| Background and objectivesThe prevalence of food allergy has increased dramatically over the past decade in the world. Recent studies report that food allergy affects4%to8%of children and3%to4%of adults, and its prevalence appears to be on the increase. Allergic diseases cause human health problems and affect social economy greatly, while the effect of current therapeutic remedies on allergic diseases is still unsatisfactory and the pathogenesis of food allergy is to be further understood.Food allergy is an immune response with the damage of oral tolerance and Th2activated in the intestinal mucosa. Studies have found that the breach of oral tolerance can be caused by a variety of mechanisms:including the T cell clones incompetence and lack, and regulatory T cells (Treg) which plays a importment role in producing antigenic specificity tolerance dendritic cell(TolDC). The TolDC has been proved to play a key role in activating the immune response and maintaining immune tolerance. Studies show that TolDC highly express IL-10, TGF-β, retinoic acid, indole2,3dioxygenase, low expression of MHC II and total stimulus molecule, through Foxp3+regulatory T cells (Treg) to suppress the immune response. TolDC play a key role in in suppressing the immune response and the maintainance of oral tolerance by improving the generation of Treg. Intestinal tract is one of the largest surface area of immune organs, and intestinal epithelial cells (IEC) are the first to contact with the external antigen. By limiting the penetration of luminal bacteria and dietary allergens, a series of the surface molecule in the surface plays an important role in maintaining the stability of the gut environment. IEC can prompt TolDC differentiation, and compared with other organs of the body, there are more TolDC in the intestinal lamina propria.As a member of the dimer polypeptide growth factor family, Transforming growth factor beta (TGF-β) involves in cellular functions regulation, proliferation, differentiation and migration. In the known three isomers (TGF-β1, TGF-β2, TGF-β3) of TGF-β, TGF-β1is mainly expressed in the immune system and TGF-β1regulates the immune function mainly by integrating with its receptor; it mainly exists in inactive state and will be activated before they can reach their potential complex biological function. Research suggests that integrin αVβ6plays an important role in the activation of TGF-β1. αVβ6is a protein composed by a and β subunits. Epithelial cells of healthy adults can express integrin αVβ6, while its expression increase greatly in inflammation, wound repair and cancer. Studies figure out that αVβ6, by combing with inactive TGF-β carboxyl terminal amino acid sequence can prompt the activation of TGF-β. Ovalbumin (ovalbumin and OVA) is commonly used in food antigen model because of its antigenicity.In our previous studies, we have established a classical mouse intestinal OVA allergy model though treating the mice OVA and adjuvant by gavage. Other study showed that a mouse intestinal food antigen tolerance model could be induced and established by challenging OVA alone, while its mechanism needs further studied. Our previous experiments by cell culture in vitro showed that the intestinal epithelial cell-derived exosomes contain the integrin αVβ6and food antigens, can increase the expression of TGF-P in bone marrow-derived dendritic cells and transformed it to TolDC, but whether the integrin αVβ6from intestinal epithelial cells plays an important role in the producing of intestinal TolDC remains to be further studied. In this study, first we will establish an OVA tolerance mice model in vivo, and further evaluate the status of TolDC in the intestinal mucosa by the intervention of intestinal integration αVβ6. The results in this study showed that anti αVβ6antibodies could block the TolDC production in OVA tolerance mice’s intestine. Intestinal epithelial origined integrin αVβ6could increase the expression of TGF-β1in intestinal DC, and turn it into an TolDC. This may indicate that integration αVβ6plays an important role in the intestinal immune tolerance, this study provides new theoretical basis for food allergic disease.MethodsThirty Balb/c male/female mice, were randomly divided into three groups:blank control group, OVA(ovalbumin) group, OVA+αVβ6antibody group. OVA group, OVA lavage (in reference with normal saline lavage blank control group) for a total of five consecutive days, OVA+αVβ6antibodies group, intraperitoneal injection of αVβ6antibody, the OVA lavage after30minutes same as that of OVA group.Took Lamina propria mononuclear cells (LPMC) of jejunum and isolated DC cells by Immunomagnetic. Ratio changes of TolDC(CD11c+TGF-β1+) were measured by flow cytometry. CD11c+and TGF-β1+distribution of jejunum was detected by double immunofluorescent staining (IF). and the changes of TGF-β1(Transforming growth factor-β) protein level was determined by Western-bolt. The experimental data were analyzed by SPSS17.0, p<0.05significant difference.Results1. Separating mice intestinal LPMC from each group, immunomagnetic separate intestinal DC cell. flow cytometry analysis by anti-CD11c antibody and TGF-β1antibody staining, the results showed that compared with the blank control group, TGF-β1+in LPMC from OVA challenged mice intestine mucosa increased significantly (p<0.05); while TGF-β1+in OVA+αVβ6antibody group decreased significantly (p<0.05). Compared with the OVA group, TGF-β1+of OVA+αVβ6antiboby group decreased significantly (p<0.05).2. After gavage by OVA, the TolDC cell in OVA group was (13.0±1.76), which increased significantly than that in control group (4.9±1.66,p<0.05), however the CD11c+DC cell of this group was (5.0±1.83), which was significantly lower than that in blank control group (8.1±1.91,p<0.05); The TolDC cell in OVA+anti αVβ6antibody group is (13.0±1.76), which was no significantly change compared with the blank control group (4.9±1.66,p>0.05), and the CD11c+DC cell count of this group was (11.0±2.16), which was no significantly change compared with the blank control group(8.1±1.915,p>0.05)3. Compared with the blank control group (0.129±0.069), TGF-β1expression in OVA group (0.236±0.049) was significantly increased (p<0.05). TGF-β1expression in OVA group was higher than that in OVA+αVβ6antibody group (0.174±0.075)(p<0.05).Conclusions1. CD11c positive cells originated from intestinal lamina propria mononuclear cells (LPMC) were detected by flow cytometry.That the expression of CD11c+and TGF-β1+increased in OVA groups, the expression of TolDC decreased after antagonising by αVβ6antibody.2. The distribution density of TGF-β1+cells in jejunum increased and TolDC increased after stimulating by OVA; however, the density of TolDC decreased after antagonising by αVβ6antibody.The expression of TGF-β1protein increased after stimulating by OVA; however the expression TGF-β1protein decreased after antagonising by αVβ6antibody. The experiment results indicate:the TGF-β1transformation is mainly through the regulation of αVβ6.3. Intestinal epithelial original integrin αVβ6is necessary to increase the TGF-β1expression in DC and turn DC into TolDC, thus αVβ6plays an important role in the intestinal immune tolerance. |
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