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. 2019 Jan 4;124(1):94-100.
doi: 10.1161/CIRCRESAHA.118.313234.

A Proinflammatory Gut Microbiota Increases Systemic Inflammation and Accelerates Atherosclerosis

Eelke Brandsma  1 , Niels J Kloosterhuis  1 , Mirjam Koster  1   2 , Daphne C Dekker  1 , Marion J J Gijbels  3   4 , Saskia van der Velden  3 , Melany Ríos-Morales  1 , Martijn J R van Faassen  5 , Marco G Loreti  1 , Alain de Bruin  1   2 , Jingyuan Fu  1   6 , Folkert Kuipers  1   5 , Barbara M Bakker  1 , Marit Westerterp  1 , Menno P J de Winther  3   7 , Marten H Hofker  1 , Bart van de Sluis  1 , Debby P Y Koonen  1
Affiliations

A Proinflammatory Gut Microbiota Increases Systemic Inflammation and Accelerates Atherosclerosis

Eelke Brandsma et al. Circ Res. .

Abstract

Rationale: Several studies have suggested a role for the gut microbiota in inflammation and atherogenesis. A causal relation relationship between gut microbiota, inflammation, and atherosclerosis has not been explored previously.

Objective: Here, we investigated whether a proinflammatory microbiota from Caspase1-/- ( Casp1-/-) mice accelerates atherogenesis in Ldlr-/- mice.

Method and results: We treated female Ldlr-/- mice with antibiotics and subsequently transplanted them with fecal microbiota from Casp1-/- mice based on a cohousing approach. Autologous transplantation of fecal microbiota of Ldlr-/- mice served as control. Mice were cohoused for 8 or 13 weeks and fed chow or high-fat cholesterol-rich diet. Fecal samples were collected, and factors related to inflammation, metabolism, intestinal health, and atherosclerotic phenotypes were measured. Unweighted Unifrac distances of 16S rDNA (ribosomal DNA) sequences confirmed the introduction of the Casp1-/- and Ldlr-/- microbiota into Ldlr-/- mice (referred to as Ldlr-/-( Casp1-/-) or Ldlr-/-( Ldlr-/-) mice). Analysis of atherosclerotic lesion size in the aortic root demonstrated a significant 29% increase in plaque size in 13-week high-fat cholesterol-fed Ldlr-/-( Casp1-/-) mice compared with Ldlr-/-( Ldlr-/-) mice. We found increased numbers of circulating monocytes and neutrophils and elevated proinflammatory cytokine levels in plasma in high-fat cholesterol-fed Ldlr-/-( Casp1-/-) compared with Ldlr-/-( Ldlr-/-) mice. Neutrophil accumulation in the aortic root of Ldlr-/-( Casp1-/-) mice was enhanced compared with Ldlr-/-( Ldlr-/-) mice. 16S-rDNA-encoding sequence analysis in feces identified a significant reduction in the short-chain fatty acid-producing taxonomies Akkermansia, Christensenellaceae, Clostridium, and Odoribacter in Ldlr-/-( Casp1-/-) mice. Consistent with these findings, cumulative concentrations of the anti-inflammatory short-chain fatty acids propionate, acetate and butyrate in the cecum were significantly reduced in 13-week high-fat cholesterol-fed Ldlr-/-( Casp1-/-) compared with Ldlr-/-( Ldlr-/-) mice.

Conclusions: Introduction of the proinflammatory Casp1-/- microbiota into Ldlr-/- mice enhances systemic inflammation and accelerates atherogenesis.

Keywords: atherosclerosis; cholesterol; diet; fatty acids, volatile; feces; inflammation.

