Fecal Protease Research Articles

Detecting Colorectal Neoplasia Using Specific Fecal Fluorogenic Protease-Sensitive Substrates: A Pilot Study.

identification and removal of advanced adenomas (AA) reduce colorectal cancer (CRC) incidence and potentially mortality. CRC screening often uses fecal immunochemical testing to select high-risk individuals for colonoscopy, despite its low sensitivity for AA and relatively high false-positivity rate. Previous studies have linked proteases to CRC development through their ability to facilitate angiogenesis and immunoregulation. This study aims to identify colorectal neoplasia-associated proteases and their substrates as a potential noninvasive screening test, introducing an innovative application of fecal protease profiling, which has previously been limited to tissue samples. eighteen fluorogenic substrates were designed based on literature. Proteolytic degradation of these substrates was measured in fecal samples of patients with CRC (n = 12), AA (n = 9), nonadvanced adenomas (n = 10), and controls (n = 14). Substrate degradation was correlated to a matched human proteome data set, and underlying proteases were identified based on their recognition patterns. Experiments with protease inhibitors and ZnCl2 were performed to further characterize the involved proteases. in total, 7 of the 18 substrates tested showed a significantly decreased proteolytic degradation in feces from patients with any colorectal neoplasia compared to the control group. The l-aspartic acid-l-glutamic acid substrate (ED) showed significantly decreased degradation in AA and CRC patients. ED degradation significantly decreased with the addition of ZnCl2 and the cysteine protease inhibitor NEM. we successfully developed colorectal neoplasia-specific fluorogenic substrates, highlighting the ED substrate as a potential substrate for the detection of AA and CRC. Although the responsible proteases require further identification, our results suggest an association with calcium-dependent cysteine proteases.

Effects of a Protease Inhibitor Camostat Mesilate on Gut Microbial Function in Patients with Irritable Bowel Syndrome: A Pilot Randomized Placebo-Controlled Study

Introduction: Increased fecal protease activity, which may induce visceral hypersensitivity, has been observed in patients with irritable bowel syndrome (IBS). Serine proteases modulate FK506 binding protein (FKBP)-type peptidylprolyl cis-trans isomerase (PPIase) activity associated with immune and glucocorticoid receptor functions. The aim was to investigate whether camostat mesilate (CM), a serine protease inhibitor, modifies fecal bacterial function related to FKBP-type PPIases in patients with IBS. Methods: Randomly assigned 16 patients with IBS received 200 mg po tid of CM and 16 patients received placebo for 14 days. Self-reported adequate relief (AR) as a primary endpoint, IBS Symptom Severity Scale (IBS-SSS), and colonic motor and pain thresholds to colorectal distention were assessed before and after treatment. The fecal bacterial content was inferred from 16S rRNA gene sequence data using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States and the Kyoto Encyclopedia of Genes and Genomes database. Results: CM significantly increased the relative abundance of Streptococcus and the functional abundances of serine protease and FKBP-type PPIase FkpA, FklB and SlyD more than placebo after treatment. CM treatment was not superior to placebo in proportion of AR although colonic motor response partially changed. Conclusion: CM modulated the fecal microbiome composition and functional potentials that are related to FKBP-type PPIase activity in IBS patients. These findings suggest that protease inhibitors may modify gut microbial function along with abnormal immunological and/or stress responses that underlie pathophysiology of IBS.

