HFD aggravated the arthritis and atherosclerosis by altering the intestinal status and gut microbiota

Abstract

Rheumatoid arthritis (RA) and cardiovascular disease (CVD) are both the chronic inflammatory disease. To investigate the influence of secondary atherosclerosis on arthritis mice, we treated the ApoE^-/- mice with K/BxN serum and high fat diet (HFD), and subsequently assessed the phenotypes as well as immune profiles of K/BxN serum and HFD induced ApoE^-/- mice. We found that HFD treatment aggravated the hyperlipidemia, atherosclerotic lesions, ankle swelling and arthropathy of mice. We further demonstrated that HFD altered the gut microbiota and metabolism, intestinal homeostasis and Th17/Treg cell balance in lamina propria lymphocytes. Moreover, HFD decreased the number of Peyer' s patches and altered the expression profiling of gut immune cells. In addition, HFD increased the number of aortic leukocytes and macrophages, then aggravated the atherosclerosis in aorta, which led to greater inflammation in mice aorta and aortic root. Collectively, our study indicated that HFD aggravated the arthritis and atherosclerosis, which may be contributed by microbiota dysbiosis, the intestinal permeability and disrupted immunological homeostasis.

Keywords: Arthritis combined with atherosclerosis; Gut microbiota; Inflammation; Intestinal status.

Fig. 1

HFD aggravated the ankle swelling and arthropathy of arthritis mice combined with atherosclerosis. A The ankle width and clinical score of mice. B HE staining of representative ankle joint specimens in four groups. H = Hyperplasia, B = Bone marrow, C = Cartilage. Data were presented as mean ± SD. Asterisk denoted statistically significant differences: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Representative results of two independent experiments were shown

Fig. 2

HFD worsened the atherosclerosis of arthritis mice combined with atherosclerosis. A The atherosclerotic plaques in cross-sectional area of the aortic root stained by oil red O and quantification of atherosclerosis per aortic root in four groups. B The content of total cholesterol (µg/µL) in serum of four groups. Data were presented as mean ± SD. Asterisk denoted statistically significant differences: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Representative results of two independent experiments were shown

Fig. 3

HFD generated greater inflammation in aorta, aortic root and serum of arthritis combined with atherosclerosis mice. A Flow cytometry gating strategy and the percent of vascular CD45⁺ leukocytes of aorta in four groups. B Flow cytometry gating strategy and the percent of vascular CD68⁺ macrophages of aorta in four groups. C Immunofluorescent staining and the mean IOD of CD11c. D Immunofluorescent staining and the mean IOD of IgG. E The content of serum cytokines (pg/mL). Data were represented as mean ± SD. Asterisk denoted statistically significant differences: *P < 0.05, **P < 0.01, ***P < 0.001. Representative results of two independent experiments were shown

Fig. 4

HFD increased the abundance of gut Burkholderiaceae and Rhodanobacter, decreased the abundance of gut Marvinbryantia and Lactobacillus, altered the intestinal homeostasis in arthritis mice combined with atherosclerosis. A Relative abundance of intestinal microbiota in four groups in phylum by 16S rDNA sequencing. B PCA plots of mice intestinal microbiota in four groups. C Comparison of different species among four groups in genus by MetaStat analysis. R1: arthritis mice combined with atherosclerosis (+ K/BxN serum + HFD), R2: atherosclerosis mice (−K/BxN serum + HFD), R3: arthritis mice (+ K/BxN serum-HFD), R4: normal mice (−K/BxN serum-HFD). D Firmicutes/Bacteroidetes ratio of mice and human gut microbiota in four groups. Data are presented as mean ± SD. Asterisk denotes statistically significant differences: *P < 0.05, **P < 0.01, ***P < 0.001. Representative results of two independent experiments are shown

Fig. 5

The gut microbiota of RA patients combined with atherosclerosis were altered. A Principal coordinate analysis (PCoA) among four groups of patients by 16S rDNA sequencing. B Multivariate statistical analysis of Metastat among four groups of patients. RS0: healthy human, RS1: RA patients, RS2: CVD patients, RS3: RA patients combined with CVD. Representative results of two independent experiments were shown

