Intestinal organoids as a replacement strategy to unravel early host intestinal epithelia interactions with whipworms

Trichuriasis is a major Neglected Tropical Disease affecting mainly children from low and middle income countries. This disease is characterized by abdominal pain, diarrhea and anemia and it is linked with physical and intellectual growth retardation. Trichuriasis is caused by infection with whipworms, which occurs upon ingestion of eggs of these parasites present on food or water. In the gut, the eggs hatch, liberating larvae that then burrow through the gut lining (epithelium). The initial stages of the epithelium infection by the larvae are thought to be crucial in determining if the parasite is expelled or if it remains in the gut causing a chronic disease. However, up to now, the details of these initial interactions between the whipworm and the gut epithelium are not understood, hampering the development of therapies to eradicate and vaccines to prevent whipworm infections.

Presently, studies on interactions between gut epithelium and whipworms are done by infecting mice with mouse whipworms and require large numbers of animals. The high dependency on the use of mice to do research in trichuriasis affects the size of the community of scientists that investigate this disease and also may compromise our understanding of human infections. My goal in this project will be to develop a new model to study whipworm-gut epithelium interactions using miniature (mini) guts. Mini-guts (also called organoids) are 3D cell clusters generated from gut tissue that have similar characteristics and function to the gut. Mini-guts can be kept alive in the lab for long time, and they can also survive freezing and thawing, allowing to do many experiments using the same material. Recently, I have established a method to inject the mini-guts with whipworm larvae that I propose as a model to replace mouse-whipworm infections. Besides the replacement of mouse use in trichuriasis research, this technique has additional advantages: first, it will allow me to more precisely look at how whipworms interact with the gut cells since I will be able to watch the infection live; and second, in the future, using human mini-guts and human whipworms we will understand much better the human disease. Ultimately, I want to identify the very first steps that occur during the initial contact and infection of whipworm larvae in the gut. To do so, using mini-guts I propose to do assays including microscopy and sequencing and test the changes that happen during infection on the whipworm and the gut epithelium.

This project will benefit both scientists and the society in general as it will: 1) provide a new model to study Trichuriasis replacing mice infections, 2) will increase our knowledge on whipworm-gut interactions and 3) in the future, it will help to develop vaccines and discover drugs to fight whipworm infections in low and middle income countries thus improving the quality of life of their children.

Aim: Early host intestinal epithelial cells (IECs)-whipworm (Trichuris sp) interactions and their implications in the immune response that determine the expulsion or establishment of the parasites in their host are not understood. This research project aims to investigate and understand these interactions in detail, and in so doing, demonstrate a viable alternative to the infection of mice with T. muris. To achieve this, I will exploit intestinal organoids as a replacement model of the murine infections that are currently used.

Objectives and Methodology: Specifically, I will: First, refine and validate the organoid-whipworm larvae infection model, by optimisation of the protocols to generate of mouse caecal organoid lines and to infect them by microinjection of T. muris larvae; second, identify the IEC-type targeted by the parasite by visualising active infection using diverse microscopic techniques; third, conduct transcriptomic, proteomic, flow cytometry and cytokine analysis to discover host IECs-whipworm interactions and evaluate IECs responses to whipworm larvae infection; fourthly, characterize the role and the mechanisms of action in the early host-parasite interaction of several novel IECs genes using mutant organoids; and finally, pilot the use of human organoids as a T.trichiura infection model. The organoid model will be validated by comparison with data already obtained from murine infections.

Scientific opportunities: This project will result in the implementation of a novel model to study Trichuris infections, will generate fundamental data on host-whipworms interactions and also support future efforts to control these parasites by the identification of potential new therapeutic targets.

Trichuriasis affects millions of people from low and middle income countries and is linked to physical and intellectual growth retardation. Trichuriasis is a major Neglected Tropical Disease, a status that is reflected in the small scientific community studying the etiology of the disease and designing new anti-parasitic interventions. One of the major drawbacks in Trichuris research is the dependency on the use of animal models since the human-infecting whipworm cannot be maintained in the lab and is only accessible (as eggs) from deworming studies. This research project proposes an innovative use of intestinal organoids as a model to study host intestinal epithelium-whipworms interactions replacing the current T. muris mouse infection model. Thus, the main area of impact for the proposed work will be in the 3Rs. On site, in a typical experiment and based on statistical power calculations, we currently use 6-8 mice per group and sex. Two types of Trichuris experiments are performed: a large T. muris challenge screen of ~1500 mutant mice per year, seeking to link infection phenotypes to host genetics; and mechanistic studies, aiming to understand the host response to whipworm infection including early host intestinal epithelial cells (IECs)-whipworm interactions. Organoids derived from a single mouse can be expanded and cryopreserved, allowing the performance of several experiments using the same starting material. Thus, using organoids for studies on IECs-whipworms interactions will no longer require any mice allowing a replacement of the large numbers of mice presently utilized on these investigations. Moreover, organoid-based screens may be a good alternative in mouse-based screens searching for host genes governing the interplay of whipworms with epithelium. Additionally, in the long term, the organoid system (from intestine, lung and liver origin) could have a further 3Rs impact by providing a valuable resource for studies on host interactions with other helminth parasites, which currently heavily rely on the use of animals. Consequently, the successful deployment of organoids will potentially have a huge long-term impact on mouse numbers used in helminth research, translating into a vast 3Rs impact.

The project will also impact diverse scientific communities. First, groups studying Trichuris will benefit from the enhancement in the understanding of early host-whipworm interactions and the introduction of a novel model to study whipworm infections including the human infective species T. trichiura. Thus, I foresee an increase in the size of the Trichuris research that is better aligned with the disease impact. Second, research on other helminth parasites will potentially profit from the knowledge, methodologies to study host-parasite interactions (as RNAseq and proteomics) and analytical approaches that will be developed here. Finally, advances in the understanding of the whipworm-IECs interplay could be exploited to understand other intestinal inflammatory diseases that have similarities with trichuriasis and to comprehend the mechanisms that underlie emergent therapies for these diseases that exploit intestinal dwelling helminths.

Ultimately, I expect this research will be translated into wider benefits to the general public. This project will potentially: 1) raise awareness of the about whipworm infection aiming to improve sanitation practices to stop its transmission and 2) will generate knowledge and screening tools that can be translated in the development of vaccines and discovery of new drugs for Trichuriasis treatment. Hence, through the development of novel anti-parasitic interventions and engagement strategies to prevent Trichuris transmission, the results of this project will potentially have a socio-economic development and education impact in endemic countries where trichuriasis is an important cause of growth stunting, intellectual retardation and cognitive and educational deficits.