Besides their long-known critical role in embryonic growth and in cancer development and progression, erythropoietin-producing hepatocellular carcinoma type B (EphB) receptor tyrosine kinases and their ephrin-B ligands are involved in the modulation of immune responses and in remodeling and maintaining the integrity of the intestinal epithelial layer. These processes are critically involved in the pathogenesis of inflammatory-based disorders of the gut, like inflammatory bowel diseases (IBDs). Accordingly, our aim was to investigate the role of the EphB/ephrin-B system in intestinal inflammation by assessing the local and systemic effects produced by its pharmacological manipulation in 2,4,6-trinitrobenzenesulfonic acid (TNBS)- (Th1-dependent model) and dextran sulphate sodium (DSS)- (innate response model) induced colitis in mice. To this purpose, we administered chimeric Fc-conjugated proteins, allegedly able to uni-directionally activate either forward (ephrin-B1-Fc) or reverse (EphB1-Fc) signaling, and the soluble monomeric EphB4 extracellular domain protein, that, simultaneously interfering with both signaling pathways, acts as EphB/ephrin-B antagonist.The blockade of the EphB/ephrin-B forward signaling by EphB4 and EphB1-Fc was ineffective against DSS-induced colitis while it evoked remarkable beneficial effects against TNBS colitis: it counteracted all the evaluated inflammatory responses and the changes elicited on splenic T lymphocytes subpopulations, without preventing the appearance of a splice variant of ephrin-B2 gene elicited by the haptenating agent in the colon. Interestingly, EphB4, preferentially displacing EphB4/ephrin-B2 interaction over EphB1/ephrin-B1 binding, was able to promote Tumor Necrosis Factor alpha (TNFα) release by splenic mononuclear cells in vitro . On the whole, the collected results point to a potential role of the EphB/ephrin-B system as a pharmacological target in intestinal inflammatory disorders and suggest that the therapeutic efficacy of its blockade seemingly works through the modulation of immune responses, independent of the changes at the transcriptional and translational level of EphB4 and ephrin-B2 genes.

Background and Aims Thrombin levels in the colon of Crohn’s disease patients have recently been found to be elevated 100-fold compared to 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 sulfonic 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 sulfonic 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.

Cystic fibrosis (CF) is a multi-organ protein misfolding disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). In addition to respiratory impairment due to mucus accumulation, viruses, bacteria and their co-infections are recognized triggers of acute pulmonary exacerbations, accelerating disease progression, and increasing hospitalization and mortality rate. Treatment complexity increases with the age of patients, as do the number and severity of side effects, drug-drug interactions and costs. Simplifying the therapeutic regimen represents therefore one of the key priorities of CF treatment. We have recently reported the discovery of multitarget compounds able to "kill two birds with one stone" by targeting F508del-CFTR and PI4KIIIβ and thus acting simultaneously as mild correctors and broad-spectrum picornavirus inhibitors. Starting from the previously identified multitarget hits, we report herein the synthesis and biological profiling of new bithiazole derivatives to elucidate the structural requirements to improve F508del-CFTR correction and antiviral potencies. The most promising compound 23a inhibited PI4KIIIβ and selected picornaviruses (EV71, CVB3, hRV02), showed good F508del-CFTR correction potency, additivity and possible synergy with lumacaftor (VX809) at low micromolar concentration. In addition, it was well tolerated in vivo by C57BL/6 mice with no sign of acute toxicity and histological alterations in key biodistribution organs.

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Mesenteric ischemia/reperfusion (I/R), following the transient deprivation of blood flow to the gut, triggers an acute flogistic process involving the disruption of endothelial and epithelial barriers integrity, the activation of immune cells, and the abundant release of inflammatory mediators. Among them, the lipid mediator sphingosine-1-phosphate (S1P) is involved in maintaining epithelial and endothelial barrier integrity and in governing the migration of immune cells through the interaction with S1P1–5 receptors. Therefore, the present work aims to investigate the involvement of S1P signaling in intestinal I/R-induced injury by studying the effects of FTY720, the non-selective S1P1,3–5 agonist, and comparing them with the responses to ozanimod, selective S1P1,5 agonist, in a murine model of gut I/R. Intestinal edema, gut and lung neutrophil infiltration, and oxidative stress were evaluated through biochemical and morphological assays. The collected results highlight the protective action of FTY720 against the inflammatory cascade elicited by mesenteric I/R injury, mainly through the control of vascular barrier integrity. While these beneficial effects were mimicked by ozanimod and can be therefore attributed largely to the effects exerted by FTY720 on S1P1, the recruitment of myeloid cells to the injured areas, limited by FTY720 but not by ozanimod, rather suggests the involvement of other receptor subtypes.

