Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies

Tue Bjerg Bennike; Thomas Gelsing Carlsen; Torkell Ellingsen; Ole Kristian Bonderup; Henning Glerup; Martin Bøgsted; Gunna Christiansen; Svend Birkelund; Allan Stensballe; Vibeke Andersen

doi:10.1097/MIB.0000000000000460

Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies

Bennike, Tue Bjerg; Carlsen, Thomas Gelsing; Ellingsen, Torkell; Bonderup, Ole Kristian; Glerup, Henning; Bøgsted, Martin; Christiansen, Gunna; Birkelund, Svend; Stensballe, Allan; Andersen, Vibeke 2015-05-19 00:00:00 ORIGINAL ARTICLE Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies ,†,‡ § Tue Bjerg Bennike, PhD,* Thomas Gelsing Carlsen, MS,* Torkell Ellingsen, MD, PhD, k,¶ k ,†† Ole Kristian Bonderup, MD, PhD, Henning Glerup, MD, PhD, Martin Bøgsted, PhD,** ‡‡ Gunna Christiansen, MD, DMSc, Svend Birkelund, MD, PhD, DMSc,* Allan Stensballe, PhD,* †,‡,§§ and Vibeke Andersen, MD, PhD Background: The etiology of the inﬂammatory bowel diseases, including ulcerative colitis (UC), remains incompletely explained. We hypothesized that an analysis of the UC colon proteome could reveal novel insights into the disease etiology. Methods: Mucosal colon biopsies were taken by endoscopy from noninﬂamed tissue of 10 patients with UC and 10 controls. The biopsies were either snap-frozen for protein analysis or prepared for histology. The protein content of the biopsies was characterized by high-throughput gel-free quantitative proteomics, and biopsy histology was analyzed by light microscopy and confocal microscopy. Results: We identiﬁed and quantiﬁed 5711 different proteins with proteomics. The abundance of the proteins calprotectin and lactotransferrin in the tissue correlated with the degree of tissue inﬂammation as determined by histology. However, fecal calprotectin did not correlate. Forty-six proteins were measured with a statistically signiﬁcant differences in abundances between the UC colon tissue and controls. Eleven of the proteins with increased abundances in the UC biopsies were associated with neutrophils and neutrophil extracellular traps. The ﬁndings were validated by microscopy, where an increased abundance of neutrophils and the presence of neutrophil extracellular traps by extracellular DNA present in the UC colon tissue were conﬁrmed. Conclusions: Neutrophils, induced neutrophil extracellular traps, and several proteins that play a part in innate immunity are all increased in abundance in the morphologically normal colon mucosa from patients with UC. The increased abundance of these antimicrobial compounds points to the stimulation of the innate immune system in the etiology of UC. (Inﬂamm Bowel Dis 2015;21:2052–2067) Key Words: ulcerative colitis, neutrophil extracellular traps, inﬂammatory bowel diseases, proteomics, microscopy lcerative colitis (UC), 1 of the 2 major forms of inﬂamma- society due to lost labor and expenses to the health care sys- 2–4 U tory bowel diseases (IBDs), is an important health problem. tem. The etiology of IBD remains incompletely explained but The incidence rate varies between the geographic regions, and in involves genetic and environmental factors. Genome-wide asso- Europe, annual incidence rates between 0.6 and 24.3 per 10 ciation studies have reported 133 loci to be associated with UC, inhabitants and prevalences rates between 4.9 and 505 per 10 many of which are associated with defects in the immune sys- inhabitants have been reported. Ulcerative colitis has a great tem. Current knowledge supports that UC is caused by an inap- impact on the quality of life of the affected individuals and for propriate immune response to the commensal microorganisms Received for publication February 26, 2015; Accepted March 27, 2015. From the *Department of Health Science and Technology, Aalborg University, Aalborg, Denmark; Organ Center, Hospital of Southern Jutland, Aabenraa, Denmark; ‡ § Institute of Regional Health Research-Center Soenderjylland, University of Southern Denmark, Odense, Denmark; Department of Rheumatology, Odense University Hospital, k ¶ Odense, Denmark; Diagnostic Center, Section of Gastroenterology, Regional Hospital Silkeborg, Silkeborg, Denmark; University Research Clinic for Innovative Patient †† Pathways, Aarhus University, Aarhus, Denmark; **Department of Clinical Medicine, Aalborg University, Aalborg, Denmark; Department of Haematology, Aalborg ‡‡ §§ University Hospital, Aalborg, Denmark; Department of Biomedicine, Aarhus University, Aarhus, Denmark; and Department of Internal Medicine, Regional Hospital Viborg, Viborg, Denmark. Supported by grants from Knud and Edith Eriksens Memorial Foundation and Ferring (V.A.); Obelske Family Foundation and the Svend Andersen Foundation (A.S.); Knud Hojgaards Foundation Denmark, the Lundbeck Foundation Denmark, the Oticon Foundation Denmark, and the Otto Monsteds Foundation Denmark (T.B.B.). The authors have no conﬂicts of interest to disclose. Reprints: Tue Bjerg Bennike, PhD, Department of Health Science and Technology, Aalborg University, Fredrik Bajers vej 3B, 9220 Aalborg, Denmark (e-mail: [email protected]). Copyright © 2015 Crohn’s & Colitis Foundation of America, Inc. This is an open-access article distributed under the terms of the Creative Commons Attribution- NonCommercial-NoDerivatives 3.0 License, where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially. DOI 10.1097/MIB.0000000000000460 Published online 19 May 2015. 2052 www.ibdjournal.org Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Neutrophil Extracellular Traps in UC living in the gastrointestinal tract, collectively termed the gut of 339 proteins. Several of the proteins were involved in inﬂam- 5–10 microbiota. mation and cytoskeleton rearrangement. However, based on the Ulcerative colitis is characterized by superﬁcial inﬂamma- number of visualized protein spots from the gel-based studies, we tory changes limited to the mucosa and submucosa of the colon. can expect the colonic biopsies to contain different proteins in the Markers of inﬂammation obtainable in feces or serum include C- thousands. The gel-free LC-MS study has, therefore, merely iden- reactive protein in serum and the proteins lactotransferrin and tiﬁed a fraction of the proteins, and high-throughput proteomics 11,12 technologies can be used to further identify and quantify disease- calprotectin in feces. C-reactive protein may be increased dur- speciﬁc proteins. We performed a proteomics study of morpho- ing the active phase of UC and is mainly produced in the liver in logically normal intestinal tissue from patients with UC, and we response to stimulation by proinﬂammatory mediators produced found it important to use a current state-of-the-art proteomics at the site of inﬂammation. Lactotransferrin is involved in the platform for high-throughput protein identiﬁcation and quantita- mucosal innate immune system response. It is an iron-binding tion in the colon mucosa. glycoprotein with antimicrobial properties and is expressed by 13,14 activated neutrophils and is released by the injured tissue. Calprotectin is a complex between the 2 proteins S100-A8 and MATERIALS AND METHODS S100-A9 and is involved in the recruitment of leukocytes. Addi- tionally, calprotectin has antimicrobial activity toward bacteria Study Cohort and fungi and composes up to 60% of the soluble cytosol proteins The study cohort size was determined based on a power 15,16 in human neutrophils. Increased abundance of fecal calprotec- analysis. Initially, we assumed that approximately 6000 t tests tin is a sensitive marker for intestinal inﬂammation but has also were to be performed in the study with an effect chance of been seen with the use of nonsteroidal anti-inﬂammatory drugs 0.01. Based on this, 10 subjects in each group to be compared and increasing age. The abundance of all 3 proteins increases in yields an estimated power of 95% to detect an effect size of 2.5 response to intestinal inﬂammation. when correlating with Benjamini and Hochberg. Therefore, 10 Accordingly, diagnostic and prognostic markers for IBD, patients with UC for whom endoscopy was planned and 10 which would be valuable tools that could signiﬁcantly improve healthy subjects were recruited at the outpatients clinic, Diagnos- treatment outcome, are currently missing. In this project, we tic center Regional Hospital Silkeborg in the period from 2012 to investigate protein changes in the intestinal tissue, in contrast to 2013. Diagnosis of UC was based on standard clinical, endo- proteins released to the bloodstream or feces. The aim was to scopic, and histological criteria, and an infectious etiology was excluded. Information on diagnosis, medication, most recent increase our knowledge of the UC etiology and to identify fecal calprotectin measurement, and smoking habits was recorded markers, which could be translated to clinical use. Several studies from the patient records. have investigated protein changes in the colon mucosa tissue. Written informed consent was obtained from all partic- However, so far mainly gel-based techniques have been used ipants before participation in the study, and the project was where the proteins are separating based on mass and charge approved by The Regional Scientiﬁc Ethical Committee (isoelectric point). Subsequently, the proteins are stained and (S-20120204) and the Danish Data Protection Agency (2008- quantiﬁed, and up to hundreds of protein spots on the gel can be 58-035). identiﬁed by mass spectrometry (MS). One such study com- pared colonic biopsies from 4 patients with UC and 5 controls Samples Collection and identiﬁed 19 proteins with different abundance between the 2 Biopsies for the histological evaluation of disease severity groups, indicating that mitochondrial dysfunction might be were sampled from descending colon, sigmoid colon, and rectum. involved in the UC etiology. A more recent study compared Three biopsies were taken from each location. The biopsies were inﬂamed and noninﬂamed colon biopsies from 20 patients with immediately placed on mixed cellulose ester ﬁlters (Advantec, UC and identiﬁed 43 proteins that differentiated between the 2 Japan), ﬁxed in phosphate-buffered 4% formaldehyde, and stored conditions, many of which were involved in energy metabolism at room temperature until tissue sample preparation 24 hours later. and oxidative stress. The 2-dimensional gel-based techniques Colon mucosal biopsies for MS were sampled 40 cm from the allow for visual identiﬁcation of regulated proteins and protein anus by sigmoidoscopy. Patient biopsies were taken from products that differ between different samples by image analysis macroscopically normal tissue, and all controls had normal followed by MS protein identiﬁcation. However, 2-dimensional ﬁndings at sigmoidoscopy. The biopsies were immediately trans- gel-based techniques are laborious and are limited to the identiﬁ- ferred to cryotubes and snap-frozen in liquid nitrogen followed by cation of protein spots in the hundreds. The identiﬁcation of non- storage at 21408C until proteomics sample preparation. changing proteins is for that reason often omitted from the 11,19 analysis. Sample Preparation for Histology In a recent study of patients with UC by Han et al, a gel- The formalin-ﬁxated biopsies were parafﬁn embedded, free liquid chromatography (LC)-MS technique is used to analyze sliced in 10 mm, deparafﬁnized with tissue clear (Sakura, AJ protein changes in colonic biopsies, which led to the identiﬁcation Alphen aan den Rijn, The Netherlands), and stained with www.ibdjournal.org 2053 Bennike et al Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 hematoxylin and eosin (H&E) (Sigma-Aldrich, St. Louis, MO) triethylammonium bicarbonate, pH 8.5), using intense shaking on or incubated with 2 mM TO-PRO-3 (Thermo Scientiﬁc, Waltham, a Precellys 24 homogenizer (Berting Technologies, Rockville, MA) in phosphate-buffered saline for 30 minutes, washed 3 times MD). The protein concentrations of the lysates were estimated by in phosphate-buffered saline, and mounted. measuring absorbance at 280 nm (A280) using a NanoDrop 1000 The H&E-stained slices were investigated with UV-Vis spectrophotometer (Thermo Scientiﬁc). To increase the a DM5500B microscopy (Leica Camera, Solms, Germany) equip- accuracy of the A280 estimations, the concentration of 4 biopsy ped with PL Floutar ·16/0.5, and ·40/1.00 objectives and Retiga lysates were determined using a bicinchoninic acid assay with 2000R CCD Camera (Qimaging, Canada), and the TO-PRO-3 bovine serum albumin as standard, measured using an Inﬁnite microscopy was performed on a SP5 confocal microscope (Leica microplate reader (Tecan, Männedorf, Switzerland). All A280 Camera) with a PL Floutar ·16/0.5, HCX PL APO ·63/1.40, and measurements were postprocessing calibrated using the bicincho- HCX PL APO ·100/1.47 objectives. The pictures were processed ninic acid assay protein determinations. with ImageJ with the LOCI Tools plugin. Protein digestion was performed using a modiﬁed ﬁlter-aided 26–28 sample preparation protocol. A biopsy lysate volume corre- Colon Inﬂammation Grade Score sponding to 100 mg of total solubilized protein was prepared for As part of the routine patient care at Regional Hospital LC-MS analysis for each biopsy. Proteins were reduced for 30 mi- Silkeborg, pathologists performed histological descriptions of nutes at room temperature by adding tris(2-carboxyethyl)phosphine formalin-ﬁxed parafﬁn-embedded H&E-stained biopsies from de- (Thermo Scientiﬁc) to a ﬁnal concentration of 10 mM. The reduced scending colon, sigmoid colon, and rectum. The biopsies were protein solution was transferred to a 30-kDa molecular weight characterized and assigned a colon inﬂammation grade score cutoff spin-ﬁlter (Millipore, Billerica, MA), and buffer exchange (0 ¼ no disease activity, 1 ¼ light activity, 2 ¼ moderate activity, was facilitated between all steps by centrifugation at 15,000g for and 3 ¼ severe activity) based on the histological descriptions 15 minutes at 48C. The proteins were alkylated using 100 mLof 18,25 (Table 1). The sum of scores from the descending colon, 50 mM 2-iodoacetamide (Sigma-Aldrich) in lysis buffer. Two sigmoid colon, and rectum were squared to yield patient colon micrograms of sequencing grade modiﬁed trypsin (Promega, inﬂammation grade scores. The scores were compared with the Madison, WI) dissolved in 50 mL of digestion buffer (0.5% sodium relative abundance of known inﬂammatory proteins in the tissue, deoxycholate, 50 mM triethylammonium bicarbonate, pH 8.5) was and Pearson’s correlation coefﬁcients were calculated. The histo- added to the spin-ﬁlter, and the proteins were digested to peptides logical evaluation was supervised by a specialist in human pathol- overnight at 378C. The peptide material was eluted from the spin- ogy, blinded for the result of the proteomic analyses. ﬁlter and puriﬁed by phase inversions. One milliliter of ethyl ace- tate with 1% formic acid was added to the peptide material, the tube was vortexed, centrifuged at 15,000g for 5 minutes to obtain phase Sample Preparation for Proteomics 26,29 separation, and the aqueous phase with peptides was recovered. The intestinal biopsies and lysates were kept on ice when The ﬁnal peptide eluent was dried down in a vacuum centrifuge possible. Each biopsy was homogenized with steel beads in overnight and stored at 2808C until time of analysis. 0.5 mL of cold lysis buffer (5% sodium deoxycholate, 50 mM TABLE 1. Disease Activity of Patients with UC Histological Grading Colon Inﬂammation F-Cal F-Cal and Biopsy Patient Descendens Sigmoideum Rectum Grade Score (mg/g) Time Difference (d) Year of Diagnosis UC_1 2 2 2 36 1285 7 2008 UC_2 0 0 1 1 889 20 2010 UC_3 1 2 2 25 1343 0 2003 UC_4 0 0 1 1 .3600 0 2010 UC_5 0 0 0 0 ,30 28 2009 UC_6 0 2 0 4 705 0 2006 UC_7 2 1 3 36 575 117 2008 UC_8 0 2 0 4 163 37 2011 UC_9 2 0 0 4 ,30 4 2000 UC_10 2 1 1.5 20.25 2060 5 2010 The histological grading of biopsies from colon descendens, colon sigmoideum, and rectum is used to calculate the colon inﬂammation grade score. Colon inﬂammation grade score ¼ (descendens score + sigmoideum score + rectum score) . Additionally, the measured fecal calprotectin (F-Cal) and the time difference between the measured F-Cal and the biopsy extraction are given. 2054 www.ibdjournal.org Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Neutrophil Extracellular Traps in UC peptides and proteins to below 1% false discovery rate. For Proteomics—Analysis quantiﬁed proteins, additional ﬁltering of the protein and Before LC-MS analysis, the dry peptide product was reconstituted in 20 mL of 2% acetonitrile and 0.1% formic acid. peptide data was done to ensure high-quality quantitative data: Five micrograms of peptide material as determined by A280 (1) The quantitation of any protein was required to be based on was analyzed per LC-MS analysis. All biopsies were analyzed at least 2 sequence-unique quantiﬁable peptides. (2) The in triplicates and in a random order of biopsy analysis. sequence-unique peptides were required to be quantiﬁable in The peptides were analyzed by LC-MS using an at least half of the UC biopsies or the control biopsies. (3) All UltiMate 3000 UPLC system (Thermo Scientiﬁc) coupled biopsies had been analyzed in triplicates on the LC-MS online to a Q Exactive mass spectrometer (Thermo Scientiﬁc) system. To remove replicate analyses with poor repeatability, using a trapping column setup. The peptide material was all measured protein abundances were log2 transformed and loaded onto a 2-cm trap column packed with Acclaim the repeated analyses were investigated individually for the PepMap100 C18, 5 mm 100 Å material (Thermo Scientiﬁc). biopsies by scatter plots. Ideally, the repeated analysis of Subsequently, the peptides were separated using a 50-cm ana- a biopsy should yield identical protein abundances, repre- lytical column packed with Acclaim PepMap100 C18, 2 mm sented by a Pearson’s correlation coefﬁcient of 1 in the scatter 100 Å material (Thermo Scientiﬁc). The peptides were eluted plots, indicating a perfect correlation. Individual replicate from the column with a gradient of 96% solvent A (0.1% analyses with a Pearson’s correlation less than 0.95 were formic acid) and 4% solvent B (0.1% formic acid in acetoni- removed from further analysis, to ensure that only highly trile), which was increased to 8% solvent B on a 5-minute reproducible replicates were included in the ﬁnal data set. ramp gradient and subsequently to 30% solvent B on a 225- (4) To further identify replicate outliers, a principle compo- minute ramp gradient, with a constant ﬂow rate of 300 nL/min. nent analysis (PCA) was performed in Perseus v1.4.1.3. The The column was washed with 90% solvent B for 10 minutes input for the PCA was all measured protein abundances in all and equilibrated for 40 minutes, resulting in a total analysis replicates. For the purpose of conducting PCA, missing values time of 290 minutes per LC-MS analysis. Both columns were (i.e., proteins where a quantitation value was not obtained for kept at 408C. The eluting peptides were introduced directly a given replicate analysis) were replaced with values from into the mass spectrometer by a picotip emitter for nanoﬂow a normal distribution (width 0.3 and down shift 1.8) to simu- ionization (New Objective, Woburn, MA). The mass spec- late signals from low abundant proteins. The grouping of the trometer was operated in positive mode using a data- replicates on the calculated scores plots was investigated. dependent acquisition method. A full MS scan in the mass Gene ontology annotations were imported from Uniprot range of m/z 400 to 1200 was acquired at a resolution of Knowledgebase for all quantiﬁed proteins using STRAP 70,000. In each cycle, the mass spectrometer would trigger up to 12 MS/MS acquisitions of eluting ions based on highest v1.5 to classify the proteins. signal intensity for fragmentation. The MS/MS scans were acquired with a dynamic mass range at a resolution of 17,500. Proteomics—Statistical Analysis The precursor ions were isolated using a quadrupole isolation win- The repeated measurements of protein abundances were dow of 1.6 m/z and fragmented using high-energy collision with combined in Perseus v1.4.1.3 by taking the median biopsy wise, a normalized collision energy of 27. Fragmented ions were dynam- and the biopsies were grouped according to disease state: 10 ically added to an exclusion list for 30 seconds. biopsies from patients with UC and 10 from controls. To identify proteins with a statistically signiﬁcant mean abundance change Proteomics—Raw File Analysis between the UC group and the control group, 2-sided t tests were The measured peptide signal intensities from the full MS performed. As several thousand t tests were performed, we 31,32 scan were integrated using MaxQuant 1.4.1.2 software and applied permutation-based false-positive control to correct for used to calculate the relative protein quantities (label-free multiple hypothesis testing (parameters: s0 ¼ 1, 250 randomiza- quantitation). The fragment scans were searched against a Uni- 37–39 tions). Up to 15% of the statistically signiﬁcantly changing prot database containing all reviewed Homo sapiens proteins proteins were allowed to be false positives (i.e., the measured (downloaded October, 8, 2013, containing 20,277 entries). average protein abundance is not statistically signiﬁcantly differ- The following abundant peptide modiﬁcations were included ent between the UC group and the control group) to ensure suf- in the analysis: carbamidomethylated cysteine residues (ﬁxed), ﬁcient input for the downstream protein functional analysis. acetylation of N peptides from the N-terminal of proteins (var- Proteins displaying a statistically signiﬁcant mean abundance iable), oxidation of methionine (variable), and deamidation of 33,34 change were further investigated using SPSS statistics v22 asparginine and glutamine residues (variable). (IBM, Armonk, NY). The MS data have been deposited to the Proteomics—Protein Data Processing ProteomeXchange Consortium (http://proteomecentral.proteo- A target-decoy fragment spectra search strategy was mexchange.org) by means of the PRIDE partner repository with 40,41 applied and used to adjust the false discovery rate of identiﬁed the data set identiﬁer PXD001608. www.ibdjournal.org 2055 Bennike et al Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 TABLE 2. Study Cohort Information and Analysis ID Age Gender Smoking Smoking History Medicine (per day) UC_1 27 Male Never Asacol (5-aminosalicylic acid) 1600 + 800 + 1600 mg UC_2 45 Male Stopped 2009 Asacol (5-aminosalicylic acid) 1600 mg · 2 Asacol (5-aminosalicylic acid) suppository 500 mg UC_3 44 Male Stopped 2000 Imurel (azathioprine) 150 mg · 1 UC_4 45 Female Stopped 2005 Pentasa (5-aminosalicylic acid) suppository 1 g UC_5 26 Female 10 cigarettes per day Current Mesasal (5-aminosalicylic acid) suppository 500 mg UC_6 70 Female Stopped 1998 Mesasal (5-aminosalicylic acid) suppository 500 mg UC_7 62 Male Stopped 2009 Imurel (Azathioprin) 100 mg; Pentasa (5-aminosalicylic acid) 2000 mg UC_8 36 Male Never Asacol (5-aminosalicylic acid) 1600 mg · 2 Asacol (5-aminosalicylic acid) suppository 500 mg UC_9 68 Female 10 cigarettes per day Current Asacol (5-aminosalicylic acid) 1600 mg · 2 UC_10 22 Female Never Asacol (5-aminosalicylic acid) 1600 mg · 2 Asacol (5-aminosalicylic acid) suppository 500 mg Pred-Clysma (glucocorticoid) 24 mg Ctrl_1 54 Male Never Ctrl_2 27 Male Never NovoRapid (insulin) Ctrl_3 42 Male Never Ctrl_4 48 Male Never Ctrl_5 54 Female Stopped 1992 Ctrl_6 27 Female Stopped 2011 Ctrl_7 42 Female Never Ctrl_8 23 Male Never Ctrl_9 28 Male Never Ctrl_10 31 Male Stopped 2008 Age and gender were not found to be statistically signiﬁcantly different (P . 