Alexis Goichon

Wafa Bahlouli

Ibtissem Ghouzali

Philippe Chan

David Vaudry

Pierre Déchelotte

Philippe Ducrotté

Moïse Coëffier
email: moise.coeffier@univ-rouen.fr

Its Relevance for Irritable Bowel Syndrome

Keywords: Colonic proteome, Immunoproteasome, Irritable bowel syndrome, Water avoidance stress

INTRODUCTION

Irritable bowel syndrome (IBS) is a functional gastrointestinal (GI) disorder that affects 5-20% of the general population in industrialized countries. [START_REF] Pluart | Functional Gastrointestinal Disorders in 35,447 Adults and Their Association with Body Mass Index[END_REF]2 It is characterized by chronic abdominal pain associated with diarrhea and/or constipation, bloating and urgency. [START_REF] Drossman | AGA Technical Review on Irritable Bowel Syndrome[END_REF] Recent studies have shown that IBS-associated symptoms, such as visceral hypersensitivity, low-grade inflammation or pain, may be linked to disturbances in intestinal barrier function. [START_REF] Simrén | Quality of Life in Inflammatory Bowel Disease in Remission: The Impact of IBS-like Symptoms and Associated Psychological Factors[END_REF][START_REF] Camilleri | Intestinal Barrier Function in Health and Gastrointestinal Disease[END_REF][START_REF] Zhou | New Insights into Visceral Hypersensitivity--Clinical Implications in IBS[END_REF][START_REF] Spiller | Increased Rectal Mucosal Enteroendocrine Cells, T Lymphocytes, and Increased Gut Permeability Following Acute Campylobacter Enteritis and in Post-Dysenteric Irritable Bowel Syndrome[END_REF][START_REF] Dunlop | Abnormal Intestinal Permeability in Subgroups of Diarrhea-Predominant Irritable Bowel Syndromes[END_REF][START_REF] Zhou | Intestinal Membrane Permeability and Hypersensitivity in the Irritable Bowel Syndrome[END_REF] Alterations were observed in the expression and distribution of intestinal tight junctions (TJs), apical protein structures that control paracellular permeability. [START_REF] Coëffier | Increased Proteasome-Mediated Degradation of Occludin in Irritable Bowel Syndrome[END_REF][START_REF] Bertiaux-Vandaële | The Expression and the Cellular Distribution of the Tight Junction Proteins Are Altered in Irritable Bowel Syndrome Patients with Differences According to the Disease Subtype[END_REF][START_REF] Martínez | The Jejunum of Diarrhea-Predominant Irritable Bowel Syndrome Shows Molecular Alterations in the Tight Junction Signaling Pathway That Are Associated with Mucosal Pathobiology and Clinical Manifestations[END_REF] Indeed, IBS patients with diarrheapredominant symptoms (IBS-D) exhibited an increased permeability both in colon [START_REF] Zhou | MicroRNA-29a Regulates Intestinal Membrane Permeability in Patients with Irritable Bowel Syndrome[END_REF] and jejunum [START_REF] Martínez | The Jejunum of Diarrhea-Predominant Irritable Bowel Syndrome Shows Molecular Alterations in the Tight Junction Signaling Pathway That Are Associated with Mucosal Pathobiology and Clinical Manifestations[END_REF] , and a reduced expression of several TJ proteins such as occludin, zonula occludens-1 (ZO-1) and claudin-1. [START_REF] Bertiaux-Vandaële | The Expression and the Cellular Distribution of the Tight Junction Proteins Are Altered in Irritable Bowel Syndrome Patients with Differences According to the Disease Subtype[END_REF][START_REF] Martínez | The Jejunum of Diarrhea-Predominant Irritable Bowel Syndrome Shows Molecular Alterations in the Tight Junction Signaling Pathway That Are Associated with Mucosal Pathobiology and Clinical Manifestations[END_REF] Emerging evidence supports the concept of altered colonic ubiquitineproteasome system (UPS) in IBS patients that could be responsible of intestinal barrier dysfunction. Interestingly, occludin has been reported as a functional target of the E3 ubiquitinprotein ligase Itch [START_REF] Traweger | The Tight Junction-Specific Protein Occludin Is a Functional Target of the E3 Ubiquitin-Protein Ligase Itch[END_REF] and we have previously shown that the low expression of occludin in the colonic mucosa of IBS patients was related to its higher degradation by UPS. [START_REF] Bertiaux-Vandaële | The Expression and the Cellular Distribution of the Tight Junction Proteins Are Altered in Irritable Bowel Syndrome Patients with Differences According to the Disease Subtype[END_REF] UPS is a major pathway of proteolysis in eukaryotic cells and may contribute to the regulation of many intestinal biological processes, such as inflammatory response, cell proliferation and apoptosis. [START_REF] Coëffier | Increased Proteasome-Mediated Degradation of Occludin in Irritable Bowel Syndrome[END_REF][START_REF] Visekruna | Proteasome-Mediated Degradation of IkappaBalpha and Processing of P105 in Crohn Disease and Ulcerative Colitis[END_REF][START_REF] Leblond | Regulation of Proteolysis by Cytokines in the Human Intestinal Epithelial Cell Line HCT-8: Role of IFNgamma[END_REF] Targeted proteins by UPS are polyubiquitinated and then processed by the constitutive 20S proteasome, which comprises three catalytic subunits named β1, β2, and β5. In mammals, the exposure of cells to inflammatory cytokines such as IFN-γ or TNF-α leads to the formation of an immunoproteasome that can be distinguished from its constitutive homologue through assembly of the catalytic immunosubunits named LMP2 (β1i), MECL-1 (β2i) and LMP7 (β5i). [START_REF] Kloetzel | Antigen Processing by the Proteasome[END_REF] It has been reported an increased expression of immunoproteasome subunits in intestinal epithelial cell lines stimulated by IFN-γ. [START_REF] Visekruna | Proteasome-Mediated Degradation of IkappaBalpha and Processing of P105 in Crohn Disease and Ulcerative Colitis[END_REF][START_REF] Leblond | Regulation of Proteolysis by Cytokines in the Human Intestinal Epithelial Cell Line HCT-8: Role of IFNgamma[END_REF] In vivo, the immunosubunits β1i and β2i were strongly upregulated in colon tissues of IBS [START_REF] Coëffier | Increased Proteasome-Mediated Degradation of Occludin in Irritable Bowel Syndrome[END_REF] and Crohn's disease (CD) patients, [START_REF] Visekruna | Proteasome-Mediated Degradation of IkappaBalpha and Processing of P105 in Crohn Disease and Ulcerative Colitis[END_REF] but not in patients with ulcerative colitis. Proteasome inhibitors such as bortezomib, MG132 and PR-957 have been developed and have achieved success in preventing experimental colitis in mice. [START_REF] Inoue | The Effect of Proteasome Inhibitor MG132 on Experimental Inflammatory Bowel Disease[END_REF][START_REF] Schmidt | Targeting the Proteasome: Partial Inhibition of the Proteasome by Bortezomib or Deletion of the Immunosubunit LMP7 Attenuates Experimental Colitis[END_REF][START_REF] Basler | Prevention of Experimental Colitis by a Selective Inhibitor of the Immunoproteasome[END_REF] Similar effects have been observed with mice deficient in subunit β5i. [START_REF] Schmidt | Targeting the Proteasome: Partial Inhibition of the Proteasome by Bortezomib or Deletion of the Immunosubunit LMP7 Attenuates Experimental Colitis[END_REF] More recently, we showed a link between proteasome and intestinal permeability in mice. In this latter study, both pharmacological immunoproteasome inhibition and β2i subunit deficiency limited colonic hyperpermeability and improved occludin expression. [START_REF] Ghouzali | Targeting Immunoproteasome and Glutamine Supplementation Prevent Intestinal Hyperpermeability[END_REF] To better understand the underlying mechanisms, we thus aimed (i) to evaluate the colonic proteome in wild-type and β2i immunoproteasome subunit knock-out (β2i -/-) mice during water avoidance stress (WAS) and (ii) to investigate the colonic expression of 49 ubiquitinated proteins in IBS-D patients.

MATERIAL AND METHODS

Animals

Male C57Bl/6 knock-out mice for MECL1 gene (or β2i proteasome subunit) were a kind gift from Professors Marcus Groettrup and Mickael Basler of Konstanz University (Germany) with the permission of Professor John Monaco from Cincinnati University (Ohio). The protocol was approved by CENOMEXA, the Ethics Committee of Normandy for Animal Experimentation (acceptance number: N/04-11-12/27/11-15). In addition, all experiments were performed in accordance with relevant guidelines and regulations (Official Journal of the European Community L 358, 18/12/1986) and followed ARRIVE guidelines. [START_REF] Kilkenny | Survey of the Quality of Experimental Design, Statistical Analysis and Reporting of Research Using Animals[END_REF]

Water avoidance stress

The water avoidance stress (WAS) is a murine model of chronic stress that induces several symptoms associated to IBS. In particular, WAS increased visceral sensibility, colonic motility, intestinal permeability, pro-inflammatory cytokine production and mast cell count. [START_REF] Ghouzali | Targeting Immunoproteasome and Glutamine Supplementation Prevent Intestinal Hyperpermeability[END_REF][START_REF] Bradesi | Repeated Exposure to Water Avoidance Stress in Rats: A New Model for Sustained Visceral Hyperalgesia[END_REF][START_REF] Lee | Repeated Water Avoidance Stress Alters Mucosal Mast Cell Counts, Interleukin-1β Levels with Sex Differences in the Distal Colon of Wistar Rats[END_REF][START_REF] Larauche | Repeated Psychological Stress-Induced Alterations of Visceral Sensitivity and Colonic Motor Functions in Mice: Influence of Surgery and Postoperative Single Housing on Visceromotor Responses[END_REF] After one week of acclimatization at 23°C (12-hour light-dark cycle; dark phase: 8:00 PM -8:00 AM), WAS model was performed during 1 hour per day during 10 consecutive days as previously described. [START_REF] Ghouzali | Targeting Immunoproteasome and Glutamine Supplementation Prevent Intestinal Hyperpermeability[END_REF] After the last WAS procedure, mice were euthanized. Colonic samples were either taken, washed with ice-cold PBS and immediately frozen until further analysis or immediately used to evaluate paracellular permeability.

