Chicken Egg Yolk Antibodies Modulate The Intestinal Immune Response In Mice Challenged With Salmonella Typhimurium

Pathogen invasion can induce immune response and activate the lymphocytes and other immune cells, and thus gives rise to the inflammatory response. However, the over activated immune system may cause the reduction of feed intake and growth, which is commonly referred to immunological stress. So how to alleviate immunological stress has very important significance. Research have shown that antibiotic can significantly improves animal performance and health status. However, the use and abuse of antibiotic have caused lots of problems, such as drug residues in animal products and bacterial resistance. Therefore, it is urgent to develop alternatives to antibiotics that can effectively control animal diseases without food safety problems. Many products have been regarded as potential alternatives to antibiotics. Among those, chicken egg yolk immunoglobulin (IgY) is a highly attractive product due to its safety and specificity which presents a promising approach for controlling of animal diseases. However, the mechanisms of IgY for protecting infectious diseases has not been well documented. Thus, we need further study to know whether IgY could modulate mucosal immune response in mice challenged with Salmonella typhimurium. On the basis of previous study, we evaluated the effects of IgY on the T lymphocytes of intestinal mucosa of mice challenged with Salmonella typhimurium. Furthermore, we study the influences of IgY on the activity of macrophage and the maturation of dendritic cell.To evaluate the effects of IgY on the T lymphocytes of intestinal mucosa of mice challenged with Salmonella typhimurium, Kunming mice were randomly divided into four groups of 10 animals each, and given 5 g/L streptomycin for 2 days followed by one day without streptomycin. Three groups were challenged intraperitoneally (IP) twice with 0.4 mL of S. typhimurium at a concentration of 109 cfu mL-1 at 0 and 24 h of the experiment, and the other group of animal were given PBS. Twenty four hours after the second challenge with S. typhimurium, the mice in the three treatment group were injected IP once a day for 3 consecutive days with 0.4 mL of specific IgY, non-specific IgY at a concentration of 20 mg mL-1 or 0.4 mL of PBS, respectively. The mice in control group received no treatment. On day 7, the lymphocyte populations of peyer’s patches, lamina propria and epithelial layer were analyzed by flow cytometry. After challenging with S. typhimurium, the survival, body weight and mental state were observed. The mortality of mice reach 60% and peyer’s patches was splenomega. In Peyer’s patches, the percentage of CD3+T cells and CD8+T cells increased 10% and 14%. In intestinal intraepithelial compartment, S. typhimurium administration shafted the percentages of T cells. In particular, the percentages of CD8+ and TCRγδ+ lymphocytes increased by 18% and 16% respectively. In the lamina propria compartment, S. typhimurium administration increased the levels of CD4+ and CD8+ lymphocytes by 5% and 12% respectively. Oral administration of specific IgY remitted the increase as a result of challenge. The mental state was improved and the mortality was decreased. Specific IgY reduced the effects of S. typhimurium in Peyer’s patches. In particular, specific IgY significantly decreased the CD3+ lymphocyte populations by 6.5%. In the intraepithelial compartment, the percentages of CD8+ and TCRy8+ lymphocytes were significantly decreased by 12% and 10% after treatment with specific IgY. Specific IgY could diminish the increase induced by S. typhimurium treatment in the lamina propria compartment. For instance, the percentage of CD4+ and CD8+ lymphocytes were decreased by 4% and 12%. In summary, specific IgY could regulate the percentages of lymphocyte populations and modulate the degree of activation of the immune response. Those effects indicate that IgY could protect intestinal from the inflammation caused by major pathogens.The influence of specific IgY on the phagocytic activity and NO production of macrophages were analyzed in vitro. The macrophages were incubated with S. typhimurium and specific IgY or nonspecific IgY at 37℃ for 3 h and cooled on ice to stop phagocytosis. The phagocytosis of S. typhimurium by RAW264.7 cells was tested by flow cytometry and confocal laser scanning microscopy. The phagocytic percentages of RAW264.7 cells were significantly increased by 20% and 33% after treatmentwith specific or nonspecific IgY at a concentration of 5 mg mL-1. It suggested that both non-specific and specific IgY enhanced the phagocytosis of S. typhimurium by RAW264.7 cells, and specific IgY treatment was better than the non-specific IgY (P<0.05). The level of NO was measured by ELISA. The results indicate that S. typhimurium challenge significantly potentiated production of NO compared to control. And, specific IgY reduced the level of NO (from 26 to 14 μmol/L).Bone marrow cells from the femurs and tibias of Kunming mice were generated with the method described previously and were induced into bone marrow dendritic cells (BMDCs). The dendritic cells were incubated with S. typhimurium and specific IgY or nonspecific IgY at 37℃ for 3 h, and then the dendritic cells were collected for analysis of phenotypic marker by flow cytometry. Our results showed that S. typhimurium caused the apoptosis of BMDCs by down-regulating MHC Ⅱ, CD40, CD80, CD83 and CD86. Importantly, treatment with specific IgY reduced the apoptosis of BMDCs and enhanced the maturation of BMDCs as evidenced by up-regulated expression of MHC Ⅱ, CD40, CD80, CD83 and CD86 molecules on BMDCs.