Gut taste receptor type 1 member 3 is an intrinsic regulator of Western diet-induced intestinal inflammation

Study design

The objective of the study was to evaluate the influence of long-term WD intake on intestinal inflammation and investigate possible mechanisms by which WD intake could affect IBD development. To this end, mice were fed normal diet (ND) or WD for 10 weeks, and bowel inflammation was evaluated through pathohistological and infiltrated inflammatory cell assessments. To identify the mechanisms by which intestinal inflammation is prompted by WD, RNA-seq was performed on the inflamed intestinal tissues. Because the results revealed increased expression of the nutrient-sensing taste receptor TAS1R3 in inflamed bowel tissues, we hypothesized that nutrient-induced TAS1R3 modulation is central to regulating intestinal inflammation. We first determined the dietary ligand(s) responsible for strongly activating TAS1R3 using in vitro assessment of TAS1R3-expressing enteroendocrine cells (EECs) to confirm whether TAS1R3 could trigger intestinal inflammation, and then assessed the downstream molecular changes. To understand the role of TAS1R3 in WD-induced intestinal inflammation, we investigated changes in intestinal gene expression profiles, inflammatory cell infiltration in intestinal tissues, and the gut microbiome of Tas1r3-deficient and littermate wild-type mice fed WD. Finally, we confirmed the expression of TAS1R3 and downstream players in intestinal biopsies of patients with IBD ex vivo to demonstrate their relevance to disease. All mice were assigned randomly to treatment groups, and animals demonstrating sickness or severe stress were euthanized and excluded. Otherwise, all data were included in the study. The experimental protocol was approved by the Seoul National University Institutional Animal Care and Usage Committee (approval SNU-181001-2).

Mice

C57BL/6 (JAX 000664) and Tas1r3tm1Csz (JAX 013066) mice were obtained from the Jackson Laboratory (West Grove, PA, USA). Tas1r3-knockout (Tas1r3−/−) mice were backcrossed to C57BL/6 mice for at least seven generations. The animals were genotyped using standard PCR. The mice (5–6 weeks old; 23–25 g) were randomly assigned to groups. Age- and weight-matched male and female littermates were used as controls. Tas1r3−/− and wild-type littermate control (Tas1r3+/+) mice were housed under constant temperature (23 ± 2 °C) and humidity (55–60%) conditions in a specific pathogen-free animal facility. All mice were housed in the same room to minimize environmental effects.

Diets

To imitate the human WD, characterized by a high-fat content and sugary drinks, mice were fed high-fat diet and sucrose solution. The high-fat diet (60%; D12492) and matching normal diet (ND; D12450J) pellets were purchased from Research Diets (New Brunswick, NJ, USA). The sugar concentration in the sucrose solution was determined, as described in a previous rodent study [22]. Two independent series of experiments were conducted, and mice were randomly assigned as follows:

In experiment 1, two groups of mice were used: (i) ND—mice administered ND with plain water (control group; n = 10 mice) and (ii) WD—mice administered the high-fat diet with sucrose solution (n = 10 mice).

In experiment 2, four groups of mice were used: (i) Tas1r3+/+ mice who were administered ND (n = 10 mice), (ii) Tas1r3−/− mice who were administered ND (n = 10 mice), (iii) Tas1r3+/+ mice who were administered WD (n = 10 mice), and (iv) Tas1r3−/− mice who were administered WD (n = 10 mice). Food and water were supplied ad libitum for 10 weeks. The body weight, as well as food and water intake, were monitored weekly.

All mice used in the experiments were randomly assigned to each group, and random numbers generated using SPSS software (version 18.0; SPSS Inc., Chicago, IL, USA) were assigned with a unique code linking to the individual animal.

Histology

After 10 weeks of diet induction or 7 days of 2% dextran sulfate sodium (molecular weight: 36,000–50,000 kDa; Cat# 02160110-CF; MP Biomedicals, Santa Ana, CA, USA) induction, all mice were anesthetized and euthanized by intraperitoneal injection of 20% urethane (U2500; Sigma-Aldrich, St. Louis, MO, USA), and the entire intestine was removed and opened longitudinally. Small and large intestinal tissues were fixed overnight in 10% formalin and embedded in paraffin. The tissue blocks were cut into Sects. (4–6 μm thick) that were mounted on glass slides, stained with hematoxylin and eosin, and photographed under a Nikon Eclipse TE2000-U microscope (Tokyo, Japan) equipped with a QImaging digital camera (Teledyne QImaging, Surrey, BC, Canada). H&E-stained intestinal sections were coded for blind microscopic assessment of inflammation. Sections coded for assessment of inflammation were scored by two blinded investigators as described previously (n = 10 mice/group) [23].