Fig. 6

HFD activated the intestinal metabolites of primary bile acid biosynthesis, aldosterone synthesis and secretion, and purine metabolism in arthritis combined with atherosclerosis mice. A The association analysis between 16S sequencing and metabolites analysis. B Volcano plot visualizing metabolites separation and KEGG pathway enrichment analysis for common differential metabolites in the arthritis combined with atherosclerosis mice and atherosclerosis mice by metabolites analysis. C Volcano plot visualizing metabolites separation and KEGG pathway enrichment analysis for common differential metabolites in the arthritis combined with atherosclerosis mice and arthritis mice. D Volcano plot visualizing metabolites separation and KEGG pathway enrichment analysis for common differential metabolites in the arthritis combined with atherosclerosis mice and normal mice. In every figures “A” represented arthritis combined with atherosclerosis mice (+ K/BxN serum + HFD), “B” represented atherosclerosis mice (−K/BxN serum + HFD), “C” represented arthritis mice (+ K/BxN serum-HFD), “D” represented normal mice (−K/BxN serum-HFD). Red dots indicated the up-regulated metabolites, green dots indicated the down-regulated metabolites, gray dots indicated no significant difference in the figures of volcano plot. Representative results of two independent experiments were shown

Fig. 7

HFD activated the colonic DEGs of IL-17 signaling pathway, increased the Th17 and decreased the Treg lamina propria lymphocytes in arthritis combined with atherosclerosis mice. A Volcano plot visualizing differential expression genes in colons and KEGG pathway enrichment analysis in the arthritis combined with atherosclerosis mice and atherosclerosis mice. B Volcano plot visualizing differential expression genes in colons and KEGG pathway enrichment analysis in the arthritis combined with atherosclerosis mice and arthritis mice. C Flow cytometry gating strategy and the percent of Th17 cells (CD4⁺ IL-17A⁺). D Flow cytometry gating strategy and the percent of Treg cells (CD4⁺ FoxP3⁺). E Volcano plot visualizing differential expression genes in colons and KEGG pathway enrichment analysis in the arthritis combined with atherosclerosis mice and normal mice. VPA: arthritis combined with atherosclerosis mice (+ K/BxN serum + HFD), VPB: atherosclerosis mice (−K/BxN serum + HFD), VPC: arthritis mice (+ K/BxN serum-HFD), VPD: normal mice (−K/BxN serum-HFD). Representative results of two independent experiments were shown

Fig. 8

HFD decreased the number of Peyer’ s patches and the mature B cells in Peyer’ s patches, increased the B1 cells in arthritis combined with atherosclerosis mice. A Flow cytometry gating strategy and the percentage of B1 cells (CD19⁺B220⁻) of Peyer’s patches lymphocytes in four groups. B Flow cytometry gating strategy and the percentage of mature B cells (CD19⁺B220⁺IgD⁺IgM⁺) and immature B cells (CD19⁺B220⁺IgD⁻IgM⁻) of peyer’s patches lymphocytes in four groups. C The number of peyer’s patches in four groups. Data were represented as mean ± SD. Asterisk denoted statistically significant differences: *P < 0.05, **P < 0.01, ***P < 0.001. Representative results of two independent experiments were shown

Fig. 9

HFD increased the CD3⁺/CXCR3⁺ cells, CD68⁺ macrophages, CD3⁺ T cells in lamina propria lymphocytes in arthritis mice combined with atherosclerosis. A Flow cytometry gating strategy and the percentage of CD3⁺/CXCR3⁺ cells of lamina propria lymphocytes in four groups. B Flow cytometry gating strategy and the percentage of CD68⁺ macrophages of lamina propria lymphocytes in four groups. C Flow cytometry gating strategy and the percentage of CD3⁺ T cells of lamina propria lymphocytes in four groups. Data were represented as mean ± SD. Asterisk denoted statistically significant differences: *P < 0.05, **P < 0.01, ***P < 0.001. Representative results of two independent experiments were shown