CD103+ DC在鞭毛素作为鼻腔粘膜佐剂增强IgA应答过程中的作用机制研究

It is well known that flagellin could promote efficiently mucosal IgA production when it was used as an intranasal adjuvant. Accordingly, flagellin has become a novel and promising mucosal adjuvant candidate. DC plays a key role in the mechanism of flagellin induced mucosal IgA production but the mechanism hasn't been characterized. CD103+ DC is a new-found DC subset in the past decade, and pulmonary CD103+ DCs are the major contributors to present antigen and activate immune response; and intestinal CD103+ DCs play a key role in intestinal IgA production through TGF-β/Treg pathway. Our previous study found that CD103+ DC also resided in nasal mucosa of normal mice. Moreover, these results also showed that the frequency of CD103+ DC in nasal mucosa was changed and the numbers of Treg in neck lymph nodes were increased after flagellin intranasal immunization. Therefore, we hypothesized that CD103+ DC might be held responsible for enhancing of mucosal IgA responses by flagellin through intranasal immunization. This study will analyze the role of CD103+ DC in the uptake, carrying of flagellin thus in the initiation of adaptive immune response and focus on evaluating the role of flagellin-conditioned CD103+ DC in the induction of IgA class switching through TGF-β/Treg pathway, which is advantageous to apply and optimize flagellin-based intranasal adjuvant and mucosal vaccine.

鞭毛素作为鼻腔粘膜佐剂能有效增强粘膜IgA应答及疫苗保护效应，已成为一种极具临床应用前景的新型粘膜佐剂。研究表明DC在鞭毛素的佐剂效应中发挥重要作用，但具体机制尚不清楚。CD103+ DC是近十年新发现的一个DC亚群，是肺部递呈抗原、激活免疫应答的主要DC亚群，且能通过TGF-β/ Treg通路在肠粘膜IgA应答中发挥关键作用。我们的前期研究显示正常小鼠鼻粘膜同样包含CD103+ DC，鞭毛素滴鼻免疫后可对其产生影响，并上调颈部淋巴结中的Treg。我们推断CD103+ DC在鞭毛素滴鼻免疫增强IgA应答中发挥关键作用。本课题将深入研究鞭毛素滴鼻免疫过程中CD103+ DC在抗原有效摄取、传递，免疫应答增强中的作用，并重点探索鞭毛素调节下的CD103+ DC与TGF-β/Treg通路的关系，阐明其增强IgA类别转换的机制，为优化和应用基于鞭毛素分子的鼻腔粘膜佐剂和粘膜疫苗奠定理论基础。

鞭毛素(FliC)作为鼻腔粘膜佐剂能有效增强粘膜IgA应答及疫苗保护效应，极具临床应用前景。DC是专职的抗原递呈细胞，鼻粘膜上皮细胞(NEC)是鼻腔免疫过程中的哨兵细胞，研究表明DC和NEC在FliC的粘膜佐剂活性中均发挥重要作用，然而FliC如何调节NEC、DC及后续的IgA应答并不清楚。本项目旨在利用体外细胞系统、体内动物实验研究FliC活化的NEC如何调节DC功能及增强IgA应答的机制。我们发现FliC滴鼻免疫能够以TLR5依赖的方式有效上调鼻部及其引流淋巴结(cervical lymphoid node, CLN) DCs表面CD80、CD86分子的表达，并增强DC介导的naïve B细胞的IgA应答。体外实验证明，FliC对呼吸道DCs的直接刺激，不足以有效的活化DC并介导后续的IgA应答。利用小鼠原代鼻腔上皮细胞（mouse NECs, mNECs）体外培养系统，我们发现FliC刺激后的mNECs能够有效活化呼吸道DC，且经这种方式活化的DC能增强naïve B细胞的IgA应答。在此过程中，mNECs TLR5通路的活化，而非DC本身TLR5通路的活化，是FliC有效调节呼吸道DCs的关键。为进一步确定FliC调节呼吸道DCs的关键因子，利用细胞因子芯片及定量ELISA实验，我们发现FliC活化的野生型NEC大量分泌GM-CSF、IL-6和TSLP等细胞因子。利用细胞因子的中和抗体的体内及体外封闭实验，我们进一步发现TLR5激活的mNECs分泌的GM-CSF是活化呼吸道DCs并介导后续IgA应答所必需的关键因子。综上所述，本研究证明在鼻腔免疫过程中，FliC通过激活NECs的TLR5通路从而间接调节呼吸道DC的功能并增强后续的IgA应答，且此过程中NECs分泌的GM-CSF是关键的调节因子。本研究为深入认识FliC的免疫调节功能、粘膜佐剂机制以及相关粘膜疫苗的设计奠定了理论基础。