Animal Model

Wild-type mice on a 129S1/SvimJ background were raised at the University of Alberta and housed under conventional conditions. At 6–8 weeks of age, female mice were placed on either a chow diet (CH) (LabDiet 5001) or a high sugar diet (HS) (50% Sucrose; Harlan Teklad AIN76A) for a period of two days. Fiber content was 5% (cellulose) and 5.3% (crude fiber) in the HS and chow diets respectively. Mice from the same litter were randomized to separate cages and groups and each experiment was repeated twice. After two days on the different diets, mice were treated with a low concentration of dextran sodium sulfate (DSS 3%; MW 35–45,000 kDa; MP Biomedicals) added to their drinking water (Day 0). After five days, DSS was removed and replaced with regular drinking water. Mice were sacrificed on Day 10 and tissues collected and snap frozen for further study (Fig. 1). Body weight, stool consistency, and fecal occult blood (FOB) were measured daily. FOB positivity was determined using the Hemoccult (Beckman Coulter) test. A disease activity index (DAI) was used that included percentage weight loss, stool consistency, and blood in stool. Each parameter was measured on a scale of 0–4 for a total DAI ranging from 0–12. The weight and length of colon and the weight of the cecum was measured at the time of sacrifice. To determine the role of acetate, a parallel group of mice received acetate (NaAc:300 mM) in the drinking water throughout the experiment beginning at day -2 when the mice were switched to the different diets. All experiments were repeated in cohorts of mice from different litters. Animal use protocols were approved by the animal care committee at the University of Alberta and all experiments were carried out in accordance with the relevant guidelines and regulations.

Figure 1 Experimental Design. Female mice on a 129S1/SvimJ background were raised on chow until 6–8 wks of age and then placed on either a chow diet (CH) (LabDiet 5001) or a high- sugar diet (HS) (50% Sucrose; Harlan Teklad AIN76A) (Day -2). After two days on the diet, mice were treated with dextran sodium sulfate (DSS 3%; MW 35–45,000 kDa; MP Biomedicals) added to their drinking water (Day 0). After five days, DSS was removed and replaced with regular drinking water for a further 5 days. Mice were sacrificed on the tenth day and tissues collected and snap frozen for further study. Weight and stools were collected on day -3 prior to the diet switch. The effect of two days on the diet (Day 0) on stool and cecal microbiome, colonic cytokine concentrations, cecal SCFA levels, gut permeability, and serum LPS was assessed. Body weight, stool consistency, and fecal occult blood (FOB) were measured daily from day 0 (start of DSS) to day 10. A parallel group of mice received acetate (NaAc:300 mM) in the drinking water throughout the experiment beginning at day -2 when the mice were switched to the different diets. Full size image

Histological injury

Colons were flushed with phosphate-buffered saline and immediately fixed in neutral buffered formalin (10%vol:vol). The fixed samples were processed with the use of standard paraffin-embedded histologic methods and hematoxylin and eosin staining. Disease scoring was based on a scoring method that included measurement of epithelial hyperplasia (0–3), enterocyte injury (0–3), and the presence of lymphocytes and neutrophils (0–4) in the lamina propria24. Total histologic score was calculated as the sum of the individual variables and had a maximum score of 10.

Preparation of bone-marrow derived macrophages

Wild-type mice were placed on chow (n = 4) or high sugar diet (n = 4) for two days. At sacrifice, femurs and tibias from each mouse were removed and cleaned, ends of bones snipped and bone marrow flushed with 10 mL of magnesium and calcium free (MCF) phosphate buffered saline (PBS), then pooled for each mouse25. Pooled cells were centrifuged at 1000 rpm for 5 minutes and supernatant removed. The pellet was reconstituted in 2 mL of macrophage complete media. Bone marrow cells were enumerated using a Coulter counter and 2 × 106 cells plated in 10 mL of macrophage complete media (MCM) which included Dulbecco’s modified eagle media (DMEM/F12), 10% heat inactivated fetal bovine serum (FBS), penicillin (100 U/ml), L-glutamine (10 mM), 100 ug/ml streptomycin and 20% macrophage colony-stimulating factor (M-CSF) obtained from L929 cell line. Cells were incubated at 37 °C and 5% CO 2 for 7 days. On day 7, cells were washed with warm M-CSF PBS and then scraped and pooled into a 50 mL conical tube. Five mL of cold DMEM/F12-10 media was added to each conical and centrifuged for 10 minutes at 400 g and 4 °C. Cells were counted and 1 × 105 cells plated into each well. Cells ± lipopolysaccharide (LPS;10 /µg/ml) were incubated at 37 °C and 5% CO 2 incubator for 24 hours.

