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Proteomic Analysis of Colon Tissue from Interleukin-10 Gene-Deficient

We measured proteomic changes in the interleukin 10 gene-deficient (Il10−/−) mouse model of inflammatory bowel disease. Inflammation was associate...
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Proteomic Analysis of Colon Tissue from Interleukin-10 Gene-Deficient Mice Fed Polyunsaturated Fatty Acids with Comparison to Transcriptomic Analysis Janine M. Cooney,*,† Matthew P. G. Barnett,‡ Diane Brewster,† Bianca Knoch,‡,||,^ Warren C. McNabb,§,r William A. Laing,†,# and Nicole C. Roy‡,r,# †

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Biological Chemistry & Bioactives, Food Innovation, The New Zealand Institute for Plant & Food Research Ltd, Ruakura Private Bag 3123, Waikato Mail Centre, Hamilton 3240, New Zealand ‡ Food Nutrition & Health, Food & Bio-based Products, and §AgResearch, Palmerston North 4442, New Zealand Institute of Food, Nutrition & Human Health, and rRiddet Institute, Massey University, Palmerston North 4442, New Zealand

bS Supporting Information ABSTRACT: Inflammatory bowel disease (IBD) is characterized by intestinal inflammation and is believed to involve complex interactions between genetic, immunological, and environmental factors. We measured changes in the proteome associated with bacterially induced intestinal inflammation in the interleukin 10 gene-deficient (Il10/) mouse model of IBD, established effects of the dietary polyunsaturated fatty acids (PUFAs) n-3 eicosapentaenoic acid (EPA) and n-6 arachidonic acid (AA) on protein expression (using oleic acid as a control fatty acid), and compared these changes with previously observed transcriptome changes in the same model. Ingenuity pathways analysis of proteomics data showed bacterially induced inflammation was associated with reduced expression of proteins from pathways of metabolism and digestion/absorption/excretion of nutrients/ions, and increased expression of cellular stress and immune response proteins. Both PUFA treatments showed anti-inflammatory activity; EPA appeared to act via the PPARα pathway, whereas AA appeared to increase energy metabolism and cytoskeletal organization and reduce cellular stress responses, possibly enabling a more robust response to inflammation. While there was agreement between proteomic and transcriptomic data with respect to pathways, there was limited concordance between individual gene and protein data, reflecting the importance of having both gene and protein data to better understand complex diseases such as IBD. KEYWORDS: colitis, inflammation, polyunsaturated fatty acids, Il10/ mice, proteomics, transcriptomics

’ INTRODUCTION Inflammation is a complex and precisely regulated host response to harmful stimuli such as foreign substances, invading microorganisms, or damaged tissue resulting from an insult such as infection. Acute inflammation acts to remove these harmful stimuli and initiate the healing process and is an essential mechanism for survival. However, if the acute process is not appropriately controlled, the result is chronic inflammation, which is a characteristic of several diseases, including atherosclerosis, rheumatoid arthritis, and inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC). While the exact etiology and pathogenesis of IBD is still unclear, it appears to involve complex interactions between the immune system, the environment, and a number of host susceptibility genes.1 Twin studies reveal that genetic susceptibility can be impacted by diet and environment. One important environmental factor appears to be the presence of normal intestinal bacteria, to which an inappropriate response occurs, and it is this r 2011 American Chemical Society

response that may trigger the chronic inflammation. As advances in the discovery of genes associated with IBD have progressed, so to has the concept of genenutrition based therapy for prevention or treatment of IBD.2 It has been well established that both n-3 and n-6 polyunsaturated fatty acids (PUFAs), as precursors of potent lipid mediators, play an important role in the regulation of inflammation.3 While it is generally accepted n-3 PUFAs such as eicosapentaenoic acid (EPA) have anti-inflammatory properties due to their ability to inhibit the formation of n-6 PUFA-derived pro-inflammatory eicosanoids via the cyclooxygenase (COX) and lipoxygenase (LOX) pathways,3,4 the activity of the n-6 PUFA arachidonic acid (AA) is more controversial, with variable results reported in terms of its effects on intestinal inflammation.57 More recently, it was recognized that fatty acids can act as hormones which Received: August 22, 2011 Published: November 22, 2011 1065