Immunohistochemistry and immunofluorescence

Serial sections (4–6 μm thick) were prepared from formalin-fixed paraffin-embedded specimens and mounted on silane-coated slides (Dako Japan Co., Ltd., Kyoto, Japan). Tissues were deparaffinized and rehydrated through a graded xylene and alcohol series, placed in citrate-buffered solution (pH 6.0) (C9999; Sigma-Aldrich), and heated in a microwave oven to 100 °C for 20 min for antigen retrieval. After washing with phosphate-buffered saline, the slides were incubated with phosphate-buffered saline-Tween with 1% bovine serum albumin (Sigma-Aldrich) for 1 h, incubated overnight at 4 °C with anti-TAS1R3 antibodies (1:100) (Cat#OSR00184W, RRID: AB_2271552; Invitrogen, Carlsbad, CA, USA) or pan-leukocytes (CD45; 1:100) (Cat#ab10558, RRID: AB_442810; Abcam, Cambridge, UK), and developed using a Mouse and Rabbit-Specific HRP/DAB Detection IHC Kit (Cat#ab64264; Abcam), according to the manufacturer’s instructions. Primary antibodies used for small or large intestine immunofluorescence staining included mouse anti-CD4 (Cat#14–0041-86, RRID: AB_467065; eBioscience, San Diego, CA, USA), rat anti-CD8 (Cat#ab22378, RRID: AB_447033; Abcam), and mouse anti-CD11b (Cat#14–0112-82, RRID: AB_467108; eBioscience), to label different types of immune cells in intestinal tissues. Secondary and secondary-conjugated primary antibodies used for small intestine and colon immunofluorescence staining included Alexa Fluor 488 anti-mouse/human CD11b (Cat#101219, RRID: AB_493545; BioLegend, San Diego, CA, USA), Alexa Fluor 594 donkey anti-rabbit (Cat#A21207, RRID: AB_141637; Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor 488 donkey anti-mouse (Cat#A21202, RRID: AB_141607; Thermo Fisher Scientific), and Alexa Fluor 594 donkey anti-rat (Cat#A21209, RRID: AB_2535795; Thermo Fisher Scientific). Immunoreactivity was visualized using a confocal microscope system (LSM 510; Carl Zeiss, Oberkochen, Germany). Images were captured at 20X magnification using an EVOS FL Cell Imaging System (Thermo Fisher Scientific) and a BZ-X710 All-in-One Fluorescence Microscope (Keyence, Osaka, Japan), and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Tissue-infiltrated cells are expressed as the ratio of the stained area to the total area of the measured tissue region (n = 10 mice/group).

Enterocyte cell culture and GLP-1 secretion assay

Human enteroendocrine NCI-H716 cells (CCL-251; American Type Culture Collection, Manassas, VA, USA) were maintained in suspension culture at 37 °C and 5% CO2, according to the supplier’s protocol. The culture medium was RPMI-1640 (Invitrogen) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin. Two days prior to the experiments, 1 × 106 cells were seeded in 24-well culture plates precoated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA), as described previously [24]. On the day of the experiments, the supernatants were replaced with medium containing 10 mM glucose (Cat# G7021), 10 mM fructose (Cat# F3510), 10 mM sucrose (glucose + fructose), and/or 10 μM palmitate (Cat# P5585). In the TAS1R3 antagonist experiment, NCI-H716 cells were pretreated with the TAS1R3 antagonist, lactisole (2.5 mM) (Cat# M6546) for 30 min and stimulated with medium containing fructose (10 mM), glucose (10 mM), and palmitate (10 μM) for 12 h. In the PPARγ antagonist experiment, NCI-H716 cells were transfected with TAS1R3 siRNA or control siRNA for 48 h, and then stimulated with the PPARγ antagonist, GW9662 (Cat# M6191) (10 μM), in the presence of fructose (10 mM), glucose (10 mM), and palmitate (10 μM) for 12 h. The cells were incubated for 2, 4, 12, and 24 h at 37 °C with or without different test agents and inhibitors. Glucose, fructose, palmitate, lactisole, and GW9662 were purchased from Sigma-Aldrich. Following incubation, the medium was collected, centrifuged at 1,000 × g for 10 min at 4 °C, to remove any floating cells, and frozen at -20 °C for subsequent biochemical analysis. GLP-1 was measured using a commercial GLP-1 (active) Enzyme-Linked Immunosorbent Assay Kit (Cat#EGLP-35 K; Millipore, Billerica, MA, USA), according to the manufacturer’s protocol (n = 3/group).

siRNA knockdown

siRNA duplexes for TAS1R3 were synthesized by Bioneer (Daejeon, South Korea). Scrambled negative control siRNA was also purchased from Bioneer. For knockdown experiments, 5 × 105 endocrine differentiated NCI-H716 cells were plated into 6-well plates and cultured for 48 h. TAS1R3 (10 nM) or control (10 nM) siRNA was transfected into the cells using Lipofectamine RNAiMAX Reagent (Invitrogen). After 48 h of transfection, the cells were induced with medium containing 10 mM glucose, 10 mM fructose, and 10 μM palmitate for 12 h (n = 3/group). The target sequences of the siRNA are listed in Additional file 1 (Table S1).

