Experimental Study On Mechanism Of Immune Tolerance In The CE-induced Allergic Reactions

Objective: Cystic echinococcosis(CE) is a zoonotic disease, and CE-induced anaphylactic shock is a serious complication which is responsible for over 20% of all sudden death cases in CE patients. It has been confirmed that immune tolerance exists in CE patients, but this condition would change after hydatid cyst fluid spill. The mechanism of anaphylactic shock remains unknown. In this research, we investigated the characteristics of specific molecular pathways by improving evaluation system of allergies and optimizing the animal model of CE-induced allergy. The molecular system is so called ’immune tolerance mediation network’ with Treg cells as the core. We also explored the mechanisms of the effect of dexamethasone on CE-induced allergic reactions. Elucidating the intrinsic mechanism underlying immune tolerance in CE may be helpful for designing protocols for early screening, intervention and prevention of the anaphylactic response in CE patients. Methods: 1) Sheep-derived E. granulosus cyst fluid(sheep hydatid fluid, SHF) and protoscolex were separated from the livers of sheep infected with E. granulosus under aseptic conditions. In the infected groups, all the mice were injected percutaneously with 2000 protoscoleces from E.granulosus. The sensitization was induced by intraperitoneally injecting SHF with dose of 0.1ml/10 g. Allergic symptoms, rectal temperature, serum antibody levels, lung HE staining, alveolar lavage fluid leukocyte count, histamine levels and PAF-AH levels were used as a series of markers for an optimized comprehensive allergic evaluation system; 2) BALB/c mice were randomized into group A(healthy control), group B(e.g infection) and group C(cystic fluid sensitization). DCs and Treg cells were detected by flow cytometry. The levels of IgE, IgG, IgG1 and cytokines(IL-10, IL-13, and TGF-β1, IL-17A) in serum were quantified by enzyme-linked immunosorbent assay; 3) C57BL/6 mice were randomized into uninfected(healthy control) group, untreated group and DEX group. Mice were intraperitoneally injected with a protoscolex suspension(0.2 mL/mouse) containing 2000 protoscoleces to induce echinococcosis. The sensitization was induced by intraperitoneally injecting SHF with dose of 0.1 ml/10 g. The DEX group was treated with 10 mg/kg dexamethasone by intraperitoneal injection 30 min before challenging. The comprehensive allergic evaluation system was used to evaluate the model. Treg cells were detected by flow cytometry. The levels of IgE, IgG, IgG1 and cytokines(IL-10, IL-13, and TGF-β1) in serum were quantified by enzyme-linked immunosorbent assay. Results: 1) The successful rate was 75% and 65% in CE infected BALB/c mice and C57BL/6 mice, respectively. The successful rate was 93.33% and 85.62% in sensitization model of BALB/c mice and C57BL/6 mice, respectively. Allergic mice presented different severity of symptoms and decreased rectal temperature. HE staining in lung showed different pathology, including inflammatory cells in BALF, as well as increased levels of histamine and IgE、IgG、IgG1. The model of CE-induced allergic reaction in BALB/c mice and C57BL/6 mice were built up successfully; 2) Finally, IgE levels were significantly higher in both groups inoculated with E. granulosus as compared to the healthy control group(both(P < 0.01), and sensitized mice had higher IgE levels as compared with those with e.g infection alone(P<0.05). After sensitization, inflammatory cells in lung and BALF increased, the levels of histamine and IL-13 or IL-17 increased(P<0.05). Compared with healthy control group, the levels of IL-10, TGF-β1 and CD4+CD25+Foxp3+Treg/CD4+ cell ratio increased in e.g infection group, declined in sensitized group(P<0.001), which present a change of increasing and decreasing. No significant differences were observed among the groups in the proportion of CD4+CD25+Foxp3-cells or CD4+Foxp3+CD25- relative to CD4+cells, CD11c+1A/IE+CD86+or CD11c+IA/IE+CD80+ cells relative to CD11c+ cells and the ratio of CD4/CD8(all P<0.05); 3) 12.5% mice in DEX group and 37.5% mice in sensitization group appear severe allergic reactions. Compared with the sensitization group, inflammatory cells in lung and BALF were reduced, histamine expression was reduced, the levels of IgE and IL-13 in serum were decreased(P<0.001) in DEX group. Serum levels of IL-10, TGF-β1 and the proportion of CD4+CD25+Foxp3+Treg cells changing trend were same. All decreased in sensitization group, increased in DEX group. Conclusions: 1) A comprehensive allergic evaluation system optimized the mice model of CE-induced allergic reactions, which lay the foundation for exploring mechanism of immune tolerance in CE-induced allergic reactions;2) After e.g infection, the status of immune tolerance was maintained through ’immune tolerance control network’ with Treg cells as the core. HCF made the network imbalance and induced allergic reactions; 3) After e.g infection, the Treg cells group maintains immune tolerance function through high expression of CD25 and Foxp3, increased IL-10 and TGF-β1 levels, and decreased Th2 cytokines IL-13 levels. After Cystic fluid sensitization, the status of immune tolerance changed, the proportion of Treg cells declined, IL-10 and TGF-β1 levels decreased,IL-13 and IL-17 levels increased, induced allergic reactions; 4) DEX can reduce allergic reaction by reducing IgE levels, decline inflammatory cells effusion in lung and BALF, reduced histamine expression in target organs during CE-induced allergic reactions; 5) DEX can stabilize the ’immune tolerance control network’ by adjusting the proportion and function of Treg cells, with the mechanism of increased levels of IL-10 & TGF-β1, and inhibition of Th2 cytokines IL-13.