Peripheral Opioid Receptor Blockade Enhances Epithelial Damage in Piroxicam-Accelerated Colitis in IL-10-Deficient Mice.

Mucosal CD4+ T lymphocytes display a potent opioid-mediated analgesic activity in interleukin (IL)-10 knockout mouse model of inflammatory bowel diseases (IBD). Considering that endogenous opioids may also exhibit anti-inflammatory activities in the periphery, we examined the consequences of a peripheral opioid receptor blockade by naloxone-methiodide, a general opioid receptor antagonist unable to cross the blood-brain barrier, on the development of piroxicam-accelerated colitis in IL-10-deficient (IL-10-/-) mice. Here, we show that IL-10-deficient mice treated with piroxicam exhibited significant alterations of the intestinal barrier function, including permeability, inflammation-related bioactive lipid mediators, and mucosal CD4+ T lymphocyte subsets. Opioid receptor antagonization in the periphery had virtually no effect on colitis severity but significantly worsened epithelial cell apoptosis and intestinal permeability. Thus, although the endogenous opioid tone is not sufficient to reduce the severity of colitis significantly, it substantially contributes to the protection of the physical integrity of the epithelial barrier.

Characterization of Human Colon Organoids From Inflammatory Bowel Disease Patients.

Inflammatory Bowel Diseases (IBD) are chronic inflammatory disorders, where epithelial defects drive, at least in part, some of the pathology. We reconstituted human intestinal epithelial organ, by using three-dimension culture of human colon organoids. Our aim was to characterize morphological and functional phenotypes of control (non-IBD) organoids, compared to inflamed organoids from IBD patients. The results generated describe the epithelial defects associated with IBD in primary organoid cultures, and evaluate the use of this model for pharmacological testing of anti-inflammatory approaches. Human colonic tissues were obtained from either surgical resections or biopsies, all harvested in non-inflammatory zones. Crypts were isolated from controls (non-IBD) and IBD patients and were cultured up to 12-days. Morphological (size, budding formation, polarization, luminal content), cell composition (proliferation, differentiation, immaturity markers expression), and functional (chemokine and tight junction protein expression) parameters were measured by immunohistochemistry, RT-qPCR or western-blot. The effects of inflammatory cocktail or anti-inflammatory treatments were studied in controls and IBD organoid cultures respectively. Organoid cultures from controls or IBD patients had the same cell composition after 10 to 12-days of culture, but IBD organoid cultures showed an inflammatory phenotype with decreased size and budding capacity, increased cell death, luminal debris, and inverted polarization. Tight junction proteins were also significantly decreased in IBD organoid cultures. Inflammatory cytokine cocktail reproduced this inflammatory phenotype in non-IBD organoids. Clinically used treatments (5-ASA, glucocorticoids, anti-TNF) reduced some, but not all parameters. Inflammatory phenotype is associated with IBD epithelium, and can be studied in organoid cultures. This model constitutes a reliable human pre-clinical model to investigate new strategies targeting epithelial repair.

Protease-Activated Receptors in the Intestine: Focus on Inflammation and Cancer.

Protease-activated receptors (PARs) belong to the G protein-coupled receptor (GPCR) family. Compared to other GPCRs, the specificity of the four PARs is the lack of physiologically soluble ligands able to induce their activation. Indeed, PARs are physiologically activated after proteolytic cleavage of their N-terminal domain by proteases. The resulting N-terminal end becomes a tethered activation ligand that interact with the extracellular loop 2 domain and thus induce PAR signal. PARs expression is ubiquitous and these receptors have been largely described in chronic inflammatory diseases and cancer. In this review, after describing their discovery, structure, mechanisms of activation, we then focus on the roles of PARs in the intestine and the two main diseases affecting the organ, namely inflammatory bowel diseases and cancer.

Thrombin modifies growth, proliferation and apoptosis of human colon organoids: a protease-activated receptor 1- and protease-activated receptor 4-dependent mechanism.

BACKGROUND AND PURPOSE: Thrombin is massively released upon tissue damage associated with bleeding or chronic inflammation. The effects of this thrombin on tissue regrowth and repair has been scarcely addressed and only in cancer cell lines. Hence, the purpose of the present study was to determine thrombin's pharmacological effects on human intestinal epithelium growth, proliferation and apoptosis, using three-dimensional cultures of human colon organoids. EXPERIMENTAL APPROACH: Crypts were isolated from human colonic resections and cultured for 6 days, forming human colon organoids. Cultured organoids were exposed to 10 and 50 mU·mL-1 of thrombin, in the presence or not of protease-activated receptor (PAR) antagonists. Organoid morphology, metabolism, proliferation and apoptosis were followed. KEY RESULTS: Thrombin favoured organoid maturation leading to a decreased number of immature cystic structures and a concomitant increased number of larger structures releasing cell debris and apoptotic cells. The size of budding structures, metabolic activity and proliferation were significantly reduced in organoid cultures exposed to thrombin, while apoptosis was dramatically increased. Both PAR1 and PAR4 antagonists inhibited apoptosis regardless of thrombin doses. Thrombin-induced inhibition of proliferation and metabolic activity were reversed by PAR4 antagonist for thrombin's lowest dose and by PAR1 antagonist for thrombin's highest dose. CONCLUSIONS AND IMPLICATIONS: Overall, our data suggest that the presence of thrombin in the vicinity of human colon epithelial cells favours their maturation at the expense of their regenerative capacities. Our data point to thrombin and its two receptors PAR1 and PAR4 as potential molecular targets for epithelial repair therapies.