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Journal of Proteome Research control the activity of key transcription factors, including activating peroxisome proliferator-activated receptors (PPARs), and can consequently function as metabolic regulators.8 In addition to their effects on the up-regulation of genes involved in lipid metabolism, the PPARα and -γ isoforms in the intestine can be activated by PUFAs to ameliorate intestinal inflammation,9 through down-regulation of the expression of genes involved in inflammation, although the precise mechanism remains to be elucidated. The effects of dietary n-3 PUFA in intervention studies with IBD patients have recently been reviewed.10 Despite positive results being reported in some of the studies, it was concluded that convincing evidence, directly relevant to human IBD, was limited. Possible reasons given for the apparent lack of activity in some of the n-3 PUFA supplementation trials included the general limitations of studies with free living humans and the lack of stratification for genetic variability. The complexity of, and potential genetic component(s) contributing to, IBD makes gene knockout animal models such as the interleukin 10 gene-deficient (Il10/) mouse important tools with which to understand disease pathogenesis, as well as understand the potential interaction of dietary compounds with genotype and how this might impact on disease symptoms.1113 IL10 is an anti-inflammatory cytokine which acts to control inflammation initiated and perpetuated by pro-inflammatory mediators via JAK1/STAT3- and p38-MAPK mediated pathways. Il10/ mice develop chronic colonic inflammation that mimics many characteristics of human IBD13 in that they overexpress cytokines such as tumor necrosis factor (TNF) and interferon gamma (IFNG) in response to lipopolysaccharide (LPS) from normal intestinal bacteria. Our previous studies focused on histological and gene expression changes associated with colon inflammation using a bacterially inoculated Il10/ mouse model of IBD14 and the effects of dietary intervention with PUFAs (EPA15 and AA16). Here we describe the effects of dietary PUFA intervention on the proteome of the colon. Furthermore, we examine whether proteomic changes are associated with the observed reduction in inflammation and are consistent with the changes observed at the transcript level.

’ EXPERIMENTAL SECTION Materials

This experiment was reviewed and approved by the AgResearch Ruakura Animal Ethics Committee in Hamilton, New Zealand, according to the New Zealand Animal Protection Act (1960) and Animal Protection Regulations (1987) and amendments. Animals, experimental design, diets, and tissue collection have been described previously15,16 and are summarized in Figure 1. Briefly, 24 male Il10/ (Strain Name: B6.129P2Il10/J) and 24 male C57 control (Strain Name: C57BL/6J) mice, purchased from The Jackson Laboratory (Bar Harbor, ME, USA), were 35 days of age at the start of the experiment and were housed individually in standard shoebox size cages under conventional conditions (22 C, 50% relative humidity, and a 12 h lightdark cycle). After 4 days, all mice were inoculated orally with normal intestinal bacteria, including pure strains of Enterococcus faecalis and E. faecium (EF), to obtain a more consistent and reproducible intestinal inflammation.14,17 Mice were randomly assigned to one of four diets prepared inhouse with 6 Il10/ and 6 C57 mice in each of the four diet groups. Diet 1 was the AIN-76A rodent diet (lipid content 5%

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Figure 1. Overall study design. Il10/ and C57 mice were fed AIN76A diet from the time of arrival (35 days of age). After 4 days, all mice were inoculated orally with bacteria commonly found in the intestinal lumen, to obtain a more consistent and reproducible intestinal inflammation, as previously described,14,17 and were then randomly divided into four treatment groups with six animals of each genotype per group. One group of mice was maintained on the AIN-76A diet; the remainder were subsequently fed diets based on AIN-76A but containing purified ethyl esters of either oleic acid (OA), eicosapentaenoic acid (EPA), or arachidonic acid (AA). Inoculation was performed at approximately 5.5 weeks (38 days) of age, and tissue sampling at 11 weeks (77 days) of age.

corn oil) and was used as a control (referred to as AIN-76A); the other three diets were based on the composition of AIN-76A. Diet 2 was a fatty acid control diet containing 3.7% oleic acid (OA) ethyl ester (NuCheck, Inc., Elysian, MN, USA) (referred to as OA), which has no effect on the tissue content of EPA or on eicosanoid biosynthesis18 and which we have previously shown to be an appropriate control fatty acid diet for experiments of this type.19 The two experimental diets contained either 3.7% EPA ethyl ester (Brainfats Biotechnology Limited, Auckland, NZ) (referred to as EPA) or 3.7% AA ethyl ester (NuCheck, Inc.) (referred to as AA). The three fatty acid supplemented diets contained 1% corn oil supplemented with sufficient purified linoleic and α-linolenic acid to meet the nutritional requirements of mice for these essential fatty acids. All diets were isocaloric and contained 5% w/w fat in total, varying only in the composition of the lipid component; all met the nutritional requirements of laboratory mice.20 The fatty acid composition of the diets was determined in accordance with established methods,21,22 and diets were divided into daily proportions and stored at 85 C until used. Freshly thawed diet (3.54.0 g/mouse) was provided daily to reduce lipid oxidation. Throughout the experimental period, food intake was estimated by collecting and weighing uneaten food. Rather than ad libitum feeding, the food given to each mouse was adjusted daily to equal the mean amount of feed consumed by all Il10/ mice on the previous day. Water was provided ad libitum and refreshed twice a week. All mice were weighed three times a week and carefully monitored for disease symptoms, including weight loss, soft feces, and inactivity. At 77 days of age, by which time bacterially inoculated Il10/ mice show consistent colon inflammation,23 samples were collected as follows: All mice were randomly divided into two sampling days and three staggered groups of 4 mice within each day. 1066