Industrial diet intervention modulates the interplay between gut microbiota and host in semi-stray dogs

BackgroundThe gut microbiota and derived metabolites play a key role in regulating host physiology. Diet is identified as a key regulatory factor of the microbiota composition and, potentially, of subsequent functionalities. Demonstrating the role of diet may be complex as most human studies are cross-sectional and dietary intervention is often accompanied by hygienic changes. The objective of the present study was to investigate the impact of an industrial diet on the modulation of the microbiota and targeted functionalities using a canine “natural” model.ResultsWe carried out a controlled dietary trial in a cohort of Tunisian semi-stray dogs. We made a transition from a natural diet to an industrial kibble diet and monitored the composition of the fecal microbiota, the concentration of short-chain fatty acids (SCFA) and bile acids (BAs), and protease activities. We demonstrated that dietary change significantly decreased fecal primary bile acids levels and protease activities. Interestingly, correlation analyses demonstrated that variation of specific microbial genera were associated with modulated physiological parameters.ConclusionsOur study reveals that an industrial diet induces beneficial changes in microbial composition and functions characterised by increased diversity, synthesis of SCFA and secondary bile acids production, stressing the key role of the diet-microbiota-dog crosstalk.

A14 ABLATION OF THE INTESTINAL EPITHELIAL SIALOME PREDISPOSES TO SPONTANEOUS COLITIS AND COLITIS-ASSOCIATED TUMORIGENESIS VIA MUCUS-DEPENDENT AND -INDEPENDENT MECHANISMS IN MICE

Abstract Background Recent work suggests epithelial sialylation (capping of glycoconjugates with the acidic sugar sialic acid) can protect the gut mucosa from inflammatory bowel disease by promoting mucus function. However, these studies are based on partial sialylation knockouts, potentially underestimating the role of glycoconjugate sialylation in mucosal homeostasis. Aims We sought to uncover how the complete intestinal epithelial sialome (the total glycan sialylation repetoire) regulates colitis susceptibility in vivo. Methods We generated a model of total gut epithelial sialylation deficiency by conditionally and inducibly (via tamoxifen, TM) targeting the CMP-Sia transporter, Slc35a1, in epithelial cells using Cre-Lox technology (Slc35a1f/f; VillinCre(ERT2) or (TM-)IEC Slc35a1-/- mice). We analyzed phenotypes clinically, histologicaly, and via multi-omic apporaches at baseline and in response to challenge. Results We confirmed the ablation of mucus sialylation via lectin staining, epithelial Slc35a1 gene expression, and O-glycomics of extracted mucins. In contrast to partial sialylation deficiencies, we found that IEC Slc35a1-/- mice developed mild spontaneous microbiota-dependent chronic colitis that was associated with tumorigenesis in the rectum in 30% of aged mice (>12 mo.). TM-IEC Slc35a1-/- mice developed less severe spontaneous colitis, but were susceptible to acute injury with 1% DSS. Ablation of the gut epithelial sialome led to reduced mucus thickness on fecal sections and within tissues, although the b1/b2 barrier layers were still intact. TM-IEC Slc35a1-/- mice showed altered microbiota composition with an increase in Clostridium spp. at the expense of a global reduction in the abundance of at least 20 unique taxa; however metabolomic analysis did not show any significant differences in short-chain fatty acid levels. IEC Slc35a1-/- Muc2 was more susceptible to fecal protease degradation vs.WT Muc2. While no spontaneous phenotype was observed in the small intestine (SI), treatment with 5-fluorouracil led to worsened SI mucositis and morbidity vs. WT littermates. This was traced to impaired stem cell activities via reduced Lgr5+ cell representation in small intestinal crypts in IEC Slc35a1-/-;Lgr5GFP mice. Conclusions Ablation of the intestinal epithelial sialome impacts the mucus-microbiota-stem cell axis with consequences on colonic mucosal homeostasis based on the type of injury. At baseline, loss of sialylation impairs mucus stability and the stem cell niche leading to microbiota-dependent spontaneous colitis and tumorigenesis. However, these same epithelial defects impair epithelial renewal and worsen colitis following acute injury likely due to the cytotoxic environment caused by the chemical agent. Funding Agencies CCC

Abstract PO-023: Impaired adipose anabolism drives fat wasting in pancreatic cancer cachexia

