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.
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.
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 PAR.sub.2 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 PAR.sub.2 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. PAR.sub.2 activation was triggered by the addition of SLIGRL agonist peptide in the culture medium. PAR.sub.2-deficient mice were used to study the impact of PAR.sub.2 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 PAR.sub.2 activation by specific PAR.sub.2 agonist peptide. The proliferation of male epithelial cells was lower compared to females but was specifically increased in PAR.sub.2 knockout male cells. PAR.sub.2 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 PAR.sub.2-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 PAR.sub.2-dependent regulation of primitive cells of the colon crypt. Keywords: Colon primitive cells, Sexual dimorphism, Protease-activated receptor
Darier disease (DD) is a severe dominant genetic skin disorder characterized by the loss of cell-to-cell adhesion and abnormal keratinization. The defective gene, ATP2A2, encodes sarco/endoplasmic ...reticulum (ER) Ca2+-ATPase isoform 2 (SERCA2), a Ca2+-ATPase pump of the ER. Here we show that Darier keratinocytes (DKs) display biochemical and morphological hallmarks of constitutive ER stress with increased sensitivity to ER stressors. Desmosome and adherens junctions (AJs) displayed features of immature adhesion complexes: expression of desmosomal cadherins (desmoglein 3 (Dsg3) and desmocollin 3 (Dsc3)) and desmoplakin was impaired at the plasma membrane, as well as E-cadherin, β-, α-, and p120-catenin staining. Dsg3, Dsc3, and E-cadherin showed perinuclear staining and co-immunostaining with ER markers, indicative of ER retention. Consistent with these abnormalities, intercellular adhesion strength was reduced as shown by a dispase mechanical dissociation assay. Exposure of normal keratinocytes to the SERCA2 inhibitor thapsigargin recapitulated these abnormalities, supporting the role of loss of SERCA2 function in impaired desmosome and AJ formation. Remarkably, treatment of DKs with the orphan drug Miglustat, a pharmacological chaperone, restored mature AJ and desmosome formation, and improved adhesion strength. These results point to an important contribution of ER stress in DD pathogenesis and provide the basis for future clinical evaluation of Miglustat in Darier patients.
The importance of extracellular calcium in epidermal differentiation and intra-epidermal cohesion has been recognized for many years. Darier disease (DD) was the first genetic skin disease caused by ...abnormal epidermal calcium homeostasis to be identified. DD is characterized by loss of cell-to-cell adhesion and abnormal keratinization. DD is caused by genetic defects in ATP2A2 encoding the sarco/endoplasmic reticulum Ca2+-ATPase isoform 2 (SERCA2). SERCA2 is a calcium pump of the endoplasmic reticulum (ER) transporting Ca2+ from the cytosol to the lumen of ER. ATP2A2 mutations lead to loss of Ca2+ transport by SERCA2 resulting in decreased ER Ca2+ concentration in Darier keratinocytes. Here, we review the role of SERCA2 pumps and calcium in normal epidermis, and we discuss the consequences of ATP2A2 mutations on Ca2+ signaling in DD. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.
► Mutations in ATP2A2 encoding the sarco/endoplasmic reticulum Ca2+-ATPase isoform 2 (SERCA2). ► Loss of Ca2+ transport from the cytosol to the lumen of the ER. ► Depleted ER Ca2+ stores ► Loss of cell-to-cell adhesion and abnormal keratinisation.
Abstract In order to survive and replicate, Salmonella has evolved mechanisms to gain access to intestinal epithelial cells of the crypt. However, the impact of Salmonella Typhimurium on stem cells ...and progenitors, which are responsible for the ability of the intestinal epithelium to renew and protect itself, remains unclear. Given that intestinal organoids growth is sustained by stem cells and progenitors activity, we have used this model to document the effects of Salmonella Typhimurium infection on epithelial proliferation and differentiation, and compared it to an in vivo model of Salmonella infection in mice. Among gut segments, the caecum was preferentially targeted by Salmonella . Analysis of infected crypts and organoids demonstrated increased length and size, respectively. mRNA transcription profiles of infected crypts and organoids pointed to upregulated EGFR-dependent signals, associated with a decrease in secretory cell lineage differentiation. To conclude, we show that organoids are suited to mimic the impact of Salmonella on stem cells and progenitors cells, carrying a great potential to drastically reduce the use of animals for scientific studies on that topic. In both models, the EGFR pathway, crucial to stem cells and progenitors proliferation and differentiation, is dysregulated by Salmonella, suggesting that repeated infections might have consequences on crypt integrity and further oncogenesis.
