High-fat-diet-associated intestinal microbiota exacerbates psoriasis-like inflammation by enhancing systemic γδ T cell IL-17 production

Abstract

Although it is known that psoriasis is strongly associated with obesity, the mechanistic connection between diet and skin lesions is not well established. Herein, we showed that only dietary fat, not carbohydrates or proteins, exacerbates psoriatic disease. Enhanced psoriatic skin inflammation was associated with changes in the intestinal mucus layer and microbiota composition by high-fat diet (HFD). Change of intestinal microbiota by vancomycin treatment effectively blocked activation of psoriatic skin inflammation by HFD, inhibited the systemic interleukin-17 (IL-17) response, and led to increased mucophilic bacterial species such as Akkermansia muciniphila. By using IL-17 reporter mice, we could show that HFD facilitates IL-17-mediated γδ T cell response in the spleen. Notably, oral gavage with live or heat-killed A. muciniphila effectively inhibited HFD-induced enhancement of psoriatic disease. In conclusion, HFD exacerbates psoriatic skin inflammation through changing the mucus barrier and the intestine microbial composition, which leads to an enhanced systemic IL-17 response.

Keywords: CP: Immunology; CP: Microbiology; high-fat diet; psoriasis; γδT cell.

Graphical abstract

Figure 1

High-fat diet exacerbates psoriatic skin inflammation C57BL/6 mice were fed a normal diet (2.9% or 3.3% fat content) or high-fat diet (11.3%, 35.7%, or 79.2% fat content) for 7–10 weeks. Mice were topically treated with imiquimod (IMQ) cream on one ear for the last 3 days of the experiment, while the contralateral ear remained unchallenged (n = 5–10 per group). (A) Ear thickness measured by caliper and quantification of epidermal thickness in histopathology sections shown. Bars show the mean ± SD. p values were calculated by analysis of variance followed by Jonckheere-Terpstra test. Representative H&E staining of IMQ-treated ears are also shown. (B) Correlation between fat, sugar, and protein contents of the food and ear thickness. Spearman’s rank correlation coefficient (rS) and p values are shown. (C) Numbers of immune cells in IMQ-challenged and unchallenged contralateral ears of mice fed with normal diet (ND; 3.3% fat) or high-fat diet (HFD; 35.7% fat) (n = 10). Bars show the mean ± SD. p values were calculated by unpaired Student’s t test.

Figure 2

Induction of γδ T lymphocytes by HFD (A) IL-17 serum levels in C57BL/6 mice fed an ND (3.3% fat) or HFD (35.7% fat) after the 3 day challenge with IMQ (n = 60). (B and C) IL-17 production from different organs (B) and contribution of organs to IL-17 production (C) in IL-17 reporter mice fed an ND or HFD 3 days after being challenged with IMQ (n = 5–9). IL-17 production is defined as FITC-mean fluorescence intensity (MFI) ×IL-17A⁺ cell number. (D) Representative dot plot showing IL-17⁺ γδ⁺ T lymphocytes pregated on CD3⁺ T cells in the spleen of mice fed an ND or HFD and challenged with IMQ. (E and F) Number (E) and level (F) of IL-17 production (MFI) of IL-17⁺ γδ and αβ T lymphocytes in the spleen (n = 7–9). (G) Contribution of IL-17-expressing cells to IL-17 production in the spleen. Bars show the mean ± SD. p values were calculated by Welch’s t test (A) or unpaired Student’s t tests (B, E, and F) and are shown above each group.

Figure 3

HFD reduces mucin expression in the intestine C57BL/6 mice were fed an ND (3.3% fat) or HFD (35.7% fat) for 8 weeks and ear challenged with IMQ or without IMQ. (A) Alcian blue periodic acid-Schiff (AB-PAS) staining of the ileum. Purple layers on the edge of the villus represents the mucin layer (black arrowhead), and mucin-producing cells are depicted by the blue-stained cells. (A and B) Representative pictures and thickness of PAS layers are shown (A: n = 5; B: n = 10). (B) Representative pictures of Mucin 2 staining in the ileum. Red: mucin 2; blue: DAPI. (C) Quantitative real-time PCR for mucin mRNA expression in the first half of ileum (n = 10). Bars in the graphs show the mean ± SD. p values were calculated by unpaired Student’s t test.

