Studies On The Mucosal Immune Mechanism Mediated By Dendritic Cells Stimulated With Porcine Rotavirus

Rotavirus（RV）infection is a global heath problem,researchers from all over the world had numerous studies on infection characteristics,immune response and vaccine development of rotavirus.Evidence is accumulating that RV infection of the body’s first invasion of intestinal epithelial cells and triggers mucosal immune responses,however,little is known about the mucosal immune mechanisms.Mucosal immune is a bridge connecting innate and adaptive immune,and dendritic cells（DCs）which are professional antigen-presenting cells（APCs）serve as the key immune molecules.We utilized PRV to stimulate mouse bone marrow-derived dendritic cells（BMDCs）and gastric BALB/c mice,explored the Toll-like receptors which is regarded as one typical PAMP on the surface of DCs.Furthermore,we explored the signal transduction mechanism in TRIF^-/-mice and BMDCs pretreated with Pepinh-TRIF in vivo.The interaction between BMDCs and PRV was investigated in vitro using flow cytometry,Quantitative real-time PCR,ELISA and western blotting analysis.The experimental results showed:（1）PRV could induce the activation and maturation of BMDCs,up-regulated the levels of MHC II and costimulatory molecules CD40,CD80 and CD86.（2）Toll-like receptor 2（TLR2）and TLR3（rather than TLR4 and TLR8）were involved in immune responses after PRV stimulation.（3）TLR2 and TLR3 got involved in the activation of NF-κB and cytokine（IL-6,IL-10,IL-12,IFN-γ）secretion by BMDCs through TRAF3 or TRAF6.（4）The PRV-stimulated BMDCs also promote the proliferation of T lymphocytes and induce the differentiation of na?ve T cells into Th1 cells.BALB/c mice were divided randomly into three groups:PBS control,PRV and poly（I:C）group.The activation,subgroup differentiation and cytokines secretion of DCs infected with PRV were investigated.The experimental results showed:（1）The expression of MHC II and costimulatory molecules（CD40,CD80 and CD86）increased in DCs of spleen and MLN.The differentiation of CD11c⁺CD11b⁺CD8α⁺,CD11c⁺CD11b⁺CD8α^-,CD11c⁺B220⁺CD8α⁺and CD11c⁺B220⁺CD8α^-was observed.（2）The mRNA levels of TLR2,TLR3,MyD88 and NF-κB in MLN and small intestine increased gradually with time elapsed after PRV infection.（3）In the early stage of viral infection,IL-10 and IL-6 secretion took up the majority in spleen and MLN,but Th1 cytokines（IL-12 and IFN-γ）were secreted mainly in late stage.The interaction between Pepinh-TRIF pretreatement BMDCs and PRV was investigated using flow cytometry,Quantitative real-time PCR and ELISA in vitro.The experimental results indicated:（1）Blocking TRIF signaling could down-regulate the levels of MHC II,CD40,CD80and CD86,then inhibited activation and maturation of BMDCs.（2）Down-regulated TRAF3 and TRAF6 could inhibit NF-κB activation,weakened the ability to induce the T lymphocytes proliferation after blocking TRIF signaling.（3）Blocking TRIF signaling could inhibit cytokines secretion of BMDCs and weak the differentiation ability of T lymphocytes by BMDCs.BALB/c and TRIF^-/-mice were divided randomly into six groups:TRIF^-/-PBS control,TRIF^-/-PRV,TRIF^-/-poly（I:C）,WT PBS control,WT PRV and WT poly（I:C）group.The activation,subgroup differentiation and cytokine secretion of DCs of TRIF deletion infected with PRV were investigated.The experimental results indicated:（1）Differentiation ablity of DCs into CD11c⁺CD11b⁺CD8α⁺DCs,CD11c⁺CD11b⁺CD8α^-DCs,CD11c⁺B220⁺CD8α⁺DCs and CD11c⁺B220⁺CD8α^-DCs was attenuated.Activation and maturation of DCs in MLN were inhibited to some certain extent.（2）Inhibited the expression levels of TRAF3 and NF-κB mRNA,while MyD88 and TRAF6 mRNA expression were hardly influenced.（3）The secretion of IL-6,IL-10,IL-12 and IFN-γwere abated in MLN of TRIF^-/-mice infected with PRV which affected the activation of T lymphocytes.The research deeply explored the mechanism for the interaction between PRV and DCs.PRV could trigger DCs to activate NF-κB through TLR2/MyD88 signaling pathway or TLR3/TRIF signaling pathways.PRV is also involved in IL-6,IL-10,IL-12 and IFN-γsecretion in the immune response.Blocking TRIF signaling or TRIF deletion could inhibit the activation and maturation of DCs stimulated with PRV,inhibited the TRAF3/NF-κB signaling pathway and hardly influenced MyD88/TRAF6 signaling pathway.Further studied on the mucosal immune mechanisms of DCs stimulated with PRV is of a great significance for understanding the PRV infection pathogenesis,then we could contrive an effective prevention against PRV infection.