Unit of Immunology, University Hospital La Paz Institute for Health Research (IdiPaz), Madrid, Spain.
Department of Products, Institute of Food Science, Technology and Nutrition (ICTAN), Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.
PLoS Negl Trop Dis. 2020 Jul 6;14(7):e0008462. doi: 10.1371/journal.pntd.0008462. eCollection 2020 Jul.
Anisakis spp. are nematode parasites found in a wide range of marine organisms. Human beings may accidentally become infected, showing the symptoms of anisakiasis and allergic responses. There has been evidence of increased intestinal permeability in A. simplex-sensitized subjects and that specific IgE titres increase in some allergic patients when fishery products are re-introduced into their diet. The aims of this work were to study the effect of A. simplex crude extract on the intestinal integrity and permeability by using Caco-2 cell monolayer. To analyse the capacity of Ani s 4 allergen to cross the epithelial barrier.
METHODOLOGY/PRINCIPAL FINDINGS: Cellular bioenergetics, transepithelial electrical resistance, viability, permeability, reactive oxygen species generation and immunofluorescent staining of tight junction proteins were analysed. A. simplex crude extract compromises the Caco-2 cell monolayer integrity in a dose-dependent manner. This effect is detected at 1 hour of culture and integrity is recovered after 24 hours of culture. The epithelial barrier disruption is accompanied by an increase in paracellular permeability and reactive oxygen species production and by a delocalization of occludin and zonula occludens-1. Finally, Ani s 4, a thermostable and resistant to digestion allergen with cystatin activity, is able to cross the epithelial barrier in Caco-2 monolayer and reach a cumulative mean percentage of 22.7% of total concentration in the basolateral side after 24 hours of culture.
CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that A. simplex induces an early and reversible alteration of integrity and permeability of Caco-2 cell monolayer and that an underlying mechanism of this effect would involve the oxidative stress and disruption of epithelial tight junctions. Additionally, it has been shown that Ani s 4 allergen is able to cross the epithelial barrier. These findings could explain the increased intestinal permeability observed in Anisakis-sensitized patients, the changes over time in IgE sensitization to A. simplex allergens, and the specific IgE persistence in Anisakis allergy.
异尖线虫属是一种广泛存在于海洋生物中的线虫寄生虫。人类可能会意外感染，表现出异尖线虫病和过敏反应的症状。有证据表明，在 A. simplex 致敏的受试者中，肠道通透性增加，并且当渔业产品重新引入饮食时，一些过敏患者的特异性 IgE 滴度增加。本工作的目的是研究 A. simplex 粗提物对 Caco-2 细胞单层肠道完整性和通透性的影响。分析 Ani s 4 过敏原穿过上皮屏障的能力。
方法/主要发现：分析细胞生物能学、跨上皮电阻、活力、通透性、活性氧生成和紧密连接蛋白的免疫荧光染色。A. simplex 粗提物以剂量依赖的方式破坏 Caco-2 细胞单层的完整性。这种作用在培养 1 小时时被检测到，并且在培养 24 小时后完整性得到恢复。上皮屏障破坏伴随着细胞旁通透性的增加和活性氧的产生，以及紧密连接蛋白 occludin 和 zonula occludens-1 的定位。最后，Ani s 4 是一种耐热且抗消化的过敏原，具有半胱氨酸蛋白酶抑制剂活性，能够穿过 Caco-2 单层上皮屏障，在培养 24 小时后，到达基底外侧侧的总浓度的 22.7%的累积平均值。
结论/意义：我们的结果表明，A. simplex 诱导 Caco-2 细胞单层完整性和通透性的早期和可逆改变，并且这种作用的潜在机制涉及氧化应激和上皮紧密连接的破坏。此外，已经表明 Ani s 4 过敏原能够穿过上皮屏障。这些发现可以解释在 Anisakis 致敏患者中观察到的肠道通透性增加、对 A. simplex 过敏原的 IgE 敏感性随时间的变化以及 Anisakis 过敏中特异性 IgE 的持续存在。
