Dysregulated protease activity is often implicated in the initiation of inflammation and immune cell recruitment in gastrointestinal inflammatory diseases. Using N-terminomics/TAILS (terminal amine isotopic labeling of substrates), we compared proteases, along with their substrates and inhibitors, between colonic mucosal biopsies of healthy patients and those with ulcerative colitis (UC). Among the 1642 N-termini enriched using TAILS, increased endogenous processing of proteins was identified in UC compared to healthy patients. Changes in the reactome pathways for proteins associated with metabolism, adherens junction proteins (E-cadherin, liver-intestinal cadherin, catenin alpha-1, and catenin delta-1), and neutrophil degranulation were identified between the two groups. Increased neutrophil infiltration and distinct proteases observed in ulcerative colitis may result in extensive break down, altered processing, or increased remodeling of adherens junctions and other cellular functions. Analysis of the preferred proteolytic cleavage sites indicated that the majority of proteolytic activity and processing comes from host proteases, but that key microbial proteases may also play a role in maintaining homeostasis. Thus, the identification of distinct proteases and processing of their substrates improves the understanding of dysregulated proteolysis in normal intestinal physiology and ulcerative colitis.P roteases are implicated in key functions during gastrointestinal (GI) homeostasis, and dysregulated proteolysis is one of the contributing factors of gastrointestinal inflammatory diseases. 1−3 Upon recruitment to the site of inflammation, activated immune cells produce various intraand extra-cellular proteases including neutrophil elastase (NE), matriptase, cathepsins, caspases, and matrix metalloproteinases, which all play roles in modulating the host response. 4−7 The shift in the balance between regulated and dysregulated proteolysis in GI inflammation is not fully understood, and the full repertoire of proteases, their substrates, and their inhibitors has not been examined in detail. Transcript analyses have been performed on samples from patients with inflammatory bowel diseases, 8 but mRNA levels often do not correlate with protein levels. 9 Furthermore, transcriptomics does not assess whether a substrate is cleaved or not by a

Background Fibrocytes are hematopoietic cells with features of mesenchymal cells found in the circulation and inflammatory sites implicated in promoting fibrosis in many fibroinflammatory diseases. However, their role(s) in the development of intestinal fibrosis is poorly understood. Here, we investigated a potential role of fibrocytes in the development of fibrosis in Crohn’s disease (CD) and sought factors that may impact their development and function. Methods Plasma and mononuclear cells were collected from patients with and without fibrostenotic CD. Fibrocytes defined as CD11b+, CD34+, and Collagen 1+ were correlated with clinical assessments of fibrosis, including evaluation using intestinal ultrasound. We measured the levels of relevant circulating molecules via Luminex and studied the effect of patient plasma proteins on fibrocyte differentiation. Results Fibrocyte numbers were increased in CD patients with stricturing Crohn’s disease compared with patients with an inflammatory phenotype (P = .0013), with strong correlation between fibrocyte numbers and acoustic radiation force impulse (ARFI), a measure of bowel elasticity on intestinal ultrasound (R = .8383, P = .0127). Fibrostenotic plasma was a more potent inducer of fibrocyte differentiation in both primary human monocytes and cell line and contained increased levels of cytokines implicated in fibrocyte differentiation compared with plasma from inflammatory patients. Interestingly, increased fibrocyte numbers at time of ultrasound were associated with escalation of medical therapy and endoscopic/surgical management of small bowel strictures at 30 months follow-up. Conclusions Circulating fibrocytes strongly correlate with fibrostenotic disease in CD, and they may serve as predictors for escalation of medical +/- surgical therapy.

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Matrix metalloproteinases (MMPs) modify bioactive factors via selective processing or degradation resulting in tumour-promoting or tumour-suppressive effects, such as those by MMP8 in various cancers. We mapped the substrates of MMP8 to elucidate its previously shown tumour-protective role in oral tongue squamous cell carcinoma (OTSCC). MMP8 overexpressing (+) HSC-3 cells, previously demonstrated to have reduced migration and invasion, showed enhanced cell-cell adhesion. By analysing the secretomes of MMP8 + and control cells with terminal amine isotopic labelling of substrates (TAILS) coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS), we identified 36 potential substrates of MMP8, including FXYD domain-containing ion transport regulator 5 (FXYD5). An anti-adhesive glycoprotein FXYD5 has been previously shown to predict poor survival in OTSCC. Cleavage of FXYD5 by MMP8 was confirmed using recombinant proteins. Furthermore, we detected a loss of FXYD5 levels on cell membrane of MMP8 + cells, which was rescued by inhibition of the proteolytic activity of MMP8. Silencing (si) FXYD5 increased the cell-cell adhesion of control but not that of MMP8 + cells. siFXYD5 diminished the viability and motility of HSC-3 cells independent of MMP8 and similar effects were seen in another tongue cancer cell line, SCC-25. FXYD5 is a novel substrate of MMP8 and reducing FXYD5 levels either with siRNA or cleavage by MMP8 increases cell adhesion leading to reduced motility. FXYD5 being a known prognostic factor in OTSCC, our findings strengthen its potential as a therapeutic target.