Enzyme-linked immunosorbent assay

Enzyme-Linked Immunosorbent Assay (ELISA) Kits specific for human TNF-α (Cat# ab181421) and IL-8 (Cat# ab46032) were purchased from Abcam. TNF-α and IL-8 in the cell-conditioned medium were quantified using Enzyme-Linked Immunosorbent Assay, according to the manufacturer’s instructions. Briefly, the samples were diluted with assay buffer and added to microwells precoated with anti-human TNF-α or IL-8 antibodies, followed by incubation with an antibody cocktail and 3,3′,5,5′-tetramethylbenzidine substrate. Conjugated enzyme activity was detected by measuring absorbance at 450 nm (n = 3/group).

RNA isolation and quantitative reverse transcription-PCR

Total RNA was extracted from the ileum tissue of WD- or DSS-induced wild-type and knockout mice and NCI-H716 cells. After harvest, the ileal and cell extracts were immediately snap-frozen by immersion in liquid nitrogen and stored at –80 °C until RNA extraction. Total RNA was isolated using RNAqueous (Cat# AM1914; Ambion, Austin, TX, USA), according to the manufacturer’s instructions. RNA extraction involved a DNase treatment step. RNA was quantified using a NanoDrop 2000/2000c Spectrophotometer (Thermo Fisher Scientific), and 1 μg of RNA from each sample was used for cDNA synthesis (Cat# 11917010; Invitrogen). Quantitative reverse transcription-PCR was performed using StepOnePlus (Real-time PCR System; Applied Biosystems, Foster City, CA, USA) and SYBRGreen (Cat# 4367659; Applied Biosystems). The relative quantification of gene expression was performed using the 2−△△Ct method. The cycle threshold values of the genes of interest were normalized to that of Gapdh (n = 3–10 mice/group). The primers used for quantitative reverse-transcription-PCR are listed in Additional file 1 (Table S2).

RNA-sequencing (RNA-seq)

Isolated RNA (1000 ng) was used as input for library generation. Intact mRNA was isolated from total RNA using a Dynabeads mRNA DIRECT Micro Kit (Ambion). The total mRNA samples were depleted of up to 99.9% of 5S, 5.8S, 18S, and 28S rRNA using the RiboMinus Eukaryote System v2 (Life Technologies, Carlsbad, CA, USA). Barcoded cDNA libraries were prepared from the rRNA-depleted mRNA samples and constructed using the Ion Total RNA Seq Kit v2 (Life Technologies). Whole-transcriptome libraries were diluted to 100 pM and amplified with ion sphere particles by emulsion PCR using an Ion One Touch 2 System (Life Technologies) and Ion PI Hi-Q OT2 200 Kit (Cat# A26434; Life Technologies). Template-positive ion sphere particles were enriched using the Ion OneTouch Enrichment System (Life Technologies), in which biotinylated adaptor sequences were selected by binding to streptavidin-conjugated beads. The template-positive ion sphere particles were sequenced using the Ion PI Hi-Q Sequencing 200 Kit (Cat# A26433; Life Technologies). Sequencing primers were annealed to template fragments attached to the ion sphere particles, and the template-positive ion sphere particle samples were loaded onto a chip from the Ion PI Chip Kit v3 (Cat# A26771; Life Technologies) and incubated with polymerase. Finally, the chip was placed on an Ion Proton System (Life Technologies) for ion semiconductor sequencing, which is based on the principle of hydrogen ion release detection when nucleotides are incorporated into the growing DNA template (n = 6 mice/group). All procedures were performed according to the manufacturer’s instructions.

