Dendritic Cells Plays A Pivotal Role In The Immune Response To Brucella Vaccine

Brucella.spp.is a facultative intracellular bacterium that causes brucellosis in a variety of animals and undulant fever in humans.Brucella spp.infects its host through mucosa of digestive tract,respiratory tract,urogenital tract,and skin wounds.As frequently described,whatever the species considered,Brucella spp.has strongly proliferated inside macrophages by inhibiting phagosome-lysosome fusion.Once inside macrophages,pathogens can diminish or abrogate their antigen presentation capacity,thus reducing the T cell-mediated immune responses,inducing chronic brucellosis.Thus,the important subject is to help domestic animals mount a solid immune response.It has been well-documented that initiation of adaptive immunity requires presentation of antigens by dendritic cells(DCs).DCs can cross present antigens.And this leads to the conclusion that states DCs are the most powerful antigen-presenting cells in the body.However,it is unclear that if DCs is capable of processing and presenting Brucella antigen in vivo.To address the issue of whether vaginal mucosa DCs take active part in the antigen processing and presentation of B.suis antigen,BALB/c mice were inoculated intravaginlly with Brucella vaccine at different time points.The draining lymph nodes-iliac lymph node (ILN)were harvested as usual.The dynamics of vaginal mucosa DCs after intravaginlly challenging with Brucella vaccine was immunohistochemically revealed.Compared with the PBS goup,it was clearly shown that number of DCs appearing in cortex area of ILN increased as early as 12h after inoculation.As time goes on,DCs further imigrated, peaking at 48h,indicative of the numbers of DCs in ILN obviously increased and gathered toward medulla area.DCs concentrated in cluster appearance near the medulla.The distributing area of DCs had enlarged,but the density of DCs had decreased and at 72h.Accordingly,the serum levels of IFN-γwere determined with ELISA at different timepoints when BALB/c mice were inoculated intravaginlly with the Brucella vaccine. The results showed that the serum levels of IFN-γin vaccinated mice the serum slightly raised(P＞0.05)at 12h and 24h post-vaccination.At 48hpost-vaccination,in contrast to the control group,the content of IFN-γin the serum was increased considerably (22.2140±2.3056pg/mL,P＜0.05).At 72h post-vaccination,the content of IFN-γin the serum reached its peaking stage and have a significant difference to the control group (23.6507±1.4727 pg/mL,P＜0.01).These results indicated that vaginal DCs were the antigen presenting cells which present brucella antigen to naive T lymphocytes,leading to the release of IFN-γ. Brucella spp.is an intracellular pathogen that preferentially infects macrophages.The macrophages in vaginal mucosa of BALB/cmice are deleted by intravaginally administration of clodronate liposome in order to test our hypothesis that DCs is better to capture the brucella antigen upon the depletion of macrophage.Mice injected with the same volume of empty liposomes as a control.Then mice were inoculated intravaginlly with Brucella vaccine at different time points.The ILN were harvested as usual.The dynamics of vaginal mucosa DCs after intravaginlly challenging with Brucella vaccine was immunohistochemi-cally revealed.In control group,it was clearly shown that number of DCs appearing in cortex area of draining lymph node increased as early as 12h after inoculation,compared with the PBS group.DCs scattered in the paracortical area at 24h.As time goes on,DCs further imigrated,peaking at 48h,indicative of the numbers of DCs in ILN obviously increased and gathered toward medulla area.At 72h post-vaccination,the quantity of DCs declined remarkably and scattered either in the cortex or the medulla.To our surprise,in experiment group,compared with the PBS group,the number of DCs has hardly changed at 12h,24h, 48h and 72h.Accordingly,the serum levels of IFN-γwere determined with ELISA at different timepoints when BALB/c mice were inoculated intravaginlly with the Brucella vaccine.In control group,the results showed that the serum levels of IFN-γin vaccinated mice slightly raised(P＞0.05)at 12h post-vaccination.At 24h post-vaccination,in contrast to the PBS group,the content of IFN-γin the serum was increased considerably(48.7508±2.4373 pg/mL,P＜0.05).At 48h post-vaccination,the content of IFN-γin the serum reached its peaking stage and have a significant difference to the PBS group(52.3050±2.3262 pg/mL,P＜0.01).Although the content of IFN-γin the serum declined at 72h post-vaccination,having significan difference(51.5511±2.0772 pg/mL,P＜0.05).In experiment group,although the results showed that the serum levels of IFN-γin vaccinated mice have slightly raised at 12h,24h,48h and 72h,there is no significan difference (P＞0.05)in contrast to the PBS group.The levels of IL-4 results show that Compare with PBS group the serum levels of IL-4 in control and experiment group vaccinated mice slightly raised(P＞0.05)at 12h,24h,48h and 72h post-vaccination.In vitro experiment,macrophage typically is plused with B.suis at 500 CFU per cell. When bacteria were phagocytized by macrophages,B.suis survives within these cells at 2h after pulse.And then the number of intracellular bacteria in murine macrophages increased between 2h and 12h.At 12h after pulse,the macrophages emerge phenomenon of leaking cytoplasmic content,rupturing of cell membrane and karyon pyknosis.It means that macrophage may relay brucella antigen to DCs by cell fraction or vesicle.It can be concluded that the dynamics of DCs in vaginal mucosa is dependent on macrophages when challenged with brucella antigen in vivo.Futhermore the DCs actived the naive T lymphocytes and initiated the Thl immune response.