Immune Activation Of Salmonella Outer Membrane Vesicles On Chicken Mononuclear Phagocytes

Salmonella can cause diseases in poultry,causing significant damage to the poultry industry.The infected eggs and poultry meat can cause food-borne diseases in humans through the food chain.Prevention and reduction of Salmonella infections in poultry can improve poultry health and reduce Salmonella-induced disease in humans.At present,antibiotics are used in the treatment of Salmonella.With the emergence of multi-drug resistant strains,it is more difficult to treat Salmonella.Thus,it is necessary to find new prevention and control measures.Outer membrane vesicles（OMVs）can stimulate host innate immune cells,enhance host immunity and reduce pathogenic infection.OMVs secreted by Salmonella have the ability to reduce the intensity of Salmonella infection in poultry,but the mechanism remains unclear.Macrophages and monocytes are important mononuclear phagocytes involved in the innate immune response.However,the effect of Salmonella OMVs on immune activation of chicken mononuclear phagocytes is unknown.Therefore,the OMVs of chicken-derived Salmonella were first isolated in this study,and then the immune activation effects of OMVs and their lipopolysaccharide（LPS）and proteins on chicken macrophage-like HD11 cells and monocytes were explored.Finally,protein composition of OMVs was further analyzed by protein mass spectrometry.The results from this study have supported to use Salmonella OMVs as potential immune activators in chickens.The results are as follows:1.Isolation,purification and characterization of chicken-derived Salmonella OMVs.In the experiment,three strains of CVCC542,SAL A,and SAL B were selected for OMVs extraction.With the increase of culture time,the number of OMVs also increased.OMVs were extracted from Salmonella with a culture time of 12 h and a culture volume of 2 L,which could satisfy the subsequent experiments.Density gradient centrifugation showed the highest number of OMVs in the 25%centrifugation layer.The laser nanoparticle analyzer showed that the size of OMVs ranged from 50 to 300 nm,with the peak size at126 nm.Scanning electron microscope（SEM）and transmission electron microscope（TEM）also showed that the size of Salmonella OMVs was around 100 nm.The protein concentrations of Salmonella CVCC542,SALA and SALB derived OMVs were 1342.15μg,1365.62μg and 1726.46μg per 10¹² particles,respectively,and the lipopolysaccharide concentrations were 1038.19μg,958.49μg and 1211.96μg per 10¹² particles respectively.The corresponding lipopolysaccharide contents perμg protein were 0.78μg,0.71μg and0.73μg,respectively.The results of this experiment show that the OMVs of Salmonella are successfully isolated.2.Immune activation of chicken bone marrow-derived monocytes and HD11macrophages by Salmonella OMVs.In this study,the effect of stimulation of chicken-derived Salmonella typhimurium CVCC542 OMVs on the phagocytosis of chicken bone marrow-derived monocytes and macrophage-like cells HD11 was detected.Compared with the PBS（control group）,both 200 ng/m L LPS in the positive control group and the OMVs group containing 200 ng/m L LPS could promote the formation of chicken bone marrow-derived mononuclear phagocytes and chicken HD11 dendrites,and promote expression of LITAF,IL-6 and i NOS m RNA,indicating that Salmonella OMVs activate bone marrow-derived mononuclear phagocytes and chicken macrophages.Phagocytosis assays also showed that OMVs could enhance the phagocytosis of FITC-Dextran and FITC-Salmonella by HD11.Colony forming unit（CFU）results also showed that OMVs stimulated more phagocytosis of bacteria after HD11 activation.In addition,the internalization assay of OMVs by HD11 cells also showed that OMVs were taken up by HD11 after co-incubating with HD11 for 8 h,16 h and 24 h.The experiments in this chapter show that OMVs can be internalized by HD11 cells,can activate chicken bone marrow-derived mononuclear phagocytes and HD11 cells and can enhance HD11phagocytic activities.3.Effects of inhibition of LPS and proteins from Salmonella OMVs on chicken mononuclear phagocyte activation.Salmonella CVCC542 OMVs activate chicken mononuclear phagocytes mainly through LPS and protein.The purpose of this experiment was to examine the effects of LPS and membrane proteins carried by OMVs on the differentiation and phagocytosis of chicken macrophages and monocytes.Salmonella OMVs were treated with polymyxin B（PMB）or proteinase K（PK）.Then,and the inflammatory factor-stimulating ability and phagocytosis of the treated OMVs on HD11cells and spleen monocytes were observed.Both treatments were able to reduce the phagocytosis of FITC-Salmonella by HD11 cells and splenic monocytes and the secretion of IL-1β,LITAF and IL-6 inflammatory factors.In addition,OMVs treatment enhanced the expression of TLR2 and TLR4 in HD11 cells.These results indicate that inhibition of Salmonella OMVs lipopolysaccharides and proteins affects immune activation in chicken macrophages and spleen monocytes.4.Analysis of Salmonella outer membrane vesicles protein composition.OMVs proteins are involved in activating chicken immune cells.So,we asked what were the main proteins of OMVs,and what were the changes of protein profiles before and after proteinase K treatment of OMVs.The protein fraction of Salmonella OMVs was analyzed by liquid chromatography-mass spectrometry,and a total of 251 proteins were identified.Cell-PLoc-2 was used to predict the subcellular structure of the proteins.Among these proteinase K untreated OMVs,the number of proteins located in the cytoplasm,inner membrane,inner membrane cytoplasm,extracellular,periplasmic,outer membrane protein and others（%）were 99（43%）,42（18%）,32（14%）,23（10%）,13（6%）,12（5%）and 9（4%）.In these proteinase K-treated OMVs proteins,the number of proteins located in the outer membrane,cytoplasm,inner membrane and cytoplasm,and the periplasm（%）were respectively 12（28%）,10（23%）,10（23%）,10（23%）,and 1（3%）.According to GO analysis in Uni Prot,proteinase K-untreated OMVs proteins were mainly classified into cellular component metabolism,transport activity and cellular structure.Proteinase K-treated OMVs proteins are mainly classified into transport activity,cellular component metabolism,protein folding and degradation.The results show that OMVs protected the outer membrane proteins Omp A,Omp D,Omp F and membrane lipoprotein Lpp from being degraded by proteinase K,while flagellin was degraded.In conclusion,this study successfully isolated the OMVs of Salmonella from chickens,which carried abundant MAMPs such as LPS and proteins.Polysaccharides and proteins activate chicken mononuclear phagocytes and enhance their phagocytosis of bacteria.OMVs have potentials as chicken immune activators.This is of great significance for preventing Salmonella infection to chickens and for ensuring poultry health and human health.