肠道菌群介导的维生素D影响婴幼儿食物过敏的前瞻性研究及作用机制

Infants and young children are at high risk of food allergy with the incidence as high as 10%. Studies suggested that children with either VitD deficiency or excess supplementation may increase the risk of food allergy. But few researches have been made on the basis of prospective study to elucidate the association between the relationship and the mechanism. Our previous study found that VitD deficiency reduced the content of intestinal lactobacillus. It has been proved that lactobacillus can stimulate dendritic cells (DC) in lamina propria of intestine, and through the TLRs signaling pathway and thus differentiate into tolerant DC inducting oral tolerance. In addition, it is known that antimicrobial peptide, whose gene promoter sequence contains VitD elements, is a type of important polypeptide for the regulation of intestinal microbiota. Accordingly, based on the previous research results, we hypothesize that VitD deficiency (or in excess) reduces the intestinal lactobacillus by affecting the antimicrobial peptides, which affects the combination of the lactobacillus with TLR2 and TLR4 receptors on the surface of DC, inhibits DC from differentiating into tolerance DC and deceases the numbers of CD103+ tolerance DC. As a result, the numbers of Foxp3+ Treg cells will decrease and promote differentiation of T cell into Th2. The damage of oral tolerance leads to the occurrence of food allergy. This project will build the first cohort of VitD and food allergy among infants and young children. On the basis of animal experiments, firstly the research is to focus on the mechanism of the influence of VitD on intestinal microbiota and intestinal multistage immune regulation pathways which jointly stimulate food allergy. Furthermore, the appropriate VitD level will be assessed for infants and young children.

婴幼儿是食物过敏高危人群，发病率约为10%。研究提示：VitD缺乏或过量均可能增加食物过敏风险。但是前瞻性研究两者关联和机制极少。我们前期研究发现：VitD缺乏使肠道乳酸杆菌减少。文献已证实：乳酸杆菌可通过TLRs信号途径刺激肠道黏膜固有层树突状细胞（DC）向耐受性DC分化，诱导口服免疫耐受。此外，也已明确：抗菌肽作为一种调整肠道菌群的重要多肽，其基因启动子序列含有VitD作用元件。据此，基于前期研究，我们推测：VitD缺乏（过量）影响抗菌肽使乳酸杆菌减少，影响乳酸杆菌与DC表面TLR2、TLR4受体的结合，抑制DC 向耐受性DC分化，CD103+耐受性DC减少，使Foxp3+Treg细胞减少，促进T细胞向Th2分化，口服耐受受损致食物过敏。本课题将建立首个VitD与食物过敏队列，结合动物实验，率先研究VitD影响肠道菌群、肠道多级免疫效应激发食物过敏机制，并评估婴幼儿适宜VitD水平。

婴幼儿是过敏性疾病高危人群，患病率约为30-40%。研究提示维生素D（VitD）缺乏或过量均可能增加过敏性疾病发生风险，但是前瞻性研究两者关联和机制极少。本项目基于上海社区医院开展了婴幼儿队列，采用HPLC-MS/MS技术检测6月龄和12月龄婴幼儿25-(OH)D水平，并随访0-2岁婴幼儿过敏性疾病的发生。结果显示，6月龄时VitD缺乏或不足[25-(OH)D<30ng/mL]可显著增加0-2岁婴幼儿过敏性疾病发生的风险。通过平滑曲线拟合和阈值分析发现，当25-(OH)D<30ng/mL时，0-2岁过敏性疾病的发生率最高，当25-(OH)D≥30ng/mL时，过敏性疾病随着25-(OH)D浓度的升高呈逐渐下降趋势。与6月龄时25-(OH)D水平充足组（≥30ng/mL）相比，VitD缺乏或不足组（<30ng/mL）婴儿在6月龄，12月龄和24月龄时发生过敏性疾病的风险均显著增加（OR=1.91, 95%CI:1.51-2.42；OR=2.02, 95%CI:1.55-2.63；OR=1.78, 95%CI:1.25-2.54）。采用16sRNA测序技术，进一步分析6月龄和12月龄VitD 缺乏组和VitD充足组婴儿肠道微生物菌群特点和组成，结果显示6月龄两组婴幼儿肠道微生物组成存在显著差异，其中反映肠道菌群失调或肠道微生物群落结构不稳定性的变形杆菌门（Proteobacteria）在VitD缺乏组中表达丰度上调。建立VitD缺乏的Balb/c小鼠模型并采用鸡卵清白蛋白（OVA）诱发过敏反应，研究VitD影响肠道菌群而致食物过敏的机制。结果发现，VitD缺乏组小鼠肠道抗菌肽蛋白（rCRAMP）表达量显著下降（p<0.05），肠道双歧杆菌（Bifidobacterium）、乳酸菌（Lactobacillus）相对丰度降低，并且肠粘膜固有层淋巴细胞Th1/Th2比率降低、Foxp3+Treg细胞数量降低、Th17细胞数量增加，以及对应细胞因子（IL-6、TNF-a，CRP）也发生改变；而当Vit D缺乏后进行补充则能部分逆转上述变化，证实了VitD通过影响抗菌肽的合成干扰肠道菌群进而致过敏性疾病的发生。综上，本课题建立了VitD与过敏性疾病队列，结合动物实验率先研究了VitD对肠道菌群的影响及其潜在的激发肠道多级免疫效应的食物过敏机制。