(861) Morphologic and immune characterization of threespine stickleback (Gasterosteus aculeatus) gastrointestinal tract for making accurate in vitro models

Introduction/Rationale: In vitro intestinal culture systems are powerful models to study mucosal immunity and host-pathogen/ commensal interactions. However, existing cell line-based models are limited as they can only represent one intestinal region, making representative organ modeling difficult. As our aim is to develop an accurate culture model of the entire threespine stickleback (Gasterosteus aculeatus) intestine, an in-depth intestinal characterization of both stromal and immune cell populations is required.

Methods: H&E and Alcian blue staining was used to visualize intestinal morphology and goblet cell abundance, respectively. Neutrophilic granulocytes were visualized with myeloperoxidase staining. RNA-ISH was also used detect lymphocyte (IgM+ and CD4+ cells) abundance and distribution within the intestine. Quantification was performed using Fiji software.

Results: Visualization of all intestinal regions (proximal, mid, hind, and rectum) indicated villi length and complexity (villi branching) decreased moving from the proximal to hind intestine. Conversely, goblet cell density increased distally along the intestine. Myloperoxidase positive cells were distributed equally across all regions of the intestine. Additionally, RNA-ISH revealed that lymphocytes did not form lymphoid structures, but were diffusely distributed along the villi in the epithelial layer and lamina propria and primarily consisted of CD4+ cells.

Conclusion: This work establishes a baseline for normal stickleback intestinal morphology and cell composition for each region, and provides guidance for the generation of region-specific in vitro models. Our ongoing work will establish stickleback intestinal cell lines and transwells systems which represent the heterogeneous composition and physiology of these anatomical zones.
Future work will use these tools to model host-microbiota interactions and quantifying morphologic and immune cell population changes during Schistocephalus solidus infection, a natural helminth of threespine stickleback.