Purified rutin and rutin-rich asparagus attenuates disease severity and tissue damage following dextran sodium sulfate-induced colitis

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1 2396 Mol. Nutr. Food Res. 2016, 60, DOI /mnfr RESEARCH ARTICLE Purified rutin and rutin-rich asparagus attenuates disease severity and tissue damage following dextran sodium sulfate-induced colitis Krista A. Power 1,2,JeniferT.Lu 1,2, Jennifer M. Monk 1,2, Dion Lepp 1, Wenqing Wu 1, Claire Zhang 1,2, Ronghua Liu 1, Rong Tsao 1, Lindsay E. Robinson 2, Geoffrey A. Wood 3 and David J. Wolyn 4 1 Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, ON, Canada 2 Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON, Canada 3 Department of Pathobiology, University of Guelph, Guelph, ON, Canada 4 Department of Plant Agriculture, University of Guelph, Guelph, ON, Canada Scope: This study investigated the effects of cooked whole asparagus (ASP) versus its equivalent level of purified flavonoid glycoside, rutin (RUT), on dextran sodium sulfate (DSS)-induced colitis and subsequent colitis recovery in mice. Methods and results: C57BL/6 male mice were fed an AIN-93G basal diet (BD), or BD supplemented with 2% cooked ASP or 0.025% RUT for 2 wks prior to and during colitis induction with 2% DSS in water for 7 days, followed by 5 days colitis recovery. In colitic mice, both ASP and RUT upregulated mediators of improved barrier integrity and enhanced mucosal injury repair (e.g. Muc1, IL-22, Rho-A, Rac1,andReg3 ), increased the proportion of mouse survival, and improved disease activity index. RUT had the greatest effect in attenuating DSS-induced colonic damage indicated by increased crypt and goblet cell restitution, reduced colonic myeloperoxidase, as well as attenuated DSS-induced microbial dysbiosis (reduced Enterobacteriaceae and Bacteroides, and increased unassigned Clostridales, Oscillospira, Lactobacillus,andBifidobacterium). Conclusion: These findings demonstrate that dietary cooked ASP and its flavonoid glycoside, RUT, may be useful in attenuating colitis severity by modulating the colonic microenvironment resulting in reduced colonic inflammation, promotion of colonic mucosal injury repair, and attenuation of colitis-associated microbial dysbiosis. Received: November 10, 2015 Revised: June 7, 2016 Accepted: June 15, 2016 Keywords: Asparagus / Colitis / Microbiota / Quercetin / Rutin Additional supporting information may be found in the online version of this article at the publisher s web-site 1 Introduction The colonic microenvironment is made up of both host and microbial factors including a tightly regulated intact mucosal Correspondence: KristaA.Power krista.power@agr.gc.ca Abbreviations: AB, Alcian Blue/nuclear fast red; ANOVA, analysis of variance; ASP, asparagus; BD, basal diet; BW, body weight; DAI, disease activity index; DSS, dextran sodium sulfate; FRAP, ferric reducing/antioxidant power; H&E, hematoxylin & eosin; IBD, inflammatory bowel disease; MPO, myeloperoxidase; ORAC, oxygen radical absorbance capacity; OTU, operational taxonomy unit; RUT, rutin epithelial barrier and a balanced gut microbial community [1]. A symbiotic relationship between the gut microbiota and the host immune system preserves the integrity of the intestinal barrier and minimizes inappropriate inflammatory responses and resulting tissue damage, which can promote the development of chronic gut-related diseases [1 4]. Ulcerative colitis is a form of inflammatory bowel disease (IBD) in which microbial dysbiosis and dysregulated inflammation in the colon are associated with immune cell hyperactivation, enhanced production of destructive inflammatory mediators (e.g. reactive oxygen species, cytokines), and epithelial barrier damage and dysfunction (ulcerations, erosions, loss of mucus-producing goblet cells) [5, 6]. Because of the remitting and relapsing nature of IBD, therapies target

2 Mol. Nutr. Food Res. 2016, 60, inflammatory and immune responses, as well as those which promote epithelial injury repair, to help heal mucosal damage, re-establish colonic homeostasis, and stimulate disease remission [5 8]. Dietary constituents that escape digestion and absorption in the upper gut can travel to the large intestine and interact with the colonic microenvironment (i.e. microbiota community and colonic mucosa), ultimately impacting the risk of gut-associated chronic diseases, such as IBD, colon cancer, and obesity [3, 4, 9]. Flavonols (e.g. isorhamnetin, kaempferol, and quercetin) are a subclass of flavonoid phytochemicals found ubiquitously in fruits, vegetables, and beverages of which daily intake levels vary between mg/day, with quercetin being the most abundant contributor to total flavonol intake [10, 11]. Quercetin has demonstrated colonic anti-inflammatory [12 14] and epithelial barrier protective effects [15 18], thus making it a potential therapeutic agent for IBD patients. Quercetin is rapidly absorbed in the upper gut, however, consumption of its glycosylated forms (e.g. rutin (quercetin 3-O-glucorhamnoside), quercitrin (quercetin 3-Orhamnoside), quercetin-3-glucosides) escape upper gut absorption and are delivered to the colon where they are metabolized by the colonic microbiota to release quercetin locally, which has been shown to reduce colonic damage and inflammation in a number of animal models of IBD [12, 13, 19 21]. Furthermore, quercetin bioavailability can depend on which glycosylated form is consumed in the diet (e.g. glucosides versus rhamnosides) [22 24], as well as the presence of other dietary factors, such as dietary fiber, which can enhance intestinal microbial activity and increase quercetin bioavailability [24 28]. Therefore, to promote increased dietary quercetin intake for IBD patients, it is important to ensure that its beneficial effects can be obtained through consumption of quercetin glycoside-rich foods. Asparagus officinalis L. is one of the richest dietary sources of rutin (quercetin 3-O-glucorhamnoside; mg/kg) [29, 30], however, no studies to date have examined its potential to attenuate colitis severity. Extracts from asparagus (ASP) have colon cancer inhibitory effects in vitro and in vivo [31] and induce anti-inflammatory effects in vitro [32], thereby demonstrating its potential to influence the colonic microenvironment and reduce colon-associated disease risk. Amongst 23 commonly consumed vegetables, ASP has the greatest antioxidative potential [30], which in addition to rutin (RUT), may be attributed to increased carotenoids and vitamin C concentrations [33]. ASP also contains dietary fiber and oligosaccharides [34] which may impact the colonic microbial composition