Thrombin is a serine protease produced by the intestinal epithelium that contributes to mucosal homeostasis by limiting microbial encroachment. In Crohn's disease, mucosal thrombin activity is markedly increased, suggesting a role in disease pathophysiology. We hypothesized that excessive luminal thrombin disrupts host-microbiota interactions and promotes a pathogenic microbial phenotype. Thrombin levels were significantly elevated in fecal samples from a subset of Crohn's disease patients compared with healthy controls. Ex vivo experiments using human colonic biopsy-derived microbiota, cultured as polymicrobial biofilms, revealed that thrombin exposure disrupted biofilm integrity, enriched the protein-rich extracellular matrix, and promoted bacterial dispersal. Dispersed bacteria displayed enhanced adhesion to intestinal epithelial cells and triggered inflammatory and antimicrobial responses through Toll-Like Receptor 5 signaling. Although the global taxonomic composition showed limited changes, metatranscriptomic analyses demonstrated distinct microbial activity profiles in the presence of thrombin. In vivo, intracolonic thrombin altered mucosal biofilm organization in mice, and the transfer of thrombin-exposed mucosal microbiota to germ-free recipients induced mucosal inflammation and bacterial translocation. Finally, in a rat model of colitis, pharmacological inhibition of thrombin activity restored mucosal biofilm architecture and reduced tissue damage. These findings identify thrombin as a host-associated modulator of microbial functional dysbiosis in patients with Crohn's disease. By driving a shift toward a pathogenic microbial phenotype, excessive thrombin links epithelial activity with altered host-microbiota interactions and mucosal inflammation. Thrombin measurement in fecal samples may represent a non-invasive marker of microbiota pathogenicity, and therapeutic targeting of thrombin offers a promising complementary strategy to immune-directed treatments in Crohn's disease.

BACKGROUND:Postoperative Ileus (POI) is an iatrogenic complication characterized by a temporary paralysis of gastrointestinal transit, leading to food intolerance, nausea, vomiting, and thus prolonged hospitalization. The severity of POI is influenced by surgical trauma, particularly in intestinal surgeries, which have a high complication rate. To date, no animal model has precisely replicated POI in the context of digestive sutures or anastomoses, procedures common in human digestive resections. METHODS:To induce POI, mice underwent different surgeries. The surgical procedure involved a midline laparotomy, externalization of the small intestine following external manipulation through moistened cotton applicators, mimicking the surgeon's action when searching for an intestinal lesion. An ileo-ileal anastomosis was also performed to ensure relevance to human surgical intervention. Intestinal transit was measured by assessing gastric emptying, gastrointestinal transit time (gavage with charcoal, fecal output), and gut motility (isotonic contraction). Postoperative inflammation is assessed in different tissue layers and areas of theintestine. KEY RESULTS:The externalization of the small intestine with caecum and manipulation for 10 min induced a pathological postoperative ileus. This simple gesture induced a decrease in intestinal transit comparable to the surgical intervention, ileo-ileal anastomosis. The model showed decreased gastric emptying and reduced ileal muscle contraction, accompanied by neutrophil and monocyte/macrophage infiltration in the external muscularis. CONCLUSIONS AND INFERENCES:The developed procedure enables inducing postoperative ileus in mice in a very simple and reproducible way that does not require any specific equipment, mimics clinical practice, and reproduces traits of human pathology.

BACKGROUND AND PURPOSE:Proteolytic balance is dysregulated in many diseases, with proteases playing critical roles in pathological pathways. A high level of Trypsin-3 expression has been implicated as a significant mediator of tumour progression and metastasis, and this protease is associated with poor prognosis for patients in various cancers. Therefore, Trypsin-3 inhibition has emerged as a promising therapeutic target. However, no physiological or pharmacological inhibitor has yet been described that specifically targets Trypsin-3. A major challenge in developing a druggable inhibitor for this protease lies in achieving selectivity, as proteases belong to a large enzymatic family with close homologues that share similarities in the three-dimensional folding of their active conformation. EXPERIMENTAL APPROACH:An advanced screening strategy of a large library of synthetic humanised nanobodies was employed to isolate highly selective recombinant antibodies targeting the active conformation of Trypsin-3. Among five hits, we combined two domains with distinct paratopes and inhibitory mechanisms to generate a macrodrug candidate capable to efficiently block Trypsin-3 activity. KEY RESULTS:This bispecific nanobody demonstrated exceptionally high selectivity and affinity for Trypsin-3 in vitro, as well as a strong ability to inhibit cancer cell migration ex vivo for the PC-3 cancer cell line. CONCLUSIONS AND IMPLICATIONS:This study underscores the versatility and potential of synthetic nanobody engineering in the development of highly selective protease inhibitors, paving the way for their consideration as drug candidates for clinical development.

