Mucosal Immunity Research Group, State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China; and.
Institute of Virology, University Hospital of Essen, Essen, Germany.
J Leukoc Biol. 2017 Sep;102(3):575-587. doi: 10.1189/jlb.3HI0816-368RR. Epub 2017 May 18.
Flagellin, as a TLR5 agonist, is an established mucosal adjuvant for enhancing mucosal IgA responses by i.n. immunization. Nasal epithelial cells (NECs) are the first sentinel cells to be exposed to antigen and adjuvant in i.n. immunization, and it is suggested that they play an important role in the mucosal adjuvant activity of flagellin. However, the molecular mechanism leading to modulation and the response by flagellin-activated NECs remain unknown. We aimed to identify the soluble mediator(s) from flagellin-activated NECs that modulate the functions of airway dendritic cells (DCs) and enhance subsequent IgA response. In vitro studies showed that compared with the TLR4 agonist LPS, flagellin directly triggered slight up-regulation of CD80 on airway DCs but was insufficient to affect CD86 expression and DC-mediated IgA response. With the use of an in vitro system for culturing mouse primary NECs (mNECs), we demonstrated that flagellin-activated mNECs could functionally modulate airway DCs, which manifested as significant up-regulation of CD80/CD86 and enhancement of IgA production. The functional modulation of airway DCs was dependent on TLR5 activation of mNECs rather than direct TLR5 activation of airway DCs. With the use of cytokine array and antibody-blocking assays, we further identified that GM-CSF, a cytokine secreted from TLR5-activated mNECs, contributes to the activation of mNECs to airway DCs and subsequent IgA enhancement. In vivo blocking experiments confirmed that GM-CSF is an important factor in recombinant flagellin derived from (FliC)-induced airway DC activation and antigen-specific IgA enhancement. Our data directly demonstrate that nasal epithelial GM-CSF contributes to TLR5-mediated modulation of airway DCs and a subsequent IgA response.
鞭毛蛋白作为 TLR5 的激动剂，通过鼻内免疫已被确立为增强黏膜 IgA 应答的黏膜佐剂。鼻腔上皮细胞（NECs）是首先接触到抗原和佐剂的哨兵细胞，据推测它们在鞭毛蛋白的黏膜佐剂活性中发挥重要作用。然而，导致调节的分子机制以及鞭毛蛋白激活的 NECs 的反应仍然未知。我们的目的是鉴定来自鞭毛蛋白激活的 NECs 的可溶性介质（s），这些介质可调节气道树突状细胞（DCs）的功能并增强随后的 IgA 应答。体外研究表明，与 TLR4 激动剂 LPS 相比，鞭毛蛋白直接轻微上调气道 DCs 的 CD80，但不足以影响 CD86 表达和 DC 介导的 IgA 反应。使用体外培养小鼠原代 NECs（mNECs）的系统，我们证明鞭毛蛋白激活的 mNECs 可以对气道 DCs 进行功能调节，表现为 CD80/CD86 的显著上调和 IgA 产生的增强。气道 DCs 的功能调节依赖于 mNECs 中 TLR5 的激活，而不是气道 DCs 中 TLR5 的直接激活。通过使用细胞因子阵列和抗体阻断试验，我们进一步确定 TLR5 激活的 mNECs 分泌的细胞因子 GM-CSF 有助于 mNECs 对气道 DCs 的激活以及随后的 IgA 增强。体内阻断实验证实 GM-CSF 是重组来自 FliC 的鞭毛蛋白诱导的气道 DC 激活和抗原特异性 IgA 增强的重要因素。我们的数据直接证明，鼻上皮 GM-CSF 有助于 TLR5 介导的气道 DC 调节和随后的 IgA 反应。
