Modulation of host intestinal epithelium by gastrointestinal nematode secreted extracellular vesicles

Abstract

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.