Proteolytic activity in the intestine is detected both in the lumen and in tissues in physiological conditions. It is increased in both compartments in pathological situations such as Inflammatory ...Bowel Disease (IBD), Irritable Bowel Syndrome (IBS) or colorectal cancer. The increased activity can be due to an overexpression and/or release of proteases, but also to a decreased expression of natural endogenous protease inhibitors. We originally aimed at identifying the disease-associated proteases and their inhibitors that are present in the intestine. We identified by in situ zymography techniques that most of the proteolytic activity detected in the intestine is present in the mucosa, associated with intestinal epithelium. We thus focused on intestinal epithelium, investigating the expression and function of epithelial proteases and protease inhibitors in health and disease. We have identified forms of elastases, trypsins, chymotrypsin, membrane-bound proteases or kallikreins in human intestinal epithelium. Some are up-regulated in disease (IBD and IBS in particular). We demonstrated that these epithelial proteases exert a number of functions in the intestine: they modify barrier function, cytokine, chemokine and mucus secretion, they are able to degrade immunoglobulins, they signal to adjacent cells, and in particular to enteric neurons. In parallel, we have demonstrated that epithelial protease inhibitors such as members of the Trappin family are protective against inflammation and are down-regulated in IBD. Epithelial proteases and protease inhibitors appear as major actors of mucosal homeostasis. Their functional study highlights new possible therapeutic intervention for intestinal pathologies.
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.
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.
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.
Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.
Abstract only
Background
Current therapies for Inflammatory Bowel Disease (IBD) are unsatisfactory for proper tissue healing. Serine proteases belong to locally produced host factors that can fuel ...inflammatory processes in tissue from IBD patients, in part through activation of Protease‐Activated Receptors (PAR). We have recently discovered that intestinal epithelium was able to produce active thrombin, suggesting that mucosa itself could be an important source of high thrombin in IBD.
Objectives
We first aimed to determine whether mucosal thrombin was upregulated in animal models of colitis and in tissues from IBD patients. We then determined whether local thrombin upregulation could contribute to local tissue malfunctions. Finally, we evaluated therapeutic feasibility of local delivering of either direct thrombin inhibitors or PAR antagonist in animal models of colitis.
Methods
Colonic tissue samples were obtained from diagnosed IBD patients undergoing colonoscopy at the Toulouse Hospital. Colitis was induced by administering trinitrobenzene sulfonic acid (TNBS) in the colon of Wistar rats or C57Bl6 mice. Human tissue collection and animal procedures received ethical approval from local ethic committees. Thrombin (100 U/ml, 10 days), direct thrombin inhibitor (dabigatran, 1 μg/kg, 4 days) and PAR1 antagonist (Vorapaxar, 2.5 mg/kg, 7 days) were administered in the colon of healthy or TNBS animals under light anesthesia. At time of the sacrifice, colonic tissues were harvested and disease severity was assessed. Thrombin expression was detected using PCR, western blot and immunofluorescence. Thrombin activity was quantified in tissue supernatants using specific enzymatic assays.
Results
We confirmed an increased thrombin protein expression in human mucosal tissue by immunofluorescence and western blots. We found that some, but not all, forms of active thrombin were upregulated, particularly in tissues from Crohn’s disease patients. As observed in human, we found that increased thrombin mRNA expression and activity is also a feature of colitis in animal models of colitis. We demonstrated
in vivo
that colonic exposure to high dose of active thrombin can cause mucosal damage and tissue dysfunctions. Specific inhibition of thrombin activity, and PAR1 antagonists prevent some intestinal damage in TNBS colitis.
Conclusions
In this study, using both animal models and human IBD tissues, we showed that upregulation of mucosal thrombin alone can lead to inflammatory insults. We propose that targeting downstream events from high thrombin activity, rather than inhibiting thrombin directly, might be a better option for IBD because mucosal thrombin at low dose plays an important role on maintaining tissue homeostasis. Considering these promising preclinical results on PAR1 antagonist, future clinical studies in IBD patients could therefore be rapidly envisioned, particularly in patients with the strongest upregulation of thrombin activity.
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