The Role Of Dendritic Cells In Early Life Allergens Immune Intervention

Backgroud and objectiveAllergic rhinitis (AR) has become a global health problem with a huge social burden. However, the pathogenesis of AR has not yet been fully elucidated, treatments are also limited. Therefore, AR early prevention is a hot topic in the current researches. Allergens immune intervention in early life is one of remarkable aspects of the area.In clinical cases, allergen specific immunotherapy is performed with different dose of allergen stimulation. The large dose of allergen induced immune tolerance, but small dose of allergen has no significant effect on AR symptom. It suggests that the stimulation of different doses of allergen may lead to different immune effect. In recent years, some studies show that environmental exposure early life may decide the development of children allergic diseases. Epidemiological survey found that exposure to high levels of allergens early life may have a protective effect in the future. However, it is not clear about the certain mechanism, and lack of researches in this area. As we all know, allergens are captured and processed by antigen presenting cells when they enter human body, then antigenic peptides are presented to the naive T cell, thus mediated T cell differentiation. Some researches show that dendritic cell (DC) present different status of differentiation and maturation when it is stimulated by external factors and local microenvironment. Different dose of allergen, which also is a kind of external interference factors, should impact on differentiation and maturation of DC. Some of the latest researches also show that different differentiation and maturation of DC has a great influence on the direction of T cell differentiation. So, we believe that DC may play a key role in early life allergen interventions.In our presumption, dose level of allergen is the only different factor, but T cell immune memory may skew to different direction. However, only if the allergen be presented by DC, it can trigger the subsequent reaction. Therefore, it is very likely that the immune intervention with different doses of allergens affect different differentiation and maturation of DC, and then impact on the differentiation of T cells and immune effects in the later. Antigen presenting cells (APC, mainly DC) and CD4+T helper cells are rich in nasal mucosa. And the function balance disorder of T cell subsets is closely related to the pathogenesis of AR. Therefore, as the most important professional antigen presenting cells, DC should play an important role in the pathogenesis of AR. It is reported that exposure to high level of allergen in early life may have protective effect on allergic diseases. On the basis of this phenomenon, we assume that active immune intervention with certain dose of allergen in early life, thus cause specific immune protective effect and prevent the corresponding allergen sensitization in the future, then suppress the occurrence of allergic rhinitis.This paper will explore the effects of the different doses of allergen (ovalbulin, OVA) exposure in early life on adult immune status in animal model, and then study the effect of different doses of allergen on differentiation and maturation of DC in vitro and subsequent influenc on the differentiation direction of naive T cell. The study may offers a theoretical basis for allergen immune intervention in early life.Metholds1. Effect of neonatal stimulatione by different doses of OVA on immune status of adult miceBALB/c mice were stimulated by different doses of OVA (10μg,1000μg) at the1st,8th,15th days after their birth, followed by challeng with OVA at the5th week as following, then killed and obtained samples one week later. Control groups were stimulated by normal saline or aluminum hydroxide, and then challenged by normal saline. Serum specific IgE, IgG1and IgG2a antibodies against ovalbumin were analyzed by ELISA. The cytokines, IL-4, IFN-y, and IL-10in the supernatant of cultured spleen mononuclear cells were quantified by ELISA.2. Effect of different doses of OVA on differentiation and maturation of DCBone marrow cells were obtained from female BALB/c mice. Bone Marrow-Derived Dendritic Cells (BMDCs) were induced and cultured by using combined GM-CSF+IL-4in vitro. Six days later, immaturate DC were stimulated by different doses of OVA (0,0.1,1,10,100,1000,10000μg/ml) for24hours. The expression of costimulatory molecules CD40, CD80, CD86and MHC Ⅱ molecule were analyzed by flow cytometry.3. Effect of different differentiation and maturation of DC on differentiation of naive T cellBone marrow cells were obtained from female BALB/c mice. BMDCs were induced and cultured by using combined GM-CSF+IL-4in vitro. Six days later, immaturate DC were stimulated by different doses of OVA (0,10,1000,10000μg/ml) for24hours. The naive T cells were selected form lymph node cells of female BALB/c mice by using immunomagnetic negative selection. BMDCs were harvested and co-cultured with the separated naive T cells at7th day. The expression of CD4+Foxp3+, CD4+T-bet+and CD4+Gata3+cells were analyzed by flow cytometry.Results1. Effect of neonatal stimulation by different doses of OVA on immune status of adult miceCompared with control groups, mice were found significant higher level of serum OVA-specific IgE after stimulated by small dose of OVA, even significant higher than large dose groups. However, there were no significant difference between OVA-specific IgE level of control groups and large dose groups. Compared with small dose groups, large dose groups produced relatively lower level of OVA-specific IgG1and higher IgG2a. At the same time, comparison of cytokines in the supernatant of cultured spleen cells was the approximate trend. Smal dose groups appeared Th2skewed reaction (lower IFN-gamma/IL-4ratio), and high dose groups appeared Th1skewed reaction (higher IFN-gamma/IL-4ratio).2. Effect of different doses of OVA on differentiation and maturation of DC After6days of culture, CD11c+cells were greater than80%. There was no significant different expression of dendritic cells phenotype, such as CD40, CD80, CD86and MHC Ⅱ, between the cells stimulated by different lower doses of OVA (0,0.1,1and10μg/ml). But when the doses of OVA≥100μg/ml, the phenotypic expression were increased significantly and correlated with the level of OVA doses positively.3. Effect of different differentiation and maturation of DC on differentiation of naive T cellCD4+CD62L+cells were selected and greater than92%. After72hours of mixed lymphocyte culture,0and10μg groups had significant higher proportion of CD4+Gata-3+cells;1000μg groups had significant higher proportion of CD4+T-bet+cells; while CD4+Foxp3+cells of10000μg groups were increased significantly, both CD4+Gata-3+and CD4+T-bet+cells were obviously decreased at the same time.ConclusionIn conclusion, we found that allergen intervention with small dose of OVA early life leads to mice specific immune response skewed to Th2advantage in the future; while intervention with large dose of OVA leads to specific immune response skewed to Thl advantage. In a certain level range, allergen stimulation with different doses of OVA causes different differentiation and maturation of DC, and then induces the different differentiation of naive T cells. Our foundings confirm the previous view, as descried previously, that high level of allergen exposure in early life has a protective effect on allergic diseases. And the foundings reveal furtherly that DC might be the possible target of protective effect induced by high level of allergen immne intervention. Our studies may provide a new idea for the prevention and treatment of allergic rhinitis or allergic disease.