0.05) between the patients with UC (n ¼ 10) and the controls (n ¼ 10) as determined by 2-sided t tests. were obtained in parallel, we expect these biopsies to have the RESULTS same characteristics. Patient Material Proteomics Veriﬁcation The recorded medical information (Table 2) revealed 9 pa- Before homogenization of the biopsies for proteomics tients receiving 5-aminosalicylic acid, 2 patients receiving azathio- sample preparation, the average wet weight of the biopsies was prine, and 1 patient receiving glucocorticoid treatment, whereas 1 measured to be 4.9 mg (3.6–5.9 mg; 25th–75th percentiles). Fol- control received insulin treatment. The study cohort age, gender, lowing homogenization of the biopsies in lysis buffer, the protein and current smoking status was not found to be statistically differ- concentration of the lysate was measured and the average ex- ent between the patients with UC and controls. However, the num- tracted protein yield, calculated from the biopsy wet weight, ber of former smokers was increased in the UC group. was 10% (9%–11%; 25th–75th percentiles), indicating an efﬁ- cient and reproducible homogenization. Histological Analysis The proteins in each homogenized sample were digested to To assess cell density and tissue morphology in the peptides using the ﬁlter-aided sample preparation protocol, and extracted biopsies, we investigated the H&E-stained sections by the complex peptide material was analyzed by LC-MS/MS with light microscopy (Fig. 1). The crypt architecture was preserved in 31,32 label-free quantitation. During the 290-minute analysis of all control samples and all UC biopsies. However, we observed an each homogenized and digested sample, the MS system detected increased number of stained cell nuclei between the intestinal peptide elusion from the LC column and selected up to 12 pre- crypts in the biopsies from patients with UC compared with con- cursor ions per MS cycle for fragmentation (Fig. 2). By bioinfor- trols. The majority of cells between the intestinal crypts had seg- matics analysis, the proteins were subsequently identiﬁed and mented cell nucleus, a characteristic of polymorphonuclear 42,43 quantiﬁed. To investigate the repeatability of the LC-MS analysis, neutrophils (PMNs). As the biopsies for the proteome analysis 2056 www.ibdjournal.org Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Neutrophil Extracellular Traps in UC of variables to a smaller number of groups (principle compo- nents), which can be visualized on scores plots and used to interpret the variance in a complex data set, like that of high-throughput proteomics. All measured protein abundances in all LC-MS analyses were used as input for the PCA, and 2- dimensional scores plots were constructed (Fig. 3B). The scores plot describes how the replicates group relative to one another, based on the differences in measured abundances of all proteins. In effect, replicates where similar protein abundances have been measured will be close in space on the scores plot, relative to the other replicates, and the PCA scores plot can thus be used to identify outliers. The PCA, thereby, includes the variance between all biopsies, unlike the scatter plots that only take the variance in the repeated analysis of the individual biopsies into account. Principal component 1 and principle component 2 rep- resent the largest and second largest variance in the protein abun- dance data set, respectively, and explain 35.4% of the variance in the protein abundance data set. In all cases, the scores plot sepa- rates all biopsies from one another, in contrast to the repeated triplicate analysis of each biopsies, which group together for each individual biopsy (Fig. 3B, circles). This indicates that the mea- sured protein abundances are more similar when the same biopsy is analyzed repeatedly than when different biopsies are analyzed. The variance in protein abundances between the different biop- sies, therefore, is larger than the technical variance when the same biopsy is analyzed repeatedly, as expected for sensitive methods that validate the methodology. Proteomics—Data Analysis Applying our strict criteria, 5711 quantiﬁable proteins remained postﬁltering. The quantiﬁable proteins were classiﬁed FIGURE 1. Biopsy histology analysis. A, Overview of the human small by biological function, subcellular location, and molecular and large intestine. B, Cross section of a human colon. Representative function, using available data from Uniprot Knowledgebase and H&E-stained colon biopsy slices from (C) control Ctrl_10 and (D) gene ontology database (Fig. 4). The analysis of the biological patient UC_05. The crypt architecture is in both slices preserved. processes performed by the proteins revealed that 68% of the However, an increased number of stained cell nuclei surrounding the quantiﬁed proteins were involved in cellular processes, such as crypts (red arrows) are apparent in the UC biopsies compared with the control biopsies (scale bars 100 mm). intracellular communication; 55% of the proteins were involved in modulation of the frequency, rate, or extent of any biological process, quality, or function; and 29% were involved in metabolic the technical triplicate analyses of each biopsy were analyzed by processes, which include anabolism and catabolism by which scatter plots, plotting the abundance of each quantiﬁed protein in living organisms transform chemical substances. We next inves- one replicate against the same protein abundance as determined in tigated the cellular component of the proteins, that is, which parts other replicates, biopsy wise. Ideally, the repeated analysis should of a cell or its extracellular environment the protein has been yield identical protein abundances, represented by a Pearson’s reported to be present. A somewhat even distribution was found correlation coefﬁcient of 1 (Fig. 3A). Of the 60 LC-MS analyses, between proteins associated with the cell cytoplasm, the cell only 1 replicate from UC_4 and 1 from UC_5 had coefﬁcients less nucleus, and the extracellular environment of the cell with 48%, than 0.95 and the 2 outlying replicates were removed from further 42%, and 35%, respectively. Finally, we investigated which pri- analysis. The result demonstrates a high degree of technical mary molecular functions the proteins took part of. The analysis repeatability of the method, that is, a low variance of the measured revealed that 68% of the proteins were involved in selective, non- protein abundances when the same biopsy is analyzed repeatedly. covalent binding with other molecules, and 41% were involved in To investigate how the measured protein abundances varied biologically catalyzed reactions. Based on the protein classiﬁca- between all replicates and identify which replicates yielded sim- tion analysis, the quantiﬁed proteins originate from all cellular ilar protein abundances, a PCA was performed. A PCA is a sta- compartments, indicating that no systematic loss of proteins has tistical analysis technique that allows for reducing a large number occurred using the protocol and thus an unbiased analysis. www.ibdjournal.org 2057 Bennike et al Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 FIGURE 2. Representative 2-dimensional plot of the liquid chromatography-mass spectrometry analysis of patient UC_9 illustrating the complexity of the analyzed peptide material. On the x-axis, the intensities of the peptide ion signals of the 19,549 acquired full MS scans are plotted, with a color gradient from white to green. The time of analysis is given on the y-axis. Each full MS scan was succeeded by up to 12 MS/MS scans of ions detected on the full MS scan and each cycle time was approximately 1 second; + indicates that the given ion signal was selected for MS/MS analysis, which came to 119,560 MS/MS scans in this 1 replicate. Next, we analyzed the quantitative information obtained that, on average, was different between the UC biopsies and con- about the proteins in each biopsy. The measured protein trol biopsies by a factor of 2.8 or more, which is referred to as the abundances in the biopsies from the patient with UC were fold change. Of the 46 proteins, 33 proteins were more abundant compared with the abundances in the biopsies from the control in the UC biopsies and 13 proteins were less abundant (Table 3). group, and t tests were performed to identify proteins with a sta- The protein with the largest mean fold abundance change between tistically signiﬁcant mean abundance change between the 2 the UC and control biopsies was lactotransferrin, which was 219 groups. Forty-six proteins demonstrated a statistical signiﬁcant times more abundant in the UC group. To further validate the mean abundance change between the UC biopsies and the control measured protein abundances, we correlated the relative abun- biopsies. All the 46 proteins were measured with an abundance dance of lactotransferrin and calprotectin with the severity of FIGURE 3. Proteomics data veriﬁcation. A, Representative scatter plot of the triplicate biopsy analysis of UC_1 of the 5711 quantiﬁed proteins. The log2 transformed protein abundances of all proteins in replicates 1, 2, and 3 are plotted against one another on the x- and y-axes, respectively. Ideally, the measurements should yield identical protein abundances, represented by a Pearson’s correlation coefﬁcients (R) of 1. B, PCA scores plot of principle components 1 and 2 of the protein abundances as measured in the UC replicates (+) and control replicates (h). Technical replicates (encircled) group together in all cases, demonstrating a high degree of technical repeatability of the method. The study cohort subject ID is given as numbers (e.g., red 3 at [+] equals UC_3) (Table 2). 2058 www.ibdjournal.org Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Neutrophil Extracellular Traps in UC FIGURE 4. Gene ontology characterization of the 5711 quantiﬁed proteins in terms of (A) biological processes performed by the proteins, (B) cellular component to which the proteins are associated, and (C) the primary molecular functions of the proteins. tissue inﬂammation in the patients with UC, as determined by investigated the TO-PRO-3 DNA–stained biopsy sections by con- histology. Both proteins are used to monitor UC disease activity. focal microscopy. The increased density of cell nuclei observed Biopsies from descending colon, sigmoid colon, and rectum were on the H&E-stained sections was again observed (Fig. 7), as was assigned a colon inﬂammation grade score based on histology the segmented multilobulated nuclei, a characteristic of PMNs descriptions (Table 1). A good correlation was found between (Fig. 8A). The PMNs were observed abundantly in biopsies the colon inﬂammation grade scores and the relative abundance from the UC group and occasionally in the control group. In of calprotectin and lactotransferrin in the tissue, with Pearson’s addition to intact PMNs, we observed neutrophils that had correlation coefﬁcients of 0.84 and 0.91 for S100-A8 and excreted nuclear DNA into the extracellular environment forming S100-A9 protein, respectively, and 0.82 for lactotransferrin NETs at 2 distinct locations in biopsies from UC_4 (Fig. 8B). (Fig. 5). Finally, we correlated the relative abundance of calpro- tectin in the tissue to the most recent measure of fecal calprotectin as determined by routine tests. However, no correlation could be DISCUSSION found, and a Pearson’s correlation coefﬁcient of 0.33 was calcu- The main result of this study is the identiﬁcation of several lated (data not shown). novel proteins, which are present in increased abundance in the Eleven of the 46 proteins with statistically signiﬁcantly UC colonic tissue. Eleven of the proteins were associated with altered abundance in the UC biopsies are present in neutrophils neutrophils and NETs, and the increased presence of both in the and have been associated with the formation of neutrophil UC colonic tissue was veriﬁed with light microscopy and extracellular traps (NETs) (Table 3, asterisk indicates protein confocal microscopy. names). NETs are a method of action by PMNs and are com- Several studies have successfully analyzed different aspects posed of extracellular ﬁbrillar networks mainly of relaxed DNA of UC using genomic and transcriptomic techniques. However, and also contain proteins and other effector molecules, some of sequencing techniques do not directly yield information about the which have antimicrobial and antibody-like properties. NETs are abundance of the end product: the proteins, which are a combi- released from PMNs in response to inﬂammatory stimuli, trap- nation of the newly synthesized and the degraded protein. In our ping the invading microorganisms, and facilitates interaction proteome analysis of colon mucosa biopsies from 10 patients with 45–47 with other effector molecules that kill the microorganisms. UC and 10 controls, we were able to quantify 5711 proteins in the All 11 NET-associated proteins were found with increased abun- colon mucosa biopsies, and the data quality was ensured by scat- dance in the UC biopsies, and on average, the proteins were 42.2 ter plots and PCA. In previous studies, the amounts of calprotectin times (P , 0.0005) more abundant in the UC biopsies (Fig. 6). and lactotransferrin in feces have been found to correlate well with the degree of neutrophil inﬁltration and inﬂammation of 11,15 Confocal Laser Scanning Microscopy the intestine. We found that the abundance of these proteins To investigate if the increased abundance of PMN- in the colonic tissue correlated well with the colon inﬂammation associated proteins was a result of an increased abundance of grade scores, providing validation to the proteomics protein abun- PMNs, and if the PMNs were intact or found as NETs, we dance data and the methodology. However, we found that the www.ibdjournal.org 2059 Bennike et al Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 2060 www.ibdjournal.org TABLE 3. List of the 46 Proteins with a Statistically Signiﬁcant Mean Abundance Change Between the UC Biopsies and Controls Fold Peptides (Unique), Change Uniprot Acc. Protein Name Protein Function Seq. Cov. MW pI 219.2 P02788 Lactotransferrin* Iron-binding protein with antimicrobial properties include bacteriostasis 51 (49), 72.7% 76,165 8.47 92.1 P14780 Matrix metalloproteinase-9* Proteolysis of the extracellular matrix and leukocyte migration 30 (30), 51.8% 66,609 5.44 63.3 P05164 Myeloperoxidase* Part of the defense system of PMN; responsible for microbicidal activity 48 (41), 59.2% 66,107 9.22 against a wide range of organisms 31.4 P24158 Myeloblastin PMN protease; degrades elastin, ﬁbronectin, laminin, vitronectin, and collagen 7 (7), 36.3% 24,247 7.79 31.3 P08246 Neutrophil elastase* Modiﬁes functions of natural killer cells, monocytes, and granulocytes 11 (11), 54.7% 25,561 9.89 18.0 Q9NRD8 Dual oxidase 2 Lactoperoxidase-mediated antimicrobial defense at the surface of mucosa 42 (42), 33.6% 172,843 7.92 16.7 P11215 Integrin alpha-M Adhesive interactions of monocytes, macrophages, and granulocytes 34 (33), 37.7% 125,471 6.75 16.3 P06702 Protein S100-A9* One of the subunits in calprotectin; regulates inﬂammatory processes and 10 (10), 78.9% 13,110 5.71 immune response; Induce neutrophil chemotaxis, adhesion, increase bactericidal activity and degranulation 13.7 P80188 Neutrophil gelatinase-associated Apoptosis, innate immunity, and renal development 12 (12), 63.6% 20,547 9.02 lipocalin 11.8 P80511 Protein S100-A12* Regulates inﬂammatory processes and immune responses; recruitment of 5 (5), 35.9% 10,443 5.80 leukocytes, promotion of cytokine and chemokine production, and regulation of leukocyte adhesion and migration; antifungal and antimicrobial activity 10.6 P22894 Neutrophil collagenase Degrade ﬁbrillar collagens 14 (14), 37.7% 41,937 5.49 9.9 P59666 Neutrophil defensin 3* Fungicide, antiviral, and antimicrobial activity against bacteria 5 (5), 26.6% 3,492 8.33 9.4 P08637 Low-afﬁnity immunoglobulin Binds monomeric, complexed, or aggregated IgG; mediates antibody- 2 (2), 8.3% 27,324 8.24 gamma Fc region receptor dependent responses (e.g., phagocytosis) III-A 9.2 P52790 Hexokinase-3 Metabolic processes; phosphorylation of hexoses 21 (18), 31.3% 99,025 5.23 8.7 O75594 Peptidoglycan recognition Innate immunity; has bactericidal activity 5 (5), 41.3% 19,434 8.23 protein 1 8.5 P36222 Chitinase-3-like protein 1 Regulates inﬂammatory cell apoptosis, dendritic cell accumulation, and 14 (14), 44.9% 40,488 8.64 macrophage differentiation; facilitates invasion of pathogenic enteric bacteria into colonic mucosa 7.8 Q06141 Regenerating islet-derived Antimicrobial activity against gram-positive bacteria 4 (4), 36.6% 16,566 7.83 protein 3-alpha 7.3 Q1HG44 Dual oxidase maturation factor 2 Required for dual oxidase 2 maturation and transport to the plasma membrane 4 (4), 16.9% 34,786 8.51 6.6 Q05315 Galectin-10* Regulates immune responses; recognition of cell surface glycans 11 (11), 67.6% 16,321 6.80 6.6 P35228 Nitric oxide synthase, inducible Produces tumoricidal and bactericidal nitric oxide in macrophages 42 (41), 45.4% 131,117 8.20 6.2 P16050 Arachidonate 15-lipoxygenase Regulates macrophage function and epithelial wound healing in the cornea; 25 (25), 52.1% 74,673 6.14 may favor clearance of apoptotic cells during inﬂammation 5.9 P12724 Eosinophil cationic protein* Antibacterial activity, including cytoplasmic membrane depolarization 10 (10), 46.9% 15,518 10.47 5.7 Q8IX19 Mast cell-expressed membrane Integral component of membrane in mast cells (part of the immune system) 3 (3), 15.5% 21,228 9.03 protein 1 Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Neutrophil Extracellular Traps in UC www.ibdjournal.org 2061 TABLE 3 (Continued) Fold Peptides (Unique), Change Uniprot Acc. Protein Name Protein Function Seq. Cov. MW pI 5.1 P10153 Nonsecretory ribonuclease Pyrimidine-speciﬁc nuclease; selective chemotactic for dendritic cells 8 (8), 44.1% 15,463 9.20 5.0 P11678 Eosinophil peroxidase Mediates tyrosine nitration of secondary granule proteins in mature eosinophils 51 (44), 56.8% 81,040 10.70 4.5 P41218 Myeloid cell nuclear differentiation Plays a role in the granulocyte/monocyte response to interferon 17 (17), 39.1% 45,836 9.76 antigen* 4.4 P06310 Ig kappa chain V-II region RPMI Immunoregulatory interactions between a lymphoid and a nonlymphoid cell 4 (3), 37.6% 12,585 9.07 4.3 Q8TCU6 Phosphatidylinositol- Neutrophil chemotaxis; may function downstream of heterotrimeric G proteins 8 (8), 6.4% 186,203 6.03 3,4,5-trisphosphate–dependent in neutrophils Rac exchanger 4.0 P01040 Cystatin-A Intracellular thiol proteinase inhibitor; desmosome-mediated cell–cell adhesion 2 (2), 30.6% 10,875 5.39 in the lower epidermis levels 4.0 Q96Q80 Derlin-3 Functional component of endoplasmic reticulum-associated degradation for 4 (3), 26.8% 26,678 8.64 misfolded lumenal glycoproteins 3.9 Q7Z7N9 Transmembrane protein 179B Integral component of membrane 2 (2), 8.2% 23,550 8.10 3.8 Q70J99 Protein unc-13 homolog D Regulates secretory lysosome exocytosis in mast cells and cytotoxic granule 16 (16), 24.7% 123,281 6.19 exocytosis in lymphocytes; required for granule maturation and docking 3.5 P08311 Cathepsin G* Serine protease; cleaves complement C3; has antibacterial activity against the 16 (16), 54.1% 26,757 11.37 gram-negative bacterium Pseudomonas aeruginosa 0.4 P29966 Myristoylated alanine-rich Filamentous actin cross-linking protein; binds calmodulin, actin, and synapsin 11 (11), 49.1% 31,423 4.46 C-kinase substrate 0.3 P00326 Alcohol dehydrogenase 1C Oxidation of alcohol 27 (10), 64.8% 39,868 8.63 0.3 P20039 HLA class II histocompatibility Binds peptides derived from antigens that access the endocytic route of 9 (2), 40.6% 27,230 6.49 antigen, DRB1-11 beta chain antigen-presenting cells and presents them on the cell surface for recognition 0.3 P10082 Peptide YY Gut peptide; inhibits exocrine pancreatic secretion, and jejunal and colonic 3 (3), 29.9% 4,410 8.34 mobility; has vasoconstrictory action 0.3 Q12805 EGF-containing ﬁbulin-like May play a role in cell adhesion and migration; binds the EGF receptor, 15 (15), 43.0% 52,765 4.85 extracellular matrix protein 1 inducing phosphorylation and activation 0.3 Q9HBI6 Phylloquinone omega-hydroxylase Oxidizes a variety of compounds, including fatty acids and xenobiotics; may 11 (3), 26.7% 60,146 6.26 CYP4F11 play a role in blood coagulation 0.3 P15502 Elastin Major structural protein of tissues; regulates proliferation and organization of 6 (6), 17.7% 65,721 10.32 vascular smooth muscles 0.3 P10915 Hyaluronan and proteoglycan link Stabilizes the aggregates of proteoglycan monomers with hyaluronic acid in 14 (13), 48.6% 38,840 6.85 protein 1 the extracellular cartilage matrix 0.2 Q86Y39 NADH dehydrogenase Subunit of the mitochondrial membrane respiratory chain NADH 7 (7), 58.2% 14,721 8.95 (ubiquinone) 1 alpha dehydrogenase, thought not to be involved in catalysis subcomplex subunit 11 Bennike et al Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 FIGURE 5. Correlation analysis of inﬂammatory proteins. Colon inﬂammation grade scores and the protein abundance in the colon tissue of the inﬂammatory proteins lactotransferrin (d), S100-A8 (-), and S100-A9 (:) (S100-A8 and S100-A9 protein constitute calpro- tectin). amount of fecal calprotectin did not correlate with the colon inﬂammation grade scores or the measured amount of calprotectin in the tissue (data not shown), which have been reported by others. A likely explanation is that the measure of fecal calpro- tectin and the biopsy extraction was not conducted in parallel in the present study, and only for 3 of the 10 patients with UC were the fecal calprotectin measures obtained on the day. Additionally, fecal calprotectin is a marker for inﬂammation in the entire intes- tinal system, in contrast to the colon inﬂammation grade score and the proteomics analysis where the transverse colon or the FIGURE 6. Fold change of statistically signiﬁcantly changing NET- associated proteins between biopsies from the patients with UC and controls. The 11 proteins are on average 42.2 times (P , 0.0005) more abundant in the biopsies from the UC group compared with the biopsies from the control group. Center line shows the median, box limits indicate the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, crosses represent sample means, and the circle represents the fold change of lactotransferrin that lies outside the whiskers. 2062 www.ibdjournal.org TABLE 3 (Continued) Fold Peptides (Unique), Change Uniprot Acc. Protein Name Protein Function Seq. Cov. MW pI 0.2 P35558 Phosphoenolpyruvate carboxykinase, Catalyzes conversion of oxaloacetate to phosphoenolpyruvate, the rate-limiting 38 (34), 71.4% 69,195 5.8 cytosolic (GTP) step in the metabolic pathway that produces glucose from lactate and other precursors from the citric acid cycle 0.2 Q6PIJ6 F-box only protein 38 Thought to recognize and bind to some phosphorylated proteins and promotes 6 (6), 7.0% 133,944 5.92 their ubiquitination and degradation 0.2 Q07654 Trefoil factor 3 Involved in the maintenance and repair of the intestinal mucosa; promotes the 3 (3), 38.8% 6580 5.13 mobility of epithelial cells in healing 0.2 Q14508 WAP four-disulﬁde core domain Broad range protease inhibitor 4 (4), 45.2% 10,036 4.50 protein 2 Fold change .1 indicates increased mean abundance in the UC biopsies compared with controls. *Protein has been associated with NETs. The number of identiﬁed peptides, the resulting sequence coverage (seq. cov), and the number of unique peptides used for the quantitation are listed for each protein. Protein functions have been downloaded from Uniprot Knowledgebase, and molecular weight (MW) in daltons and isoelectric point (pI) have been downloaded from ExPASy. EGF, epidermal growth factor; GTP, guanosine triphosphate. Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Neutrophil Extracellular Traps in UC FIGURE 7. Confocal microscopic images of TO-PRO-3–stained biopsy slices (scale bars 100 mm): (A) Ctrl_5 and (B) UC_3. An increased number of stained cell nuclei surrounding the intestinal crypts are in general apparent in the UC biopsies compared with the control biopsies. ascending colon was not considered. This is likely why, for example, proteins were more abundant. These proteins were identiﬁed and patient UC_4 treated with suppository Pentasa had a colon inﬂam- quantiﬁed in the present study; however, we could not conﬁrm the mation grade score of 1, indicating for the most part healthy looking increased abundance. However, our study found several proteins biopsies. However, .3600 mg of fecal calprotectin per gram stools associated with the innate immune system to be increased in abun- wasmeasuredindicatinginﬂammation in the transverse colon or the dance in the UC colonic tissue. The differences between ﬁndings in ascending colon, too far from the rectum for the treatment with sup- the 2 studies are mainly ascribed to differences in the obtained patient pository Pentasa to have an effect. Finally, it has been reported that material and study design. The advantages of a state-of-the-art instru- fecal calprotectin can be increased 3 months before a disease ﬂare. ment and bioinformatics platform are demonstrated in the increase As the patients in this study were not followed up after the biopsy was from up to 339 proteins in the study by Han et al to the 5711 taken, it cannot be excluded that these patients later could have devel- quantiﬁable proteins in the present study, thus providing information oped disease ﬂares, which caused the elevated fecal calprotectin. on the regulation of a great number of proteins and thus cellular Of the 5711 quantiﬁable proteins, 46 proteins were functions. A potential drawback of the high number of quantiﬁable measured to have a statistically signiﬁcant change in abundance proteins is that a large number of t tests need to be performed during between the biopsies from the patients with UC and the controls. Of the statistical analysis. To avoid a high number of false positives these, 33 proteins were more abundant in the UC biopsies. Several of among the proteins found to be statistically signiﬁcant, multiple the proteins with a statistically signiﬁcant abundance increase were hypothesis testing must be applied, which increases the requirements involved in inﬂammation. On comparison with the only other gel-free for the P-values. As a consequence, when many proteins are quan- LC-MS study conducted, we found several proteins that were tiﬁed and analyzed, the statistical analysis results in the need for determined to be more abundant in the UC colon tissue in both a higher number of included subjects in the study. As an example, studies, namely, Protein S100-A9 (calprotectin), neutrophil elastase, an overabundance of the enzyme indoleamine-2,3-dioxygene has myeloperoxidase, neutrophil defensin, and cathepsin G. These con- been found in intestinal mucosal tissue from patients with IBD com- sistent ﬁndings validate the study. However, the study by Han pared with normal mucosa, hypothesizing an involvement of the 20 50,51 et al additionally reported that a number of histones and ﬁbrinogen Kynurenine pathway of tryptophan metabolism in the IBDs. www.ibdjournal.org 2063 Bennike et al Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 FIGURE 8. Confocal microscopy images of TO-PRO-3–stained biopsy slices from the UC group (scale bars 100 mm): (A) UC_6 with an intact neutrophil, with segmented multiobulated-shaped nucleus; (B) UC_4 with NET formation by excretion of the neutrophil DNA. 11,19 Indoleamine-2,3-dioxygenase is an intracellular enzyme that catalyzes detectable with 2-dimensional gel studies (Table 3). This in- the production of Kynurenine from the amino acid