and activity (e.g. production of SCFAs), as well as potentially influencing quercetin bioavailability, and IBD symptoms and severity [25, 26, 35, 36]. The overall objective of this study was to determine the potential for dietary ASP to attenuate dextran sodium sulfate (DSS)-induced colonic damage and inflammation in mice which may prove beneficial for IBD patients as a dietary complementary therapy. Furthermore, this study set out to compare the effects of dietary ASP to its equivalent level of dietary purified RUT, to determine if protective effects previously demonstrated with RUT [12, 19, 20], can be obtained through consumption of a RUT-rich food. 2 Materials and methods 2.1 ASP preparation, nutritional composition, and RUT concentration Twenty-five kilograms of ASP (Asparagus officinalis; cultivar Guelph Millennium; Dalton White farms, ON, Canada) spears (top 7 cm; most edible portion and contains the highest RUT concentration compared to the bottom stem portion [37]) were washed in cold dh 2 O, steamed for 5 min, freezedried (Guelph Food and Technology Centre, ON, Canada), blended, and stored at 80 C until further use (Supporting Information Fig. 1). Macronutrients, total dietary and soluble fiber were analyzed in the freeze-dried ASP by Maxxam Analytics (Mississauga, ON, Canada): 5.5% fat, 45.6% protein, 15.8% available carbohydrate, 21.7% fiber (13.8% insoluble and 7.9% soluble), 8.5% ash, and 3% moisture. To determine the RUT concentration, phenolic compounds were extracted from the freeze-dried ASP and separated by HPLC as previously described [29] with slight modification. RUT and quercetin in samples were identified by comparing their retention times and absorbance spectra with the standards, and quantified based on their peak areas at 360 nm. RUT and quercetin standards were purchased from Sigma (Sigma Chemicals, USA), and all samples were analyzed in triplicate. The concentration of RUT in our ASP was found to be 0.84/100 g and quercetin was not detected (Supporting Information Fig. 1). 2.2 Experimental diets Experimental diets were prepared by Harlan Laboratories, Inc. (Madison, WI, USA) and included an AIN-93G basal diet (BD) [38], BD supplemented with 2% freeze-dried ASP, and BD supplemented with 0.025% RUT (equivalent RUT level in the ASP diet; cat# A , VWR, ON, Canada). The 2% level of ASP supplementation was chosen from previously conducted pilot trials testing palatability of various supplementation levels, which showed that diet intake was not affected by 2% ASP supplementation, but 6% resulted in reduced dietary intake suggesting reduced palatability/taste aversion by mice. All experimental diets had equivalent macronutrient levels and were isocaloric (Table 1). Furthermore, the antioxidant potential of the experimental diets was measured by oxygen radical absorbance capacity (ORAC) and ferric reducing/antioxidant power (FRAP) assays as described previously [38]. As shown in Table 1, dietary FRAP and ORAC were both increased in the RUT and ASP diets compared to BD, with ASP having the highest antioxidant potential. ASP contains an array of antioxidant compounds

3 2398 K. A. Power et al. Mol. Nutr. Food Res. 2016, 60, Table 1. Composition and antioxidant potential of the experimental diets Ingredient (g/kg) BD RUT ASP RUT trihydrate ASP flour Casein L-Cystine Sucrose Corn starch Maltodextrin Corn oil Cellulose Mineral mix Vitamin mix Choline bitartrate TBHQ Protein (% kcal) CHO (% kcal) Fat (% kcal) ORAC ( mol TE/g DW) FRAP ( mol AAE/g DW) Antioxidant potential 2.20 ± 0.49 c 4.55 ± 0.09 b 9.00 ± 0.84 a ± 1.05 c ± 0.92 b ± 2.56 a Three experimental diets: AIN-93G BD, BD supplemented with 2% ASP, and BD supplemented with 0.025% RUT were prepared in accordance with the AIN-93G diet formulation. Differences in ORAC and FRAP levels between diets were determined by oneway ANOVA (SNK post hoc test); Within a row, means with different letters are significantly different (p < 0.05). Values are mean ± SEM. AAE, acetic acid equivalent; DW, dry weight; TBHQ, tertiary butylhydroquinone; TE, trolox equivalent. (e.g. phenolic acids, carotenoids) in addition to RUT [3], which may explain these ORAC and FRAP results. 2.3 Study design Five-week-old C57BL/6 male mice were purchased from Charles River (Portage, MI, USA), housed three to four per cage and acclimatized for 1 wk with ad libitum access to BD. Mice were divided into three treatment groups: BD (n = 35); ASP (n = 33); and RUT (n = 30), such that the mean body weight (BW) between each group did not differ (p > 0.05). Each group consumed their respective diet for 2 wks, during which, food intake and BW were recorded, and after which, fresh urine and feces were collected and stored at 80 C for later assessment of microbial community structure and activity. A subset of mice (n = 6/group selected from four to five cages) were euthanized to study the effects of diet on biomarkers of colon barrier integrity in healthy (pre-dss) mice. Colitis was induced in the remaining mice by administering 2% w/v DSS (MP Biomedicals, USA, MW ) in autoclaved water for 7 days, after which a subset of DSSexposed mice were sacrificed (n = 11 12/group) to evaluate dietary effects on biomarkers of acute colitis. The remaining mice were switched to DSS-free autoclaved drinking water for 5 days in order to initiate colitis recovery (n = 12 15/group). Food intake, water/dss consumption, and disease activity index (DAI; BW loss, stool consistency [diarrhea], and stool blood scores) as described previously [38]) were recorded daily during colitis induction and recovery phases of the experiment. A subset of BD-fed mice (n = 6) received DSS-free autoclaved water and served as healthy controls throughout the colitis experiment. Any mice experiencing a BW loss of >20% and/or having a DAI score of 3, for more than two consecutive days, were removed from the study and euthanized. All animal protocols were approved by the Animal Care and Use Committee (University of Guelph; AUP # 10R067). All mice (Pre-DSS and post-dss) were euthanized by cervical dislocation, blood was collected by cardiac puncture, and serum was stored at 80 C. The colon and cecum were removed intact, length and weight recorded, and luminal contents collected and stored at 80 C. After removing feces, colons were weighed, and 1 cm distal and proximal segments were formalin-fixed and the remainder was stored at 80 C. 2.4 Colon histology and immunohistochemistry In DSS-exposed mice, histological damage scores were assigned to paraffin-embedded hematoxylin & eosin (H&E) and Alcian Blue/Nuclear Fast red (AB)-stained distal colon crosssections using the scoring system previously