Molecular Mechanisms Of Antigen Processing In Mouse Dendritc Cell For Inactivated Foot-and-Mouth Disease Virus

Objective: Foot-and-mouth disease (FMD) caused by foot-and-mouth disease virus(FMDV) is a highly contagious vesicular disease of cloven- hoofed animals, such as pigs, cattle, and sheep. It was documented that humans were sporadically infected by FMDV. The mechanisms that animals mount immune responses against FMDV antigens have not yet been elucidated. It has been well-documented that initiation of adaptive immunity requires presentation of antigens by dendritic cells (DC). DC is able to cross present antigens. And this leads to the conclusion that states DC are the most powerful antigen-presenting cells in the body. However, it is unclear if DC is capable of processing and presenting FMDV antigen onto T lymphocytes in the draining lymph nodes.Methods: FMDV infects animals usually by way of digestive tract, respiratory tract, and wounded skin. In an infected animal and vaccinated one, FMDVs were replicated in great deal at pharynx. So, pharynx is regarded as the most important organ in host defence. There is a growing body of evidence that peripheral DC migrate into the draining lymph nodes immediately after encountering the invading pathogens. However, no datum is available that states the dynamics of FMDV antigen-bearing DC in vivo.Cell-mediated immune response and humoral immune response are the two arms of adaptive immunity. In case of protein antigens, it has been well-demonstrated that the antibody response is almost always dependent on effector CD4+T cells. The crucial role of CD8+T cells in limiting virus infection is always seen in animal studies. And the elimination of infected cells without the destruction of healthy tissue requires the cytotoxic mechanisms of CD8+T cells to be both powerful and accurately targeted. Thus, CD4+ and CD8+ T cells were taken into the experiment as the recipients of antigenic peptides presented by MoDC and the exclusive indicators of antigen presenting pathways mediated by class I MHC molecule and class II MHC molecule, respectively.Migration of pharyngeal DCTo address the issue of whether pharyngeal DC take active part in the antigen processing and presentation of FMDV, BALB/c mice were inoculated orally with the inactivated FMDV vaccine deprive of adjuvant at different time points. The draining lymph nodes were harvested as usual. The dynamics of pharyngeal DC was immunohistochemically investigated after orally adminisdrated with inactivated FMDV.Cell-mediated immune response to inactivated FMDV in mouseThirty six BALB/c mice were randomly grouped into six groups of 6 mice each. Five groups of BALB/c mice were inoculated orally with the inactivated FMDV vaccine. As control, the mice of sixth group were orally administered with steriled phosphate-buffered saline (PBS) and the blood samples were collected at 24h after oral administration. Peripheral blood were harvested at 24h(the first group), 48h(the second group), 72h(the third group), 96h(the fourth group)and 120h(the fiveth group)after vaccination. Serum samples from whole blood were examined for the presence of IFN-γusing a commercially available ELISA kit according to manufacturer's instructions. Generation of monocyte-derived dendritic cells(MoDC)Peripheral blood mononuclear cells (PBMCs) were used as a source of DC precursors and isolated on Ficoll density gradients by using LSM and then cultured for 1d with 10000 U/ml penicillin-streptomycin and 10% heat inactivated foetal calf serum in RPMI-1640. The adherent fraction was cultured in RPMI 1640 medium supplemented with 20 ng/ml recombinant mouse IL-4, 20ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF), 10000 U/ml penicillin - streptomycin and 10% heat inactivated foetal calf serum to generate immature DCs. DCs were harvested after incubation for 5 days at 37℃in 5% CO2. To identify the resulting MoDC, immunocytochemistry and flow cytometry were performed. Ubiquitination of inactivated FMDV antigens in MoDCBriefly, inactivated FMDV-pulsed MoDCs were solubilized by incubation in lysis-buffer. The lysate samples were centrifuged and the resultant solubilized fractions were analyzed by western blotting. The blots were incubated with two anti-ubiquitinated protein antibodies (clone FK1 and clone FK2) following manufacturer's instruction.Inactivated FMDV-pulsed MoDC initiate T cell responses in vitroTo determine the ability of the FMDV-pulsed MoDC is able to mount specific T cell responses,MoDCs were pulseded for 4h with inactivated FMDV (2PFU/cell) and cocultured with T cell preparation of cervial lymph node (DCs:T cells=1:5). To block the activation of CD4+T cells, purified anti-mouse CD4 antibodies were applied to the co-culture system. This procejure leads to CD8+ T cells available for the test of ubiquitin-proteasome MHC-I pathway. To test the responsiveness of CD4+ T cells, purified anti-mouse CD8a antibodies were applied to the co-culture system to block the antigen recognition by TCR on CD8+T cells. CD4+T cells and/or CD8+ T cells were triggered with FMDV-pulsed MoDC for 9h, 12h, 24h, and 48h, respectively. IFN-γcontents in culture supernatants were assayed by using ELISA kits according to manufacturer's instruction.Inhibition studies of antigen presentationMoDCs were incubated with lactacystin (10μM) and chloroquine (100μM) for 2h at 37℃, respectively, and then pulsed with inactivated FMDV for 4h. The FMDV-pulsed MoDC were cocultured with CD8+ T cells (in the presence of chloroquine) or CD4+ T cells (in the presence of lactacystin). Release of the IFN-γeither from effector CD4+T cells or from activated CD8+T cells were assayed at 9h, 12h, 24h, and 48h by using ELISA kits according to manufacturer's instructions.Results: Pharyngeal DC is the antigen–presenting cells of inactivated FMDVIn contrast to the control mice, many DC were present in cortex of lymph node at 24h post-vaccination. The number of DC was dramatically increased at 48h post-vaccination and the accumulation was predominantly detected in paracortical area. The migration of DC reached their peaking stage in paracortical area at 72h post-vaccination. At 96h post-vaccination, the quantity of DC declined remarkably and scattered either in the cortex or the medulla. DC were found to emerge again primarily in cortex at 120h post-vaccination. This data indicate that pharyngeal DC is the antigen-presenting cells of inactivated FMDV.Cell-mediated immune response to inactivated FMDV in mouseAccordingly, the serum levels of IFN-γwere determined with ELISA at different time points after BALB/c mice were inoculated orally with the inactivated FMDV vaccine. The results showed that the serum levels of IFN-γin vaccinated mice slightly raisedat 24h post-vaccination, but no significant difference with comparation to control mice (10.1568±0.4689 vs 10.0512±0.0684, P>0.05). At 48h post- vaccination, in contrast to the control mice, the serum level of IFN-γwas considerably increased(10.7161±0.2199 vs 10.0748±0.0571, P<0.01). The content of IFN-γin the serum reached its peaking stage at 72h and has a significant difference to the control mice(11.6784±0.4590 vs 10.1249±0.1498, P<0.01). The content of IFN-γin the serum declined at 96h post- vaccination, having no significant difference at 120h post-vaccination (10.5285±1.3903 vs 10.0534±0.1543, P>0.05). These results indicate that pharyngeal DC were the antigen presenting cells which present FMDV antigen to na?ve T lymphocytes in lymph node, where the activation of T cells occurred, leading to the rapid release of IFN-γ. Generation of monocyte-derived dendritic cells(MoDC)To elucidate the mechanism of processing of FMDV antigen in DC, monocyte-derived dendritic cells (MoDC) of BALB/c mouse were generated, and the resulting MoDC were identified by immunohistochemistry and FACS analysis. More than 99% of generated MoDC express the specific markers, such as S-100, CD11c, and MHC-II molecule.Ubiquitination of inactivated FMDV antigens in MoDCThe cell lysates of FMDV-pulsed DCs were subjected to SDS-PAGE and immunoblotted with two anti-ubiquitinated protein antibodies(clone FK1 and clone FK2). It was clearly shown that the ubiquitinated FMDV protein of approximately 75kD was detected at 0.5h, 3h, 6h, 10h, and 20h immediately after FMDV- loading onto MoDCs. At 0.5h of antigen pulse, the content of ubiquitinated FMDV antigens increased considerably. However, the content of ubiquitinated proteins did not show any recognizable fluctuation from then on.Inactivated FMDV-pulsed MoDC initiate T cell responses in vitroBoth CD4+T cells and CD8+T cells contributed to the release IFN-γwhen cocultured with inactivated FMDV-pulsed MoDC. But CD8+T cell response was slightly behind the onset of CD4+T cell response in terms of the release of IFN-γ. To identify their relative contribution in the IFN-γresponse, FMDV-pulsed MoDC was pretreated with chloroquine, a lysosome inhibitor, and lactacystin, a proteasome inhibitor, respectively. Both inhibitors dramatically downregulated the IFN-γrelease from CD4+ T cells. Chloroquine significantly inhibited the IFN-γrelease from CD8+ T cells. By contrast, the capacity of CD8+T cells to secrete IFN-γwas potentially augmented when proteasome in MoDC was specifically inhibited by lactacystein. However, IFN-γrelease from CD8+ T cells was considerably inhibited when chloroquine and lactacystin were applied to coculture system simultaneously.Conclusion: It can be concluded from the data mentioned above that pharyngeal dendritic cell is the antigen presenting cell of inactivated FMDV when administered orally. The pharyngeal dendritic cells migrate into the cervical lymph nodes where to initiate cell-mediated immune response which is characteristic of IFN-γrelease.Inactivated FMDV in dendritic cells was polyubiquitinated and/or monoubiquitinated, but the dominant ubiquitination is monoubiquitinated. Consequently, FMDV antigens were degraded both in the endosome- lysosome pathway and ubiquitin- proteasome pathway. The resulting epitopes were in bulk presented via MHC-II molecule for the recognition by TCR on CD4+T cells, leading to the activation of CD4+T cells. Some epitopes were loaded onto MHC-I molecule to initiate CD8+T cell response later. Furthermore, DC predominantly cross-presented FMDV antigenic peptides for CD8+T cells in the way of MHC-I molecules that were loaded with peptides from lysosomal degradation. By contrast, FMDV-pulsed DC presented exogenous antigens by way of MHC-II molecules and triggered CD4+T cells to preferentially differentiate into Th1 subset. Notebally, CD8+T cell response is drastically characteristic of response exhaustion as DC did during the migration into the draining lymph node.Taken together, these findings show for the first time that the DC is the potential APC for the inactivated FMDV. The protein antigens of inactivated FMDV are recognized by the ubiquitinating system in dendritic cells and processed and presented by DC via both the classical major histocompatibility complex (MHC) classⅡand cross- presentation passways.