Modulation of host intestinal epithelium by gastrointestinal nematode secreted extracellular vesicles

Helminths have co-evolved alongside their hosts for millions of years and haveadapted eloquent mechanisms that allow them to reside in the host withoutcausing significant pathology, or elimination. The ability of these parasites tomanipulate their specialised host is reflected by their continued persistence asa global health concern, with ~1 billion people infected with soil transmittedhelminths (STHs). Helminth infections have long been associated with reducedallergic and autoimmune diseases leading to the hypothesis that helminthsuppress the host immune system, and this has been confirmed in both animalmodels and controlled human infection studies. Many studies have shown thatmany suppressive effects of infection on the host immune system can beattributed to helminth excreted/secreted products (ES). A small but growing listof individual molecules from helminth ES have been characterised, and themechanism of action elucidated. For example, multiple helminth species havebeen identified to secrete TGFβ mimic proteins that can bind host TGFβreceptor and induce T-regulatory (Treg) immunosuppressive cells. However,the full repertoire of helminth secreted molecules that modulate the host ishypothesised to be far from complete. Our lab discovered the presence of extracellular vesicles (EVs) within ES fromthe mouse infective helminth Heligmosomoides polygyrus bakeri (H. bakeri).EVs are lipid bilayer enclosed nanoparticles that carry proteins, lipids andnucleic acids and are released ubiquitously by all cells and organisms studiedto date. In mammalian systems EVs provide a mechanism of communicationbetween near or distal cells. In the context of host-pathogen dynamics it isproposed that EVs could play a role in enabling parasites to condition theirenvironment during infection. There is mounting evidence of host-parasite EVmediated modulation occurrence between plants and colonising fungal cells,bacteria and mammalian host cells, and several parasites and theirmammalian hosts including several helminth species. During H. bakeri infection host immune suppression is thought to primarilyoccur during the adult stage of infection when the parasite resides in the lumenof the duodenum in close proximity to the intestinal epithelium. The intestinalepithelium plays an integral role both in helminth detection, and in mediatingparasite clearance. Therefore, I hypothesised that adult H. bakeri EVs targetthe intestinal epithelium and directly modulate this tissue. The goal of thisthesis was to determine the role of helminth EVs in infection dynamics andhost modulation in the intestinal epithelium. I aimed to address whether H.bakeri EVs enter the intestinal epithelium, whether uptake is targeted to aspecific cell type and how these effects the function of this tissue usingintestinal organoid models. To address these aims development and refinement of methods for high purityEV preparations and EV labelling was required in order to directly implicate H.bakeri EVs as the causative agent in host responses. In Chapter 3, I comparedvarious combinational approaches to EV isolation and improved the purity ofour EV and EV depleted HES preparations. I then assessed EV preparationsusing cryoEM which furthered our understanding of the morphology anddiversity of H bakeri secreted EVs. I trialled multiple labelling methodologiesand found a low-background labelling method that allowed high confidenceidentification of uptake for subsequent chapters. However, I later discoveredthat the majority of labelling techniques trialled had variable labelling efficiencywith low proportions of EVs labelled; this is a caveat to consider wheninterpreting results using labelled EVs. To understand how H. bakeri EVs interact with the intestinal epithelium Ideveloped methods to grow small intestinal 2-D organoids (enteroids) whichare in vitro cultures that reconstitute the intestinal epithelium (Chapter 4). 2-Denteroids have greater cellular complexity as compared to a homogenous cellline and allow us to address the question of cell type specificity for uptake. 2-D enteroid cultures maintained cellular polarisation and differentiated into 6-7major cell types of the intestinal epithelium. In Chapter 5, I demonstrated by using fluorescently labelled EVs that H. bakeriEVs enter organoid cells, however at a lower proportion than I see side-by-side for our cell line cultures. This led to the hypothesis that H. bakeri EVscould target specific cellular populations within the intestinal epithelium. Toidentify whether uptake of H. bakeri EVs occurs in a targeted fashion byspecific cell types I performed microscopy experiments aiming to co-localiseEVs with certain cell types. Microscopy approaches did not provide a definitiveanswer to the question of whether uptake is cell type specific. Next, I modifiedthe cellular proportion of our 2-D enteroids to identify whether this altered theproportion of EV uptake. Goblet and tuft cells are specialised cells of theepithelium that are strongly induced during helminth infection and mediatehelminth clearance; I reasoned that H. bakeri EVs may specifically enter andmodulate these cell types. Organoid cultures that were enriched in goblet andtuft cells showed no enhanced ability to take up EVs, suggesting that neithergoblet nor tuft cells are specifically targeted over other cell types; however, thisdata does not rule out that H. bakeri EVs can enter these cell types andmodulate them. Whether cell type specificity exists for the uptake of H. bakeriEVs within the intestinal epithelium remains unclear and is still an active areaof investigation. To understand how EV treatment of 2-D enteroids altered host geneexpression in Chapter 6, I performed RNA sequencing (RNA seq) andcharacterised the transcriptional changes within 2-D enteroids to H. bakeri EVsor EV depleted ES after 24 h. Genes critical for maintenance of stem cells, cellcycle and antimicrobial defence were downregulated by H. bakeri EVs. Withinthe intestinal epithelium only a proportion of the cells are mitotic, thereforechanges in cell cycle suggest a modulation of either stem cells of Transit-amplifying (TA) cells. I also identified several changes in cell type restricted genes expressed specifically by stem cells, Paneth cells, TA cells orEnteroendocrine cells (EECs). I now hypothesise EVs specifically modify thesecell types. To define the cell type specific responses after EV or EV depletedES treatment I performed single cell RNA seq, unfortunately the quality of ourcontrol sample made interpreting these results difficult. However, these dataserve as conformation of the cellular composition of our 2-D enteroids model. In addition, I also utilised our 2-D organoid model to perform novel co-culturedexperiments with live adult or larval stage 4 (L4) H. bakeri and performedtranscriptional analysis of the host epithelium under these conditions. Thesedata allow us to uncouple the impact of infection with whole parasites on theintestinal epithelium from any immune driven changes in the epithelium thatoccur in vivo. These data also serve as a comparison between host effectsattributed specifically to H. bakeri EVs, and changes induced by the wholeparasite. In summary, this thesis contributes new knowledge to our understanding of H.bakeri interactions with the intestinal epithelium in the absence of host immunedriven responses and distinguishes the role of secreted H. bakeri EVs inmodulating this tissue. I determined that H. bakeri EVs enter host epithelialcells in 2-D enteroids, but whether this is specifically targeted to certainsubpopulations remains elusive. I characterised the host gene expressionchanges upon H. bakeri EV treatment in 2-D enteroids, these findings furtherour understanding as a field of which host genes and pathways are targetedby H. bakeri. In the future, this thesis along with continued research, couldhave important implications for helminth eradication. Conversely, where H.bakeri EVs suppress specific genes or pathways involved in diseases of theintestinal epithelium such as ulcerative colitis, Crohn's disease, oradenocarcinoma, they could provide novel strategies for therapeutics