described [38]. All images were captured using a BX51 microscope (Olympus America, Inc., USA) equipped with an Olympus DP72 Digital Camera System. From healthy mice (Pre-DSS), formalinfixed paraffin-embedded cross-sections (5 m) from the proximal and distal colon were stained with H&E or AB, to assess effects of experimental diets on baseline colon health parameters. For crypt height measurements, H&E stained proximal and distal segments were assessed using 20 fully elongated and intact crypt structures from five to six mice/group at 200x magnification using the National Institute of Health ImageJ software. Colon crypt mucus content was measured in AB-stained colon cross-sections using the National Institute of Health ImageJ software. Briefly, images captured at 200x magnification were first RGB color split and the red channel was used to quantify intensity of the AB-stained areas (upper threshold set at 75 for each image). AB-stained areas (lumen mucus excluded) were corrected for total crypt area and presented as mucus content/ m 2. Cell proliferation and apoptosis were measured by immunohistochemistry in distal colon cross-sections from mice following 7 days of DSS. Colon cross-sections (5 m) were deparaffinised and rehydrated, and antigens retrieved by microwave in citrate buffer (ph = 6). Endogenous peroxidase activity was quenched in 3% H 2 O 2 and nonspecific binding was blocked using 5% goat serum (#G9223) in TBST (Cell Signaling Technology) for 1 h at room temperature. Sections were incubated with primary antibody (1:500;

4 Mol. Nutr. Food Res. 2016, 60, Anti-Ki-67 polyclonal antibody [Millipore Ltd., #AB9260]; or 1:300 cleaved caspase-3 [Asp175] antibody [Cell Signaling Technology, MA, USA; # 9661]) overnight at 4 C inahumidified chamber, followed by incubation with SignalStain R Boost IHC Detection Reagent (HRP, Mouse) as indicated by the manufacturer (Cell Signaling Technology, #8114). Slides were then incubated DAB+ solution (DAKO Canada Inc., #K3468) for 1.5 min for Ki-67 and 7 min for cleaved caspase-3, counterstained with hematoxylin, dehydrated and mounted with Permount (Fisher Scientific, #SP-15). At least 1000 epithelial cells from two different sections/mouse (n = 8 9 mice/group) were counted and the % Ki-67+ stained cells are presented as proliferation index. The number of cleaved caspase-3+ cells within the epithelium were counted throughout the cross-section (n = 2 cross-sections from nine mice/group), corrected for epithelial area (mm 2 ), and expressed as apoptosis index. All images were assessed at 400x magnification using a BX51 microscope (Olympus America) equipped with an Olympus DP72 Digital Camera System. Bio-Rad protein assay (Bio-Rad). Myeloperoxidase (MPO) levels were measured by ELISA (Hycult Biotech, Plymouth Meeting, PA, USA) following the manufacturer s instructions with final values corrected for tissue weight. Activated (i.e. GTP-bound) RhoA and Rac1 were measured by G-LISA (Cytoskeleton Inc., Denver, CO, USA) using 12 g of protein/assay with absorbance read at 490 nm. 2.7 Cytokine analysis Serum levels of IL-10, IL-1, IL-6, IL-17A TNF-, andifn- were measured following acute colitis and the colitis recovery time points using the Bio-PlexPro Th17 6-plex assay (Bio-Rad) and Bio-Plex 200 System (Bio-Rad) following the manufacturer s instructions. 2.5 Colon mrna expression The mouse inflammatory response and autoimmunity RT 2 profiler PCR array (PAMM-077Z; Qiagen, Mississauga, ON, Canada) was used to determine the expression of 84 key genes involved in autoimmune and inflammatory immune responses in proximal-mid colon samples following acute colitis induction (i.e. mice sacrificed after 7 days of DSS exposure). Colon RNA was isolated by Trizol/chloroform extraction and purified using the RNeasy kit (Qiagen, Canada). Samples were DNase treated using the RNase-Free DNase kit (Qiagen, Canada) and RNA quality was assessed using Experion RNA Analysis kits (Bio-Rad, Canada). Total RNA (1 g) was converted to cdna using the RT2 First Strand kit (Qiagen) and quantitative real-time PCR was assessed on the ViiA7 Real-Time PCR System (Life Technologies). mrna expression was calculated using the 2 (40-CT) method and normalized to the expression of the housekeeping gene B2M (beta 2 microglobulin). Following colitis recovery (7 days DSS + 5 days H 2 O), colon RNA was isolated as described above and RNA (1 g) was converted to cdna using High Capacity cdna Reverse Transcription kit and real-time PCR analysis was performed using a 7900HT Fast Real Time PCR system (Applied Biosystems, Forest City, CA, USA). Gene expression was calculated as described in this section (above) and normalized to the expression level of the housekeeping gene (RPLP0). Primer sequences are reported in Supporting Information Table Colonic myeloperoxidase and RhoA and Rac1 activity assays Colon tissue was homogenized as described [38] and protein concentrations determined using the colorimetric 2.8 Colonic microbial activity Fecal and urinary RUT and quercetin analyses Freeze-dried fecal pellets ( g) were extracted with 500 L of methanol/h 2 O/formic acid = 80:19.9:0.1 v/v/v, and 5 L of 0.5 mg/ml naringenin (95% purity; Sigma Chemicals) was added to each tube as the internal standard (final concentration 5 g/ml). The samples were vortexed and placed on a rocker for 4 h; then centrifuged at g for 10 min. The supernatants were transferred into clean tubes and centrifuged one more time. Supernatants were then analyzed by HPLC as described previously [30]. Mouse urine was prepared according to the method described in Shimoi et al. [39] with some modifications. An internal standard of 10 ppm (final concentration) naringenin (95% purity; Sigma Chemicals) was added to each 150 L sample of mouse urine. The urine was acidified with the same volume of 1 M sodium acetate buffer (ph 4.5) and preincubated for 2 min at 37 C. Then, each sample was incubated for 20 min at 37 C with 100 Lof -glucuronidase/sulfatase ( units/ml and units/ml, respectively, dissolved in 50 mm sodium acetate buffer; Type H-1, partially purified powder from Sigma Chemicals). After incubation, 100 L of 0.01 M oxalic acid was added and the mixtures were centrifuged at 6800 rcf for 5 min. Supernatants were applied to pretreated Sep-Pak C18 cartridges, washed with 3 ml of 0.01 M oxalic acid and 3 ml of dh 2 O, and eluted with 3 ml of 100% MeOH. The eluate was evaporated with a Savant SpeedVac concentrator with solvent compatible Labconco CentriVap cold trap and vacuum pump. The dried residue was dissolved in 100 L 80% MeOH, centrifuged ( rcf), and the supernatant was then analyzed by HPLC as described previously [29].

