Effects Of Major Outer Membrane Proteins Of Brucella On The Maturity And Antigen Presentation Of Dendritic Cells

Object:Brucellosis is one of the most common zoonotic diseases in the world,which seriously infected the development of animal husbandry production in China.Dendritic cells(DCs)were considered to be the most effective professional antigen presenting cells(APCs)and played an important role in the interaction between pathogens and host immunity.This study was to investigate how the main outer membrane protein of Brucella regulates the maturation of DCs,the efficiency of antigen presentation,and the ability to activate primary T lymphocytes,thereby affecting the host's innate and acquired immunity against Brucella.Which will lay a theoretical foundation for the rational design of the vaccine against Brucella and a better understanding of the pathogenic mechanism of Brucella and the protective immune response mechanism of the host.Methods:(1)the experimental group was divided into: Brucella major outer membrane proteins stimulation group;Brucella LPS stimulation group;E.coli LPS stimulation group stimulated mice BMDCs,respectively.The expression of co-stimulatory molecules and MHC molecules was analyzed by Flow cytometry.The secretion of related cytokines was detected by ELISA.The transcription level of TLRs mRNA was detected by qRT-PCR,and the ability of mouse BMDCs to activate the proliferation of allogeneic mouse splenic T cells were determined by MLRs.(2)The mouse BMDCs were infected with Brucella outer membrane protein gene deletion mutants and vaccine strain RB51,respectively.And CD40,CD80,and CD83 were detected by flow cytometry.The expression of co-stimulatory molecules and MHC molecules was analyzed by Flow cytometry.The secretion of related cytokines was detected by ELISA.The transcription level of TLRs mRNA was detected by qRT-PCR,and the ability of mouse BMDCs to activate the proliferation of allogeneic mouse splenic T cells were determined by MLRs.(3)The BMDCs were infected with Brucella major outer membrane protein-deleted strain,Brucella parental strain 2308,and Brucella vaccine strain RB51.The intracellular survival and replication was detected by CFU counts,and the effect of Brucella outer membrane protein on the apoptosis of mouse BMDCs was detected by flow cytometry.Results:(1)Flow cytometry results showed that the expression of co-stimulatory molecules and MHC molecules on the surface of BMDCs were significantly increased after the stimulation with outer membrane proteins(OMP10,OMP19 and BP26),while the expression of some surface molecules was significantly reduced after OMP25 and OMP31 stimulation of BMDCs.ELISA results showed that the secretion of IL-12,IL-6,INF-? and TNF-? in the culture supernatant of BMDCs stimulated by outer membrane proteins(OMP10,OMP19 and BP26)was significantly increased,IL-10 and IL-4 were The secretion was significantly reduced,and the results after stimulation with OMP25 and OMP31 were opposite.At the same time,qRT-PCR results confirmed that TLRs were expressed in different degrees in each group of treated BMDCs;The results of MLRs showed that BMDCs stimulated by outer membrane proteins(OMP10,OMP19 and BP26)promoted the proliferation of splenic T cells in allogeneic mice,while OMP25 and OMP31 stimulated the proliferation of T cells.(2)Flow cytometry showed that the expression of BMDCs on the cell surface was significantly increased after infection with the outer membrane protein-deleted strain and the vaccine strain RB51,and the expression of the cell surface molecules of the BMDCs was significantly decreased after the parental strain 2308 infection.ELISA results showed that the deletion strain and vaccine strain RB51 infected mouse BMDCs,IL-12,IL-6,INF-?,TNF-? secretion was significantly increased,IL-10 and IL-4 secretion Significantly decreased,the secretion of IL-12,IL-6,INF-?,and TNF-? in BMDCs was significantly reduced after infection with parental strain 2308,and the secretion of IL-10 and IL-4 was significantly increased.The results of MLRs showed that the infection of BMDCs by the outer membrane protein-deleted strain and the vaccine strain RB51 significantly increased the proliferation efficiency of splenic T cells in allogeneic mice,while the proliferation of T lymphocytes was inhibited by the BMDCs in the parental strain 2308.(3)CFU results showed that the number of CFUs in the outer membrane protein-deleted strains of BMDCs showed a decreasing trend after infection,and CFU of the vaccine strain RB51 increased significantly at 6 h after infection,then showed a downward trend,and there was a slight recovery after 48 h after infection(but statistics The difference between studies is not significant).The number of CFU in BMDCs infected with virulent strain 2308 decreased significantly at 6 h after infection,and then gradually increased,and then decreased at 48 h after infection.Apoptosis was detected by flow cytometry.It was found that all groups of BMDCs showed higher apoptotic rates,and virulent strain 2308 induced higher levels of apoptosis.Conclusion: The results of this study indicate that Brucella outer membrane proteins(OMP10,OMP19,BP26)and outer membrane protein-deleted strains and vaccine strains can induce maturation and antigen presentation of mouse BMDC,and induce lower levels of apoptosis,helping host cells recognize antigens and thus clear pathogens.The TLRs-mediated signaling pathway is involved in the maturation and antigen presentation of BMDCs induced by Brucella outer membrane proteins.In order to laying a foundation for designing a rational vaccine against Brucella,As well as better understanding of Brucella's pathogenesis and protective immune response mechanisms in the host.