Effects Of Digestive Peptides From Bovine Allergen On Intestinal Mucosal Immune Cells And Proliferation Of CD4~+ T Cells In Vitro

Milk is a kind resource of food protein for infant and young children.However,some of them suffer from milk allergy which affect their live quality.In general,milk contains more than 30 kinds of protein,all of which are potential allergenic.Among them,casein,?-lactoglobulin and?-lactalbumin are regarded as three main allergens.It was reported that about 82%of allergic patients are sensitive to?-lactoglobulin.In our study,whole milk and infant formula were digested in simulated gastric fluid and intestinal fluid in vitro,respectively.The sequence alignment of digested peptides and the amino acid sequence of?-lactoglobulin were carried out and then combined with result of T cell epitope prediction to screen 5 peptides as candidates.On the based of this,the effects of digestive peptides on the maturation,proliferation and differentiation of mouse intestinal immune cells and CD4⁺T cells were detected in order to verifying accuracy of 5 T cell epitope-digested peptides and exploring the change of intestinal immune cells in milk allergy.The main methods,results and conclusions of this study are as follows.1.To predict Th epitope of?-lactoglobulin by online website tool of NetMHC?II?/pan,SYFPEITHI,and IEDB,six T cell epitope regions of bovine?-lactoglobulin were obtained,being AA_1-21,AA_23-61,AA_71-103,AA_107-119,AA_123-140,and AA_148-160.In addition,we simulated gastric and gastrointestinal digestion of pure milk and infant formula,respectively.The result from Tricine-SDS-PAGE electrophoresis showed that BLG and ALA were more resistant to digestion in simulated infants'digestionand the digested peptides?<10kDa?were detected by liquid chromatography-mass spectrometry.The sequence of 730 digested peptides were obtained.Among them,there were 109 digested peptides derived from?-lactoglobulin?Score range>20?,their molecular weight was among 813.42Da-2218.22Da.Their regions of these peptides by alignment were located in AA_1-21,AA_23-61,AA_71-103,AA_107-119,AA_123-140,and AA_148-16048-160 of?-lactoglobulin.Combining the information of predicted results and the digestive peptide sequence of BLG,five T cell dominant regions were chosen and synthesized:f1(AA_1-14),f2(AA_24-35),f3(AA_40-60),f4(AA_82-101),and f5(AA_123-139).2.To obtain specific immune cells,a mouse model sensitized by BLG sensitization was constructed,After oral challenge,the mice were sacrificed and serum was collected by centrifugation.The serum levels of IgE,IgG and IgA were determined by indirect ELISA.The results showed that there were no significant differences in BLG-specific IgG antibodies between groups C,M and F?p>0.05?.The level of IgE antibody in group F was significantly higher than that of group C and M?p<0.05?,and the level of IgA antibody appeared a significant difference in the three groups?p<0.05?.3.The mouse lamina propria cells were obtained and the phenotype of dendritic cells in lamina propria cells was detected by flow cytometry.The results showed that the proportion of CD11c⁺MHC II⁺cells in M and F group accounted for more than 80%.which indicated dendritic cells dominated in lamina propria cells in experimental animal.In addition,the proportion of CD103⁺CD11c⁺cells in the lamina propria cells of group C,M and F was 46.25±1.63,58.95±4.45,and 74.00±3.68,respectively.4.The digested peptides were co-incubated with LP cells,and the expression of MHC II,CD80 and CD86 on the surface of LPDC cells was determined by flow cytometry.The results showed that the effects of peptides f1-f6 on CD80 and CD86 expression levels were consistent.Peptide f3 significantly stimulated the maturation of LPDCs,while peptide f1 and f4 delayed the maturation of LPDCs.5.In further,the differentiation of T cells in MLN were moniotered by flow cytometry after stimulation by the peptides.The effect of peptides on CD4⁺T cells and CD8⁺T cell subsets were not obviously different.For the differentiation of Th subpopulations,of the C group,the digestive peptides were down-regulated in the expression levels of Th1 and Th2.In group M and F,peptide f1,f4 and f5 increased the expression of Th1cells,and the f1 and f4 peptide segments also increased the expression of Th2 cells.6.CD4⁺T cells in spleen cells from non-sensitized mice were sorted by CD4 positive sorting magnetic beads and the purity of CD4⁺cells reached 96.9%by flow cytometry,which can be used for the subsequent T preparation proliferation test.CD4⁺T cellswere stained with CFSE dye,stimulated with peptide and LPDC cells for 72h.The proportion of T cell proliferation,LPDC cell surface molecules and the level of cytokines were determined.The results showed that f1-f5 peptides could stimulate T cell proliferation,while the expression levels of MHC II,CD80,CD86 and CD209 remained the same or decreased compared with the peptide-free treatment,indicating that the selected peptides did not promote the maturation of LPDC cells in sensitization phase.In the effect on T cell differentiation,the f1 peptide segment inhibited the differentiation of Th1 and Th2 cells,promoted the differentiation of Treg cells.