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Figure 2. Overall approach and main outcomes of the study, and overlaps between effects of EPA and AA dietary interventions. Histological evaluation of colon tissue collected from Il10/ and C57 mice (as described in the Experimental Section and shown in more detail in Figure 1) showed a decrease in intestinal inflammation associated with feeding of both EPA and AA (panel 2; Supporting Information Figure 1). Subsequent microarray (panel 3) and proteomics (panel 4) analyses identified genes and proteins associated with this reduction of inflammation. Integration of gene and protein data (5) identified key pathways associated with inflammation (panel 6) and with the reduction of inflammation in response to both EPA and AA (panel 7). Note that panels 13 refer to work which has previously been reported,15,16 while panels 47 show the work described in the current paper.

On the day prior to sampling, mice were fasted overnight for 14 h and then food was reoffered for 2 h and again removed for the 2 hours immediately before sampling, to minimize the variation in time between the last food intake and tissue sampling.24 Mice were euthanized by CO2 asphyxiation and cervical dislocation, and the intestine was quickly removed, cut open lengthwise, flushed with 0.9% NaCl to remove digesta, and divided into duodenum, jejunum, ileum, and colon. A piece of each section was stored at room temperature in 10% phosphate buffered formalin for histological analysis. Tissue samples were snap frozen in liquid nitrogen and stored at 85 C for gene and protein profiling. Sample Pools for Differential In-Gel Expression

Gels for proteomic analysis were run as duplicate biological replicates (pool 1 and pool 2 for each diet group) using the same pooled samples that were used previously in gene expression studies. Briefly, an equimolar pool of colon RNA extracts from 23 mice per treatment was made to minimize individual variation for microarray analysis, as previously described.15,16 Protein samples (derived from the same TRIzol extract as the RNA) from the same mice were pooled for proteomics analysis— this enabled direct comparisons between gene and protein

expression data. For the purpose of this comparison, differentially expressed proteins were only flagged as significant where the abundance fold-change for each biological replicate changed in the same direction (i.e., either up- or down-regulated) and either both gave a value >|1.5|-fold or one biological replicate gave a value of >|2|-fold difference and the other biological replicate gave one of >|1.3|-fold. Proteins of interest were excised, digested with trypsin, and identified by LC-MS/MS, as described below. To determine the effect of a PUFA diet on the expression of proteins involved in colon inflammation, direct differential in-gel expression comparisons were carried out between genotype (Il10 / vs C57) for each diet. To determine the effect of dietary PUFA intervention on protein expression within a genotype, direct differential in-gel expression comparisons were carried out (dietary group OA, EPA, or AA vs AIN-76A) for both Il10/ and C57 mice. Two-Dimensional Gel Electrophoresis for Differential In-Gel Expression

Samples were prepared as previously described.23 Briefly, pooled aliquots of protein, 50 μg per treatment, were labeled with 200 1067

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Journal of Proteome Research pmol CyDyes (GE Healthcare) as per manufacturer’s instructions. In all cases, “controls” (C57 mice or AIN-76A-fed mice) were labeled with Cy5 and “test” samples were labeled with Cy2. Each control and test was mixed to give 100 μg of protein and run on the same first dimension strip. The first dimension, equilibration, second dimension, scanning, staining, analysis, and digestion were performed as previously described.23 LC-MS Analysis of Peptides

Tryptic peptides were separated and analyzed using an Ettan multidimensional liquid chromatography system (GE Healthcare) coupled to an LTQ linear ion trap mass spectrometer with a nanospray ionization interface (ThermoQuest, Finnigan, San Jose, CA, USA). Samples (2 μL) were injected onto a 300 μm i.d.  5 mm trap column (Zorbax 300-SB C18) for in-line desalting and were separated on a nanoscale reverse phase chromatography column, 75 μm i.d.  150 mm, 3 μm (LC Packings, San Francisco, CA, USA), in high-throughput configuration at 280 nL/min with a linear gradient from 0 to 60% B over 50 min (A: 0.1% formic acid; B: 84% acetonitrile and 0.1% formic acid). Data were acquired using a top 3 experiment in data-dependent mode with dynamic exclusion enabled. Database Searching and Data Interpretation

MS/MS data were analyzed using TurboSEQUEST protein identification software,25,26 and spectra were searched against the National Center for Biotechnology Information (NCBI) Mus musculus database. Modifications were set to allow for differential oxidation modification of 16 Da to methionine residues and static carboxyamidomethylation modification of 57 Da to cysteine residues. The criteria used for a positive peptide identification for a doubly charged peptide were a correlation factor (XCorr) > 2.0, a delta cross-correlation factor (dCn) > 0.1 (indicating a significant difference between the best match reported and the next best match), and a high preliminary scoring (Sp). For triply charged peptides, the correlation factor threshold was set at 2.5. All matched peptides were confirmed by visual examination of the spectra. Functional Analysis

