Fig 1.
Decrease of Paneth cells in mice fed a high-fat diet (HFD).
(A) Hematoxylin and eosin (H&E) staining of the small intestine showing typical eosinophilic granules of Paneth cells at the base of the crypts (upper row, arrows), and a Periodic acid-Schiff (PAS)/Alcian blue-stained section showing purple Paneth cell granules (lower row, arrow heads). Mice were fed either a normal chow diet (NCD) or a HFD for 15 weeks. (B) Quantification of the Paneth cell area was conducted as described in the Materials and methods (n = 5 each). (C) Confocal microscopic images of Paneth cells at the base of the crypts stained with anti-lysozyme Ab. (D) Quantification of lysozyme content per crypt was performed as described in the Materials and methods (n = 5 each). (E) Immunohistochemistry using anti-procryptdin Ab. (F) RT-qPCR analysis of antimicrobial peptides (AMPs) produced at the bottom of the crypts was performed using RNA isolated by laser capture microdissection (n = 5 each). Original magnification for all images is ×100, except the bottom row of (C) (×1,000) and the bottom row of (E) (×200). Scale bar, 100 μm. All values are expressed as the means ± SEM. A student’s t-test was used to compare values between two groups. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 2.
Paneth cell death and impaired Paneth cell function in mice fed a high-fat diet (HFD).
(A) Crypt cell death in vivo. Crypts isolated from mice fed a normal chow diet (NCD) or a HFD were dissociated into single cells and stained using the Annexin V-FITC Apoptosis Detection Kit for flow cytometric analysis of cell death (upper panel). The percentage of 7-ADD-stained cells was compared between groups (lower panel). (B) CD24+ Paneth cell death in HFD-fed mice. Isolated crypts were dissociated into single cell suspensions, and CD24+ Paneth cell death was determined using an Annexin V-FITC Apoptosis Detection Kit for flow cytometric analysis of cell death gated on CD24 (upper panel). The percentage of 7-ADD-stained cells among CD24+ Paneth cells was compared between groups (lower panel). (C) Crypt cell death ex vivo. Crypts isolated from NCD-or HFD-fed mice were cultured ex vivo for 18 h, and DNA oligonucleosome contents in the cell lysate were measured (n = 3). (D) Crypts were stimulated with carbachol (CCh) for 30 min, and the supernatants were subjected to an immunoblot (IB) assay using anti-lysozyme Ab. (E) Bactericidal activity of intestinal crypt supernatant against Salmonella typhimurium. Data are expressed as the percentage of killed bacteria relative to unexposed bacteria (n = 3 each). All values are expressed as the means ± SEM. A student’s t-test was used to compare values between two groups. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3.
Decrease of goblet cells in mice fed a high-fat diet (HFD).
(A and B) Changes in goblet cells in HFD-fed mice. Representative PAS/Alcian blue-stained sections of the distal ileum from NCD- or HFD-fed mice showing goblet cells (blue) (A) and quantification of goblet cell number (n = 5 each) (B). (C and D) Changes in Mucin-2 expression. Confocal microscopic images of the distal ileum following immunofluorescence staining using anti-Mucin-2 mAb (C) and relative expression of Mucin-2 determined by RT-qPCR (n = 5 each) (D). (E) Immunohistochemistry using anti-Notch intracellular domain (NICD) and phospho-S6 Abs. Original magnification for all images is ×100, except (E) (×200). Scale bar, 100 μm. All values are expressed as the means ± SEM. A student’s t-test was used to compare values between two groups. *P < 0.05 and ***P < 0.001.
Fig 4.
Disruption of the gut barrier in high-fat diet (HFD)-fed mice.
(A) RT-qPCR analysis of Occludin and Zo-1 using RNA from the distal ileum of normal chow diet (NCD)- or HFD-fed mice (n = 5 each). (B) Representative immunohistochemistry of distal ileal sections using anti-Occludin or -Zo-1 mAb. (C and D) RhoA expression in the intestine of mice fed NCD or HFD. mRNA expression (C) and representative immunohistochemistry (D) of RhoA in the small intestine. (E) Serum levels of FITC 4 h after oral FITC-dextran administration to NCD- or HFD-fed mice by oral gavage (n = 5 each). Original magnification ×200 for all images. Scale bar, 100 μm. All values are expressed as the means ± SEM. A student’s t-test was used to compare values between two groups. *P < 0.05, **P < 0.01.
Fig 5.
Aggravated experimental colitis in high-fat diet (HFD)-fed mice.
(A) Mice fed normal chow diet (NCD) or HFD for 15 weeks were either untreated or treated with 3% dextran sodium sulfate (DSS) for 5 days, and monitored for colitis progression. Body weight is presented as the percentage of the initial weight. (B) Colon lengths measured on day 7 of DSS administration (left). Representative images of the colon are shown (right). (C) Representative H&E sections of the cecum, the ascending colon (A-), transverse colon (T-), and descending (D-) colon on day 7 of DSS administration to NCD-fed (left column) or HFD-fed (middle column) mice (arrowheads, ulceration; asterisks, inflammation; arrows, regeneration of crypts). Colitis scoring on day 7 of DSS administration (right column). Original magnification for all images is ×100, except the top row of (C) (×10). Scale bar, 100 μm. All values are expressed as the means ± SEM. A student’s t-test was used to compare values between two groups. *P < 0.05, **P < 0.01, ***P < 0.001 (n = 5 each).
Fig 6.
Flow cytometric analysis of cells in the colonic epithelium of high-fat diet (HFD)-fed mice treated with dextran sodium sulfate (DSS).
