Takai Toshiro, Chen Xue, Xie Yang, Vu Anh Tuan, Le Tuan Anh, Kinoshita Hirokazu, Kawasaki Junko, Kamijo Seiji, Hara Mutsuko, Ushio Hiroko, Baba Tadashi, Hiramatsu Keiichi, Ikeda Shigaku, Ogawa Hideoki, Okumura Ko
Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan; Department of Dermatology, Peking University People's Hospital, Beijing, China.
Methods Enzymol. 2014;535:371-87. doi: 10.1016/B978-0-12-397925-4.00021-3.
The skin epidermis and mucosal epithelia (airway, ocular tissues, gut, and so on) are located at the interface between the body and environment and have critical roles in the response to various stimuli. Thymic stromal lymphopoietin (TSLP), a cytokine expressed mainly by epidermal keratinocytes (KCs) and mucosal epithelial cells, is a critical factor linking the innate response at barrier surfaces to Th2-skewed acquired immune response. TSLP is highly expressed in skin lesions of atopic dermatitis patients. Here, we describe on Toll-like receptor (TLR)-mediated induction of TSLP expression in primary cultured human KCs, placing emphasis on experimental methods used in our studies. Double-stranded RNA (TLR3 ligand), flagellin (TLR5 ligand), and diacylated lipopeptide (TLR2-TLR6 ligand) stimulated human KCs to express TSLP and Staphylococcus aureus membranes did so via the TLR2-TLR6 pathway. Atopic cytokine milieu upregulated the TLR-mediated induction of TSLP. Culturing in the absence of glucocorticoid before stimulation enhanced the TSLP expression. Extracellular double-stranded RNA induced TSLP via endosomal acidification- and NF-κB-dependent pathway. Specific measurement of the long TSLP transcript, which contributes to the production of the TSLP protein, rather than total or the short transcript is useful for accurate detection of functional human TSLP gene expression. The results suggest that environment-, infection-, and/or self-derived TLR ligands contribute to the initiation and/or amplification of Th2-type skin inflammation including atopic dermatitis and atopic march through the induction of TSLP expression in KCs and include information helpful for understanding the role of the gene-environment interaction relevant in allergic diseases.
皮肤表皮和黏膜上皮（气道、眼组织、肠道等）位于机体与环境的界面，在对各种刺激的反应中起关键作用。胸腺基质淋巴细胞生成素（TSLP）是一种主要由表皮角质形成细胞（KC）和黏膜上皮细胞表达的细胞因子，是连接屏障表面固有反应与Th2偏向性获得性免疫反应的关键因子。TSLP在特应性皮炎患者的皮肤病变中高度表达。在此，我们描述了Toll样受体（TLR）介导的原代培养人KC中TSLP表达的诱导，重点介绍了我们研究中使用的实验方法。双链RNA（TLR3配体）、鞭毛蛋白（TLR5配体）和二酰化脂肽（TLR2-TLR6配体）刺激人KC表达TSLP，金黄色葡萄球菌膜通过TLR2-TLR6途径诱导TSLP表达。特应性细胞因子环境上调了TLR介导的TSLP诱导。刺激前在无糖皮质激素的条件下培养可增强TSLP表达。细胞外双链RNA通过内体酸化和NF-κB依赖途径诱导TSLP。特异性检测有助于TSLP蛋白产生的长TSLP转录本，而不是总转录本或短转录本，对于准确检测功能性人TSLP基因表达很有用。结果表明，环境、感染和/或自身来源的TLR配体通过诱导KC中TSLP表达，促进包括特应性皮炎和特应性进程在内的Th2型皮肤炎症的起始和/或放大，并包含有助于理解与过敏性疾病相关的基因-环境相互作用作用的信息。
