Filaggrin-deficient rats generated using zinc-finger nucleases spontaneously exhibit dry scaly skin
Filaggrin-deficient rats generated using zinc-finger nucleases spontaneously exhibit dry scaly skin
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使用锌指核酸酶产生的丝聚蛋白缺陷大鼠自发地表现出干燥的鳞状皮肤
DOI：
10.1016/j.alit.2022.04.001
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发表时间：
2022
影响因子：
6.8
通讯作者：
Kabashima Kenji
中科院分区：
文献类型：
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作者：
Nakashima Chisa;Doi Hiromi;Nakajima Saeko;Mashimo Tomoji;Oga Toru;Ishida-Yamamoto Akemi;Honda Tetsuya;Ishida Yoshihiro;Otsuka Atsushi;Kabashima Kenji
聚丝蛋白（FLG）是参与皮肤屏障功能的关键蛋白之一，已知在特应性皮炎（AD）患者的表皮中减少。1,2先前的一项研究表明，flg缺失小鼠表现为皮肤干燥鳞片状，聚丝蛋白降解产物天然保湿因子（NMF）减少；但经皮失水（TEWL）正常。另一方面，已知纯合子Flg缺陷患者表现出TEWL的增加，4突出表明Flg缺陷在小鼠和人类之间的耐受性并不相同。大鼠皮肤是啮齿类动物中与人类皮肤结构最相似的，例如药物通过大鼠皮肤的渗透性与人类皮肤对药物的渗透性相关。在这项研究中，我们使用锌指核酸酶（ZFNs）技术新生成并表征了Flg敲除（Flg À/À）大鼠。6为了生成Flg À/À大鼠，我们使用常规的ZFN试剂6（图1a）靶向大鼠Flg基因的第二外显子，并通过免疫组化染色生成Flg À/À大鼠（图1b）。与野生型幼崽相比，新生Flg À/À大鼠表现出干燥、有光泽的鳞状皮肤，尾巴上有收缩环，背部有突出的表皮嵴，周围有深沟（图1c）。我们知道，侧聚蛋白沉积在角蛋白纤维上，是角透明素颗粒的主要成分，含有侧聚蛋白的F颗粒就是角透明素颗粒。在这项研究中，我们证明了通过电子显微镜在野生型大鼠和Flg þ/À大鼠中观察到正常的大F颗粒（图1 D）。Flg À/À大鼠的生长受到轻微限制（图2a），但Flg À/À大鼠和野生型大鼠的毛发生长正常，Flg À/À大鼠的躯干皮肤表型在毛发生长后变得不那么明显（数据未显示）。人们普遍认为TEWL升高表明角质层（SC）屏障功能障碍。TEWL因此被用作评估AD患者皮肤干燥和SC屏障功能的替代标志物。与Flg À/À小鼠的正常TEWL结果相比，我们发现Flg À/À大鼠出生后3天的TEWL与野生型大鼠相比有所增加（图2b）。此外，体内共聚焦拉曼显微光谱分析显示，NMF显著下降，但神经酰胺没有下降，神经酰胺已知会影响皮肤屏障功能和TEWL（图2 C， D）。然而，随着这些老鼠的成长，这些发现变得不那么明显；8周龄野生型和Flg À/À大鼠TEWL无显著差异（图2e）。电镜分析显示，8周龄野生型和Flg À/À大鼠的角膜透明素颗粒和致密角质层形态相似（图2f）。最后，我们用维生素D3类似物MC903诱导ad样皮炎模型。9野生型和Flg À/À大鼠的临床评分无显著差异（图2d）。苏木精和伊红染色显示表皮厚度无显著差异（图2d）。利用ZFN技术成功生成Flg À/À大鼠。先前的报告显示，新生Flg À/À小鼠的TEWL值没有改变；3然而，本研究中Flg À/À大鼠TEWL自发增加。Flg À/À大鼠与小鼠结果的差异可能源于动物物种的差异。在FLG突变诱导的寻常性鱼鳞病和AD患者中，有报道称TEWL增加。4,8在这方面，Flg À/À大鼠可以用于分析皮肤的屏障功能。此外，我们认为，出生后不久的生长迟缓是……
Filaggrin (FLG) is one of key proteins involved in the skin barrier function that is known to be decreased in the epidermis of atopic dermatitis (AD) patients. 1, 2 A previous study demonstrated that Flg-null mice exhibited dry scaly skin and decreased natural moisturizing factors (NMF) that are filaggrin degradation products; however transepidermal water loss (TEWL) was normal. 3 On the other hand, homozygous Flg-deficient patients are known to exhibit an increase in TEWL, 4 highlighting that Flg deficiency is not tolerated equally between mice and humans. Rat skin is the most structurally similar to human skin among rodents, eg the skin permeability of drugs through rat skin correlates with human skin for drug permeation. 5 In this study, we newly generated and characterized Flg knockout (Flg À/À) rats using the Zinc-finger nucleases (ZFNs) technology. 6 To generate Flg À/À rats, we targeted the second exon of rat Flg gene using conventional ZFN reagents 6 (Fig. 1 A) and generated Flg À/À rats confirmed by immunohistochemical staining (Fig. 1 B). Neonatal Flg À/À rats exhibited dry scaly skin with a shiny tone, constriction rings on the tail, and prominent areas of crista cutis surrounded by deep furrows on the back when compared with wild-type littermates (Fig. 1 C). It is known that profilaggrin is deposited on keratin fibers and exists as a major component of keratohyalin granules, and that F granules containing profilaggrin are keratohyalin granules. 7 In this study, we have demonstrated that normal large F granules were observed in wild-type rats and Flg þ/À rats by electron microscopy (Fig. 1 D). Mild growth restriction was observed in Flg À/À rats (Fig. 2 A), but both Flg À/À rats and wild-type rats exhibited normal hair growth, and the trunk skin phenotype of Flg À/À rats became less obvious after hair outgrowth (data not shown). It is generally agreed that increased TEWL is indicative of stratum corneum (SC) barrier dysfunction. TEWL is thus used as a surrogate marker to assess skin dryness and SC barrier function in patients with AD. 8 In contrast to the results of normal TEWL in Flg À/À mice, 3 we found an increase in TEWL 3 days after birth in Flg À/À rats compared to wild-type rats (Fig. 2 B). In addition, in vivo confocal Raman microspectroscopic analysis revealed a significant decrease in NMF but not ceramide that is known to affect the skin barrier function and TEWL (Fig. 2 C, D). However, as these rats grew, these findings became less apparent; there was no significant difference in TEWL between 8-week-old wild-type and Flg À/À rats (Fig. 2 E). Electron microscopic analysis revealed that both 8-week-old wild-type and Flg À/À rats exhibited similar morphologies of keratohyaline granules and compact stratum corneum (Fig. 2 F). Finally, we used AD-like dermatitis model induced by a vitamin D3 analogue MC903. 9 There was no significant difference in clinical scores between wild-type and Flg À/À rats (Fig. 2 D). Hematoxylin and eosin staining revealed no significant difference in epidermal thickness (Fig. 2 D).We successfully generated Flg À/À rats by using the ZFN technology. A previous report showed that neonatal Flg À/À mice displayed unaltered TEWL values; 3 however, Flg À/À rats showed an increase in TEWL spontaneously in this study. Discrepancy in the results between Flg À/À rats and mice may be derived from differences in animal species. In patients with ichthyosis vulgaris and AD that can be induced by FLG mutations, an increase in TEWL has been reported. 4, 8 In this regard, Flg À/À rats can be useful for analyzing the barrier function of the skin. In addition, we consider that growth retardation in the immediate postnatal period is …