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.

Alterations of gut microbiota have been implicated in a broad variety of intestinal diseases. The mechanisms whereby this may occur remains elusive. Gut mucosal microbiota is naturally organized as a polymicrobial biofilm, separated from the intestinal epithelium by a sterile mucus layer. We recently discovered that intestinal epithelium releases active thrombin, which plays a key role in segregation of intestinal biofilms from host tissue. Furthermore, we detected an upregulation of active thrombin in inflamed human patients and in models of colitis. Objectives Our study objective was to determine whether exposure to high thrombin, such as this occurring during inflammation, will alter commensal microbiota biofilms and promote the dispersion of bacteria predisposed to damage the intestinal epithelium. Methods Microbiota extracted from healthy human colon biopsies were seeded into the Calgary Biofilm Device and polystyrene coupons to develop, a multispecies anaerobic biofilm. Biofilms were exposed to various concentrations of thrombin (10 to 1000 Unit/ml). Dispersed bacteria released from thrombin‐treated biofilms were collected and their composition was assessed by 16S sequencing. These bacteria were apically exposed to human epithelial monolayers on transwells (Caco2 and HT29MTX). Adhesion (90 minutes), invasion (gentamicin assay, 3 hours) and translocation (4 hours) to basolateral side was quantified by plating on agar. Transwells were processed for fluorescent in situ hybridization for bacteria staining and phalloidin antibody for host cell cytoskeleton. Motility phenotype of biofilmdispersed bacteria was assessed on soft agarose gels (swarming and swimming). Mice (B6) were treated intracolonically with thrombin (5U per day for 10 days) or boiled thrombin (similar dose). Rats (Wistar) were treated with TNBS to induce colitis, and were treated for 3 days intracolonically with dabigatran (thrombin inhibitor, 1 μg/kg) or vehicle. Results Biofilm‐dispersed bacteria from thrombin‐treated biofilms attached more importantly to the epithelial monolayers compared to untreated biofilm. 3D reconstruction images confirmed such thrombin‐induced phenotype. Thrombin alters swarming and swimming motility in soft agarose gel. 16S analysis further precise the specific composition of biofilm‐dispersed bacteria after thrombin exposure. In mice, intrarectal administration of thrombin caused alterations of gut microbiota biofilms structure (16S sequencing) and organization ( in situ imaging of gut microbiota). During colitis in rats, local inhibition of thrombin activity prevented gut microbiota biofilms alterations associated with colitis (in situ imaging of gut microbiota and 16S analysis). Conclusions These data suggest that high concentration of thrombin released at gut mucosal surface during inflammation alters gut biofilm organization and modifies the phenotype of biofilm‐dispersed bacteria, which were able to invade and cross the epithelial barrier, thus increasing their likelihood to trigger inflammatory flares.

Eph receptors, comprising A and B classes, interact with cell-bound ephrins generating bidirectional signaling. Although mainly related to carcinogenesis and organogenesis, the role of Eph/ephrin system in inflammation is growingly acknowledged. Recently, we showed that EphA/ephrin-A proteins can modulate the acute inflammatory responses induced by mesenteric ischemia/reperfusion, while beneficial effects were granted by EphB4, acting as EphB/ephrin-B antagonist, in a murine model of Crohn’s disease (CD). Accordingly, we now aim to evaluate the effects of UniPR1331, a pan-Eph/ephrin antagonist, in TNBS-induced colitis and to ascertain whether UniPR1331 effects can be attributed to A- or B-type signaling interference. The potential anti-inflammatory action of UniPR1331 was compared to those of the recombinant proteins EphA2, a purported EphA/ephrin-A antagonist, and of ephrin-A1-Fc and EphA2-Fc, supposedly activating forward and reverse EphA/ephrin-A signaling, in murine TNBS-induced colitis and in stimulated cultured mononuclear splenocytes. UniPR1331 antagonized the inflammatory responses both in vivo, mimicking EphB4 protection, and in vitro; EphA/ephrin-A proteins were inactive or only weakly effective. Our findings represent a further proof-of-concept that blockade of EphB/ephrin-B signaling is a promising pharmacological strategy for CD management and highlight UniPR1331 as a novel drug candidate, seemingly working through the modulation of immune responses.