Intestinal permeability

Colonic permeability was evaluated by measuring paracellular flux of FITC-dextran (4 kDa) through colonic samples mounted in Ussing chambers as previously described. [START_REF] Jésus | Alteration of Intestinal Barrier Function during Activity-Based Anorexia in Mice[END_REF] Briefly, FITCdextran (5 mg/ml) was placed in the mucosal side. After 3 hours at 37°C, medium from the serosal side was removed and stored at -80°C. The fluorescence level of FITC-dextran was measured in 96-well black plate with spectrometer Chameleon V (excitation at 485 nm, emission at 535 nm; Hidex, Turku, Finland).

qRT-PCR

Mucosal total RNA was extracted from samples as previously described. [START_REF] Coëffier | Enteral Glutamine Stimulates Protein Synthesis and Decreases Ubiquitin MRNA Level in Human Gut Mucosa[END_REF] After reverse transcription of 1.5 µg total RNA into cDNA by using 200 units of SuperScript™ II Reverse Transcriptase (LifeTechnologies, Cergy-Pontoise, France), qPCR was performed by SYBR™ Green technology on BioRad CFX96 real time PCR system (BioRad Laboratories, Marnes la Coquette, France) in duplicate for each sample. GAPDH was used as the endogenous reference gene. Specific primers are displayed in Table S-1.

Proteasome activities

Proteasome activities were evaluated on colonic mucosa homogenized in ice-cold lysis buffer containing 30 mM Tris-HCl (pH 7.2), 1 mM dithiothreitol, 1% Triton X-100 by spectrofluorimetry on microtiter plate fluorometer Mithras LB 940 (Berthold Technologies, Bad Wildbad, Germany) as previously described. 10

Protein extraction for comparative proteome and ubiquitome analysis

Colonic mucosa were homogenized, as previously described, 28,29 in ice-cold lysis buffer containing 7 M urea, 2 M thiourea, 4% (wt:vol) CHAPS, 50 mM dithiothreitol, 25 mM spermine tetrahydrochloride, 1X protease inhibitor cocktail (P2714; Sigma Aldrich) and 0.5% (vol:vol) IPG buffer with pH 3-10 non-linear (GE Healthcare). The protein content was determined by using 2D Quant kit (GE Healthcare).

Comparative proteome analysis

Total protein extract (40 µg for silver staining or 500 µg for CBB G-250 staining) was used to rehydrate Immobiline DryStrip gels (pH 3-10 NL 18 cm; GE Healthcare). Proteins were resolved in the first dimension by isoelectric focusing for a total of 50 000 V-h by using the Ettan IPGphor 3 (GE Healthcare). After equilibration of the strips, the second dimension was performed on 8-16% polyacrylamide gradient gels (20 cm x 18 cm x 1 mm) and was run on an Ettan Daltsix vertical system (GE Healthcare). The 2D gels were silver-stained (PlusOne Silver Staining kit; GE Healthcare) for image analysis or dyed following the CBB G-250 staining method for mass spectrometric analysis. [START_REF] Goichon | Effects of an Enteral Glucose Supply on Protein Synthesis, Proteolytic Pathways, and Proteome in Human Duodenal Mucosa[END_REF]

(i) 2D image Analysis

Silver stained gels were scanned with an ImageScanner II (GE Healthcare) previously calibrated by a greyscale marker (Kodak) and 2D images were captured with Labscan 6.00 software (GE Healthcare). Differential analysis was realized by using ImageMaster 2D Platinum v7.0 software (GE Healthcare) for spot detection, background subtraction, spot volume normalisation and differences in spot expression among the groups and/or each experiment. Each tissue sample was subjected to 2DE three times to minimize run-to-run variation, and each set of three gels was compared by using ImageMaster to confirm the nonappearance of statistically differential spots within the set of gels. The most representative silver-staining gels (gel migration, spot definition, and spot number) of each sample (n=5 per group) was used to identify differential colonic protein expression. The expression level was determined by the relative volume of each spot in the gel and expressed as %volume, calculated as spot volume/Σvolumes of all spots resolved in the gel. Variations in abundance, corresponding to normalized spot volume, were calculated as the ratio of average values of %volume for each group. Only spots with a %volume variation >1.4 were included in analysis. Statistical differences between the groups were determined using ANOVA (significance level P values < 0.05).

(ii) In-gel trypsin digestion and protein identification by LC-ESI-MS/MS

Protein spots of interest were manually excised from colloidal Coomassie blue-stained 2D gels and digested. Briefly, gel fragments were washed three times for 20 min in destaining solution containing 50% (v/v) methanol and 50 mM ammonium bicarbonate. The excised spots were then air-dried for 1 h, which was followed by in-gel digestion in 30 µL of a digestion buffer containing 50 mM ammonium bicarbonate and 6 ng/µL sequencing grade bovine trypsin (Roche Diagnostics, Meylan, France). After overnight digestion, the aliquots were treated with 50 % (v/v) acetonitrile and 5 % (v/v) formic acid. Speed-vas-dried peptide extracts were resuspended in 10 µL of 5 % (v/v) acetonitrile and 0.1 % (v/v) formic acid and then analyzed with a nanoflow liquid chromatograph LC1200 system coupled to a Q-TOF 6550 mass spectrometer equipped with a nanospray source and an HPLC-chip cube interface (Agilent Technologies). A 20-min linear gradient (3-75% acetonitrile in 0.1% formic acid), at a flow rate of 350 nL/min, was used to separate peptides on a polaris-HR-Chip C18 column (150 mm long × 75 µm inner diameter).

Full autoMS1 scans from 290 to 1700 m/z and autoMS2 from 59 to 1700 m/z were recorded. In every cycle, a maximum of 5 precursors sort by charge state (2+ preferred and single-charged ions excluded) were isolated and fragmented in the collision cell that was automatically adjusted depending on the m/z. Active exclusion of these precursors was enabled after 1 spectra within 0.13 min, and the absolute threshold for precursor selection was set to 1000 counts (relative threshold 0.001%). For protein identification, MS/MS peak lists were extracted and compared with the protein databases using the MASCOT Daemon version 2.4.0 (Matrix Science, London, UK) search engine. The MASCOT searches were performed in Mus musculus database as previously described. [START_REF] Goichon | Enteral Delivery of Proteins Enhances the Expression of Proteins Involved in the Cytoskeleton and Protein Biosynthesis in Human Duodenal Mucosa[END_REF] Protein hits were automatically validated if they satisfied one of the following criteria: identification with at least two top ranking peptides (bold and red) each with a MASCOT score of more than 41 (P < 0.001), or at least two top ranking peptides (bold and red) each with a MASCOT score of more than 31 (P < 0.01). To evaluate false-positive rates, all the initial database searches were performed using the "decoy" option of MASCOT. Results were considered relevant if the false-positive rate never exceeded 1%. The mass spectrometry data were deposited in the ProteomeXchange Consortium via the PRIDE partner repository (https://www.ebi.ac.uk/pride/archive/) with the data set identifier PXD011199 and 10.6019/PXD011199. [START_REF] Vizcaíno | Update of the PRIDE Database and Its Related Tools[END_REF]

Patients

Fourteen patients, who met the Rome III criteria for IBS with predominant diarrhea and seen in the Department of Gastroenterology of the Rouen University Hospital, were included between December 2012 and July 2016 (10 women/4 men, aged 41.2 ± 11.2 years). IBS patients with the following criteria were excluded: (i) patients with IBS predominant constipation or alternating constipation and diarrhea, (ii) blood dyscrasia, (iii) personal history of auto-immune disease, (iv) personal history of inflammatory bowel disease, (v) personal history of digestive cancer, or (vi) macroscopic (except for diverticulosis, hyperplasic polyp, and angiodysplasia) and histological abnormality of the colonic mucosa. Fourteen control patients (8 women/6 men, aged 55 ± 17.4 years) were subjects admitted in the Department of Gastroenterology of the Rouen University Hospital for a colonoscopy indicated within the framework of screening (anemia, personal or family history of polyp, family history of colorectal cancer, rectal bleeding, screening campaign).

To be selected, control subjects had to fulfill all of the following criteria: (i) macroscopically and histological normal colonic mucosa (diverticulosis, hyperplasic polyp, and angiodysplasia were tolerated), (ii) lack of any digestive symptom, and (iii) lack of any organic or functional bowel disease. A complete colonoscopy was performed under general anesthesia after large bowel cleansing with 4L of a macrogol solution. Biopsies were taken in the descending colon in all cases. Two biopsies for each patient were analyzed by a pathologist in order to exclude any histological abnormality. Written informed consent was obtained from each patient. The protocol was conducted in accordance with the Declaration of Helsinki and European GCP for biomedical research.

Ubiquitome analysis

Expression of 49 ubiquitinated proteins was assessed using the commercially Proteome Profiler TM Human Ubiquitin Array kit (R&D Systems, Abingdon, UK) according to the manufacturer's instructions. Briefly, 300 µg of colonic mucosal proteins were loaded on membranes previously coated with specific antibodies directed against ubiquitinated proteins.

After incubation of these membranes with specific buffers and wash steps, ubiquitinated proteins were detected using ECL reagents (GE Healthcare). Each spot was quantified by densitometry using ImageScanner III and ImageQuant TL software (GE Healthcare). Only spots with a %Vol variation ratio greater than 1.5 were considered relevant.

Gene ontology-based analysis

The PANTHER classification system (version 10.1, http://www.pantherdb.org) was used to categorize the identified proteins according to their molecular function and biological process.

To identify significantly enriched biological themes and functional groups in the list of identified proteins, functional annotation clustering analysis was performed using the DAVID Gene Ontology (GO) tool version 6.7 [START_REF] Huang | Systematic and Integrative Analysis of Large Gene Lists Using DAVID Bioinformatics Resources[END_REF] as previously described [START_REF] Goichon | Enteral Delivery of Proteins Enhances the Expression of Proteins Involved in the Cytoskeleton and Protein Biosynthesis in Human Duodenal Mucosa[END_REF] with minor modifications: the reference dataset were Mus musculus or Homo sapiens, and P < 0.05 for Benjamini-Hochberg correction was considered significant.