Figure 4

Intestinal microbiome affects the exacerbation of psoriatic skin inflammation by HFD C57BL/6 mice were fed an ND (3.3% fat) or HFD (35.7% fat) for 8 weeks and challenged with IMQ. 0.5 mg/dL vancomycin (VCM) was administrated as drinking water for the last 2 weeks. (A) Ear thickness measured by caliper (n = 5–15). Epidermal thickness in H&E sections (n = 5–10). (B) Pathway analysis of serum molecules measured by liquid chromatography mass spectrometry. Shown are pathways upregulated in the HFD group but decreased by VCM treatment (n = 10). (C) Quantitative real-time PCR of Il17a expression in the skin (n = 5). (D) Circulating IL-17 was measured in the serum by ELISA (HFD; n = 60, HFD + VCM; n = 15). (E) Immune cell numbers in the total ear measured by flow cytometry (n = 5). Bars show the mean ± SD. p values were calculated by analysis of variance followed by Sidak’s post hoc tests for comparison of multiple groups or by unpaired Student’s t test for comparison of two groups.

Figure 5

Akkermansia muciniphila regulates the intestinal homeostasis (A and B) C57BL/6 mice were fed an ND (3.3% fat) or HFD (35.7% fat) for 8 weeks and challenged with IMQ. 0.5 mg/dL VCM was administrated in drinking water for the last 2 weeks. (A) Microbiome sequencing analysis of the bacterial 16S rDNA in the intestine (n = 5). (B) Quantitative real-time PCR of 16S rDNA of Akkermansia muciniphila (Akk) in the intestine (n = 5–10). (C–F) C57BL/6 mice were fed an ND (3.3% fat) or HFD (35.7% fat) for 8 weeks and challenged with IMQ. VCM in drinking water and live or heat-killed Akk oral gavage were given for the last 2 weeks. (C) Shannon index values indicated gut microbiota diversity. (D) Microbiome sequencing analysis of the bacterial 16S rDNA in the ileum at the level of phylum and genus (n = 5). (E) Quantitative real-time PCR of mucins of the first half of ileum (n = 6). (F) AB-PAS staining of the ileum, and quantification of the PAS layer and the goblet cells/villus (n = 5–12). Bars show the mean ± SD. p values were calculated by analysis of variance followed by Sidak’s post hoc tests for comparison of multiple groups or by unpaired Student’s t test for comparison of two groups.

Figure 6

Akk suppresses psoriatic skin inflammation and systemic IL-17 induced by HFD C57BL/6 mice were fed an ND (3.3% fat) or HFD (35.7% fat) for 8 weeks and challenged with IMQ. Oral gavages of live or heat-killed Akk were done for the last 2 weeks. (A) Representative H&E staining of the IMQ-treated ears from HFD and HFD-live Akk-treated mice. (B) Ear thickness was quantified in these different groups (n = 8–20). (C and D) Comparison of the proportion of PMA/ionomycin-stimulated IL-17A⁺ T cells, γδ T cells, and IL-17⁺ γδ T cells in mLNs from each group (n = 5–6). (E and F) Flow cytometric analysis of the IL-17⁺ T lymphocytes in the spleen. The boxed population shows the proportion of CD3 T cells (E). Proportion (%) of IL-17⁺ T lymphocytes (F) (n = 5). Data are presented as the mean ± SD. p values were calculated by analysis of variance followed by Sidak’s post hoc tests for comparison of groups indicated.

Figure 7

Akk reduced the increased ear IL-17 levels from HFD (A) Representative dot plot showing IL-17⁺ γδ T lymphocytes pregated on CD3⁺ T cells in the whole IMQ-treated ear of ND- and HFD-fed mice with or without live or heat-killed Akk gavage. (B) Flow cytometric analysis of the IL-17⁺ γδ T lymphocytes in the IMQ-treated ear skin. Histogram of IL-17A-GFP population, collective MFI, and number of IL-17⁺ γδ T lymphocytes in the whole IMQ-treated ear skin (n = 4–6). (C) Fold induction of Il17a mRNA in the ear skin, as analyzed by qRT-PCR (n = 5). (D) ELISA quantification of IL-17A concentration (pg/mL) in serum mice from the different groups (n = 5). (E) Immunofluorescence for IL-17A (red) and DNA (DAPI, blue) and quantification of IL-17 cells/field in the IMQ-treated ear with or without HFD/AKK (n = 4–5). Data are presented as the mean ± SD. p values were calculated by analysis of variance followed by Sidak’s post hoc tests for comparison of groups indicated.