Immunological Mechanisms Of Egg Ovalbumin Anaphylaxis Affected By Staphylococcus Enterotoxin B

Food allergy affects about 10% of the people,impairs human health,and brings economic and social burdens.It has also become a global public health and food safety issue.Researches on food allergy risk factors will be helpful to improve food safety management and reveal the mechanism of food allergy.Protein-rich foods like eggs,milk,and aquatic products are the main allergenic foods,which are also easily contaminated by bacteria and their toxins during husbandry and processing,leading to food safety incidents.Allergic foods and bacterial contamination are at risk of coexposure.These bacteria toxins could lead to damages of the intestinal mucosal barrier and imbalance of the local immunity,thus affecting immune cells’ recognition and uptake to allergens.And it makes bacteria toxins as a kind of potential risk factor for food allergy.Therefore,it is great scientific significance to explore the influence of bacterial toxin on the development of food allergies.And it also can contribute to the improvement of food safety management.Eggs are one of the prime allergic foods recognized by FAO/WHO and are easily contaminated by Staphylococcus aureus enterotoxin B(SEB).The influence and mechanism of SEB contamination in egg products on food allergy are worthy of scientific and systematic research.The ovalbumin(OVA)and SEB y was selected as the research objects in this study.Firstly,the effects of SEB on sensitization and challenge stage of OVA food allergy were studied through animal models.The impact of SEB on the Th immune balance of OVA food allergy was studied by evaluation of Th immune balance.Then,the changes in the cecum flora community and function were analyzed to determine the effects of SEB on OVA food allergy intestinal flora.Various cell models were constructed to explore the impact of SEB on the innate immune response to OVA.Because of the activation of SEB on Th cells,the role of SEB superantigen in OVA food allergy was also explored.Finally,the dose effect of SEB on OVA Th immune response was analyzed.The effects of SEB on OVA food allergy and its immunological mechanism were elucidated through the multilevel and progressive evaluation of the changes in the immune system response to OVA under exposure to SEB.It will provide a scientific basis for finally revealing the correlation between bacterial toxin exposure and food allergy and its mechanism of action.The main methods,results and conclusions of this study are as follows:1.A mouse model of SEB exposure affecting OVA sensitization was established.After oral exposure to OVA and different doses of SEB,mice were challenged with OVA.Through results of clinical symptoms of food allergy,intestinal barrier damage in tissue sections,serum-specific antibody levels by ELISA,and local and peripheral immune cell community changes in mice by flow cytometry,we determined that SEB co-exposure did not increase Ig E secretion,but significantly increased OVAspecific Ig G and Ig G1 secretion,increased IL-13,IL-10 and IFN-γ secretion levels during splenic cell restimulation,and increased the number of mast cells in the lamina propria from 14% to 25%.The expression of IL-33 receptor protein was increased from 10% to 18%.These results showed that SEB co-exposure did not lead to establishing Ig E-mediated OVA food allergy but enhanced Th2 immunity,mast cell proliferation,and innate immune cell activation.It suggested that SEB coexposure still has the potential risk of promoting the development of food allergy.2.Mice were sensitized with intraperitoneal injection of OVA and aluminum adjuvant and then challenged with OVA and different doses of SEB.To determine whether co-exposure to SEB during the activation phase promoted OVA food allergy symptoms,specific Ig E levels,intestinal damage,and changes in intestinal mucosal immune cell responses to OVA in mice.It was observed that the co-exposure of 1μg/ml SEB during the food allergy phase resulted in a 50% more severe food allergy clinical symptom score,0.3℃ more decrease in body temperature,and 120%increased diarrhoea rate score in OVA-sensitized mice compared to OVA group.The results showed that co-exposure to lower dose SEB during the challenges aggravated the clinical symptoms,intestinal damage,and immune imbalance in OVA-sensitized mice.3.Spleen cell restimulation,ELISA and flow cytometry were further used to evaluate the effects of SEB co-exposure at the activation stage on the function of DC cells and the direction of Th cell differentiation in spleen and mesenteric lymph nodes(MLN).Th2 cells