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

瘤胃吸虫(Rf)是一种吸虫寄生虫，在气候变化的情况下感染反刍动物，其流行率正在上升。严重感染未成熟的RF可导致动物发病和死亡。由于与其他更具致病性的胃肠道蠕虫相比，RF感染的出现时间相对较晚，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，有能力调节宿主的免疫反应，使它们受益，从而在宿主中存活更长时间。在一些寄生虫物种中已经证明的一个重要机制是使用细胞外小泡(EV)，它具有运输寄生虫衍生的具有生物活性的货物分子的能力，并在宿主中引起免疫调节变化。成体RF已知能产生和释放EV(Huson等人，2018年)，含有先前在相关物种中被确定为免疫调节剂的蛋白质，并且也有能力调节瘤胃内的细菌种群(Allen等人。2021年)。然而，这些EV用来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为了在受控的实验环境中发展对分子宿主寄生虫相互作用的理解，从而允许减少、改进和取代动物研究。细胞培养模型通常在体外使用，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体外模型的有用工具。这些外植体模型也是一种新的蠕虫感染方法，但目前还没有用于蠕虫的研究。为了开始阐明RF EVS在宿主免疫反应中的作用，本研究旨在进一步建立与RF易感部位(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发展我们对RF在宿主内的免疫调节作用的理解。本项目将完成了解宿主对RF的免疫反应的关键工作，这对于了解与更具致病性的肝吸虫混合感染的免疫反应是至关重要的。本研究将检验的假设是：1.成年RF产生的EVS将调节瘤胃组织外植体和上皮细胞以及牛巨噬细胞的免疫特异性细胞系的宿主免疫反应。2.在EV中识别的特定预测免疫相关蛋白将产生与整个EV研究中观察到的一样的免疫调节反应。(Lead：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年；使用RT-PCR进行靶向基因表达)，以及评估细胞活性和代谢对EV刺激的反应。(Lead：QUB)3.合成成体RF EVS组成性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，评估免疫应答，以更好地了解EVS中生物活性分子的作用机制。4.评估EV刺激牛巨噬细胞的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。(销售线索：Au)

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)