Department of Biochemistry, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
The Second Affiliated Hospital, The State Key Laboratory of Respiratory Disease, Guangdong Provincial Key Laboratory of Allergy & Clinical Immunology, Sino-French Hoffmann Institute, Guangzhou Medical University, Guangzhou 510260, China.
Int Immunopharmacol. 2018 Sep;62:139-146. doi: 10.1016/j.intimp.2018.05.013. Epub 2018 Jul 10.
Mal f 1, the first allergen cloned from Malassezia furfur, has positive IgE reactivity in sera from atopic dermatitis (AD) patients. The mechanism by which Mal f 1 induces the maturation of human dendritic cells (DCs) and maintains the symptoms of AD is not well understood.
The present study aims to explore the activation profile of THP-1 derived dendritic cells (TDDCs) stimulated by recombinant Mal f 1, as well as to explore the IgE-binding ability of rMal f 1 and its correlation with IgE-binding activity of complete allergens of M. furfur.
rMal f 1 was produced by expression in E. coli and purification with affinity chromatography. The ability of rMal f 1 and ImmunoCAP complete allergens of M. furfur to bind to serum specific IgE was assayed in parallel by ELISA and immunoblotting. Immature TDDCs were stimulated with rMal f 1 or an enzyme-digested product of rMal f 1. The expression levels of markers, CD83, CD80, CD86, and HLA-DR, were investigated by flow cytometry. The levels of interleukin (IL)-6, IL-10, IL-12p70 and tumor necrosis factor (TNF)-α in culture supernatants were determined by ELISA.
Eighteen patient sera were identified that reacted positively to the complete allergens of M. furfur as determined by ImmunoCAP and also showed positive responses to rMal f 1. Five patient sera were identified that had no reaction to ImmunoCAP complete allergens of M. furfur and also exhibited negative response to rMal f 1. All sera, except for one, had no reaction to the unrelated allergen Bet v 1. rMal f 1 upregulated the maturation surface marker CD83 on TDDCs. In addition, rMal f 1 also induced high levels of CD80 and CD86. Increased expression of HLA-DR, a first signal for T cell activation, was observed. Secretion of IL-6, TNF-α and IL-10 by TDDCs increased significantly (P < 0.0001 for IL-6, P < 0.01 for TNF-α and P < 0.05 for IL-10) after stimulation by rMal f 1, while the IL-12p70 level was unaltered.
We have shown that rMal f 1 has ideal IgE binding ability and good correlation with binding activity to M. furfur. Moreover, we have revealed a hitherto unknown DC activation profile after rMal f 1 stimulation whereby TNF-α, IL-6, and IL-10 were significantly increased and IL-12 was unaltered, suggesting that rMal f 1 can predispose a DC bias toward the T22/T17 pathway beyond the routine IgE-dependent T2 pathway, thus providing intriguing clues for clinical treatment involving both pathways.
从糠秕马拉色菌中克隆的第一个过敏原 Mal f 1，在特应性皮炎（AD）患者的血清中具有阳性 IgE 反应性。Mal f 1 诱导人树突状细胞（DC）成熟并维持 AD 症状的机制尚不清楚。
本研究旨在探讨重组 Mal f 1 刺激 THP-1 衍生的树突状细胞（TDDC）的激活谱，并探讨 rMal f 1 的 IgE 结合能力及其与糠秕马拉色菌完整过敏原的 IgE 结合活性的相关性。
通过在大肠杆菌中表达和亲和层析纯化生产 rMal f 1。通过 ELISA 和免疫印迹平行测定 rMal f 1 和糠秕马拉色菌完整过敏原与血清特异性 IgE 的结合能力。用 rMal f 1 或 rMal f 1 的酶消化产物刺激未成熟的 TDDC。通过流式细胞术检测标记物 CD83、CD80、CD86 和 HLA-DR 的表达水平。通过 ELISA 测定培养上清液中白细胞介素（IL）-6、IL-10、IL-12p70 和肿瘤坏死因子（TNF）-α 的水平。
通过 ImmunoCAP 鉴定出 18 份患者血清对糠秕马拉色菌完整过敏原呈阳性反应，并且对 rMal f 1 也呈阳性反应。鉴定出 5 份患者血清对糠秕马拉色菌完整过敏原无反应，对 rMal f 1 也无反应。除了 1 份血清外，所有血清对无关过敏原 Bet v 1 均无反应。rMal f 1 上调 TDDC 的成熟表面标记物 CD83。此外，rMal f 1 还诱导高水平的 CD80 和 CD86。观察到 HLA-DR 的表达增加，这是 T 细胞激活的第一个信号。rMal f 1 刺激后 TDDC 中 IL-6、TNF-α 和 IL-10 的分泌显著增加（IL-6 为 P<0.0001，TNF-α 为 P<0.01，IL-10 为 P<0.05），而 IL-12p70 水平不变。
我们已经表明，rMal f 1 具有理想的 IgE 结合能力，并且与糠秕马拉色菌的结合活性具有良好的相关性。此外，我们揭示了 rMal f 1 刺激后的未知树突状细胞激活谱，其中 TNF-α、IL-6 和 IL-10 显著增加，而 IL-12 不变，这表明 rMal f 1 可以使 DC 向 T22/T17 途径倾斜，超出常规的 IgE 依赖性 T2 途径，从而为涉及两条途径的临床治疗提供了有趣的线索。