Bioinformatics analysis of RNA-seq data

Raw RNA-seq reads were split into individual samples based on their barcodes and quality controlled using the FASTQC tool. The reads were analyzed using Partek Flow software (Partek, St. Louis, MO, USA) [25]. Briefly, the reads were mapped to Genome Reference Consortium Mouse Build 38 (mm10) using STAR 2.5.3a aligner. Quantification was performed using the transcript model Ensembl Transcripts release 91. Differential expression analysis was conducted using R package DESeq2. Genes with an absolute fold-change of ≥ 2 and a false discovery rate (FDR) < 0.01 were considered as differentially expressed genes (DEGs). The DEGs were subjected to ingenuity pathway analysis using IPA software (Qiagen, Hilden, Germany) [26]. Hierarchical clustering and biological classification analyses were also performed. Fisher’s exact test was used to determine the significance of the enrichment of specific biological processes among the DEGs. Hierarchical clustering analysis was performed using Genesis v1.7.545 based on a Pearson correlation distance matrix and average linkage algorithm.

Protein extraction and western blotting

Approximately 0.1 g of terminal ileum tissue isolated from Tas1r3+/+ or Tas1r3−/− mice was transferred to 1 mL Tissue Extraction Reagent 1 (Cat# FNN0071; Invitrogen) with 1/1,000 protease inhibitor (Cat# P-2714; Sigma-Aldrich, St. Louis, MO, USA) and homogenized. NCI-H716 cells were also lysed using RIPA buffer and homogenized. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Cat# 23227; Thermo Fisher Scientific) with bovine serum albumin as the standard. Aliquots of each protein lysate (20 μg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane, blocked for 1 h at 24 °C with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween 20, and incubated with monoclonal rabbit anti-mTOR (1:1,000) (Cat# 2972S, RRID: AB_330978), rabbit anti-phospho-mTOR (1:1,000) (Cat# 2971S, RRID: AB_330970), and rabbit anti-PPARgamma (1:1,000) (Cat# 2443S, RRID: AB_823598) primary antibodies from Cell Signaling Technology (Danvers, MA, USA), and rabbit anti-Occludin (1:2,000) (Cat# ab216327, RRID:AB_2737295) and rabbit anti-Claudin-1 (1:2,000) (Cat# ab180158) primary antibodies from Abcam. Mouse anti-α-tubulin (1:10,000) (Cat# T5168, RRID: AB_477579) from Sigma was used as a control. Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:5,000) (Cat# 7074S, RRID: AB_2099233) from Cell Signaling Technology and goat anti-mouse secondary antibodies (1:10,000) (Cat# G21040, RRID: AB_2536527) from Invitrogen were used for detection. The target proteins were detected using enhanced chemiluminescence western blot detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ, USA) (n = 6 mice/group).

16S rRNA gene sequencing

Fecal samples were frozen immediately after collection and stored at –80 °C. Total bacterial DNA was isolated from fecal samples using a QIAamp Fast DNA Stool Mini Kit (Cat# 51604; Qiagen), according to the manufacturer’s protocol for pathogen detection, with slight modifications. Briefly, approximately 200 mg of fecal material was homogenized for 1 min at 30 Hz with 5-mm sterilized steel beads in ASL buffer using a TissueLyser bead mill (Qiagen). The suspension was heated at 95 °C to lyse gram-positive bacterial cells. In the final incubation step, we extended the elution time from 1 to 5 min to increase the DNA yield. A 16S rRNA sequencing library was constructed, according to the 16S metagenomics sequencing library preparation protocol (Illumina, San Diego, CA, USA), targeting the V3 and V4 hypervariable regions of the 16S rRNA gene. KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, MA, USA) and the Agencourt AMPure XP System (Beckman Coulter Genomics, Brea, CA, USA) were used to amplify and purify the PCR products, respectively. The amplicons were sequenced in paired-end mode (PE275) using a MiSeq System (Illumina) (n = 10–14 mice/group).

16S rRNA-seq analysis

Paired sequences were dereplicated using the QIIME pipeline [27], and de novo and reference-based chimeras were removed using UCHIME software. Sequences from all samples were merged and sorted based on their relative abundances, and then closed operational taxonomic unit selection was performed using a 99% similarity threshold, followed by stringent taxonomic assignment using Silva v1.19. Based on the operational taxonomic unit abundance matrix and respective taxonomic classifications, feature abundance matrices were calculated at different taxonomic levels (from genus to phylum). To estimate the species diversity within samples (α-diversity), Shannon’s and Pielou’s indices were calculated using the R package phyloseq, after rarefaction. For comparisons among samples (β-diversity), Bray–Curtis and weighted principal coordinate analysis (PCoA) UniFrac dissimilarities were determined using phyloseq, based on profiles normalized to sample depth. Linear discriminant analysis effect size analysis was conducted online using the Galaxy workflow framework [28] to identify differentially abundant bacterial genera.

