壳聚糖作用于肠道CD11b+DC增强B细胞分泌特异性SIgA的分子机制

Whether mucosal SIgA level is correlated with anti-microb immunoprotection and the mechanism underlying the induction of SIgA through mucosal B cell class switch recombination (CSR) remains unclear. Mucosal DC has been postulated to facilitate the IgA CSR of mucosal B cells especially in a T-independently fashion. By using chitosan as mucosal delivery carrier, our previous study demonstrated that intranasal immunization with chitosan formulated DNA vaccine significantly enhanced intestinal SIgA production and contributed to the protection against Coxsackievirus B3. To elucidate the underlying cellular and molecular mechanism of mucosal SIgA production through gut B cell CSR, we focused our research on 3 mucosal DC subsets, CD103+DC, CD11b+DC and TipDC, which are postulated to enhance IgA CSR by B cells through direct cytokine stimulation or indirect CD4+Th cell priming. In this study, using CVB3-induced myocarditis as animal model; the dynamic alteration of intestinal CD103+DC, CD11b+DC and TipDC, SIgA level as long with onset of myocarditis would be first studied. Then in an in vitro B-DC-Th-cytokine coculture model, DC's ability to trigger B cell SIgA CSR in presence of antigen, BAFF & APRIL and Th cells would be carefully evaluated. In vivo transfer of purified DC subsets would be performed to see their interaction with B/Th cells and role in the gut SIgA synthesis. This study is aimed to clarify the key mechanism of selective stimulation of mucosal SIgA production in the context of mucosal DC subsets by chitosan, and may give valuable clues as well as insights into future designing of mucosal vaccines.

对早期抗感染保护具有关键作用的黏膜B细胞SIgA类别转换的细胞与分子免疫学机制尚未阐明。基于我们前期研究发现的壳聚糖（chitosan）递送的DNA疫苗经滴鼻免疫可显著诱导肠道SIgA且与增强的免疫保护相关的基础上，拟深入研究肠道B细胞进行SIgA类别转换的DC调控机制。推测肠道DC亚群经chitosan固有免疫激活通过分泌BAFF、APRIL等以T细胞不依赖方式直接激活B或通过激活CD4+Th细胞间接促进黏膜下B细胞类别转换（CSR）。拟在chitosan-DNA免疫小鼠预防病毒性心肌炎模型中，动态观测肠道DC亚群（CD103+DC,CD11b+DC,TipDC）、SIgA水平及免疫保护的相关性；体外以B-DC-Th-CK共培养，鉴定壳聚糖促进IgA CSR的肠道DC亚群的表型和特征及作用分子机制，解析壳聚糖选择性增强黏膜SIgA的机制，为新型黏膜疫苗设计奠定思路。

诱导分泌型IgA（Secretory IgA, SIgA）在肠道等黏膜局部抗感染免疫中发挥重要作用，是新型黏膜疫苗设计的关键目标。我们前期以壳聚糖（chitosan）组装DNA制备纳米颗粒疫苗，经滴鼻免疫显著提高肠道特异性SIgA而显著增强抗病毒免疫保护。本研究以小鼠肠道为主要研究对象，深入探索了chitosan作为黏膜疫苗载体及佐剂通过调节肠道DC表型及功能、进而增强B细胞SIgA类别转换的细胞与分子机制。结果发现：1）chitosan-DNA滴鼻可显著增强肠道SIgA水平、特异性SIgA+B细胞比例及关键转录酶AID和转录中间体的上调表达。2）chitosan-DNA可显著提高MLN肠系膜淋巴结（MLN）中CD103+DC和pDC的比例、数目及成熟度。3）chitosan通过上调pDC表面mAPRIL表达经与同样被上调的B细胞表面受体TACI相互作用，而显著促进肠道SIgA类别转换。4）chitosan经由CD103+DC促进SIgA类别转换依赖chitosan上调的肠道Th17应答。本研究首次证明黏膜载体及佐剂chitosan通过激活MLN内pDC 经由mAPRIL-TACI轴增强肠道B细胞发生SIgA类别转换的细胞与分子机制，为chitosan及其衍生物更好地应用于新型黏膜疫苗体系奠定理论和应用基础。