Dissertation committee:
Κόλλιας Γεώργιος, Καθηγητής, Τμήμα Ιατρικής, ΕΚΠΑ
Σουγιουλτζής Σταύρος, Αναπληρωτής Καθηγητής, Τμήμα Ιατρικής, ΕΚΠΑ
Χατζηγεωργίου Αντώνιος, Επίκουρος Καθηγητής, Τμήμα Ιατρικής, ΕΚΠΑ
Σφηκάκης Πέτρος, Καθηγητής, Τμήμα Ιατρικής, ΕΚΠΑ
Μπάμιας Γεώργιος, Αναπληρωτής Καθηγητής, Τμήμα Ιατρικής, ΕΚΠΑ
Τσουμακίδου Μαρία, Ερευνήτρια Β’, BSRC "Alexander Fleming"
Κολιαράκη Βασιλική, Ερευνήτρια Γ’, BSRC "Alexander Fleming"
Summary:
The collapse of the intestinal epithelial barrier homeostasis, also known as the leaky- gut syndrome, has been associated with many chronic inflammatory conditions, including the Inflammatory Bowel Disease (IBD). IBD is characterized by chronic inflammation of different areas of the gastrointestinal tract, with intermittent periods of remission and relapse of the disease activity. On the very early phase of IBD, the disruption of the intestinal epithelial barrier integrity allows the commensal bacteria and antigens to translocate in the submucosal area and trigger the reaction of the innate immune system. Despite the fact that the causality of the disease has not been fully deciphered, it is known that both environmental and genetic factors contribute to its initiation.
Corticotropin Releasing Hormone (CRH) is the hormone responsible for the mitigation of the stress response, with a well-documented immunoregulatory action on both systemic and tissue specific level. Here, we aim to assess the role of CRH in the regeneration of the intestinal epithelium upon exposure to injury insults. For this purpose, we applied the DSS induced experimental colitis model in Crh-/- mice. We show that Crh-/- mice exert severe acute inflammatory response, followed by increased lethality and impaired tissue regeneration after the removal of DSS. Moreover, we detect increased expression of IL-6, as well as an altered composition of the gut microbiome under baseline conditions, indicating the presence of a dysbiotic environment.
Acknowledging the implication of autophagy and inflammasome in the communication of the intestinal microflora with the epithelial barrier, we next assessed the interaction of CRH with these cellular mechanisms. Interestingly, the levels of LC3, a well-established marker indicating activation of autophagy, revealed increased autophagy levels under baseline conditions and in the absence of CRH, sustained over the course of the experimental disease. Inhibition of autophagy, using the pharmacological agent 3-Methyladenine, resulted in increased survival and limited immune response of the Crh-/- mice upon termination of the DSS treatment. In parallel, we confirmed the negative interaction between CRH and the autophagy signaling pathways in vitro using amino acids deprivation in Mouse Embryonic Fibroblasts (MEFs) and LPS treatment of the mouse macrophages cell line, Raw264.7.
Aligned with the previous findings was the increased expression of the inflammasome component NLRP6, responsible for sensing of microbial metabolites, and its downstream targets under baseline conditions. Administration of antibiotics for 10 days prior and during the DSS treatment, abrogated the increased lethality of Crh-/- mice and significantly ameliorated the tissue regeneration.
Based on our findings, we propose that CRH controls fundamental homeostatic mechanisms of the intestinal tissue under baseline and inflammatory conditions. Taking also into account its increased expression in the colon tissue of IBD patients, we underlie the significance of the CRH signaling pathway for the identification of therapeutic targets at different stages of the human disease. However, considering the limited translatability of the animal-based studies from bench to the patient bedside, we attempted to establish a platform that would allow for the ex vivo culture and experimentation on human derived colonic epithelial cells.
Hence, we combined recent advancements of the organoids and Organ-on-Chip technology to establish the Colon Intestine-Chip. The newly developed platform emulates the mechanical forces and the extracellular matrix components of the human tissue. Transcriptomic analysis, using RNA sequencing, revealed the significance of endothelium on the structural and functional maturation of intestinal epithelium ex vivo. The Colon Intestine-Chip allowed the multi-lineage differentiation of the biopsy derived colonoids, whereas its periodic exposure to air, reminiscent of the periodicity of the food chyme transition, enhanced the differentiation of Goblet cells.
We provide evidence that the Colon Intestine-Chip platform responds to interferon gamma (IFNγ), a prototype cytokine utilized to model inflammation-induced barrier disruption, by induction of apoptosis and reorganization of the apical junctional complexes as shown with other systems. The IFNγ mediated cytokine secretion across the three healthy donors applied, reflects the expected donor-to-donor variability, supporting the potential use of the Colon Intestine-Chip as a precision medicine tool. We also describe the mechanism of action of interleukin 22 (IL-22) on mature, organoid-derived intestinal epithelial cells that is consistent with barrier disruption in contrast to the previously described barrier protective action. Overall, we propose the Colon Intestine-Chip as a promising human organoid-derived platform to decipher mechanisms driving the development of leaky gut in patients and enable their translation for this unmet medical need.
Keywords:
DSS colitis, Corticotropin releasing hormone (CRH), Organ-on-Chip, Organoids, Intestinal epithelial barrier