Measurement of tissue cytokines

Snap frozen colonic tissue was homogenized in PBS containing 0.05% Tween 20. Homogenates were centrifuged at 9600 × g for 10 minutes. IFN-γ, IL-1β, IL-10, IL-12 p70, IL-2, IL-4, IL-5, KC/GRO (keratinocyte chemoattractant/human growth-regulated oncogene), IL-6, and TNF-α were evaluated using the Proinflammatory Panel 1 V-PLEX Mouse Kit (Meso Scale Discovery, Rockville, MD) as per manufacturer’s protocol. TGF-β was measured using ELISA duo set (R&D Systems, Inc., Minneapolis, MN). Cytokine levels were corrected for tissue weight.

Microbiome analysis

Cecums were collected at sacrifice and immediately snap frozen in liquid nitrogen and stored at −80 °C until processed for either microbial composition or SCFA. Freshly voided stools were collected and frozen. For sequencing, DNA was extracted using AquaStool and cleaned using ethanol precipitation. Microbial composition was assessed using Illumina’s established 16S rRNA amplicon sequencing method and the MiSeq sequencing platform. Briefly, a segment of the V3 and V4 region of the 16S gene was amplified with gene specific primers (aligning to 341 bp and 805 bp in the gene) that also included an adapter sequence overhang: Bact_16s_ILL1_341mF 5-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3, Bact_16s_ILL1_805mR 5- GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3. This PCR reaction was cycled 25 times and the resulting reaction purified using bead-based clean-up followed by an 8 cycle PCR reaction using Illumina’s proprietary bar-coding primers that also align to the adapter sequence. After a second clean-up the bar-coded libraries were diluted, denatured, pooled and run using a V3 300 bp reagent cartridge on the MiSeq system. Bacterial composition was estimated using Quantitative Insights into Microbial Ecology (QIIME 1.9.1) pipelines26. QIIME was used to de-multiplex the barcoded reads and perform chimera filtering. Filtered sequence reads were grouped into OTUs at a sequence similarity level of 97%, which approximates species-level phylotypes. Taxonomy of the OTUs was assigned and sequences were aligned with RDP classifier and Pynast27. Alpha diversities of each microbial community were calculated using the Shannon diversity metric.

Assessment of gut permeability

At sacrifice, a 10 cm portion of small intestine was excised distally from 10 cm below the ligament of Treitz and flushed with ice-cold phosphate-buffered saline (PBS). The intestinal segment was ligated at one end and a tube was inserted to add 400 µl of fluorescein isothiocyanate (FITC)/rhodamine dextran (40 µg/mL FITC-labelled 4 kDa dextran, and 40 µg/mL rhodamine labelled 70 kDa dextran in Hank’s Buffered Salt Solution). A ligature was applied as the tube was removed. The length and width of the segment was measured and then submerged in 10 mL of HBSS maintained at 37 °C and percolated with carbogen (95% O 2 , 5% CO 2 ). Samples (100 µl) were collected from surrounding media at baseline and every ten minutes for thirty minutes. Fluorescence was measured using a SpectraMax M3 spectrophotometer (Molecular Devices, USA) fluorescence at 438ex/544em for FITC and once at 520ex/590em for rhodamine. A leakage ratio was calculated for each time point by normalizing dextran (µg) to the gut volume then dividing the 4 kDa dextran by 70 kDa dextran to obtain a ratio. Serum LPS measurement was carried out using the Pyrochrome Limulus Amoebocyte Lysate (LAL) assay according to manufacturer’s instructions (Associates of Cape Cod Incorporated).

Measurements of short-chain fatty acids (SCFA)

The concentrations of SCFA in cecal contents were determined using gas chromatography. Stool (0.2 g) was homogenized in 800 uL of 0.1 N hydrochloric acid. Phosphoric acid (200 µl of 25%) was then added and the sample centrifiuged at 3 000 × g for ten minutes. Supernatant was added to internal standard solution (150 mg of 4-methyl-valeric acid, S381810, Sigma-Aldrich) and 5% phosphoric acid in a glass chromatography tube, mixed well, and kept at room temperature for 30 min. The supernatant was analyzed for SCFA using a Varian model 3400 Gas Chromatograph (Varian, Walnut Creek, CA) with a Stabilwax-DA column (30-m × 0.25-mm i.d.; Restek, Bellefonte, PA). A flame-ionization detector was used with an injector temperature of 170 °C and a detector temperature of 190 °C.

Statistical analysis

All data is presented as the mean ± SEM. Statistical analysis was performed using STATA v13.1. Differences between group means were evaluated by using one-way analysis of variance with Tukey post hoc test to correct for multiple comparisons. White’s nonparametric t-test was used to compare microbial communities between groups, with Benjamini-Hochberg test applied to assess for false discovery rate. Principal component analysis (PCA) plots of bacterial populations were created using Metaboanalyst 3.0. Significance was defined as p < 0.05.