Western blot analysis

20 µg of total protein extracts were separated by 4-20% SDS-PAGE gels and electrotransferred onto nitrocellulose membrane (GE Healthcare) as previously described. [START_REF] Goichon | Effects of an Enteral Glucose Supply on Protein Synthesis, Proteolytic Pathways, and Proteome in Human Duodenal Mucosa[END_REF] Membrane was blocked with 3% (wt:vol) BSA in TBS-T solution [10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.2% (vol:vol) Tween 20] and then incubated with corresponding primary antibodies overnight at 4°C: mouse monoclonal antibodies anti-UCH-L3 (sc-100340, 1:2500); goat polyclonal antibodies anti-GDH1 (sc-160382, 1:1000) or anti-HSP60 (sc-1052, 1:3000); rabbit monoclonal antibodies anti-Ub (sc-9133, 1:1000) from Santa Cruz Biotechnology. Membrane was washed three times for 10 min with TBS-T and then incubated with appropriate horseradish peroxidasecoupled secondary antibodies [HRP-goat anti-mouse IgG (sc-2005, 1:5000; Santa Cruz Biotechnology), HRP-donkey anti-goat IgG (sc-2020, 1:5000; Santa Cruz Biotechnology), HRP-swine anti-rabbit IgG (P0399, 1:5000; Dako)] for 1 h at room temperature. After 2 washes with TBS-T, immunocomplexes were revealed by using ECL detection reagents (GE Healthcare). Protein bands were quantified by densitometry with ImageScanner III and ImageQuant TL software (GE Healthcare). The blot was stripped and reblotted with a mouse monoclonal antibody anti β actin (A5441, 1:5000; Sigma Aldrich) also used as a loading control.

Detection of carbonylated Proteins

Carbonylated proteins were detected and analyzed following derivatization of protein carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH), using the OxyBlot kit (Millipore, Billerica, MA). Briefly, colonic proteins were incubated with DNPH for 15 min at room temperature to convert carbonyl to hydrazone derivatives. The samples were neutralized and applied on SDS-PAGE and transferred to nitrocellulose membranes. Immunodetection was performed with a primary antibody directed against dinitrophenylhydrazone.

Statistical analysis

Results were compared in GraphPad Prism 5.0 (GraphPad software Inc, San Diego, CA, USA) by two-way ANOVA (stress x β2i -/-) with Bonferroni post-tests. To data from patients, t-test or Mann-Whitney test were used as appropriate. For all tests, P < 0.05 was considered significant.

RESULTS

Immunoproteasome subunit beta2i deficiency prevents hyperpermeability and proinflammatory cytokine production in the colonic tissue of stressed mice

The WAS model increased fecal pellet output from day 1 to day 10 both in wild-type (wt) and β2i -/-mice (Figure S-1A). However, β2i -/-WAS mice showed lower fecal pellet output than wt WAS mice. In β2i -/-mice, trypsin-like activity was not affected by WAS (Figure S-1B).

Interestingly, colonic mRNA levels for TNF-α and IL-1β were not altered in β2i -/-WAS mice (Figure S-1C). Gut barrier function was assessed by paracellular permeability of FITC-dextran.

WAS induced enhanced passage of FITC-dextran across the colonic mucosa of wt mice (Figure S-1D). Interestingly, FITC-dextran flux was markedly reduced in β2i -/-WAS mice compared to wt WAS mice (Figure S-1D). These data confirm that WAS did not affect colonic permeability in β2i -/-mice, as previously described. [START_REF] Ghouzali | Targeting Immunoproteasome and Glutamine Supplementation Prevent Intestinal Hyperpermeability[END_REF]

WAS and immunoproteasome subunit beta2i deficiency alter colonic proteome in mice

To evaluate colonic proteome and then identify altered biological processes in wt and β2i /-mice both in control and WAS conditions, we used a proteomic approach combining 2DE mapping and mass spectrometry identification. Image analysis detected approximately 2100 protein spots per 2D gel, out of which 75% were successfully matched throughout all gels. Representative 2D gels from both conditions are shown in Figure 1A. Fifty protein spots were differentially and significantly expressed between the four mice groups (at least ± 1.4 fold change; two-way ANOVA, p<0.05). These protein spots were analyzed by using liquid chromatography coupled with electrospray ionization MS/MS, and 36 spots (72%) were identified (Table 1). Overall, 33 non-redundant proteins were obtained. Indeed, two proteins, named MICOS complex subunit Mic60 (spots 2 and 4, Figure 1A) and protein disulfide-isomerase precursor (spots 14, 16 and 17; Figure 1A), were detected at multiple spot positions that could be due to protein isoforms, i.e. difference in maturation state, degradation and/or post-translational modifications. Among the identified proteins, 22 proteins were downregulated in the colonic mucosa of wt WAS mice compared with wt mice, while only 6 proteins were affected by WAS in β2i -/-mice. Interestingly, among these 6 proteins, only 2 were downregulated. In addition, we also observed that 7 proteins were differentially expressed in β2i -/-WAS mice compared to wt WAS mice. In this latter case, proteasome subunit alpha type-3 (PSMA3), proteasome subunit alpha type-5 (PSAM5) and ubiquitin carboxyl-terminal hydrolase isozyme L3 (UCH-L3) were up-regulated in the colonic mucosa of β2i -/-WAS mice (range from 2.20 to 3.80-fold), whereas proteasome subunit alpha type-4 (PSMA4), alpha-enloase 1 (ENO1), 60 kDa heat shock protein (HSP60) and glutamate dehydrogenase 1 (GDH1) showed a reduced expression (with fold changes from 1.48 to 1.86).

To validate the data from proteomics experiments, three proteins were randomly selected to verify the expression level via Western blot analysis (Figure 2A). The colonic expression levels of GDH1 and HSP60 were downregulated in β2i -/-WAS mice compared to wt WAS mice, whereas UCH-L3 expression was increased, which is consistent with the results given by 2-DE analysis, and thus confirmed the proteomic findings.

WAS and immunoproteasome subunit beta2i deficiency modify expression of protein involved in mitochondrial and energy metabolism in the colon

Panther Gene Ontology (GO) analysis allowed us to elucidate the different functions and processes in which the 33 proteins identified are putatively involved. According to the molecular function analysis (Figure 1B), most of the proteins were related with catalytic activity (53.1%) and binding (18.8%). Concerning biological processes, the identified proteins were mainly involved in metabolic process (45.5%, Figure 1B). To expand the GO analysis, we next analyzed the proteomic data with DAVID functional annotation tool. DAVID uses GO and other data sources to cluster proteins based on the shared annotations to similarity clusters. DAVID analysis identified 3 significant annotation clusters (Table 2), of which the highest ranked cluster had an enrichment score (ES) of 4.89 and was represented by 3 terms relating to mitochondrion.

The mitochondrial cluster accounted for 33% of the identified proteins and included: ATP synthase subunit beta (Atp5b), Elongation factor G1 (EF-G1mt), Elongation factor G2 (EF-G2mt), Electron transfer flavoprotein subunit alpha (ETFA), GDH1, HSP60, Heat shock protein 75 (HSP75), Isovaleryl-CoA dehydrogenase (IVD), MICOS complex subunit (Mic60), 3mercaptopyruvate sulfurtransferase (MST), Vacuolar protein sorting-associating protein 4B (Vsp4b). The expression levels of Atp5b, EF-G1mt, EF-G2mt, HSP75, IVD, Mic60, MST and Vsp4b were downregulated in the colonic mucosa of wt mice during WAS (Figure 3). It was also observed a decreased expression of pyruvate kinase (PKM), adenylosuccinate synthetase isozyme (Adss2) and adenosine kinase (AK), which are involved in energy metabolism (Figure 3). These data suggest a significant decrease of mitochondrial activity and energy metabolism in the colonic mucosa during WAS. Invalidation of β2i expression alone induced similar modifications of colonic proteome except for Vsp4b, Atp5b and AK (Table 1). Interestingly, Vsp4b, Atp5b and AK remained unaffected in β2i -/-WAS mice compared with β2i -/-control mice. In addition, in β2i -/-mice, WAS altered alpha-enolase-1 (ENO1), which was downregulated, and Uridine phosphorylase 1 (UPase 1; Table 1). By comparing the response of wt and β2i -/-mice to WAS, we observed a reduction of ENO1, HSP60 and GDH1 expression in β2i -/-WAS mice (Table 1 and Figure 3).

WAS and immunoproteasome subunit beta2i deficiency alter ubiquitin-proteasome system in the colon.

We also observed that proteasome subunits were significantly enriched among identified proteins. The proteasome cluster (ES = 2.06, Table 2) was made up from three proteasome alpha subunits (PSMA3/4/5) and 26S protease regulatory subunit S10B (PSMC6). The expression levels of PSCM6 and two others UPS related-proteins, namely pre-mRNA-processing factor (Prpf19) and protein deglycase (DJ-1), were decreased in the colonic mucosa of wt mice during WAS (Figure 4). Interestingly, β2i -/-WAS mice exhibited an increased expression of PSMA3 and PSMA5 as compared to β2i -/-control mice, and of PSMA3, PSMA5 and UCH-L3 when compared to wt WAS mice (Table 1 and Figure 4). By contrast, PSMA4 were downregulated while Prpf19 and DJ-1 remained unaffected in β2i -/-WAS mice. UCH-L3 is a deubiquitinating enzyme (DUB) that controls levels of cellular ubiquitin through processing of ubiquitinated proteins. We thus evaluated the level of ubiquitinated proteins in the colonic mucosa and we observed that both wt and β2i -/-control mice exhibited a reduction of ubiquitinated protein levels in response to WAS (Figure 2B). Interestingly, β2i -/-WAS mice exhibited lower ubiquitinated protein level than wt WAS mice. All these data suggest that (i) WAS affected UPS and (ii) that subunit β2i deficiency may regulate the degradation of specific proteins.

Ubiquitination of inflammation-related proteins is modulated in the colonic mucosa of IBS-D patients

Because stressed mice exhibited UPS changes in their colonic mucosa, we investigated in IBS-D patients the putative changes of ubiquitination of colonic proteins. For this purpose, we used macroarrays directed against 49 ubiquitinated proteins. Among these 49 proteins, the expression of 7 ubiquitinated proteins was significant decreased in the colonic mucosa of IBS-D patients compared with control patients (Figure 5 Interestingly, enrichment analysis of KEGG pathways in DAVID showed that A20, COX-2, IRF3 and TRAF6 were involved in the NF-κB signaling pathway (39.7-fold enrichment, p=0.031). All these data suggest that ubiquitination of these proteins may participate in the pathophysiology of IBS-D, which is commonly described as a low grade inflammatory state, by regulating apoptosis and disrupting UPS mechanism.

DISCUSSION

In the present study, we provide evidence that the ubiquitin-proteasome system contributes to the pathophysiology of IBS. Indeed, we report that colonic proteome is altered in stressed mice and that β2i subunit deficiency modifies the proteome response that is associated with a limitation of colonic hyperpermeability. Interestingly, we show specific alterations of proteins involved in UPS, mitochondrial and energy metabolism. We also report changes in the pattern of colonic ubiquitome in IBS-D patients, and particularly a reduced expression of ubiquitinated proteins involved in the NF-κB signaling pathway.