in MLN and spleen of mice in the SEB group were increased by 3-4 times,and 1 ng/ml SEB could increase the secretion level of Th2 immune factor in spleen cells in response to OVA by 3-10 times.The results showed that SEB could promote the activation of DC-T cells,affect Th immune balance,and inhibit immune tolerance in mice during the development of OVA food allergy.And the promotion of Th2 cell differentiation is dominate by the SEB exposure dose.Lower dose SEB exposure makes Th immune direction to Th2 direction,which may be the critical mechanism of SEB co-exposure to promote the development of OVA food allergy and aggravate the symptoms of food allergy.4.16 S high-throughput sequencing technology and gene function annotation database was used to evaluate whether co-exposure of SEB during the food allergy would changes the intestinal microecology and function of mice.The analysis of high-throughput sequencing results found that OVA food allergy resulted in the deterioration of intestinal microecology and the imbalance of microflora structure in mice.The co-exposure to SEB and OVA aggravated the decrease of intestinal microflora diversity in mice caused by OVA food allergy,and beta diversity decreased by 14%.The changes in intestinal microflora mainly focused on increasing the proportion of Prevotella and Povotella.It also leads to the decline of flora function in immunity,intestinal repair,transport and other aspects.5.Small intestinal epithelial cells,ILC2 s,and DCs were isolated and cultured in vitro and stimulated with SEB and OVA,respectively.The expression of immune factors and changes in cell function was evaluated by Flow cytometry and ELISA.SEB could increase OVA transport rate through Caco-2 cell monolayer by about 3-4times,the level of IL-33 secretion in epithelial cells by about 26%,and the level of IL-4 secretion in ILC2 cells by order of magnitude.These results indicated that costimulation of SEB could promote the transport rate of OVA through intestinal barrier and boost innate immunity,including epithelium and ILC2 cells.Thus,SEB may promote the development of food allergy through the intestinal epithelial-earlywarning factor-ILC2-Th2 immune pathway and the uptake of OVA by DC cells and expressing surface costimulatory signals.6.Na?ve Th cells were separated and co-cultured with bone marrow-derived DC(BMDC)cells to construct a co-culture model of DC-T cells.The proportion of Th1/2/reg cell differentiation was evaluated by flow cytometry,and possible activation pathways in BMDC cells were analyzed by RNA-seq technique.The results suggest that SEB can activate and promote Th2 cell differentiation through DC-T cells crosstalk,which also is a critical mechanism of OVA food allergy.The NF-κB pathway and C-type receptor signaling pathway may be the possible pathways that SEB promotes the immune response of DC cells to OVA.7.Focusing on the DC-T co-culture model,1,10,100 ng/ml SEB and OVA were co-stimulated at different stages of DC-T cell co-culture.The differentiation trend of Th cells was analyzed by flow cytometry and ELISA.The results showed that in wild-type Th cells and OVA-specific transgenic mice(DO11.10)derived Th cells,the stimulation of OVA and higher doses of SEB(10 and 100 ng/ml)could promote the differentiation of Th cells towards Th1/2/reg direction and related to the co-culture with BMDCs.The promotion of Th1/2 immune balance by SEB costimulation showed that low dose SEB promoted Th2,while high dose upregulated Th1 and inhibited Th2.In DO11.10 Th cells,the proportion of Th1 cells was positively correlated with the dose of SEB co-stimulation,up to 32%.In comparison,the balance of Th2 cells was about 7% at a low dose.It may be one of the reasons why SEB promoted OVA food allergy more significantly in mice with the low dose.In conclusion,SEB cannot directly establish Ig E-mediated food allergy but can activate innate intestinal immunity and increase the risk of food allergy.During challenges,SEB could aggravate the clinical symptoms of food allergy,promote Th2 immunity,and further destroy the homeostasis of intestinal flora.In vitro experiments revealed that SEB could promote intestinal barrier transport,innate immunity,and T immune response to OVA.Especially in Th cell activation,it showed a dose-effect relationship that lower SEB inclined to Th2 and higher inclined to Th1.This paper illustrated the relationship between SEB and OVA food allergy and its mechanism and confirmed the promotion of SEB contamination in food to OVA food allergy development.It benefits the final disclosure of bacterial toxin exposure and food allergy correlation and mechanism.