Identification Of Tolerated Epitope Peptides From The Digestion Of Dairy Products And The Induction Of Immune Tolerance In Cow’s Milk Allergy

Cow’s milk are one of the eight allergic foods.It is reported that cow’s milk contains more than 30 allergen proteins,all of which have potential sensitization properties.As is well known,yogurts fermented from cow’s milk not only have high nutritional value and good flavor,but also are popular.At the same time,the structure of allergens during fermentation can be changed to reduce its allergenicity.It is recognized as the safe,edible dairy product with the lowest allergenicity.In addition,yogurts are easier to digest,and most Ig E mediated cow’s milk allergy（CMA）patients can tolerate yogurt.Therefore,exploring the digestive characteristics of yogurt and its induced tolerance mechanism can provide theoretical and scientific basis for the development of hypoallergenic dairy products.In the paper,yogurts were used as the research object to detect the changes in the main allergen proteins and their digestive characteristics,and explores the changes in the degradation of allergens and the binding ability of specific antibodies.On the basis,the sequence,function,and allergenicity of its peptides and the peptides of digestive products are characterized.At the same time,bioinformatics methods are used to determine the epitopes information of the main allergens in the residual peptides,including T cell epitope analysis and Ig E epitope analysis.And the screening of tolerance T cell epitopes was carried out through in vitro experiments on mouse sensitized immune cells.Based on these data,we obtained immune tolerance candidate peptides,and combined with the immune tolerance model of mice cow’s milk allergy（CMA）.Then the candidate peptides was validated in the animal models,and explored the changes in humoral and cellular immunology,as well as the metabolic and molecular mechanisms.The main methods,results,and conclusions of the study are as follows:1.Six commercial yogurts（Gu Mi yogurt,JSD yogurt,HR yogurt,HN yogurt,JA yogurt,and HS yogurt）were selected to analyze the degradation and allergenicity of the main allergens.SDS-PAGE and RP-HPLC were used to analyze the changes of main allergen proteins.Western blot and ELISA were used to identify the Ig G and Ig E binding ability of major allergens.The peptides in yogurts were identified by liquid chromatography mass spectrometry（LC-MS/MS）and the epitopes of major allergens were analyzed.Gu Mi yogurt had the lowest binding capacity for Ig G and Ig E.Through LC-MS/MS detection,it was found that 17 peptides of the main allergens in Gu Mi yogurt were identified,and the prediction of epitopes revealed that 6 peptides did not have the potential allergenicity.At the same time,among all the peptides,6 Ig E epitopes of BLG and 14 Ig E epitopes of casein were hydrolyzed and destroyed.In addition,13peptides were located within the spatial structure of proteins,which suggested that these peptides might be wrapped up and not easily interrupted.2.CMA is triggered by digested peptides,so the residual epitopes after digestion are the material basis for allergenicity.Due to the characteristics of digestion and absorption of yogurts,further research was conducted on its digestive peptides.In the section,yogurt and cow’s milk were digested in vitro by simulating the gastrointestinal digestion of infants and adults.Tricine-SDS-PAGE,RP-HPLC,and indirect ELISA were used to explore the changes in the digestive stability and Ig E binding ability of milk proteins under different conditions.Compared to cow’s milk,yogurt was more easily digested,and its Ig E binding ability was significantly reduced by 54.53%.At the same time,the digestive ability of adults was stronger than that of infants.LC-MS/MS was used to identify the peptides of the main allergens in the digestion products of yogurt,and the transport and absorption ability to Caco2 cells was detected.The peptides were analyzed for T-cell epitopes,Ig E epitopes,and other related information through bioinformatics.By analyzing the peptides in the digestion products,113 peptides were obtained in major allergens,of which 38 peptides were present in all digestion products under different conditions,which indicated that these peptides were more resistant to digestion.9,3,and 26 of these 38 peptides are derived from BLG,ALA,and casein.11peptides were contained T cell epitopes,no Ig E epitopes,and couldn’t induce the produce of IL4 through bioinformatics analysis.These epitopes may induce immune tolerance in CMA,providing a basis for subsequent research.3.In this section,the identification of tolerance epitope peptides was conducted through in vitro cell experiments.Based on the main allergen peptides detected in gastrointestinal digestion products,peptides containing 13-20 amino acids in the main allergen（excluding the reported peptides containing Ig E epitopes and predicted B-cell epitopes）were selected as candidate peptides.Then,mesenteric lymph nodes（MLN）and spleen tissue from the intestinal tract of allergic mice were isolated and ground into a single cell suspension.The candidate peptides were used to co-culture with spleen cells and MLN,respectively.The supernatant was used to detect related cytokines.Flow cytometry was used to identify the change of dendritic cells（DC）and the