Effects Of Procine Epidemic Diarrhea Virus Influenced The Immune Function Of Porcine Dendritic Cells

Porcine epidemic diarrhea virus(PEDV)is the causative agent of porcine epidemic diarrhea(PED),an acute and highly contagious enteric disease in swine characterized by severe enteritis,vomiting and watery diarrhea,which approach 80%-100%mortality in suckling piglets less than ten days of age.Even infected piglets that recovered from PEDV infections,they could have impaired the immune function and cause the secondary viral or bacterial infection.The outbreak of PED resulting in tremendous economic losses.The target organ of PEDV is piglet small intestinal,and there are many dendritic cells(DCs)distributed beneath the intestinal epithelium and act as a portal for virus invasion.DCs are the most potent antigen-presenting cells and the key initiators of adaptive immune responses to play a central role in the intestinal mucosa.In this study,we investigated how attenuated PEDV(CV777)and inactivated PEDV affects the function of porcine monocyte-derived dendritic cells(Mo-DCs)and intestinal DCs.Our findings may assist in the development of specific vaccine strategies and therapeutic approaches against this pathogen.1 Generation of Mo-DCsDCs are the most potent antigen-presenting cells and the key initiators of adaptive immune responses to play a central role in the intestinal mucosa.As the key initiators,they can present antigen to trigger T cell responses against an array of invading pathogens.So it's important to establish a method for induction of porcine DCs.Porcine monocytes were cultured for 6 days using RPMI 1640 media with GM-CSF and IL-4 to allow them to differentiate into immature Mo-DCs.Cell phenotypes were then evaluated using microscopy.Immature Mo-DCs were semi-suspended or suspended with a round appearance with dendritic processes.Immature Mo-DCs stimulated with LPS for 24h were larger and irregularly shaped,occurred as single cells or clusters,and formed long pseudopodia,all of which are characteristics expected for DCs morphology.A phenotypic evaluation of immature Mo-DCs and mature Mo-DCs was also conducted using flow cytometric analysis.Cell phenotype was evaluated with SWC3a~+CDla~+,SWC3a~+CD80/86~+and SWC3a~+SLA-II-DR~+.Using these reagents,17.15%,62.02%,and 49.13%of immature Mo-DCs display the respective markers,and 25.30%,89.44%,and 62.73%of mature Mo-DCs display the respective markers.Because the generated cells were confirmed to be Mo-DCs,they were used in subsequent experiments.2 Effects of attenuated and inactivated PEDV influenced the immune function of porcine Mo-DCs in vitroThe target organ of PEDV is piglet small intestinal,and there are many dendritic cells(DCs)distributed beneath the intestinal epithelium and act as a portal for virus invasion.DCs are the most potent antigen-presenting cells and the key initiators of adaptive immune responses to play a central role in the intestinal mucosa.In order to investigate Effects of attenuated and inactivated PEDV influenced the immune function of porcine Mo-DCs in vitro,the real-time PCR and viral titer were used to detect the susceptibility of Mo-DCs to attenuated PEDV.Thereafter,the phenotype markers changes of Mo-DCs were tested by flow cytometric analysis,and the expression of cytokines on attenuated and inactivated PEDV effected the Mo-DCs was determine by ELISA.Finally,the presenting-antigen ability of Mo-DCs was detected by mixed lymphocyte reaction.The results proved that Mo-DCs could infect attenuated PEDV at lower level,and the immature Mo-DCs infectivity was higher than mature Mo-DCs.the expression of CDla~+,CD80/86~+ and SLA-II-DR~+ in immature and mature Mo-DCs effected with attenuated and inactivated PEDV were significantly higher than control group(p<0.05).The secretion of IL-12 and IFN-y in attenuated and inactivated PEDV effected immature Mo-DCs was significantly higher than control group(p<0.05).Immature and mature Mo-DCs effected with attenuated and inactivated PEDV could stimulate T cell proliferation to some extent.These results indicated that attenuated and inactivated PEDV could improve the functions of Mo-DCs,such as the uptake and presenting antigen,cytokine secretion and the ability to stimulate T cell proliferation.3 Effects of attenuated and inactivated PEDV influenced the immune function of porcine Mo-DCs in vivoIn order to investigate Effects of attenuated and inactivated PEDV influenced the immune function of porcine Mo-DCs in vivo,we injected the attenuated and inactivated PEDV into the intestinal lumen of piglets with the intestinal ligation surgery.The immunofluorescence and immunohistochemistry were carried out to detect the change number of SWC3a~+SLA-II-DR~+ DCs,CDllb~+CD16~+DCs,CD3~+,CD4~+ and CD8~+ T cells in intestinal villi and lamina propria at different times,which helped us have a better understanding of the uptake and presenting function in porcine intestinal DCs.The results proved that at the injection of PEDV 3 h,the number of SWC3a~+ SLA-?-DR~+ DCs and CD11b~+CD16~+DCs in intestinal villi rapidly increased(p<0.05),at the injection of PEDV 48 h,the number of SWC3a~+SLA-?-DR~+ DCs and CD11b~+CD16~+DCs in lamina propria increased(p<0.05).And the number of CD3~+,CD4~+ and CD8~+ T cells in intestinal villi and lamina propria were significantly increased at the injection of attenuated and inactivated PEDV 15 min,3 h and 48 h(p<0.05).The results above demonstrate that the ability to uptake and present antigen have an increaseing with the time after piglets injected with attenuated and inactivated PEDV.For further study the effects of attenuated and inactivated PEDV on immune function such as antigen-present of porcine intestinal DCs,the study injected model antigen,inactivate Escherichia coli K88,into the piglets intestinal lumen with the intestinal ligation surgery 48 h after fed attenuated and inactivated PEDV.The immunofluorescence and Immunohistochemistry were carried out to detected the change number of SWC3a~+SLA-?-DR~+ DCs,CD11b~+CD16~+DCs,CD3~+,CD4~+ and CD8~+ T cells in intestinal villi and lamina propria at different times.The results showed that 3 h after piglets injected with inactivate Escherichia coli K88,there is a decrease on the number of SWC3a~+SLA-DR-?~+ DCs and CDllb~+CD16~+ DCs in intestinal villi(p<0.05).But the number of SWC3a~+SLA-DR-?~+ DCs and CD11b~+CD16~+ DCs in intestinal villi and lamina propria in all treated groups obviously increased(p<0.05).This showed that the DCs in intestinal villi may have a migrate to the intestinal lamina propria.Whlie when 15 min and 3 h after attenuated and inactivated PEDV treated-piglets injected with inactivate Escherichia coli K88,all the number of CD3~+,CD4~+ and CD8~+ T cells in intestinal villi and lamina propria obviously increased(p<0.05).These results indicated that attenuated and inactivated PEDV could improve the functions of Mo-DCs.Our findings may assist in the development of specific vaccine strategies and therapeutic approaches against this pathogen.