Functional classification of proteins was based on Gene Ontology using the mouse genome database (http://www.informatics.jax.org), the Kyoto Encyclopedia of Genes and Genomes (KEGG) encyclopedia (http://www.genome.ad.jp/kegg/genes.html), and Ingenuity Pathways Analysis (IPA) (Ingenuity Systems, http://www.ingenuity.com). IPA mapped each differentially expressed protein to its corresponding gene object (e.g., genes, mRNAs, and proteins) in the Ingenuity Pathways Knowledge Base (IPKB). These genes, called “focus genes”, were overlaid onto a global molecular network developed from information contained in the IPKB and were used as the starting point for generating biological networks. The functional analysis of a network identified the biological functions and/or canonical pathways that were most significant to the genes in the network.

’ RESULTS Previously reported data established that dietary EPA15 and AA16 both reduced total colon inflammation in the Il10/ mouse model without changing the histological injury score in the C57 mice (Figure 2, panel 2; Supplementary Figure 1). In order to establish changes in protein expression associated with these changes in inflammation, differential expression comparisons using DIGE technology and LCMS peptide identification

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were carried out between genotypes (Il10/ vs C57) for each diet (AIN76A, OA, EPA, or AA) and within genotypes (dietary group OA, EPA, or AA vs AIN-76A) for both Il10/ and C57 mice (Figure 1; Figure 2, panel 4). Comparison between Il10/ and C57 Mice within Diets: The Inflammatory Proteome

We identified 172 protein spot-feature changes that were consistently detected across all diet treatments. The 104 protein spot-features that met the threshold criteria were selected for identification. These spot-features represented 77 unique proteins (excluding multiple isoforms due to post-translational modifications) and are shown in the gel image depicted in Supporting Information Figure 2 and listed in Supporting Information Table 1. In five cases, two proteins were identified in the same 2-D gel spotfeature. Differentially expressed proteins between genotypes (Il10/ vs C57) across diets and classified according to cellular processes and biological functions are shown in Figure 3. IPA was able to map 105 proteins (75 unique, i.e., excluding multiple isoforms due to post-translational modifications) to their corresponding gene objects in the IPKB. Seventy-two unique “focus genes” were network eligible, and 72 were eligible for function/pathways/lists analysis. For Il10/ mice compared with C57 mice, a widespread down-regulation of metabolism-related proteins was observed, indicating a reduced functionality of numerous biochemical pathways. This included carbohydrate metabolism (e.g., butanoate, propanoate, pyruvate, ascorbate, and aldarate metabolism; glycolysis/gluconeogenesis, pentose phosphate pathway, citrate cycle), amino acid metabolism (valine, leucine, and isoleucine degradation; arginine and proline metabolism; cysteine metabolism), energy metabolism (nitrogen and sulfur metabolism), lipid metabolism (fatty acid metabolism; bile acid biosynthesis; synthesis and degradation of ketone bodies), and xenobiotic biodegradation and metabolism. This was the case for each of the four diets, although the genotype effect was less consistent in the two PUFA diets, particularly EPA (AIN-76A 65 metabolism-related proteins differentially expressed in Il10/ compared with C57 mice; OA 61; EPA; 37; AA 47; Figure 3). Proteins associated with LPS/IL-1 mediated inhibition of retinoid X receptor (RXR) function were also downregulated. Up-regulated cellular processes in the Il10/compared with the C57 mice included functions associated with cytoskeletal assembly and organization, immune responses, acute phase response signaling, hypoxia, NRF2-mediated oxidative stress response, intracellular and secondary messenger signaling, nucleic acid binding and protein translation, folding, sorting, and degradation. Several proteins associated with apoptosis were upregulated while others were down-regulated. Effect of PUFAs on Inflammation and Colon Protein Expression

We identified 80 significantly consistent protein spot-feature changes within the pooled biological replicates for either Il10/ or C57 mice across diets (dietary group OA, EPA, or AA vs AIN76A), of which 41 met the threshold criteria and were selected for identification. These spot-features represented 37 unique proteins (excluding multiple isoforms due to post-translational modifications) and are shown in the gel image depicted in Supporting Information Figure 3 and listed in Supporting Information Table 2. In five cases, multiple proteins were identified in the same 2-D gel spot-feature. 1068

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Figure 3. Continued

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Figure 3. Continued

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Figure 3. Heat map showing the expression levels and associated pathways of proteins differentially or nondifferentially (nc) expressed in the colon of interleukin-10 gene-deficient (Il10/) compared with C57 wild-type mice across all diets. Some of the proteins were associated with multiple biological processes and are therefore classified into several functional groups, as indicated by an asterisk.