5 2400 K. A. Power et al. Mol. Nutr. Food Res. 2016, 60, Fecal SCFA analyses SCFA concentrations were measured (acetic acid, propionic acid, butyric acid, and valeric acid) from fecal samples ( mg) collected from mice prior to DSS exposure (i.e. healthy mice) by GC as described previously [40]. 2.9 Colonic microbial community structure S rrna gene library preparation and sequencing Genomic DNA was extracted from freshly defecated and snap frozen feces (n = 8 mice/group) prior to DSS exposure (effect of diet alone) or from the colon contents collected from colitic mice sacrificed after 7 days DSS + 5 days dh 2 O (n = 8 9/group), and their corresponding aged-matched healthy controls (n = 6), using the QiaAmp DNA stool mini kit (Qiagen, Valencia, CA, USA). Sequencing libraries of the 16S V3-4 region were prepared according to the Illumina 16S Metagenomic Sequencing Library Preparation Guide Rev. B. Briefly, primers Bakt_341F (5 -CCTACGGGNGGCWGCAG-3 ) and Bakt_805R (5 - GACTACHVGGGTATCTAATCC-3 ) containing 5 Illumina overhang adapter sequences (5 TCGTCGGCAGCGTCAGA TGTGTATAAGAGACAG and 5 GTCTCGTGGGCTCGGAG ATGTGTATAAGAGACAG, respectively) were used to amplify an 550 bp fragment of the 16S rrna V3-4 region [41]. Each reaction contained 12.5 ng of template DNA, 200 nm each primer and 1X KAPA HiFi HotStart ReadyMix (VWR, Mississauga, ON, Canada) in a 25 L volume, which was amplified under the following conditions: 95 C for3min, 25 cycles of 95 C for 30 s, 55 C for 30 s, and 72 C for 30 s, followed by 72 C for 5 min. PCR products were purified with Ampure XP beads (Beckman Coulter, Mississauga, ON, Canada) and sequencing adapters containing 8-bp indices were added to the 3 and 5 ends by PCR using the Nextera XT Index kit (Illumina, San Diego, CA, USA) in a 50 L reaction containing 5 L PCR amplicon, 5 L each indexing primer, and 25 L 2x KAPA HiFi HotStart ReadyMix under the following conditions: 95 C for 3 min, eight cycles of 95 C for 30 s, 55 C for 30 s, and 72 C for 30 s, followed by 72 Cfor 5 min. Following purification with Ampure XP beads, the amplicons were quantified using the Quant-iT PicoGreen double-stranded DNA assay (Invitrogen/Life Technologies Inc., Burlington, ON, Canada) and equimolar ratios were pooled and combined with 5% equimolar PhiX DNA (Illumina) for sequencing on one flow-cell of a MiSeq instrument, using the MiSeq 600-cycle v3 kit (Illumina) Sequence processing and diversity analysis The resulting fastq files containing 300 bp dual-indexed paired-end reads were processed with Qiime version [42]. The paired-end reads were first joined by aligning overlapping sequences with fastq-join [43], and then quality filtered and demultiplexed in Qiime using default settings. Specifically, reads with less than three consecutive bases with a quality score (q) < 3 were removed and the remaining reads were then trimmed to the last base with q > 3. Reads < 75% of the original length, or with one or more ambiguous bases, were also removed. The remaining reads were clustered at 97% identity with UCLUST [44] and operational taxonomy units (OTUs) were picked using an open-reference approach with the GreenGenes database (gg_otus_13_8) [45] as a reference. OTUs representing <0.005% of the population were removed from the resulting OTU table and taxonomy was assigned by the Ribosomal Database Project classifier [46]. The sequences were aligned against the GreenGenes core set with PyNast [47] and a phylogenetic tree constructed with FastTree [48]. -Diversity metrics were then calculated by Qiime and UniFrac [49] and -diversity was assessed by PERMANOVA followed by principal coordinates analysis. Significant differences (p < 0.05; FDR < 0.1) in OTU counts between the different diets were determined by the Kruskal Wallis test followed by Dunn s multiple comparison test using the R package dunn.test version Statistical analyses All values are expressed as means ± SEM. Food and water intakes, BW, and DAI scores were analyzed by two-way analysis of variance (ANOVA) and all other data were analyzed by oneway ANOVA followed by either SNK or Dunn s post hoc tests using Sigma Plot 12.0 (Systat Software Inc., USA) with a significant difference of p < Kaplan Meier survival curves were generated using GraphPad Prism 5.03 (GraphPad Software Inc., USA) and analyzed by the Log-Rank (Mantel Cox) 2 test. Survival curves of the DSS-exposed treatment groups were considered significantly different from healthy controls if the p-value was less than the Bonferroni-corrected threshold (p < 0.017). 3 Results 3.1 Dietary ASP modulates the baseline colonic microbial environment in healthy mice Mice fed RUT or ASP diets had similar weight gain, diet intake, and liver and spleen weights indicating comparable RUT exposure between the experimental groups, as well as similar health status (Supporting Information Fig. 2). Although the primary objective of this study was to determine the therapeutic potential of ASP and RUT to attenuate DSS-induced mucosal damage and inflammation, mice were fed RUT and ASP diets for 2 wks prior to DSS exposure, therefore, we also studied the impact of the diets on the baseline colonic microenvironment. Proximal and distal colon crypt height and

6 Mol. Nutr. Food Res. 2016, 60, Figure 1. Effects of dietary RUT and ASP on colonic microbial activity and community structure in healthy mice. (A) Fecal samples were collected from mice (eight to nine per group) fed BD, BD % RUT, or BD + 2% ASP and analyzed for SCFA concentrations. Values are means ± SEM. Data were analyzed by one-way ANOVA followed by SNK post hoc test. Bars not sharing a lower case letter differ (p < 0.05). Urine (B) and fecal (C) samples were collected from mice fed BD, RUT, or ASP and quercetin and RUT levels were measured. Quercetin was not detected in BD urine samples or any fecal samples, and RUT was not detected in any urine samples or in the BD feces. Quercetin and RUT concentrations between ASP and RUT were compared by Student s t-test; **p = ; ***p < (D) Bacterial communities were clustered using principal coordinate analysis (PCoA) of unweighted UniFrac distance matrices. The first three principal components (PC1, PC2, and PC3) are plotted for each mouse. Percent of dataset variability explained by each principal component is shown in brackets in the axes titles. Each spot represents one mouse and each group is denoted by a different color (blue, BD; yellow, RUT; green, ASP). (E) Microbiota taxa composition at the genus level (legend includes taxa representing >1% total composition in at least one group). crypt mucus content did not differ between