Functional and molecular analysis of the epithelial barrier of porcine Peyer's patches follicle-associated epithelium

小肠黏膜的Peyer’s patches （PP）负责肠腔的免疫监视。PP被滤泡相关上皮（FAE）覆盖，FAE具有特殊的屏障特性，可避免免疫细胞和抗原的过度接触。这种屏障功能是由紧密连接蛋白（TJ）提供的，它可以受到内源性和外源性因素的影响。在第一个资助期内，我们表征了猪PP的上皮屏障功能，分析了几种化合物（包括肿瘤坏死因子α、槲皮素、小檗碱和caprate）的作用，并进一步开发了单细胞和信号分析的细胞培养系统和方案。从目前的技术状况来看，出现了一些新的假设和实验挑战，这些将在第二个资助期得到解决。一个主要方面是PP在不同发育阶段对猪肠道食源性和炎症效应物的特异性敏感性，以及细胞模型的分析。因此，在第二个资助期，研究将扩展到以下方面：(i)目前在（病理）生理学，治疗和预防方面讨论的新化合物（次生植物化合物，包括羟基肉桂酸和大麻素，细胞因子），（ii）组织（仔猪年龄段），以及（iii）细胞模型，包括共培养肠细胞和建立的异源表达系统非洲爪蟾卵母细胞进行更详细的分子研究。这一重点将继续允许一种富有成效的、假设驱动的方法，并将进一步将分子功能TJ研究转移到该物种的PP-FAE。因此，本项目的目的是(i)分析一系列可能的屏障效应对PP-FAE的屏障增强和扰动机制，（ii）关注不同年龄组作用的相关性，以及（iii）利用细胞模型进一步分析体外信号传导和TJ蛋白相互作用。本研究继续并扩展了广泛的电生理、分子和免疫组织化学技术的使用，以确定猪PP中TJ蛋白的生理和病理生理相关机制。特别关注屏障功能和信号传导将加强和扩展猪免疫学和上皮生理学界面的新创新研究领域，从而开发出新的潜在治疗和预防策略。

Peyer’s patches (PP) of the small intestinal mucosa are responsible for immune surveillance of the intestinal lumen. PP are covered by the follicle associated epithelium (FAE), which has specific barrier properties to avoid excessive contact of immune cells and antigens. This barrier function is provided by tight junction (TJ) proteins, which can be affected by endogenous and exogenous factors, though. During the first funding period, we have characterized the epithelial barrier function of porcine PP, analyzed effects of several compounds including tumor necrosis factor α, quercetin, berberine, and caprate, and further developed cell culture systems and protocols for single cell and signaling analysis. From the current state of the art, a number of new hypotheses and experimental challenges emerged, which will be addressed in the second funding period. A main aspect is the specific susceptibility of PP for foodborne and inflammatory effectors in porcine intestine during different stages of development, and analysis in cell models. Thus in the second funding period, studies will be extended regarding (i) new compounds which are currently discussed in context with (patho)physiology, therapy and prevention (secondary plant compounds including hydroxycinnamic acids and cannabinoids, cytokines), (ii) tissues (age groups of piglets), and (iii) cell models, including co-cultured intestinal cells and the established heterologous expression system Xenopus laevis oocyte for even more detailed molecular studies.This focus will continue to allow a productive, hypothesis-driven approach and will further transfer molecular-functional TJ research to the PP-FAE of this species. Therefore, aims of this project are (i) to analyze mechanisms of barrier strengthening and of perturbation in PP-FAE by a series of presumable barrier effectors, (ii) to focus on the relevance of the effects in different age groups, and (iii) to employ the cell models for further analyses of signaling and TJ protein interaction, in vitro. This study continues and extends the use of a broad spectrum of electrophysiological, molecular and immunohistochemical techniques to identify physiologically and pathophysiologically relevant mechanisms of TJ proteins in porcine PP. The special focus on barrier function and signaling will strengthen and extend the new innovative research field at the interface of pig immunology and epithelial physiology to allow a development of novel potentially therapeutic and preventive strategies.