Abstract Purpose/Objectives: The disease associated wasting condition, cachexia, is a common complication of pancreatic ductal adenocarcinoma (PDAC) that impacts quality of life and portends poor survival. Cachexia remains refractory to nutritional supplementation, but the mechanisms underlying this phenotype are unclear. By examining the adipose response to different nutritional contexts in cachectic mice, we sought to understand the relative contributions of enhanced catabolism and impaired anabolism on adipose tissue wasting. Materials/Methods: Adult C57BL/6J mice received orthotopic PDAC tumor injections (KrasG12D; p53R172H/+; Pdx1-cre) or sham injections. Mice were fasted 24 h or fasted and refed 24 h at mid-cachexia time point, and gross fat pads were weighed to assess anabolic potential. 3T3-L1 cells were treated with PDAC cell conditioned media during or after differentiation to model cachexia in vitro. Lipolytic rate was quantified as normalized quantity of glycerol released from inguinal (iWAT) and gonadal (gWAT) fat pads ex vivo or differentiated 3T3-L1 cells in vitro. Lipogenesis was measured in vitro using Oil-red-O staining. Adipose tissue mRNA levels were measured using bulk RNAseq and confirmed with qPCR. Malabsorption was assessed via fecal protease activity and total fecal protein and lipid content. Results: Adipose tissue in PDAC and control mice were equally sensitive to fasting/food restriction. Both iWAT and gWAT fat pads from PDAC mice showed decreased lipolysis ex vivo, associated with decreased lipase (Atgl & Lipe) mRNA expression. In the fast/refeed paradigm, PDAC mice were unable to restore both iWAT and gWAT mass after refeed, in contrast to control mice. We found no evidence of malabsorption at this timepoint by any measure. RNAseq on gWAT revealed 614 differentially expressed genes between PDAC and control mice. Downregulated (n=111) genes were most closely associated with adipogenesis (adj p<.05), and expression of adipogenesis master regulators Pparg and Cebpa reduced in WAT from PDAC mice. Upregulated genes were associated with increased RELA and NFkB activity (adj p<5 × 10−22) and inflammatory response hallmarks. Treatment of 3T3-L1 cells with PDAC conditioned media phenocopied in vivo model, showing impaired lipolysis and lipogenesis, associated with decreased expression of lipolytic, adipogenic, and lipogenic genes. Conclusions: Adipose tissue wasting in PDAC cachexia can result from impaired anabolism in the absence of enhanced lipolysis or malabsorption. This deficit in adipogenesis is mediated by a cancer cell secreted product and is associated with increased inflammatory signaling in adipocytes, most likely via RELA and NFkB activity. Restoring adipose anabolic potential may provide a novel approach to cachexia treatment in PDAC. Citation Format: Katherine Pelz, Grace McCarthy, Heike Mendez, Samantha Z. Brown, Jonathan R. Brody, Aaron J. Grossberg. Impaired adipose anabolism drives fat wasting in pancreatic cancer cachexia [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-023.

Use of an emollient-containing diaper and pH-buffered wipe regimen restores skin pH and reduces residual enzymatic activity.

Background/ObjectivesDiaper dermatitis is one of the most frequent skin conditions affecting infants and is associated with elevated skin pH, exposure to urine and feces, and increased fecal protease and lipase activity, resulting in stratum corneum barrier damage and increased risk of infection. The study aim was to determine the impact of two diaper and wipe regimens on newborn infant skin pH and residual enzyme activity after stool cleaning.MethodsTwo diaper and wipe regimens were compared in a randomized, single‐blinded crossover study. Regimen A paired an emollient‐containing diaper with an acidic, pH‐buffered wipe. Regimen B was a non‐emollient diaper and wipe with limited buffering capacity. A 3‐day washout period preceded each 3‐day regimen use period. Skin pH at the perianal/buttocks interface (PBI), genital region, and undiapered chest control were measured at baseline and day 3. Skin swabs were collected for residual enzyme activity after a stool cleaning event.ResultsDiapered skin pH at the PBI was similar to undiapered skin after 3 days of use for Regimen A, while PBI pH for Regimen B was elevated versus control. PBI pH was lower for Regimen A versus Regimen B. After a stool cleaning, PBI skin pH for Regimen A was lower immediately and had lower residual enzyme activity versus Regimen B (P < .05), and the pH‐lowering effect was sustained up to 60 minutes.ConclusionsThese results suggest that the use of an emollient‐containing diaper with a pH‐buffered wipe creates conditions favorable to optimum diapered skin health.