tryptophan. The cludes protein S100-A12, which was found to be 11.8-fold enzyme is regulated by gamma-interferon of the immune system. increased in the UC colonic tissue and also has been found ele- The protein in our study was detected with a 1.8 mean fold abundance vated in the serum of children with IBD. Interestingly, the pres- increase in the UC biopsies compared with controls but not statistically ent study is the ﬁrst MS-driven proteomics study to measure an signiﬁcant. This indicates that a larger study cohort could have iden- increased abundance of lactotransferrin in the UC colonic tissue. tiﬁed a higher number of statistically signiﬁcantly changed proteins. The increased abundance of fecal lactotransferrin is used as Gel-free quantitative LC-MS studies hold a number of a marker for inﬂammation, and the protein should have been 11,55 advantages over 1- and 2-dimensional gel-based approaches, includ- detectable using 2-dimensional gel-based analysis. However, ing increased sensitivity, increased dynamic range, and identiﬁcation it is possible that the relative abundance of this protein remains and quantiﬁcation of proteins in the thousands instead of hundreds. too low for gel-based studies. Nonetheless, the protein abundance However, the gel-free approaches such as the present study are limited in the colonic tissue correlates well with the abundance of cal- 11,19 in the ability to detect protein fragment products. The increased protectin in the tissue, and the colon inﬂammation grade scores in 11,55 sensitivity of the gel-free approaches becomes apparent when consid- this study, as expected. Another protein involved in inﬂam- ering that the gel-free LC-MS method usedinthisstudy analyzed 5 mation and found with increased abundance in the UC biopsies is mgofthe ﬁnal material compared with the hundreds of microgram myeloblastin. Myeloblastin is a protease found in neutrophils and material used for the 2-dimensional gel analysis, which allowed us to was found to be 31.4-fold more abundant in the UC colonic 17,53 conduct repeated analysis of each biopsy. Two-dimensional gel biopsies compared with controls. The protease has speciﬁcity studies are usually limited to proteins within a molecular mass range toward, among others, the protein elastin, which accordingly of 15 to 200 kDa and isoelectric point of the proteins in the range of 3 has a decreased abundance in the UC biopsies, with a 1/3.3- to 10. These limitations do not apply for gel-free approaches, and fold change. Another interesting protein found with 8.7-fold applying these criteria to the 46 proteins detected with a statistically increase in abundance in the UC colonic biopsies is peptidoglycan signiﬁcant change in this study 30.4% (14 proteins) would not be recognition protein 1. The protein is part of the innate immune 2064 www.ibdjournal.org Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Neutrophil Extracellular Traps in UC systems recognition of microorganisms and mediates its effect by found as NETs. We stained the biopsy sections with a DNA stain binding bacterial peptidoglycans, which triggers a signaling cas- and investigated the sections by confocal microscopy, which al- 56,57 cade. This is the ﬁrst study to ﬁnd an increased abundance of lowed us to achieve a higher resolution than with light micros- peptidoglycan recognition protein 1 in UC colonic tissue. copy. Again, the segmented cell nucleus of the PMNs was Hexokinase-3 is an intracellular enzyme that phosphorylates glu- apparent, and additionally, in biopsies from UC_4, we observed cose into glucose-6-phosphate and, together with hexokinase 1, 2, NETs at 2 distinct locations. The proteomics data and microscopy 58,59 and 4, is a central rate-limiting step in glucose metabolism. data demonstrate the increased abundance of PMNs in UC colon Not much is known about the regulation of hexokinase 3; how- tissue and NETs in biopsies from UC_4. This study is the ﬁrst to ever, the 9.2-fold increased abundance of this enzyme in the UC directly observe NETs in UC colon tissue. Interestingly, the abun- biopsies indicates that glucose metabolism might be increased in dances of several NET-associated histone proteins (H1e, H2A, the inﬂamed tissue compared with controls. H2B, H3, and H4) were not found with increased abundance in The increased abundance of inﬂammation-associated pro- the UC biopsies. Additionally, histones H2A and H3 were found teins points to an activated innate immune system in the UC to be more abundant in the UC colonic tissue by Han et al, colon, even in colonic tissue without visible inﬂammation. It indicating that NETs could have been present in the colonic tissue should be noted that we do not know the impact of the medication obtained in this study as well. However, as histone proteins are an on the protein regulations. However, as most of the statistically essential part of nearly all cells, a relative increase in the abun- signiﬁcant changed proteins are involved in inﬂammatory pro- dance of these proteins between the UC biopsies and controls as cesses, these protein changes are likely caused by the disease a result of the presence of NETs might be too small to be detected. development. Additionally, the number of former smokers was NETs are part of the innate immune system’s response to 47,68 increased in the UC group compared with controls. The impact of invading pathogens. Additionally, several of proteins found to cigarette smoking on the etiology of UC has been vastly debated be more abundant in the UC colon tissue are directly involved in 60–65 over the years. Current knowledge supports that the risk for the mucosal innate immune system response. However, the cause developing UC is highest 2 to 5 years after smoking cessation and for the immune response remains unknown, and no indications of remains elevated for more than 20 years, and patients with UC are bacteria or epithelial cell disruption were detected by the histol- more prone to disease ﬂairs if they quit smoking. However, the ogy analysis, which should have been visible using confocal subject is still debated and the dose–response effect remains to be microscopy. It cannot be ruled out that the immune reactions properly determined. Based on the present study, we cannot could be caused by bacterial components capable of crossing assess the impact of former smoking on the protein data. the intestinal barrier of epithelial cells, which is known to be To evaluate the morphology of the tissue, the biopsies were compromised in IBD where an increased epithelial permeability 10,69,70 sectioned and H&E stained, and the histological evaluation was has been described. However, it has been demonstrated that done by light microscopy. The intestinal crypts were preserved in aberrant formation of NETs might be involved in the pathogenesis all biopsies, in agreement with the biopsies being taken from of autoimmunity and additionally might be formed during a num- 71–73 morphologically normal tissue. However, an increased abundance ber of activities, including exercise. Therefore, based on the of cell nuclei in the colon mucosal tissue was observed between obtained data in this study, we can conclude that a chronic inﬂam- the intestinal crypts, with segmented cell nuclei, a characteristic of mation is present in the tissue, even though the surface appears PMNs. Based on the proteomics data and light microscopy, we normal. However, we cannot point to the origin of the inﬂamma- here verify the increased density of PMNs in the UC colonic tion or the target of the immune response. tissue. This has been reported by other groups as well, but this Our ﬁndings demonstrate that even though remission has is the ﬁrst study to support the light microscopy identiﬁcation of been achieved on the surface of the UC colonic tissue, a chronic 66,67 18 the PMNs with MS techniques. PMNs are essential for the condition is still present within the tissue. Poulsen et al per- innate immunity and are the most abundant leukocyte in plasma. formed a 2-dimensional proteomics study of 20 patients with In response to inﬂammatory stimuli, they migrate from plasma to UC where inﬂamed and noninﬂamed tissue from the same patient the affected tissue where they take part in the ﬁrst line of innate with UC were compared, in contrast to the present study where immune response. The effect is mediated through the release of noninﬂamed tissue from several patients with UC was compared 18 18 enzymes from the granules and the production of compounds with with controls. Interestingly, the study by Poulsen et al found antimicrobial potential and inﬂammation regulatory effects. One that in contrast to the macroscopically different tissue, the pro- of the compounds is NET. PMNs can disintegrate their cell mem- teome of visually inﬂamed and noninﬂamed tissue was only brane and release NETs to capture and kill invasive microorgan- slightly different. Of the 43 protein spots identiﬁed with a different 47,68 isms. The NETs are composed of relaxed DNA to which abundance, the protein displaying the largest fold change was histones and other effector molecules are bound. Eleven of the glycerol-3-phosphate dehydrogenase, which was increased by 46 proteins with statistically signiﬁcantly altered abundance in the 3.3-fold in the inﬂamed UC tissue compared with noninﬂamed UC biopsies are associated with NET formation, and all were tissue, which is a minor change compared with the up to 219-fold measured with increased abundance. As no proteins unique to changes measured in the present study. Combined with the anal- NETs are known, we investigated if the PMNs were intact or ysis of differences in colonic proteome between patients with UC www.ibdjournal.org 2065 Bennike et al Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 and controls, we can conclude that the difference in proteome REFERENCES 1. Molodecky NA, Soon IS, Rabi DM, et al. Increasing incidence and prev- between visually inﬂamed and noninﬂamed UC colonic tissue is alence of the inﬂammatory bowel diseases with time, based on systematic vastly smaller than the interpersonal difference between patients review. Gastroenterology. 2012;142:46–54.e42. with UC and controls. This indicates that changes are present in 2. Odes S, Vardi H, Friger M, et al. Cost analysis and cost determinants in a European inﬂammatory bowel disease inception cohort with 10 years of the UC colonic tissue regardless of visible surface inﬂammation, follow-up evaluation. Gastroenterology. 2006;131:719–728. even in well-medicated patients with UC. A 2012 Danish 3. Jess T, Frisch M, Simonsen J. Trends in overall and cause-speciﬁc mor- population-based cohort study of 300 patients with UC in the tality among patients with inﬂammatory bowel disease from 1982 to 2010. period of 2003 to 2011, found that even though intensiﬁed immu- Clin Gastroenterol Hepatol. 2013;11:43–48. 4. Jussila A, Virta LJ, Pukkala E, et al. Mortality and causes of death in nomodulation therapy is being used in Denmark, the ﬁrst time patients with inﬂammatory bowel disease: a nationwide register study in recurrence rate, hospitalization rate, and surgical rates for patients Finland. J Crohns Colitis. 2014;8:1088–1096. with UC have not decreased. This observation supports the 5. Khor B, Gardet A, Xavier RJ. Genetics and pathogenesis of inﬂammatory bowel disease. Nature. 2011;474:307–317. hypothesis that the administered treatment mostly is symptomatic. 6. Iskandar HN, Ciorba MA. Biomarkers in inﬂammatory bowel disease: However, it is worth mentioning that a study by Reich et al, in current practices and recent advances. Transl Res. 2012;159:313–325. which 481 patients with UC undergoing colectomy in the period 7. Ehrentraut SF, Colgan SP. Implications of protein post-translational mod- iﬁcations in IBD. Inﬂamm Bowel Dis. 2012;18:1378–1388. 1998 to 2011 were included, showed that following the approval of 8. Thompson AI, Lees CW. Genetics of ulcerative colitis. Inﬂamm Bowel anti–tumor necrosis factor treatment for UC in 2005, the colectomy Dis. 2011;17:831–848. rate has dropped. None of the patients included in this study had 9. Jostins L, Ripke S, Weersma RK, et al. Host-microbe interactions have shaped the genetic architecture of inﬂammatory bowel disease. Nature. active disease or was under treatment with anti-tumor necrosis 2012;491:119–124. factor substances. Nonetheless, based on our study, we can con- 10. Xavier RJ, Podolsky DK. Unravelling the pathogenesis of inﬂammatory clude that chronic inﬂammation remains in the UC colonic tissue. bowel disease. Nature. 2007;448:427–434. 11. Bennike T, Birkelund S, Stensballe A, et al. Biomarkers in inﬂammatory In conclusion, we have found an increased abundance of bowel diseases: current status and proteomics identiﬁcation strategies. PMNs in the UC colonic tissue, using high-throughput proteo- World J Gastroenterol. 2014;20:3231–3244. mics, light microscopy, and confocal microscopy. Additionally, 12. Tontini GE, Vecchi M, Pastorelli L, et al. Differential diagnosis in inﬂam- matory bowel disease colitis: state of the art and future perspectives. we have for the ﬁrst time observed NETs, detected by proteomics World J Gastroenterol. 2015;21:21–46. and veriﬁed by light microscopy at 2 distinct locations in a biopsy 13. Kane SV, Sandborn WJ, Rufo PA, et al. Fecal lactoferrin is a sensitive and from a patient with UC. Our ﬁndings demonstrate an activated speciﬁc marker in identifying intestinal inﬂammation. Am J Gastroenterol. 2003;98:1309–1314. innate immune system in well-medicated patients with UC 14. Mendoza JL, Abreu MT. Biological markers in inﬂammatory bowel dis- without disease activity and the presence of chronic inﬂammation ease: practical consideration for clinicians. Gastroenterol Clin Biol. 2009; in the morphologically normal tissue. Additionally, through 33(suppl 3):S158–S173. 15. Johne B, Fagerhol MK, Lyberg T, et al. Functional and clinical aspects of a comparison with other studies, we can conclude that the the myelomonocyte protein calprotectin. Mol Pathol. 1997;50:113–123. proteome of inﬂamed and noninﬂamed tissue from the same 16. Steinbakk M, Naess-Andresen C-F, Fagerhol MK, et al. Antimicrobial patient is much more similar compared with the proteome of actions of calcium binding leucocyte L1 protein, calprotectin. Lancet. 1990;336:763–765. different individuals, indicating that a chronic condition is present 17. Hsieh SY, Shih TC, Yeh CY, et al. Comparative proteomic studies on the even in UC colonic tissue that appears normal during endoscopy. pathogenesis of human ulcerative colitis. Proteomics. 2006;6:5322–5331. However, no indications of bacterial inﬁltrations in the tissue were 18. Poulsen NA, Andersen V, Møller JC, et al. Comparative analysis of in- ﬂamed and non-inﬂamed colon biopsies reveals strong proteomic inﬂam- found in the study. To increase our understanding of the UC and mation proﬁle in patients with ulcerative colitis. BMC Gastroenterol. IBD etiology, future studies should aim at investigating the 2012;12:76. etiology of the immune reaction in the UC colonic tissue. 19. Camerini S, Mauri P. The role of protein and peptide separation before mass spectrometry analysis in clinical proteomics. J Chromatogr A. 2015; 1381:1–12. 20. Han NY, Choi W, Park JM, et al. Label-free quantiﬁcation for discovering ACKNOWLEDGMENTS novel biomarkers in the diagnosis and assessment of disease activity in inﬂammatory bowel disease. J Dig Dis. 2013;14:166–174. Knud and Edith Eriksens Memorial Foundation and Ferring 21. Krzywinski M, Altman N. Points of signiﬁcance: comparing samples— are acknowledged for grants, enabling the collection of the part II. Nat Methods. 2014;11:355–356. biological sample material (V.A.). The Obelske Family Founda- 22. Dignass A, Eliakim R, Magro F, et al. Second European evidence-based consensus on the diagnosis and management of ulcerative colitis part 1: tion and the Svend Andersen Foundation are acknowledged for deﬁnitions and diagnosis. J Crohns Colitis. 2012;6:965–990. grants supporting the analytical platform (A.S.). Knud Hojgaards 23. Fernández-Bañares F, Casalots J, Salas A, et al. Paucicellular lymphocytic Foundation Denmark, the Lundbeck Foundation Denmark, the colitis: is it a minor form of lymphocytic colitis? A clinical pathological and immunological study. Am J Gastroenterol. 2009;104:1189–1198. Oticon Foundation Denmark, and the Otto Monsteds Foundation 24. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years Denmark are acknowledged for grants supporting the collabora- of image analysis. Nat Methods. 2012;9:671–675. tion (T.B.B.). Additionally, the authors would like to thank 25. Hanauer SB, Robinson M, Pruitt R, et al. Budesonide enema for the Kasper B. Lauridsen for help establishing the patient cohort, Lau treatment of active, distal ulcerative colitis and proctitis: a dose-ranging study. Gastroenterology. 1998;115:525–532. Sennels for help with the data analysis, Professor Mogens Vyberg 26. Leon IR, Schwammle V, Jensen ON, et al. Quantitative assessment of in- for help interpreting the histology descriptions, and the PRIDE solution digestion efﬁciency identiﬁes optimal protocols for unbiased pro- team for making the proteomics data publically available. tein analysis. Mol Cell Proteomics. 2013;12:2992–3005. 2066 www.ibdjournal.org Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Neutrophil Extracellular Traps in UC 27. Wisniewski JR, Zougman A, Nagaraj N, et al. Universal sample prepara- 51. Munn DH, Mellor AL. Indoleamine 2,3 dioxygenase and metabolic con- tion method for proteome analysis. Nat Methods. 2009;6:359–362. trol of immune responses. Trends Immunol. 2013;34:137–143. 28. Manza LL, Stamer SL, Ham AJL, et al. Sample preparation and digestion 52. Schmidt SV, Schultze JL. New insights into IDO biology in bacterial and for proteomic analyses using spin ﬁlters. Proteomics. 2005;5:1742–1745. viral infections. Front Immunol. 2014;5:384. 29. Masuda T, Tomita M, Ishihama Y. Phase transfer surfactant-aided trypsin 53. Shkoda A, Werner T, Daniel H, et al. Differential protein expression digestion for membrane proteome analysis. JProteomeRes. 2008;7:731–740. proﬁle in the intestinal epithelium from patients with inﬂammatory bowel 30. Urbaniak GC, Plous S. Research Randomizer (Version 4.0) [computer disease. J Proteome Res. 2007;6:1114–1125. software]. 2013. Available at: http://www.randomizer.org/. Accessed 54. Leach ST, Yang Z, Messina I, et al. Serum and mucosal S100 proteins, October 10, 2013. calprotectin (S100A8/S100A9) and S100A12, are elevated at diagnosis in 31. Cox J, Mann M. MaxQuant enables high peptide identiﬁcation rates, children with inﬂammatory bowel disease. Scand J Gastroenterol. 2007; individualized p.p.b.-range mass accuracies and proteome-wide protein 42:1321–1331. quantiﬁcation. Nat Biotech. 2008;26:1367–1372. 55. Lehmann FS, Burri E, Beglinger C. The role and utility of faecal 32. Cox J, Neuhauser N, Michalski A, et al. Andromeda: a peptide search markers in inﬂammatory bowel disease. Therap Adv Gastroenterol. engine integrated into the MaxQuant environment. J Proteome Res. 2011; 2015;8:23–36. 10:1794–1805. 56. Lu X, Wang M, Qi J, et al. Peptidoglycan recognition proteins are a new 33. Bennike T, Ayturk U, Haslauer CM, et al. A normative study of the class of human bactericidal proteins. J Biol Chem. 2006;281:5895–5907. synovial ﬂuid proteome from healthy porcine knee joints. J Proteome 57. Liu C, Xu Z, Gupta D, et al. Peptidoglycan recognition proteins a novel Res. 2014;13:4377–4387. family of four human innate immunity pattern recognition molecules. 34. Bennike T, Lauridsen KB, Olesen MK, et al. Optimizing the identiﬁcation J Biol Chem. 2001;276:34686–34694. of citrullinated peptides by mass spectrometry: utilizing the inability of 58. Furuta H, Nishi S, Le Beau MM, et al. Sequence of human hexokinase III trypsin to cleave after citrullinated amino acids. J Proteomics Bioinform. cDNA and assignment of the human hexokinase III gene (HK3) to chro- 2013;06:288–295. mosome band 5q35.2 by ﬂuorescence in situ hybridization. Genomics. 35. Deeb SJ, D’Souza RCJ, Cox J, et al. Super-silac allows classiﬁcation of 1996;36:206–209. diffuse large B-cell lymphoma subtypes by their protein expression pro- 59. Lowes W, Walker M, Alberti KG, et al. Hexokinase isoenzymes in normal ﬁles. Mol Cell Proteomics. 2012;11:77–89. and cirrhotic human liver: suppression of glucokinase in cirrhosis. Bio- 36. Bhatia VN, Perlman DH, Costello CE, et al. Software tool for researching chim Biophys Acta. 1998;1379:134–142. annotations of proteins: open-source protein annotation software with data 60. Rosenfeld G, Bressler B. The truth about cigarette smoking and the risk of visualization. Anal Chem. 2009;81:9819–9823. inﬂammatory bowel disease. Am J Gastroenterol. 2012;107:1407–1408. 37. Tusher VG, Tibshirani R, Chu G. Signiﬁcance analysis of microarrays 61. Higuchi LM, Khalili H, Chan AT, et al. A Prospective study of cigarette applied to the ionizing radiation response. Proc Natl Acad Sci U S A. smoking and the risk of inﬂammatory bowel disease in women. Am J 2001;98:5116–5121. Gastroenterol. 2012;107:1399–1406. 38. Hubner NC, Bird AW, Cox J, et al. Quantitative proteomics combined 62. Abraham C, Cho JH. Inﬂammatory bowel disease. N Engl J Med. 2009; with BAC TransgeneOmics reveals in vivo protein interactions. J Cell 361:2066–2078. Biol. 2010;189:739–754. 63. Mahid SS, Minor KS, Soto RE, et al. Smoking and inﬂammatory bowel 39. Murgia M, Nagaraj N, Deshmukh AS, et al. Single muscle ﬁber proteo- disease: a meta-analysis. Mayo Clin Proc. 2006;81:1462–1471. mics reveals unexpected mitochondrial specialization. EMBO Rep. 2015; 64. García Rodríguez LA, González-Pérez A, Johansson S, et al. Risk factors 16:387–395. for inﬂammatory bowel disease in the general population. Aliment Phar- 40. Vizcaíno JA, Deutsch EW, Wang R, et al. ProteomeXchange provides macol Ther. 2005;22:309–315. globally coordinated proteomics data submission and dissemination. Nat 65. Lindberg E, Tysk C, Andersson K, et al. Smoking and inﬂammatory Biotechnol. 2014;32:223–226. bowel disease. A case control study. Gut. 1988;29:352–357. 41. Vizcaíno JA, Côté RG, Csordas A, et al. The PRoteomics IDEntiﬁcations 66. Khor TS, Fujita H, Nagata K, et al. Biopsy interpretation of colonic (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res. biopsies when inﬂammatory bowel disease is excluded. J Gastroenterol. 2013;41:D1063–D1069. 2012;47:226–248. 42. Fournier BM, Parkos CA. The role of neutrophils during intestinal inﬂam- 67. Geboes K. Histopathology of Crohn’s disease and ulcerative colitis. In- mation. Mucosal Immunol. 2012;5:354–366. ﬂamm Bowel Dis. 2003:255–276. 43. Brinkmann V, Zychlinsky A. Neutrophil extracellular traps: is immunity 68. Branzk N, Lubojemska A, Hardison SE, et al. Neutrophils sense microbe the second function of chromatin? J Cell Biol. 2012;198:773–783. size and selectively release neutrophil extracellular traps in response to 44. Rao PK, Li Q. Principal component analysis of proteome dynamics in large pathogens. Nat Immunol. 2014;15:1017–1025. iron-starved mycobacterium tuberculosis. J Proteomics Bioinform. 2009; 69. Fava F, Danese S. Intestinal microbiota in inﬂammatory bowel disease: 2:19–31. friend of foe? World J Gastroenterol. 2011;17:557–566. 45. O’Donoghue AJ, Jin Y, Knudsen GM, et al. Global substrate proﬁling of 70. Tlaskalová-Hogenová H, Stepánková R, Hudcovic T, et al. Commensal proteases in human neutrophil extracellular traps reveals consensus motif bacteria (normal microﬂora), mucosal immunity and chronic inﬂammatory predominantly contributed by Elastase Glogauer M, ed. PLoS One. 2013; and autoimmune diseases. Immunol Lett. 2004;93:97–108. 8:e75141. 71. Beiter T, Fragasso A, Hartl D, et al. Neutrophil extracellular traps: a walk 46. Mantovani A, Cassatella MA, Costantini C, et al. Neutrophils in the on the wild side of exercise immunology. Sports Med. 2015;45:625–640. activation and regulation of innate and adaptive immunity. Nat Rev Im- 72. Beiter T, Fragasso A, Hudemann J, et al. Neutrophils release extracel- munol. 2011;11:519–531. lular DNA traps in response to exercise. J Appl Physiol (1985). 2014; 47. Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular 117:325–333. traps kill bacteria. Science. 2004;303:1532–1535. 73. Stephan A, Fabri M. The NET, the trap, and the pathogen: neutrophil 48. Schoepfer AM, Beglinger C, Straumann A, et al. Fecal calprotectin more extracellular traps in cutaneous immunity. Exp Dermatol. 2014;24: accurately reﬂects endoscopic activity ulcerative colitis than Lichtiger 161–166. Index, C-reactive protein platelets, hemoglobin, and blood leukocytes. 74. Vester-Andersen MK, Vind I, Prosberg MV, et al. Hospitalisation, surgi- Inﬂamm Bowel Dis. 2013;19:332–341. cal and medical recurrence rates in inﬂammatory bowel disease 2003– 49. Vos MD, Louis EJ, Jahnsen J, et al. Consecutive fecal calprotectin meas- 2011—a Danish population-based cohort study. J Crohns Colitis. 2014;8: urements predict relapse patients ulcerative colitis receiving inﬂiximab 1675–1683. maintenance therapy. Inﬂamm Bowel Dis. 2013;19:2111–2117. 75. Reich KM, Chang H-J, Rezaie A, et al. The incidence rate of colectomy 50. Barceló-Batllori S, André M, Servis C, et al. Proteomic analysis of cyto- for medically refractory ulcerative colitis has declined in parallel with kine induced proteins in human intestinal epithelial cells: implications for increasing anti-TNF use: a time-trend study. Aliment Pharmacol Ther. inﬂammatory bowel diseases. Proteomics. 2002;2:551–560. 2014;40:629–638. www.ibdjournal.org 2067 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Inflammatory Bowel Diseases Pubmed Central http://www.deepdyve.com/lp/pubmed-central/neutrophil-extracellular-traps-in-ulcerative-colitis-a-proteome-0n2neQGn80

