Prenatal stress induces changes in PAR2- and M3-dependent regulation of colon primitive cells.

Prenatal stress is associated with a high risk of developing adult intestinal pathologies, such as irritable bowel syndrome, chronic inflammation, and cancer. Although epithelial stem cells and progenitors have been implicated in intestinal pathophysiology, how prenatal stress could impact their functions is still unknown. We have investigated the proliferative and differentiation capacities of primitive cells using epithelial crypts isolated from colons of adult male and female mice whose mothers have been stressed during late gestation. Our results show that stem cell/progenitor proliferation and differentiation in vitro are negatively impacted by prenatal stress in male progeny. This is promoted by a reinforcement of the negative proliferative/differentiation control by the protease-activated receptor 2 (PAR2) and the muscarinic receptor 3 (M3), two G protein-coupled receptors present in the crypt. Conversely, prenatal stress does not change in vitro proliferation of colon primitive cells in female progeny. Importantly, this maintenance is associated with a functional switch in the M3 negative control of colonoid growth, becoming proliferative after prenatal stress. In addition, the proliferative role of PAR2 specific to females is maintained under prenatal stress, even though PAR2-targeted stress signals Dusp6 and activated GSK3ß are increased, reaching the levels of males. An epithelial serine protease could play a critical role in the activation of the survival kinase GSK3ß in colonoids from prenatally stressed female progeny. Altogether, our results show that following prenatal stress, colon primitive cells cope with stress through sexually dimorphic mechanisms that could pave the way to dysregulated crypt regeneration and intestinal pathologies.NEW & NOTEWORTHY Primitive cells isolated from mouse colon following prenatal stress and exposed to additional stress conditions such as in vitro culture, present sexually dimorphic mechanisms based on PAR2- and M3-dependent regulation of proliferation and differentiation. Whereas prenatal stress reinforces the physiological negative control exerted by PAR2 and M3 in crypts from males, in females, it induces a switch in M3- and PAR2-dependent regulation leading to a resistant and proliferative phenotype of progenitor.

Chymotrypsin activity signals to intestinal epithelium by protease-activated receptor-dependent mechanisms.

BACKGROUND AND PURPOSE: Chymotrypsin is a pancreatic protease secreted into the lumen of the small intestine to digest food proteins. We hypothesized that chymotrypsin activity may be found close to epithelial cells and that chymotrypsin signals to them via protease-activated receptors (PARs). We deciphered molecular pharmacological mechanisms and gene expression regulation for chymotrypsin signalling in intestinal epithelial cells. EXPERIMENTAL APPROACH: The presence and activity of chymotrypsin were evaluated by Western blot and enzymatic activity tests in the luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N-terminally tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. The chymotrypsin signalling mechanism was investigated in CMT93 intestinal epithelial cells by calcium mobilization assays and Western blot analyses of (ERK1/2) phosphorylation. The transcriptional consequences of chymotrypsin signalling were analysed on colonic organoids. KEY RESULTS: We found that chymotrypsin was present and active in the vicinity of the colonic epithelium. Molecular pharmacological studies have shown that chymotrypsin cleaves both PAR1 and PAR2 receptors. Chymotrypsin activated calcium and ERK1/2 signalling pathways through PAR2, and this pathway promoted interleukin-10 (IL-10) up-regulation in colonic organoids. In contrast, chymotrypsin disarmed PAR1, preventing further activation by its canonical agonist, thrombin. CONCLUSION AND IMPLICATIONS: Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.

Epithelial production of elastase is increased in inflammatory bowel disease and causes mucosal inflammation.

Imbalance between proteases and their inhibitors plays a crucial role in the development of Inflammatory Bowel Diseases (IBD). Increased elastolytic activity is observed in the colon of patients suffering from IBD. Here, we aimed at identifying the players involved in elastolytic hyperactivity associated with IBD and their contribution to the disease. We revealed that epithelial cells are a major source of elastolytic activity in healthy human colonic tissues and this activity is greatly increased in IBD patients, both in diseased and distant sites of inflammation. This study identified a previously unrevealed production of elastase 2A (ELA2A) by colonic epithelial cells, which was enhanced in IBD patients. We demonstrated that ELA2A hyperactivity is sufficient to lead to a leaky epithelial barrier. Epithelial ELA2A hyperactivity also modified the cytokine gene expression profile with an increase of pro-inflammatory cytokine transcripts, while reducing the expression of pro-resolving and repair factor genes. ELA2A thus appears as a novel actor produced by intestinal epithelial cells, which can drive inflammation and loss of barrier function, two essentials pathophysiological hallmarks of IBD. Targeting ELA2A hyperactivity should thus be considered as a potential target for IBD treatment.