Chronic inflammation is a common trait in the pathogenesis of several diseases of the gut, including inflammatory bowel disease and celiac disease. Control of the inflammatory response is crucial in these pathologies to avoid tissue destruction and loss of intestinal function. Over the last 50 years, the identification of the mechanisms and mediators involved in the acute phase of the inflammatory response, which is characterized by massive leukocyte recruitment, has led to a number of therapeutic options. New drugs targeting inflammatory flares are still under development. However, interest on the other end of the spectrum-the resolution and repair phases-has emerged, as promoting tissue functional repair may maintain remission and counteract the chronicity of the disease. This review aims to discuss the current and future pharmacological approaches to the treatment of chronic intestinal inflammation and the restoration of functional tissues.

Pathophysiological mechanisms of unhealthy aging, particularly the transition from robustness to frailty, remain poorly understood. Despite extensive microbiome research on taxonomy, the behavior of early prefrail gut bacteria in their natural community-host mucosal tissue context remains unexplored. Using fecal samples from the INSPIRE-T aging human cohort, we characterized gut microbiota phenotype during prefrailty stages using a polymicrobial biofilm model. Results revealed that prefrail-derived biofilms exhibited distinct taxonomic and physical alterations, enhanced dispersal, and increased epithelial virulence compared to robust counterparts. Multiparametric analyses linked biofilm characteristics to clinical traits, suggesting their potential as aging status indicators. Polyphenol-rich grape pomace extract partially reversed prefrail biofilm alterations and reduced proinflammatory prefrail biofilm responses in vitro. Microbiota from prefrail-aged mice induced colon damage in antibiotic-treated recipients, establishing a prefrail microbiome-inflammation causality. Overall, the findings identified novel prefrail microbiome characteristics, established causal inflammatory links, and supported microbiota-targeted geroprotective interventions for the prefrail populations.

Abstract Background Dysregulated proteolytic activity (PA) has been detected in intestinal biopsy samples of individuals with inflammatory bowel disease (IBD) compared to healthy controls. It is known that increased microbial fecal PA precedes ulcerative colitis (UC) diagnosis in an at-risk population. Intestinal inflammation in UC is characterized by continuous ulceration, inflammation in Crohn’s disease (CD) is patchy in nature. Sites of inflammation in CD has been associated with altered mucosa-associated microbes and higher permeability, suggesting these could be localized sites of increased microbial PA. Aims To investigate whether inflamed mucosal areas in CD are associated with proteolytic microbiota and increased permeability. Methods CD patients in flare (n=23) were recruited at McMaster University Medical Centre and matched (or paired) biopsies were collected at colonoscopy from inflamed and adjacent (within 5 cm) non-inflamed sites. Biopsy mucosal-to-serosal 51Cr-EDTA flux rate and tissue conductance were measured with Ussing chambers. Total proteolytic (trypsin), elastase-like (FITC-elastin), and mucolytic (0.05% mucin plates) activities were also assessed in biopsies from both sites and in situ zymography was used to measure and localize elastase-like activity in biopsy samples. Mucosal brushing samples from the same participants were also collected and total RNA purified for metatranscriptomic analysis to characterize active microbial pathways with a focus on identifying highly expressed microbial proteases. Results Paracellular permeability and tissue conductance were greater in biopsies from inflamed areas than in biopsies from matched non-inflamed areas (p<0.001). Elastase-like and mucolytic activities were higher in inflamed areas compared to matched non-inflamed areas (p<0.05 and p=0.04, respectively), no significance was detected in total proteolytic activity. In situ zymography revealed greater elastase-like activity in inflamed areas compared to non-inflamed areas (p=0.04), localized to both the epithelial and submucosa layers in inflamed tissues but only to the epithelial layer in matched non-inflamed tissue. Preliminary analysis of the metatranscriptomics results suggests that specific microbial proteases may be increased at inflamed sites in CD. Conclusions Our results suggest that localised variations in tissue inflammation, PA and mucosal permeability in CD may be causally linked, providing a rationale for the patchy distribution of mucosal injury. We, therefore, hypothesize that microbial elastase-like activity promotes mucosal inflammation through barrier disruption. This mechanism could be targeted locally to prevent or modulate microbially associated PA with the aim of enhancing mucosal healing in IBD. Funding Agencies:

STUDY QUESTION:Does a human fallopian tube (HFT) organoid model offer a favourable apical environment for human sperm survival and motility? SUMMARY ANSWER:After differentiation, the apical compartment of a new HFT organoid model provides a favourable environment for sperm motility, which is better than commercial media. WHAT IS KNOWN ALREADY:HFTs are the site of major events that are crucial for achieving an ongoing pregnancy, such as gamete survival and competence, fertilization steps, and preimplantation embryo development. In order to better understand the tubal physiology and tubal factors involved in these reproductive functions, and to improve still suboptimal in vitro conditions for gamete preparation and embryo culture during IVF, we sought to develop an HFT organoid model from isolated adult stem cells to allow spermatozoa co-culture in the apical compartment. STUDY DESIGN, SIZE, DURATION:Over a 2-year period, fallopian tube tissues were collected for organoid culture purposes from 10 'donor' patients undergoing bilateral salpingectomy by laparoscopy for definitive sterilization. After tissue digestion, isolated cells from the isthmus and ampulla regions were separately seeded in 3D Matrigel and cultured with conventional growth factors for organoid culture and specific factors for differentiation of the female genital tract. PARTICIPANTS/MATERIALS, SETTING, METHODS:HFT organoids were characterized by light microscopy, scanning and transmission electron microscopy, immunofluorescence, and transcriptome analysis. Following simultaneous organoid culture on specific inserts, spermatozoa from five donors were placed either in control media or in the apical compartment of colon or HFT organoids (isthmus and ampulla separately) for 96 h. Vitality and motility and kinematic parameters were assessed at 0, 48, and 96 h on 200 spermatozoa in each condition and in duplicate and compared using the Wilcoxon test. MAIN RESULTS AND THE ROLE OF CHANCE:Specific fallopian tube differentiation of our model was confirmed by immunofluorescence, transcriptome analysis, and electron microscopy observations that exhibited ciliated and secretory cells. We succeeded in releasing spermatozoa in the apical compartment of HFT organoids and in recovering them for sperm analysis. Sperm vitality values were similar in HFT organoids and in commercial sperm media. We demonstrated a superiority of the HFT organoid apical compartment for sperm motility compared with other controls (colon organoids, organoid culture media, and conventional commercial sperm fertilization media). At 48 h of incubation, progressive sperm motility was higher in the apical compartment of HFT organoids (ampulla 31% ± 17, isthmus 29% ± 15) than in commercial fertilization media (15% ± 15) (P < 0.05) and compared with all other conditions. At 96 h, progressive sperm motility was almost nil (<1%) in all conditions except for spermatozoa in HFT organoids (P < 0.05): 12% ± 15 and 13% ± 17 in ampulla and isthmus organoids, respectively. Computer-assisted sperm analysis (CASA) analysis also showed that the organoids were able to maintain significantly higher levels of kinematic parameters (curvilinear velocity, average path velocity, straight linear velocity, and amplitude of lateral movement of the head) and therefore more efficient mobility compared with commercial IVF media. LARGE SCALE DATA:N/A. LIMITATIONS, REASONS FOR CAUTION:This was an in vitro study in which conditions of organoid culture could not exactly mimic the in vivo environment of the extracellular matrix and vascularization of fallopian tubes. WIDER IMPLICATIONS OF THE FINDINGS:This work opens up perspectives for better understanding of HFT physiology. For the first time, it highlights the possibility of developing HFT organoids for reproductive purposes. In the future, it could help us to improve gamete fertilizing abilities and embryo culture conditions during human ARTs. STUDY FUNDING/COMPETING INTEREST(S):This study was funded by a grant from the Occitanie region, and by financial allocations from the DEFE and IRSD research teams. The authors have no conflicts of interest to report.

Extracellular proteases, originating from the host or the microbiota, are key signalling molecules involved in cellular communication with the environment. They signal through a wide array of mechanisms, ranging from receptor activation to protein transformation and even degradation. Protease signals are irreversible, as it involves the cleavage of proteins. Therefore, proteases are tightly controlled, and must be understood within the context of the complex networks in which they operate — their activity is tightly regulated by access to specific substrates and the presence of inhibitors. The intestine is particularly exposed to extracellular proteases, which have major roles in gut physiology: digestion, food antigen processing, barrier function, epithelial renewal and microbiome homeostasis. Dysregulated proteolytic balance is associated with intestinal pathologies including inflammatory bowel disease, irritable bowel syndrome, coeliac disease and colorectal cancer. Extracellular proteases are major contributors to a number of gut dysfunctions, including microbiota dysbiosis, barrier dysfunction, matrix remodelling, activation of mucosal immunity and nociceptive or motility abnormalities. Consequently, proteolytic homeostasis at the intestinal mucosa surface has become a goal for intestinal health, and new therapeutic options targeting the interplay among proteases, their inhibitors and their substrates have been explored. In this Review, Deraison and Vergnolle describe the role of extracellular proteases and protease inhibitors in gastrointestinal physiology and in diseases including inflammatory bowel disease, irritable bowel syndrome and colorectal cancer. They also explore emerging therapeutic options for restoring proteolytic balance.

