The Antigen-presenting Machanisms Of Dendritic Cells Pulsed With Recombinant VP1-VP4Protein Of FMDV

Foot-and-mouth disease (FMD) is an acute, febrile and highly contagious viral diseaseof cloven-hoofed animals caused by FMD virus. There is no safe and effective controlapproach for this disease yet. Experimental results show that dendritic cells (DCs) were notlargely susceptible to infection by FMDV. However, binding specific antibodies to FMDVcan not only infect DCs, resulting in the loss of antigen-presenting function, but also causethe high levels of DCs’ death, while exposing DCs to immune complex containinginactivated FMDV resulted in significantly increased T cell stimulation. Therefore,attention has been shifted onto the mechanisms of cellular immune response to FMDV. Theinitiation of cellular immune response needs DCs as antigen presenting cells, but suchstudies have not been documented at present.To address these issues mentioned above, pET32a-VP1-VP4prokaryotic expressionsystem was constructed. Recombinant VP1-VP4protein was obtained and labeled withFITC. Bone marrow-derived dendritic cells (BMDCs) of BALB/c mice were pretreatedwith different combinations of mannose receptor (MR) inhibitor, scavenger receptor (SR)inhibitor, macropinocytosis inhibitor, Hsp90inhibitor, proteasome inhibitor, lysosomalinhibitor, and recycling endosome inhibitor, respectively, and then pulsed with purifiedVP1-VP4protein. Confocal microscopy, flow cytometry and ELISA were employed toanalyze the dynamics of VP1-VP4uptaking, processing and presenting in BMDCs.Observation with confocal microscope showed that VP1-VP4-FITC was internalizedby BMDCs and stored in early endosomes. The amount of intracellular antigen increasedwith time and endosome was enlarged gradually. However, endosomes containingVP1-VP4were dramatically reduced when BMDCs macropinocytosis was inhibited,indicating that macropinocytosis was the main way of FMDV VP1-VP4internalization.Enhanced VP1-VP4internalization was found at different time points after administrationof MR and/or SR inhibitor, which indicated that the VP1-VP4recognition by MR or SR onBMDCs is able to suppress antigen uptake. VP1-VP4-FITC antigen accumulated inlysosomes when hydrolysis of VP1-VP4in lysosomes was inhibited. These resultsshowed that the lysosomal processing of VP1-VP4protein in the BMDCs plays animportant role. Of noting, VP1-VP4accumulation within BMDCs increased obviously when proteasome or Hsp90was inhibited. And this pattern of gathering increased moresignificantly when blocked Hsp90and proteasome simultaneously, even appeared singlegiant vesicle in one whole cell. This implies that Hsp90played a vital role in thetransportation of VP1-VP4from endosomal compartments into cytosol.BMDCs pulsed with VP1-VP4protein were co-cultured with T lymphocytes, anddetermined by flow cytometry. The results showed that MHC II positive proportionincreased while CD11c positive proportion had a small decline, indicating that BMDCscould be activated by VP1-VP4. When lysosomal enzymes were inhibited, MHC IIexpression had limited variation while CD11c expression was reduced. Importantly, almostall BMDCs expressed MHC II molecules after the block of recycling endosome. In thegroup which recycling endosomes and lysosomal enzymes were blocked at the same time,BMDCs presented much higher level of MHC II molecules, while CD11c expressionobviously increased. Sueprisingly, BMDCs from all groups expressed very low level ofCD80, indicating that some other pathway may participate in the degradation of VP1-VP4in BMDCs.ELISA assay results showed that the supernatant of BMDCs paused with VP1-VP4and co-cultured with T cells contained great quantity of IFN-γ and IL-17, but the contentsof IL-4and TGF-β1were very low. IFN-γ levels decreased strikingly (P<0.01) regardlessof inhibiting Hsp90, preteasomme or lysosome, indicating that they all playedcorresponding roles in VP1-VP4antigen presention. Notably, BMDCs pretreated with200nmol/mL of novobiocin became powerful to stimulate T cell release of higher IFN-γ levelcomparing with those pretreated with1μmol/mL (P<0.01), but perform the contrary resultwhen the proteasome inhibitor added at the same time. Collectively, VP1-VP4storage inendosomal compartments can be degraded and presented through the lysosomal pathway,producing high IFN-γ level. Primaquine blocks the recycling endosome transporting to cellsurface. On the premise of inhibited proteasomes, BMDCs pretreated with100nmol/mL ofprimaquine released much less IFN-γ than the BMDCs pretreated with50nmol/mL ofprimaquine. These data indicated the importance of cross-presenting pathway in BMDCspresenting FMDV VP1-VP4protein.IL-17appeared extremely high concentration except the groups pretreated withchloroquine and almost peaked within48h. But the contents of IL-17were equipotentbetween these groups at time points72h and96h (P＞0.05). Otherwise the contents ofIL-17were consistent with the contents of IFN-γ. Notably, the concentration of IFN-γpresented the trend of rising within96h while IL-17peaked within48h. From these compelling and unique data, a highlighting conclusion should be drawnthat BMDCs internalized VP1-VP4antigen mainly by macropinocytosis and endosomescontaining VP1-VP4protein generated intracellularly. VP1-VP4protein can be degraded inlysosomes which are fused with endosomes. Antigenic epitopes generated here loaded ontoMHC II molecules and then presented to CD4+T lymphocytes. VP1-VP4protein stored inendosomes could also be transported to cytosol by Hsp90for proteasome degradation.Epitopes here associated with MHC I molecules which then presented to CD8+Tlymphocytes. Endosomes also presented VP1-VP4to T cells by recycling mechanism.Activated T cells generated vigerous levels of IFN-γ and IL-17, indicating both Th1response and Th17response were induced.