombinant vector was identified usingdouble-digestion and DNA sequencing. Then we transformed the recombinant vectorinto competent E.coli DH5α cells, screened positive clones, extracted plasmid, andpurified it. The pMB-HA-MARCH1expression vectors and siRNA were transfectedinto DC of MODS1d group. The morphology, viability and apoptosis rate of DC, theexpression of MHC-Ⅱ proteins on DC, the proliferative activity of T cells and thelevels of IL-2, IFN-γ and TNF-α secreted by T cells were detected by MTT, PIstaining, flow cytometry, Real-Time PCR, co-immunoprecipitation and Western Blotin MODS1d group, MARCH1siRNA knock down group and MARCH1overexpression group.Result:The number of CD11c+DC in the course of MODS was graduallyreduced. The expression of CD86and MHC-Ⅱ proteins on DC, the levels of IL-12and TNF-α secreted by DC, the proliferative activity of T cells and the levels of IL-2and IFN-γ secreted by T cells were increased first, and reached a maximum value inthe MODS1d group, thereafter decreased to the lowest point in the MODS period, butthe mRNA levels of MHC-Ⅱ and ubiquitin were not significant. The changes of E3ubiquitin ligase enzyme MARCH1were extremely significant. The levels of mRNAand protein expression of MARCH1were decreased first and reached a minimumvalue in the MODS1d group, then after a slight increasing trend they were decreasedagain in the MODS10d group. The trend of MHC-Ⅱ and MARCH1was roughly theopposite in the course of MODS. Compared with normal control group the degree ofspleen DC MHC-Ⅱ protein ubiquitination in the MODS1d group was significantlylower. The change of apoptosis rate of DC in the groups treated by MG132was notsignificant. The expression of MHC-Ⅱ proteins on DC, the proliferative activity of Tcells and the levels of IL-2, IFN-γ and TNF-α secreted by T cells in the MODS1dgroup and MG132treated normal control group were higher than the normal controlgroup, where the former is slightly higher than the latter, and the MODS1d grouptreated by MG132was the highest. We successfully constructed the eukaryoticexpression vector pMB HA-MARCH1of the E3ubiquitin ligase MARCH1, and synthesized three effective siRNA of MARCH1. We interfered DC in the MODS1dgroup with overexpression MARCH1and siRNA, and found that the morphology,vitality, and apoptosis rates of DC in the three groups had no significant changes.Thedegree of ubiquitination of MHC-Ⅱ was increased, and the expression of MHC-Ⅱproteins on DC, the proliferative activity of T cells and the levels of IL-2, IFN-γ andTNF-α secreted by T cells were reduced by interfered DC in the MODS1d group withoverexpression MARCH1. While the degree of ubiquitination of MHC-Ⅱ was reduced,and the expression of MHC-Ⅱ proteins on DC, the proliferative activity of T cells andthe levels of IL-2, IFN-γ and TNF-α secreted by T cells were increased by interferedDC in the MODS1d group with MARCH1siRNA.Conclusion:1,The number of DC was decreased, but the antigen-presentingability and the capability to activate T cell-mediated immune response of DC wereenhanced during the first strike period of MODS; the number and the immunefunctions of DC dropped to the lowest and the organism was in a state of immunesuppression during the MODS period.2,MARCH1can degrade the expression ofMHC-Ⅱ on spleen DC by ubiquitination in the course of MODS.3,The change ofMHC-Ⅱ on spleen DC in MODS was really ubiquitin-dependent.4,We successfullyconstructed the eukaryotic expression vector pMB HA-MARCH1of the E3ubiquitinligase MARCH1, and synthesized three effective siRNA of MARCH1.5,Interferingthe expression of MARCH1can change the expression of MHC-Ⅱ on DC and theimmune function of DC by degrading MHC-Ⅱ through ubiquitination in the firststrike period of MODS. It suggested that we can use MARCH1as a potentialtherapeutic target for MODS.