Effect Of Vitamin A On Dendritic Cells And Its Role In Mucosal Immunity

Effect of vitamin A on dendritic cells and its role in mucosal immunityObjectives: (1): In order to clarify the mechanism that how vitamin A enhances the anti-infectious immunity in children, we investigated the effect of vitamin A on dendritic cells (DCs), the most potent antigen-presenting cells, to probe into its role at the start step of immune response. By cell culture in vitro we studied the affection of all-trans retinoic acid on the differentiation, maturation, and functions of DCs derived from cord blood monocytes. The Ro 41-5253, a specific antagonist for retinoic acid receptor a, was used to demonstrate the pathway of retinoic acid functions; (2): To research the effects and mechanisms of vitamin A on mucosal immunity, we established an animal model, the rat with vitamin A deficiency (VAD), to observe the affections of vitamin A on DCs distribution, maturation, antigen recognition and cytokines production. The study will provide theoretical and experimental evidences for the reasonable supply of vitamin A in clinical practice.Methods: (1). Monocytes were separated from 18 cord blood samples, and DCs were induced from monocytes by culture in vitro. Cell culture was divided into 4 groups: control group; retinoic acid group (RA group); RARα antagonist group (RO group) and RA+RO group. Expression of surface molecules, CD1α, CD83, HLA-DR on DCs were measured by flow cytometry. To evaluate the differentiation and maturation of DCs, the percentage of immature DCs (CD1α+HLA-DR+) and the mature DCs (CD83+HLA-DR+) were counted, meanwhile the fluorescence intensity of HLA-DR was detected. To observe the influence of RA or / and RO on the stimulating ability of DCs to allo-genetic T cells proliferation, mixed lymphocyte reaction (MLR) was used. Cytokines were measured in protein level by ELISA and in mRNA levels by RT-quantity PCR to decipher the role of RA or / and RO in the regulation of Th1/Th2 balance by DCs. (2). The rat model with VAD was established and by use of immunohistochemical, RT-quantity PCR and ELISA methods, to detect the number of DCs (OX-62 positive cells), the maturation of DCs (the positive areas of CD80 and CCR7), the pathogen recognition receptors (the positive areas of TLR2 and of TLR4) and TLRs signal transduction (the mRNA level of MyD88), the protein levels and mRNA levels of cytokines, and the expressions of retinoic receptors (RAR α,β,γ/RXRα,β,γ) in mucosa.Results: Cell culture data showed that RA inhabited the differentiation andmaturation of DCs from cord blood monocytes, suppressed allo-T cell proliferation induced by DCs, and down-regulated Th1 cytokine (IL-12, IFN-γ), up- regulated Th2 cytokines (IL-4, IL-10). However, when Ro 41-5253 was added into cell culture, all these actions of RA on DCs were reversed. The results from animal experiment showed that: 1. the numbers of DCs in trachea and distal ileum mucosa were increased in VAD group. 2. The expressions of TLR2 and MyD88 in trachea of VAD rats were up-regulated, but the expression of TLR4 was not influenced; the increased expressions of TLR2 and TLR4 in Peyer's patches of ileum were also found in the VAD group. 3. There was not marked change in the expression of CD80 and CCR7 in trachea mucosa, but the expression of CCR7 in Peyer's patches of ileum was increased in VAD rats; 4. In VAD group, IL-12 production was raised in trachea and distal ileum mucosa, but IFN-γ production was either no change (in trachea mucosa) or decreased (in ileum mucosa). Th2 cytokines were reduced in mucosa from VAD rats; especially in trachea (IL-4 and IL-6 were diminished significantly). Moreover, the production of IL-10 protein was declined in trachea and ileum mucosa of VAD rats. All six subtypes of retinoic acid receptors existed on the trachea and ileum mucosa, but only the expression of retinoic acid receptor alpha was down-regulated in VAD group.Conclusion: 1.Retinoic acid inhibits the differentiation and maturation of DCs derived from cord blood monocytes, reduces the ability to stimulating allo-T lymphocytes proliferation by DCs, and makes immune response bias to Th2, which contributes to antibody production and enhanced humoral immunity. 2. RARα plays a very important role in the regulation of DCs by retinoic acid. 3. VAD increases the number and the maturation of mucosal dendritic cells, enhances pathogens recognition and strengthenes their signal transduction. 4. VAD decreases Th2 cytokines in thachea mucosa and reduces Th1 cytokine (IFN-γ) and the regulating cytokine (IL-10) in ileum mucosa, which may contribute to the impaired mucosal immunity in rat. 5. All six subtypes of retinoic acid receptors exist in the trachea and ileum mucosa on different levels, but only the expression of retinoic acid receptor alpha is down-regulated by VAD.