Previous studies clearly demonstrated that UPS may be involved in the regulation of inflammatory response [START_REF] Bertrand | Evaluation of Ubiquitinated Proteins by Proteomics Reveals the Role of the Ubiquitin Proteasome System in the Regulation of Grp75 and Grp78 Chaperone Proteins during Intestinal Inflammation[END_REF] or gut barrier function [START_REF] Coëffier | Increased Proteasome-Mediated Degradation of Occludin in Irritable Bowel Syndrome[END_REF] . We and others have reported the formation of an alternative proteasome called immunoproteasome in the colonic mucosa of IBS [START_REF] Coëffier | Increased Proteasome-Mediated Degradation of Occludin in Irritable Bowel Syndrome[END_REF] and CD [START_REF] Visekruna | Proteasome-Mediated Degradation of IkappaBalpha and Processing of P105 in Crohn Disease and Ulcerative Colitis[END_REF][START_REF] Visekruna | Comparative Expression Analysis and Characterization of 20S Proteasomes in Human Intestinal Tissues: The Proteasome Pattern as Diagnostic Tool for IBD Patients[END_REF] patients. It was also shown that proteasome composition and activity were different between CD and IBS patients. [START_REF] Coëffier | Increased Proteasome-Mediated Degradation of Occludin in Irritable Bowel Syndrome[END_REF] Indeed, trypsin-like activity was increased in the colonic mucosa of IBS patients [START_REF] Rolland-Fourcade | Epithelial Expression and Function of Trypsin-3 in Irritable Bowel Syndrome[END_REF] but not in CD patients. By contrast, chymotrypsin-like activity remained unchanged in IBS patients whereas it was upregulated in CD patients. In the present study, we used a stress model (the water avoidance stress "WAS" model) able to reproduce certain aspects of IBS such as visceral hypersensibility or intestinal hyperpermeability. Indeed, stress contributes to the development and aggravation of IBS symptoms. [START_REF] Moloney | Stress-Induced Visceral Pain: Toward Animal Models of Irritable-Bowel Syndrome and Associated Comorbidities[END_REF] WAS mice exhibited an increase colonic permeability, trypsin-like activity and β2i/β2 subunit ratio, [START_REF] Ghouzali | Targeting Immunoproteasome and Glutamine Supplementation Prevent Intestinal Hyperpermeability[END_REF] that was previously observed in IBS-D patients. [START_REF] Coëffier | Increased Proteasome-Mediated Degradation of Occludin in Irritable Bowel Syndrome[END_REF] In our study, we showed that β2i subunit deficiency increased expression of proteasome subunits (PSMA3/5) in stressed mice, whereas PSMA4 and ubiquitinated proteins was downregulated. The shift from constitutive to immunoproteasome could be responsible for the decrease of ubiquitinated proteins after WAS since Seifert et al. showed that immunoproteasome activity is higher than constitutive proteasome activity. [START_REF] Seifert | Immunoproteasomes Preserve Protein Homeostasis upon Interferon-Induced Oxidative Stress[END_REF] Nevertheless, Nathan et al. have reported that immunoproteasomes and constitutive proteasomes degraded ubiquitinated proteins at similar rates following IFNγ-treatment. [START_REF] Nathan | Immuno-and Constitutive Proteasomes Do Not Differ in Their Abilities to Degrade Ubiquitinated Proteins[END_REF] Other processes like ubiquitination and deubiquitination may also be involved. UCH-L3, which was increased in β2i -/- mice in the present study, is a deubiquitinating enzyme performing hydrolysis of the bond at the C terminus of the ubiquitin and triming down the polyubiquitin chain into monomers. UCH-L3 knock-out mice led to marked accumulation of ubiquitinated proteins in the skeletal muscles. [START_REF] Setsuie | Skeletal Muscles of Uchl3 Knockout Mice Show Polyubiquitinated Protein Accumulation and Stress Responses[END_REF] However, the effect of UCH-L3 overexpression on ubiquitinated protein level remains unknown. Thus, the underlying mechanisms responsible of the ubiqtuinated protein level changes need to be deeply explored. UCH-L3 also processed an ubiquitin-like substrate called NEDD8 and regulated smad1 ubiquitination in osteoblast differentiation. [START_REF] Kim | Ubiquitin C-Terminal Hydrolase-L3 Regulates Smad1 Ubiquitination and Osteoblast Differentiation[END_REF] Interestingly, Sano et al. have reported that UCH-L3 prevented mitochondrial oxidative stress-related apoptosis in photoreceptor cells. [START_REF] Sano | Photoreceptor Cell Apoptosis in the Retinal Degeneration of Uchl3-Deficient Mice[END_REF] UCH-L3 may then affect mitochondrial structure and the levels of reactive oxygen species (ROS). By contrast, it was considered as negative regulators of apoptosis in cancer. [START_REF] Song | Ubiquitin C-Terminal Hydrolase-L3 Regulates EMT Process and Cancer Metastasis in Prostate Cell Lines[END_REF][START_REF] Rolén | Activity Profiling of Deubiquitinating Enzymes in Cervical Carcinoma Biopsies and Cell Lines[END_REF][START_REF] Miyoshi | High Expression of Ubiquitin Carboxy-Terminal Hydrolase-L1 and -L3 MRNA Predicts Early Recurrence in Patients with Invasive Breast Cancer[END_REF] More interestingly, Zhao et al. have recently underlined a relationship between UCH-L3 and inflammatory response. Indeed, overexpression of UCH-L3 inhibited the activity of cellular SCF β-TrCP ligase E3 through a limitation of cullin-1 neddylation. [START_REF] Zhao | Ubiquitin C-Terminal Hydrolase-L3 Promotes Interferon Antiviral Activity by Stabilizing Type I-Interferon Receptor[END_REF] SCF β-TrCP ligase E3 induces ubiquitination and degradation of phosphorylated substrates including I-κB, the inhibitor of NF-κB. [START_REF] Kanarek | Regulation of NF-ΚB by Ubiquitination and Degradation of the IκBs[END_REF] Previous papers reported that NF-κB activity can also be limited by glutamine through the regulation of I-κB ubiquitination [START_REF] Bertrand | Glutamine Enema Regulates Colonic Ubiquitinated Proteins but Not Proteasome Activities during TNBS-Induced Colitis Leading to Increased Mitochondrial Activity[END_REF][START_REF] Coëffier | Potential for Amino Acids Supplementation during Inflammatory Bowel Diseases[END_REF] and particularly by the regulation of cullin-1 neddylation. [START_REF] Singleton | Glutamine Attenuates Inflammation and NF-KappaB Activation via Cullin-1 Deneddylation[END_REF] Interestingly, in the present study, we showed that β2i subunit deficiency leads to a reduced expression of GDH1 in the colonic mucosa of WAS mice. GDH1 is a metabolic enzyme mainly located in the mitochondria that catalyzes the reversible NAD (P)+-linked oxidative deamination of glutamate into α-ketoglutarate and ammonia. This reaction is the final step of glutaminolysis after the conversion of glutamine to glutamate by glutaminase. In inflammatory experimental conditions, glutamine supplementation improves gut barrier function [START_REF] Beutheu | Glutamine Supplementation, but Not Combined Glutamine and Arginine Supplementation, Improves Gut Barrier Function during Chemotherapy-Induced Intestinal Mucositis in Rats[END_REF][START_REF] Beutheu | Glutamine and Arginine Improve Permeability and Tight Junction Protein Expression in Methotrexate-Treated Caco-2 Cells[END_REF][START_REF] Choi | The Effect of Oral Glutamine on 5-Fluorouracil/Leucovorin-Induced Mucositis/Stomatitis Assessed by Intestinal Permeability Test[END_REF][START_REF] Daniele | Oral Glutamine in the Prevention of Fluorouracil Induced Intestinal Toxicity: A Double Blind, Placebo Controlled, Randomised Trial[END_REF] , regulates UPS [START_REF] Bertrand | Glutamine Enema Regulates Colonic Ubiquitinated Proteins but Not Proteasome Activities during TNBS-Induced Colitis Leading to Increased Mitochondrial Activity[END_REF][START_REF] Singleton | Glutamine Attenuates Inflammation and NF-KappaB Activation via Cullin-1 Deneddylation[END_REF][START_REF] Hubert-Buron | Glutamine Pretreatment Reduces IL-8 Production in Human Intestinal Epithelial Cells by Limiting IkappaBalpha Ubiquitination[END_REF] and restores colonic TJ protein expression. [START_REF] Beutheu | Glutamine Supplementation, but Not Combined Glutamine and Arginine Supplementation, Improves Gut Barrier Function during Chemotherapy-Induced Intestinal Mucositis in Rats[END_REF][START_REF] Beutheu | Glutamine and Arginine Improve Permeability and Tight Junction Protein Expression in Methotrexate-Treated Caco-2 Cells[END_REF][START_REF] Bertrand | Glutamine Restores Tight Junction Protein Claudin-1 Expression in Colonic Mucosa of Patients With Diarrhea-Predominant Irritable Bowel Syndrome[END_REF] We have recently demonstrated that glutamine supplementation prevented colonic hyperpermeability and reduced proinflammatory cytokine production in WAS mice. [START_REF] Ghouzali | Targeting Immunoproteasome and Glutamine Supplementation Prevent Intestinal Hyperpermeability[END_REF] Zhou et al. have recently reported that reduced expression of glutamine synthase in colon tissues of IBS-D patients may lead to decreased claudin-1 expression, and consequently increased intestinal permeability. [START_REF] Zhou | MicroRNA 29 Targets Nuclear Factor-ΚB-Repressing Factor and Claudin 1 to Increase Intestinal Permeability[END_REF] In the same way, we have shown ex vivo that glutamine supplementation increased claudin-1 expression in the colonic mucosa of IBS-D patients. [START_REF] Bertrand | Glutamine Restores Tight Junction Protein Claudin-1 Expression in Colonic Mucosa of Patients With Diarrhea-Predominant Irritable Bowel Syndrome[END_REF] In a randomized controlled trial, Zhou et al recently showed the beneficial effect of glutamine supplementation on intestinal permeability and gastrointestinal symptoms in post-infectious IBS patients. [START_REF] Zhou | Randomised Placebo-Controlled Trial of Dietary Glutamine Supplements for Postinfectious Irritable Bowel Syndrome[END_REF] All these data are in accordance with previous studies showing that glutamine supplementation or deprivation is able to modify TJ protein expression and influence intestinal permeability. [START_REF] Beutheu | Glutamine and Arginine Improve Permeability and Tight Junction Protein Expression in Methotrexate-Treated Caco-2 Cells[END_REF][START_REF] Li | Glutamine Deprivation Alters Intestinal Tight Junctions via a PI3-K/Akt Mediated Pathway in Caco-2 Cells[END_REF] The mechanisms of action for glutamine remain not completely understood and have been previously discussed. [START_REF] Coëffier | Intestinal Permeability in Patients with Diarrhea-Predominant Irritable Bowel Syndrome: Is There a Place for Glutamine Supplementation[END_REF][START_REF] Zhou | To PMID 25277410[END_REF] In our model, we can thus speculate that immunoproteasome inhibition induces higher glutamine content and overexpression of UCH-L3 leading to limitation of inflammatory response and improved barrier function (Figure 6). The assessment of colonic mucosal glutamine content in β2i -/-WAS mice should be thus further investigated.