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Journal of Proteome Research Functional analyses of differentially expressed proteins within genotype across diets for both Il10/ and C57 mice classified according to cellular processes and biological functions are shown in Figure 4. IPA was able to map 47 proteins (36 were unique, i.e. excluding multiple isoforms due to post-translational modifications) to their corresponding gene objects in the IPKB. Fifteen unique “focus genes” were network eligible and 15 were eligible for function/ pathways/lists analysis. Some of the proteins were associated with multiple biological processes and are therefore classified into several functional groups. Dietary Intervention with OA

A diet enriched with the monounsaturated fatty acid OA was included as the fatty acid control diet. We have previously shown that this is an appropriate control diet showing few differences at either the transcriptome or proteome level when compared with AIN-76A.19 Briefly, OA supplementation resulted in varied effects on metabolism in Il10/ mice: proteins involved in lipid metabolism were up-regulated, while there was both up- and down-regulation of proteins associated with energy and carbohydrate metabolism. OA had no effect on proteins involved in energy metabolism

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in Il10/ mice, while the only metabolism-related protein affected by this diet in C57 mice was a fatty acid binding protein, FABP4. Signaling processes and stress and inflammatory/immune response proteins were generally up-regulated by OA in both C57 and Il10/ mice, as were cytoskeletal proteins in the C57 mice. In the case of Il10/ mice, both up- (MYL6, TMP3) and down- (ACTG1, KRT19, TAGLN) regulation was observed in response to OA when compared with AIN-76A. No other effects of OA were observed in C57 mice, while in Il10/ mice one protein associated with translation, EIF5A, was up-regulated in response to OA. Dietary Intervention with EPA

Supplementation with dietary EPA generally decreased or did not change the expression of metabolism-related proteins in Il10/ mice compared with those fed the control AIN-76A diet. For C57 mice using the same direct diet comparison, a similar pattern was noted for proteins involved in carbohydrate and amino acid metabolism; however, proteins associated with energy metabolism were both up- and down-regulated, and proteins associated with lipid metabolism were generally up-regulated.

Figure 4. Continued 1072

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Figure 4. Heat map showing the expression levels and associated pathways of proteins differentially or nondifferentially (nc) expressed in the colon of C57 wild-type mice or interleukin-10 gene-deficient (Il10/) mice across OA, EPA, or AA diets compared with an AIN76A diet. Some of the proteins were associated with multiple biological processes and are therefore classified into several functional groups, as indicated by an asterisk.

Signaling pathways associated with hypoxia signaling in the cardiovascular system (HSP90AB1, LDHA) and HIF1A signaling (LDHA) were down-regulated in Il10/ mice fed an EPA rich diet compared with Il10/ mice fed the control AIN-76A diet. For C57 mice using the same direct diet comparison, no change was observed. Nitric oxide signaling in the cardiovascular system was up-regulated (CALM1) for both Il10/ and C57 mice and down-regulated (HSP90AB1) for Il10/ mice only. Intracellular and secondary messenger signaling processes involved in calcium signaling (CALM1, MYL6, TPM1, TPM2, TPM3), phospholipase C and protein kinase signaling (CALM1, MYL6), and Rho signaling (MSN, MYL6) were up-regulated similarly in Il10/ and C57 mice in response to EPA supplementation.

Glucocorticoid receptor signaling (ANXA1, HSP90AB1) was down-regulated in Il10/ mice but unaffected for C57 mice. ILK signaling (MYL6, NACA) was also up-regulated similarly in Il10/ and C57 mice in response to EPA supplementation. Signaling pathways that were varyingly modulated included those associated with apoptosis, such as apoptosis signaling (LMNA) and tight junction signaling (MYL6), which were upregulated for both Il10/ and C57 mice, and aryl hydrocarbon signaling (HSP90AB1), which was down-regulated for Il10/ mice only. Up-regulated processes by EPA for Il10/ mice also included those involved with cellular immune response such as leukocyte extravasation signaling (MSN, MYL6) and calcium-induced T lymphocyte apoptosis (CALM1). For C57 mice, leukocyte 1073

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Journal of Proteome Research extravasation signaling was varyingly modulated (ACTG1, MYL6) and calcium-induced T lymphocyte apoptosis (CALM1) was also up-regulated. Processes in Il10/ mice for which EPA had varying effects included those associated with inflammation and inflammatory response, for which several proteins were down-regulated (ANXA1, ANXA2) and one was up-regulated (FABP4), and cellular mediated immune response cell movement, where two proteins were down-regulated (ANXA1, ANXA2) and five proteins were upregulated (FABP4, MSN, TPM1, TPM2, TPM3). Cellular mediated immune response cell movement was also up-regulated for C57 mice (FABP4, FABP6, TPM1, TPM2, TPM3). Dietary supplementation with EPA led to the up-regulation of a number of proteins associated with cellular assembly and organizational processes, such as formation of microfilaments and microtube bundles and actin binding (TPM1, TPM2, TPM3) and regulation of actin-based motility (MYL6) for both Il10/ and C57 mice. For C57 mice, two proteins important in the organization of the sarcomere (ACTG1, KRT19) and one involved in actin cross-linking (TAGLN) were down-regulated, but this effect was not observed for Il10/ mice. One protein involved in the organization of collagen fibrils (ANXA2) was down-regulated for Il10/ mice but not for C57 mice. Other processes up-regulated for both Il10/ and C57 mice in response to dietary supplementation with EPA included protein translation (EIF5A), while, for Il10 / mice only, protein folding, sorting and degradation (CALU), and calciumactivated chloride conductance (CCLA3) were also upregulated. Dietary Intervention with AA

