Ruminating over host-parasite interaction models for fluke driven immune responses

瘤胃吸虫（RF）是一种感染反刍家畜的吸虫寄生虫，由于气候变化，其流行率正在上升。未成熟RF的严重感染可导致动物发病率和死亡率。与其他致病性更高的胃肠道蠕虫相比，RF感染的出现相对较晚，因此关于寄生虫如何与宿主相互作用的知识有限，特别是在宿主免疫反应方面。众所周知，相关的致病蠕虫，如肝吸虫，具有调节宿主免疫反应的能力，使其在宿主中存活更长时间。在几种寄生虫中已证实的一个重要机制是利用细胞外囊泡（EVs），它具有运输寄生虫衍生的货物分子的能力，这些分子具有生物活性，并在宿主中引起免疫调节变化。已知成年RF可以产生和释放ev (Huson等人，2018)，其中含有先前在相关物种中被鉴定为免疫调节剂的蛋白质，并且还具有调节瘤胃内细菌种群的能力（Allen等人，2021）。然而，这些ev调节宿主组织免疫反应的机制尚不清楚。动物疾病的体外模型是在受控的实验环境中发展对分子宿主和寄生虫相互作用的理解所必需的，从而允许减少、改进和替代动物研究。细胞培养模型通常用于体外，但不能考虑组织内的多种细胞类型。因此，组织外植体模型是提供离体模型的有用工具。这些外植体模型也是蠕虫感染的一种新方法，但目前尚未用于蠕虫研究。为了开始阐明RF ev在宿主免疫应答中的作用，本研究旨在进一步建立与RF偏好部位（反刍动物胃肠道）相关的新的体外模型，并利用已建立的物种特异性细胞培养模型来加深我们对RF在宿主内免疫调节作用的理解。该项目将完成了解宿主对RF免疫反应的关键工作，这将对了解与致病性更高的肝吸虫合并感染的免疫反应至关重要。在本研究中需要检验的假设是：1。成体RF产生的ev可调节瘤胃组织外植体和上皮细胞以及牛巨噬细胞免疫特异性细胞系的宿主免疫反应。2. 在EV中发现的特异性预测免疫相关蛋白将产生免疫调节反应，就像在整个EV研究中观察到的那样。为了进一步调查这些假设，将完成以下目标：1。建立牛瘤胃体外外植体组织模型，成功维持宿主-寄生虫相互作用短期（最多72小时）（例如维持组织结构和活力），并用成体瘤胃的ev刺激，通过细胞因子分泌和多组学（转录组学和蛋白质组学分析）评估组织中的免疫反应。(铅:AU) 2。在已建立的瘤胃上皮细胞模型中验证目标1的结果（Ji等人，2021；使用RT-PCR检测靶向基因表达），并评估细胞活力和代谢对EV刺激的响应。(铅:QUB) 3。合成由成年RF ev组成表达的关键免疫调节蛋白，在瘤胃组织外植体/上皮细胞模型中进行测试，并评估免疫反应，以更好地了解ev内生物活性分子的作用机制。（主讲：蛋白质合成上的QUB和实验工作上的AU）评估受成年RF ev刺激的牛巨噬细胞免疫参数随时间的变化，以更好地了解即时和长期免疫反应。(铅:非盟)

Rumen fluke (RF), Calicophoron daubneyi, are trematode parasites that infect ruminant livestock and given a changing climate, are increasing in prevalence. Heavy infections with immature RF can result in animal morbidity and mortality. Due to the relatively recent emergence of RF infections compared to other more pathogenic gastrointestinal helminths, there is limited knowledge as to how the parasite interacts with the host, especially in terms of the host immune response. It is well known that related pathogenic helminths, such as liver fluke, have the ability to modulate host immune responses to their benefit, allowing longer survival in the host. One important mechanism that has been demonstrated in several parasitic species is the use of extracellular vesicles (EVs), which have the ability to transport parasite-derived cargo molecules, which are bioactive, and cause immunomodulatory changes in the host. Adult RF are known to produce and release EVs (Huson et al., 2018), containing proteins previously identified as immune modulators in related species and also have the ability to modulate bacterial populations within the rumen (Allen et al. 2021). However, the mechanism used by these EVs to modulate the host tissue immune responses is unknown. In vitro models of animal diseases are desirable to develop an understanding of molecular host parasite interactions within a controlled experimental environment, allowing the reduction, refinement and replacement of animal studies. Cell culture models are commonly used in vitro, but do not account for multiple cell types within a tissue. As such, tissue explant models are a useful tool for providing ex vivo models. These explant models are also a novel method for helminth infections, but at present have not been utilised for helminth studies. To begin to elucidate the role of RF EVs on the host immune response, the present study aims to further establish new in vitro models that are relevant to RF predilection sites (ruminant gastrointestinal tract) and utilise established species-specific cell culture models to develop our understanding of RF immunomodulatory roles within the host. This project will complete key work to understand the host immune response to RF, which will be essential to understanding immune responses in co-infections with the more pathogenic liver fluke.The hypotheses to be tested in this studentship are: 1. EVs produced by adult RF will modulate the host immune response in rumen tissue explants and epithelial cells, as well as an immune specific cell line of bovine macrophages. 2. Specific predicted immune related proteins identified in EVs will produce immunomodulatory responses like that observed in whole EV studies. To investigate these hypotheses further, the following objectives will be completed: 1.Establish an in vitro explant tissue model of the bovine rumen for host-parasite interactions that can be maintained for a short term (up to 72 hours) successfully (e.g. tissue architecture and viability maintained) and be stimulated with EVs from adult RF to assess immune responses in the tissue via cytokine secretion and polyomics (transcriptome and proteome profiling). (Lead:AU)2.Verify results from objective 1 in an established model of rumen epithelial cells (Ji et al., 2021; using RT-PCR for targeted gene expression), as well as assessing cell viability and metabolism in response to EV stimulation. (Lead:QUB)3.Synthesise key immune modulator proteins constitutively expressed by adult RF EVs to test in the rumen tissue explant/epithelial cell model and assess immune responses to better understand mechanisms of action of bioactive molecules within EVs. (Lead:QUB on synthesis of proteins and AU on experimental work)4.Assess changes in immune parameters in bovine macrophages stimulated with EVs from adult RF over time to better understand immediate and longer-term immune responses. (Lead:AU)