To confirm the putative role of proteasome system in the pathophysiology of IBS, we have investigated in IBS-D patients the changes of ubiquitination rate of colonic proteins. 21 Interestingly, we observed alterations of ubiquitome, and particularly a reduced expression of 7 ubiquitinated proteins involved in the NF-κB signaling pathway. Cleynen et al. previously reported that adherent-invasive Escherichia coli (AIEC) was able to regulate UPS leading to a decrease of NF-κB negative regulator CYLD in intestinal epithelial cells. [START_REF] Cleynen | Genetic and Microbial[END_REF] In patients with IBS-D, Zhou et al. recently underlined that colonic hyperpermeability caused by TNF-α may be due to activation of NF-kB expression and disassembly of claudin-1. [START_REF] Zhou | MicroRNA 29 Targets Nuclear Factor-ΚB-Repressing Factor and Claudin 1 to Increase Intestinal Permeability[END_REF] All of these data suggest potential relationship between UPS, glutamine content, inflammatory response and TJ protein expression in the pathophysiology of IBS. Further investigations should be done to deeply explore these mechanisms.

In conclusion, we provide evidence that (i) β2i subunit deficiency modified colonic UPS function in stressed mice that was associated with an improvement of intestinal hyperpermeability, (ii) IBS-D patients exhibited altered colonic ubiquitome leading to altered NF-κB pathway. Therefore, immunoproteasome targeting represents a new therapeutic strategy for the treatment of IBS-D patients with intestinal hyperpermeability. Table S-1. Sequences of used primers for qPCR

ASSOCIATED CONTENT Supporting Information

ACKNOWLEDGMENTS

We thank the staff of the Endoscopy Unit for their assistance with the endoscopic procedure. We dedicate this work to the memory of Prof. Philippe Ducrotté, our colleague and friend, who passed away on December 30, 2017. This work was supported by a Cephalon Research Prize (to IG), by INSERM and the University of Rouen Normandy and co-supported by European Union and Normandie Regional Council (to WB and for equipments). Europe gets involved in Normandie with European Regional Development Fund (ERDF). These funders did not participate in the design, implementation, analysis and interpretation of the data.

AUTHOR CONTRIBUTIONS:

AG, IG, WB, PC performed experiments and analyses. AG and MC designed experiments. PDu performed colonoscopy and sampling. AG, DV, PDe, PDu and MC interpreted the data. AG and MC wrote the main manuscript text and prepared figures. All authors reviewed the manuscript.

DISCLOSURE STATEMENT:

The authors declare that they have no conflict of interest.

FIGURE LEGENDS:

Figure 1. Colonic proteome alterations in wild-type (wt) and β2i -/-mice during WAS (A) Silver-stained 2-dimensional gel images representing colonic proteome from wt control (Ct), β2i -/-Ct, wt WAS and β2i -/-WAS mice. For isoelectric focusing, 40 µg of total colonic proteins was separated onto pH 3-10 NL IPG strip (18 cm). SDS-PAGE was performed 4-16 % gel and proteins were revealed with silver staining. The molecular weight of each protein was determined by standard markers. Differentially expressed proteins (> 1.4 fold difference; circled spots with a number) were determined by statistical analysis (two-way ANOVA, P < 0.05).

Protein spots identification is displayed in Table 1. (B) Pie chart depicting the functional classification of proteins differentially expressed between the four groups mice. Using the online resource PANTHER classification system, proteins of interest were categorized into the broad GO classes biological process and molecular function. For each category, the number of identified proteins and their percentage of the total number of proteins in the pie chart are indicated for each GO term.

Figure 2. Expression levels of GDH1, HSP60, UCH-L3 and ubiquitinated proteins in the colonic mucosa of β2i -/-WAS mice

Representative immunoblots and densitometric analysis of GDH-1 (A), HSP60 (A), UCH-L3 (A), ubiquitinated protein (B) and β-actin levels in the colonic mucosa of wild-type (wt) or β2i knock-out (β2i -/-) mice subjected to WAS (1 h/day for 10 days) or not (Control; Ct). Values are means ± sem (n = 5 per group). Open and closed bars represent control and WAS mice, respectively. Two-way ANOVA P values: (A. GDH1) P(WAS) = 0.0363, P(β2i -/-) = 0.0305, p(interaction) = 0.1709; (A. HSP60) P(WAS) = 0.0013, P(β2i -/-) = 0.0388, P(interaction) = 0.0805; (A. UCH-L3) P(WAS) = 0.1797, P(β2i -/-) = 0.0019, P(interaction) = 0.9172; (B) P(WAS) < 0.0001, P(β2i -/-) = 0.0243, P(interaction) = 0.1393. *, P < 0.05 vs. wt Ct; #, P < 0.05 vs. wt WAS; and $, P < 0.05 vs. β2i -/-Ct. GDH-1, Glutamate dehydrogenase 1; HSP60, 60 kDa heat shock protein; UCH-L3, Ubiquitin carboxyl-terminal hydrolase isozyme L3.

Figure 3. Schema of mitochondrial and energy metabolism alterations in the colonic

mucosa WAS induces protein expression changes in the colon tissue of wt mice (in red) and β2i - /-mice (in mauve). These changes are different from those induced by β2i subunit deficiency during WAS (in green). Proteins in red, mauve or green are downregulated in the colonic mucosa of mice, excepted for UPase 1: Adss2, Adenylosuccinate synthetase isozyme 2; AK, Adenosine kinase; Atp5b, ATP synthase subunit beta; EF-G1mt, Elongation factor G1; EF-G2mt, Elongation factor G2; ENO1, alpha-enolase 1; GDH1, Glutamate dehydrogenase 1 ; HSP60, 60 kDa heat shock protein; HSP75, Heat shock protein 75; IVD, isovaleryl-CoA dehydrogenase; Mic60, MICOS complex subunit; MST, 3-mercaptopyruvate sulfurtransferase; PKM, Pyruvate kinase; Vsp4b, Vacuolar protein sorting-associating protein 4B. These modifications suggest a significant decrease of mitochondrial activity and energy metabolism in the colonic mucosa of wt mice during WAS. Interestingly, these metabolic disturbances were minored in β2i -/-WAS mice.

Figure 4. Schema of ubiquitin-proteasome system (UPS) alterations in the colonic mucosa

WAS induces protein expression changes in the colon tissue of wt mice (in red) and β2i -/-mice (in mauve). These changes are different from those induced by β2i subunit deficiency during WAS (in green). The external and internal rings constitute the 20S proteasome. The lid and base constitute the 19S regulatory complex. In some cases, it can be replaced by the PA28 or 11S regulatory complex, constituted of a single ring of 7 subunits. Proteins in red, mauve or green are upregulated (plus sign, +) or downregulated (minus sign, -) in the colonic mucosa of mice: DJ-1, Protein deglycase; Prp19, Pre-mRNA-processing factor 19; PSMA3, Proteasome subunit alpha type-3; PSMA4, Proteasome subunit alpha type-4; PSMA5, Proteasome subunit alpha type-5; PSMC6, 26S protease regulatory subunit S10B; UCH-L3, Ubiquitin carboxyl-terminal hydrolase isozyme L3. These modifications suggest altered UPS function in the colonic mucosa of wt mice during WAS. It was also observed disturbances in UPS in β2i -/-WAS mice. In this case, immunoproteasome subunit beta2i deficiency induces increased colonic expression of UCH-L3, PSMA3 and PSMA5 in stressed mice, while PSMA4 were downregulated. α, proteasome alpha subunit; β, proteasome beta subunit, DUBs, deubiquitinating enzymes; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; Ub, Ubiquitin. b MASCOT, algorithm that uses mass spectrometric data to identify proteins from primary sequence databases.

c Number of peptides matched.

d Average spot volume ratio between two groups. +, upregulated; -, downregulated; NS, not significant. 1 Fold enrichment of the cluster.

2 P value from modified Fisher's exact test (EASE score).

  ): TNF-α-induced protein 3 (A20), Prostaglandin G/H synthase 2 (COX-2), Heat shock 70 kDa protein (HSP70), Heat shock protein HSP 90 (HSP90), Interferon regulatory factor 3 (IRF3), Nuclear factor erythroid 2-related factor 2 (Nrf2), TNF receptor-associated factor 6 (TRAF-6). Nevertheless, the total expression of ubiquitinated proteins was not significantly different between control and IBS-D patients (Figure S-2A).

Figure S- 1 .

 1 Figure S-1. Colonic permeability and cytokine production in stressed mice.

Figure S- 2 .

 2 Figure S-2. Expression levels of ubiquitinated and carbonylated proteins in colonic mucosa of

  -α-induced protein 3; Adss2, Adenylosuccinate synthetase isozyme; AK, Adenosine kinase; Atp5b, ATP synthase subunit beta; CD, Crohn's disease; COX-2, Prostaglandin G/H synthase 2; EF-G1mt, Elongation factor G1; EF-G2mt, Elongation factor G2; ENOA, Alphaenolase-1; ETFA, Electron transfer flavoprotein subunit alpha; GDH1, Glutamate dehydrogenase 1; GO, Panther Gene Ontology; HSP60, 60 kDa heat shock protein; HSP70, Heat shock 70 kDa protein; HSP75, Heat shock protein 75 kDa; HSP90, Heat shock protein 90 kDa; IBS, irritable bowel syndrome; IBS-D, diarrhea-predominant irritable bowel syndrome; IVD, Isovaleryl-CoA dehydrogenase; IRF3, Interferon regulatory factor 3; Mic60, MICOS complex subunit; MST, 3mercaptopyruvate sulfurtransferase; Nrf2, Nuclear factor erythroid 2-related factor 2; NF-κB, nuclear factor-kappa B; PKM, Pyruvate kinase; Prpf19, Pre-mRNA-processing factor; PSMA3/4/5, Proteasome alpha subunits; PSMC6, 26S Protease regulatory subunit S10B; TJs, tight junctions; TRAF-6, TNF receptor-associated factor 6; UCH-L3, Ubiquitin carboxylterminal hydrolase isozyme L3; UPase 1, Uridine phosphorylase 1; UPS, ubiquitine-proteasome system ; Vsp4b, Vacuolar protein sorting-associating protein 4B; WAS, water avoidance stress; wt, wild-type. Factors Modulating the Ubiquitin Proteasome System in Inflammatory Bowel Disease. Gut 2014, 63 (8), 1265-1274.