Supplementation with dietary AA generally increased the expression levels of metabolism-related proteins for Il10/ mice compared with those fed the control AIN76A diet. Pathways affected included those involved in carbohydrate metabolism (e.g., pyruvate and glyoxylate/dicarboxylate metabolism; glycolysis/gluconeogenesis), amino acid metabolism (ALDH1B1, a protein which plays a role in the metabolism of a variety of amino acids and that was up-regulated), energy metabolism (in which both up- and down-regulated proteins were observed), and lipid metabolism. These pathways were largely unaffected for C57 mice using the same direct diet comparison. Other generally up-regulated cellular processes included functions associated with cytoskeletal assembly and organization, stress and immune/ inflammatory response, LPS/IL-1 mediated inhibition of RXR function, intracellular and secondary messenger signaling (calcium, Rho, and protein kinase signaling), and apoptosis (LMNA). Of these, only intracellular and secondary messenger signaling processes were up-regulated to the same extent in C57 mice using the same direct diet comparison.

’ DISCUSSION We describe the effects of dietary EPA and AA supplementation in Il10/ mice on the inflamed colon proteome, and we discuss these with respect to previously identified patterns of transcript expression (Supporting Information Figures 46).15,16 The experimental design and key findings are summarized in Figure 2. Inflammation was associated with down-regulation of proteins involved with metabolism and digestion/absorption/excretion of nutrients/ions and with up-regulation of cellular stress and immune response proteins. The anti-inflammatory activity of EPA appeared to be via the PPARa pathway, whereas AA

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appeared to increase energy metabolism and cytoskeletal organization and to reduce cellular stress responses, suggesting that this diet may enable a better response to inflammatory triggers. The Inflammatory Proteome: Comparing Inflamed Il10/ Mice with C57 Mice

This revealed down-regulation of proteins involved in carbohydrate, lipid, amino acid, and energy metabolism in inflamed mice, suggesting impaired functionality of multiple metabolic pathways or redirection of resources to the priority of fighting infection/inflammation. This observation is consistent with both human27 and animal28,29 studies that suggest chronic intestinal inflammation represents an energy deficiency disease. Key metabolic processes associated with maintenance of energy homeostasis, such as glycolysis/gluconeogenesis and fatty acid β-oxidation, were affected in our study, supporting the hypothesis that mucosal cells have inadequate energy resources during inflammation.28 We observed reduced expression of multiple transcripts15 (Supporting Information Figure 4) and proteins related to digestion/absorption/excretion of nutrients/ions, such as members of the solute carrier (Slc) family and ATP-binding cassette (Abc) subfamily. This is consistent with the observation that inhibition of metabolism and energy deficiency in the intestinal mucosa could affect other energy-consuming processes such as selective permeability and epithelial nutrient transport.30 Furthermore, reduction in xenobiotic processing (e.g., Abc family and cytochrome P450s) may exacerbate the outcome by allowing more toxic and/or pro-inflammatory compounds into the cell (similar to the Mdr1a mouse model of IBD31). In the present study, besides the overall reduction in energy-dependent processes, several cytoskeletal proteins important to maintenance of membrane integrity and function were down-regulated (VIL1, KRT8), as were transcripts coding for claudin 8 (Cldn8) and junctional adhesion molecule 3 (Jam3),15 both required for the maintenance of epithelial tight junctions. Several cellular stress and immune response proteins were highly up-regulated in inflamed Il10/ mice. Pancreatitis associated protein (REG3B) and S100 calcium binding protein A9 (S100A9) have been listed in a recent patent application as fecal markers of inflammation in IBD patients (WO/2010/062663).32 S100A9 expression was highly up-regulated in Il10/ mice in the AIN-76A and OA groups, while REG3B protein expression was up-regulated in Il10/ mice in all four diet groups. It has been suggested that REB3B down-regulates NF-KB, suppresses pro-inflammatory signals,33,34 and is involved in the innate immune response to bacterial colonization of the intestinal tract.35 Up-regulated S100A9 activates the p38MAPK cascade and NF-KB or calcium-dependent signal transduction and is involved in the production of inflammatory cytokines, in cell migration, and in calcium homeostasis. This was reflected in the inflammatory transcriptome analysis which showed altered expression of numerous cellular and humoral immune response and cytokine signaling genes.15 As expected with a dysfunctional IL10 signaling pathway, pro-inflammatory cytokines (e.g., Il1a, Il1b, Tnfa) and chemokine receptor (e.g., Ccr5) genes in the IL10 signaling pathway were up-regulated. A growing body of evidence suggests that metabolic, ER, and oxidative stress and the inflammatory response are linked.36 Upregulation of PDIA3 and PDIA4 isomerize enzymes, and induction of heat shock proteins such as HSP90B1, HSPA5, and HSPE1, in Il10/ mice may be related to ER stress. Heat shock proteins are a key element in restoring ER homeostasis by triggering unfolded protein response (UPR) pathways. Inflammatory 1074