Loading next page...

References (153)

T. Khor, H. Fujita, K. Nagata, M. Shimizu, G. Lauwers (2012)
Biopsy interpretation of colonic biopsies when inflammatory bowel disease is excluded
Journal of Gastroenterology, 47
(BeiterTFragassoAHudemannJ Neutrophils release extracellular DNA traps in response to exercise. J Appl Physiol (1985). 2014;117:325–333.24833781)
BeiterTFragassoAHudemannJ Neutrophils release extracellular DNA traps in response to exercise. J Appl Physiol (1985). 2014;117:325–333.24833781
BeiterTFragassoAHudemannJ Neutrophils release extracellular DNA traps in response to exercise. J Appl Physiol (1985). 2014;117:325–333.24833781, BeiterTFragassoAHudemannJ Neutrophils release extracellular DNA traps in response to exercise. J Appl Physiol (1985). 2014;117:325–333.24833781
(ManzaLLStamerSLHamAJL Sample preparation and digestion for proteomic analyses using spin filters. Proteomics. 2005;5:1742–1745.15761957)
ManzaLLStamerSLHamAJL Sample preparation and digestion for proteomic analyses using spin filters. Proteomics. 2005;5:1742–1745.15761957
ManzaLLStamerSLHamAJL Sample preparation and digestion for proteomic analyses using spin filters. Proteomics. 2005;5:1742–1745.15761957, ManzaLLStamerSLHamAJL Sample preparation and digestion for proteomic analyses using spin filters. Proteomics. 2005;5:1742–1745.15761957
(CoxJMannM MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotech. 2008;26:1367–1372.)
CoxJMannM MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotech. 2008;26:1367–1372.
CoxJMannM MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotech. 2008;26:1367–1372., CoxJMannM MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotech. 2008;26:1367–1372.
K. Eboes (2003)
Histopathology of Crohn's Disease and Ulcerative Colitis
Xiaofeng Lu, Minhui Wang, Jin Qi, Haitao Wang, Xinna Li, D. Gupta, R. Dziarski (2006)
Peptidoglycan Recognition Proteins Are a New Class of Human Bactericidal Proteins*
Journal of Biological Chemistry, 281
Linda Manza, Sheryl Stamer, A. Ham, S. Codreanu, Daniel Liebler (2005)
Sample preparation and digestion for proteomic analyses using spin filters
PROTEOMICS, 5
Nora Branzk, A. Lubojemska, S. Hardison, Qian Wang, M. Gutierrez, Gordon Brown, V. Papayannopoulos (2014)
Neutrophils sense microbial size and selectively release neutrophil extracellular traps in response to large pathogens
Nature immunology, 15
(KrzywinskiMAltmanN Points of significance: comparing samples—part II. Nat Methods. 2014;11:355–356.)
KrzywinskiMAltmanN Points of significance: comparing samples—part II. Nat Methods. 2014;11:355–356.
KrzywinskiMAltmanN Points of significance: comparing samples—part II. Nat Methods. 2014;11:355–356., KrzywinskiMAltmanN Points of significance: comparing samples—part II. Nat Methods. 2014;11:355–356.
(BranzkNLubojemskaAHardisonSE Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nat Immunol. 2014;15:1017–1025.25217981)
BranzkNLubojemskaAHardisonSE Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nat Immunol. 2014;15:1017–1025.25217981
BranzkNLubojemskaAHardisonSE Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nat Immunol. 2014;15:1017–1025.25217981, BranzkNLubojemskaAHardisonSE Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nat Immunol. 2014;15:1017–1025.25217981
(DeebSJD'SouzaRCJCoxJ Super-silac allows classification of diffuse large B-cell lymphoma subtypes by their protein expression profiles. Mol Cell Proteomics. 2012;11:77–89.22442255)
DeebSJD'SouzaRCJCoxJ Super-silac allows classification of diffuse large B-cell lymphoma subtypes by their protein expression profiles. Mol Cell Proteomics. 2012;11:77–89.22442255
DeebSJD'SouzaRCJCoxJ Super-silac allows classification of diffuse large B-cell lymphoma subtypes by their protein expression profiles. Mol Cell Proteomics. 2012;11:77–89.22442255, DeebSJD'SouzaRCJCoxJ Super-silac allows classification of diffuse large B-cell lymphoma subtypes by their protein expression profiles. Mol Cell Proteomics. 2012;11:77–89.22442255
(AbrahamCChoJH Inflammatory bowel disease. N Engl J Med. 2009;361:2066–2078.19923578)
AbrahamCChoJH Inflammatory bowel disease. N Engl J Med. 2009;361:2066–2078.19923578
AbrahamCChoJH Inflammatory bowel disease. N Engl J Med. 2009;361:2066–2078.19923578, AbrahamCChoJH Inflammatory bowel disease. N Engl J Med. 2009;361:2066–2078.19923578
W. Lowes, M. Walker, K. Alberti, L. Agius (1998)
Hexokinase isoenzymes in normal and cirrhotic human liver: suppression of glucokinase in cirrhosis.
Biochimica et biophysica acta, 1379 1
K. Geboes (2008)
What histologic features best differentiate Crohn's disease from ulcerative colitis?
Inflammatory bowel diseases, 14 Suppl 2
(Barceló-BatlloriSAndréMServisC Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells: implications for inflammatory bowel diseases. Proteomics. 2002;2:551–560.11987129)
Barceló-BatlloriSAndréMServisC Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells: implications for inflammatory bowel diseases. Proteomics. 2002;2:551–560.11987129
Barceló-BatlloriSAndréMServisC Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells: implications for inflammatory bowel diseases. Proteomics. 2002;2:551–560.11987129, Barceló-BatlloriSAndréMServisC Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells: implications for inflammatory bowel diseases. Proteomics. 2002;2:551–560.11987129
(LuXWangMQiJ Peptidoglycan recognition proteins are a new class of human bactericidal proteins. J Biol Chem. 2006;281:5895–5907.16354652)
LuXWangMQiJ Peptidoglycan recognition proteins are a new class of human bactericidal proteins. J Biol Chem. 2006;281:5895–5907.16354652
LuXWangMQiJ Peptidoglycan recognition proteins are a new class of human bactericidal proteins. J Biol Chem. 2006;281:5895–5907.16354652, LuXWangMQiJ Peptidoglycan recognition proteins are a new class of human bactericidal proteins. J Biol Chem. 2006;281:5895–5907.16354652
(StephanAFabriM The NET, the trap, and the pathogen: neutrophil extracellular traps in cutaneous immunity. Exp Dermatol. 2014;24:161–166.25421224)
StephanAFabriM The NET, the trap, and the pathogen: neutrophil extracellular traps in cutaneous immunity. Exp Dermatol. 2014;24:161–166.25421224
StephanAFabriM The NET, the trap, and the pathogen: neutrophil extracellular traps in cutaneous immunity. Exp Dermatol. 2014;24:161–166.25421224, StephanAFabriM The NET, the trap, and the pathogen: neutrophil extracellular traps in cutaneous immunity. Exp Dermatol. 2014;24:161–166.25421224
J. Cox, M. Mann (2008)
MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification
Nature Biotechnology, 26
T. Bennike, Kasper Lauridsen, M. Olesen, V. Andersen, S. Birkelund, A. Stensballe (2013)
Optimizing the Identification of Citrullinated Peptides by Mass Spectrometry: Utilizing the Inability of Trypsin to Cleave after Citrullinated Amino Acids
Journal of Proteomics & Bioinformatics, 6
H. Tlaskalova-Hogenova, R. Štěpánková, T. Hudcovic, L. Tučková, B. Cukrowska, R. Lodinová-Žádníková, H. Kozakova, P. Rossmann, J. Bartova, D. Sokol, D. Funda, D. Borovská, Z. Reháková, J. Šinkora, Jaroslav Hofman, P. Drastich, A. Kokesova (2004)
Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases.
Immunology letters, 93 2-3
E. Szigethy, L. McLafferty, A. Goyal (2011)
Inflammatory bowel disease.
Pediatric clinics of North America, 58 4
(CoxJNeuhauserNMichalskiA Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res. 2011;10:1794–1805.21254760)
CoxJNeuhauserNMichalskiA Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res. 2011;10:1794–1805.21254760
CoxJNeuhauserNMichalskiA Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res. 2011;10:1794–1805.21254760, CoxJNeuhauserNMichalskiA Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res. 2011;10:1794–1805.21254760
F. Fava, S. Danese (2011)
Intestinal microbiota in inflammatory bowel disease: friend of foe?
World journal of gastroenterology, 17 5
(MasudaTTomitaMIshihamaY Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J Proteome Res. 2008;7:731–740.18183947)
MasudaTTomitaMIshihamaY Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J Proteome Res. 2008;7:731–740.18183947
MasudaTTomitaMIshihamaY Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J Proteome Res. 2008;7:731–740.18183947, MasudaTTomitaMIshihamaY Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J Proteome Res. 2008;7:731–740.18183947
A. O’Donoghue, Ye Jin, G. Knudsen, N. Perera, D. Jenne, John Murphy, C. Craik, T. Hermiston (2013)
Global Substrate Profiling of Proteases in Human Neutrophil Extracellular Traps Reveals Consensus Motif Predominantly Contributed by Elastase
PLoS ONE, 8
A. Dignass, R. Eliakim, F. Magro, C. Maaser, Y. Chowers, K. Geboes, G. Mantzaris, W. Reinisch, J. Colombel, S. Vermeire, S. Travis, J. Lindsay, G. Assche (2012)
Second European evidence-based consensus on the diagnosis and management of ulcerative colitis part 1: definitions and diagnosis.
Journal of Crohn's & colitis, 6 10
F. Fernández-Bañares, J. Casalots, A. Salas, M. Esteve, M. Rosinach, M. Forné, C. Loras, R. Santaolalla, J. Espinós, J. Viver (2009)
Paucicellular Lymphocytic Colitis: Is It a Minor Form of Lymphocytic Colitis? A Clinical Pathological and Immunological Study
The American Journal of Gastroenterology, 104
S. Leach, Zheng Yang, I. Messina, Changjie Song, C. Geczy, A. Cunningham, A. Day (2007)
Serum and mucosal S100 proteins, calprotectin (S100A8/S100A9) and S100A12, are elevated at diagnosis in children with inflammatory bowel disease
Scandinavian Journal of Gastroenterology, 42
Oussema Souiai, F. Guerfali, Slimane Miled, C. Brun, Alia Benkahla (2001)
In silico prediction of protein-protein interactions in human macrophages
BMC Research Notes, 7
F. Lehmann, E. Burri, C. Beglinger (2015)
The role and utility of faecal markers in inflammatory bowel disease
Therapeutic Advances in Gastroenterology, 8
(BrinkmannVReichardUGoosmannC Neutrophil extracellular traps kill bacteria. Science. 2004;303:1532–1535.15001782)
BrinkmannVReichardUGoosmannC Neutrophil extracellular traps kill bacteria. Science. 2004;303:1532–1535.15001782
BrinkmannVReichardUGoosmannC Neutrophil extracellular traps kill bacteria. Science. 2004;303:1532–1535.15001782, BrinkmannVReichardUGoosmannC Neutrophil extracellular traps kill bacteria. Science. 2004;303:1532–1535.15001782
M. Murgia, Nagarjuna Nagaraj, A. Deshmukh, Marlis Zeiler, Pasqua Cancellara, Irene Moretti, C. Reggiani, S. Schiaffino, M. Mann (2015)
Single muscle fiber proteomics reveals unexpected mitochondrial specialization
EMBO Reports, 16
M. Krzywinski, Naomi Altman (2014)
Points of significance: Comparing samples—part I
Nature Methods, 11
B. Johne, M. Fagerhol, T. Lyberg, H. Prydz, P. Brandtzaeg, C. Naess-Andresen, I. Dale (1997)
Functional and clinical aspects of the myelomonocyte protein calprotectin.
Molecular Pathology, 50
(MantovaniACassatellaMACostantiniC Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol. 2011;11:519–531.21785456)
MantovaniACassatellaMACostantiniC Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol. 2011;11:519–531.21785456
MantovaniACassatellaMACostantiniC Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol. 2011;11:519–531.21785456, MantovaniACassatellaMACostantiniC Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol. 2011;11:519–531.21785456
S. Barceló-Batllori, M. André, C. Servis, N. Lévy, O. Takikawa, P. Michetti, M. Reymond, E. Felley-Bosco (2002)
Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells: Implications for inflammatory bowel diseases
PROTEOMICS, 2
(2013)
Version 4.0) [computer software]. 2013. Available at: http://www.randomizer.org
(KhorTSFujitaHNagataK Biopsy interpretation of colonic biopsies when inflammatory bowel disease is excluded. J Gastroenterol. 2012;47:226–248.22322659)
KhorTSFujitaHNagataK Biopsy interpretation of colonic biopsies when inflammatory bowel disease is excluded. J Gastroenterol. 2012;47:226–248.22322659
KhorTSFujitaHNagataK Biopsy interpretation of colonic biopsies when inflammatory bowel disease is excluded. J Gastroenterol. 2012;47:226–248.22322659, KhorTSFujitaHNagataK Biopsy interpretation of colonic biopsies when inflammatory bowel disease is excluded. J Gastroenterol. 2012;47:226–248.22322659
G. Rosenfeld, B. Bressler (2012)
Editorial: The Truth About Cigarette Smoking and the Risk of Inflammatory Bowel Disease
The American Journal of Gastroenterology, 107
(CameriniSMauriP The role of protein and peptide separation before mass spectrometry analysis in clinical proteomics. J Chromatogr A. 2015;1381:1–12.25618357)
CameriniSMauriP The role of protein and peptide separation before mass spectrometry analysis in clinical proteomics. J Chromatogr A. 2015;1381:1–12.25618357
CameriniSMauriP The role of protein and peptide separation before mass spectrometry analysis in clinical proteomics. J Chromatogr A. 2015;1381:1–12.25618357, CameriniSMauriP The role of protein and peptide separation before mass spectrometry analysis in clinical proteomics. J Chromatogr A. 2015;1381:1–12.25618357
L. Jostins, S. Ripke, R. Weersma, R. Duerr, D. McGovern, Ken Hui, James Lee, L. Schumm, Y. Sharma, Carl Anderson, J. Essers, M. Mitrovič, Kaida Ning, I. Cleynen, E. Théâtre, Sarah Spain, Soumya Raychaudhuri, P. Goyette, Zhi Wei, Clara Abraham, J. Achkar, Tariq Ahmad, L. Amininejad, Ashwin Ananthakrishnan, Vibeke Andersen, Jane Andrews, L. Baidoo, T. Balschun, P. Bampton, Alain Bitton, G. Boucher, S. Brand, Carsten Büning, A. Cohain, Sven Cichon, Mauro D’Amato, Dirk Jong, Kathy Devaney, Marla Dubinsky, C. Edwards, D. Ellinghaus, Lynnette Ferguson, Denis Franchimont, K. Fransén, R. Gearry, Michel Georges, C. Gieger, J. Glas, T. Haritunians, A. Hart, C. Hawkey, M. Hedl, Xinli Hu, Tom Karlsen, L. Kupčinskas, S. Kugathasan, A. Latiano, D. Laukens, I. Lawrance, Charlie Lees, É. Louis, G. Mahy, John Mansfield, Angharad Morgan, C. Mowat, W. Newman, O. Palmieri, C. Ponsioen, U. Potocnik, N. Prescott, Miguel Regueiro, Jerome Rotter, Richard Russell, J. Sanderson, Miquel Sans, J. Satsangi, Stefan Schreiber, L. Simms, Jurgita Šventoraitytė, S. Targan, Kent Taylor, M. Tremelling, H Verspaget, M. Vos, C. Wijmenga, D. Wilson, J. Winkelmann, R. Xavier, Sebastian Zeissig, Bin Zhang, Clarence Zhang, Hongyu Zhao, M. Silverberg, V. Annese, H. Hakonarson, S. Brant, G. Radford-Smith, Christopher Mathew, J. Rioux, Eric Schadt, M. Daly, A. Franke, M. Parkes, S. Vermeire, J. Barrett, Judy Cho (2012)
Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease
Nature, 491
(LehmannFSBurriEBeglingerC The role and utility of faecal markers in inflammatory bowel disease. Therap Adv Gastroenterol. 2015;8:23–36.)
LehmannFSBurriEBeglingerC The role and utility of faecal markers in inflammatory bowel disease. Therap Adv Gastroenterol. 2015;8:23–36.
LehmannFSBurriEBeglingerC The role and utility of faecal markers in inflammatory bowel disease. Therap Adv Gastroenterol. 2015;8:23–36., LehmannFSBurriEBeglingerC The role and utility of faecal markers in inflammatory bowel disease. Therap Adv Gastroenterol. 2015;8:23–36.
(García RodríguezLAGonzález-PérezAJohanssonS Risk factors for inflammatory bowel disease in the general population. Aliment Pharmacol Ther. 2005;22:309–315.16097997)
García RodríguezLAGonzález-PérezAJohanssonS Risk factors for inflammatory bowel disease in the general population. Aliment Pharmacol Ther. 2005;22:309–315.16097997
García RodríguezLAGonzález-PérezAJohanssonS Risk factors for inflammatory bowel disease in the general population. Aliment Pharmacol Ther. 2005;22:309–315.16097997, García RodríguezLAGonzález-PérezAJohanssonS Risk factors for inflammatory bowel disease in the general population. Aliment Pharmacol Ther. 2005;22:309–315.16097997
A. Shkoda, Tanja Werner, H. Daniel, M. Gunckel, G. Rogler, D. Haller (2007)
Differential protein expression profile in the intestinal epithelium from patients with inflammatory bowel disease.
Journal of proteome research, 6 3
J. Vizcaíno, R. Côté, A. Csordas, José Dianes, A. Fabregat, Joseph Foster, J. Griss, E. Alpi, Melih Birim, Javier Contell, Gavin O'Kelly, Andreas Schoenegger, David Ovelleiro, Yasset Pérez-Riverol, F. Reisinger, D. Rios, Rui Wang, H. Hermjakob (2012)
The Proteomics Identifications (PRIDE) database and associated tools: status in 2013
Nucleic Acids Research, 41
(XavierRJPodolskyDK Unravelling the pathogenesis of inflammatory bowel disease. Nature. 2007;448:427–434.17653185)
XavierRJPodolskyDK Unravelling the pathogenesis of inflammatory bowel disease. Nature. 2007;448:427–434.17653185
XavierRJPodolskyDK Unravelling the pathogenesis of inflammatory bowel disease. Nature. 2007;448:427–434.17653185, XavierRJPodolskyDK Unravelling the pathogenesis of inflammatory bowel disease. Nature. 2007;448:427–434.17653185
(JussilaAVirtaLJPukkalaE Mortality and causes of death in patients with inflammatory bowel disease: a nationwide register study in Finland. J Crohns Colitis. 2014;8:1088–1096.24630486)
JussilaAVirtaLJPukkalaE Mortality and causes of death in patients with inflammatory bowel disease: a nationwide register study in Finland. J Crohns Colitis. 2014;8:1088–1096.24630486
JussilaAVirtaLJPukkalaE Mortality and causes of death in patients with inflammatory bowel disease: a nationwide register study in Finland. J Crohns Colitis. 2014;8:1088–1096.24630486, JussilaAVirtaLJPukkalaE Mortality and causes of death in patients with inflammatory bowel disease: a nationwide register study in Finland. J Crohns Colitis. 2014;8:1088–1096.24630486
(JohneBFagerholMKLybergT Functional and clinical aspects of the myelomonocyte protein calprotectin. Mol Pathol. 1997;50:113–123.9292145)
JohneBFagerholMKLybergT Functional and clinical aspects of the myelomonocyte protein calprotectin. Mol Pathol. 1997;50:113–123.9292145
JohneBFagerholMKLybergT Functional and clinical aspects of the myelomonocyte protein calprotectin. Mol Pathol. 1997;50:113–123.9292145, JohneBFagerholMKLybergT Functional and clinical aspects of the myelomonocyte protein calprotectin. Mol Pathol. 1997;50:113–123.9292145
(LeonIRSchwammleVJensenON Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis. Mol Cell Proteomics. 2013;12:2992–3005.23792921)
LeonIRSchwammleVJensenON Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis. Mol Cell Proteomics. 2013;12:2992–3005.23792921
LeonIRSchwammleVJensenON Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis. Mol Cell Proteomics. 2013;12:2992–3005.23792921, LeonIRSchwammleVJensenON Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis. Mol Cell Proteomics. 2013;12:2992–3005.23792921
(HsiehSYShihTCYehCY Comparative proteomic studies on the pathogenesis of human ulcerative colitis. Proteomics. 2006;6:5322–5331.16947118)
HsiehSYShihTCYehCY Comparative proteomic studies on the pathogenesis of human ulcerative colitis. Proteomics. 2006;6:5322–5331.16947118
HsiehSYShihTCYehCY Comparative proteomic studies on the pathogenesis of human ulcerative colitis. Proteomics. 2006;6:5322–5331.16947118, HsiehSYShihTCYehCY Comparative proteomic studies on the pathogenesis of human ulcerative colitis. Proteomics. 2006;6:5322–5331.16947118
(BrinkmannVZychlinskyA Neutrophil extracellular traps: is immunity the second function of chromatin? J Cell Biol. 2012;198:773–783.22945932)
BrinkmannVZychlinskyA Neutrophil extracellular traps: is immunity the second function of chromatin? J Cell Biol. 2012;198:773–783.22945932
BrinkmannVZychlinskyA Neutrophil extracellular traps: is immunity the second function of chromatin? J Cell Biol. 2012;198:773–783.22945932, BrinkmannVZychlinskyA Neutrophil extracellular traps: is immunity the second function of chromatin? J Cell Biol. 2012;198:773–783.22945932
(LowesWWalkerMAlbertiKG Hexokinase isoenzymes in normal and cirrhotic human liver: suppression of glucokinase in cirrhosis. Biochim Biophys Acta. 1998;1379:134–142.9468341)
LowesWWalkerMAlbertiKG Hexokinase isoenzymes in normal and cirrhotic human liver: suppression of glucokinase in cirrhosis. Biochim Biophys Acta. 1998;1379:134–142.9468341
LowesWWalkerMAlbertiKG Hexokinase isoenzymes in normal and cirrhotic human liver: suppression of glucokinase in cirrhosis. Biochim Biophys Acta. 1998;1379:134–142.9468341, LowesWWalkerMAlbertiKG Hexokinase isoenzymes in normal and cirrhotic human liver: suppression of glucokinase in cirrhosis. Biochim Biophys Acta. 1998;1379:134–142.9468341
S. Camerini, P. Mauri (2015)
The role of protein and peptide separation before mass spectrometry analysis in clinical proteomics.
Journal of chromatography. A, 1381
J. Vizcaíno, E. Deutsch, Rui Wang, A. Csordas, F. Reisinger, D. Rios, José Dianes, Zhi Sun, T. Farrah, N. Bandeira, P. Binz, I. Xenarios, M. Eisenacher, Gerhard Mayer, L. Gatto, A. Campos, R. Chalkley, Hans-Joachim Kraus, J. Albar, S. Martínez-Bartolomé, R. Apweiler, G. Omenn, L. Martens, A. Jones, H. Hermjakob (2014)
ProteomeXchange provides globally co-ordinated proteomics data submission and dissemination
Nature biotechnology, 32
(HubnerNCBirdAWCoxJ Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions. J Cell Biol. 2010;189:739–754.20479470)
HubnerNCBirdAWCoxJ Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions. J Cell Biol. 2010;189:739–754.20479470
HubnerNCBirdAWCoxJ Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions. J Cell Biol. 2010;189:739–754.20479470, HubnerNCBirdAWCoxJ Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions. J Cell Biol. 2010;189:739–754.20479470
R. Xavier, D. Podolsky (2007)
Unravelling the pathogenesis of inflammatory bowel disease
Nature, 448
(FurutaHNishiSLe BeauMM Sequence of human hexokinase III cDNA and assignment of the human hexokinase III gene (HK3) to chromosome band 5q35.2 by fluorescence in situ hybridization. Genomics. 1996;36:206–209.8812439)
FurutaHNishiSLe BeauMM Sequence of human hexokinase III cDNA and assignment of the human hexokinase III gene (HK3) to chromosome band 5q35.2 by fluorescence in situ hybridization. Genomics. 1996;36:206–209.8812439
FurutaHNishiSLe BeauMM Sequence of human hexokinase III cDNA and assignment of the human hexokinase III gene (HK3) to chromosome band 5q35.2 by fluorescence in situ hybridization. Genomics. 1996;36:206–209.8812439, FurutaHNishiSLe BeauMM Sequence of human hexokinase III cDNA and assignment of the human hexokinase III gene (HK3) to chromosome band 5q35.2 by fluorescence in situ hybridization. Genomics. 1996;36:206–209.8812439
Vivek Bhatia, David Perlman, C. Costello, M. McComb (2009)
Software tool for researching annotations of proteins: open-source protein annotation software with data visualization.
Analytical chemistry, 81 23
S. Odes, H. Vardi, M. Friger, F. Wolters, M. Russel, L. Riis, P. Munkholm, P. Politi, E. Tsianos, J. Clofent, S. Vermeire, E. Monteiro, I. Mouzas, G. Fornaciari, J. Sijbrandij, C. Limonard, G. Zeijl, C. O'Morain, B. Moum, M. Vatn, R. Stockbrugger (2006)
Cost analysis and cost determinants in a European inflammatory bowel disease inception cohort with 10 years of follow-up evaluation.
Gastroenterology, 131 3
(LiuCXuZGuptaD Peptidoglycan recognition proteins a novel family of four human innate immunity pattern recognition molecules. J Biol Chem. 2001;276:34686–34694.11461926)
LiuCXuZGuptaD Peptidoglycan recognition proteins a novel family of four human innate immunity pattern recognition molecules. J Biol Chem. 2001;276:34686–34694.11461926
LiuCXuZGuptaD Peptidoglycan recognition proteins a novel family of four human innate immunity pattern recognition molecules. J Biol Chem. 2001;276:34686–34694.11461926, LiuCXuZGuptaD Peptidoglycan recognition proteins a novel family of four human innate immunity pattern recognition molecules. J Biol Chem. 2001;276:34686–34694.11461926
Chao Liu, Zhaojun Xu, D. Gupta, R. Dziarski (2001)
Peptidoglycan Recognition Proteins
The Journal of Biological Chemistry, 276
(FavaFDaneseS Intestinal microbiota in inflammatory bowel disease: friend of foe? World J Gastroenterol. 2011;17:557–566.21350704)
FavaFDaneseS Intestinal microbiota in inflammatory bowel disease: friend of foe? World J Gastroenterol. 2011;17:557–566.21350704
FavaFDaneseS Intestinal microbiota in inflammatory bowel disease: friend of foe? World J Gastroenterol. 2011;17:557–566.21350704, FavaFDaneseS Intestinal microbiota in inflammatory bowel disease: friend of foe? World J Gastroenterol. 2011;17:557–566.21350704
L. Higuchi, H. Khalili, A. Chan, J. Richter, A. Bousvaros, C. Fuchs (2012)
A Prospective Study of Cigarette Smoking and the Risk of Inflammatory Bowel Disease in Women
The American Journal of Gastroenterology, 107
(LeachSTYangZMessinaI Serum and mucosal S100 proteins, calprotectin (S100A8/S100A9) and S100A12, are elevated at diagnosis in children with inflammatory bowel disease. Scand J Gastroenterol. 2007;42:1321–1331.17852869)
LeachSTYangZMessinaI Serum and mucosal S100 proteins, calprotectin (S100A8/S100A9) and S100A12, are elevated at diagnosis in children with inflammatory bowel disease. Scand J Gastroenterol. 2007;42:1321–1331.17852869
LeachSTYangZMessinaI Serum and mucosal S100 proteins, calprotectin (S100A8/S100A9) and S100A12, are elevated at diagnosis in children with inflammatory bowel disease. Scand J Gastroenterol. 2007;42:1321–1331.17852869, LeachSTYangZMessinaI Serum and mucosal S100 proteins, calprotectin (S100A8/S100A9) and S100A12, are elevated at diagnosis in children with inflammatory bowel disease. Scand J Gastroenterol. 2007;42:1321–1331.17852869
Alexander Stephan, M. Fabri (2015)
The NET, the trap and the pathogen: neutrophil extracellular traps in cutaneous immunity
Experimental Dermatology, 24
V. Brinkmann, U. Reichard, C. Goosmann, B. Fauler, Yvonne Uhlemann, D. Weiss, Y. Weinrauch, A. Zychlinsky (2004)
Neutrophil Extracellular Traps Kill Bacteria
Science, 303
M. Vos, E. Louis, J. Jahnsen, J. Vandervoort, M. Noman, O. Dewit, G. D'Haens, D. Franchimont, F. Baert, R. Torp, M. Henriksen, P. Potvin, P. Hootegem, P. Hindryckx, T. Moreels, A. Collard, L. Karlsen, E. Kittang, G. Lambrecht, T. Grimstad, J. Koch, I. Lygren, Jean-Claude Coche, F. Mana, A. Gossum, J. Belaiche, M. Cool, F. Fontaine, J. Maisin, V. Muls, B. Neuville, D. Staessen, G. Assche, Thomas Lange, I. Solberg, B. Cruyssen, S. Vermeire (2013)
Consecutive Fecal Calprotectin Measurements to Predict Relapse in Patients with Ulcerative Colitis Receiving Infliximab Maintenance Therapy
Inflammatory Bowel Diseases, 19
N. Molodecky, I. Soon, D. Rabi, W. Ghali, Mollie Ferris, G. Chernoff, E. Benchimol, R. Panaccione, Subrata Ghosh, H. Barkema, G. Kaplan (2012)
Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review.
Gastroenterology, 142 1
(TontiniGEVecchiMPastorelliL Differential diagnosis in inflammatory bowel disease colitis: state of the art and future perspectives. World J Gastroenterol. 2015;21:21–46.25574078)
TontiniGEVecchiMPastorelliL Differential diagnosis in inflammatory bowel disease colitis: state of the art and future perspectives. World J Gastroenterol. 2015;21:21–46.25574078
TontiniGEVecchiMPastorelliL Differential diagnosis in inflammatory bowel disease colitis: state of the art and future perspectives. World J Gastroenterol. 2015;21:21–46.25574078, TontiniGEVecchiMPastorelliL Differential diagnosis in inflammatory bowel disease colitis: state of the art and future perspectives. World J Gastroenterol. 2015;21:21–46.25574078
Na-Young Han, Wooseok Choi, Jong-Moon Park, Eun Kim, Hookeun Lee, K. Hahm (2013)
Label‐free quantification for discovering novel biomarkers in the diagnosis and assessment of disease activity in inflammatory bowel disease
Journal of Digestive Diseases, 14
(HiguchiLMKhaliliHChanAT A Prospective study of cigarette smoking and the risk of inflammatory bowel disease in women. Am J Gastroenterol. 2012;107:1399–1406.22777340)
HiguchiLMKhaliliHChanAT A Prospective study of cigarette smoking and the risk of inflammatory bowel disease in women. Am J Gastroenterol. 2012;107:1399–1406.22777340
HiguchiLMKhaliliHChanAT A Prospective study of cigarette smoking and the risk of inflammatory bowel disease in women. Am J Gastroenterol. 2012;107:1399–1406.22777340, HiguchiLMKhaliliHChanAT A Prospective study of cigarette smoking and the risk of inflammatory bowel disease in women. Am J Gastroenterol. 2012;107:1399–1406.22777340
A. Jussila, L. Virta, E. Pukkala, M. Färkkilä (2014)
Mortality and causes of death in patients with inflammatory bowel disease: a nationwide register study in Finland.
Journal of Crohn's & colitis, 8 9
(Vester-AndersenMKVindIProsbergMV Hospitalisation, surgical and medical recurrence rates in inflammatory bowel disease 2003–2011—a Danish population-based cohort study. J Crohns Colitis. 2014;8:1675–1683.25154681)
Vester-AndersenMKVindIProsbergMV Hospitalisation, surgical and medical recurrence rates in inflammatory bowel disease 2003–2011—a Danish population-based cohort study. J Crohns Colitis. 2014;8:1675–1683.25154681
Vester-AndersenMKVindIProsbergMV Hospitalisation, surgical and medical recurrence rates in inflammatory bowel disease 2003–2011—a Danish population-based cohort study. J Crohns Colitis. 2014;8:1675–1683.25154681, Vester-AndersenMKVindIProsbergMV Hospitalisation, surgical and medical recurrence rates in inflammatory bowel disease 2003–2011—a Danish population-based cohort study. J Crohns Colitis. 2014;8:1675–1683.25154681
(DignassAEliakimRMagroF Second European evidence-based consensus on the diagnosis and management of ulcerative colitis part 1: definitions and diagnosis. J Crohns Colitis. 2012;6:965–990.23040452)
DignassAEliakimRMagroF Second European evidence-based consensus on the diagnosis and management of ulcerative colitis part 1: definitions and diagnosis. J Crohns Colitis. 2012;6:965–990.23040452
DignassAEliakimRMagroF Second European evidence-based consensus on the diagnosis and management of ulcerative colitis part 1: definitions and diagnosis. J Crohns Colitis. 2012;6:965–990.23040452, DignassAEliakimRMagroF Second European evidence-based consensus on the diagnosis and management of ulcerative colitis part 1: definitions and diagnosis. J Crohns Colitis. 2012;6:965–990.23040452
(Fernández-BañaresFCasalotsJSalasA Paucicellular lymphocytic colitis: is it a minor form of lymphocytic colitis? A clinical pathological and immunological study. Am J Gastroenterol. 2009;104:1189–1198.19352342)
Fernández-BañaresFCasalotsJSalasA Paucicellular lymphocytic colitis: is it a minor form of lymphocytic colitis? A clinical pathological and immunological study. Am J Gastroenterol. 2009;104:1189–1198.19352342
Fernández-BañaresFCasalotsJSalasA Paucicellular lymphocytic colitis: is it a minor form of lymphocytic colitis? A clinical pathological and immunological study. Am J Gastroenterol. 2009;104:1189–1198.19352342, Fernández-BañaresFCasalotsJSalasA Paucicellular lymphocytic colitis: is it a minor form of lymphocytic colitis? A clinical pathological and immunological study. Am J Gastroenterol. 2009;104:1189–1198.19352342
(Tlaskalová-HogenováHŠtěpánkováRHudcovicT Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases. Immunol Lett. 2004;93:97–108.15158604)
Tlaskalová-HogenováHŠtěpánkováRHudcovicT Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases. Immunol Lett. 2004;93:97–108.15158604
