Testing direct effects of antibiotic alternatives on the intestinal epithelium using intestinal organoids

B. van der Hee*

*Corresponding author for this work

Research output: Contribution to conferenceAbstract

Abstract

The impact of antibacterial drugs as treatment of infection has been pivotal in animal husbandry, but the past decades have shown that bacterial susceptibility to these is rapidly declining. This alarming rate of multidrug resistant bacteria necessitates searching for novel antimicrobials to combat infections. However, exploration of new antimicrobials could pose previously unidentified risks for the host. For example, recent observations have shown that prolonged exposure of mammalian cells to antibacterial drugs decreases oxidative capacity of cells, meaning impact on cellular metabolism and energy provision. It is thought that the resemblance of mitochondria to bacteria could be a contributing cofactor in this process. Another example is treatment with macrolides have shown optic nerve cytotoxicity in patients, whereas fluoroquinolones were associated with diminished cell proliferation in cell culture. Therefore, it is important to assess how novel antimicrobials impact on host biology to verify their safety on a cellular level.

Cytotoxicity and other metabolic effects of compounds such as antibacterial drugs are commonly tested in cell culture-based assays. The use of these cell lines has been vital for dose-dependent drug research, but it is becoming increasingly clear that they often display non-translational cellular behavior. For instance, cell models could acquire genetic mutations, chromosome rearrangements, and aneuploidy, which could affect results and subsequently conclusions on drug efficacy. Other cell models, like explants or primary cell cultures, are short-lived and mainly consist of one cell type. These generally do not mimic the diverse interplay between specialized cell types of a tissue, for example in the gastrointestinal tract. We therefore investigated using intestinal organoids as culture model, due to their resemblance to the structural, functional, and cellular complexity of their derived organ.

Within the intestinal crypt, stem cells reside to give rise to the single-cell layer epithelium that functions as absorptive and digestive system, but also as gatekeeper to foreign antigens. By isolating these stem cells, we could grow small organ-like structures that spatially organize and differentiate into organoids. These could be developed from fresh slaughter material but also from frozen biopsies stored longer than a year. For instance, organoids from the jejunum resembled their host-derived epithelium and tissue location at a transcriptional level and provided a more comprehensive model than the conventionally used cell line IPEC-J2. We also observed location-specific regulation when comparing to other small intestinal locations. As an example, ileum organoids were more involved in innate immune signaling, whereas the jejunum organoids had higher transcription of absorptive and digestive processes. However, the three-dimensional geometry limits direct access to the luminal surface for compound interaction studies. This is important, as it is known that polarized cells show different vectorial organization of surface receptors and transporters, which is then important for translational purposes.

We therefore developed optimized procedures for a 2-dimensional monolayer platform by fully dissociating differentiated 3-dimensional organoids. These monolayers still displayed intricate differentiation to specialized cell types, e.g., mucus-producing goblet cells and absorptive enterocytes, and are amenable to compound interaction studies in conventional cell culture plates. Generating organoids from genetically distinct animals enables us to study the direct interaction of previous and novel antibacterial drug effects in vitro on different tissues and allows us to predict biological and pleotropic implications of drugs more accurately. Moreover, they can also be used to test other types of treatments to prevent bacterial adhesion with short interfering RNAs (siRNA), receptor priming using bacterial metabolites, or hereditary polymorphisms associated with disease resistance.
Original languageEnglish
Pages71
Number of pages1
Publication statusPublished - 9 Jun 2021
Event5th International Conference on Responsible Use of Antibiotics in Animals: Towards Sustainable Solutions - Virtual
Duration: 7 Jun 20219 Jun 2021
https://www.responsibleantibioticuse.org/

Conference

Conference5th International Conference on Responsible Use of Antibiotics in Animals
Period7/06/219/06/21
Internet address

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