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Journal of Proteome Research cytokines can induce ER stress by triggering calcium signals and accumulation of ROS (reactive oxygen species) associated with ER folding and mitochondrial dysfunction (i.e., reduced ATP production). ER stress and activation of the UPR can promote the inflammatory response, antioxidative stress response, apoptosis, and other stress pathways leading to a “stress-inflammation” interactive loop.36 Effect of Dietary PUFAs on the Oxidative and Cellular Stress Response

Reduced expression of ANXA1 and ANXA2 in Il10/ mice fed either EPA or AA-supplemented diets may be associated with reduction of inflammation. Increased expression and secretion of ANXA1 has been reported in inflamed mucosal tissues in rodent models of colitis and in human UC.3739 Annexins A1 and A2 have also been reported to mediate the expression of a number of matrix metallopeptidases (MMPs). In the current study, changes in annexin expression were not observed at the transcript level, but dietary AA did decrease Mmp13 gene expression.16 Overexpression of MMP-7 and MMP-13 is associated with intestinal inflammation and fibrosis,40 while the mRNA levels of several MMPs have been shown to be significantly increased in the inflamed colonic mucosa of patients with IBD.41 Dietary EPA reduced the expression of the heat shock protein HSP90AB1 in Il10/ mice. HSP90 interacts with PPARs, repressing both PPARα and PPARβ activity.42 The observed reduction of HSP90AB1 expression may result in decreased interaction with PPARα, a consequent increase in availability and/or activity of PPARα, and thus, enhanced expression of its target genes. Furthermore, inhibition of HSP90 appears to promote T cell tolerance;43 reduction of HSP90AB1 in EPAfed Il10/ mice may be a mechanism by which EPA partially suppresses an inappropriate immune response. This finding is consistent with the observation that dietary EPA reversed the decrease in colon Ppara gene expression levels observed in OA-fed Il10/ mice.15 Of particular note, the expression of REG3B, one protein associated with the inflammatory proteome, was down-regulated by AA. Reg3b and the related Reg3g genes are induced during development of intestinal inflammation in response to intestinal bacteria. The observed REG3B reduction in Il10/ mice fed the AA diet may be a reflection of the reduction in inflammation. Although other proteins associated with the inflammatory proteome were not modulated by dietary AA, several proinflammatory marker genes, such as Tnf, Il1a, Il1b, and Il1r2, were down-regulated16 (Supporting Information Figure 6). Effect of Dietary PUFAs on Metabolism

As expected, dietary supplementation with either EPA or AA modulated the expression of proteins associated with fatty acid metabolism. Both PUFA and the OA control diet up-regulated fatty acid binding protein (FAB) expression for both Il10/ and C57 mice in comparison with those fed the AIN-76A diet. Gene expression of several FAB isoforms is modulated by the lipid content of the diet, enabling control of the cellular FA flux. This positive regulatory loop is thought to be essential in maintaining the integrity of the intestinal mucosa by acting as a buffer against the cytotoxic effects of free cellular FAs.44 In the present study, while changes to typical fatty acid responsive genes were observed for both dietary EPA and AA supplementation,15,16 changes in protein expression were predominately limited to FABs. We have previously reported that dietary EPA reversed the decrease in colon Ppara gene expression observed in OA-fed

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Il10/ mice.15 Many of the genes and proteins involved in lipid metabolism that showed decreased expression during the inflammatory response are regulated by nuclear hormone receptors such as PPARα. Activation of the Ppara gene by EPA in Il10/ mice may play a role in the observed up-regulation of genes associated with fatty acid β-oxidation and down-regulation of genes associated with fatty acid synthesis. Dietary AA supplementation did not modulate PPARα at a transcript or protein level in our study; however, decreased colonic expression of genes involved with cholesterol or fatty acid synthesis, including Srebf1, Acaca, and Hmgcr, was observed for Il10/ mice fed AA.16 In addition to the observed changes in fatty acid metabolism, there was up-regulation of proteins involved in energy homeostasis, in particular glycolysis (ALDH1B1, PKM2), the citrate cycle (ACO2), oxidative phosphorylation (ATP5A1, ATP5B, UQCRFS1), and nitrogen metabolism (CAR3). This was not observed in response to EPA. As chronic intestinal inflammation may represent an energy deficiency disease, the general up-regulation of metabolism observed in AA-fed mice could enable a more robust response to the inflammatory triggers, although it is not clear by which mechanism(s). Effect of PUFA on Cytoskeletal Assembly and Organization