Tlaskalová-HogenováHŠtěpánkováRHudcovicT Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases. Immunol Lett. 2004;93:97–108.15158604, Tlaskalová-HogenováHŠtěpánkováRHudcovicT Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases. Immunol Lett. 2004;93:97–108.15158604
S. Hsieh, Tsung-Chieh Shih, Chien‐Yuh Yeh, Chun‐Jung Lin, yun-Ying Chou, Ying-Shiung Lee (2006)
Comparative proteomic studies on the pathogenesis of human ulcerative colitis
PROTEOMICS, 6
(ReichKMChangH-JRezaieA The incidence rate of colectomy for medically refractory ulcerative colitis has declined in parallel with increasing anti-TNF use: a time-trend study. Aliment Pharmacol Ther. 2014;40:629–638.25039715)
ReichKMChangH-JRezaieA The incidence rate of colectomy for medically refractory ulcerative colitis has declined in parallel with increasing anti-TNF use: a time-trend study. Aliment Pharmacol Ther. 2014;40:629–638.25039715
ReichKMChangH-JRezaieA The incidence rate of colectomy for medically refractory ulcerative colitis has declined in parallel with increasing anti-TNF use: a time-trend study. Aliment Pharmacol Ther. 2014;40:629–638.25039715, ReichKMChangH-JRezaieA The incidence rate of colectomy for medically refractory ulcerative colitis has declined in parallel with increasing anti-TNF use: a time-trend study. Aliment Pharmacol Ther. 2014;40:629–638.25039715
(HanNYChoiWParkJM Label-free quantification for discovering novel biomarkers in the diagnosis and assessment of disease activity in inflammatory bowel disease. J Dig Dis. 2013;14:166–174.23320753)
HanNYChoiWParkJM Label-free quantification for discovering novel biomarkers in the diagnosis and assessment of disease activity in inflammatory bowel disease. J Dig Dis. 2013;14:166–174.23320753
HanNYChoiWParkJM Label-free quantification for discovering novel biomarkers in the diagnosis and assessment of disease activity in inflammatory bowel disease. J Dig Dis. 2013;14:166–174.23320753, HanNYChoiWParkJM Label-free quantification for discovering novel biomarkers in the diagnosis and assessment of disease activity in inflammatory bowel disease. J Dig Dis. 2013;14:166–174.23320753
J. Cox, Nadin Neuhauser, Annette Michalski, R. Scheltema, J. Olsen, M. Mann (2011)
Andromeda: a peptide search engine integrated into the MaxQuant environment.
Journal of proteome research, 10 4
(RosenfeldGBresslerB The truth about cigarette smoking and the risk of inflammatory bowel disease. Am J Gastroenterol. 2012;107:1407–1408.22951878)
RosenfeldGBresslerB The truth about cigarette smoking and the risk of inflammatory bowel disease. Am J Gastroenterol. 2012;107:1407–1408.22951878
RosenfeldGBresslerB The truth about cigarette smoking and the risk of inflammatory bowel disease. Am J Gastroenterol. 2012;107:1407–1408.22951878, RosenfeldGBresslerB The truth about cigarette smoking and the risk of inflammatory bowel disease. Am J Gastroenterol. 2012;107:1407–1408.22951878
A. Thompson, C. Lees (2011)
Genetics of ulcerative colitis
Inflammatory Bowel Diseases, 17
S. Deeb, Rochelle D’Souza, J. Cox, M. Schmidt-Supprian, M. Mann (2012)
Super-SILAC Allows Classification of Diffuse Large B-cell Lymphoma Subtypes by Their Protein Expression Profiles*
Molecular & Cellular Proteomics : MCP, 11
(SchmidtSVSchultzeJL New insights into IDO biology in bacterial and viral infections. Front Immunol. 2014;5:384.25157255)
SchmidtSVSchultzeJL New insights into IDO biology in bacterial and viral infections. Front Immunol. 2014;5:384.25157255
SchmidtSVSchultzeJL New insights into IDO biology in bacterial and viral infections. Front Immunol. 2014;5:384.25157255, SchmidtSVSchultzeJL New insights into IDO biology in bacterial and viral infections. Front Immunol. 2014;5:384.25157255
S. Kane, W. Sandborn, P. Rufo, A. Zholudev, J. Boone, D. Lyerly, M. Camilleri, S. Hanauer (2003)
Fecal Lactoferrin Is a Sensitive and Specific Marker in Identifying Intestinal Inflammation
American Journal of Gastroenterology, 98
T. Beiter, Annunziata Fragasso, D. Hartl, A. Nieß (2015)
Neutrophil Extracellular Traps: A Walk on the Wild Side of Exercise Immunology
Sports Medicine, 45
G. Tontini, M. Vecchi, L. Pastorelli, M. Neurath, H. Neumann (2015)
Differential diagnosis in inflammatory bowel disease colitis: state of the art and future perspectives.
World journal of gastroenterology, 21 1
(BhatiaVNPerlmanDHCostelloCE Software tool for researching annotations of proteins: open-source protein annotation software with data visualization. Anal Chem. 2009;81:9819–9823.19839595)
BhatiaVNPerlmanDHCostelloCE Software tool for researching annotations of proteins: open-source protein annotation software with data visualization. Anal Chem. 2009;81:9819–9823.19839595
BhatiaVNPerlmanDHCostelloCE Software tool for researching annotations of proteins: open-source protein annotation software with data visualization. Anal Chem. 2009;81:9819–9823.19839595, BhatiaVNPerlmanDHCostelloCE Software tool for researching annotations of proteins: open-source protein annotation software with data visualization. Anal Chem. 2009;81:9819–9823.19839595
(RaoPKLiQ Principal component analysis of proteome dynamics in iron-starved mycobacterium tuberculosis. J Proteomics Bioinform. 2009;2:19–31.19436767)
RaoPKLiQ Principal component analysis of proteome dynamics in iron-starved mycobacterium tuberculosis. J Proteomics Bioinform. 2009;2:19–31.19436767
RaoPKLiQ Principal component analysis of proteome dynamics in iron-starved mycobacterium tuberculosis. J Proteomics Bioinform. 2009;2:19–31.19436767, RaoPKLiQ Principal component analysis of proteome dynamics in iron-starved mycobacterium tuberculosis. J Proteomics Bioinform. 2009;2:19–31.19436767
D. Munn, A. Mellor (2013)
Indoleamine 2,3 dioxygenase and metabolic control of immune responses.
Trends in immunology, 34 3
J. Mendoza, M. Abreu (2009)
Biological markers in inflammatory bowel disease: practical consideration for clinicians.
Gastroenterologie clinique et biologique, 33 Suppl 3
(SchoepferAMBeglingerCStraumannA Fecal calprotectin more accurately reflects endoscopic activity ulcerative colitis than Lichtiger Index, C-reactive protein platelets, hemoglobin, and blood leukocytes. Inflamm Bowel Dis. 2013;19:332–341.23328771)
SchoepferAMBeglingerCStraumannA Fecal calprotectin more accurately reflects endoscopic activity ulcerative colitis than Lichtiger Index, C-reactive protein platelets, hemoglobin, and blood leukocytes. Inflamm Bowel Dis. 2013;19:332–341.23328771
SchoepferAMBeglingerCStraumannA Fecal calprotectin more accurately reflects endoscopic activity ulcerative colitis than Lichtiger Index, C-reactive protein platelets, hemoglobin, and blood leukocytes. Inflamm Bowel Dis. 2013;19:332–341.23328771, SchoepferAMBeglingerCStraumannA Fecal calprotectin more accurately reflects endoscopic activity ulcerative colitis than Lichtiger Index, C-reactive protein platelets, hemoglobin, and blood leukocytes. Inflamm Bowel Dis. 2013;19:332–341.23328771
(BennikeTLauridsenKBOlesenMK Optimizing the identification of citrullinated peptides by mass spectrometry: utilizing the inability of trypsin to cleave after citrullinated amino acids. J Proteomics Bioinform. 2013;06:288–295.)
BennikeTLauridsenKBOlesenMK Optimizing the identification of citrullinated peptides by mass spectrometry: utilizing the inability of trypsin to cleave after citrullinated amino acids. J Proteomics Bioinform. 2013;06:288–295.
BennikeTLauridsenKBOlesenMK Optimizing the identification of citrullinated peptides by mass spectrometry: utilizing the inability of trypsin to cleave after citrullinated amino acids. J Proteomics Bioinform. 2013;06:288–295., BennikeTLauridsenKBOlesenMK Optimizing the identification of citrullinated peptides by mass spectrometry: utilizing the inability of trypsin to cleave after citrullinated amino acids. J Proteomics Bioinform. 2013;06:288–295.
K. Reich, Hsiu-Ju Chang, Ali Rezaie, Haili Wang, Karen Goodman, G. Kaplan, Lawrence Svenson, Gordon Lees, Richard Fedorak, K. Kroeker (2014)
The incidence rate of colectomy for medically refractory ulcerative colitis has declined in parallel with increasing anti‐TNF use: a time‐trend study
Alimentary Pharmacology & Therapeutics, 40
(JessTFrischMSimonsenJ Trends in overall and cause-specific mortality among patients with inflammatory bowel disease from 1982 to 2010. Clin Gastroenterol Hepatol. 2013;11:43–48.23022699)
JessTFrischMSimonsenJ Trends in overall and cause-specific mortality among patients with inflammatory bowel disease from 1982 to 2010. Clin Gastroenterol Hepatol. 2013;11:43–48.23022699
JessTFrischMSimonsenJ Trends in overall and cause-specific mortality among patients with inflammatory bowel disease from 1982 to 2010. Clin Gastroenterol Hepatol. 2013;11:43–48.23022699, JessTFrischMSimonsenJ Trends in overall and cause-specific mortality among patients with inflammatory bowel disease from 1982 to 2010. Clin Gastroenterol Hepatol. 2013;11:43–48.23022699
(2015)
Inflamm Bowel Dis Neutrophil Extracellular Traps in UC
(FournierBMParkosCA The role of neutrophils during intestinal inflammation. Mucosal Immunol. 2012;5:354–366.22491176)
FournierBMParkosCA The role of neutrophils during intestinal inflammation. Mucosal Immunol. 2012;5:354–366.22491176
FournierBMParkosCA The role of neutrophils during intestinal inflammation. Mucosal Immunol. 2012;5:354–366.22491176, FournierBMParkosCA The role of neutrophils during intestinal inflammation. Mucosal Immunol. 2012;5:354–366.22491176
S. Hanauer, Malcolm Robinson, R. Pruitt, A. Lazenby, T. Persson, L. Nilsson, K. Walton-Bowen, L. Haskell, J. Levine (1998)
Budesonide enema for the treatment of active, distal ulcerative colitis and proctitis: a dose-ranging study. U.S. Budesonide enema study group.
Gastroenterology, 115 3
(IskandarHNCiorbaMA Biomarkers in inflammatory bowel disease: current practices and recent advances. Transl Res. 2012;159:313–325.22424434)
IskandarHNCiorbaMA Biomarkers in inflammatory bowel disease: current practices and recent advances. Transl Res. 2012;159:313–325.22424434
IskandarHNCiorbaMA Biomarkers in inflammatory bowel disease: current practices and recent advances. Transl Res. 2012;159:313–325.22424434, IskandarHNCiorbaMA Biomarkers in inflammatory bowel disease: current practices and recent advances. Transl Res. 2012;159:313–325.22424434
E. Lindberg, C. Tysk, K. Andersson, G. Järnerot (1988)
Smoking and inflammatory bowel disease. A case control study.
Gut, 29
T. Beiter, Annunziata Fragasso, Jens Hudemann, M. Schild, J. Steinacker, F. Mooren, A. Nieß (2014)
Neutrophils release extracellular DNA traps in response to exercise.
Journal of applied physiology, 117 3
S. Schmidt, J. Schultze (2014)
New Insights into IDO Biology in Bacterial and Viral Infections
Frontiers in Immunology, 5
(BennikeTAyturkUHaslauerCM A normative study of the synovial fluid proteome from healthy porcine knee joints. J Proteome Res. 2014;13:4377–4387.25160569)
BennikeTAyturkUHaslauerCM A normative study of the synovial fluid proteome from healthy porcine knee joints. J Proteome Res. 2014;13:4377–4387.25160569
BennikeTAyturkUHaslauerCM A normative study of the synovial fluid proteome from healthy porcine knee joints. J Proteome Res. 2014;13:4377–4387.25160569, BennikeTAyturkUHaslauerCM A normative study of the synovial fluid proteome from healthy porcine knee joints. J Proteome Res. 2014;13:4377–4387.25160569
(MurgiaMNagarajNDeshmukhAS Single muscle fiber proteomics reveals unexpected mitochondrial specialization. EMBO Rep. 2015;16:387–395.25643707)
MurgiaMNagarajNDeshmukhAS Single muscle fiber proteomics reveals unexpected mitochondrial specialization. EMBO Rep. 2015;16:387–395.25643707
MurgiaMNagarajNDeshmukhAS Single muscle fiber proteomics reveals unexpected mitochondrial specialization. EMBO Rep. 2015;16:387–395.25643707, MurgiaMNagarajNDeshmukhAS Single muscle fiber proteomics reveals unexpected mitochondrial specialization. EMBO Rep. 2015;16:387–395.25643707
L. Rodríguez, A. Gonzáléz-Pérez, S. Johansson, S. Johansson, M. Wallander, M. Wallander (2005)
Risk factors for inflammatory bowel disease in the general population
Alimentary Pharmacology & Therapeutics, 22
A. Mantovani, M. Cassatella, C. Costantini, S. Jaillon (2011)
Neutrophils in the activation and regulation of innate and adaptive immunity
Nature Reviews Immunology, 11
Ta-Chiang Liu, T. Stappenbeck (2016)
Genetics and Pathogenesis of Inflammatory Bowel Disease.
Annual review of pathology, 11
I. León, V. Schwämmle, O. Jensen, R. Sprenger (2013)
Quantitative Assessment of In-solution Digestion Efficiency Identifies Optimal Protocols for Unbiased Protein Analysis*
Molecular & Cellular Proteomics, 12
V. Brinkmann, A. Zychlinsky (2012)
Neutrophil extracellular traps: Is immunity the second function of chromatin?
The Journal of Cell Biology, 198
T. Bennike, S. Birkelund, A. Stensballe, V. Andersen (2014)
Biomarkers in inflammatory bowel diseases: current status and proteomics identification strategies.
World journal of gastroenterology, 20 12
(OdesSVardiHFrigerM Cost analysis and cost determinants in a European inflammatory bowel disease inception cohort with 10 years of follow-up evaluation. Gastroenterology. 2006;131:719–728.16952541)
OdesSVardiHFrigerM Cost analysis and cost determinants in a European inflammatory bowel disease inception cohort with 10 years of follow-up evaluation. Gastroenterology. 2006;131:719–728.16952541
OdesSVardiHFrigerM Cost analysis and cost determinants in a European inflammatory bowel disease inception cohort with 10 years of follow-up evaluation. Gastroenterology. 2006;131:719–728.16952541, OdesSVardiHFrigerM Cost analysis and cost determinants in a European inflammatory bowel disease inception cohort with 10 years of follow-up evaluation. Gastroenterology. 2006;131:719–728.16952541
C. Schneider, W. Rasband, K. Eliceiri (2012)
NIH Image to ImageJ: 25 years of image analysis
Nature Methods, 9
(LindbergETyskCAnderssonK Smoking and inflammatory bowel disease. A case control study. Gut. 1988;29:352–357.3356367)
LindbergETyskCAnderssonK Smoking and inflammatory bowel disease. A case control study. Gut. 1988;29:352–357.3356367
LindbergETyskCAnderssonK Smoking and inflammatory bowel disease. A case control study. Gut. 1988;29:352–357.3356367, LindbergETyskCAnderssonK Smoking and inflammatory bowel disease. A case control study. Gut. 1988;29:352–357.3356367
(TusherVGTibshiraniRChuG Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A. 2001;98:5116–5121.11309499)
TusherVGTibshiraniRChuG Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A. 2001;98:5116–5121.11309499
TusherVGTibshiraniRChuG Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A. 2001;98:5116–5121.11309499, TusherVGTibshiraniRChuG Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A. 2001;98:5116–5121.11309499
(KhorBGardetAXavierRJ Genetics and pathogenesis of inflammatory bowel disease. Nature. 2011;474:307–317.21677747)
KhorBGardetAXavierRJ Genetics and pathogenesis of inflammatory bowel disease. Nature. 2011;474:307–317.21677747
KhorBGardetAXavierRJ Genetics and pathogenesis of inflammatory bowel disease. Nature. 2011;474:307–317.21677747, KhorBGardetAXavierRJ Genetics and pathogenesis of inflammatory bowel disease. Nature. 2011;474:307–317.21677747
(VizcaínoJADeutschEWWangR ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat Biotechnol. 2014;32:223–226.24727771)
VizcaínoJADeutschEWWangR ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat Biotechnol. 2014;32:223–226.24727771
VizcaínoJADeutschEWWangR ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat Biotechnol. 2014;32:223–226.24727771, VizcaínoJADeutschEWWangR ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat Biotechnol. 2014;32:223–226.24727771
(EhrentrautSFColganSP Implications of protein post-translational modifications in IBD. Inflamm Bowel Dis. 2012;18:1378–1388.22223542)
EhrentrautSFColganSP Implications of protein post-translational modifications in IBD. Inflamm Bowel Dis. 2012;18:1378–1388.22223542
EhrentrautSFColganSP Implications of protein post-translational modifications in IBD. Inflamm Bowel Dis. 2012;18:1378–1388.22223542, EhrentrautSFColganSP Implications of protein post-translational modifications in IBD. Inflamm Bowel Dis. 2012;18:1378–1388.22223542
(MolodeckyNASoonISRabiDM Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology. 2012;142:46–54.e42.22001864)
MolodeckyNASoonISRabiDM Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology. 2012;142:46–54.e42.22001864
MolodeckyNASoonISRabiDM Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology. 2012;142:46–54.e42.22001864, MolodeckyNASoonISRabiDM Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology. 2012;142:46–54.e42.22001864
(VizcaínoJACôtéRGCsordasA The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res. 2013;41:D1063–D1069.23203882)
VizcaínoJACôtéRGCsordasA The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res. 2013;41:D1063–D1069.23203882
VizcaínoJACôtéRGCsordasA The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res. 2013;41:D1063–D1069.23203882, VizcaínoJACôtéRGCsordasA The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res. 2013;41:D1063–D1069.23203882
(BennikeTBirkelundSStensballeA Biomarkers in inflammatory bowel diseases: current status and proteomics identification strategies. World J Gastroenterol. 2014;20:3231–3244.24696607)
BennikeTBirkelundSStensballeA Biomarkers in inflammatory bowel diseases: current status and proteomics identification strategies. World J Gastroenterol. 2014;20:3231–3244.24696607
BennikeTBirkelundSStensballeA Biomarkers in inflammatory bowel diseases: current status and proteomics identification strategies. World J Gastroenterol. 2014;20:3231–3244.24696607, BennikeTBirkelundSStensballeA Biomarkers in inflammatory bowel diseases: current status and proteomics identification strategies. World J Gastroenterol. 2014;20:3231–3244.24696607
(MendozaJLAbreuMT Biological markers in inflammatory bowel disease: practical consideration for clinicians. Gastroenterol Clin Biol. 2009;33(suppl 3):S158–S173.20117339)
MendozaJLAbreuMT Biological markers in inflammatory bowel disease: practical consideration for clinicians. Gastroenterol Clin Biol. 2009;33(suppl 3):S158–S173.20117339
MendozaJLAbreuMT Biological markers in inflammatory bowel disease: practical consideration for clinicians. Gastroenterol Clin Biol. 2009;33(suppl 3):S158–S173.20117339, MendozaJLAbreuMT Biological markers in inflammatory bowel disease: practical consideration for clinicians. Gastroenterol Clin Biol. 2009;33(suppl 3):S158–S173.20117339
(VosMDLouisEJJahnsenJ Consecutive fecal calprotectin measurements predict relapse patients ulcerative colitis receiving infliximab maintenance therapy. Inflamm Bowel Dis. 2013;19:2111–2117.23883959)
VosMDLouisEJJahnsenJ Consecutive fecal calprotectin measurements predict relapse patients ulcerative colitis receiving infliximab maintenance therapy. Inflamm Bowel Dis. 2013;19:2111–2117.23883959
VosMDLouisEJJahnsenJ Consecutive fecal calprotectin measurements predict relapse patients ulcerative colitis receiving infliximab maintenance therapy. Inflamm Bowel Dis. 2013;19:2111–2117.23883959, VosMDLouisEJJahnsenJ Consecutive fecal calprotectin measurements predict relapse patients ulcerative colitis receiving infliximab maintenance therapy. Inflamm Bowel Dis. 2013;19:2111–2117.23883959
H. Iskandar, M. Ciorba (2012)
Biomarkers in inflammatory bowel disease: current practices and recent advances.
Translational research : the journal of laboratory and clinical medicine, 159 4
(ThompsonAILeesCW Genetics of ulcerative colitis. Inflamm Bowel Dis. 2011;17:831–848.21319274)
ThompsonAILeesCW Genetics of ulcerative colitis. Inflamm Bowel Dis. 2011;17:831–848.21319274
ThompsonAILeesCW Genetics of ulcerative colitis. Inflamm Bowel Dis. 2011;17:831–848.21319274, ThompsonAILeesCW Genetics of ulcerative colitis. Inflamm Bowel Dis. 2011;17:831–848.21319274
Takeshi Masuda, M. Tomita, Y. Ishihama (2008)
Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis.
Journal of proteome research, 7 2
(BeiterTFragassoAHartlD Neutrophil extracellular traps: a walk on the wild side of exercise immunology. Sports Med. 2015;45:625–640.25504501)
BeiterTFragassoAHartlD Neutrophil extracellular traps: a walk on the wild side of exercise immunology. Sports Med. 2015;45:625–640.25504501
BeiterTFragassoAHartlD Neutrophil extracellular traps: a walk on the wild side of exercise immunology. Sports Med. 2015;45:625–640.25504501, BeiterTFragassoAHartlD Neutrophil extracellular traps: a walk on the wild side of exercise immunology. Sports Med. 2015;45:625–640.25504501
(MahidSSMinorKSSotoRE Smoking and inflammatory bowel disease: a meta-analysis. Mayo Clin Proc. 2006;81:1462–1471.17120402)
MahidSSMinorKSSotoRE Smoking and inflammatory bowel disease: a meta-analysis. Mayo Clin Proc. 2006;81:1462–1471.17120402
MahidSSMinorKSSotoRE Smoking and inflammatory bowel disease: a meta-analysis. Mayo Clin Proc. 2006;81:1462–1471.17120402, MahidSSMinorKSSotoRE Smoking and inflammatory bowel disease: a meta-analysis. Mayo Clin Proc. 2006;81:1462–1471.17120402
(SchneiderCARasbandWSEliceiriKW NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–675.22930834)
SchneiderCARasbandWSEliceiriKW NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–675.22930834
SchneiderCARasbandWSEliceiriKW NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–675.22930834, SchneiderCARasbandWSEliceiriKW NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–675.22930834
B. Fournier, C. Parkos (2012)
The role of neutrophils during intestinal inflammation
Mucosal Immunology, 5
(PoulsenNAAndersenVMøllerJC Comparative analysis of inflamed and non-inflamed colon biopsies reveals strong proteomic inflammation profile in patients with ulcerative colitis. BMC Gastroenterol. 2012;12:76.22726388)
PoulsenNAAndersenVMøllerJC Comparative analysis of inflamed and non-inflamed colon biopsies reveals strong proteomic inflammation profile in patients with ulcerative colitis. BMC Gastroenterol. 2012;12:76.22726388
PoulsenNAAndersenVMøllerJC Comparative analysis of inflamed and non-inflamed colon biopsies reveals strong proteomic inflammation profile in patients with ulcerative colitis. BMC Gastroenterol. 2012;12:76.22726388, PoulsenNAAndersenVMøllerJC Comparative analysis of inflamed and non-inflamed colon biopsies reveals strong proteomic inflammation profile in patients with ulcerative colitis. BMC Gastroenterol. 2012;12:76.22726388
(KaneSVSandbornWJRufoPA Fecal lactoferrin is a sensitive and specific marker in identifying intestinal inflammation. Am J Gastroenterol. 2003;98:1309–1314.12818275)
KaneSVSandbornWJRufoPA Fecal lactoferrin is a sensitive and specific marker in identifying intestinal inflammation. Am J Gastroenterol. 2003;98:1309–1314.12818275
KaneSVSandbornWJRufoPA Fecal lactoferrin is a sensitive and specific marker in identifying intestinal inflammation. Am J Gastroenterol. 2003;98:1309–1314.12818275, KaneSVSandbornWJRufoPA Fecal lactoferrin is a sensitive and specific marker in identifying intestinal inflammation. Am J Gastroenterol. 2003;98:1309–1314.12818275
(2013)
Research Randomizer (Version 4.0) [computer software
Prahlad Rao, Qingbo Li (2009)
Principal Component Analysis of Proteome Dynamics in Iron-starved Mycobacterium Tuberculosis.
Journal of proteomics & bioinformatics, 2 1
N. Hubner, A. Bird, J. Cox, Bianca Splettstoesser, Peter Bandilla, I. Poser, A. Hyman, M. Mann (2010)
Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions
The Journal of Cell Biology, 189
(JostinsLRipkeSWeersmaRK Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature. 2012;491:119–124.23128233)
JostinsLRipkeSWeersmaRK Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature. 2012;491:119–124.23128233
JostinsLRipkeSWeersmaRK Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature. 2012;491:119–124.23128233, JostinsLRipkeSWeersmaRK Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature. 2012;491:119–124.23128233
M. Vester-Andersen, I. Vind, M. Prosberg, Bo Bengtsson, Thomas Blixt, P. Munkholm, M. Andersson, T. Jess, F. Bendtsen (2014)
Hospitalisation, surgical and medical recurrence rates in inflammatory bowel disease 2003-2011—a Danish population-based cohort study.
Journal of Crohn's & colitis, 8 12
(HanauerSBRobinsonMPruittR Budesonide enema for the treatment of active, distal ulcerative colitis and proctitis: a dose-ranging study. Gastroenterology. 1998;115:525–532.9721148)
HanauerSBRobinsonMPruittR Budesonide enema for the treatment of active, distal ulcerative colitis and proctitis: a dose-ranging study. Gastroenterology. 1998;115:525–532.9721148
HanauerSBRobinsonMPruittR Budesonide enema for the treatment of active, distal ulcerative colitis and proctitis: a dose-ranging study. Gastroenterology. 1998;115:525–532.9721148, HanauerSBRobinsonMPruittR Budesonide enema for the treatment of active, distal ulcerative colitis and proctitis: a dose-ranging study. Gastroenterology. 1998;115:525–532.9721148
(WisniewskiJRZougmanANagarajN Universal sample preparation method for proteome analysis. Nat Methods. 2009;6:359–362.19377485)
WisniewskiJRZougmanANagarajN Universal sample preparation method for proteome analysis. Nat Methods. 2009;6:359–362.19377485
WisniewskiJRZougmanANagarajN Universal sample preparation method for proteome analysis. Nat Methods. 2009;6:359–362.19377485, WisniewskiJRZougmanANagarajN Universal sample preparation method for proteome analysis. Nat Methods. 2009;6:359–362.19377485
(ShkodaAWernerTDanielH Differential protein expression profile in the intestinal epithelium from patients with inflammatory bowel disease. J Proteome Res. 2007;6:1114–1125.17330946)
ShkodaAWernerTDanielH Differential protein expression profile in the intestinal epithelium from patients with inflammatory bowel disease. J Proteome Res. 2007;6:1114–1125.17330946
ShkodaAWernerTDanielH Differential protein expression profile in the intestinal epithelium from patients with inflammatory bowel disease. J Proteome Res. 2007;6:1114–1125.17330946, ShkodaAWernerTDanielH Differential protein expression profile in the intestinal epithelium from patients with inflammatory bowel disease. J Proteome Res. 2007;6:1114–1125.17330946
T. Bennike, U. Ayturk, C. Haslauer, John Froehlich, B. Proffen, Omar Barnaby, S. Birkelund, M. Murray, M. Warman, A. Stensballe, H. Steen (2014)
A Normative Study of the Synovial Fluid Proteome from Healthy Porcine Knee Joints
Journal of Proteome Research, 13
A. Schoepfer, C. Beglinger, A. Straumann, E. Safroneeva, Y. Romero, D. Armstrong, Carsten Schmidt, M. Trummler, V. Pittet, S. Vavricka (2013)
Fecal Calprotectin More Accurately Reflects Endoscopic Activity of Ulcerative Colitis than the Lichtiger Index, C-reactive Protein, Platelets, Hemoglobin, and Blood Leukocytes
Inflammatory Bowel Diseases, 19
C. Eyers (2009)
Universal sample preparation method for proteome analysis
M. Steinbakk, C. Naess-Andresen, M. Fagerhol, E. Lingaas, I. Dale, P. Brandtzaeg (1990)
Antimicrobial actions of calcium binding leucocyte L1 protein, calprotectin
The Lancet, 336
S. Ehrentraut, S. Colgan (2012)
Implications of Protein Post‐Translational Modifications in IBD
Inflammatory Bowel Diseases, 18
(O'DonoghueAJJinYKnudsenGM Global substrate profiling of proteases in human neutrophil extracellular traps reveals consensus motif predominantly contributed by Elastase Glogauer M, ed. PLoS One. 2013;8:e75141.24073241)
O'DonoghueAJJinYKnudsenGM Global substrate profiling of proteases in human neutrophil extracellular traps reveals consensus motif predominantly contributed by Elastase Glogauer M, ed. PLoS One. 2013;8:e75141.24073241
O'DonoghueAJJinYKnudsenGM Global substrate profiling of proteases in human neutrophil extracellular traps reveals consensus motif predominantly contributed by Elastase Glogauer M, ed. PLoS One. 2013;8:e75141.24073241, O'DonoghueAJJinYKnudsenGM Global substrate profiling of proteases in human neutrophil extracellular traps reveals consensus motif predominantly contributed by Elastase Glogauer M, ed. PLoS One. 2013;8:e75141.24073241
T. Jess, M. Frisch, J. Simonsen (2013)
Trends in overall and cause-specific mortality among patients with inflammatory bowel disease from 1982 to 2010.
Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association, 11 1
(SteinbakkMNaess-AndresenC-FFagerholMK Antimicrobial actions of calcium binding leucocyte L1 protein, calprotectin. Lancet. 1990;336:763–765.1976144)
SteinbakkMNaess-AndresenC-FFagerholMK Antimicrobial actions of calcium binding leucocyte L1 protein, calprotectin. Lancet. 1990;336:763–765.1976144
SteinbakkMNaess-AndresenC-FFagerholMK Antimicrobial actions of calcium binding leucocyte L1 protein, calprotectin. Lancet. 1990;336:763–765.1976144, SteinbakkMNaess-AndresenC-FFagerholMK Antimicrobial actions of calcium binding leucocyte L1 protein, calprotectin. Lancet. 1990;336:763–765.1976144
(UrbaniakGCPlousS Research Randomizer (Version 4.0) [computer software]. 2013 Available at: http://www.randomizer.org/. Accessed October 10, 2013.)
UrbaniakGCPlousS Research Randomizer (Version 4.0) [computer software]. 2013 Available at: http://www.randomizer.org/. Accessed October 10, 2013.
UrbaniakGCPlousS Research Randomizer (Version 4.0) [computer software]. 2013 Available at: http://www.randomizer.org/. Accessed October 10, 2013., UrbaniakGCPlousS Research Randomizer (Version 4.0) [computer software]. 2013 Available at: http://www.randomizer.org/. Accessed October 10, 2013.
(GeboesK Histopathology of Crohn's disease and ulcerative colitis. Inflamm Bowel Dis. 2003:255–276.)
GeboesK Histopathology of Crohn's disease and ulcerative colitis. Inflamm Bowel Dis. 2003:255–276.
GeboesK Histopathology of Crohn's disease and ulcerative colitis. Inflamm Bowel Dis. 2003:255–276., GeboesK Histopathology of Crohn's disease and ulcerative colitis. Inflamm Bowel Dis. 2003:255–276.
Hiroto Furuta, Shigeo Nishi, Shigeo Nishi, M. Beau, A. Fernald, Hideki Yano, Graeme Bell, Graeme Bell (1996)
Sequence of human hexokinase III cDNA and assignment of the human hexokinase III gene (HK3) to chromosome band 5q35.2 by fluorescence in situ hybridization.
Genomics, 36 1
S. Mahid, K. Minor, Roberto Soto, C. Hornung, S. Galandiuk (2006)
Smoking and inflammatory bowel disease: a meta-analysis.
Mayo Clinic proceedings, 81 11
N. Poulsen, V. Andersen, J. Møller, H. Møller, F. Jessen, S. Purup, L. Larsen (2012)
Comparative analysis of inflamed and non-inflamed colon biopsies reveals strong proteomic inflammation profile in patients with ulcerative colitis
BMC Gastroenterology, 12
(MunnDHMellorAL Indoleamine 2,3 dioxygenase and metabolic control of immune responses. Trends Immunol. 2013;34:137–143.23103127)
MunnDHMellorAL Indoleamine 2,3 dioxygenase and metabolic control of immune responses. Trends Immunol. 2013;34:137–143.23103127
MunnDHMellorAL Indoleamine 2,3 dioxygenase and metabolic control of immune responses. Trends Immunol. 2013;34:137–143.23103127, MunnDHMellorAL Indoleamine 2,3 dioxygenase and metabolic control of immune responses. Trends Immunol. 2013;34:137–143.23103127