differentiation of CD4⁺T cell.The tolerant T cell epitopes were obtained.Based on all the results,it was found that peptides BLG AA71-83,BLG AA136-148,andα_S1-casein AA120-132increased the level of Treg and might contain potentially tolerant T cell epitopes.The identified tolerance T cell epitopes provide a basis for further validation in vivo experiments.4.To explore the role of T cell epitopes in inducing tolerance,the oral tolerance model was constructed based on the CMA model.Tolerance was induced by whey and whey hydrolysates,and the optimal tolerance model was selected.Compared to the allergic group,it was found through temperature and symptom scores that the symptom scores of other intervention groups were significantly reduced,and the temperature also significantly returned to the normal level of mice.By detecting the changes in the specific antibodies Ig E,Ig G,Ig G1,Ig G2a,and Ig A antibodies to BLG in the serum of mice.It was found that the level of Ig E in other tolerance groups were significantly reduced,while the Ig G,Ig G1,Ig G2a,and Ig A antibodies in the whey group were significantly reduced.The level of m MCP-1 also significantly decreased in all intervention groups.The cytokines IL4,IL5,IL13,and IL-6 secreted by spleen cells were significantly decreased.In addition,flow cytometry also found that Th2 and Th17cells in spleen cells were significantly downregulated,while Treg cells were significantly upregulated.In addition,the tolerance type CD103⁺DC in MLN cells significantly increased in intervention groups.Moreover,there was no significant difference in relevant tolerance indicators between whey and different whey hydrolysates groups,indicating that both whey and different whey hydrolysates can induce tolerance in CMA.5.Based on the above screened T cell epitope peptides and the constructed tolerance model,four BLG peptides,BLG AA 2-14（IVTQTMKGLDIQK）,BLG AA84-101（IDALNEKVLVLDTDYKK）,BLG AA 71-83（IIAEKTKIPAVFK）,and BLG AA 136-148（FDKALKALPMHIR）,were selected for tolerance induction.In this section,high and low doses of mixed tolerance epitope peptides would be selected to induce tolerance in the CMA model of mouse.The allergic symptoms score and body temperature in mice were detected,and verify the immune tolerance ability through relevant immunological indicators.Compared to the allergic group,the results showed a significant recovery in body temperature and symptoms.The level of Ig E was significant decreased in both the oral low concentration and high concentration peptides groups,and significant decreased in the level of m MCP-1 in the low concentration peptides groups.The cytokine IL4 secreted by spleen cells was significantly decreased in peptides intervention groups,and IL-6 was also significantly decreased in the intragastric intervention groups.Th2 and Th17 cells in spleen cells also significantly decreased in intervention groups,and Treg cells also significantly increased.The levels of Th2 and Th1 cells in MLN cells have significantly decreased,indicating that the intervention of peptides induced the development of tolerance in CMA.However,some indicators could be affected by peptide concentration and intervention methods,and the specific mechanisms need to be further explored.6.Based on the above evaluation of the tolerance ability of tolerance epitope peptides to CMA of mice,we also conducted exploration in three aspects about intestinal flora,CD4⁺T cell transcriptome,and serum metabolomics.The results showed that peptides mainly regulate specific intestinal bacteria（Lachnospiraceae NK4A136,Muribaceae,Enterohabdus）to induce the development of tolerance.These bacteria also have significant correlations with the key metabolites in serum.For example,there was a significant negative correlation between Lachnospiracea and the metabolite L-Tryptophan,a significant negative correlation between Murbaculaceae and the metabolite PI（18:0/20:4（5Z,8Z,11Z,14Z））.Enterohabdus was significantly positively correlated with metabolites L-Isoleucine and L-Phenylalanine.The intervention of peptides was affected by key metabolites DG（18:0/20:4（8Z,11Z,14Z,17Z）/0:0）,PW-C003586HMDB0007535,Gamma-Glu-leu,3-Methylindole,5-Hydroxyindoleacetylglycine,Racemetirosine,Sulfolithocholylglycine.And the regulatory pathways were enriched in allergy related pathways such as Th1 and Th2cell differentiation,NF-κB signaling pathway,Th17 cell differentiation,B cell receptor signaling pathway,T cell receptor signaling pathway.At the same time,transcriptional genomic assay also indicated that key differential genes such as Niban2,Mcam,Mpzl2,Cpd,Igkv6-13,Ighv7-3,Ighv4-1,Igkv8-19,Ighv4-2,Ighv7-4,Igkv4-74 could regulate antigen processing and presentation,cytokine receptor interactions,and regulate immune system processes,signal transduction,the cell proliferation and differentiation,and other pathways.The results of functional enrichment indicated that peptide intervention could significantly alter immune related functions,including cell activation regulation,B cell signaling pathway,immunoglobulin production and receptor binding,immune response regulation,immune response,and other functions.Based on the above analysis,it was shown that tolerance peptides played a important role in the tolerance induction of CMA through significant regulation of related signaling pathways by key metabolites and genes.