Supplementary Material for: Gastrointestinal Immune Response to the Shrimp Allergen Tropomyosin: Histological and Immunological Analysis in an Animal Model of Shrimp Tropomyosin Hypersensitivity

<b><i>Background:</i></b> Shellfish hypersensitivity is among the most common food allergies. A murine model of IgE-mediated shrimp allergy has been established in our laboratory. The aim of this study is to determine the intestinal histological changes and cytokine expression profile of this model sensitized with the major shellfish allergen tropomyosin. <b><i>Methods:</i></b> Female Balb/c mice orally sensitized and challenged with recombinant tropomyosin were sacrificed. Continuous sections of duodenum, jejunum and ileum were prepared using the Swiss roll technique for histological and immunological analysis. Duodenal epithelial cell apoptosis and migration were examined. mRNA expression of IL-4, IL-6, IL-10, IL-13, IL-18 and IFN-γ in intestinal tissue was measured via RT-PCR. <b><i>Results:</i></b> In tropomyosin-sensitized and challenged mice, an increased number of eosinophils, mast cells and goblet cells was found 24 h after challenge. There were also increased mast cell and goblet cell numbers at 72 h after challenge, but the level of eosinophils decreased. Differences compared with control mice are most prominent at the duodenum compared to the distal regions. In addition, TUNEL assay indicates a significantly higher apoptosis rate in sensitized mice sacrificed 72 h after challenge, and mRNA expression showed a biased Th2/Th1 cytokine profile and a higher level of murine mast cell protease 1. <b><i>Conclusions:</i></b> This study documented a multitude of histological and immunological changes in the gut in a murine model of shrimp allergy. Even without repetitive intragastric challenge, shrimp tropomyosin induces an increase in the number of inflammatory cells to varying degrees within the small intestine. This model provides an important tool for testing new therapeutic interventions.

<b><i>背景：</i></b> 甲壳类动物超敏反应是最常见的食物过敏类型之一。本实验室已成功构建IgE介导的虾类过敏小鼠模型。本研究旨在探究该以主要甲壳类过敏原原肌球蛋白（tropomyosin）致敏的小鼠模型的肠道组织学变化与细胞因子表达谱。<b><i>方法：</i></b> 对经重组原肌球蛋白口服致敏并激发的雌性Balb/c小鼠实施安乐死。采用瑞士卷（Swiss roll）技术制备十二指肠、空肠及回肠的连续切片，用于组织学与免疫学分析。检测十二指肠上皮细胞的凋亡与迁移情况。通过逆转录聚合酶链式反应（RT-PCR）检测肠道组织中IL-4、IL-6、IL-10、IL-13、IL-18及干扰素-γ（IFN-γ）的mRNA表达水平。<b><i>结果：</i></b> 在经原肌球蛋白致敏并激发的小鼠中，激发后24小时可见嗜酸性粒细胞、肥大细胞及杯状细胞数量增多。激发后72小时，肥大细胞与杯状细胞数量仍维持升高，但嗜酸性粒细胞水平有所下降。相较于对照组小鼠，上述差异在十二指肠部位最为显著，远较远端肠道突出。此外，末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定（TUNEL assay）结果显示，于激发后72小时处死的致敏小鼠，其上皮细胞凋亡率显著升高；mRNA表达分析显示，细胞因子谱呈现Th2/Th1偏态分布，且小鼠肥大细胞蛋白酶1（murine mast cell protease 1）水平更高。<b><i>结论：</i></b> 本研究阐明了虾类过敏小鼠模型肠道内存在多种组织学与免疫学改变。即便未进行反复的胃内激发，虾类原肌球蛋白仍可在小肠内不同程度地诱导炎症细胞数量增多。该模型可为新型治疗干预手段的研发提供重要的实验工具。