Celiac disease (CeD) is a gluten-induced autoimmune disorder causing small intestinal inflammation and histological damage in genetically predisposed subjects (HLA DQ2/DQ8). The stem cell-mediated intestinal epithelium is a rapidly self-renewing tissue separated from the mesenchyme. Within the mesenchymal niche, fibroblasts (FBs) maintain gut development and tissue homeostasis by secreting critical growth factors, cytokines, and extracellular matrix (ECM) proteins. This study aimed at evaluating the interplay between intestinal FBs and epithelial cells (IECs), utilizing primary FBs and organoids derived from duodenal endoscopic biopsies of healthy controls and CeD patients.
Intestinal FBs and organoids from healthy controls and celiac subjects were grown to investigate the levels of cytokines, chemokines, and growth factors in their culture media (CM) by Bio-Plex analysis. Markers for stress (p-p38/p-MKK), autophagy (LC3-II/p62), and inflammation (p-NF-κB) were evaluated by Western blot. CM conditioned by FBs from GCD-CeD patients was used to treat control (CTR) organoids to test its ability to modulate stress, block autophagic flux, and induce inflammation, as assessed by Western blot analysis.
Bio-Plex analysis revealed that 17/27 analytes were increased in CM from GCD-CeD organoids, while 11/27 were increased in GCD-CeD FBs, compared to their respective CTRs. Cytokines IL-4 and IL-15, as well as chemokines G-CSF and MIP-1β, were expressed by both organoids and FBs. In GCD-CeD FBs, we observed increased p-p38/p-MKK (p<0.05), ROS, dNTPs, and lipid accumulation (p<0.001), indicating clear stress induction. Autophagic flux was impaired, with a decreased LC3-II/LC3-I ratio (p<0.001) and accumulated p62 (p<0.001). Additionally, p-NF-κB levels were significantly higher than CTRs (p<0.05). Notably, control organoids treated with GCD-CeD FB-CM showed reduced autophagic flux alongside increased stress and inflammatory markers.
These results indicate that fibroblasts exhibit a defect in stress response, autophagic flux, and inflammation, and are capable of altering control organoids, suggesting an interplay between epithelial and mesenchymal cells in CeD.