dietary groups (Supporting Information Fig. 3), indicating similar colon barrier architecture between dietary groups. On the other hand, diet-induced effects were observed on the colonic microbial activity and community structure. Total and individual fecal SCFA concentrations (acetic, butyric, and valeric acids) were increased in mice fed ASP, compared to BD and RUT, suggesting an increase in microbial activity in ASP-fed mice (Fig. 1). Enhanced microbial activity in ASP-fed mice was also indicated following measurement of fecal and urinary RUT and quercetin levels (Fig. 1). Quercetin is the microbialderived metabolite of RUT, and although RUT and ASP experimental diets contained the same level of RUT, ASP-fed mice had lower fecal RUT concentrations and higher urinary quercetin concentrations, suggesting a higher microbial conversion of RUT to quercetin following ASP consumption compared to mice consuming purified RUT. Of note, RUT was not detected in urine, and quercetin was not detected in fecal samples, from any dietary group. To determine if the baseline microbial community structure was modulated in the RUT and ASP dietary groups, we sequenced 16S rrna gene libraries prepared from total fecal DNA and performed diversity analyses. Following trimming, assembly, and quality filtering, sequences of 600 bp were subsampled from each 16S library and used to assess the microbial community structure. While the taxa richness was not modulated in the RUT and ASP groups ( -diversity; PD Whole tree: BD = ± 1.31; RUT = ± 1.40; ASP = ± 1.57; BD versus RUT: p = 0.29; BD versus ASP: p = 0.74), -diversity (unweighted Unifrac) was

7 2402 K. A. Power et al. Mol. Nutr. Food Res. 2016, 60, Table 2. Relative abundance of fecal microbial community members in healthy mice after RUT and ASP dietary intervention Taxonomic level Abundance (%) BD RUT ASP Bacteroidetes (p) ± ± ± 2.34 Prevotellaceae (f); Prevotella (g) 0.02 ± 0.01 b 0.02 ± 0.01 b 0.21 ± 0.04 a Bacteroidaceae (f); Bacteroides (g) ± 1.00 a 9.32 ± 0.67 b 6.50 ± 0.76 c Firmicutes (p) ± ± ± 2.67 Lachnospiraceae (f); Dorea (g) ± 0.01 b ± 0.01 a ± 0.01 b Verrucomicrobia (p) 3.28 ± ± ± 0.78 Deferribacteres (p) 1.31 ± ± ± 0.25 Proteobacteria (p) 0.31 ± ± ± 0.02 Alcaligenaceae (f); Sutterella (g) 0.13 ± 0.05 a 0.18 ± 0.04 a 0.03 ± 0.01 b Actinobacteria (p) 0.24 ± ± ± 0.03 Tenericutes (p) 0.17 ± ± ± 0.14 Relative abundance (mean ± SEM) of fecal bacterial taxa at the phylum level (p) and corresponding family (f) and genus (g) members that were significantly different between dietary groups (p < 0.05; 10% FDR); ASP, asparagus diets. significantly modulated by diet (p = ). Bacterial communities were grouped using principal coordinate analysis of the unweighted Unifrac distance matrix, which clustered according to diet (Fig. 1). The phylum level was dominated by members of Firmicutes and Bacteroidetes, while the remaining species were members of Verrucomicrobia, Deferribacteres, Proteobacteria, Actinobacteria,andTenericutes, the abundance of which did not differ between dietary groups (Table 2). However, shifts in genus members within these phyla were observed in the ASP and RUT groups. As shown in Fig. 1 and Table 2, within the Bacteroidetes phylum, genus member Prevotella increased while Bacteroides decreased, and the Proteobacteria genus member Sutterella decreased in the ASP group. Similarly, in the RUT group, a decrease in Bacteroides was observed, however this decrease was less than that seen in the ASP group. Furthermore, the Firmicutes genus member Dorea was decreased in the RUT group (Table 2). Collectively, these results indicate that the colonic microbial community structure and activity was modulated in the ASP group, while the RUT group had minimal shifts in the microbial community structure compared to BD. 3.2 Dietary RUT and ASP attenuates disease severity and enhance survival following DSS-induced colitis Colitis severity was assessed by monitoring the DAI (average of BW loss, stool blood, and stool consistency scores) during the 7-day colitis induction phase, as well as the subsequent 5-day colitis recovery phase. As shown in Fig. 2, DSS-induced BW loss started between DSS days 6 7, and continued throughout the recovery phase in the BD group, while the RUT group had significantly less BW loss compared to the BD group during the last 3 days of colitis recovery. This was also reflected in the DAI score, which shows that the BD group had the highest DAI scores throughout the recovery period (indicative of more severe clinical symptoms), while the RUT group had the lowest, suggesting an attenuation of colitis severity, in particular during the injury-repair phase of the experiment (Fig. 2). DAI in the ASP and BD groups did not differ during the colitis induction phase, but DAI in the ASP group was reduced at day 4 of the recovery phase indicating an attenuation of colitis severity; however, the effects of ASP were weaker than that induced by RUT. Water intake did not differ between groups either during the DSS colitis induction phase or the recovery phase, indicating equal DSS exposure across groups (Fig. 2). Diet intake did not differ between groups during the colitis induction or recovery phases, with the exception of day 4 of recovery where the RUT group consumed more diet compared to the BD group (Fig. 2), suggesting an improvement in health status in this group. During the recovery period, the survival rate was notably different between groups (Supporting Information Fig. 4). As mentioned in Section 2.3, mice were removed from the study and terminated if their BW loss exceeded 20% of their initial BW and if they experienced excessive diarrhea and stool blood loss for two consecutive days. Based on these parameters, 50% (6 out of 12) of the mice in the BD group remained in the study to the end of the recovery period, while 73% (11 out of 15) and 85% (11 out of 13), remained in the study from the ASP and RUT groups, respectively. This resulted in a significant difference in survival between the DSS group (p = ) and the healthy unchallenged controls (100% survival), while the survival of the ASP (p = 0.059) and RUT (p = 0.17) groups did not differ from that of healthy controls. 3.3 Dietary RUT attenuates DSS-induced colonic damage and inflammation Mice were sacrificed at two time points; (i) after 7 days of 2% DSS exposure (colitis induction), and (ii) after a subsequent

