The Role of Caffeine and Other Xanthine Metabolites in the Modulation of Colonic Inflammation

Inflammatory Bowel Disease (IBD) is a chronic autoimmune inflammatory condition that primarily affects the lower gastrointestinal tract. It is pathologically characterized by disruptions in epithelial integrity, immune system dysregulation, and features of an autoinflammatory syndrome [1]. Caffeine, a methylxanthine compound, is among the most commonly consumed bioactive substances globally, and is found not only in coffee but also in tea, soft drinks, and even some medications [2]. Despite the widespread intake of caffeine and its metabolites, their specific effects on intestinal inflammation and epithelial barrier dynamics are still not completely understood [3]. This study aimed to evaluate the influence of caffeine and its major metabolites – paraxanthine, theobromine, and theophylline – on inflammatory signaling and epithelial integrity in gut epithelial models, with the goal of elucidating their potential implications for gastrointestinal health, especially in relation to Inflammatory Bowel Disease. A concentration-dependent screening was first performed using Caco-2 cells to determine bioactivity. Metabolites selected from this screening were subsequently evaluated in inflamed 3D intestinal epithelial organoids derived from ulcerative colitis patient (n = 1) and non-IBD control (n = 1). Inflammation was induced over a 24-hour period using a combination of TNF-α and IFN-γ cytokines. To assess the impact on inflammatory activity and epithelial barrier integrity, targeted gene expression analysis was performed using RT-PCR. Additionally, in order to test the antioxidant properties of the coffee [3], the production of reactive oxygen species (ROS) was evaluated through flow cytometry and the DHE assay. The initial analysis revealed that caffeine (affecting TNF-α, CXCL9, and IL18; p < 0.05) and its metabolite paraxanthine (influencing TNF-α; p < 0.05) significantly modulated the expression of genes related to inflammatory cytokines. Considering expression of tight junction genes, paraxanthine notably increased OCLN, TJP1, and CLDN1 levels (p < 0.05), while caffeine – only CLDN1 (p < 0.05). These findings have led to more detailed investigation using 3D intestinal organoid models. However, in the inflamed organoid model, neither caffeine nor paraxanthine was able to reduce the expression of genes encoding inflammatory cytokines. Nevertheless, both metabolites significantly reinstated the expression of genes encoding tight junction proteins – OCLN and TJP1 (p < 0.05). Additionally, caffeine and paraxanthine lowered the production of reactive oxygen species by 25% and 8%, respectively. Methylxanthine caffeine and its main metabolite paraxanthine may support gut epithelial barrier function by promoting the restoration of tight junction protein gene expression, leading to the modulation of colonic inflammation.