Figure 5 .

 5 Figure 5. Expression of ubiquitinated proteins in the colonic mucosa of IBS-D patients. The

Figure 6 .

 6 Figure 6. Speculative schema of interactions between UCH-L3, glutamine content and

3 P

 3 value adjusted for multiple testing according to the Benjamini-Hochberg model.[START_REF] Simrén | Quality of Life in Inflammatory Bowel Disease in Remission: The Impact of IBS-like Symptoms and Associated Psychological Factors[END_REF] Genes encoding the differentially expressed proteins in Mus Musculus: ADSS -Adenylosuccinate Synthase; ATP5B -ATP Synthase, H+ Transporting, Mitochondrial F1 Complex, Beta Polypeptide; EEF2 -Eukaryotic Translation Elongation Factor 2; ETFA -Electron Transfer Flavoprotein Alpha Subunit; GFM1 -G Elongation Factor Mitochondrial 1; GFM2 -Elongation Factor Mitochondrial 2; TRAP1 -Heat shock protein 75 kDa, mitochondrial; GLUD1 -Glutamate Dehydrogenase 1; HSPD1-Heat Shock Protein Family D (Hsp60) Member 1; IMMT -Inner Membrane Mitochondrial Protein; IVD -Isovaleryl-CoA Dehydrogenase; MPST -Mercaptopyruvate Sulfurtransferase; PSMA3 -Proteasome Subunit Alpha 3; PSMA4 -Proteasome Subunit Alpha 4; PSMA5 -Proteasome Subunit Alpha 5; PSMC6 -Proteasome 26S Subunit, ATPase 6; TUBB5 -Tubb5 tubulin, beta 5 class I; VSP4B -Vacuolar protein sorting-associated protein 4B.

Table 1 . Colonic protein expression changes in wild-type (wt) and β2i immunoproteasome subunit knock-out (β2i -/-) mice during water avoidance stress (WAS) or no stress (Ct) Spot number Swiss-Prot accession number Protein name (short name) Theoretical a Mascot Score b , Sequence coverage (%), peptide hit c Fold change d Two-way ANOVA: P(WAS), P(β2i -/-),

 1

	P(interaction)

Table 1 . (Continued) Spot number Swiss-Prot accession number Protein name (short name) Theoretical a Mascot Score b Sequence coverage (%) peptide hit c Fold change d Two-way ANOVA: P(WAS), P(β2i -/-), P(interaction) pI Mr (kDa) wt Ct/ wt WAS wt Ct/ β2i -/-Ct β2i -/-Ct / β2i -/-WAS wt WAS / β2i -/-WAS

 1

	26	_	Undetermined	_	_	_	NS	NS	+1.59	+1.67	0.0277, 0.0127, 0.0312
	27	_	Undetermined	_	_	_	-2.01	-1.70	NS	NS	0.0015, 0.0159, 0.0318
	28	_	Undetermined	_	_	_	NS	-1.60	NS	NS	0.249, 0.0452, 0.03630
	29	_	Undetermined	_	_	_	-1.65	-1.83	NS	NS	0.080, 0.0286, 0.11610
	30	P17182	Alpha-enolase 1 (Eno1)	6.37	47.1	773, 26%, 9	NS	NS	-1.55	-1.65	0.030, 0.0078, 0.13510
	31	P55264	Adenosine kinase (AK)	5.84	40.1	162, 16%, 5	-1.76	NS	NS	NS	0.0188, 0.1506, 0.2865
	32	P46664	Adenylosuccinate synthetase isozyme 2 (Adss2)	5.98	50.0	111.0, 7%, 3	-1.45	-1.55	NS	NS	0.1908, 0.0202, 0.0465
	33	_	Undetermined	_	_	_	-2.46	-2.31	NS	NS	0.0341, 0.0588, 0.0010
	34	P62334	26S protease regulatory subunit S10B (Psmc6)	7.10	44.1	404, 30%, 8	-1.47	NS	NS	NS	0.0078, 0.0359, 0.1776
	35	_	Undetermined	_	_	_	-2.57	-2.41	NS	NS	0.0096, 0.0187, 0.0002
	36	Q9JHI5	isovaleryl-CoA dehydrogenase (IVD)	8.53	46.3	350, 20%, 7	-1.56	-1.73	NS	NS	0.118, 0.0354, 0.07550
	37	Q9CQM9 Glutaredoxin-3 (Glrx3)	5.42	37.8	93.0, 7%, 2	-1.64	-1.76	NS	NS	0.2368, 0.0992, 0.0010
	38	Q9JMD3	PCTP-like protein (PCTP-L)	6.67	32.9	169.0, 8%, 2	NS	NS	NS	NS	0.0224, 0.0711, 0.6146
	39	_	Undetermined	_	_	_	-1.67	NS	-1.84	NS	0.0002, 0.9421, 0.7163
	40	_	Undetermined	_	_	_	NS	+2.35	NS	NS	0.1041, 0.0075, 0.1830
	41	_	Undetermined	_	_	_	-1.97	-1.85	NS	NS	0.1486, 0.2342, 0.0151
	42	P52624	Uridine phosphorylase 1 (UPase 1)	6.12	34.1	101.0, 6%, 2	NS	NS	+3.59	NS	0.0096, 0.2894, 0.2429
	43	Q99J99	3-mercaptopyruvate sulfurtransferase (MST)	6.11	33.0	416, 36%, 9	-1.51	-1.44	NS	NS	0.0075, 0.0178, 0.2104
	44	Q99LC5	Electron transfer flavoprotein subunit alpha (Alpha-ETF)	8.62	35.0	878, 49%, 13	NS	-1.50	NS	NS	0.0393, 0.0059, 0.4695
	45	O70435	Proteasome subunit alpha type-3 (Psma3)	5.29	28.4	80.0, 10%, 2	NS	+5.06	+1.72	+3.80	0.0152, < 0.0001, 0.211
	46	Q9R1P0	Proteasome subunit alpha type-4 (Psma4)	7.59	29.5	231, 19%; 4	NS	NS	NS	-1.70	0.204, 0.0167, 0.32380
	47	Q9JKB1	Ub carboxyl-terminal hydrolase isozyme L3 (Uchl3)	4.96	26.1	142, 16%, 3	NS	NS	NS	+2.35	0.7986, 0.0105, 0.0445
	48	Q9Z2U1	Proteasome subunit alpha type-5 (Psma5)	4.74	26.4	87.0, 7%, 2	NS	NS	+1.85	+2.22	0.2666, 0.0385, 0.0004
	49	Q99LX0	Protein deglycase DJ-1 (DJ-1)	6.32	20.0	178, 12%, 3	-1.54	-1.70	NS	NS	0.0036, 0.0008, 0.4190
	50	Q3THE2	Myosin regulatory light chain 12B (Myl12b)	4.71	19.8	105, 12%, 3	NS	NS	NS	NS	0.0519, 0.0117, 0.7918

a Theoretical molecular mass (Mr) and isoelectric point (pI) calculated from the mature protein and amino acid sequence, respectively.

Table 2 . DAVID functional annotation clusters generated from the list of colonic proteins differentially expressed between the four mice groups

 2

	Annotation category	Term	Enrichment Score 1	Protein count	P value 2 Benjamini 3	Related genes 4
	Annotation Cluster 1 SP_PIR_KEYWORDS	Mitochondrion	4.89	11	7.0E-6 2.2E-4	ATP5B, ETFA, GFM1, GFM2, GLUD1,
						HSPD1, IMMT, IVD, MPST, TRAP1, VSP4B
	SP_PIR_KEYWORDS	Transit peptide		8	1.3E-5 3.1E-4	ATP5B, ETFA, GFM1, GFM2, GLUD1,
						HSPD1, IVD, TRAP1
	UP_SEQ_FEATURE	Transit peptide: Mitochondrion		8	2.4E-5 2.5E-3	ATP5B, ETFA, GFM1, GFM2, GLUD1,
						HSPD1, IVD, TRAP1
	Annotation Cluster 2 INTERPRO	Proteasome, alpha-subunit	2.06	3	8.2E-5 9.2E-3	PSMA3, PSMA4, PSMA5
	SP_PIR_KEYWORDS	Proteasome		4	1.2E-4 1.9E-3	PSMA3, PSMA4, PSMA5, PSMC6
	INTERPRO	Proteasome, subunit alpha/beta		3	5.5E-4 3.1E-2	PSMA3, PSMA4, PSMA5
	KEGG_PATHWAY	Proteasome		4	6.2E-4 1.9E-2	PSMA3, PSMA4, PSMA5, PSMC6
	SP_PIR_KEYWORDS	Threonine protease		3	6.4E-4 6.0E-3	PSMA3, PSMA4, PSMA5
	Annotation Cluster 3 SP_PIR_KEYWORDS	gtp-binding	1.94	6	2.2E-4 2.3E-3	ADSS, EEF2, GFM1, GFM2, GLUD1,TUBB5
	PANTHER_MF_ALL	Translation elongation factor		3	5.3E-4 2.6E-2	EEF2, GFM1, GFM2


E Torres

J Desquines

S Guilbert

P Lacote

M.-C Baietto

M Coret

M Blat

A Ambard

OXYGEN SEGREGATION IN PRE-HYDRIDED ZIRCALOY-4 CLADDING DURING A SIMULATED LOCA TRANSIENT

Oxygen and hydrogen distributions are key elements influencing the residual ductility of zirconium-based nuclear fuel cladding during the quench phase following a Loss Of Coolant Accident (LOCA). During the high temperature oxidation, a complex partitioning of the alloying elements is observed. A finite-difference code for solving the oxygen diffusion equations has been developed by IRSN to predict the oxygen profile within the samples. The comparison between the calculations and the experimental results in the mixed α+β region shows that the oxygen diffusion is not accurately predicted by the existing modeling. This work aims at determining the key parameters controlling the average oxygen profile within the sample in the two-phase regions at 1200°C. High temperature steam oxidation tests interrupted by water quench were performed using pre-hydrided Zircaloy-4 samples. Experimental oxygen distribution was measured by Electron Probe Micro-Analysis (EPMA). The phase distributions within the cladding thickness, was measured using image analysis to determine the radial profile of α(O) phase fraction. It is further demonstrated and experimentally checked that the α-phase fraction in these regions follows a diffusion-like radial profile. A new phase fraction modeling is then proposed in the cladding metallic part during steam oxidation. The modeling results are compared to a large set of experiments including the influence of exposure duration and hydrogen content. Another key outcome from this modeling is that oxygen average profile is straightforward derived from the proposed modeling.