Publisher: Pubmed Central
Copyright: Copyright © 2015 Crohn's & Colitis Foundation of America, Inc.
ISSN: 1078-0998
eISSN: 1536-4844
DOI: 10.1097/MIB.0000000000000460
Publisher site: See Article on Publisher Site

Abstract

ORIGINAL ARTICLE Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies ,†,‡ § Tue Bjerg Bennike, PhD,* Thomas Gelsing Carlsen, MS,* Torkell Ellingsen, MD, PhD, k,¶ k ,†† Ole Kristian Bonderup, MD, PhD, Henning Glerup, MD, PhD, Martin Bøgsted, PhD,** ‡‡ Gunna Christiansen, MD, DMSc, Svend Birkelund, MD, PhD, DMSc,* Allan Stensballe, PhD,* †,‡,§§ and Vibeke Andersen, MD, PhD Background: The etiology of the inﬂammatory bowel diseases, including ulcerative colitis (UC), remains incompletely explained. We hypothesized that an analysis of the UC colon proteome could reveal novel insights into the disease etiology. Methods: Mucosal colon biopsies were taken by endoscopy from noninﬂamed tissue of 10 patients with UC and 10 controls. The biopsies were either snap-frozen for protein analysis or prepared for histology. The protein content of the biopsies was characterized by high-throughput gel-free quantitative proteomics, and biopsy histology was analyzed by light microscopy and confocal microscopy. Results: We identiﬁed and quantiﬁed 5711 different proteins with proteomics. The abundance of the proteins calprotectin and lactotransferrin in the tissue correlated with the degree of tissue inﬂammation as determined by histology. However, fecal calprotectin did not correlate. Forty-six proteins were measured with a statistically signiﬁcant differences in abundances between the UC colon tissue and controls. Eleven of the proteins with increased abundances in the UC biopsies were associated with neutrophils and neutrophil extracellular traps. The ﬁndings were validated by microscopy, where an increased abundance of neutrophils and the presence of neutrophil extracellular traps by extracellular DNA present in the UC colon tissue were conﬁrmed. Conclusions: Neutrophils, induced neutrophil extracellular traps, and several proteins that play a part in innate immunity are all increased in abundance in the morphologically normal colon mucosa from patients with UC. The increased abundance of these antimicrobial compounds points to the stimulation of the innate immune system in the etiology of UC. (Inﬂamm Bowel Dis 2015;21:2052–2067) Key Words: ulcerative colitis, neutrophil extracellular traps, inﬂammatory bowel diseases, proteomics, microscopy lcerative colitis (UC), 1 of the 2 major forms of inﬂamma- society due to lost labor and expenses to the health care sys- 2–4 U tory bowel diseases (IBDs), is an important health problem. tem. The etiology of IBD remains incompletely explained but The incidence rate varies between the geographic regions, and in involves genetic and environmental factors. Genome-wide asso- Europe, annual incidence rates between 0.6 and 24.3 per 10 ciation studies have reported 133 loci to be associated with UC, inhabitants and prevalences rates between 4.9 and 505 per 10 many of which are associated with defects in the immune sys- inhabitants have been reported. Ulcerative colitis has a great tem. Current knowledge supports that UC is caused by an inap- impact on the quality of life of the affected individuals and for propriate immune response to the commensal microorganisms Received for publication February 26, 2015; Accepted March 27, 2015. From the *Department of Health Science and Technology, Aalborg University, Aalborg, Denmark; Organ Center, Hospital of Southern Jutland, Aabenraa, Denmark; ‡ § Institute of Regional Health Research-Center Soenderjylland, University of Southern Denmark, Odense, Denmark; Department of Rheumatology, Odense University Hospital, k ¶ Odense, Denmark; Diagnostic Center, Section of Gastroenterology, Regional Hospital Silkeborg, Silkeborg, Denmark; University Research Clinic for Innovative Patient †† Pathways, Aarhus University, Aarhus, Denmark; **Department of Clinical Medicine, Aalborg University, Aalborg, Denmark; Department of Haematology, Aalborg ‡‡ §§ University Hospital, Aalborg, Denmark; Department of Biomedicine, Aarhus University, Aarhus, Denmark; and Department of Internal Medicine, Regional Hospital Viborg, Viborg, Denmark. Supported by grants from Knud and Edith Eriksens Memorial Foundation and Ferring (V.A.); Obelske Family Foundation and the Svend Andersen Foundation (A.S.); Knud Hojgaards Foundation Denmark, the Lundbeck Foundation Denmark, the Oticon Foundation Denmark, and the Otto Monsteds Foundation Denmark (T.B.B.). The authors have no conﬂicts of interest to disclose. Reprints: Tue Bjerg Bennike, PhD, Department of Health Science and Technology, Aalborg University, Fredrik Bajers vej 3B, 9220 Aalborg, Denmark (e-mail: [email protected]). Copyright © 2015 Crohn’s & Colitis Foundation of America, Inc. This is an open-access article distributed under the terms of the Creative Commons Attribution- NonCommercial-NoDerivatives 3.0 License, where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially. DOI 10.1097/MIB.0000000000000460 Published online 19 May 2015. 2052 www.ibdjournal.org Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Neutrophil Extracellular Traps in UC living in the gastrointestinal tract, collectively termed the gut of 339 proteins. Several of the proteins were involved in inﬂam- 5–10 microbiota. mation and cytoskeleton rearrangement. However, based on the Ulcerative colitis is characterized by superﬁcial inﬂamma- number of visualized protein spots from the gel-based studies, we tory changes limited to the mucosa and submucosa of the colon. can expect the colonic biopsies to contain different proteins in the Markers of inﬂammation obtainable in feces or serum include C- thousands. The gel-free LC-MS study has, therefore, merely iden- reactive protein in serum and the proteins lactotransferrin and tiﬁed a fraction of the proteins, and high-throughput proteomics 11,12 technologies can be used to further identify and quantify disease- calprotectin in feces. C-reactive protein may be increased dur- speciﬁc proteins. We performed a proteomics study of morpho- ing the active phase of UC and is mainly produced in the liver in logically normal intestinal tissue from patients with UC, and we response to stimulation by proinﬂammatory mediators produced found it important to use a current state-of-the-art proteomics at the site of inﬂammation. Lactotransferrin is involved in the platform for high-throughput protein identiﬁcation and quantita- mucosal innate immune system response. It is an iron-binding tion in the colon mucosa. glycoprotein with antimicrobial properties and is expressed by 13,14 activated neutrophils and is released by the injured tissue. Calprotectin is a complex between the 2 proteins S100-A8 and MATERIALS AND METHODS S100-A9 and is involved in the recruitment of leukocytes. Addi- tionally, calprotectin has antimicrobial activity toward bacteria Study Cohort and fungi and composes up to 60% of the soluble cytosol proteins The study cohort size was determined based on a power 15,16 in human neutrophils. Increased abundance of fecal calprotec- analysis. Initially, we assumed that approximately 6000 t tests tin is a sensitive marker for intestinal inﬂammation but has also were to be performed in the study with an effect chance of been seen with the use of nonsteroidal anti-inﬂammatory drugs 0.01. Based on this, 10 subjects in each group to be compared and increasing age. The abundance of all 3 proteins increases in yields an estimated power of 95% to detect an effect size of 2.5 response to intestinal inﬂammation. when correlating with Benjamini and Hochberg. Therefore, 10 Accordingly, diagnostic and prognostic markers for IBD, patients with UC for whom endoscopy was planned and 10 which would be valuable tools that could signiﬁcantly improve healthy subjects were recruited at the outpatients clinic, Diagnos- treatment outcome, are currently missing. In this project, we tic center Regional Hospital Silkeborg in the period from 2012 to investigate protein changes in the intestinal tissue, in contrast to 2013. Diagnosis of UC was based on standard clinical, endo- proteins released to the bloodstream or feces. The aim was to scopic, and histological criteria, and an infectious etiology was excluded. Information on diagnosis, medication, most recent increase our knowledge of the UC etiology and to identify fecal calprotectin measurement, and smoking habits was recorded markers, which could be translated to clinical use. Several studies from the patient records. have investigated protein changes in the colon mucosa tissue. Written informed consent was obtained from all partic- However, so far mainly gel-based techniques have been used ipants before participation in the study, and the project was where the proteins are separating based on mass and charge approved by The Regional Scientiﬁc Ethical Committee (isoelectric point). Subsequently, the proteins are stained and (S-20120204) and the Danish Data Protection Agency (2008- quantiﬁed, and up to hundreds of protein spots on the gel can be 58-035). identiﬁed by mass spectrometry (MS). One such study com- pared colonic biopsies from 4 patients with UC and 5 controls Samples Collection and identiﬁed 19 proteins with different abundance between the 2 Biopsies for the histological evaluation of disease severity groups, indicating that mitochondrial dysfunction might be were sampled from descending colon, sigmoid colon, and rectum. involved in the UC etiology. A more recent study compared Three biopsies were taken from each location. The biopsies were inﬂamed and noninﬂamed colon biopsies from 20 patients with immediately placed on mixed cellulose ester ﬁlters (Advantec, UC and identiﬁed 43 proteins that differentiated between the 2 Japan), ﬁxed in phosphate-buffered 4% formaldehyde, and stored conditions, many of which were involved in energy metabolism at room temperature until tissue sample preparation 24 hours later. and oxidative stress. The 2-dimensional gel-based techniques Colon mucosal biopsies for MS were sampled 40 cm from the allow for visual identiﬁcation of regulated proteins and protein anus by sigmoidoscopy. Patient biopsies were taken from products that differ between different samples by image analysis macroscopically normal tissue, and all controls had normal followed by MS protein identiﬁcation. However, 2-dimensional ﬁndings at sigmoidoscopy. The biopsies were immediately trans- gel-based techniques are laborious and are limited to the identiﬁ- ferred to cryotubes and snap-frozen in liquid nitrogen followed by cation of protein spots in the hundreds. The identiﬁcation of non- storage at 21408C until proteomics sample preparation. changing proteins is for that reason often omitted from the 11,19 analysis. Sample Preparation for Histology In a recent study of patients with UC by Han et al, a gel- The formalin-ﬁxated biopsies were parafﬁn embedded, free liquid chromatography (LC)-MS technique is used to analyze sliced in 10 mm, deparafﬁnized with tissue clear (Sakura, AJ protein changes in colonic biopsies, which led to the identiﬁcation Alphen aan den Rijn, The Netherlands), and stained with www.ibdjournal.org 2053 Bennike et al Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 hematoxylin and eosin (H&E) (Sigma-Aldrich, St. Louis, MO) triethylammonium bicarbonate, pH 8.5), using intense shaking on or incubated with 2 mM TO-PRO-3 (Thermo Scientiﬁc, Waltham, a Precellys 24 homogenizer (Berting Technologies, Rockville, MA) in phosphate-buffered saline for 30 minutes, washed 3 times MD). The protein concentrations of the lysates were estimated by in phosphate-buffered saline, and mounted. measuring absorbance at 280 nm (A280) using a NanoDrop 1000 The H&E-stained slices were investigated with UV-Vis spectrophotometer (Thermo Scientiﬁc). To increase the a DM5500B microscopy (Leica Camera, Solms, Germany) equip- accuracy of the A280 estimations, the concentration of 4 biopsy ped with PL Floutar ·16/0.5, and ·40/1.00 objectives and Retiga lysates were determined using a bicinchoninic acid assay with 2000R CCD Camera (Qimaging, Canada), and the TO-PRO-3 bovine serum albumin as standard, measured using an Inﬁnite microscopy was performed on a SP5 confocal microscope (Leica microplate reader (Tecan, Männedorf, Switzerland). All A280 Camera) with a PL Floutar ·16/0.5, HCX PL APO ·63/1.40, and measurements were postprocessing calibrated using the bicincho- HCX PL APO ·100/1.47 objectives. The pictures were processed ninic acid assay protein determinations. with ImageJ with the LOCI Tools plugin. Protein digestion was performed using a modiﬁed ﬁlter-aided 26–28 sample preparation protocol. A biopsy lysate volume corre- Colon Inﬂammation Grade Score sponding to 100 mg of total solubilized protein was prepared for As part of the routine patient care at Regional Hospital LC-MS analysis for each biopsy. Proteins were reduced for 30 mi- Silkeborg, pathologists performed histological descriptions of nutes at room temperature by adding tris(2-carboxyethyl)phosphine formalin-ﬁxed parafﬁn-embedded H&E-stained biopsies from de- (Thermo Scientiﬁc) to a ﬁnal concentration of 10 mM. The reduced scending colon, sigmoid colon, and rectum. The biopsies were protein solution was transferred to a 30-kDa molecular weight characterized and assigned a colon inﬂammation grade score cutoff spin-ﬁlter (Millipore, Billerica, MA), and buffer exchange (0 ¼ no disease activity, 1 ¼ light activity, 2 ¼ moderate activity, was facilitated between all steps by centrifugation at 15,000g for and 3 ¼ severe activity) based on the histological descriptions 15 minutes at 48C. The proteins were alkylated using 100 mLof 18,25 (Table 1). The sum of scores from the descending colon, 50 mM 2-iodoacetamide (Sigma-Aldrich) in lysis buffer. Two sigmoid colon, and rectum were squared to yield patient colon micrograms of sequencing grade modiﬁed trypsin (Promega, inﬂammation grade scores. The scores were compared with the Madison, WI) dissolved in 50 mL of digestion buffer (0.5% sodium relative abundance of known inﬂammatory proteins in the tissue, deoxycholate, 50 mM triethylammonium bicarbonate, pH 8.5) was and Pearson’s correlation coefﬁcients were calculated. The histo- added to the spin-ﬁlter, and the proteins were digested to peptides logical evaluation was supervised by a specialist in human pathol- overnight at 378C. The peptide material was eluted from the spin- ogy, blinded for the result of the proteomic analyses. ﬁlter and puriﬁed by phase inversions. One milliliter of ethyl ace- tate with 1% formic acid was added to the peptide material, the tube was vortexed, centrifuged at 15,000g for 5 minutes to obtain phase Sample Preparation for Proteomics 26,29 separation, and the aqueous phase with peptides was recovered. The intestinal biopsies and lysates were kept on ice when The ﬁnal peptide eluent was dried down in a vacuum centrifuge possible. Each biopsy was homogenized with steel beads in overnight and stored at 2808C until time of analysis. 0.5 mL of cold lysis buffer (5% sodium deoxycholate, 50 mM TABLE 1. Disease Activity of Patients with UC Histological Grading Colon Inﬂammation F-Cal F-Cal and Biopsy Patient Descendens Sigmoideum Rectum Grade Score (mg/g) Time Difference (d) Year of Diagnosis UC_1 2 2 2 36 1285 7 2008 UC_2 0 0 1 1 889 20 2010 UC_3 1 2 2 25 1343 0 2003 UC_4 0 0 1 1 .3600 0 2010 UC_5 0 0 0 0 ,30 28 2009 UC_6 0 2 0 4 705 0 2006 UC_7 2 1 3 36 575 117 2008 UC_8 0 2 0 4 163 37 2011 UC_9 2 0 0 4 ,30 4 2000 UC_10 2 1 1.5 20.25 2060 5 2010 The histological grading of biopsies from colon descendens, colon sigmoideum, and rectum is used to calculate the colon inﬂammation grade score. Colon inﬂammation grade score ¼ (descendens score + sigmoideum score + rectum score) . Additionally, the measured fecal calprotectin (F-Cal) and the time difference between the measured F-Cal and the biopsy extraction are given. 2054 www.ibdjournal.org Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Neutrophil Extracellular Traps in UC peptides and proteins to below 1% false discovery rate. For Proteomics—Analysis quantiﬁed proteins, additional ﬁltering of the protein and Before LC-MS analysis, the dry peptide product was reconstituted in 20 mL of 2% acetonitrile and 0.1% formic acid. peptide data was done to ensure high-quality quantitative data: Five micrograms of peptide material as determined by A280 (1) The quantitation of any protein was required to be based on was analyzed per LC-MS analysis. All biopsies were analyzed at least 2 sequence-unique quantiﬁable peptides. (2) The in triplicates and in a random order of biopsy analysis. sequence-unique peptides were required to be quantiﬁable in The peptides were analyzed by LC-MS using an at least half of the UC biopsies or the control biopsies. (3) All UltiMate 3000 UPLC system (Thermo Scientiﬁc) coupled biopsies had been analyzed in triplicates on the LC-MS online to a Q Exactive mass spectrometer (Thermo Scientiﬁc) system. To remove replicate analyses with poor repeatability, using a trapping column setup. The peptide material was all measured protein abundances were log2 transformed and loaded onto a 2-cm trap column packed with Acclaim the repeated analyses were investigated individually for the PepMap100 C18, 5 mm 100 Å material (Thermo Scientiﬁc). biopsies by scatter plots. Ideally, the repeated analysis of Subsequently, the peptides were separated using a 50-cm ana- a biopsy should yield identical protein abundances, repre- lytical column packed with Acclaim PepMap100 C18, 2 mm sented by a Pearson’s correlation coefﬁcient of 1 in the scatter 100 Å material (Thermo Scientiﬁc). The peptides were eluted plots, indicating a perfect correlation. Individual replicate from the column with a gradient of 96% solvent A (0.1% analyses with a Pearson’s correlation less than 0.95 were formic acid) and 4% solvent B (0.1% formic acid in acetoni- removed from further analysis, to ensure that only highly trile), which was increased to 8% solvent B on a 5-minute reproducible replicates were included in the ﬁnal data set. ramp gradient and subsequently to 30% solvent B on a 225- (4) To further identify replicate outliers, a principle compo- minute ramp gradient, with a constant ﬂow rate of 300 nL/min. nent analysis (PCA) was performed in Perseus v1.4.1.3. The The column was washed with 90% solvent B for 10 minutes input for the PCA was all measured protein abundances in all and equilibrated for 40 minutes, resulting in a total analysis replicates. For the purpose of conducting PCA, missing values time of 290 minutes per LC-MS analysis. Both columns were (i.e., proteins where a quantitation value was not obtained for kept at 408C. The eluting peptides were introduced directly a given replicate analysis) were replaced with values from into the mass spectrometer by a picotip emitter for nanoﬂow a normal distribution (width 0.3 and down shift 1.8) to simu- ionization (New Objective, Woburn, MA). The mass spec- late signals from low abundant proteins. The grouping of the trometer was operated in positive mode using a data- replicates on the calculated scores plots was investigated. dependent acquisition method. A full MS scan in the mass Gene ontology annotations were imported from Uniprot range of m/z 400 to 1200 was acquired at a resolution of Knowledgebase for all quantiﬁed proteins using STRAP 70,000. In each cycle, the mass spectrometer would trigger up to 12 MS/MS acquisitions of eluting ions based on highest v1.5 to classify the proteins. signal intensity for fragmentation. The MS/MS scans were acquired with a dynamic mass range at a resolution of 17,500. Proteomics—Statistical Analysis The precursor ions were isolated using a quadrupole isolation win- The repeated measurements of protein abundances were dow of 1.6 m/z and fragmented using high-energy collision with combined in Perseus v1.4.1.3 by taking the median biopsy wise, a normalized collision energy of 27. Fragmented ions were dynam- and the biopsies were grouped according to disease state: 10 ically added to an exclusion list for 30 seconds. biopsies from patients with UC and 10 from controls. To identify proteins with a statistically signiﬁcant mean abundance change Proteomics—Raw File Analysis between the UC group and the control group, 2-sided t tests were The measured peptide signal intensities from the full MS performed. As several thousand t tests were performed, we 31,32 scan were integrated using MaxQuant 1.4.1.2 software and applied permutation-based false-positive control to correct for used to calculate the relative protein quantities (label-free multiple hypothesis testing (parameters: s0 ¼ 1, 250 randomiza- quantitation). The fragment scans were searched against a Uni- 37–39 tions). Up to 15% of the statistically signiﬁcantly changing prot database containing all reviewed Homo sapiens proteins proteins were allowed to be false positives (i.e., the measured (downloaded October, 8, 2013, containing 20,277 entries). average protein abundance is not statistically signiﬁcantly differ- The following abundant peptide modiﬁcations were included ent between the UC group and the control group) to ensure suf- in the analysis: carbamidomethylated cysteine residues (ﬁxed), ﬁcient input for the downstream protein functional analysis. acetylation of N peptides from the N-terminal of proteins (var- Proteins displaying a statistically signiﬁcant mean abundance iable), oxidation of methionine (variable), and deamidation of 33,34 change were further investigated using SPSS statistics v22 asparginine and glutamine residues (variable). (IBM, Armonk, NY). The MS data have been deposited to the Proteomics—Protein Data Processing ProteomeXchange Consortium (http://proteomecentral.proteo- A target-decoy fragment spectra search strategy was mexchange.org) by means of the PRIDE partner repository with 40,41 applied and used to adjust the false discovery rate of identiﬁed the data set identiﬁer PXD001608. www.ibdjournal.org 2055 Bennike et al Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 TABLE 2. Study Cohort Information and Analysis ID Age Gender Smoking Smoking History Medicine (per day) UC_1 27 Male Never Asacol (5-aminosalicylic acid) 1600 + 800 + 1600 mg UC_2 45 Male Stopped 2009 Asacol (5-aminosalicylic acid) 1600 mg · 2 Asacol (5-aminosalicylic acid) suppository 500 mg UC_3 44 Male Stopped 2000 Imurel (azathioprine) 150 mg · 1 UC_4 45 Female Stopped 2005 Pentasa (5-aminosalicylic acid) suppository 1 g UC_5 26 Female 10 cigarettes per day Current Mesasal (5-aminosalicylic acid) suppository 500 mg UC_6 70 Female Stopped 1998 Mesasal (5-aminosalicylic acid) suppository 500 mg UC_7 62 Male Stopped 2009 Imurel (Azathioprin) 100 mg; Pentasa (5-aminosalicylic acid) 2000 mg UC_8 36 Male Never Asacol (5-aminosalicylic acid) 1600 mg · 2 Asacol (5-aminosalicylic acid) suppository 500 mg UC_9 68 Female 10 cigarettes per day Current Asacol (5-aminosalicylic acid) 1600 mg · 2 UC_10 22 Female Never Asacol (5-aminosalicylic acid) 1600 mg · 2 Asacol (5-aminosalicylic acid) suppository 500 mg Pred-Clysma (glucocorticoid) 24 mg Ctrl_1 54 Male Never Ctrl_2 27 Male Never NovoRapid (insulin) Ctrl_3 42 Male Never Ctrl_4 48 Male Never Ctrl_5 54 Female Stopped 1992 Ctrl_6 27 Female Stopped 2011 Ctrl_7 42 Female Never Ctrl_8 23 Male Never Ctrl_9 28 Male Never Ctrl_10 31 Male Stopped 2008 Age and gender were not found to be statistically signiﬁcantly different (P . 