Special considerations for studies of extracellular vesicles from parasitic helminths: A community-led roadmap to increase rigour and reproducibility

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved

Development of caecaloids to study host-pathogen interactions: new insights into immunoregulatory functions of Trichuris muris extracellular vesicles in the caecum.

The caecum, an intestinal appendage in the junction of the small and large intestines, displays a unique epithelium that serves as an exclusive niche for a range of pathogens including whipworms (Trichuris spp.). While protocols to grow organoids from small intestine (enteroids) and colon (colonoids) exist, the conditions to culture organoids from the caecum have yet to be described. Here, we report methods to grow, differentiate and characterise mouse adult stem cell-derived caecal organoids, termed caecaloids. We compare the cellular composition of caecaloids with that of enteroids, identifying differences in intestinal epithelial cell populations that mimic those found in the caecum and small intestine. The remarkable similarity in the intestinal epithelial cell composition and spatial conformation of caecaloids and their tissue of origin enables their use as an in vitro model to study host interactions with important caecal pathogens. Thus, exploiting this system, we investigated the responses of caecal intestinal epithelial cells to extracellular vesicles secreted/excreted by the intracellular helminth Trichuris muris. Our findings reveal novel immunoregulatory effects of whipworm extracellular vesicles on the caecal epithelium, including the downregulation of responses to nucleic acid recognition and type-I interferon signalling

Toll-like receptor 4, but not neutrophil extracellular Traps, Promote IFN Type I expression to enhance Th2 responses to Nippostrongylus brasiliensis

The induction of Th2 responses is thought to be multifactorial, and emerge from specific pathways distinct from those associated with antagonistic antibacterial or antiviral Th1 responses. Here, we show that the recognition of non-viable Nippostrongylus brasiliensis (Nb) in the skin induces a strong recruitment of monocytes and neutrophils and the release of neutrophil extracellular traps (NETs). Nb also activates toll-like receptor 4 (TLR4) signaling with expression of Ifnb transcripts in the skin and the development of an IFN type I signature on helminth antigen-bearing dendritic cells in draining lymph nodes. Co-injection of Nb together with about 10,000 Gram-negative bacteria amplified this TLR4-dependent but NET-independent IFN type I response and enhanced the development of Th2 responses. Thus, a limited activation of antibacterial signaling pathways is able to boost antihelminthic responses, suggesting a role for bacterial sensing in the optimal induction of Th2 immunity

