P0003 Differential Effects of JAK Inhibitors on Epithelial-Immune Interaction in Inflammatory Bowel Disease
Authors: Floor E , van der Kooij R , van Wijk F , Oldenburg B , Vercoulen Y 
Published: January 2025

Abstract

Background
Multiple cytokines are involved in the pathogenesis of inflammatory diseases like inflammatory bowel disease (IBD) and affect both immune and epithelial cells. JAK-STAT signaling plays an important role in immune responses by transmitting cytokine signals, resulting in altered gene expression and immune activation. To treat diseases like IBD, compounds that interfere with the JAK-STAT pathway, JAK inhibitors, have been developed to dampen inflammation and reduce cytokine signaling. However, these inhibitors' actual mode of action on the human colon has not been fully elucidated. Therefore, we developed a co-culture system to test the effect of different JAK inhibitors on epithelial-immune interactions.

Methods
Human colonoids were disrupted into single cells and seeded on Transwell® inserts in an upside-down orientation to form monolayers1,2 (Figure 1). After differentiation of the layer3, epithelial cells were either collected for qPCR analysis or fixed for immunofluorescence microscopy. To study epithelial-immune interactions co-culture experiments were performed using peripheral blood mononuclear cells (PBMCs). Cells were treated with tofacitinib, filgotinib, upadacitinib, and/or different cytokines. Epithelial cells were collected for western blot analysis and PBMCs were collected for flow cytometry analysis.

Results
Immunofluorescence staining showed a representative number of enterocytes and goblet cells in the upside-down colonoid monolayer which was confirmed by qPCR. Co-culture of epithelial cells with activated PBMCs showed induction of the JAK-STAT pathway; both phosphorylated STAT1 (pSTAT1) and STAT3 (pSTAT3) were induced. Treatment of PBMCs with tofacitinib, upadacitinib, or filgotinib completely diminished pSTAT1 and pSTAT3 induction. In epithelial cells, treatment with tofacitinib and upadacitinib completely diminished pSTAT1 and pSTAT3 induction whereas filgotinib treatment did not.

Conclusion
We validated an effective method to grow monolayers from colonoid-derived human stem cells, containing differentiated epithelial enterocytes and goblet cells (1). We confirmed that epithelial-immune interactions can be studied with this model as pSTAT1 and pSTAT3 could be induced in the epithelial layer by activated PBMCs (2). pSTAT1 and pSTAT3 were completely inhibited by all inhibitors in PBMCs while epithelial cells didn’t fully respond to filgotinib, indicating that epithelial and immune cells respond differently to the inhibitors (3). We plan to capitalize on this system by unraveling the effects of multiple IBD-related cytokines and JAK inhibitors on healthy and IBD epithelial cells to improve clinical treatment strategies.

References
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2.Jelinsky, S.A., Derksen, M., Bauman, E., Verissimo, C.S., van Dooremalen, W.T.M., Roos, J.L., Higuera Barón, C., Caballero-Franco, C., Johnson, B.G., Rooks, M.G., et al. (2023). Molecular and Functional Characterization of Human Intestinal Organoids and Monolayers for Modeling Epithelial Barrier. Inflamm Bowel Dis 29, 195–206. https://doi.org/10.1093/ibd/izac212.
3.Fujii, M., Matano, M., Toshimitsu, K., Takano, A., Mikami, Y., Nishikori, S., Sugimoto, S., and Sato, T. (2018). Human Intestinal Organoids Maintain Self-Renewal Capacity and Cellular Diversity in Niche-Inspired Culture Condition. Cell Stem Cell 23, 787-793.e6. https://doi.org/10.1016/j.stem.2018.11.016.

Figure 1. Upside-down co-culturing of colonic epithelial cells and PBMCs