(A and B) Proportions of T cell subsets. Proportions of TCRγδ and TCRαβ cells (A) and those of CD8αα and CD8αβ T cells (B) among colonic intestinal epithelial lymphocytes (IELs) gated on CD3 were determined on day 7 of DSS administration to normal chow diet (NCD) or HFD-fed mice. (C and D) Proportion of myeloid subsets. Proportions of CD11b+Ly6C+ monocytes (C) and CD11b+F4/80+ macrophages (D) in colonic epithelium on day 7 of DSS treatment were determined by flow cytometry. All values are expressed as the means ± SEM. A student’s t-test was used to compare values between two groups. *P < 0.05, **P < 0.01 (n = 5 each).
Fig 7.
Cytokines in experimental colitis of high-fat diet (HFD)-fed mice.
(A) Cytokine content in culture supernatant following overnight incubation of tissue explants was determined by ELISA on day 7 of dextran sodium sulfate (DSS) treatment. (B and C) Role of IL-6 in DSS-colitis of HFD-fed mice. DSS (3%) was administered to HFD-fed IL-6-knockout or wild-type mice for 5 days. Representative H&E sections of the colons on day 7 of DSS administration (B). Body weight is presented as a percentage of the initial body weight (C). (D) Bacterial translocation to the liver on day 7 of DSS treatment was quantified by colony-forming unit (CFU) counts (×1,000). All values are expressed as the means ± SEM. A student’s t-test was used to compare values between two groups. *P < 0.05 (n = 5 each). Original magnification, ×200. Scale bar, 100 μm.
Fig 8.
Alteration of intestinal microbiota following high-fat diet (HFD) feeding and dextran sodium sulfate (DSS) treatment.
(A) Weighted principal coordinates analysis (PCoA) plots. Each spot represents one sample, and each group of mice is denoted by a different color. (B and C) Phylum-level changes of gut microbiota following HFD feeding and DSS treatment. Pie charts showing the differences in the relative abundances (%) of bacterial phyla (B). Changes of specific phyla (C). Normal chow diet (NCD)-control (CT), NCD-fed mice without DSS treatment; NCD-DSS, NCD-fed mice with DSS treatment; HFD-CT, HFD-fed mice without DSS treatment; HFD-DSS, HFD-fed mice with DSS treatment (n = 5 each). A one-way ANOVA was used to compare values among multiple groups. If an ANOVA test showed significant differences, the Duncan post-hoc test was used to compare two specific groups. If the Duncan test did not show a significant difference (P > 0.05), the two groups were labeled with the same letter over the respective bars. P values < 0.05 were considered statistically significant.
Fig 9.
Linear discriminant analysis (LDA) effect size (LEfSe) analysis of gut microbiota changes following consumption of a high-fat diet (HFD) and dextran sodium sulfate (DSS) treatment. Effect of DSS.
(A and B) Normal chow diet (NCD)-control (CT) vs. NCD-DSS mice. (C and D) HFD-CT vs. HFD-DSS mice. The phylogenetic tree and histogram show LDA scores calculated for differences in genus-level abundance between DSS-treated and -untreated mice. NCD-CT, NCD-fed mice without DSS treatment; NCD-DSS, NCD-fed mice with DSS treatment; HFD-CT, HFD-fed mice without DSS treatment; HFD-DSS, HFD-fed mice with DSS treatment (n = 5 each). The colors represent the group in which the indicated taxa is more abundant compared to the other group. The LDA scores of the NCD-CT and HFD-CT were negative, while those of the NCD-DSS and HFD-DSS were positive. Such negativity or positivity is determined by alphabetical order of the groups, and the absolute values of the effect size indicate the scale of the difference between 2 groups regardless of the positivity or negativity.
Fig 10.
Linear discriminant analysis (LDA) effect size (LEfSe) analysis of gut microbiota changes following consumption of a high-fat diet (HFD) and dextran sodium sulfate (DSS) treatment. Effect of diet.
(A and B) Normal chow diet (NCD) control-CT vs. HFD-CT mice. (C and D) NCD-DSS vs. HFD-DSS mice. The phylogenetic tree and histogram show LDA scores calculated for differences in genus-level abundance between mice fed different diets. NCD-CT, NCD-fed mice without DSS treatment; NCD-DSS, NCD-fed mice with DSS treatment; HFD-CT, HFD-fed mice without DSS treatment; HFD-DSS, HFD-fed mice with DSS treatment (n = 5 each). As in Fig 9, the colors represent which group the taxa was more abundant compared to the other group. The LDA scores of the HFD-CT and HFD-DSS were negative (Fig 10B), while those of the NCD-CT and NCD-DSS were positive. The absolute values of the effect size indicate the scale of the difference between 2 groups regardless of the positivity or negativity.
Fig 11.
Fecal microbial transplantation (FMT) and antibiotic treatment alter colitis progression.
(A-C) Antibiotic treatment. Mice were fed normal chow diet (NCD) or high-fat diet (HFD), and an antibiotic combination (A) or a single antibiotic (B and C) was administered for 8 weeks. Subsequently, DSS-colitis was induced. (D-F) Effects of FMT. NCD- or HFD-fed mice gavaged with fecal material from the same or the other group were treated with dextran sodium sulfate (DSS) and monitored for colitis development. Body weight is presented as the percentage of the initial weight (D). Colon lengths on day 13 of DSS treatment (E, left). Images of representative colons (E, right). (n = 5 each). *P < 0.05. All values are expressed as the means ± SEM. A student’s t-test was used to compare values between two groups. A one-way ANOVA was used to compare values among multiple groups. If an ANOVA test showed significant differences, the Duncan post-hoc test was used to compare two specific groups. If the Duncan test did not show a significant difference (P > 0.05), the two groups were labeled with the same letter over the respective bars. P values < 0.05 were considered statistically significant. *P < 0.05 (n = 5 each). NS, not significant.