Introduction

The intestinal epithelium as dynamic cellular interface integrates signals from the microbial and metabolic environment to sustain tissue homeostasis. We established a clear mechanistic link between mitochondrial dysfunction of the epithelium and the development and progression of inflammatory bowel diseases (IBD). Several clinical trials targeting disease conditions associated with impaired mitochondrial function use mitochondria-targeted pharmaceutical molecules. Thus, we hypothesize that mitochondria-protective targets can be repurposed for adjuvant IBD therapy. We aim to characterize the effect of 10 metabolic drugs targeting various mitochondrial functions on IBD patient-derived organoids, as well as IBD mouse and pig models.

Aims & Methods

Cellular toxicity of the drugs Acipimox, AICAR, ALCAR, Bezafibrate, Epi-743, Idebenone, Metformin, SS-31 and TUDCA was assessed in the intestinal epithelial cell line Mode-K and murine wild-type (WT) small intestinal (SI) organoids. Furthermore, intestinal organoids from TNF^ΔARE mice, a model for Crohn's Disease (CD) like ileitis, were used to mimic IBD-related inflammatory conditions and treated with a predefined concentration of all drugs. Additionally, the corresponding TNF^ΔARE pig model and organoids generated from resected tissue of CD patients were treated with the compounds. Epithelial stemness and differentiation were analysed via imaging. Drug-related changes on mitochondrial respiration were assessed using respirometry.

Results

Cytotoxicity tests were performed in cell and WT organoid cultures to determine suitable concentration ranges for further treatment studies. Exposure of cells to Metformin inhibited cellular respiration, while Idebenone bypassed complex I inhibition and increased ATP production by 57 %. Furthermore, the 10 tested drugs differentially mediated concentration-dependent effects on seeding efficiency in murine WT organoids. To assess the rescuing capacity of the drugs in an inflammatory context, the predefined concentrations of all 10 compounds were applied to SI organoids generated from the inflamed ileum of TNF^ΔARE mice. Three drugs were excluded from further experiments based on reduced organoid seeding efficiency and de novo crypt formation. In comparison, the remaining seven drugs either did not influence organoid growth and differentiation or were able to increase them. Those seven compounds demonstrated similar results using crypts isolated from acutely inflamed ileal tissue sections of the TNF^ΔARE pig model with Epi-743, Idebenone and SS-31 showing a significant increase in de novo crypt formation. Those three drugs did not affect seeding efficiency and de novo crypt formation of human CD organoids from the resection border, while Acipimox, ALCAR, Bezafibrate and Metformin significantly reduced them. Treatment of strongly inflamed parts of resected CD patient tissue specimens with Epi-743, Idebenone and SS-31 restored growth and differentiation capacity in seeded crypts, indicating an increased stemness upon mitochondrial drug treatment. Those drugs will be tested in vivo in the DSS, TNF^ΔARE and the metabolic injury mouse model Hsp60^Δ/ΔIEC; Il10^-/- as well as the TNF^ΔARE pig model.

Conclusion

Selected drugs targeting mitochondrial functions restored seeding efficiency, differentiation, and stemness of intestinal organoids from inflamed tissue sections of TNF^ΔARE mice, TNF^ΔARE pigs, and CD patients. These findings clearly support the potential of metabolic drug repurposing for adjuvant IBD therapy and indicate putative risks for Metformin.

Introduction

Inflammatory bowel disease (IBD) is a growing global health problem with increasing prevalence. Current therapeutic strategies for IBD are mainly aimed at modulating the dysregulated immune activity in the intestinal mucosa. Targeting the JAK/STAT signalling pathway has been shown to be highly effective in ulcerative colitis, but not in Crohn's disease (CD), where efficacy was found to be insufficient in the initiation phase of a clinical trial [1]. Failure of the therapeutic response in Crohn’s disease may be influenced by the unique immune microenvironments in the small and large intestine and their characteristic cytokine profiles and immune cell compositions.

Aims & Methods

To gain deeper insight into region-specific efficacy in the inflamed ileum versus colon, this study investigates the immunomodulatory effects of filgotinib, a Jak1 inhibitor, using precision-cut intestinal slices (PCIS) from patients with Crohn's diseases. PCIS are viable, ultra-thin human tissue slices that retain the original intestinal architecture ex vivo and contain important cell types, including resident immune cells.
PCIS were prepared from resected ileal and colonic tissue of CD patients. The slices were cultured ex vivo and stimulated with concanavalin A or anti-CD3/CD28 antibodies to induce T cell-driven inflammatory responses. Filgotinib (1-100 µM) was applied to assess the immunomodulatory effects of JAK1 inhibition following Concanavalin A preactivation. To exclude treatment-related toxicity, cellular integrity was evaluated using an LDH release assay. Tissue morphology was examined by H&E staining and immunofluorescence. The immune response was assessed by multiplex ELISA of 20 cytokines.

Results

After filgotinib treatment, primary tissue slices from the ileum and colon of CD patients remained viable. Regional morphological characteristics of ileum and colon tissue were preserved after ex vivo cultivation, with prominent villi and smaller crypts in the ileum, and larger, more frequent crypts and flattened villi in the colon. Compared to non-CD tissue slices, Crohn’s disease-related changes, such as immune cell accumulation, were also observed in CD tissue slices ex vivo. Upon T cell-specific stimulation with anti-CD3/CD28 antibodies, region-specific immune reactivity was observed. Colonic PCIS showed an increased release of IL-2 (~3-fold), IL-10 (~2‑fold) and TRAIL (~4-fold) compared to ileal PCIS, indicating a stronger involvement of Th and regulatory T cells in the colon. In contrast, ileal PCIS showed higher levels of MIP-3α (2-fold) and granzyme A (~8-fold) after stimulation, suggesting increased cytotoxic T cell activity in this region. Filgotinib treatment showed region-specific differences in its ability to modulate cytokine release in PCIS of CD patients. In colonic PCIS, filgotinib reduced the induced secretion of Th17-associated cytokines such as IL-17F and IL-22 by up to 95%, whereas in ileal PCIS it showed no effect on the same cytokines.

Conclusion

In conclusion, filgotinib exhibited more potent anti‑inflammatory effect on Th17 cells in the colon, and a markedly reduced efficacy in the ileum. This study demonstrates for the first time that precision-cut intestinal slices can be used to predict region-specific drug response in inflamed intestinal tissue ex vivo, underscoring the critical importance of region-specific immune profiling in IBD and highlight the limited efficacy of JAK1 inhibition in ileum. This ex vivo, patient-derived model represents a valuable platform for evaluating localised treatment efficacy and advancing personalised therapeutic strategies in IBD.

References

[1] Rogler G. Efficacy of JAK inhibitors in Crohn's Disease. J Crohns Colitis. 2020 Aug 1;14(Supplement_2):S746-S754. doi: 10.1093/ecco-jcc/jjz186