Introduction 1.

During a Loss Of Coolant Accident, zirconium-based nuclear fuel claddings are submitted to high temperature steam oxidation before core reflooding. During the high temperature oxidation, metallurgical evolutions due to α-β phase transformations are observed in the material ( Fig. 1). A partitioning of the main chemical elements is also evidenced within the cladding thickness. This segregation is associated to oxygen diffusion and progressive transformation of the β-phase into an oxygen stabilized α(O) layer. The microstructure results in a complex distribution of oxygen and hydrogen in the two-phase constituted material. Fig. 1. Metallurgical evolutions of Zircaloy-4 alloy during a LOCA transient [START_REF] Chung | Pseudobinary Zircaloy-Oxygen phase diagram[END_REF] These phenomena have a direct impact on the material mechanical properties [START_REF] Brachet | Hydrogen content, peroxidation, and cooling scenario effects on postquench microstructure and mechanical properties of Zircaloy-4 and M5® alloys in LOCA conditions[END_REF]. It was frequently reported that oxygen and hydrogen contents in the prior-β phase or in the α+β phase had a combined influence on the cladding post quenching embrittlement [START_REF] Brachet | Hydrogen content, peroxidation, and cooling scenario effects on postquench microstructure and mechanical properties of Zircaloy-4 and M5® alloys in LOCA conditions[END_REF][START_REF] Brachet | Influence of hydrogen content on the α/β phase transformation and on the thermal-mechanical behavior of Zy-4, M4 and M5 alloys during the first phase of LOCA transient[END_REF][START_REF] Waeckel | Fuel Safety Research at EDF[END_REF][START_REF] Billone | Cladding embrittlement during postulated Loss-of-Coolant Accidents[END_REF][START_REF] Sawatsky | A proposed criterion for the oxygen embrittlement of Zircaloy-4 fuel cladding[END_REF][START_REF] Chung | Mechanical properties of Zircaloy containing oxygen[END_REF]. It is thus necessary to understand the mechanisms influencing the motion of these two chemical elements to fully describe the post-quench embrittlement.

Hydrogen distribution within the cladding was previously discussed in another paper [START_REF] Torres | Adsorption and diffusion of hydrogen in Zircaloy-4", Fontevraud 8 -Contribution of Materials Investigations and Operating Experience to LWRs' Safety, Performance and Reliability[END_REF]. In the open literature, analytical oxygen diffusion models are restricted to samples having simple geometry that is usually not satisfactory to analyze tubular geometry [START_REF] Ma | Oxidation kinetics and oxygen diffusion in low-tin Zircaloy-4 up to 1523K[END_REF]. A finitedifference software solving the oxygen diffusion equations named DIFFOX has been developed by IRSN [START_REF] Duriez | Characterization of Oxygen Distribution in LOCA Situations[END_REF]. The comparison between the calculations and the experimental results in the mixed α+β region shows that the oxygen diffusion is not accurately predicted. This work aims at determining experimentally the key parameters controlling the average oxygen profile within the sample both in the prior-β phase and the α+β region at 1200°C. This study provides improved modeling to be implemented in the diffusion softwares.

Several Zircaloy-4 samples are pre-hydrided with hydrogen contents ranging between the as-received content (~ 10 wppm) and 400 wppm. The samples are then steam oxidized at high temperature (1200°C), to simulate LOCA conditions, and then quenched in a water bath at room temperature. The microstructure of the material, especially the phase distributions within the cladding thickness, is systematically characterized by metallography. Local oxygen concentrations are specifically investigated using Electron Probe Micro-Analysis (EPMA). Experimental radial oxygen distributions can then be compared to several diffusion modeling results.

This paper describes the test devices and the experimental method supporting this study. The experimental results are presented, discussed and compared to those predicted numerically using calculations derived from the DIFFOX tool. A new modeling is thus proposed. The improved modeling is then validated by comparison to a large set of experiments including influence of exposure duration and hydrogen content.

Materials and method 2. 2.1 Materials

The specimens investigated in this study are stress-relieved annealed low tin Zircaloy-4 fuel cladding tubes provided by AREVA-NP. The tubular samples are 70 mm long, have a 9.5 mm outer diameter and a 570 µm thickness respectively. Chemical composition of the tested material is detailed in Table 1

Experiments

The 70 mm long specimens are first pre-hydrided at the École Centrale de Paris using gaseous charging at 420°C. More details about hydrogen gaseous charging are provided in a previous paper [START_REF] Torres | Hydrogen motion in Zircaloy-4 cladding during a LOCA transient[END_REF]. The tubes are then cut into 20 mm long segments for oxidation and thin ring samples both for hydrogen measurements and for metallographic analyses before oxidation. Two-sided steam oxidations are then performed in a vertical furnace at 1200°C in a mixture of argon and steam. The argon and steam flow rates are respectively 10Nl.min -1 and 500g.h -1 (50 vol% argon and 50 vol% steam) in order to prevent steam starvation and limit hydrogen pick-up. Oxidation durations are adjusted to reach ECR (Equivalent Clad Reacted) values of 15%, 20% and 25% using Cathcart-Pawel correlation assuming inner and outer diameter oxidation [START_REF] Cathcart | Zirconium metal-water oxidation kinetics IV. Reaction rate studies[END_REF]. Oxidation is interrupted by dropping the sample into a water bath at room temperature as illustrated in Fig. 2. More details about the oxidation protocol and its qualification are provided by Duriez and Guilbert [START_REF] Duriez | Characterization of Oxygen Distribution in LOCA Situations[END_REF][START_REF] Guilbert | Influence of a pre-oxide layer on oxygen diffusion and on postquench mechanical properties of Zircaloy-4 after steam oxidation at 900°C[END_REF][START_REF] Guilbert | Effect of Pre-oxide on Zircaloy-4 high-temperature steam oxidation and post-quench mechanical properties[END_REF]. Radial cross sections are prepared for hydrogen measurements and microstructure analyses. Oxygen profiles are systematically measured using EPMA on each radial cut. The microstructure of the material, especially the phase distributions within the cladding thickness, is characterized by metallography.

Fig. 2. Vertical furnace used for the steam oxidations at 1200°C.

Samples characterizations

Hydrogen measurements

The hydrogen content of the samples is measured using a Brücker ONH mat 286 by melting the sample at 2000°C in the presence of a carrier gas (argon). After calibration, the hydrogen content is determined by catharometry comparing the conductivity of the mixture with the one of pure argon. For 70 mm long tube samples, four axial locations are systematically investigated. A minimum of four analyses at each axial location are performed to check the homogeneity of hydrogen content. After measurement, the zirconia is partially melted in the graphite crucible. Brachet and Rapsaet have shown by ERDA measurements that the hydrogen content is negligible in the zirconia layer after oxidation at high temperature [START_REF] Raepsaet | Quantification and local distribution of hydrogen within Zircaloy-4 PWR nuclear fuel cladding tubes at the nuclear microprobe of the Pierre Süe Laboratory from µ-ERDA[END_REF][START_REF] Brachet | Oxygen, hydrogen and main alloying chemical elements partitioning upon alpha-beta phase transformation in zirconium alloys[END_REF]. The zirconia layer weight is thus subtracted to take into account the melted sample weight. Hydrogen is measured after gaseous charging and after steam oxidation.

Metallurgical examinations

After oxidation, two metallographic radial cross sections are cut at each end for all the tested samples. Metallurgical examinations are performed to determine the various material layers thicknesses obtained after the LOCA transient. Oxygen diffusion profiles are investigated by Electron Probe Micro Analysis (EPMA) using a CAMECA SX100 electron probe. Each radial cross section of the samples is embedded in a conductive resin along with an as-received Zircaloy-4 sample. After mechanical polishing, a chemical etching with a fluorhydric acid solution is performed before introduction into the microprobe vacuum chamber. Oxygen K line is measured with a W/Si multi-layer synthetic crystal. Oxygen calibration is performed using as-received Zircaloy-4 with homogeneous and well known oxygen contents. A typical accuracy of 0.2 wt% is expected for oxygen content measurements. All the profiles are measured across the thickness with 1 µm stage displacement step in each phase and 250 nm stage displacement step at the zirconia/α(O) and α/prior-β interfaces. Point analyses are also performed in the α(O) inclusions, the α(O) layer and in the prior-β phase. Maps are obtained from 400x300 µm 2 areas with a stage displacement step of 1 µm during 300 ms per point.

Optical microscopy was used to measure the oxide and α(O) layers thickness. The phase distribution in the metallic part of the samples is determined by image analysis based on a half ring metallography reconstruction. The metallography is first transformed into a binary image representing each of the two phases in the metal (Fig. 3). This binary image is then used to determine the average radial profile of α(O) phase fraction f(α). Results and discussion 3.

Oxidation results

The typical microstructure of Zircaloy-4 samples after steam oxidation at 1200°C is illustrated in Fig. 4. The measured values of some parameters such as the measured Equivalent Clad Reacted (ECR), initial and final hydrogen contents, α(O) and zirconia layer thicknesses are respectively reported in Table 2. The results indicate low hydrogen pickup during oxidation. A good consistency between measured ECR and its assessment using Cathcart-Pawel correlation is obtained [START_REF] Cathcart | Zirconium metal-water oxidation kinetics IV. Reaction rate studies[END_REF]. No influence of hydrogen content on the measured ECR is observed. Hydrogen has no effect on the oxide and oxygen stabilized layer growth. All the results are in agreement with the literature data [START_REF] Brachet | Hydrogen content, peroxidation, and cooling scenario effects on postquench microstructure and mechanical properties of Zircaloy-4 and M5® alloys in LOCA conditions[END_REF][START_REF] Guilbert | Influence of a pre-oxide layer on oxygen diffusion and on postquench mechanical properties of Zircaloy-4 after steam oxidation at 900°C[END_REF][START_REF] Saux | Influence of pre-transient oxide on LOCA high temperature steam oxidation and postquench mechanical properties of Zircaloy-4 and M5 TM cladding[END_REF].