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Figures

Figure 1.
Figure 1.
Transplantation of Casp1−/− microbiota into Ldlr−/− mice via a cohousing approach. Female Ldlr−/− mice aged 12 wk were exposed to fecal microbiota derived from Casp1−/− or Ldlr−/− mice for 8 or 13 wk while fed a chow diet or high-fat cholesterol (HFC) diet. A, Experimental setup of the cohousing approach. Female Ldlr−/− mice were orally gavaged with a cocktail of broad-spectrum antibiotics for a period of 10 d to suppress intestinal microbes. This was followed by daily transfer of used bedding material from cages housing nonantibiotic-treated Ldlr−/− (donor) or Casp1−/− (donor) mice to cages housing the antibiotic-treated Ldlr−/− mice for 1 wk. During this period the mice were kept on chow diet or switched to an HFC diet for the remainder of the study. The antibiotic-treated Ldlr−/− mice were then cohoused with nonantibiotic-treated Casp1−/− mice (referred to as Ldlr−/−(Casp1−/−) mice) or Ldlr−/− mice (autologous transplantation, referred to as Ldlr−/−(Ldlr−/−) mice) in a 3:2 ratio for a period of 8 or 13 wk. B, Principal coordinate analysis plot of Unweighted UniFrac distance on the basis of 16S-rDNA (ribosomal DNA)-encoding sequences in feces collected from chow- and HFC-fed Ldlr−/− mice exposed to Casp1−/− or Ldlr−/− microbiota for 13 wk. Chow: Ldlr−/− mice (donor), n=8; Ldlr−/−(Ldlr−/−) mice, n=15; Casp1−/− mice (donor), n=9; Ldlr−/−(Casp1−/−) mice, n=14. HFC: Ldlr−/− mice (donor), n=7; Ldlr−/−(Ldlr−/−) mice, n=13; Casp1−/− mice (donor), n=8; Ldlr−/−(Casp1−/−) mice, n=14. PC indicates principal coordinate.
Figure 2.
Figure 2.
Casp1−/− microbiota promotes atherosclerosis development in Ldlr−/− mice fed high-fat cholesterol (HFC) diet. A, Representative toluidine blue stained slides of the aortic root. Scale bars, 400 μm. B, Quantification of atherosclerotic root lesion area. Chow (13 wk): Ldlr−/−(Ldlr−/−) mice, n=15; Ldlr−/−(Casp1−/−) mice, n=16. HFC (8 wk): Ldlr−/−(Ldlr−/−) mice, n=19; Ldlr−/−(Casp1−/−) mice, n=19. HFC (13 wk): Ldlr−/−(Ldlr−/−) mice, n=14; Ldlr−/−(Casp1−/−) mice, n=13. In bar graphs, data represent number of observations. For the scatter plot, the midline represents the mean±SEM. *P<0.05 by unpaired 1-tailed Student t test.
Figure 3.
Figure 3.
Casp1−/− dysbiosis leads to systemic inflammation. A, Plasma cytokines at time of sacrifice. n=10 per group. B, White blood cell (WBC) count and immune subsets during week 5 of cohousing. Ldlr−/−(Ldlr−/−) mice, n=18; Ldlr−/−(Casp1−/−) mice, n=17. C–D, Female Ldlr−/− mice aged 12 wk were exposed to fecal microbiota derived from Casp1−/− or Ldlr−/− mice for 13 wk while fed high-fat cholesterol (HFC) diet. C, Representative Ly6G-stained slides of the aortic root. Scale bars, 100 μm. D, Number of infiltrated neutrophils per 100.000 μm2 characterized by Ly6G-stained slides of the aortic root. Ldlr−/−(Ldlr−/−) mice, n=13; Ldlr−/−(Casp1−/−) mice, n=12. Data represent mean±SEM. *P<0.05 as determined by unpaired 1-tailed Student t test. IFN indicates interferon; and IL, interleukin.
Figure 4.
Figure 4.
Casp1−/−-induced alterations in the gut microbiota. Female Ldlr−/− mice were exposed to fecal microbiota derived from Casp1−/− or Ldlr−/− mice by means of cohousing for 13 wk while fed chow and high-fat cholesterol (HFC) diet. A–D, Abundance of microbiota taxonomies based on LEfSe analysis of 16S-rDNA (ribosomal DNA)-encoding sequences in feces collected at time of sacrifice. A, Family Akkermansia. B, Genus Christensenellaceae. C, Genus Clostridium. D, Genus Odoribacter. E, Cecum concentration of propionate, acetate, and butyrate in HFC-fed mice. A–D, Chow: Ldlr−/−(Ldlr−/−) mice, n=15; Ldlr−/−(Casp1−/−) mice, n=14; HFC: Ldlr−/−(Ldlr−/−) mice, n=13; Ldlr−/−(Casp1−/−) mice, n=14. E, Ldlr−/−(Ldlr−/−) mice, n=8; Ldlr−/−(Casp1−/−) mice, n=9. Data represent mean±SEM. *P<0.05 as determined by Kruskal-Wallis test (A–D) and unpaired 1-tailed Student t test (E). SCFA indicates short-chain-fatty acid.
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Comment in

  • Knights in Shining Armor.
    Dekker Nitert M. Dekker Nitert M. Circ Res. 2019 Jan 4;124(1):12-14. doi: 10.1161/CIRCRESAHA.118.314246. Circ Res. 2019. PMID: 30605411 No abstract available.

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