| Background and ObjectivesAs much as 2% to 6% of the population has food allergy (FA) and food antigen-related disorders, and the prevalence has risen, especially in the last 20 years. The clinical symptoms of food allergy vary from slight abdominal discomfort to life-threatening anaphylactic shock. However, the etiology and immune mechanisms of intestinal allergic diseases remain poorly understood. FA is generally regarded as a collapse of the balance between intestinal immune system and food antigens, resulting in a skewed Th2-dominant intestinal immune response. It is believed that under normal circumstances, the gut immune system is relatively hyporesponsive to food antigens and commensal bacteria, and this state of hyporesponsiveness (tolerance) is maintained by a number of important mechanisms including the integrity of gut epithelium and the presence of tolerogenic dendritic cells (DCs) and regulatory T cells.CD4+CD25+ regulatory T cells (Treg) are subset of regulatory T cells and they play an important role in maintaining the stabilization of internal homeostasis, anti-infection, oral tolerance, introduction of transplantation tolerance and autoimmune disorder. Reduction of Treg number and impairment of Treg function can breach the balance of oral tolerance. Recently the forkhead-winged helix family transcription factor, Foxp3, has been shown being specifically expressed in murine CD4+CD25+Treg cells, and appeared being a 'master gene' controlling the development and suppressive function of CD4+CD25+Treg cells, and previous studies reported that Foxp3 gene mutation or decreased expression could lead to Treg cell dysfunction. It is necessary to elucidate the immunogenesis of Treg cells in FA.The TIM (T-cell immunoglobulin domain and mucin domain) family was discovered in 2001 and received much attention due to its extensive immune regulations in allergy, autoimmunity and the balance of Th1-Th2. The TIM family consists of eight genes in mouse (TIM1-8) and three genes in human (TIM1,3, and 4). Among these members, TIM1 is constitutively expressed on CD4+ T cells, and its ligand TIM4 is expressed in DCs. The interaction of TIM1 and TIM4 could promote Th2 cell polarization. In addition, studies showed that TIM1-specific agonist antibody decreased the suppressive capacity of natural regulatory T cells, in line with a reduction in their expression of Foxp3, GITR (glucocorticoid-induced tumour-necrosis factor (TNF)-receptor-related protein), CTLA4 (cytotoxic T-lymphocyte antigen 4) and IL-10. However, the functional status of Treg cells and the impact of TIM1 on Treg cells in FA has not been reported. We speculate that Treg cells are dysfunctional and TIM4 interaction with TIM1 might attenuate the function of Treg cells and oral tolerance, which might be one of the important mechanisms leading to FA. In this study, a SEB+OVA allergy model was established, and pretreatment with anti-TIM1 and anti-TIM4 to further stuty the functional status of Treg cells and the function of TIM1.The aims are to evaluate the function of Treg cells in food allergic mice by measuring the expressions of Foxp3 mRNA in jejunum and spleen and the expressions of TGF-β1 and IL-10 in SEB+OVA sensitized mice, and to determine the influence of TIM4-TIM1 pathway on Treg cells.Materals and MethodsThirty-two BALB/c mice fed on the OVA-free diet were randomly divided into four groups, and eight mice were used for each group: A group of mice were sensitized by ip injection with SEB+OVA and the other three groups of mice were separately induced with normal saline (NS), anti-TIM1+SEB+OVA, anti-TIM4+SEB+OVA, on the 0,3rd and 9th day; and all of the mice were challenged by means of ig with SEB+ OVA (except NS) on the 7th and 14th day. Diarrhea, the signal of food allergy, was also noted by direct observation of the colon and cecum; the liquid stool observed following SEB+OVA challenge-induced diarrhea in the sensitized mice contrasts with the solid pellets seen in the distal colon of other mice in control groups. Twenty-four hours after the last gavage, the mice were killed and subjected to immunologic analyses.1 The level of total OVA sIgE and IL-4 in blood serum was analyzed by ELISA.2 The expressions of Foxp3 mRNA in jejunum and spleen were measured by RT-PCR.3 The expressions of TIM4 mRNA in jejunum were measured by RT-PCR.4 The levels of TGF-β1 and IL-10 in serum were analyzed by ELISA.5 The expressions of TGF-β1 and IL-10 in jejunum were detected by immunohistochemistry.6 The numbers of eosinophils were counted by hematoxylin-eosin staining.Data were expressed as the means±SD. 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 The levels of OVA-specific IgE and IL-4 and the numbers of eosinophils in SEB+OVA sensitization group were higher than those of contral group (all P<0.05).2 Expressions of Foxp3 mRNA in jejunum and spleen decreased significantly in SEB+OVA group compared with those of NS (0.401±0.145 vs 0.732±0.162, P=0.000; 0.407±0.082 vs 0.691±0.145, P=0.000). In contrast, the expressions of Foxp3 mRNA in jejunum and spleen were significantly higher in anti-TIM1+SEB+OVA and anti-TIM4+SEB+OVA groups compared with those of SEB+OVA group (all P<0.05).3 Expressions of TIM4 mRNA in jejunum were higher in SEB+OVA (0.615), anti-TIM1+SEB+OVA (0.606) and anti-TIM4+SEB+OVA (0.578) groups compared with those of contral group (0.485) (all P<0.05). 4 The levels of TGF-β1 in serum and jejunum in SEB+OVA sensitization group decreased significantly compared with those of NS (7859.853±126.704 vs 8342.814±488.461, P<0.05; 108.834±9.634 vs 156.298±12.002, P<0.05) Importantly, the levels of TGF-β1 in the serum and jejunum were significantly higher in anti-TIM1+SEB+OVA and anti-TIM4+SEB+OVA groups compared with SEB+OVA group (all P<0.05).5 The levels of OVA-specific IgE and IL-4 and the numbers of eosinophils in SEB+OVA sensitization group were higher than those of SEB+OVA group (all P <0.05).Conclusions1 SEB+OVA sensitization induced food allery in mice.2 The expressions of Foxp3 mRNA in the jejunum and spleen and the levels of TGF-β1 in serum and jejunum in SEB+OVA sensitization group decreased significantly compared with those treated with NS. These results manifested Treg cells were dysfunctional and its oral tolerance was impaired in FA.3 SEB+OVA sensitization increased the expressions of TIM4.4 Pretreatmented with anti-TIM1 or anti-TIM4 cound restore Treg cells' function and dampen allergic responses, suggesting the pathway of TIM4-TIM1 may play a key role in food allergy.
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