Fecal Serine Protease Profiling in Inflammatory Bowel Diseases.

Serine proteases are extensively known to play key roles in many physiological processes. However, their dysregulation is often associated to several diseases including inflammatory bowel diseases (IBD). Here, we used specific substrates to monitor fecal protease activities in a large cohort of healthy and IBD patients. Of interest, serine protease activity was 10-fold higher in IBD fecal samples compared to healthy controls. Moreover, functional analysis of these fecal proteolytic activities revealed that the most increased activities are trypsin-like, elastase-like and cathepsin G-like. We also show for the first time, an increase of proteinase 3-like activity in these samples compared to controls. Results presented here will guide further investigations to better understand the relevance of these peptidases in IBD.

The Functional Role of Fecal Microbiota Transplantation on Dextran Sulfate Sodium-Induced Colitis in Mice.

Increasingly studies revealed that dysbiosis of gut microbiota plays a pivotal role in the pathogenesis of ulcerative colitis (UC). Fecal microbiota transplantation (FMT) has drawn more and more attention and become an important therapeutic approach. This study aims to examine the facts about the effective components and look into potential mechanisms of FMT. Colitis was induced by 3% (w/v) dextran sulfate sodium (DSS) in drinking water for 7 days. Colitis mice were administered by oral gavage with fecal suspension, fecal supernatant, fecal bacteria, or boiling-killed fecal bacteria from healthy controls and the disease activity index was monitored daily. On the seventh day, mice were euthanized. The length, histological score, parameters related to inflammation, gut barrier functions of the colon, activities of digestive protease and β-glucuronidase in feces were measured. All of the four fecal components showed certain degree of efficacy in DSS-induced colitis, while transplantation of fecal suspension showed the most potent effect as demonstrated by less body weight loss, lower disease activity scores, more expression of tight junction proteins and TRAF6 and IκBα, less expression of TNF-α, IL-1β, IL-10, TLR-4, and MyD88 in gut tissue, as well as restoration of fecal β-glucuronidase and decreases in fecal digestive proteases. These results provide a novel insight into the possible mechanism of FMT and may help to improve and optimize clinical use of FMT.

Soy isoflavones and cholecalciferol reduce inflammation, and gut permeability, without any effect on antioxidant capacity in irritable bowel syndrome: A randomized clinical trial

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that is more prevalent in women. Vitamin D deficiency and hormonal disorders are also prevalent in Iranian women, and may influence the severity of clinical outcomes mediated by microinflammation, oxidative stress and intestinal permeability pathways. Our objective was to investigate the effects of co-administration of soy and vitamin D on some inflammatory, antioxidant and gut permeability markers in women with IBS. In a randomized clinical trial, women (18-75 years of age) were randomly allocated into four groups to receive soy isoflavones (40mg/day), cholecalciferol (50,000 IU/15 days), both soy isoflavones and cholecalciferol, or placebo for six weeks. The outcomes were plasma inflammatory markers, antioxidant status and fecal protease activity at week 0 and week 6. After the intervention, plasma inflammatory markers and fecal protease activity were reduced significantly in all treatment groups compared to the placebo group; however, there was no significant effect on antioxidant status. This study suggests combined supplementation of soy isoflavones and active vitamin D can improve some biochemical parameters regarding inflammation and intestinal permeability of IBS in women. Clinical.Trials.govNCT02026518.

Colonic mast cell numbers, symptom profile, and mucosal expression of elements of the epithelial barrier in irritable bowel syndrome.

