Bile acid toxicity in Paneth cells contributes to gut dysbiosis induced by high-fat feeding

High-fat feeding (HFF) leads to gut dysbiosis through unclear mechanisms. We hypothesize that bile acids secreted in response to high-fat diets (HFDs) may act on intestinal Paneth cells, leading to gut dysbiosis. We found that HFF resulted in widespread taxonomic shifts in the bacteria of the ileal mucosa, characterized by depletion of Lactobacillus and enrichment of Akkermansia muciniphila, Clostridium XIVa, Ruminococcaceae, and Lachnospiraceae, which were prevented by the bile acid binder cholestyramine. Immunohistochemistry and in situ hybridization studies showed that G protein–coupled bile acid receptor (TGR5) expressed in Paneth cells was upregulated in the rats fed HFD or normal chow supplemented with cholic acid. This was accompanied by decreased lysozyme+ Paneth cells and α-defensin 5 and 6 and increased expression of XBP-1. Pretreatment with ER stress inhibitor 4PBA or with cholestyramine prevented these changes. Ileal explants incubated with deoxycholic acid or cholic acid caused a decrease in α-defensin 5 and 6 and an increase in XBP-1, which was prevented by TGR5 antibody or 4PBA. In conclusion, this is the first demonstration to our knowledge that TGR5 is expressed in Paneth cells. HFF resulted in increased bile acid secretion and upregulation of TGR5 expression in Paneth cells. Bile acid toxicity in Paneth cells contributes to gut dysbiosis induced by HFF.


150-200g
for 3 min to separate crypts from single cells. The final pure crypts were used for Western Blot or RT-qPCR analysis.
Dissociated crypts were centrifuged at 2,500 rpm for 5 min. The cells were washed with PBS and re-suspended in Hank's Balanced Salt Solution (HBSS, Thermo Fisher Scientific) /0.3 U/ml dispase at 37°C. Shaking was performed every 2 min for 10 min.
100 mg DNaseI was then added, and the cells were passed through a 70-mm filter (BD Bioscience). Cells were centrifuged at 2,500 rpm for 5 min and re-suspended in 4 ml HBSS with 5% FBS, followed by passage through a 100-mm filter and combination with an additional 100 mg DNaseI. Isolated cells were used for immunohistochemistry or flow cytometry studies.

Immunohistochemistry
Isolated epithelial cells were put on the slides and fixed in 4% paraformaldehyde for 15 min. Segments of the terminal ileum were fixed in 10% formalin and embedded in paraffin, sectioned at 5-μm thickness. Sections of ileal tissue or isolated epithelial cells were stained according to standard immunohistochemistry protocols and manufacturer's recommendations as described previously ( 52 ) . Double immunohistochemical staining was performed against: Lysozyme (1:250, A0099, DAKO Cytomation) and TGR5 (1:200,Santa Cruz Biotechnology) or Lysozyme (1:250, A0099, DAKO Cytomation) and marker of ER stress, XBP-1 (1:300, sc-32136, Santa Cruz Biotechnology), respectively. Species-specific fluorophore-conjugated secondary antibodies Alexa Fluor 488 and Alexa Fluor 594 (Jackson ImmunoResearch Laboratories, West Grove, PA) were employed. Slides were washed with PBS and mounted with Prolong Gold Antifade reagent with DAPI (Invitrogen). Tissues were examined and photographed by use of a fluorescence microscope (Olympus BX-51, Tokyo, Japan). TGR5 and lysozyme were detected by a Leica TCS SP8 confocal laser scanning microscope (Leica TCS NT, Wetzlar, Germany).

In situ hybridization
The rats were euthanized with CO2 and perfused via the ascending aorta with phosphate buffered saline (PBS, in mmol/L: NaCl 137; KCl 2.7; Na2HPO4 10 and

Western blot analysis
The Western blot study was performed as follows: Briefly, proteins extracted from ileal crypts of rats were analyzed on Ready Gel Tris·HCl (Bio-Rad Laboratories). The tissue was homogenized in ice-cold lysis buffer. The homogenate was centrifuged at 14,000 g for 10 min. Protein samples were run on Ready Gel 7.5% or 4-20% overnight, and then washed in Tris-buffered saline for 1 h. The membranes were probed with corresponding horseradish peroxidase-conjugated secondary antibodies at 1:8,000 dilution for 1 h at room temperature, and the bands were visualized by electrochemiluminescence (PerkinElmer, Waltham, MA). Signals were quantified with use of Image J (National Institutes of Health, Bethesda, MD) and normalized by GAPDH.

RT-qPCR
Total RNAs were extracted from rat ileal crypts tissue by use of TRIzol reagent (Life The PCR conditions were as follows: one cycle at 95°C for 10 minutes, followed by 40 two-temperature cycles at 95°C for 15 seconds and 60°C for 60 seconds. PCR amplifications were performed in a total volume of 25 μl, containing iQSYBR Green supermix (Bio-Rad Laboratories). Cytokine transcript levels were normalized to that of GADPH, and relative gene expression was expressed as the fold change (2−ΔΔCt) relative to expression in the control samples.

Electron microscopy
After rats had been sacrificed, their small intestines were immediately fixed in 1.25% glutaraldehyde, 4% formaldehyde in 0.1Mcacodylate buffer at pH7.4 at room temperature for electronmicroscopy. Next, samples were rinsed in cacodylate buffer and postfixed in 1% OsO4 in 0.067 M cacodylate buffer (pH 7.4) supplemented with 1.5% potassium ferrocyanide at 4 °C for 1 hour. After a short rinse in cacodylate buffer, dehydration was carried out in graded ethanol series, followed by embedding in Epon. After staining with uranyl acetate and lead citrate, the ultrathin sections were examined with a JEOL 1400-plus transmission electron microscope.