Gastrointestinal disorders represent a range of serious, potentially life-threatening conditions that continue to be a major challenge to horse owners and the equine industry, leading to significant financial costs associated with prevention and treatment as well as the loss of horses of all ages. A major roadblock in studying and understanding disease outcomes associated with infectious causes of colic is a lack of relevant laboratory model systems in which to model intestinal infections. To overcome this obstacle, the Shaffer Laboratory developed microscopic, organ-like systems (termed ‘organoids’) from a variety of horse tissues to study bacterial and viral equine infectious diseases. Importantly, due to their unlimited self-renewal and tissue expansion, organoids bridge the gap between the laboratory and disease models, providing an attractive alternative to animal experimentation. In addition, organoids have emerged as an invaluable tool for accurately predicting drug metabolism and response, such that they represent an ideal platform for therapeutic discovery and pre-clinical development.

When generated from the intestines, mini-gut organoids (referred to as ‘enteroids’) reproduce the unique characteristics and microarchitecture of the equine gastrointestinal tract, providing a robust laboratory model to mimic the horse intestine. While enteroids grown in a three-dimensional (3D) configuration are beneficial for long-term culture and functional characterization, host-pathogen interactions are more challenging to model. For example, enteroids grow in a wide range of shapes and sizes, leading to inconsistent approximation of equine cell numbers and inaccurate or unpredictable bacteria-to-cell ratios. Moreover, pathogens invade tissues using receptors localized to the luminal host cell surface. Unfortunately, ensuring that bacteria or viruses gain access to the enteroid interior where luminal cell receptors are located is technically challenging, time-consuming and difficult to standardize. To address this challenge, our lab developed methods to manipulate enteroids to allow bacteria or viruses access to critical cell receptors. Our first approach involves imbedding parts of enteroids onto a semipermeable membrane submerged into specialized media that encourages normal cell division and architecture. In this system, enteroids self-assemble such that the luminal cell surfaces (those that would normally be on the inside of the intestine) are exposed and accessible from the top of the culture dish. This technique provides a more convenient, consistent and precise way to control equine cell numbers and allows us to easily infect enteroid-derived tissues.

Our second method involves attaching enteroid tissue onto microfluidic chip devices capable of directional fluid flow and mechanical deformation that applies physical stretch across the chip. Critically, these normal, physiologic forces generate a tissue microenvironment that more accurately mimics the directional flow of digesta traveling through the gut via wavelike peristalsis. These chips developed in our lab consist of two parallel channels separated by a semipermeable membrane that enables “cross-talk” between the interfaced gut tissue and an artificial vasculature created by an equine endothelial cell barrier. Therefore, these gut-on-a-chip models recreate a multi-tissue system similar to blood vessel lined organs in a living horse. Notably, this system affords the opportunity to study the frontline defense to infectious agents; introduction of immune cells through the endothelial channel allows us to monitor pathogen-induced cellular migration from the artificial vasculature into the gut tissue. In this configuration, immune cells exit the vasculature channel to neutralize invading pathogens in the gut tissue channel, thereby replicating host defenses elicited during infection in the horse.

Using advanced laboratory techniques, we validated and demonstrated the functionality of our enteroids. For example, we determined that, similar to tissues within the horse gastrointestinal intestinal tract, equine enteroids exhibit barrier functions, relevant brush border enzyme activities and mucus production. Further, we compared cellular differentiation patterns within enteroid systems using immunohistochemistry and advanced RNA sequencing analyses. Across our various techniques, we determined that our polarized enteroid model exhibits increased cellular differentiation compared to 3D enteroids. However, the addition of biomechanical forces (stretch and flow) in the gut-on-a-chip system demonstrated the highest level of cell type variation, reinforcing our hypothesis that microfluidic devices generate the most life-like laboratory environment in which to study equine infectious disease outcomes.

Our aim is to develop versatile enteroid models for both bacterial and viral infections. Since Salmonella is one of the most common causes of bacterial enteritis in the horse, we first used our enteroid platform to evaluate the effects of bacterial infection on host inflammatory responses. In initial studies, we analyzed how S. Typhimurium uses bacterial nanomachines to inject virulence proteins into intestinal cells to manipulate host immunity and enable colonization. Building on our success, we are using our unique models to investigate additional aspects of microbial pathogenesis and to accelerate the development of novel vaccines to combat infectious disease threats to the equine industry. For example, equine rotavirus B has emerged as a significant health and economic concern and continues to cause foal diarrhea outbreaks in Kentucky and other states including New York and Pennsylvania. We are the first laboratory to demonstrate productive equine rotavirus replication in our intestinal enteroid cultures – a critical experimental step that will enable future vaccine development.

Overall, our organoid technologies can be used to understand how pathogens interact with specific equine tissues, to explore new ways to prevent infection and to discover effective drugs to combat infectious disease. Furthermore, developing additional equine organoid models will provide the experimental foundation for numerous studies focused on tissue injury and regeneration, inflammation and infection control, pre-clinical analysis of new therapeutics, toxicology and drug metabolism studies and the identification of genetic factors that determine disease outcome in the horse. Our organoid-to-microfluidic chip pipeline is rapidly pioneering the way towards successful equine precision medicine that will allow us to develop effective medical intervention strategies in the lab. Ultimately, our platform will reduce animal experimentation and is accelerating progress in disease modeling, vaccine design and development, understanding infectious disease outcomes and investigating regenerative medicine in the context of the dynamic physiology of the horse.

Source: April 2024 Equine Disease Quarterly.

Lynn Leedhanachoke, MS, DVM, is a graduate student in UK’s Department of Veterinary Science, Gluck Equine Research Center. Carrie Shaffer, PhD, is an assistant professor, also within UK’s Department of Veterinary Science, Gluck Equine Research Center.