This study aimed to determine whether patients with IBS displayed altered mucosal mast cell (MC) numbers and proportions of MCs co-localizing with nerves compared with healthy subjects (HS) and whether these MC characteristics correlated with IBS symptoms, elements of the epithelial barrier, or visceral sensitivity. Mucosal MC characteristics were determined using immunoassay. IBS symptoms, gene expression of elements of the epithelial barrier, fecal serine protease activity, and visceral sensitivity were assessed. The MC numbers per mm2 were 2.0 (0.0-6.0) in patients with IBS (n=43) and 3.5 (1.1-9.1) in HS (n=20, P=.26). Of these, MCs were 0.0 (0.0-20) % vs 3.1 (0.0-18) % (P=.76) in IBS and HS, respectively, in co-localization with nerve fibers. MC characteristics were equivalent in the different IBS subtypes. Hierarchical cluster analysis identified two distinct groups among patients with IBS: MC high (higher MC numbers and proportions of MCs co-localizing with nerves) and MC low (lower MC numbers and proportions of MCs co-localizing with nerves). The MC high and MC low groups could not be discriminated with regard to IBS symptoms, parameters of visceral sensitivity, gene expression of elements of the epithelial barrier, and fecal protease activity. There was no evidence of increased infiltration or altered localization of MCs in the colonic mucosa of patients with IBS. These MC characteristics were not linked to global IBS symptoms or mucosal expression of elements of the epithelial barrier. These findings indicate that quantity and location of mucosal MCs are factors not involved in the pathophysiology of IBS.

Faecal Proteases from Pouchitis Patients Activate Protease Activating Receptor-2 to Disrupt the Epithelial Barrier.

The pathogenesis of pouch inflammation may involve epithelial barrier disruption. We investigated whether faecal proteolytic activity is increased during pouchitis and results in epithelial barrier dysfunction through protease activating receptor [PAR] activation, and assessed whether the intestinal microbiome may be the source of the proteases. Faecal samples were measured for protease activity using a fluorescein isothiocyanate [FITC]-casein florescence assay. Caco-2 cell monolayers were exposed to faecal supernatants to assess permeability to FITC-dextran. Tight junction protein integrity and PAR activation were assessed by immunoblot and immunofluorescence. A truncated PAR2 protein in Caco-2 cells was achieved by stable transfection using CRISPR/Cas9 plasmid. PAR2 activation in pouch biopsies was examined using antibodies directed to the N-terminus of the protein. Microbial composition was analysed based on 16S rRNA gene sequence analysis. Ten pouchitis patients, six normal pouch [NP] patients and nine healthy controls [HC] were recruited. The pouchitis patients exhibited a 5.19- and 5.35-fold higher faecal protease [FP] activity [p ≤ 0.05] compared to the NP and HC participants, respectively. The genus Haemophilus was positively associated with FP activity [R = 0.718, false discovery rate < 0.1]. Faecal supernatants from pouchitis patients activated PAR2 on Caco-2 monolayers, disrupted tight junction proteins and increased epithelial permeability. PAR2 truncation in Caco-2 abrogated faecal protease-mediated permeability. Pouch biopsies obtained from pouchitis patients, but not from NP patients, displayed PAR2 activation. Protease-producing bacteria may increase faecal proteolytic activity that results in pouch inflammation through disruption of tight junction proteins and increased epithelial permeability in a PAR2-dependent manner. This mechanism may initiate or propagate pouch inflammation.

