| BACKGROUNDWith the development and clinical application of new types of antibiotic and vaccine, the treatment of infectious diseases have achieved a great success. However, in the past a few decades, the prevalence of allergic diseases have grown obviously, as much as 15%to 20%of the population have IgE-mediated allergic reactions with exogenous antigens, and as much as 2%to 6%of the population have food allergy and allergic related symptoms. In recent years, although allergic diseases have been studied numerously, the pathogenesis and etiology of them remain unclear and the treatment of them are limited and ineffective.It is generally believed that dendritic cells and Th2 cells are critical in the allergic immune response. DCs are the most powerful antigen presenting cells (APCs) in the body, which are the only APCs that can activate the naive T cells. DCs capture and process the exogenous antigens, and present antigen information to T cells to initialize T cell-mediated immune response. The mature DCs can express high levels of MHC-Ⅰ/Ⅱmolecules and costimulatory molecules such as CD80 and CD86 which can assist DCs present antigen to T cells, and then activate T cells effectively. The naive CD4+T cells could be activated to differentiate into Thl, Th2, Th17 or Treg cells. Thl cells mainly release type 1 cytokines, such as IFN-γ. While Th2 cells mainly release IL-4, IL-5 and IL-13. Then These cytokines play an important role not only in the production of antigen-specific IgE by B cells, but also in mucus secretion, muscle contractility, and eosinophilia. Mast cells activated by IgE cross-linking release allergic mediators to initiate the hypersensitivity reactions and allergic clinical symptoms on re-exposure to specific antigens. Thus, the activation of Th2 cells is a critical step in the immune mechanisms of food allergy.Recent studies discovered that T cell immunoglobulin and mucin domains (TIMs) gene family introduced a new family of cell surface proteins. The TIMs gene family have been found including 8 members (TIMs 1-8) in mice, and including three members (TIM1,3 and 4) in humans. They have important regulatory role in the development of allergic diseases and autoimmune diseases. TIM4 is expressed on the surface of APC, especially mature DCs, and TIM1 is expressed on T cells, particularly Th2 cells. It is recently found that they are the important factors of T cells. In vitro studies have shown that the expression of TIM4 protein on DCs is related to the severity level of autoimmune diseases. TIM4 that combines with TIM1 promotes T cell proliferation, and studies suggest that TIM4 may as a new molecular to regulate T cell response. Our previous studies showed that food allergic mice existed Treg cell dysfunction, the interaction of TIM4 and TIM1 reduce the function and status of Treg cell and break the balance of intestinal immune tolerance. But whether TIM4 effect the differentiation of food antigen-specific Th2 cells keep poorly studied, more experimental evidences in vitro should be provided, and the underlying mechanism needs further study.It is supposed that concurrent exposure to both microbial product and food antigen leads to the development of food antigen-specific Th2 cells and food allergy (FA). the interaction of TIM4 and its receptor may result in the imbalance of Thl/Th2 and break immune tolerance, which is the key pathogenesis of FA. In this study, BMDC and CD4+T cells were cocultured, evaluate effective of microbial product and food antigen for CD4+T cells and the role of TIM4 in the FA. Through this study, the molecular mechanism of food allergy could be futher understood and the new theoretical basis may be provided for clinical treatment of the FA. AIMSThe expression of TIM4 mRNA was measured and the expressions of CD llc, MHC-Ⅱ, CD86 on DCs were detected to investigate the role of microbial product in the generation of TIM4 and the stimulant effect of microbial product on DCs. The splenic CD4+T cells were isolated from the allergic BALB/c mice that were sensitized together by SEB and OVA, and CD4+T cells and DCs were cultured under different conditions in vitro in order to test the cytokine levels of Thl/Th2. The role of TIM4 in CD4+T reaction and food allergy was examined by exposure to microbial product.METHODSThe bone marrow-derived DCs in BALB/C mice were isolated and cultured in vitro, cultured DCs were divided into staphylococcal enterotoxin B (SEB)-stimulated group and control group. BALB/c mice were sensitized to SEB and OVA for the allergic spleen CD4+T cells. CD4+T cells and DCs were divided into 5 groups:control group, SEB plus ovalbumin (OVA) group, SEB group, OVA group and anti-TIM4 antibody plus SEB and OVA group. The related indicators were detected.1. The expression of TIM4 mRNA on DCs was detected by RT-PCR.2. The expressions of the CDllc, MHC-Ⅱ, CD86 on DCs surface were analyzed by Flow cytometry.3. The levels of IL-4 and IFN-γin mixed cultured cells supernatant were tested by ELISA.Data were expressed as the means±D. All statistical analysis was performed using SPSS statistical version 13.0. Differences between groups were determined with one-way ANOVA; a P value of less than 0.05 was considered to be statistically significant.RESULTS1. Bone marrow cells in vitro were isolated, and were induced into dendritic cells. The characteristic sign of CD llc was measured by flow cytometry, and its purity was up to 70%. DCs showed a characteristic morphology. 2. The SEB was used to stimulate DCs. The expression of TIM4 in DCs at messenger RNA (mRNA) was increased significantly in the SEB-stimulated group compared with the control group (0.941±0.018 vs 0.422±0.083, P<0.05), and the SEB up-regulated the expression of TIM-4 in a dose-dependent manner.3. The SEB was used to stimulate DCs, and the flow cytometry was used to measure. The expression of MHC-Ⅱand costimulatory molecule CD86 on DCs significantly increased after SEB stimulation (MHC-Ⅱ:76.684%±3.1803%vs 52.984%±3.6026%, P= 0.000; CD86:89.746%±2.113%vs 67.558%±0.4341%, P= 0.000).4. Compared with control DCs co-culture with CD4+T cells, the level of IL-4 in culture medium in the SEB plus OVA group increased significantly (295.834±20.408 vs 78.335±13.109, P<0.05), level of IFN-y decreased significantly (362.109±92.271 vs 761.897±102.967, P 0.05). Levels of IL-4 and IFN-y in the SEB group and OVA group showed no significant differences with those in the control group. In contrast, the level of IL-4 was significantly lower and and that of IFN-y was significantly higher in the anti-TIM4 antibody group compared with that of SEB plus OVA group (P＜0.05). The expressions of IL-4 and IFN-y in the anti-TIM4 antibody group showed no significant differences with those in the control group, SEB group and OVA group.CONCLUSIONS1. SEB up-regulates the expression of TIM4 on DCs.2. SEB increases the expressions of MHC-Ⅱand CD86 on DCs, and enhances DCs maturation.3. Concurrent exposure to SEB and OVA in coculture of CD4+T cells and DCs are able to cause Th2 cells polarization and decreased Thl cytokine response, and trigger the subsequent development of food allergy. SEB or OVA alone does not change the levels of IL-4 and IFN-y, which suggest that SEB or OVA alone does not cause food allergic immune response.4. TIM4 antibody can inhibit Th2 cells polarization, it can effectively restore the imbalance of Thl/Th2 cells and it may cure food allergy, which also suggest that TIM4 is an important molecular in the pathogenesis of FA. |
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