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

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)