Bacterial β-glucuronidase alleviates dextran sulfate sodium-induced colitis in mice: A possible crucial new diagnostic and therapeutic target for inflammatory bowel disease

ObjectiveInflammatory bowel diseases (IBD) including ulcerative colitis and Crohn's disease are devastating diseases of the gut. At present, all the treatments are mainly targeting symptoms like inflammation. The disease remains regarded as incurable, largely due to lacking of knowledge on its etiology. Our previous studies suggested that impaired inactivation of digestive proteases by deconjugated bilirubin in experimental colitis, thus bacterial β-glucuronidase for catalyzing the reaction, may have played critical role in the pathogenesis of IBD. MethodsWe first analyzed β-glucuronidase activity in gut tissue and feces of mice by a colitis model. Then the effect of β-glucuronidase on experimental colitis was investigated in detail by administration of β-glucuronidase (from E. coli) and fecal material transplantation to mice with 3% DSS in drinking water for 7 days. ResultsMice with colitis showed unchanged activity of β-glucuronidase in colon tissue but decreased activity in feces. Treatment with bacterial β-glucuronidase at 100 U or above alleviated DSS-induced colitis as demonstrated by the less body weight loss, less disease activity score, increased expression of tight junction proteins and decreased gut permeability, decreases in MPO, TNF-α, IL-1β, TLR-4 and MyD88, and increase in IL-10 and IκBα in gut, restored fecal β-glucuronidase and gut microbiota along with decreases in fecal digestive proteases. Transplantation of fecal material from control to colitis mice showed similar effects as treatment with β-glucuronidase. ConclusionsBacterial β-glucuronidase showed strong inhibition on colitis along with the reduction in fecal digestive proteases, which may be a crucial diagnostic and therapeutic target for IBD.

Serine proteases as luminal mediators of intestinal barrier dysfunction and symptom severity in IBS

ObjectiveThe intestinal lumen contains several proteases. Our aim was to determine the role of faecal proteases in mediating barrier dysfunction and symptoms in IBS.Design39 patients with IBS and 25 healthy...

P007 Faecal protease activity as a predictor marker of disease recurrence in patients with Crohn's disease following ileocecectomy

P007 Faecal protease activity as a predictor marker of disease recurrence in patients with Crohn's disease following ileocecectomy

Protease Activated Receptor-2 Induces Immune Activation and Visceral Hypersensitivity in Post-infectious Irritable Bowel Syndrome Mice.

The role of protease activated receptor-2 (PAR-2) in the pathogenesis of abdominal pain in irritable bowel syndrome (IBS) is not well defined. To investigate the role of PAR-2-mediated visceral hypersensitivity in a post-infectious IBS (PI-IBS) mouse model. T. spiralis-infected PI-IBS mouse model was used. Fecal serine protease activity and intestinal mast cells were evaluated. Intestinal permeability was assessed by urine lactulose/mannitol ratio, and colonic expressions of PAR-2 and tight junction (TJ) proteins were examined by Western blot. Intestinal immune profile was assessed by measuring Th (T helper) 1/Th2 cytokine expression. Visceral sensitivity was evaluated by abdominal withdrawal reflex in response to colorectal distention. Colonic PAR-2 expression as well as fecal serine protease activity and intestinal mast cell counts were elevated in PI-IBS compared to the control mice. Decreased colonic TJ proteins expression, increased lactulose/mannitol ratio, elevated colonic Th1/Th2 cytokine ratio, and visceral hypersensitivity were observed in PI-IBS compared to the control mice. Administration of PAR-2 agonist in control mice demonstrated similar changes observed in PI-IBS mice, while PAR-2 antagonist normalized the increased intestinal permeability and reduced visceral hypersensitivity observed in PI-IBS mice. PAR-2 activation increases intestinal permeability leading to immune activation and visceral hypersensitivity in PI-IBS mouse model.

Increased Pancreatic Protease Activity in Response to Antibiotics Impairs Gut Barrier and Triggers Colitis.