0.05) between the patients with UC (n ¼ 10) and the controls (n ¼ 10) as determined by 2-sided t tests. were obtained in parallel, we expect these biopsies to have the RESULTS same characteristics. Patient Material Proteomics Veriﬁcation The recorded medical information (Table 2) revealed 9 pa- Before homogenization of the biopsies for proteomics tients receiving 5-aminosalicylic acid, 2 patients receiving azathio- sample preparation, the average wet weight of the biopsies was prine, and 1 patient receiving glucocorticoid treatment, whereas 1 measured to be 4.9 mg (3.6–5.9 mg; 25th–75th percentiles). Fol- control received insulin treatment. The study cohort age, gender, lowing homogenization of the biopsies in lysis buffer, the protein and current smoking status was not found to be statistically differ- concentration of the lysate was measured and the average ex- ent between the patients with UC and controls. However, the num- tracted protein yield, calculated from the biopsy wet weight, ber of former smokers was increased in the UC group. was 10% (9%–11%; 25th–75th percentiles), indicating an efﬁ- cient and reproducible homogenization. Histological Analysis The proteins in each homogenized sample were digested to To assess cell density and tissue morphology in the peptides using the ﬁlter-aided sample preparation protocol, and extracted biopsies, we investigated the H&E-stained sections by the complex peptide material was analyzed by LC-MS/MS with light microscopy (Fig. 1). The crypt architecture was preserved in 31,32 label-free quantitation. During the 290-minute analysis of all control samples and all UC biopsies. However, we observed an each homogenized and digested sample, the MS system detected increased number of stained cell nuclei between the intestinal peptide elusion from the LC column and selected up to 12 pre- crypts in the biopsies from patients with UC compared with con- cursor ions per MS cycle for fragmentation (Fig. 2). By bioinfor- trols. The majority of cells between the intestinal crypts had seg- matics analysis, the proteins were subsequently identiﬁed and mented cell nucleus, a characteristic of polymorphonuclear 42,43 quantiﬁed. To investigate the repeatability of the LC-MS analysis, neutrophils (PMNs). As the biopsies for the proteome analysis 2056 www.ibdjournal.org Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Neutrophil Extracellular Traps in UC of variables to a smaller number of groups (principle compo- nents), which can be visualized on scores plots and used to interpret the variance in a complex data set, like that of high-throughput proteomics. All measured protein abundances in all LC-MS analyses were used as input for the PCA, and 2- dimensional scores plots were constructed (Fig. 3B). The scores plot describes how the replicates group relative to one another, based on the differences in measured abundances of all proteins. In effect, replicates where similar protein abundances have been measured will be close in space on the scores plot, relative to the other replicates, and the PCA scores plot can thus be used to identify outliers. The PCA, thereby, includes the variance between all biopsies, unlike the scatter plots that only take the variance in the repeated analysis of the individual biopsies into account. Principal component 1 and principle component 2 rep- resent the largest and second largest variance in the protein abun- dance data set, respectively, and explain 35.4% of the variance in the protein abundance data set. In all cases, the scores plot sepa- rates all biopsies from one another, in contrast to the repeated triplicate analysis of each biopsies, which group together for each individual biopsy (Fig. 3B, circles). This indicates that the mea- sured protein abundances are more similar when the same biopsy is analyzed repeatedly than when different biopsies are analyzed. The variance in protein abundances between the different biop- sies, therefore, is larger than the technical variance when the same biopsy is analyzed repeatedly, as expected for sensitive methods that validate the methodology. Proteomics—Data Analysis Applying our strict criteria, 5711 quantiﬁable proteins remained postﬁltering. The quantiﬁable proteins were classiﬁed FIGURE 1. Biopsy histology analysis. A, Overview of the human small by biological function, subcellular location, and molecular and large intestine. B, Cross section of a human colon. Representative function, using available data from Uniprot Knowledgebase and H&E-stained colon biopsy slices from (C) control Ctrl_10 and (D) gene ontology database (Fig. 4). The analysis of the biological patient UC_05. The crypt architecture is in both slices preserved. processes performed by the proteins revealed that 68% of the However, an increased number of stained cell nuclei surrounding the quantiﬁed proteins were involved in cellular processes, such as crypts (red arrows) are apparent in the UC biopsies compared with the control biopsies (scale bars 100 mm). intracellular communication; 55% of the proteins were involved in modulation of the frequency, rate, or extent of any biological process, quality, or function; and 29% were involved in metabolic the technical triplicate analyses of each biopsy were analyzed by processes, which include anabolism and catabolism by which scatter plots, plotting the abundance of each quantiﬁed protein in living organisms transform chemical substances. We next inves- one replicate against the same protein abundance as determined in tigated the cellular component of the proteins, that is, which parts other replicates, biopsy wise. Ideally, the repeated analysis should of a cell or its extracellular environment the protein has been yield identical protein abundances, represented by a Pearson’s reported to be present. A somewhat even distribution was found correlation coefﬁcient of 1 (Fig. 3A). Of the 60 LC-MS analyses, between proteins associated with the cell cytoplasm, the cell only 1 replicate from UC_4 and 1 from UC_5 had coefﬁcients less nucleus, and the extracellular environment of the cell with 48%, than 0.95 and the 2 outlying replicates were removed from further 42%, and 35%, respectively. Finally, we investigated which pri- analysis. The result demonstrates a high degree of technical mary molecular functions the proteins took part of. The analysis repeatability of the method, that is, a low variance of the measured revealed that 68% of the proteins were involved in selective, non- protein abundances when the same biopsy is analyzed repeatedly. covalent binding with other molecules, and 41% were involved in To investigate how the measured protein abundances varied biologically catalyzed reactions. Based on the protein classiﬁca- between all replicates and identify which replicates yielded sim- tion analysis, the quantiﬁed proteins originate from all cellular ilar protein abundances, a PCA was performed. A PCA is a sta- compartments, indicating that no systematic loss of proteins has tistical analysis technique that allows for reducing a large number occurred using the protocol and thus an unbiased analysis. www.ibdjournal.org 2057 Bennike et al Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 FIGURE 2. Representative 2-dimensional plot of the liquid chromatography-mass spectrometry analysis of patient UC_9 illustrating the complexity of the analyzed peptide material. On the x-axis, the intensities of the peptide ion signals of the 19,549 acquired full MS scans are plotted, with a color gradient from white to green. The time of analysis is given on the y-axis. Each full MS scan was succeeded by up to 12 MS/MS scans of ions detected on the full MS scan and each cycle time was approximately 1 second; + indicates that the given ion signal was selected for MS/MS analysis, which came to 119,560 MS/MS scans in this 1 replicate. Next, we analyzed the quantitative information obtained that, on average, was different between the UC biopsies and con- about the proteins in each biopsy. The measured protein trol biopsies by a factor of 2.8 or more, which is referred to as the abundances in the biopsies from the patient with UC were fold change. Of the 46 proteins, 33 proteins were more abundant compared with the abundances in the biopsies from the control in the UC biopsies and 13 proteins were less abundant (Table 3). group, and t tests were performed to identify proteins with a sta- The protein with the largest mean fold abundance change between tistically signiﬁcant mean abundance change between the 2 the UC and control biopsies was lactotransferrin, which was 219 groups. Forty-six proteins demonstrated a statistical signiﬁcant times more abundant in the UC group. To further validate the mean abundance change between the UC biopsies and the control measured protein abundances, we correlated the relative abun- biopsies. All the 46 proteins were measured with an abundance dance of lactotransferrin and calprotectin with the severity of FIGURE 3. Proteomics data veriﬁcation. A, Representative scatter plot of the triplicate biopsy analysis of UC_1 of the 5711 quantiﬁed proteins. The log2 transformed protein abundances of all proteins in replicates 1, 2, and 3 are plotted against one another on the x- and y-axes, respectively. Ideally, the measurements should yield identical protein abundances, represented by a Pearson’s correlation coefﬁcients (R) of 1. B, PCA scores plot of principle components 1 and 2 of the protein abundances as measured in the UC replicates (+) and control replicates (h). Technical replicates (encircled) group together in all cases, demonstrating a high degree of technical repeatability of the method. The study cohort subject ID is given as numbers (e.g., red 3 at [+] equals UC_3) (Table 2). 2058 www.ibdjournal.org Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Neutrophil Extracellular Traps in UC FIGURE 4. Gene ontology characterization of the 5711 quantiﬁed proteins in terms of (A) biological processes performed by the proteins, (B) cellular component to which the proteins are associated, and (C) the primary molecular functions of the proteins. tissue inﬂammation in the patients with UC, as determined by investigated the TO-PRO-3 DNA–stained biopsy sections by con- histology. Both proteins are used to monitor UC disease activity. focal microscopy. The increased density of cell nuclei observed Biopsies from descending colon, sigmoid colon, and rectum were on the H&E-stained sections was again observed (Fig. 7), as was assigned a colon inﬂammation grade score based on histology the segmented multilobulated nuclei, a characteristic of PMNs descriptions (Table 1). A good correlation was found between (Fig. 8A). The PMNs were observed abundantly in biopsies the colon inﬂammation grade scores and the relative abundance from the UC group and occasionally in the control group. In of calprotectin and lactotransferrin in the tissue, with Pearson’s addition to intact PMNs, we observed neutrophils that had correlation coefﬁcients of 0.84 and 0.91 for S100-A8 and excreted nuclear DNA into the extracellular environment forming S100-A9 protein, respectively, and 0.82 for lactotransferrin NETs at 2 distinct locations in biopsies from UC_4 (Fig. 8B). (Fig. 5). Finally, we correlated the relative abundance of calpro- tectin in the tissue to the most recent measure of fecal calprotectin as determined by routine tests. However, no correlation could be DISCUSSION found, and a Pearson’s correlation coefﬁcient of 0.33 was calcu- The main result of this study is the identiﬁcation of several lated (data not shown). novel proteins, which are present in increased abundance in the Eleven of the 46 proteins with statistically signiﬁcantly UC colonic tissue. Eleven of the proteins were associated with altered abundance in the UC biopsies are present in neutrophils neutrophils and NETs, and the increased presence of both in the and have been associated with the formation of neutrophil UC colonic tissue was veriﬁed with light microscopy and extracellular traps (NETs) (Table 3, asterisk indicates protein confocal microscopy. names). NETs are a method of action by PMNs and are com- Several studies have successfully analyzed different aspects posed of extracellular ﬁbrillar networks mainly of relaxed DNA of UC using genomic and transcriptomic techniques. However, and also contain proteins and other effector molecules, some of sequencing techniques do not directly yield information about the which have antimicrobial and antibody-like properties. NETs are abundance of the end product: the proteins, which are a combi- released from PMNs in response to inﬂammatory stimuli, trap- nation of the newly synthesized and the degraded protein. In our ping the invading microorganisms, and facilitates interaction proteome analysis of colon mucosa biopsies from 10 patients with 45–47 with other effector molecules that kill the microorganisms. UC and 10 controls, we were able to quantify 5711 proteins in the All 11 NET-associated proteins were found with increased abun- colon mucosa biopsies, and the data quality was ensured by scat- dance in the UC biopsies, and on average, the proteins were 42.2 ter plots and PCA. In previous studies, the amounts of calprotectin times (P , 0.0005) more abundant in the UC biopsies (Fig. 6). and lactotransferrin in feces have been found to correlate well with the degree of neutrophil inﬁltration and inﬂammation of 11,15 Confocal Laser Scanning Microscopy the intestine. We found that the abundance of these proteins To investigate if the increased abundance of PMN- in the colonic tissue correlated well with the colon inﬂammation associated proteins was a result of an increased abundance of grade scores, providing validation to the proteomics protein abun- PMNs, and if the PMNs were intact or found as NETs, we dance data and the methodology. However, we found that the www.ibdjournal.org 2059 Bennike et al Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 2060 www.ibdjournal.org TABLE 3. List of the 46 Proteins with a Statistically Signiﬁcant Mean Abundance Change Between the UC Biopsies and Controls Fold Peptides (Unique), Change Uniprot Acc. Protein Name Protein Function Seq. Cov. MW pI 219.2 P02788 Lactotransferrin* Iron-binding protein with antimicrobial properties include bacteriostasis 51 (49), 72.7% 76,165 8.47 92.1 P14780 Matrix metalloproteinase-9* Proteolysis of the extracellular matrix and leukocyte migration 30 (30), 51.8% 66,609 5.44 63.3 P05164 Myeloperoxidase* Part of the defense system of PMN; responsible for microbicidal activity 48 (41), 59.2% 66,107 9.22 against a wide range of organisms 31.4 P24158 Myeloblastin PMN protease; degrades elastin, ﬁbronectin, laminin, vitronectin, and collagen 7 (7), 36.3% 24,247 7.79 31.3 P08246 Neutrophil elastase* Modiﬁes functions of natural killer cells, monocytes, and granulocytes 11 (11), 54.7% 25,561 9.89 18.0 Q9NRD8 Dual oxidase 2 Lactoperoxidase-mediated antimicrobial defense at the surface of mucosa 42 (42), 33.6% 172,843 7.92 16.7 P11215 Integrin alpha-M Adhesive interactions of monocytes, macrophages, and granulocytes 34 (33), 37.7% 125,471 6.75 16.3 P06702 Protein S100-A9* One of the subunits in calprotectin; regulates inﬂammatory processes and 10 (10), 78.9% 13,110 5.71 immune response; Induce neutrophil chemotaxis, adhesion, increase bactericidal activity and degranulation 13.7 P80188 Neutrophil gelatinase-associated Apoptosis, innate immunity, and renal development 12 (12), 63.6% 20,547 9.02 lipocalin 11.8 P80511 Protein S100-A12* Regulates inﬂammatory processes and immune responses; recruitment of 5 (5), 35.9% 10,443 5.80 leukocytes, promotion of cytokine and chemokine production, and regulation of leukocyte adhesion and migration; antifungal and antimicrobial activity 10.6 P22894 Neutrophil collagenase Degrade ﬁbrillar collagens 14 (14), 37.7% 41,937 5.49 9.9 P59666 Neutrophil defensin 3* Fungicide, antiviral, and antimicrobial activity against bacteria 5 (5), 26.6% 3,492 8.33 9.4 P08637 Low-afﬁnity immunoglobulin Binds monomeric, complexed, or aggregated IgG; mediates antibody- 2 (2), 8.3% 27,324 8.24 gamma Fc region receptor dependent responses (e.g., phagocytosis) III-A 9.2 P52790 Hexokinase-3 Metabolic processes; phosphorylation of hexoses 21 (18), 31.3% 99,025 5.23 8.7 O75594 Peptidoglycan recognition Innate immunity; has bactericidal activity 5 (5), 41.3% 19,434 8.23 protein 1 8.5 P36222 Chitinase-3-like protein 1 Regulates inﬂammatory cell apoptosis, dendritic cell accumulation, and 14 (14), 44.9% 40,488 8.64 macrophage differentiation; facilitates invasion of pathogenic enteric bacteria into colonic mucosa 7.8 Q06141 Regenerating islet-derived Antimicrobial activity against gram-positive bacteria 4 (4), 36.6% 16,566 7.83 protein 3-alpha 7.3 Q1HG44 Dual oxidase maturation factor 2 Required for dual oxidase 2 maturation and transport to the plasma membrane 4 (4), 16.9% 34,786 8.51 6.6 Q05315 Galectin-10* Regulates immune responses; recognition of cell surface glycans 11 (11), 67.6% 16,321 6.80 6.6 P35228 Nitric oxide synthase, inducible Produces tumoricidal and bactericidal nitric oxide in macrophages 42 (41), 45.4% 131,117 8.20 6.2 P16050 Arachidonate 15-lipoxygenase Regulates macrophage function and epithelial wound healing in the cornea; 25 (25), 52.1% 74,673 6.14 may favor clearance of apoptotic cells during inﬂammation 5.9 P12724 Eosinophil cationic protein* Antibacterial activity, including cytoplasmic membrane depolarization 10 (10), 46.9% 15,518 10.47 5.7 Q8IX19 Mast cell-expressed membrane Integral component of membrane in mast cells (part of the immune system) 3 (3), 15.5% 21,228 9.03 protein 1 Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Neutrophil Extracellular Traps in UC www.ibdjournal.org 2061 TABLE 3 (Continued) Fold Peptides (Unique), Change Uniprot Acc. Protein Name Protein Function Seq. Cov. MW pI 5.1 P10153 Nonsecretory ribonuclease Pyrimidine-speciﬁc nuclease; selective chemotactic for dendritic cells 8 (8), 44.1% 15,463 9.20 5.0 P11678 Eosinophil peroxidase Mediates tyrosine nitration of secondary granule proteins in mature eosinophils 51 (44), 56.8% 81,040 10.70 4.5 P41218 Myeloid cell nuclear differentiation Plays a role in the granulocyte/monocyte response to interferon 17 (17), 39.1% 45,836 9.76 antigen* 4.4 P06310 Ig kappa chain V-II region RPMI Immunoregulatory interactions between a lymphoid and a nonlymphoid cell 4 (3), 37.6% 12,585 9.07 4.3 Q8TCU6 Phosphatidylinositol- Neutrophil chemotaxis; may function downstream of heterotrimeric G proteins 8 (8), 6.4% 186,203 6.03 3,4,5-trisphosphate–dependent in neutrophils Rac exchanger 4.0 P01040 Cystatin-A Intracellular thiol proteinase inhibitor; desmosome-mediated cell–cell adhesion 2 (2), 30.6% 10,875 5.39 in the lower epidermis levels 4.0 Q96Q80 Derlin-3 Functional component of endoplasmic reticulum-associated degradation for 4 (3), 26.8% 26,678 8.64 misfolded lumenal glycoproteins 3.9 Q7Z7N9 Transmembrane protein 179B Integral component of membrane 2 (2), 8.2% 23,550 8.10 3.8 Q70J99 Protein unc-13 homolog D Regulates secretory lysosome exocytosis in mast cells and cytotoxic granule 16 (16), 24.7% 123,281 6.19 exocytosis in lymphocytes; required for granule maturation and docking 3.5 P08311 Cathepsin G* Serine protease; cleaves complement C3; has antibacterial activity against the 16 (16), 54.1% 26,757 11.37 gram-negative bacterium Pseudomonas aeruginosa 0.4 P29966 Myristoylated alanine-rich Filamentous actin cross-linking protein; binds calmodulin, actin, and synapsin 11 (11), 49.1% 31,423 4.46 C-kinase substrate 0.3 P00326 Alcohol dehydrogenase 1C Oxidation of alcohol 27 (10), 64.8% 39,868 8.63 0.3 P20039 HLA class II histocompatibility Binds peptides derived from antigens that access the endocytic route of 9 (2), 40.6% 27,230 6.49 antigen, DRB1-11 beta chain antigen-presenting cells and presents them on the cell surface for recognition 0.3 P10082 Peptide YY Gut peptide; inhibits exocrine pancreatic secretion, and jejunal and colonic 3 (3), 29.9% 4,410 8.34 mobility; has vasoconstrictory action 0.3 Q12805 EGF-containing ﬁbulin-like May play a role in cell adhesion and migration; binds the EGF receptor, 15 (15), 43.0% 52,765 4.85 extracellular matrix protein 1 inducing phosphorylation and activation 0.3 Q9HBI6 Phylloquinone omega-hydroxylase Oxidizes a variety of compounds, including fatty acids and xenobiotics; may 11 (3), 26.7% 60,146 6.26 CYP4F11 play a role in blood coagulation 0.3 P15502 Elastin Major structural protein of tissues; regulates proliferation and organization of 6 (6), 17.7% 65,721 10.32 vascular smooth muscles 0.3 P10915 Hyaluronan and proteoglycan link Stabilizes the aggregates of proteoglycan monomers with hyaluronic acid in 14 (13), 48.6% 38,840 6.85 protein 1 the extracellular cartilage matrix 0.2 Q86Y39 NADH dehydrogenase Subunit of the mitochondrial membrane respiratory chain NADH 7 (7), 58.2% 14,721 8.95 (ubiquinone) 1 alpha dehydrogenase, thought not to be involved in catalysis subcomplex subunit 11 Bennike et al Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 FIGURE 5. Correlation analysis of inﬂammatory proteins. Colon inﬂammation grade scores and the protein abundance in the colon tissue of the inﬂammatory proteins lactotransferrin (d), S100-A8 (-), and S100-A9 (:) (S100-A8 and S100-A9 protein constitute calpro- tectin). amount of fecal calprotectin did not correlate with the colon inﬂammation grade scores or the measured amount of calprotectin in the tissue (data not shown), which have been reported by others. A likely explanation is that the measure of fecal calpro- tectin and the biopsy extraction was not conducted in parallel in the present study, and only for 3 of the 10 patients with UC were the fecal calprotectin measures obtained on the day. Additionally, fecal calprotectin is a marker for inﬂammation in the entire intes- tinal system, in contrast to the colon inﬂammation grade score and the proteomics analysis where the transverse colon or the FIGURE 6. Fold change of statistically signiﬁcantly changing NET- associated proteins between biopsies from the patients with UC and controls. The 11 proteins are on average 42.2 times (P , 0.0005) more abundant in the biopsies from the UC group compared with the biopsies from the control group. Center line shows the median, box limits indicate the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, crosses represent sample means, and the circle represents the fold change of lactotransferrin that lies outside the whiskers. 2062 www.ibdjournal.org TABLE 3 (Continued) Fold Peptides (Unique), Change Uniprot Acc. Protein Name Protein Function Seq. Cov. MW pI 0.2 P35558 Phosphoenolpyruvate carboxykinase, Catalyzes conversion of oxaloacetate to phosphoenolpyruvate, the rate-limiting 38 (34), 71.4% 69,195 5.8 cytosolic (GTP) step in the metabolic pathway that produces glucose from lactate and other precursors from the citric acid cycle 0.2 Q6PIJ6 F-box only protein 38 Thought to recognize and bind to some phosphorylated proteins and promotes 6 (6), 7.0% 133,944 5.92 their ubiquitination and degradation 0.2 Q07654 Trefoil factor 3 Involved in the maintenance and repair of the intestinal mucosa; promotes the 3 (3), 38.8% 6580 5.13 mobility of epithelial cells in healing 0.2 Q14508 WAP four-disulﬁde core domain Broad range protease inhibitor 4 (4), 45.2% 10,036 4.50 protein 2 Fold change .1 indicates increased mean abundance in the UC biopsies compared with controls. *Protein has been associated with NETs. The number of identiﬁed peptides, the resulting sequence coverage (seq. cov), and the number of unique peptides used for the quantitation are listed for each protein. Protein functions have been downloaded from Uniprot Knowledgebase, and molecular weight (MW) in daltons and isoelectric point (pI) have been downloaded from ExPASy. EGF, epidermal growth factor; GTP, guanosine triphosphate. Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Neutrophil Extracellular Traps in UC FIGURE 7. Confocal microscopic images of TO-PRO-3–stained biopsy slices (scale bars 100 mm): (A) Ctrl_5 and (B) UC_3. An increased number of stained cell nuclei surrounding the intestinal crypts are in general apparent in the UC biopsies compared with the control biopsies. ascending colon was not considered. This is likely why, for example, proteins were more abundant. These proteins were identiﬁed and patient UC_4 treated with suppository Pentasa had a colon inﬂam- quantiﬁed in the present study; however, we could not conﬁrm the mation grade score of 1, indicating for the most part healthy looking increased abundance. However, our study found several proteins biopsies. However, .3600 mg of fecal calprotectin per gram stools associated with the innate immune system to be increased in abun- wasmeasuredindicatinginﬂammation in the transverse colon or the dance in the UC colonic tissue. The differences between ﬁndings in ascending colon, too far from the rectum for the treatment with sup- the 2 studies are mainly ascribed to differences in the obtained patient pository Pentasa to have an effect. Finally, it has been reported that material and study design. The advantages of a state-of-the-art instru- fecal calprotectin can be increased 3 months before a disease ﬂare. ment and bioinformatics platform are demonstrated in the increase As the patients in this study were not followed up after the biopsy was from up to 339 proteins in the study by Han et al to the 5711 taken, it cannot be excluded that these patients later could have devel- quantiﬁable proteins in the present study, thus providing information oped disease ﬂares, which caused the elevated fecal calprotectin. on the regulation of a great number of proteins and thus cellular Of the 5711 quantiﬁable proteins, 46 proteins were functions. A potential drawback of the high number of quantiﬁable measured to have a statistically signiﬁcant change in abundance proteins is that a large number of t tests need to be performed during between the biopsies from the patients with UC and the controls. Of the statistical analysis. To avoid a high number of false positives these, 33 proteins were more abundant in the UC biopsies. Several of among the proteins found to be statistically signiﬁcant, multiple the proteins with a statistically signiﬁcant abundance increase were hypothesis testing must be applied, which increases the requirements involved in inﬂammation. On comparison with the only other gel-free for the P-values. As a consequence, when many proteins are quan- LC-MS study conducted, we found several proteins that were tiﬁed and analyzed, the statistical analysis results in the need for determined to be more abundant in the UC colon tissue in both a higher number of included subjects in the study. As an example, studies, namely, Protein S100-A9 (calprotectin), neutrophil elastase, an overabundance of the enzyme indoleamine-2,3-dioxygene has myeloperoxidase, neutrophil defensin, and cathepsin G. These con- been found in intestinal mucosal tissue from patients with IBD com- sistent ﬁndings validate the study. However, the study by Han pared with normal mucosa, hypothesizing an involvement of the 20 50,51 et al additionally reported that a number of histones and ﬁbrinogen Kynurenine pathway of tryptophan metabolism in the IBDs. www.ibdjournal.org 2063 Bennike et al Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 FIGURE 8. Confocal microscopy images of TO-PRO-3–stained biopsy slices from the UC group (scale bars 100 mm): (A) UC_6 with an intact neutrophil, with segmented multiobulated-shaped nucleus; (B) UC_4 with NET formation by excretion of the neutrophil DNA. 11,19 Indoleamine-2,3-dioxygenase is an intracellular enzyme that catalyzes detectable with 2-dimensional gel studies (Table 3). This in- the production of Kynurenine from the amino acid tryptophan. The cludes protein S100-A12, which was found to be 11.8-fold enzyme is regulated by gamma-interferon of the immune system. increased in the UC colonic tissue and also has been found ele- The protein in our study was