The greatest changes in proteins associated with cytoskeletal assembly important for barrier integrity were observed in Il10/ mice in response to the AA diet. Loss of epithelial cell barrier function may play a key role in IBD development by allowing antigenic material into the submucosa from the lumen. This exposes lamina propia immune cells to na€ive antigens, eliciting an inflammatory response.45 Dietary supplementation with EPA and AA increased the transcript levels of some of the transporter and xenobiotic metabolism genes down-regulated in the inflammatory transcriptome important for barrier integrity in Il10/ mice, but it did not affect the transcript levels of key modulated tight junction genes such as claudin 8 (Cldn8) and junctional adhesion molecule 3 (Jam3) (Supporting Information Figures 5 and 6). Dietary AA increased the expression of multiple isoforms of keratin 8 (K8) and keratin 19 (K19), the major intermediate filament proteins present in intestinal epithelia, in Il10/ but not C57 mice. Many of the same protein isoforms were downregulated in the inflammatory proteome. In addition to maintenance of normal epithelial architecture, many keratins have functional roles in epithelial physiology. Recent studies have reported K8 mis-sense mutation in a subset of IBD patients,46 while K8 null FVB/N mice develop colonic hyperplasia and Th-2 mediated chronic colitis that is amenable to antibiotic treatment.47 Conclusions and Potential Relevance to Human IBD

Our study suggests that the dietary PUFAs EPA and AA may both ameliorate intestinal inflammation, although by different mechanisms. Inflammation was associated with down-regulation of metabolism and digestion/absorption/excretion of nutrients/ ions, and up-regulation of cellular stress and immune responses. Changes in transcript15 and protein expression patterns suggest that EPA exerts its anti-inflammatory effects via PPARα, at least in part by modulating the expression of HSP90AB1. In contrast, the reduction in colon inflammation by AA may be the consequence of increased energy metabolism and cytoskeletal organization, enabling a more robust response to the inflammatory triggers, or possibly by reducing the influx of antigens and xenobiotics through leaky organ and cell membranes. 1075

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Journal of Proteome Research These findings support studies suggesting that dietary PUFA supplementation, or consumption of foods rich in these compounds (for example, n-3 PUFA-rich fish), may reduce or prevent IBD symptoms and therefore play a part in an overall IBD treatment regime. However, as these observations are based on an Il10/ mouse model, these compounds may only be effective for certain IBD patients, e.g., those with genetic variants in the IL10 signaling pathway. As is the case for any animal model, care must be taken when extrapolating data to a human clinical situation. Further research is required to confirm if the effects observed in response to purified compounds can be replicated when feeding diets based on a whole food such as PUFA-rich salmon, before any dietary advice could be provided within a clinical setting. While there was agreement between proteomic and transcriptomic pathway data, individual transcript and protein data had limited concordance, reflecting the importance of having both transcript and protein data to better understand complex diseases such as IBD, and the effects that dietary interventions may have on these diseases.

’ ASSOCIATED CONTENT

bS

Supporting Information Six figures showing a summary of the colon histological injury score (HIS) in Il10/ and C57 mice in response to diets enriched with polyunsaturated fatty acids; 2D-DIGE gels; generation of biological networks of genes of the most significant pathways in the Il10/ vs C57 mice; and the effect of dietary supplementation with EPA or AA on the biological network of genes; and two tables showing proteins differentially expressed in the colon of Il10/ compared with C57 mice across all diets and in the colon of C57 or Il10/ mice fed OA, EPA, or AA diets compared with an AIN76A diet. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone: +64-7-959-4522. Fax: +64-7-959-4431. Present Addresses

^ Illawarra Health and Medical Research Institute, University of Wollongong, New South Wales 2522, Australia.

Author Contributions #

W.A.L. was responsible for leadership of the proteomics analysis, and N.C.R. for overall supervision of the design, animal study, and microarray analysis. All authors are part of Nutrigenomics New Zealand, www.nutrigenomics.co.nz.

’ ACKNOWLEDGMENT This study was part of Nutrigenomics New Zealand, a collaboration between AgResearch Limited, Plant & Food Research, and The University of Auckland primarily funded by the New Zealand Ministry of Science and Innovation (MSI). The authors acknowledge the important contributions of Ric Broadhurst, Bobby Smith (AgResearch), and Philippa Dryland (The University of Auckland) for assistance with the animal trial, Dwayne Jensen (Plant & Food Research) for LC-MS technical assistance, and Arjan Scheepens, Margot Skinner, and Matt Miller (Plant & Food Research) for valuable discussions.

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