Background & AimsAntibiotic (ABx) therapy is associated with increased risk for Crohn’s disease but underlying mechanisms are unknown. We observed high fecal serine protease activity (PA) to be a frequent side effect of ABx therapy. The aim of the present study was to unravel whether this rise in large intestinal PA may promote colitis development via detrimental effects on the large intestinal barrier.MethodsTranswell experiments were used to assess the impact of high PA in ABx-treated patients or vancomycin/metronidazole-treated mice on the epithelial barrier. Serine protease profiling was performed using liquid chromatography–mass spectrometry/mass spectrometry analysis. The impact of high large intestinal PA on the intestinal barrier in wild-type and interleukin (IL)10-/- mice and on colitis development in IL10-/- mice was investigated using vancomycin/metronidazole with or without oral serine protease inhibitor (AEBSF) treatment.ResultsThe ABx-induced, high large intestinal PA was caused by significantly increased levels of pancreatic proteases and impaired epithelial barrier integrity. In wild-type mice, the rise in PA caused a transient increase in intestinal permeability but did not affect susceptibility to chemically induced acute colitis. In IL10-/- mice, increased PA caused a consistent impairment of the intestinal barrier associated with inflammatory activation in the large intestinal tissue. In the long term, the vancomycin/metronidazole-induced lasting increase in PA aggravated colitis development in IL10-/- mice.ConclusionsHigh large intestinal PA is a frequent adverse effect of ABx therapy, which is detrimental to the large intestinal barrier and may contribute to the development of chronic intestinal inflammation in susceptible individuals.

Elevated fecal peptidase D at onset of colitis in Galphai2-/- mice, a mouse model of IBD.

BackgroundThe identification of novel fecal biomarkers in inflammatory bowel disease (IBD) is hampered by the complexity of the human fecal proteome. On the other hand, in experimental mouse models there is probably less variation. We investigated the fecal protein content in mice to identify possible biomarkers and pathogenic mechanisms.MethodsFecal samples were collected at onset of inflammation in Galphai2-/- mice, a well-described spontaneous model of chronic colitis, and from healthy littermates. The fecal proteome was analyzed by two-dimensional electrophoresis and quantitative mass spectrometry and results were then validated in a new cohort of mice.ResultsAs a potential top marker of disease, peptidase D was found at a higher ratio in Galphai2-/- mouse feces relative to controls (fold change 27; p = 0.019). Other proteins found to be enriched in Gαi2-/- mice were mainly pancreatic proteases, and proteins from plasma and blood cells. A tendency of increased calprotectin, subunit S100-A8, was also observed (fold change 21; p = 0.058). Proteases are potential activators of inflammation in the gastrointestinal tract through their interaction with the proteinase-activated receptor 2 (PAR2). Accordingly, the level of PAR2 was found to be elevated in both the colon and the pancreas of Galphai2-/- mice at different stages of disease.ConclusionsThese findings identify peptidase D, an ubiquitously expressed intracellular peptidase, as a potential novel marker of colitis. The elevated levels of fecal proteases may be involved in the pathogenesis of colitis and contribute to the clinical phenotype, possibly by activation of intestinal PAR2.

Unconjugated bilirubin ameliorates the inflammation and digestive protease increase in TNBS-induced colitis.

The authors previously demonstrated that unconjugated bilirubin (UCB) may inhibit the activities of various digestive proteases, including trypsin and chymotrypsin. The digestive proteases in the lower gut are important in the pathogenesis of inflammatory bowel diseases. The effects of UCB on the inflammation and levels of digestive proteases in feces of rats with colitis have not yet been revealed. The present study investigated the effect of UCB on the inflammatory status and levels of trypsin and chymotrypsin in the feces of rats with trinitrobenzenesulfonic acid (TNBS)-induced colitis. The data indicated that treatment with TNBS resulted in a marked reduction in weight gain, which was significantly alleviated in UCB-treated rats. Furthermore, UCB treatment alleviated the inflammation induced by TNBS, detected via macroscopic damage and microscopic inflammation scores, and pro-inflammatory markers including myeloperoxidase (MPO), tumor necrosis factor (TNF)-α and interleukin (IL)-1β. Furthermore, rats with colitis demonstrated significant increases in fecal trypsin and chymotrypsin levels, whereas UCB treatment significantly alleviated these increases. A significant positive correlation was additionally revealed among the pro-inflammatory markers (MPO, TNF-α and IL-1β) and fecal digestive proteases (trypsin and chymotrypsin) in colitis. The results of the present study demonstrated that UCB ameliorated the inflammation and digestive protease increase in TNBS-induced colitis.