detected with a 1.8 mean fold abundance vated in the serum of children with IBD. Interestingly, the pres- increase in the UC biopsies compared with controls but not statistically ent study is the ﬁrst MS-driven proteomics study to measure an signiﬁcant. This indicates that a larger study cohort could have iden- increased abundance of lactotransferrin in the UC colonic tissue. tiﬁed a higher number of statistically signiﬁcantly changed proteins. The increased abundance of fecal lactotransferrin is used as Gel-free quantitative LC-MS studies hold a number of a marker for inﬂammation, and the protein should have been 11,55 advantages over 1- and 2-dimensional gel-based approaches, includ- detectable using 2-dimensional gel-based analysis. However, ing increased sensitivity, increased dynamic range, and identiﬁcation it is possible that the relative abundance of this protein remains and quantiﬁcation of proteins in the thousands instead of hundreds. too low for gel-based studies. Nonetheless, the protein abundance However, the gel-free approaches such as the present study are limited in the colonic tissue correlates well with the abundance of cal- 11,19 in the ability to detect protein fragment products. The increased protectin in the tissue, and the colon inﬂammation grade scores in 11,55 sensitivity of the gel-free approaches becomes apparent when consid- this study, as expected. Another protein involved in inﬂam- ering that the gel-free LC-MS method usedinthisstudy analyzed 5 mation and found with increased abundance in the UC biopsies is mgofthe ﬁnal material compared with the hundreds of microgram myeloblastin. Myeloblastin is a protease found in neutrophils and material used for the 2-dimensional gel analysis, which allowed us to was found to be 31.4-fold more abundant in the UC colonic 17,53 conduct repeated analysis of each biopsy. Two-dimensional gel biopsies compared with controls. The protease has speciﬁcity studies are usually limited to proteins within a molecular mass range toward, among others, the protein elastin, which accordingly of 15 to 200 kDa and isoelectric point of the proteins in the range of 3 has a decreased abundance in the UC biopsies, with a 1/3.3- to 10. These limitations do not apply for gel-free approaches, and fold change. Another interesting protein found with 8.7-fold applying these criteria to the 46 proteins detected with a statistically increase in abundance in the UC colonic biopsies is peptidoglycan signiﬁcant change in this study 30.4% (14 proteins) would not be recognition protein 1. The protein is part of the innate immune 2064 www.ibdjournal.org Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Neutrophil Extracellular Traps in UC systems recognition of microorganisms and mediates its effect by found as NETs. We stained the biopsy sections with a DNA stain binding bacterial peptidoglycans, which triggers a signaling cas- and investigated the sections by confocal microscopy, which al- 56,57 cade. This is the ﬁrst study to ﬁnd an increased abundance of lowed us to achieve a higher resolution than with light micros- peptidoglycan recognition protein 1 in UC colonic tissue. copy. Again, the segmented cell nucleus of the PMNs was Hexokinase-3 is an intracellular enzyme that phosphorylates glu- apparent, and additionally, in biopsies from UC_4, we observed cose into glucose-6-phosphate and, together with hexokinase 1, 2, NETs at 2 distinct locations. The proteomics data and microscopy 58,59 and 4, is a central rate-limiting step in glucose metabolism. data demonstrate the increased abundance of PMNs in UC colon Not much is known about the regulation of hexokinase 3; how- tissue and NETs in biopsies from UC_4. This study is the ﬁrst to ever, the 9.2-fold increased abundance of this enzyme in the UC directly observe NETs in UC colon tissue. Interestingly, the abun- biopsies indicates that glucose metabolism might be increased in dances of several NET-associated histone proteins (H1e, H2A, the inﬂamed tissue compared with controls. H2B, H3, and H4) were not found with increased abundance in The increased abundance of inﬂammation-associated pro- the UC biopsies. Additionally, histones H2A and H3 were found teins points to an activated innate immune system in the UC to be more abundant in the UC colonic tissue by Han et al, colon, even in colonic tissue without visible inﬂammation. It indicating that NETs could have been present in the colonic tissue should be noted that we do not know the impact of the medication obtained in this study as well. However, as histone proteins are an on the protein regulations. However, as most of the statistically essential part of nearly all cells, a relative increase in the abun- signiﬁcant changed proteins are involved in inﬂammatory pro- dance of these proteins between the UC biopsies and controls as cesses, these protein changes are likely caused by the disease a result of the presence of NETs might be too small to be detected. development. Additionally, the number of former smokers was NETs are part of the innate immune system’s response to 47,68 increased in the UC group compared with controls. The impact of invading pathogens. Additionally, several of proteins found to cigarette smoking on the etiology of UC has been vastly debated be more abundant in the UC colon tissue are directly involved in 60–65 over the years. Current knowledge supports that the risk for the mucosal innate immune system response. However, the cause developing UC is highest 2 to 5 years after smoking cessation and for the immune response remains unknown, and no indications of remains elevated for more than 20 years, and patients with UC are bacteria or epithelial cell disruption were detected by the histol- more prone to disease ﬂairs if they quit smoking. However, the ogy analysis, which should have been visible using confocal subject is still debated and the dose–response effect remains to be microscopy. It cannot be ruled out that the immune reactions properly determined. Based on the present study, we cannot could be caused by bacterial components capable of crossing assess the impact of former smoking on the protein data. the intestinal barrier of epithelial cells, which is known to be To evaluate the morphology of the tissue, the biopsies were compromised in IBD where an increased epithelial permeability 10,69,70 sectioned and H&E stained, and the histological evaluation was has been described. However, it has been demonstrated that done by light microscopy. The intestinal crypts were preserved in aberrant formation of NETs might be involved in the pathogenesis all biopsies, in agreement with the biopsies being taken from of autoimmunity and additionally might be formed during a num- 71–73 morphologically normal tissue. However, an increased abundance ber of activities, including exercise. Therefore, based on the of cell nuclei in the colon mucosal tissue was observed between obtained data in this study, we can conclude that a chronic inﬂam- the intestinal crypts, with segmented cell nuclei, a characteristic of mation is present in the tissue, even though the surface appears PMNs. Based on the proteomics data and light microscopy, we normal. However, we cannot point to the origin of the inﬂamma- here verify the increased density of PMNs in the UC colonic tion or the target of the immune response. tissue. This has been reported by other groups as well, but this Our ﬁndings demonstrate that even though remission has is the ﬁrst study to support the light microscopy identiﬁcation of been achieved on the surface of the UC colonic tissue, a chronic 66,67 18 the PMNs with MS techniques. PMNs are essential for the condition is still present within the tissue. Poulsen et al per- innate immunity and are the most abundant leukocyte in plasma. formed a 2-dimensional proteomics study of 20 patients with In response to inﬂammatory stimuli, they migrate from plasma to UC where inﬂamed and noninﬂamed tissue from the same patient the affected tissue where they take part in the ﬁrst line of innate with UC were compared, in contrast to the present study where immune response. The effect is mediated through the release of noninﬂamed tissue from several patients with UC was compared 18 18 enzymes from the granules and the production of compounds with with controls. Interestingly, the study by Poulsen et al found antimicrobial potential and inﬂammation regulatory effects. One that in contrast to the macroscopically different tissue, the pro- of the compounds is NET. PMNs can disintegrate their cell mem- teome of visually inﬂamed and noninﬂamed tissue was only brane and release NETs to capture and kill invasive microorgan- slightly different. Of the 43 protein spots identiﬁed with a different 47,68 isms. The NETs are composed of relaxed DNA to which abundance, the protein displaying the largest fold change was histones and other effector molecules are bound. Eleven of the glycerol-3-phosphate dehydrogenase, which was increased by 46 proteins with statistically signiﬁcantly altered abundance in the 3.3-fold in the inﬂamed UC tissue compared with noninﬂamed UC biopsies are associated with NET formation, and all were tissue, which is a minor change compared with the up to 219-fold measured with increased abundance. As no proteins unique to changes measured in the present study. Combined with the anal- NETs are known, we investigated if the PMNs were intact or ysis of differences in colonic proteome between patients with UC www.ibdjournal.org 2065 Bennike et al Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 and controls, we can conclude that the difference in proteome REFERENCES 1. Molodecky NA, Soon IS, Rabi DM, et al. Increasing incidence and prev- between visually inﬂamed and noninﬂamed UC colonic tissue is alence of the inﬂammatory bowel diseases with time, based on systematic vastly smaller than the interpersonal difference between patients review. Gastroenterology. 2012;142:46–54.e42. with UC and controls. This indicates that changes are present in 2. Odes S, Vardi H, Friger M, et al. Cost analysis and cost determinants in a European inﬂammatory bowel disease inception cohort with 10 years of the UC colonic tissue regardless of visible surface inﬂammation, follow-up evaluation. Gastroenterology. 2006;131:719–728. even in well-medicated patients with UC. A 2012 Danish 3. Jess T, Frisch M, Simonsen J. Trends in overall and cause-speciﬁc mor- population-based cohort study of 300 patients with UC in the tality among patients with inﬂammatory bowel disease from 1982 to 2010. period of 2003 to 2011, found that even though intensiﬁed immu- Clin Gastroenterol Hepatol. 2013;11:43–48. 4. Jussila A, Virta LJ, Pukkala E, et al. Mortality and causes of death in nomodulation therapy is being used in Denmark, the ﬁrst time patients with inﬂammatory bowel disease: a nationwide register study in recurrence rate, hospitalization rate, and surgical rates for patients Finland. J Crohns Colitis. 2014;8:1088–1096. with UC have not decreased. This observation supports the 5. Khor B, Gardet A, Xavier RJ. Genetics and pathogenesis of inﬂammatory bowel disease. Nature. 2011;474:307–317. hypothesis that the administered treatment mostly is symptomatic. 6. Iskandar HN, Ciorba MA. Biomarkers in inﬂammatory bowel disease: However, it is worth mentioning that a study by Reich et al, in current practices and recent advances. Transl Res. 2012;159:313–325. which 481 patients with UC undergoing colectomy in the period 7. Ehrentraut SF, Colgan SP. Implications of protein post-translational mod- iﬁcations in IBD. Inﬂamm Bowel Dis. 2012;18:1378–1388. 1998 to 2011 were included, showed that following the approval of 8. Thompson AI, Lees CW. Genetics of ulcerative colitis. Inﬂamm Bowel anti–tumor necrosis factor treatment for UC in 2005, the colectomy Dis. 2011;17:831–848. rate has dropped. None of the patients included in this study had 9. Jostins L, Ripke S, Weersma RK, et al. Host-microbe interactions have shaped the genetic architecture of inﬂammatory bowel disease. Nature. active disease or was under treatment with anti-tumor necrosis 2012;491:119–124. factor substances. Nonetheless, based on our study, we can con- 10. Xavier RJ, Podolsky DK. Unravelling the pathogenesis of inﬂammatory clude that chronic inﬂammation remains in the UC colonic tissue. bowel disease. Nature. 2007;448:427–434. 11. Bennike T, Birkelund S, Stensballe A, et al. Biomarkers in inﬂammatory In conclusion, we have found an increased abundance of bowel diseases: current status and proteomics identiﬁcation strategies. PMNs in the UC colonic tissue, using high-throughput proteo- World J Gastroenterol. 2014;20:3231–3244. mics, light microscopy, and confocal microscopy. Additionally, 12. Tontini GE, Vecchi M, Pastorelli L, et al. Differential diagnosis in inﬂam- matory bowel disease colitis: state of the art and future perspectives. we have for the ﬁrst time observed NETs, detected by proteomics World J Gastroenterol. 2015;21:21–46. and veriﬁed by light microscopy at 2 distinct locations in a biopsy 13. Kane SV, Sandborn WJ, Rufo PA, et al. Fecal lactoferrin is a sensitive and from a patient with UC. Our ﬁndings demonstrate an activated speciﬁc marker in identifying intestinal inﬂammation. Am J Gastroenterol. 2003;98:1309–1314. innate immune system in well-medicated patients with UC 14. Mendoza JL, Abreu MT. Biological markers in inﬂammatory bowel dis- without disease activity and the presence of chronic inﬂammation ease: practical consideration for clinicians. Gastroenterol Clin Biol. 2009; in the morphologically normal tissue. Additionally, through 33(suppl 3):S158–S173. 15. Johne B, Fagerhol MK, Lyberg T, et al. Functional and clinical aspects of a comparison with other studies, we can conclude that the the myelomonocyte protein calprotectin. Mol Pathol. 1997;50:113–123. proteome of inﬂamed and noninﬂamed tissue from the same 16. Steinbakk M, Naess-Andresen C-F, Fagerhol MK, et al. Antimicrobial patient is much more similar compared with the proteome of actions of calcium binding leucocyte L1 protein, calprotectin. Lancet. 1990;336:763–765. different individuals, indicating that a chronic condition is present 17. Hsieh SY, Shih TC, Yeh CY, et al. Comparative proteomic studies on the even in UC colonic tissue that appears normal during endoscopy. pathogenesis of human ulcerative colitis. Proteomics. 2006;6:5322–5331. However, no indications of bacterial inﬁltrations in the tissue were 18. Poulsen NA, Andersen V, Møller JC, et al. Comparative analysis of in- ﬂamed and non-inﬂamed colon biopsies reveals strong proteomic inﬂam- found in the study. To increase our understanding of the UC and mation proﬁle in patients with ulcerative colitis. BMC Gastroenterol. IBD etiology, future studies should aim at investigating the 2012;12:76. etiology of the immune reaction in the UC colonic tissue. 19. Camerini S, Mauri P. The role of protein and peptide separation before mass spectrometry analysis in clinical proteomics. J Chromatogr A. 2015; 1381:1–12. 20. Han NY, Choi W, Park JM, et al. Label-free quantiﬁcation for discovering ACKNOWLEDGMENTS novel biomarkers in the diagnosis and assessment of disease activity in inﬂammatory bowel disease. J Dig Dis. 2013;14:166–174. Knud and Edith Eriksens Memorial Foundation and Ferring 21. Krzywinski M, Altman N. Points of signiﬁcance: comparing samples— are acknowledged for grants, enabling the collection of the part II. Nat Methods. 2014;11:355–356. biological sample material (V.A.). The Obelske Family Founda- 22. Dignass A, Eliakim R, Magro F, et al. Second European evidence-based consensus on the diagnosis and management of ulcerative colitis part 1: tion and the Svend Andersen Foundation are acknowledged for deﬁnitions and diagnosis. J Crohns Colitis. 2012;6:965–990. grants supporting the analytical platform (A.S.). Knud Hojgaards 23. Fernández-Bañares F, Casalots J, Salas A, et al. Paucicellular lymphocytic Foundation Denmark, the Lundbeck Foundation Denmark, the colitis: is it a minor form of lymphocytic colitis? A clinical pathological and immunological study. Am J Gastroenterol. 2009;104:1189–1198. Oticon Foundation Denmark, and the Otto Monsteds Foundation 24. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years Denmark are acknowledged for grants supporting the collabora- of image analysis. Nat Methods. 2012;9:671–675. tion (T.B.B.). Additionally, the authors would like to thank 25. Hanauer SB, Robinson M, Pruitt R, et al. Budesonide enema for the Kasper B. Lauridsen for help establishing the patient cohort, Lau treatment of active, distal ulcerative colitis and proctitis: a dose-ranging study. Gastroenterology. 1998;115:525–532. Sennels for help with the data analysis, Professor Mogens Vyberg 26. Leon IR, Schwammle V, Jensen ON, et al. Quantitative assessment of in- for help interpreting the histology descriptions, and the PRIDE solution digestion efﬁciency identiﬁes optimal protocols for unbiased pro- team for making the proteomics data publically available. tein analysis. Mol Cell Proteomics. 2013;12:2992–3005. 2066 www.ibdjournal.org Inﬂamm Bowel Dis Volume 21, Number 9, September 2015 Neutrophil Extracellular Traps in UC 27. Wisniewski JR, Zougman A, Nagaraj N, et al. Universal sample prepara- 51. Munn DH, Mellor AL. Indoleamine 2,3 dioxygenase and metabolic con- tion method for proteome analysis. Nat Methods. 2009;6:359–362. trol of immune responses. Trends Immunol. 2013;34:137–143. 28. Manza LL, Stamer SL, Ham AJL, et al. Sample preparation and digestion 52. Schmidt SV, Schultze JL. New insights into IDO biology in bacterial and for proteomic analyses using spin ﬁlters. Proteomics. 2005;5:1742–1745. viral infections. Front Immunol. 2014;5:384. 29. Masuda T, Tomita M, Ishihama Y. Phase transfer surfactant-aided trypsin 53. Shkoda A, Werner T, Daniel H, et al. Differential protein expression digestion for membrane proteome analysis. JProteomeRes. 2008;7:731–740. proﬁle in the intestinal epithelium from patients with inﬂammatory bowel 30. Urbaniak GC, Plous S. Research Randomizer (Version 4.0) [computer disease. J Proteome Res. 2007;6:1114–1125. software]. 2013. Available at: http://www.randomizer.org/. Accessed 54. Leach ST, Yang Z, Messina I, et al. Serum and mucosal S100 proteins, October 10, 2013. calprotectin (S100A8/S100A9) and S100A12, are elevated at diagnosis in 31. Cox J, Mann M. MaxQuant enables high peptide identiﬁcation rates, children with inﬂammatory bowel disease. Scand J Gastroenterol. 2007; individualized p.p.b.-range mass accuracies and proteome-wide protein 42:1321–1331. quantiﬁcation. Nat Biotech. 2008;26:1367–1372. 55. Lehmann FS, Burri E, Beglinger C. The role and utility of faecal 32. Cox J, Neuhauser N, Michalski A, et al. Andromeda: a peptide search markers in inﬂammatory bowel disease. Therap Adv Gastroenterol. engine integrated into the MaxQuant environment. J Proteome Res. 2011; 2015;8:23–36. 10:1794–1805. 56. Lu X, Wang M, Qi J, et al. Peptidoglycan recognition proteins are a new 33. Bennike T, Ayturk U, Haslauer CM, et al. A normative study of the class of human bactericidal proteins. J Biol Chem. 2006;281:5895–5907. synovial ﬂuid proteome from healthy porcine knee joints. J Proteome 57. Liu C, Xu Z, Gupta D, et al. Peptidoglycan recognition proteins a novel Res. 2014;13:4377–4387. family of four human innate immunity pattern recognition molecules. 34. Bennike T, Lauridsen KB, Olesen MK, et al. Optimizing the identiﬁcation J Biol Chem. 2001;276:34686–34694. of citrullinated peptides by mass spectrometry: utilizing the inability of 58. Furuta H, Nishi S, Le Beau MM, et al. Sequence of human hexokinase III trypsin to cleave after citrullinated amino acids. J Proteomics Bioinform. cDNA and assignment of the human hexokinase III gene (HK3) to chro- 2013;06:288–295. mosome band 5q35.2 by ﬂuorescence in situ hybridization. Genomics. 35. Deeb SJ, D’Souza RCJ, Cox J, et al. Super-silac allows classiﬁcation of 1996;36:206–209. diffuse large B-cell lymphoma subtypes by their protein expression pro- 59. Lowes W, Walker M, Alberti KG, et al. Hexokinase isoenzymes in normal ﬁles. Mol Cell Proteomics. 2012;11:77–89. and cirrhotic human liver: suppression of glucokinase in cirrhosis. Bio- 36. Bhatia VN, Perlman DH, Costello CE, et al. Software tool for researching chim Biophys Acta. 1998;1379:134–142. annotations of proteins: open-source protein annotation software with data 60. Rosenfeld G, Bressler B. The truth about cigarette smoking and the risk of visualization. Anal Chem. 2009;81:9819–9823. inﬂammatory bowel disease. Am J Gastroenterol. 2012;107:1407–1408. 37. Tusher VG, Tibshirani R, Chu G. Signiﬁcance analysis of microarrays 61. Higuchi LM, Khalili H, Chan AT, et al. A Prospective study of cigarette applied to the ionizing radiation response. Proc Natl Acad Sci U S A. smoking and the risk of inﬂammatory bowel disease in women. Am J 2001;98:5116–5121. Gastroenterol. 2012;107:1399–1406. 38. Hubner NC, Bird AW, Cox J, et al. Quantitative proteomics combined 62. Abraham C, Cho JH. Inﬂammatory bowel disease. N Engl J Med. 2009; with BAC TransgeneOmics reveals in vivo protein interactions. J Cell 361:2066–2078. Biol. 2010;189:739–754. 63. Mahid SS, Minor KS, Soto RE, et al. Smoking and inﬂammatory bowel 39. Murgia M, Nagaraj N, Deshmukh AS, et al. Single muscle ﬁber proteo- disease: a meta-analysis. Mayo Clin Proc. 2006;81:1462–1471. mics reveals unexpected mitochondrial specialization. EMBO Rep. 2015; 64. García Rodríguez LA, González-Pérez A, Johansson S, et al. Risk factors 16:387–395. for inﬂammatory bowel disease in the general population. Aliment Phar- 40. Vizcaíno JA, Deutsch EW, Wang R, et al. ProteomeXchange provides macol Ther. 2005;22:309–315. globally coordinated proteomics data submission and dissemination. Nat 65. Lindberg E, Tysk C, Andersson K, et al. Smoking and inﬂammatory Biotechnol. 2014;32:223–226. bowel disease. A case control study. Gut. 1988;29:352–357. 41. Vizcaíno JA, Côté RG, Csordas A, et al. The PRoteomics IDEntiﬁcations 66. Khor TS, Fujita H, Nagata K, et al. Biopsy interpretation of colonic (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res. biopsies when inﬂammatory bowel disease is excluded. J Gastroenterol. 2013;41:D1063–D1069. 2012;47:226–248. 42. Fournier BM, Parkos CA. The role of neutrophils during intestinal inﬂam- 67. Geboes K. Histopathology of Crohn’s disease and ulcerative colitis. In- mation. Mucosal Immunol. 2012;5:354–366. ﬂamm Bowel Dis. 2003:255–276. 43. Brinkmann V, Zychlinsky A. Neutrophil extracellular traps: is immunity 68. Branzk N, Lubojemska A, Hardison SE, et al. Neutrophils sense microbe the second function of chromatin? J Cell Biol. 2012;198:773–783. size and selectively release neutrophil extracellular traps in response to 44. Rao PK, Li Q. Principal component analysis of proteome dynamics in large pathogens. Nat Immunol. 2014;15:1017–1025. iron-starved mycobacterium tuberculosis. J Proteomics Bioinform. 2009; 69. Fava F, Danese S. Intestinal microbiota in inﬂammatory bowel disease: 2:19–31. friend of foe? World J Gastroenterol. 2011;17:557–566. 45. O’Donoghue AJ, Jin Y, Knudsen GM, et al. Global substrate proﬁling of 70. Tlaskalová-Hogenová H, Stepánková R, Hudcovic T, et al. Commensal proteases in human neutrophil extracellular traps reveals consensus motif bacteria (normal microﬂora), mucosal immunity and chronic inﬂammatory predominantly contributed by Elastase Glogauer M, ed. PLoS One. 2013; and autoimmune diseases. Immunol Lett. 2004;93:97–108. 8:e75141. 71. Beiter T, Fragasso A, Hartl D, et al. Neutrophil extracellular traps: a walk 46. Mantovani A, Cassatella MA, Costantini C, et al. Neutrophils in the on the wild side of exercise immunology. Sports Med. 2015;45:625–640. activation and regulation of innate and adaptive immunity. Nat Rev Im- 72. Beiter T, Fragasso A, Hudemann J, et al. Neutrophils release extracel- munol. 2011;11:519–531. lular DNA traps in response to exercise. J Appl Physiol (1985). 2014; 47. Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular 117:325–333. traps kill bacteria. Science. 2004;303:1532–1535. 73. Stephan A, Fabri M. The NET, the trap, and the pathogen: neutrophil 48. Schoepfer AM, Beglinger C, Straumann A, et al. Fecal calprotectin more extracellular traps in cutaneous immunity. Exp Dermatol. 2014;24: accurately reﬂects endoscopic activity ulcerative colitis than Lichtiger 161–166. Index, C-reactive protein platelets, hemoglobin, and blood leukocytes. 74. Vester-Andersen MK, Vind I, Prosberg MV, et al. Hospitalisation, surgi- Inﬂamm Bowel Dis. 2013;19:332–341. cal and medical recurrence rates in inﬂammatory bowel disease 2003– 49. Vos MD, Louis EJ, Jahnsen J, et al. Consecutive fecal calprotectin meas- 2011—a Danish population-based cohort study. J Crohns Colitis. 2014;8: urements predict relapse patients ulcerative colitis receiving inﬂiximab 1675–1683. maintenance therapy. Inﬂamm Bowel Dis. 2013;19:2111–2117. 75. Reich KM, Chang H-J, Rezaie A, et al. The incidence rate of colectomy 50. Barceló-Batllori S, André M, Servis C, et al. Proteomic analysis of cyto- for medically refractory ulcerative colitis has declined in parallel with kine induced proteins in human intestinal epithelial cells: implications for increasing anti-TNF use: a time-trend study. Aliment Pharmacol Ther. inﬂammatory bowel diseases. Proteomics. 2002;2:551–560. 2014;40:629–638. www.ibdjournal.org 2067

Journal

Inflammatory Bowel Diseases – Pubmed Central

Published: May 19, 2015

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies

Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies

Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies

References (153)

Abstract

Journal

Recommended Articles

There are no references for this article.

Our policy towards the use of cookies