Imbalances in proteolytic activity have been linked to the development of inflammatory bowel diseases (IBD) and experimental colitis. Proteases in the intestine play important roles in maintaining homeostasis, but exposure of mucosal tissues to excess proteolytic activity can promote pathology through protease-activated receptors (PARs). Previous research implicates microbial proteases in IBD, but the underlying pathways and specific interactions between microbes and PARs remain unclear. In this study, we investigated the role of microbial proteolytic activation of the external domain of PAR2 in intestinal injury using mice expressing PAR2 with a mutated N-terminal external domain that is resistant to canonical activation by proteolytic cleavage. Our findings demonstrate the key role of proteolytic cleavage of the PAR2 external domain in promoting intestinal permeability and inflammation during colitis. In wild-type mice expressing protease-sensitive PAR2, excessive inflammation leads to the expansion of bacterial taxa that cleave the external domain of PAR2, exacerbating colitis severity. In contrast, mice expressing mutated protease-resistant PAR2 exhibit attenuated colitis severity and do not experience the same proteolytic bacterial expansion. Colonization of wild-type mice with proteolytic PAR2-activating Enterococcus and Staphylococcus worsens colitis severity. Our study identifies a previously unknown interaction between proteolytic bacterial communities, which are shaped by inflammation, and the external domain of PAR2 in colitis. The findings should encourage new therapeutic developments for IBD by targeting excessive PAR2 cleavage by bacterial proteases.

Background: Several medicinal treatments for avoiding post-operative ileus (POI) after abdominal surgery have been evaluated in randomised controlled trials. This network meta-analysis aimed to explore the relative effectiveness of these different treatments on ileus outcome measures. Methods: A systematic literature review was performed to identify randomised controlled trials (RCTs) comparing treatments for post-operative ileus following abdominal surgery. A Bayesian network meta-analysis was performed. Direct and indirect comparisons of all regimens were simultaneously compared using random-effects network meta-analysis. Results: A total of 38 randomised controlled trials were included in this network meta-analysis reporting on 6371 patients. Our network meta-analysis shows that prokinetics significantly reduce the duration of first gas (Mean difference (MD) (hours) – 16; credible interval - 30, - 3.1; surface under the cumulative ranking curve (SUCRA) 0.418), duration of first bowel movements (Mean difference (MD) (hours) -25; credible interval - 39, - 11; SUCRA 0.25) and duration of post-operative hospitalisation (Mean difference (MD) (hours) – 1.9; credible interval – 3.8, - 0.040; SUCRA 0.34). Opioid antagonists are the only treatment that significantly improve the duration of food recovery (Mean difference (MD) (hours) - 19; credible interval - 26, - 14; SUCRA 0.163). Conclusion: Based on our meta-analysis, the two most consistent pharmacological treatments able to effectively reduce POI after abdominal surgery are prokinetics and opioid antagonists. The absence of clear superiority of one treatment over another highlights the limits of the pharmacological principles available.