Siropins, novel serine protease inhibitors from gut microbiota acting on human proteases involved in inflammatory bowel diseases.

BackgroundIn eukaryotes, the serpins constitute a wide family of protease inhibitors regulating many physiological pathways. Many reports stressed the key role of serpins in several human physiopathologies including mainly the inflammatory bowel diseases. In this context, eukaryotic serpins were largely studied and their use to limit inflammation was reported. In comparison to that, bacterial serpins and mainly those from human gut microbiota remain poorly studied.ResultsThe two genes encoding for putative serpins from the human gut bacterium Eubacterium sireaum, display low sequence identities. These genes were overexpressed and the encoded proteins, named Siropins, were purified. Activity studies demonstrated that both purified proteins inhibited serine proteases but surprisingly they preferentially inhibited two human serine proteases (Human Neutrophil Elastase and Proteinase3). The biochemical characterization of these Siropins revealed that Siropin 1 was the most active and stable at low pH values while Siropin 2 was more thermoactive and thermostable. Kinetic analysis allowed the determination of the stoichiometry of inhibition (SI) which was around 1 and of the association rate constants of 7.7 × 104 for the Human Neutrophil Elastase and 2.6 × 105 for the Proteinase3. Moreover, both Siropins displayed the ability to inhibit proteases usually present in fecal waters. Altogether our data indicate the high efficiency of Siropins and their probable involvement in the control of the overall intestine protease activity.ConclusionsHere we report the purification and the biochemical characterization of two novel serpins originated from Eubacterium sireaum, a human gastro-intestinal tract commensal bacteria. These proteins that we called Siropins, efficiently inhibited two human proteases reported to be associated with inflammatory bowel diseases. The determination of the biochemical properties of these enzymes revealed different temperature and pH behaviours that may reflect adaptation of this human commensal bacterium to different ecological environments. To the best of our knowledge, it is the first bacterial serpins showing an attractive inhibition of fecal proteases recovered from a mice group with chemically induced inflammation. Altogether our data highlight the interesting potential of Siropins, and serpins from the human gut microbiota in general, to be used as new alternative to face inflammatory diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0596-2) contains supplementary material, which is available to authorized users.

Testing in Microbiome-Profiling Studies with MiRKAT, the Microbiome Regression-Based Kernel Association Test

High-throughput sequencing technology has enabled population-based studies of the role of the human microbiome in disease etiology and exposure response. Distance-based analysis is a popular strategy for evaluating the overall association between microbiome diversity and outcome, wherein the phylogenetic distance between individuals' microbiome profiles is computed and tested for association via permutation. Despite their practical popularity, distance-based approaches suffer from important challenges, especially in selecting the best distance and extending the methods to alternative outcomes, such as survival outcomes. We propose the microbiome regression-based kernel association test (MiRKAT), which directly regresses the outcome on the microbiome profiles via the semi-parametric kernel machine regression framework. MiRKAT allows for easy covariate adjustment and extension to alternative outcomes while non-parametrically modeling the microbiome through a kernel that incorporates phylogenetic distance. It uses a variance-component score statistic to test for the association with analytical p value calculation. The model also allows simultaneous examination of multiple distances, alleviating the problem of choosing the best distance. Our simulations demonstrated that MiRKAT provides correctly controlled type I error and adequate power in detecting overall association. "Optimal" MiRKAT, which considers multiple candidate distances, is robust in that it suffers from little power loss in comparison to when the best distance is used and can achieve tremendous power gain in comparison to when a poor distance is chosen. Finally, we applied MiRKAT to real microbiome datasets to show that microbial communities are associated with smoking and with fecal protease levels after confounders are controlled for.