A PHYSIOLOGICALLY RELEVANT IN VITRO MODEL OF RAINBOW TROUT Oncorhynchus mykiss INTESTINAL IMMUNITY AND ITS INFLAMMATORY RESPONSIVENESS

IntroductionThe rainbow trout (Oncorhynchus mykiss) intestine is organized into two main functional districts, the proximal and distal regions, each contributing differently to nutrient absorption and mucosal immunity. Like other teleosts, trout lack organized GALT structures, resulting in a diffuse intestinal immune system largely coordinated by enterocytes[1] .

To reproduce this complexity in vitro, we established two epithelial cell lines derived from the proximal and distal intestine [2]. Building on these models, and on culture conditions already described in our previous work [3,4], we propose here a functional reinterpretation of conventional in vitro settings, defining two complementary epithelial states: a niche-like module, associated with proliferation and stemness, and a fold-like module, associated with enterocyte differentiation. Importantly, these modules represent a biologically grounded framework for interpreting epithelial phenotypes emerging under standard conditions.

Using these established systems, we explored their intrinsic immune competence under physiological, non-inflammatory conditions. We assessed the spatial distribution of IL8, IL6, IL1β, and TNFα through in situ hybridization, comparing in vitro results with the in vivo intestine to evaluate the physiological relevance of this interpretative model. Finally, we tested their responsiveness to an inflammatory stimulus (LPS) by monitoring the transcriptional activation of pro-inflammatory cytokines.

Results and discussionIn vivo, all four cytokines were detected at low levels and were mainly restricted to the intestinal mucosa. TNFα and IL8 were the most abundant markers and the only ones showing occasional strongly positive cells, whereas IL6 and IL1β were expressed only in rare cells. No major differences were observed between proximal and distal regions or between the stem cell niche and differentiated fold, indicating a relatively homogeneous distribution of the diffuse immune system along the trout gut.

In vitro, all four genes were consistently expressed in both epithelial cell lines and across the two functional modules, confirming that trout enterocytes retain intrinsic immune competence. As observed in vivo, IL8 and TNFα were the most highly expressed markers and showed occasional highly positive cells, suggesting the presence of a particularly responsive epithelial subset. Overall, the spatial and qualitative expression patterns closely mirrored the in vivo reference, supporting the physiological relevance of interpreting these culture conditions as functionally distinct epithelial states.

Upon LPS exposure, both modules showed activation of the inflammatory program, with upregulation of several pro-inflammatory cytokines. This response indicates that these epithelial systems not only preserve basal immune competence but also retain the ability to sense and respond to external stimuli. While further work is needed to dissect the specificity and magnitude of these responses, the results support the validity of this in vitro framework.

Taken together, these findings highlight that appropriate culture conditions can recapitulate distinct, physiologically meaningful epithelial states when interpreted within a coherent biological framework. This approach provides a practical and scalable platform for aquaculture applications, enabling the screening of feed ingredients and functional compounds, as well as the evaluation of their effects on intestinal immune regulation in a region-specific and physiologically relevant context.

Figure 1: Representative in situ hybridization of IL8 (red signal) in the rainbow trout intestine and in the respective in vitro models. The images include both proximal and distal regions and highlight the two functional epithelial units that characterize the trout gut: the stem cell niche and the differentiated fold. Corresponding in vitro images from the proximal and distal epithelial cell lines are shown for both the niche and fold modules. Nuclei are counterstained with DAPI (blue).

Acknowledgment

This work was supported by NUTRIsim

References

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