Does a human fallopian tube (HFT) organoid model offer a favorable apical environment for human sperm survival and motility? The apical compartment of a new model of human fallopian tube organoids provides after differentiation a favorable environment for sperm motility. Human fallopian tubes are the site of major events that are crucial for achieving an ongoing pregnancy, such as gamete survival and competence, fertilization steps, and preimplantation embryo development. In order to better understand the tubal physiology and tubal factors involved in these reproductive functions, and to improve still suboptimal in vitro conditions for gametes preparation and embryo culture during In Vitro Fertilization IVF, we sought to develop a human fallopian tube organoid model from isolated adult stem cells allowing spermatozoa coculture in the apical compartment. Over a two-year period, fallopian tube tissues were collected for organoid culture purposes from 10 “donor” patients undergoing bilateral salpingectomy by laparoscopy for definitive sterilization. After tissue digestion, adult stem cells from the isthmus and ampulla regions were separately seeded in 3D Matrigel and cultured with conventional growth factors for organoid culture and specific factors for differentiation of the female genital tract. HFT organoids were characterized by light microscopy, scanning and transmission electron microscopy, immunofluorescence and transcriptomic analysis. Following simultaneous organoid culture on specific inserts, spermatozoa from five donors were placed either in control media or in the apical compartment of colon or HFT organoids (isthmus and ampulla separately) for 96 hours. Vitality and motility were assessed at 0, 48, and 96 hours on 200 spermatozoa in each condition and in duplicate and compared using Wilcoxon test. Specific fallopian tube differentiation of our model was confirmed by immunofluorescence, transcriptome analysis and electron microscopy observations that exhibited ciliary and secretory cells. We succeeded in releasing spermatozoa in the apical compartment of HFT organoids and in recovering them for sperm analysis. Sperm vitality values are similar in HFT organoids and in commercial sperm media. We demonstrated a superiority of HFT organoid apical compartment for sperm motility compared with other controls (colon organoids, organoid culture media, and conventional commercial sperm washing and fertilization media). At 48 hours of incubation, progressive sperm motility was higher in the apical compartment of HFT organoids (ampulla 30.6% ±17.3, isthmus 29.3% ±14.8) than in commercial fertilization media (15.3% ±14.6) (p < 0.05) and compared with all other conditions. At 96 hours progressive sperm motility was almost nil (<1%) in all conditions except for spermatozoa within HFT organoids (p < 0.05): 11.5% ±14.7 and 12.9% ±16.9 in ampulla and isthmus organoids, respectively. This was an in vitro study in which conditions of organoid culture could not exactly mimic the in vivo environment of extracellular matrix and the vascularization of fallopian tubes. This work opens up perspectives for better understanding of HFT physiology. For the first time, it highlights the possibility of developing HFT organoids for reproductive purposes. In the future, it could help us to improve gametes fertilizing abilities and culture conditions of embryos during human assisted reproductive technologies. not applicable

Background and purpose Chymotrypsin is a serine protease produced by the pancreas and secreted into the lumen of the small intestine, where it digests food proteins. Due to its presence in the gut lumen, we hypothesized that chymotrypsin activity may be found close to epithelial cells and signals to them via Protease-activated receptors (PARs). We deciphered molecular pharmacology mechanisms for chymotrypsin signaling in intestinal epithelial cells. Experimental approaches The presence and activity of chymotrypsin were evaluated by western blot (WB) and enzymatic activity tests in 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. To study the pharmacology of chymotrypsin signals, we investigated calcium signaling and ERK1/2 activation using calcium mobilization assays and WB in CMT93 intestinal epithelial cells. Key results We found that chymotrypsin was present and active in the vicinity of the murine and human colonic epithelium. Molecular pharmacology studies evidenced that chymotrypsin cleaved both PARs receptors. While chymotrypsin activated calcium and ERK1/2 signaling pathways through PAR2, it disarmed PAR1, preventing further activation by its canonical agonist thrombin. CONCLUSION Our work suggests that the function of chymotrypsin in the gut lumen goes well beyond a simple digestive role. Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.

Itch is an unpleasant sensation that evokes a desire to scratch. The skin barrier is constantly exposed to mi-crobes and their products. However, the role of microbes in itch generation is unknown. Here, we show that Staphylococcus aureus, a bacterial pathogen associated with itchy skin diseases, directly activates pruricep-tor sensory neurons to drive itch. Epicutaneous S. aureus exposure causes robust itch and scratch-induced damage. By testing multiple isogenic bacterial mutants for virulence factors, we identify the S. aureus serine protease V8 as a critical mediator in evoking spontaneous itch and alloknesis. V8 cleaves proteinase-acti-vated receptor 1 (PAR1) on mouse and human sensory neurons. Targeting PAR1 through genetic deficiency, small interfering RNA (siRNA) knockdown, or pharmacological blockade decreases itch and skin damage caused by V8 and S. aureus exposure. Thus, we identify a mechanism of action for a pruritogenic bacterial factor and demonstrate the potential of inhibiting V8-PAR1 signaling to treat itch.

Elafin and its precursor trappin-2 are known for their contribution to the physiological mucosal shield against luminal microbes. Such a contribution seems to be particularly relevant in the gut, where the exposure of host tissues to heavy loads of microbes is constant and contributes to mucosa-associated pathologies. The expression of trappin-2/elafin has been shown to be differentially regulated in diseases associated with gut inflammation. Accumulating evidence has demonstrated the protective effects of trappin-2/elafin in gut intestinal disorders associated with acute or chronic inflammation, or with gluten sensitization disorders. The protective effects of trappin-2/elafin in the gut are discussed in terms of their pleiotropic modes of action: acting as protease inhibitors, transglutaminase substrates, antimicrobial peptides or as a regulator of pro-inflammatory transcription factors. Further, the question of the therapeutic potential of trappin-2/elafin delivery at the intestinal mucosa surface is raised. Whether trappin-2/elafin mucosal delivery should be considered to ensure intestinal tissue repair is also discussed.