The Function Of Different Subsets Of DCs In The Epicutaneous Sensitization Of Atopic Dermatitis

Background:Atopic dermatitis (AD) is a common, chronic, pruritic, retractable and inflammatory skin disease. It’s hard to prevent and treat since the pathogenesis of AD remains unclear. Currently, it is believed that in addition to some genetic factors, AD is also caused by environmental factors, skin barrier defects and immune unbalance of TH2/TH1cell subset. Skin associated lymphoid system (SALT) can not only perceive and deliver the stimulation and signal from outside, but also mediate and guide the subsequent immune response. It plays a significant role in the onset and maintain of the inflammation. Dendritic cells (DCs) is an important component of the SALT, which participates a variety of immune response processes and plays a key role in the processes of skin anti-infection epicutaneous sensitization and epicutaneous immune tolerance. However, the exact function and mechanism of DCs in the pathogenesis of AD has not been fully elucidated. If we can make clear the function of DCs subsets and mediate the function and activity of DCs pointedly, it is likely to change the immune response state of skin and realize the prevention and treatment of inflammatory and allergic skin disease, such as AD.The DCs of skin includes Plasmacytoid dendritic cells(pDC) and conventional dendritic cells(cDC). There are at least3subsets of cDC:Langerhans cells (LCs) in the epidermis and Langerin-positive and Langerin-negative DCs in the dermis. Different cDC subsets paly diverse roles in skin immune response. The previous studies indicated that LCs accelerate immune activation mainly. Nakajima reported that LCs can promote the epicutaneous sensitization process of protein antigens and take part in the inflammatory process of AD mouse models. Recently, an abundance of researches have shown that LCs also has the negative immunomodulatory function and suppress the CHS、rejection and other immune responses. It plays a vital role in the development of the skin homeostasis. Therefore, the definite role and mechanism of LCs in the epicutaneous sensitization of AD need further research. Currently, it is believed that Langerin+dDC and Langerin-dDC are mainly involved in the process of inflammation such as immune activity and CHS. However, the roles of Langerin+dDC and Langerin-dDC in epercutaneous sensitization of AD have not been reported yet.In recent years, the application of LC-depleted mouse models has contributed a lot to understand the role of LCs and dDC. Langerin-DTR mice were generated by using "knock-in" technology to introduce the primate receptor for diphtheria toxin(DT) into the endogenous murine langerin gene. Injection of DT leads to rapid and efficient ablation of the LCs and Langerin+dDC which express langerin. Later, the two type of langerin+cell gradually recovered at different time. The intervention into the cells recovery at different time can help us analyze the role of LCs, Langerin dDC and Langerin dDC.Epicutaneous sensitization is thought to be closely related to the pathogenesis of AD. OVA-induced epicutaneous sensitization will raise OVA specific serum IgE and IgG1levels, expression of mRNA of the cytokines IL-4, IL-5and will induce clinic skin lesion and pathology which is similar to AD. It is the preferable AD model which can reflect the epicutaneous sensitization.Objective:We use the wild-type mice and Langerin-DTR mice to establish the epicutaneous sensitization AD model and to explore the function and mechanism of devise subsets of DCs in the pathogenesis of AD.Methods:1.The development of AD mouse model:Balb/c mice were sensitized through the skin with OVA solution and saline was used as negative control. Each mouse had a total of five3-day exposures to the patch, separated by4-day intervals. The levels of serum IgE^OVA-specific IgG1and IgG2a will be measured to judge the effect of epicutaneous sensitization. After stimulate epicutaneous sensitization mouse models with OVA, clinical severity scores and total histology scores will be graded to estimate the effect of AD model. The inflammatory cells infiltrated in epidermis and dermis will be detected by flow cytometry.2. Apply the Langerin-DTR mice to analyze the roles of various DCs subsets in epicutaneous sensitization:2days and10days after DT administration, the patch tests were given to LC-depleted mice. Both Langerin-DTR mice which were injected DT but not pasted and Langerin-DTR mice which were not injected DT were used as negative control. Measure the expression levels of total IgE, OVA specific IgG1and IgG2a and compare the change of epicutaneous sensitization under the state of various DCs subsets depleted.Results: 1.The development of AD mouse model:In the OVA-induced allergic skin dermatitis model, there is a significant increase in the level of total IgE^special IgG1and IgG2a compared to the control group. And the level of these mentioned above antibody of the OVA-pasted group is close to the OVA-ip group. After stimulated, the scores of Clinical severity and histology scores of AD model also exceed the control group. It means that epicutaneous sensitization model could be successfully developed via our protocal. The phenotype of LCs under inflammatory condition was measured by flow cytometry. It is found that there is a significant increase in the number of CD11bhighlangerinhighLCs and the inflammatory cells infiltrated in dermis.2. Take advantage of the Langerin-DTR mice to analyze the roles of various DCs subsets in epicutaneous sensitization:At different times after DT administration, the patch tests were given to LC-depleted mice. It is founded that the total IgE, OVA specific IgG1and IgG2a level of the Langerin-DTR mice which are pasted2days after DT administration is much higher than the Langerin-DTR mice which were injected DT but not pasted. The result shows that Langerin-dDC takes part in the epicutaneous sensitization of AD.The expression level of total IgE, OVA specific IgGl and IgG2a in the Langerin-DTR pasted10days after DT administration exceed they expressed in the Langerin-DTR mice pasted at2days after DT administration, but it is still lower than that expressed in the Langerin-DTR mice which were not injected DT. So we can conclude that Langerin+dDC also paly a role in the process of epicutaneous sensitization and LCs may take part in the process too.Conclusions:1. Both Langerin-dDC and Langerin+dDC take part in the epicutaneous sensitization of AD model.2. LCs may participate in the epicutaneous sensitization of AD model. But the conclusion needs be further testified.