| Food allergy is recognized as a global food safety issue by the FAO/WHO and is considered a"neglected food safety’silent killer’".Currently,strict avoidance of allergenic foods is regarded as the most effective measure for preventing and managing food allergies.However,due to factors such as cross-reactivity of allergens,inadequate food allergens labeling regulations,and nutritional deficiencies resulting from avoidance diets,this passive approach often fails to effectively eliminate food allergies and may even have negative health consequences for affected individuals.The concept of oral administration of food allergen proteins to induce immune tolerance,as a causal treatment strategy,challenges traditional approaches to managing food allergies and offers a new perspective for addressing this issue.However,as an emerging treatment modality,there remains limited understanding of the factors influencing the establishment of oral allergen-induced immune tolerance and the mechanisms of immune regulation associated with it.Therefore,this study aims to elucidate the factors influencing oral allergen-induced immune tolerance and to analyze the mechanisms underlying the transition from a normal state to food allergy and then to immune tolerance,using an established immune tolerance model.Through conventional transcriptomic analysis combined with systems immunology approaches,we aim to identify the driving factors of this transition and elucidate the mechanisms of immune tolerance.Ultimately,our findings will provide a theoretical basis for guiding the development of foods that induce tolerance.The main research content and results are outlined below:(1)Food allergy models were established using three sensitization methods:oral,skin,and intraperitoneal sensitization.Food allergy symptoms and changes in body temperature were the primary criteria,supplemented by indicators such as release of food allergy mediators and alterations in immune cells,to screen for stable and reproducible food allergy models.Results revealed that the intraperitoneal sensitization followed by oral challenge induced a decrease in body temperature of approximately1°C and a diarrhea rate of 100%.Additionally,this model exhibited other characteristic features of human allergies,including elevated Ig E levels,mast cell activation,intestinal barrier disruption,and Th2 cell polarization.Based on this model,a study simulated early introduction of allergenic food in infants and toddlers to investigate the impact of oral administration of the food allergen protein OVA on inducing immune tolerance.Results showed that pre-sensitization with oral exposure to 5mg of OVA protein completely prevented the occurrence of later-stage food allergy symptoms,evidenced by normal body temperature and absence of diarrhea(100%diarrhea rate in the food allergy group vs.0%in the immune tolerance group),thus achieving immune tolerance to the allergenic protein OVA.(2)Further,replacing the oral administration of 5mg OVA with 5mg of milk allergenβ-lactoglobulin(BLG),the effects of introducing a different type of allergen before sensitization with OVA on establishing immune tolerance were explored.Results demonstrated that both OVA and BLG allergen introduction induced the generation of food antigen-specific Treg cells in mesenteric lymph nodes,upregulating by 10.6%and9.7%respectively.However,in the OVA-induced food allergy model,only the introduction of the same type of allergenic protein,OVA,could inhibit the occurrence of allergic diarrhea and body temperature decrease,while the introduction of the unrelated antigen BLG could not.Further sequencing of intestinal microbiota and metabolite analysis revealed that,compared to OVA introduction,BLG introduction led to a decrease in the abundance of short-chain fatty acid-related bacteria(such as Oscillospira and Ruminococcaceae)and a reduction in butyrate.These results demonstrate the allergen-specificity of the induction of immune tolerance by allergen introduction and elucidate the involvement of gut microbiota and metabolic dysregulation in the induction of immune tolerance to allergenic proteins.(3)Utilizing Bulk transcriptome analysis,the expression patterns of intestinal genes during the transition from normal to allergy to tolerance phenotypes were studied,and the role of the GSDMC-IL33 axis in the establishment of immune tolerance was determined.Analysis of the whole gene expression profile revealed a set of genes(Cluster 1)exhibiting a low-high-low expression pattern during the transition from normal to allergy to tolerance phenotypes.Functional enrichment analysis using GO terms identified a significant enrichment of cell pyroptosis processes.Further gene screening revealed abnormal activation of GSDMC family genes during food allergy,with m RNA expression of GSDMC2/3 genes increasing by over 200-fold.Detection of downstream molecules of GSDMC confirmed abnormal activation of the GSDMC-IL33 axis during food allergy,which could be inhibited by oral administration of OVA.(4)Food allergy and the induction of immune tolerance involve the intricate interactions of various cell subsets in the intestinal mucosal immune system.Given the limitations of bulk transcriptomics in analyzing complex tissues,this section further employs single cell RNA sequencing to finely identify and characterize intestinal mucosal cells under normal,food allergy,and immune tolerance states.Initially,utilizing the 10X Genomics single-cell sequencing platform,immune cells in the lamina propria(LP)and mesenteric lymph nodes(MLN)were subjected to single-cell sequencing,thereby delineating a single-cell atlas of murine intestinal mucosal immune cells.Subsequently,it was revealed that the LP comprised 22 cell subtypes,primarily including mast cells,T cells,B cells,ILC cells,DC cells,among others,while the MLN encompassed 18 cell subtypes,mainly consisting of T cells,B cells,and a minor population of myeloid cells.Finally,each cell subtype in the intestinal LP and MLN was meticulously identified,elucidating the heterogeneity and compositional differences of intestinal mucosal immune cells under food allergy and immune tolerance states.(5)Building upon the single cell atlas of LP and MLN immune cells,this section elucidates the key CD4~+T cell subsets and the driving factors behind the transition of these subsets that contribute to the formation of food allergy and immune tolerance.The results indicate that in the intestinal LP and Th2 cells exhibit high expression of alarm factors IL-25 and IL-33 receptors IL17RB and IL1RL1 genes,and Foxp3~+Tregs display significant Th2 characteristics,suggesting the involvement of these CD4~+T cell subsets in driving the development of food allergy.Furthermore,through clustering analysis and gene expression profiling of CD4~+Th cells in the MLN,a complex Th1/Th2 mixed pattern was observed.Subsequent flow cytometry analysis of Foxp3~+Tregs and Th cells in the MLN reveals that oral exposure to OVA before sensitization can upregulate T-bet expression in CD4~+GATA3~+Th2 cells and suppress the skewing of Treg cells towards Th2 direction,potentially mediating the formation of immune tolerance.Lastly,based on the evolution of allergic and tolerant states and pseudotime trajectory analysis,it is revealed that genes including Il1rl1,Vd200r3,Tph1,Calca,Stab2,Gata2,Ccl4,Iigp1,Irgm2,Gata3 may drive the fate determination of Th2 cells.In conclusion,this study successfully constructed a model of immune tolerance and demonstrated the allergen-specificity of the allergen protein-induced immune tolerance strategy;Furthermore,from the perspective of overall intestinal gene expression profiles and key immune cell subpopulations,we revealed the changes in the gene response patterns of intestinal mucosal cells in response to OVA stimulation and the roles of IL17RB~+IL1RL1~+Th2 cells,Foxp3~+Treg cells and CD4~+Th cells subpopulations in mediating the transition from food allergy to immune tolerance state.The results of this work interpret the role and function of intestinal mucosal immune cells in the process of immune tolerance induced by food allergen proteins to a certain extent,and provide a theoretical basis for guiding the development of food products that induce tolerance. |
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