Stool butyrate assay

Aliquots (200 mg) of fecal samples were thawed, to which 4 volumes of distilled water (800 μL) were added, followed by vortexing at room temperature for 5 min until the samples were homogenized. Then, 15 μL of 95% sulfuric acid (Sigma-Aldrich) was added to 300 μL of fecal supernatant for acidification (final 5% (v/v)), followed by stabilization for 5 min. After centrifugation at 14,000 × g for 5 min, the supernatants were transferred to a new tube. To extract volatile materials, 30 μL of 1% (v/v) internal standard (2-methylpentanoic acid; Sigma-Aldrich) and 300 μL of anhydrous ethyl ether (Sigma-Aldrich) were added to acidified fecal supernatant. The samples were vortexed for 1 min, and then centrifuged at 14,000 × g for 5 min. The upper layer was carefully transferred to a GC vial and stored at -80 °C before analysis. The 10 mM butyrate solution (Cat# T8626; Sigma-Aldrich) was used as the standard for butyrate analysis. Stool butyrate was measured using the Agilent Technologies 7890A GC System (Santa Clara, CA, USA), as described by David et al. [29] (n = 20 mice/group).

IBD gene expression profiling

To analyze the DEGs in IBD (n = 204) versus non-IBD (n = 74), mRNA microarray expression profiles were retrieved and downloaded from the Gene Expression Omnibus database [30] by searching the following keywords: “IBD,” “active ulcerative colitis,” “active Crohn’s disease,” and “Homo sapiens” (organism). The inclusion criteria were as follows: (i) intestinal tissues (not cells) from adult patients with active IBD and (ii) samples from patients with IBD who had not received any interventions or treatments. After screening, five mRNA expression datasets (GSE160804, GSE126124, GSE95095, GSE75214, and GSE53306) were selected for analysis (see Additional file 1: Table S3). The raw microarray data were downloaded from the Gene Expression Omnibus database and preprocessed using Partek Genomics Suite version 6.6 (Partek) with the robust multichip analysis algorithm, which performs background adjustment, quantile normalization, and probe summarization. GC-content correction was used, as suggested by the default pipeline of Partek Genomics Suite. To estimate the effect of the normalization procedure, expression data without normalization and with standard robust multichip analysis normalization (without GC-content correction) were also generated. Differential gene expression analysis was performed using R/Bioconductor [31, 32]. For DEG selection, an FDR P < 0.001 was considered the cutoff value.

Murine colitis model

Colitis was induced by administering 2% DSS dissolved in drinking water for 7 days. Control mice were provided with drinking water without DSS. Colitis development was evaluated by monitoring daily weight changes. Colitis severity was also scored by evaluating the clinical disease activity through daily observation of the following parameters: weight loss (0 points = no weight loss or weight gain, 1 point = 5–10% weight loss, 2 points = 11–15% weight loss, 3 points = 16–20% weight loss, 4 points =  > 21% weight loss); stool consistency (0 points = normal and well-formed, 2 points = very soft and unformed, 4 points = watery stool); and bleeding stool score (0 points = normal color stool, 2 points = reddish color stool, 4 points = bloody stool). The disease activity index was calculated based on the combined scores of weight loss, stool consistency, and bleeding, and ranged from 0 to 12. All parameters were scored from Days 0 to 7. On Day 7 after DSS-colitis induction, the mice were sacrificed, and the entire intestine was quickly removed. After determining the colon length as a marker of inflammation, the entire colon was cut open lengthways and gently flushed with sterile phosphate-buffered saline to remove any traces of feces. Small and large intestinal segments were immediately frozen in liquid nitrogen and stored at − 80 °C for subsequent extraction of total RNA. For histological analysis, intestinal segments were fixed in 10% neutral buffered formalin phosphate and stored at room temperature until inflammation was analyzed (n = 7 mice/group).

Statistical analysis

All data were analyzed using GraphPad Prism software (version 9.0; GraphPad Inc., San Diego, CA, USA). The Kolmogorov–Smirnov test was used to assess data normality. All data were found to be normally distributed. SigmaPlot 11.0 was used to estimate all sample sizes using data from previous experiments and preliminary data with α = 0.05 and β = 0.2. Unless otherwise specified, all data are expressed as means ± standard errors of the mean. Descriptive statistics are listed in Additional file 1 (Table S4). The mean values of two groups were compared using Student’s t-test, and the means of multiple groups were compared using one-way analysis of variance, followed by Bonferroni post-hoc test. All statistical analyses were two-sided, and P < 0.05 was considered significant. In gene expression analyses, significant enrichment of specific genes was determined using a right-tailed Fisher’s exact test. Correlations between the relative abundance of butyrate-producing bacteria and transcript expression of PPAR-associated molecules were analyzed using Spearman’s correlation test.

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