Oxygen distribution

Oxygen partitioning occurs within the thickness of the cladding tube during the high temperature oxidation. This segregation results from oxygen diffusion inducing a progressive transformation of the β phase into an oxygen stabilized α(O) layer [START_REF] Desgranges | Simulation of Oxygen diffusion during high temperature oxidation of Zr base Alloys[END_REF][START_REF] Brachet | Mechanical behavior at Room Temperature and Metallurgical study of low-Tin Zy-4 and M5 TM after oxidation at 1100°C and quenching[END_REF]. Oxygen segregation within the cladding samples is systematically analyzed using EPMA. Typical oxygen concentration using point measurements or mapping are illustrated in Fig. 5.

Oxygen is segregated into the α-phase consistently with its α-stabilizing properties. Oxygen concentrations are accurately determined by point analysis in each phase. The electron beam is defocused to obtain a lower sensitivity to short range discrepancies. About 10 measurement points per phase are acquired. Results are plotted in Fig. 5(b).

Oxygen concentration profiles modeling

DIFFOX is a 1D finite-difference calculation code describing several connected reaction layers used to solve the oxygen diffusion equations. It has been developed by IRSN to address LOCA induced oxidation [START_REF] Duriez | Characterization of Oxygen Distribution in LOCA Situations[END_REF]. The oxygen concentration profile is calculated by solving the 2 nd Fick Law diffusion equation in cylindrical coordinates according to Eq. 1. The left side is considered to be the oxygen accumulation and the right side the space change of the diffusion flux F r . The oxygen flux is assumed to be proportional to the oxygen gradient according to the 1 st Fick Law (Eq. 2). The motion of layer boundaries is derived from the mass balance of oxygen at the interface between two layers according to Stefan equation (Eq. 3). The oxygen diffusion coefficient is extracted from literature data for each of the α(O) phase and β-phase. Considering now the mixed α+β phase, it is assumed that the oxygen motion is described by a diffusion law applicant to the average oxygen content. Nevertheless, the equivalent diffusion coefficient has to be determined. In the DIFFOX code, this diffusion coefficient was empirically determined as well as the conditions required to form a layer containing mixed α+β phases. Where C(r,t) is the oxygen concentration, D i (T) is the oxygen diffusion coefficient in phase i (i=α, β, α+β), t is the time and r is the radius.

𝜕𝑆 𝜕𝑡 = 𝐹 + -𝐹 - 2𝜋𝑆(𝐶 + -𝐶 -) (Eq. 3)
Where S is the interface radius, F + and F -are the left and right side oxygen fluxes, C + and C - the oxygen concentrations at each side of the interface.

Measured oxygen distributions are compared to DIFFOX predictions in Fig. 6. The calculations accurately determine the α-layer thickness but strongly underestimate the oxygen content within the α(O) inclusions in the α(O)+β regions. EPMA indicates that in the α(O) inclusions and in the prior-β phase, the oxygen content is constant but different in each of the two phases. Consequently, the equivalent averaged oxygen radial profile can be determined using the α(O)-phase radial profile. The measurement of oxygen average profile combining EPMA determined concentrations in each phase and average phase radial profile are summarized in Eq. 4.

[𝑂] = 𝐶 𝛼 0 . 𝑓 𝛼 + 𝐶 𝛽 0 . 𝑓 𝛽 (Eq. 4)

A rather continuous oxygen profile is obtained between the α(O) layer and the two-phase region. Experimental and calculated profiles are illustrated in Fig. 6.

The comparison between the calculations and measurements in the mixed α+β region shows that the oxygen diffusion is not accurately predicted by the modeling tools.

Fig. 6. EPMA oxygen profiles versus Diffox calculation or measured average profile (H64-3)

Oxygen distribution in the two-phase region

Assuming that the oxygen diffusion in the α+β phase is governed by the 2 nd Fick type law, the combined Eq. 1 and Eq. 4 implies that α-phase fraction is also governed by a diffusionlike law having the same diffusion coefficient (Eq. 5).

𝑑𝑓 𝛼 𝑑𝑡 = 𝐷 𝛼+𝛽 𝜕 2 𝑓 𝛼 𝜕𝑟 2 (Eq. 5)
If the phase fraction is governed by a diffusive process, it is also interesting to consider that Eq.5 implies that oxygen distribution follows an Eq. 1 diffusion type law.

Two types of input data are thus needed for the new calculations: the equilibrium concentration at each interface and the diffusion coefficient in the two-phase region. The oxygen concentrations are relying on EPMA measurements illustrated in Fig. 5

(b).

A Crank analytical solution [START_REF]Crank -The mathematics of diffusion[END_REF] is first used to determine the concentration profile between the edges of a thick plate having constant concentration at each edge (𝑟 = ±𝑙). This solution is used to describe the phase fraction in a cladding having only an α+β region layered by α(O). Indeed, this situation corresponds to long steam exposure at elevated temperature with no remaining β-phase in the central region of the cladding. The boundary layer condition is determined assuming continuity with α(O) uniform layer (𝑓 𝛼 (±𝑙, 𝑡) = 1) at the edges of α+β region. The proposed solution is assumed to be acceptably good close to the α/α+β interface and accurate enough for a first order assessment of the oxygen diffusion coefficient 𝐷 𝛼+𝛽 .

The Crank analytical solution [START_REF]Crank -The mathematics of diffusion[END_REF] is given by Eq. 6. The diffusion coefficient 𝐷 𝛼+𝛽 is adjusted in the solution to fit measured phase fraction profile with the proposed analytical solution.

𝑓 𝛼(𝑟,𝑡) = )] 𝑛≥0 (Eq. 6)

A diffusion coefficient 𝐷 𝛼+𝛽 equal to 1.5.10 -7 cm 2 /s was obtained when simulating the measured radial distribution of phase fraction. The obtained diffusion coefficient was poorly dependent on sample average hydrogen content suggesting that the oxygen diffusion is not influenced by hydrogen.

A new model is developed including a moving interface satisfying the following conditions:

-If the oxygen concentration is less than the solubility limit in the β-phase, the α(O)phase fraction is equal to zero. Oxygen diffusion is thus homogeneous in this phase and governed by Eq. 7 with D β is equal to 1.6.10 -6 cm 2 /s [START_REF] Perkins | Oxygen diffusion in β-Zircaloy[END_REF].

𝑑[𝑂] 𝑑𝑡 = 𝐷 𝛽 𝜕 2 [𝑂] 𝜕𝑟 2 (Eq.7)
-If the oxygen concentration in the β-phase reaches the solubility limit, the phase fraction increases at the considered location and follows Eq.8. In this situation, the oxygen concentration in both the α(O) inclusions and the prior-β phase are determined relying on EPMA measurements (Fig. 5). The link between phase fraction and oxygen content is established by Eq. 4. The inclusion growth is then controlled by grain boundary diffusion of oxygen along the α(O) inclusion interface with β-phase as described by Eq. 8 where 𝐷 𝛼+𝛽 is an empirical overall diffusion coefficient for the two-phases layer. -

The α/α+β interface motion is governed by Eq.9 using experimental measurements for both α(O) and zirconia layer thicknesses. (Eq.9)

and f α (r interface , t) = 1

A good consistency between the average phases fractions measured across the sample metallic surfaces versus oxidation time is obtained for a 𝐷 𝛼+𝛽 value of 1.5.10 -7 cm 2 /s as illustrated in Fig. 7. Experiments obtained in this study but also from previous experiments conducted at IRSN with identical material are then simulated in order to validate this approach in the α+β regions. A good consistency is obtained between measured and predicted α(O)-phase fraction as illustrated in Fig. 8. The present study addresses oxygen distribution in Zircaloy-4 cladding after a LOCA steam oxidation transient. Within the samples, special attention was paid to the region with coexisting (O)+ phases. This region is not homogenous and considering 1D diffusion modeling, a special data treatment has to be performed to compare experimental data to modeling results. Using EPMA measurements, oxygen distribution within the cladding samples after steam oxidation was better characterized. Average radial profiles of oxygen have been determined assuming that the phase contents are uniform and close to equilibrium conditions. The oxygen profile is compared to diffusion simulations using the DIFFOX tool. The model prediction has shown that oxygen diffusion in the mixed phase region has to be improved. A new modeling was consequently proposed to determine the oxygen diffusion mechanisms in the two-phase region. The modeling results are satisfactorily compared to a large set of experiments. This model has been only fitted and validated at 1200°C and should be further tested at other oxidation temperatures.

Fig. 3 .

 3 Fig. 3. Example of transformation of the optical metallography into a binary image used to determine the experimental average phase fraction radial profile (sample labelled H66-7).

Fig. 4 .

 4 Fig. 4. SEM micrograph of Zircaloy-4 after oxidation at 1200°C during 400s (H60-5)

Fig. 5 .

 5 Fig. 5. Oxygen measurements obtained by EPMA of Zircaloy-4 after oxidation at 1200°C (a) Maps in the wall cladding (H64-3) and (b) oxygen concentrations in each phase

Fig. 7 .Fig. 8 .

 78 Fig. 7. Measured α(O) average phase fraction versus modeling using numerically determined oxygen diffusion coefficient in the α+β region

Table 1 .

 1 . Chemical composition of the tested Zircaloy-4

	Sn (wt%)	Fe (wt %)	Cr (wt %)	O (wt %)	H (wppm)	Zr
	1.3	0.21	0.10	0.13	10	balance

Table 2 .

 2 Oxidation results

	Sample Time	Initial hydrogen content	final hydrogen content	ZrO 2 layer thickness	α(O) layer thickness	Measured ECR	α(O) phase fraction
	(#)	s	wppm	wppm	µm	µm	%	%
	AR-1	624	11 ± 5	18 ± 2	71	92	23.6	66
	AR-2	400	11 ± 5	15 ± 2	58	79	19.6	-
	AR-3	196	11 ± 5	15 ± 2	44	59	14.7	-
	H64-3	624	144 ± 17 214 ± 19	74	96	24.2	76
	H64-5	400	147 ± 17 210 ± 20	59	81	20.2	58
	H64-7	196	173 ± 24 219 ± 26	44	59	14.9	32
	H60-3	624	305 ± 39 396 ± 35	72	100	23.8	62
	H60-5	400	287 ± 32 376 ± 34	58	80	19.0	51
	H60-7	196	305 ± 38 356 ± 34	42	59	14.1	27
	H66-3	624	92 ± 12	116 ± 10	74	104	23.9	79
	H66-5	400	86 ± 9	114 ± 11	57	82	19.8	61
	H66-7	196	92 ± 12	104 ± 11	43	58	14.3	45
	H58-3	624	149 ± 17 199 ± 17	68	92	23.8	67
	H58-5	400	157 ± 18 202 ± 18	58	78	19.7	51
	H58-7	196	165 ± 18 191 ± 18	43	59	15.1	30