Including migrant oncology patients in research : a multisite pilot randomised controlled trial testing consultation audio-recordings and question prompt lists

Background: Oncology patients who are migrants or refugees face worse outcomes due to language and communication barriers impacting care. Interventions such as consultation audio-recordings and question prompt lists may prove beneficial in mediating communication challenges. However, designing robust research inclusive of patients who do not speak English is challenging. This study therefore aimed to: a) pilot test and assess the appropriateness of the proposed research design and methods for engaging migrant populations, and b) determine whether a multi-site RCT efficacy assessment of the communication intervention utilising these methods is feasible. Methods: This study is a mixed-methods parallel-group, randomised controlled feasibility pilot trial. Feasibility outcomes comprised assessment of: i) screening and recruitment processes, ii) design and procedures, and iii) research time and costing. The communication intervention comprised audio-recordings of a key medical consultation with an interpreter, and question prompt lists and cancer information translated into Arabic, Greek, Traditional, and Simplified Chinese. Results: Assessment of feasibility parameters revealed that despite barriers, methods utilised in this study supported the inclusion of migrant oncology patients in research. A future multi-site RCT efficacy assessment of the INFORM communication intervention using these methods is feasible if recommendations to strengthen screening and recruitment are adopted. Importantly, hiring of bilingual research assistants, and engagement with community and consumer advocates is essential. Early involvement of clinical and interpreting staff as key stakeholders is likewise recommended. Conclusion: Results from this feasibility RCT help us better understand and overcome the challenges and misconceptions about including migrant patients in clinical research

MicroRNA-dependent regulation of Hox gene expression sculpts fine-grain morphological patterns in a Drosophila appendage

Disruptions of normal Hox gene expression can lead to severe morphological defects revealing a link between the regulation of Hox expression and pattern formation. Here we explore these links focusing on the impact of microRNA regulation on the expression of the Drosophila Hox gene Ultrabithorax (Ubx) during haltere development. Through the combination of bioinformatic and transcriptomic analyses we identify the miR-310/313 cluster (miR-310C) as a candidate regulator of Ubx. Several experiments confirm this. First, miR-310C and Ubx protein show complementary expression patterns in haltere imaginal discs; second, artificial activation of miR-310C expression in haltere discs leads to Ubx-like phenotypes. Third, expression of a fluorescent reporter bearing Ubx 3'UTR sequences is reduced when co-expressed with miR-310C Fourth, deletion of miR-310C leads to Ubx upregulation and changes the array of mechanosensory sensilla at the base of the haltere. Fifth, artificial increase of Ubx levels within the miR-310C expression domain phenocopies the mechanosensory defects observed in miR-310C mutants. We propose that miR-310C-mediated repression delimits Ubx fine-grain expression contributing to the sculpting of complex morphologies in the Drosophila haltere. Our work reveals a novel role of microRNA regulation in the control of Hox gene expression with impact on morphology

Eye gaze metrics reflect a shared motor representation for action observation and movement imagery

Action observation (AO) and movement imagery (MI) have been reported to share similar neural networks. This study investigated the congruency between AO and MI using the eye gaze metrics, dwell time and fixation number. A simple reach-grasp-place arm movement was observed and, in a second condition, imagined where the movement was presented from the first person perspective (1PP) and the third person perspective (3PP). Dwell time and number of fixations were calculated for whole scene and regions of interest (ROIs). For whole scene, no significant differences were found in the number of fixations for condition (AO, MI) or perspective. Dwell time, however, was significantly longer in AO than MI. For ROIs, the number of fixations was significantly greater in 1PP than 3PP. The data provide support for congruence between motor simulation states but also indicate some functional differences

Genomic analysis of estrogen cascade reveals histone variant H2A.Z associated with breast cancer progression

We demonstrate an integrated approach to the study of a transcriptional regulatory cascade involved in the progression of breast cancer and we identify a protein associated with disease progression. Using chromatin immunoprecipitation and genome tiling arrays, whole genome mapping of transcription factor-binding sites was combined with gene expression profiling to identify genes involved in the proliferative response to estrogen (E2). Using RNA interference, selected ERα and c-MYC gene targets were knocked down to identify mediators of E2-stimulated cell proliferation. Tissue microarray screening revealed that high expression of an epigenetic factor, the E2-inducible histone variant H2A.Z, is significantly associated with lymph node metastasis and decreased breast cancer survival. Detection of H2A.Z levels independently increased the prognostic power of biomarkers currently in clinical use. This integrated approach has accelerated the identification of a molecule linked to breast cancer progression, has implications for diagnostic and therapeutic interventions, and can be applied to a wide range of cancers