8 Mol. Nutr. Food Res. 2016, 60, A Diet Intake (g/mouse/day) % DSS dh 2 0 * C % BW Change % DSS dh 2 0 * * * BD ASP RUT B Water Intake (g/mouse/day) % DSS dh 2 0 D DAI % DSS dh 2 0 * * * * Figure 2. Diet and water intake, BW, and DAI during DSS-induced colitis induction and colitis recovery. Mice fed BD, BD % RUT, or BD + 2% ASP were exposed to 2% DSS in H 2 O for 7 days to induce colitis, followed by 5 days colitis recovery with DSS-free dh 2 O, and diet (A) and water intake (B) were measured daily, as well as % BW change (C) and DAI (D). All values are expressed as means ± SEM. Data were analyzed by two-way ANOVA (time and diet main effects). * = significant difference compared to BD group at the time point indicated; p < day colitis recovery period, and distal colon sections were analyzed for colitis severity (histological damage and crypt mucin content). In agreement with the DAI data (Fig. 2), there were no differences between groups in histological damage or mucin content scores after 7 days of DSS (Supporting Information Fig. 5). There were also no differences in colonic myeloperoxidase (MPO; biomarker of neutrophil infiltration), cell proliferation (Ki-67 labeling index), apoptosis (cleaved caspase-3; Supporting Information Fig. 5), or serum cytokine concentrations (IL-10, IL-1, IL-6, IL-17A TNF-,andIFN- ; Supporting Information Table 2), which were increased by DSS compared to healthy controls, but unaffected by diet. Conversely, during the colitis recovery phase, histological changes were observed between dietary groups. Five days following cessation of DSS, colon sections from mice in the BD + DSS group continued to demonstrate extensive ulcerations, immune cell infiltration, and crypt structures were absent in the majority of tissue cross-sections as shown in Fig. 3, whereas the RUT mice had less extensive histological damage and more importantly, showed evidence of crypt regeneration and goblet cell restitution. Histological damage and mucin content scores in the ASP group did not differ from the BD group. Clearly, the effects of RUT were most prominent, which was also demonstrated by colonic MPO concentrations, showing reductions in the RUT group compared to the BD and ASP groups (Fig. 3). On the other hand, similar to the effects observed after 7 days of DSS, serum cytokines remained elevated in DSS-exposed mice and were not impacted by diet during the colitis recovery phase (Supporting Information Table 2). Overall, in agreement with improved DAI and BW gain in the RUT group during the colitis recovery phase, we also observed a RUT-induced improvement in colon architecture and reduced colonic inflammation compared to BD-fed colitic mice. 3.4 Dietary RUT and ASP alter the colonic expression of mediators of immune cell chemotaxis, anti-inflammation, and injury repair after 7 days of DSS exposure To determine potential mechanisms through which ASP, and to a greater extent RUT, attenuated DSS-induced colitis (at the recovery time point: 7 days DSS + 5 days H 2 O), we assessed colonic mrna expression using the Mouse Inflammatory Response and Autoimmunity PCR arrays at the

9 2404 K. A. Power et al. Mol. Nutr. Food Res. 2016, 60, Healthy BD BD RUT ASP 7 days 2%DSS + 5 days H 2 0 BD Healthy BD RUT ASP Mucosal area (mm 2 ) 329± ± ± ±24.2 Histological Damage 0 537±40.4 a 337±32.3 b 478±32.5 a Mucin score 0.16±0.01 a 0.013±0.002 b 0.052±0.021 ab 0.015±0.003 b MPO (log U/g) 3.0±0.08 c 5.3±0.15 a 4.4±0.23 b 5.1±0.12 a Figure 3. Effects of dietary RUT and ASP on DSS-induced distal colon histological damage and MPO during colitis recovery. H&E or AB-stained distal colon cross-sections were analyzed for mucosal area, histological damage, and mucin content from mice given 2% DSS for 7 days followed by a 5-day recovery. Midcolon protein lysates were analyzed for MPO levels and presented as log MPO/g tissue. Data are means ± SEM. Data in rows not sharing a lower case letter differ between dietary groups (p < 0.05). Representative images from AB stain distal colon cross-sections (mucins stain turquoise) from DSS-exposed mice fed BD, RUT, and ASP and aged-matched healthy control (scale bar = 100 m). earlier time point: following 7 days of DSS; to gain insight into gene expression changes that might influence the recovery trajectory. As shown in Table 3, a number of genes involved in chemokine signaling chemokines (CCL5, 7, 12 and CXCL3, 9) were upregulated by both RUT and ASP compared to BD + DSS, whereas only RUT upregulated expression of CXCL5, 10 and CXCR2. In response to injury, chemokines play an important role in initiating recruitment of immune cells to areas of damage in order to establish colonic immune homeostasis and stimulate barrier repair [50, 51]. Two members of the IL-10 cytokine family, IL-10 and IL-22, were also upregulated by both RUT and ASP compared to BD + DSS. IL-10 plays an important role in immune homeostasis in order to protect the colonic epithelium from damage caused by inflammation [52 54], while IL-22 is better known for its role in initiating repair of damaged epithelium and stimulating secretion of mucin and antimicrobial peptides [5, 53 57]. Overall, during the colitis induction phase (7 days DSS), ASP and RUT enhanced the mrna expression of genes which play a role in mediating injury repair and reducing inflammation. 3.5 Dietary RUT and ASP alter the expression of mediators of colon barrier assembly and function, and colonic injury repair during the early stages of colitis recovery To support our findings that DSS-induced colonic damage and inflammation were attenuated by RUT during early colitis recovery (7 days DSS + 5 days dh 2 O; Fig. 3), we measured the expression of mediators involved in colon barrier defense, Table 3. Colon mrna expression (arbitrary units) after 7 days DSS exposure Gene BD BD + DSS RUT + DSS ASP + DSS CCL ± 0.17 c 5.16 ± 1.99 b ± 1.96 a ± 2.83 a CCL ± 0.20 c 8.35 ± 2.21 b ± 5.71 a ± 4.08 a CCL ± 0.16 c 8.20 ± 2.10 b 24.3 ± 3.06 a ± 7.50 a CXCL ± 0.07 c 0.69 ± 0.18 b 3.49 ± 1.42 a 3.12 ± 1.24 a CXCL ± 0.24 c ± 2.91 b ± 9.54 a ± 7.25 ab CXCL ± 0.06 b 0.54 ± 0.25 b 3.83 ± 1.68 a 4.11 ± 2.25 a CXCL ± 0.21 c 5.12 ± 1.88 b ± 2.19 a 6.62 ± 0.60 b CXCR ± c 0.61 ± 0.23 b 3.49 ± 1.92 a 0.15 ± 0.05 b IL ± 0.10 c 5.11 ± 1.45 b ± 6.63 a ± a IL ± 0.05 c 2.99 ± 1.16 b ± 6.96 a ± a Colon mrna expression in BD healthy controls (n = 4) and DSS-treated mice (n = 5 7/dietary group; 7 days DSS exposure). Data for each gene were normalized to the expression level of the housekeeping gene (B2M, beta 2 microglobulin), analyzed by one-way ANOVA followed by Least Squares Means post hoc test and only genes that were differentially expressed between DSS-treated dietary groups are shown. Values are means ± SEM and within each row, values not sharing a lower case letter differ (p < 0.05).

