Establishment Of An In Vitro Chicken-derived Immune Cells Culture Method And Its Application In Immune Response To Salmonella Infection

Salmonella is an important zoonotic pathogen that contains more than 2600 serotypes.Among them,Salmonella enterica serovar Enteritidis(S.Enteritidis)and S.Pullorum are the predominant serotypes that infect chickens,with strong infectivity and pathogenicity,posing a great threat to the poultry industry.Salmonella infection in poultry can induce strong humoral immune responses to clear extracellular bacteria,but clearance of intracellular Salmonella relies on the cellular immune response.Salmonella can mediate immune escape through inhibiting the T-cell immune response,promoting the persistent survival of bacteria within the host cells.Therefore,the development of vaccines or drugs for Salmonella in poultry as immunological prevention and control methods urgently requires the resolution of chicken T-cell immune response detection problems.In this study,due to the lack of commercialized reagents and antibodies for detection of chicken immune response worldwide,we established an in vitro isolation technique for chicken immune cells and an interaction model of chicken macrophages and T cells,and evaluated the application prospect of this model by detecting T-cell immune responses after Salmonella infection.In this study,primary chicken T cells with high activity were sorted using an optimized flow separation system,and the culture conditions of T cells were determined by exploring the in vitro culture temperature,serum type,serum concentration,and whether stimulants and growth factors needed to be added according to the detection of cell mortality.Under these culture conditions,an in vitro interaction model of chicken primary antigen-presenting cells(avian macrophages)and T cells was established to study the expression of cytokines and the type of T cell immune response induced by the interaction between T cells and macrophages infected with avian Salmonella,providing a new technology and method for the in vitro study of chicken immune responses induced by avian Salmonella.1.Establishment of in vitro culture technology of chicken T cells based on fluorescence activated cell sortingThe anticoagulants,concentrations of BSA in the cell washing buffer,cell sorting system,and T cell culture conditions were optimized to reduce the mortality of T cells derived from PBMCs.We first compared the effects of heparin sodium,heparin lithium,and sodium citrate on the activity of chicken PBMCs through apoptosis detection,and ultimately selected heparin lithium as the anticoagulant.The effects of different washing buffering systems on the PBMCs activity of chickens were then evaluated by flow cytometry,and the results showed that the cells were more stable in PBS containing 1%BSA.We then used a FACSAria flow cytometer to sort T cells from chicken PBMCs and compared the effects of Purity and Yield sorting modes on the purity of T cells.The results showed that higher purity T cells(up to 98.3%)were obtained in using the Purity mode than in the Yield mode,and the T cell death rate was the lowest when using PBS containing 2.5%BSA in the cell sorting system.In addition,we optimized the culture conditions for avian T cells.Firstly,it was determined that the death rate of T cells cultured at 37℃ was significantly lower than at 42℃.Secondly,the effects of adding 10%chicken serum,10%FBS,and 5%FBS+5%chicken serum to the cell culture medium on the T cells mortality were analyzed,and the results showed that using a culture medium containing 10%FBS could significantly reduce the death rate of T cells.Above all,we used heparin lithium as an anticoagulant,1%BSA PBS as the washing buffer system,2.5%BSA PBS as the cell sorting system,and Purity model for collection of avian T cells;RPMI1640+10%FBS as the cell culture medium;and 37℃ as the cell culture temperature.This section established a cell sorting method and culture condition of avian T cell,providing foundation for the establishment of in vitro avian macrophage-T cell interaction model.2.Study on immune response of primary macrophage-T cell interaction model in chickens infected with SalmonellaTo establish a macrophage-T cell interaction model in vitro,chicken primary macrophages(chMDMs)were obtained as antigen-presenting cells by adhesive cultivation of PBMCs for 48 h.The adhesion,invasion,and intracellular proliferation ability of S.Enteritidis C50041 to chMDM was determined to evaluate the possibility of chMDM as an antigen-presenting cell.The results showed that when S.Enteritidis C50041 infected macrophages with a MOI of 10.the adhesion rate was approximately 7%and the invasion rate was approximately 5%.To further identify whether the primary macrophages could be used as antigen-presenting cells in the interaction model in vitro,we compared the mRNA levels of cytokines expressed by chMDMs infected with S.Enteritidis C50041,S.Pullorum C79-13,and ipaJ-deleted strain C79-13ApSPI12 for 5 h.The mRNA levels of IL-6,iNOS,CXCLi1,and IL-1β in chMDMs infection with S.Enteritidis C50041 were significantly higher than those in cells infected with S.Pullorum C79-13,indicating that S.Enteritidis could induce a stronger inflammatory response than S.Pullorum,which is consistent with previous studies.The above studies confirmed that chicken primary macrophages can be used as antigen-presenting cells in an in vitro interaction model.Therefore,we obtained the corresponding spleen T cells from chickens inoculated with S.Enteritidis C50041 and S.Pullorum C79-13.The T cells were co-cultured with macrophages infected with the corresponding Salmonella for 72 h and subjected to detection of the mRNA levels of cytokines produced by T cells.The results showed that S.Enteritidis C50041 could induce T cells to produce high levels of IL-6,IL-1β,CXCLil,and CXCLi2,implying that S.Enteritidis can induce Thl immune responses,while S.Pullorum C79-13 can induce a high level of IL-4 on T cells,indicating that S.Pullorum mainly induce Th2-biased immune responses.Meanwhile,spleen T cells obtained from chickens infected with C79-13 and C79-13ApSPI12 were also co-cultured with macrophages,and the results showed that splenic T cells co-cultured with macrophages infected with C79-13ΔpSPI12(ipaJ-deleted strain)produced higher levels of iNOS,CXCLil,and CXCLi2,indicating that deletion of ipaJ lead to increased Thl cellular immune responses and inflammatory response,which was consistent with the previous reports that IpaJ inhibited host Thl response and inflammatory response.The above studies confirmed that the established chicken primary macrophage-T cell interaction model in this study can be used to study the immune response of chickens infected with Salmonella,providing a new technology and method for studying the avian immune responses induced by avian Salmonella.