Increased Mucosal Thrombin is Associated with Crohn's Disease and Causes Inflammatory Damage through Protease-activated Receptors Activation.

BACKGROUND AND AIMS: Thrombin levels in the colon of Crohn's disease patients have recently been found to be elevated 100-fold compared with healthy controls. Our aim was to determine whether and how dysregulated thrombin activity could contribute to local tissue malfunctions associated with Crohn's disease. METHODS: Thrombin activity was studied in tissues from Crohn's disease patients and healthy controls. Intracolonic administration of thrombin to wild-type or protease-activated receptor-deficient mice was used to assess the effects and mechanisms of local thrombin upregulation. Colitis was induced in rats and mice by the intracolonic administration of trinitrobenzene sulphonic acid. RESULTS: Active forms of thrombin were increased in Crohn's disease patient tissues. Elevated thrombin expression and activity were associated with intestinal epithelial cells. Increased thrombin activity and expression were also a feature of experimental colitis in rats. Colonic exposure to doses of active thrombin comparable to what is found in inflammatory bowel disease tissues caused mucosal damage and tissue dysfunctions in mice, through a mechanism involving both protease-activated receptors -1 and -4. Intracolonic administration of the thrombin inhibitor dabigatran, as well as inhibition of protease-activated receptor-1, prevented trinitrobenzene sulphonic acid-induced colitis in rodent models. CONCLUSIONS: Our data demonstrated that increased local thrombin activity, as it occurs in the colon of patients with inflammatory bowel disease, causes mucosal damage and inflammation. Colonic thrombin and protease-activated receptor-1 appear as possible mechanisms involved in mucosal damage and loss of function and therefore represent potential therapeutic targets for treating inflammatory bowel disease.

Sexual dimorphism in PAR2-dependent regulation of primitive colonic cells.

BACKGROUND: Sexual dimorphism in biological responses is a critical knowledge for therapeutic proposals. However, gender differences in intestinal stem cell physiology have been poorly studied. Given the important role of the protease-activated receptor PAR2 in the control of colon epithelial primitive cells and cell cycle genes, we have performed a sex-based comparison of its expression and of the effects of PAR2 activation or knockout on cell proliferation and survival functions. METHODS: Epithelial primitive cells isolated from colons from male and female mice were cultured as colonoids, and their number and size were measured. PAR2 activation was triggered by the addition of SLIGRL agonist peptide in the culture medium. PAR2-deficient mice were used to study the impact of PAR2 expression on colon epithelial cell culture and gene expression. RESULTS: Colonoids from female mice were more abundant and larger compared to males, and these differences were further increased after PAR2 activation by specific PAR2 agonist peptide. The proliferation of male epithelial cells was lower compared to females but was specifically increased in PAR2 knockout male cells. PAR2 expression was higher in male colon cells compared to females and controlled the gene expression and activation of key negative signals of the primitive cell proliferation. This PAR2-dependent brake on the proliferation of male colon primitive cells was correlated with stress resistance. CONCLUSIONS: Altogether, these data demonstrate that there is a sexual dimorphism in the PAR2-dependent regulation of primitive cells of the colon crypt.

Active thrombin produced by the intestinal epithelium controls mucosal biofilms.

Proteolytic homeostasis is important at mucosal surfaces, but its actors and their precise role in physiology are poorly understood. Here we report that healthy human and mouse colon epithelia are a major source of active thrombin. We show that mucosal thrombin is directly regulated by the presence of commensal microbiota. Specific inhibition of luminal thrombin activity causes macroscopic and microscopic damage as well as transcriptomic alterations of genes involved in host-microbiota interactions. Further, luminal thrombin inhibition impairs the spatial segregation of microbiota biofilms, allowing bacteria to invade the mucus layer and to translocate across the epithelium. Thrombin cleaves the biofilm matrix of reconstituted mucosa-associated human microbiota. Our results indicate that thrombin constrains biofilms at the intestinal mucosa. Further work is needed to test whether thrombin plays similar roles in other mucosal surfaces, given that lung, bladder and skin epithelia also express thrombin.