10 Mol. Nutr. Food Res. 2016, 60, Table 4. Colon mrna expression (arbitrary units) during colitis recovery (7 days DSS + 5 days H 2 O) Gene BD + DSS RUT + DSS ASP + DSS Mucus layer and goblet cell function Muc ± 1.29 b 17.6 ± 3.25 a 12.1 ± 1.85 a Muc ± 1.42 b 8.48 ± 1.77 a 2.31 ± 0.57 b Muc ± 0.88 b 4.56 ± 0.93 a 1.21 ± 0.26 b TFF ± 0.72 b 9.50 ± 1.30 a 8.01 ± 1.03 a Epithelial barrier assembly Occludin 5.57 ± 1.27 b ± 2.21 a 9.04 ± 1.52 a ZO ± 0.57 b 5.28 ± 0.44 a 3.74 ± 0.30 b Galectin ± 0.18 b 2.33 ± 0.26 a 3.45 ± 0.72 a Galectin ± 1.39 b ± 2.18 a ± 1.91 a Galectin ± 0.44 b 3.35 ± 0.51 a 2.90 ± 0.57 ab Wound healing Rho-A 2.61 ± 0.35 b 4.79 ± 1.10 a 5.00 ± 1.03 a Rac ± 2.64 b 14.1 ± 3.08 a 14.4 ± 3.17 a TGFβ ± 0.04 b 1.09 ± 0.32 a 0.84 ± 0.21 a IL ± 1.10 b 8.57 ± 1.95 b ± 3.09 a Reg ± 0.41 c 2.56 ± 0.47 b 6.08 ± 1.71 a TLR ± 0.20 c 3.42 ± 0.61 a 2.29 ± 0.26 b Values are means ± SEM (n = 9 11/dietary group). Data were analyzed by one-way ANOVA followed by Least Square Means post hoc test (p 0.05). Within each row, values not sharing a lower case letter differ. Data were normalized to RPLP0 expression and mrna expression was calculated according to the calculation: 2 (40-Ct). Data for Galectin-4 were log-transformed and the nontransformed means are shown. function, and integrity, as well as wound healing (Table 4). RUT increased Muc2 expression, the predominant secreted gel-forming mucin in the mucus layer [58], and mrna expression of Muc1 and Muc3, which are membrane-associated mucins that play a role in injury-repair process [59 61]. This is in agreement with our histological assessment which shows goblet cell restitution in DSS-treated mice fed RUT diet (Fig. 3). ASP also increased Muc1 mrna expression but had no effect on Muc2 or 3 expression compared to BD. On the other hand, trefoil factor 3 (TFF3) whichisagobletcellsecreted peptide known to be involved in mucosal injury repair [8, 62, 63], was upregulated by both RUT and ASP. Furthermore, genes encoding tight junction proteins, occludin and ZO-1, which assemble and maintain the structural integrity of the epithelial barrier [62], were increased by RUT, while ASP increased occludin mrna expression. RUT and ASP also enhanced mrna expression of Galectin-2 and 3, while RUT also increased Galectin-4 mrna expression. Galectins are -galactoside carbohydrate-binding lectins that mediate cell-matrix adhesion, promoting cell migration and initiating wound closure [64, 65]. Several unique and overlapping mediators of epithelial restitution and barrier assembly [5, 6], were also modulated by ASP and RUT. Members of the Rho subfamily of small GTPases, RhoA, and Rac1, which are important in cell migration, actin cytoskeleton reorganization, and formation of cellular junctions [51, 65 68], were upregulated by RUT and ASP. These findings were confirmed by increased colonic protein levels of GTP-bound (i.e. activated) RhoA and Rac1 in RUT- and ASP-fed mice compared to BD (Fig. 4). TGF-β1 mrna expression was upregulated by both RUT and ASP and plays a central or driving role in several mechanisms of epithelial restitution and wound closure following injury [5, 8, 51, 62, 69, 70]. Similar to the effect observed after 7 days of DSS, IL22 mrna expression continued to be elevated in mice fed ASP only. In connection to this, IL-22 is a strong regulator of Reg3 [55, 71], a Gram-positive antimicrobial peptide that plays a key role in the host microbial defense response and shaping the microbial community [72, 73]. ASP and RUT both significantly enhanced Reg3 mrna expression, although ASP induced greater expression levels than RUT. Furthermore, Toll-like receptor 9 (TLR9), an innate immune receptor which contributes to colonic immune homeostasis and initiates wound repair [51, 74], was significantly upregulated by RUT and ASP. In summary, these results indicate that both RUT and ASP modulate an array of genes involved in colonic barrier assembly, defense, and wound repair which may be involved in attenuating clinical symptoms, increasing survival, and promoting mucosal repair following DSS-induced colitis. 3.6 Dietary RUT and ASP modulate the DSS-altered colonic microbial community structure Colonic microbial dysbiosis has been reported following DSS exposure in C57Bl/6 mice, including shifts in the abundance in members of the Bacteroidales, Clostridiales, Enterobacteriales, Deferribacterales, andverrucomicrobiales [75 78]; changes which may mediate adverse microbial host interactions, thereby driving inflammatory responses. Therefore, we investigated whether consumption of RUT or ASP altered the microbial community structure of DSS-exposed mice, which may have played a role in reducing colitis severity. Following trimming, assembly, and quality filtering, sequences of 600 bp were subsampled from each 16S library and used to assess the microbial community structure. To determine if taxa richness was altered by our treatment groups (BD healthy controls and BD-, ASP-, and RUT-fed mice following 7 days DSS + 5 days H 2 O recovery), we performed -diversity analyses using PD Whole tree method (Fig. 5) which demonstrated significant differences between groups: BD = ± 1.46; BD + DSS = ± 2.44; RUT + DSS = ± 1.44; ASP + DSS = ± 2.43; BD versus BD + DSS: p = 0.006; BD versus RUT + DSS: p = 0.006; BD versus ASP + DSS: p = 0.006; BD + DSS versus RUT + DSS: p = 0.006; BD + DSS versus ASP + DSS: p = 0.06). These results indicate that while all DSS-exposed groups reduced taxa richness, in colitic mice fed RUT, the taxa richness was greater than those fed the BD. A similar trend was seen with ASP-fed colitic mice but this did not reach significance. Further, colonic microbial community diversity was significantly different between all groups ( -diversity [unweighted Unifrac; p = ]), as well as between

11 2406 K. A. Power et al. Mol. Nutr. Food Res. 2016, 60, A GTP-bound RhoA a a b BD RUT ASP B GTP-bound Rac1 1.5 a 1.0 a 0.5 b 0.0 BD RUT ASP 7 d 2% DSS + 5 d H d 2% DSS + 5 d H 2 0 Figure 4. The effects of RUT and ASP on colon RhoA and Rac1 activation during colitis recovery. Colon tissue samples from five to seven mice/group treated with DSS for 7 days + 5 days H 2 O (colitis recovery) were analyzed for activated (i.e. GTP-bound) protein expression of RhoA (A) and Rac1 (B). Values are means ± SEM and bars not sharing a lower case letter differ between dietary groups (p < 0.05). DSS-exposed groups (BD + DSS versus RUT + DSS, p = ; BD + DSS versus ASP + DSS, p = ). Bacterial communities were grouped using principal coordinate analysis of the unweighted Unifrac distance matrix, which clustered according to DSS exposure, as well as by diet (Fig. 5). Significant shifts in the colonic microbial community were seen in mice exposed to 2% DSS for 7 days followed by an H 2 O recovery period, compared to aged-matched non-dss treated mice. At the Phylum level, BD + DSS mice had reduced Bacteroidetes, and increased Deferribacteres and Proteobacteria, the latter of which was the dominate phylum in the BD + DSS microbial community compared to healthy controls (Fig. 5; Table 5). RUT + DSS and ASP + DSS groups did not differ significantly from BD + DSS group in these Phyla abundances; however of note, the mean abundance of Proteobacteria phylum was 50 and 40% reduced in the RUT and ASP groups, respectively, and the increased abundance of Deferribacteres by DSS, was attenuated in the ASP + DSS (OTU assigned to Mucispirillum schaedleri; Table 5 and Supporting Information Table 3). Within Bacteroidetes, several genus members were altered in the BD + DSS group including increased Bacteroides and reduced unassigned Rikenellaceae, unassigned S24-7, Prevotella, and unassigned Bacteroidales compared to BD healthy controls (Fig. 5; Supporting Information Table 3). Interestingly, the DSS-induced increase in Bacteroides abundance was attenuated in the RUT and ASP groups, such that the abundance was not different from BD healthy controls. Similarly, the DSS-induced decrease in Rikenellaceae was attenuated in the RUT group while the decrease in Prevotella was attenuated in the ASP group (Supporting Information Table 3). Numerous genus members within the Firmicutes phylum were also modulated in the DSS group compared to BD healthy controls, including increases in unassigned Enterococcaceae, unassigned Peptostreptococcaceae, and Anaerotruncus, and decreases in unassigned Mogibacteriaceae, unassigned Lachnospiraceae, rc4-4, unassigned Peptococcaceae, Ruminococcus (Ruminococcaceae family), and unassigned Clostridiales. Attenuation of DSS-induced changes in unassigned Lachnospiraceae, Ruminococcus, unassigned Clostridiales, and unassigned Enterococcaceae, were observed in the RUT group, and, to a lesser extent, the ASP group. Interestingly, the abundance of Ruminococcus (Lachnospiraceae family), Oscillospira, Lactobacillus, Streptococcus, Coprococcus, and unassigned Erysipelotrichaceae were not significantly modified in the BD + DSS group compared to healthy controls, but were increased in the RUT group, and to a lesser extent, the ASP group. Within the Proteobacteria phylum, reductions in unassigned RF32 and increases in Sutterella, unassigned Enterobacteriacea, and Enterobacteriacea Proteus genus members were found in the BD + DSS group compared to BD healthy controls. Of note, the increases in unassigned Enterobacteriacea, and Enterobacteriacea Proteus genus members were not as pronounced in the RUT and ASP groups, compared to BD health controls, however, they did not differ significantly from BD + DSS. Finally, within the Actinobacteria Phylum, the abundance of Bifidobacterium was reduced in the BD + DSS group, which was attenuated in the RUT group. 4 Discussion ASP, a commonly consumed food worldwide, contains an array of human health promoting components including antioxidant flavonoids (e.g. RUT), micronutrients, carotenoids, and dietary fiber [29,33,34], yet research demonstrating health effects of ASP are lacking. This study has demonstrated for the first time, the potential for cooked ASP spears to mitigate colonic mucosal damage and promote mucosal healing after exposure to an inflammatory insult; an effect which is likely a result of its high RUT content. The beneficial effects of RUT on colonic inflammation have been demonstrated previously [19, 20, 79 83], and is shown to be due in part to the anti-inflammatory, antioxidant, and colon barrier protective effects of its microbial-derived metabolite, quercetin [14 17]. In this study, we have demonstrated that the beneficial effects observed with purified dietary RUT can be partly obtained through consumption of a cooked RUT-rich food which enhances the physiological and clinical relevance of our findings. During colitis induction (7 days of 2% DSS), we observed an increase in DAI and BW loss (Fig. 2), mucosal damage, loss of colonic mucin content, increased colonic MPO (biomarker

12 Mol. Nutr. Food Res. 2016, 60, Figure 5. Effects of dietary RUT and ASP on DSS-induced microbial dysbiosis (7 days DSS + 5 days H 2 O). (A) Rarefaction curves comparing the -diversity (Phylogenetic diversity whole tree metric) of colon content microbiota from healthy BD-fed mice (blue), and 2% DSS-exposed mice fed BD (orange), RUT (green), or ASP (red) diets. (B) Bacterial communities were clustered using principal coordinate analysis (PCoA) of unweighted UniFrac distance matrices. The first three principal coordinates (PC1, PC2, and PC3) are plotted for each mouse. Percent of dataset variability explained by each principal coordinate is shown in brackets in the axes titles. Each spot represents one mouse and each group is denoted by a different color (blue, BD; red, DSS; yellow, RUT; green, ASP). Microbiota taxa composition at the (C) phylum and (D) genus levels. The genus (g) is given where available, otherwise the family (f) or order (o) is given. Legend includes taxa representing >1% total composition in at least one group. of neutrophil infiltration; Supporting Information Fig. 5), and increased serum inflammatory cytokines (Supporting Information Table 2), all of which were not attenuated by the RUT or ASP diets. On the other hand, at this time point both RUT and ASP enhanced colonic expression of genes involved in immune cell chemotaxis, anti-inflammation, and injury repair (Table 3) which indicates a diet-induced change in the colonic microenvironment which may alter the tissues responsiveness and severity of DSS-induced colonic damage. This was evident in the early stages of colitis recovery (5 days after cessation of DSS), where RUT- and ASP-fed mice exhibited improvements in various parameters of colitis severity compared to DSS controls, including an increased proportion of mouse survival (Supporting Information Fig. 4) and reduced DAI scores (Fig. 2). Furthermore, dietary RUT was more potent in stimulating mucosal repair as indicated histologically by the reappearance of crypt structures and goblet cells; important components of the colonic epithelial barrier (Fig. 3). Although there was evidence of crypt structures in colons of ASP-fed mice, the results were not significantly different from BD-fed DSS controls (Fig. 3). In support of this, both RUT and ASP enhanced expression of genes involved in colonic epithelial barrier assembly and function, and gene and protein mediators involved in mucosal injury repair (Table 4; Fig. 4). Of particular interest, IL-22 is a cytokine recently identified as an important mediator of wound healing, in part through its ability to stimulate Muc expression (required to re-establish the protective mucus barrier) and expression of antimicrobial peptides (to help control microbial interaction with the colonic mucosa) [53 57]. Both RUT and ASP upregulated IL22 mrna (Tables 3 and 4), as well as expression