Indoleamine 2,3-dioxygenase-1 is protective in atherosclerosis and its metabolites provide new opportunities for drug development

Abstract

Atherosclerosis is the major cause of cardiovascular disease (CVD), the leading cause of death worldwide. Despite much focus on lipid abnormalities in atherosclerosis, it is clear that the immune system also has important pro- and antiatherogenic functions. The enzyme indoleamine-2,3-dioxygenase (IDO) catalyses degradation of the essential amino acid tryptophan into immunomodulatory metabolites. How IDO deficiency affects immune responses during atherogenesis is unknown and we explored potential mechanisms in models of murine and human atherosclerosis. IDO deficiency in hypercholesterolemic ApoE(-/-) mice caused a significant increase in lesion size and surrogate markers of plaque vulnerability. No significant changes in cholesterol levels were observed but decreases in IL-10 production were found in the peripheral blood, spleen and lymph node B cells of IDO-deficient compared with IDO-competent ApoE(-/-) mice. 3,4,-Dimethoxycinnamoyl anthranilic acid (3,4-DAA), an orally active synthetic derivative of the tryptophan metabolite anthranilic acid, but not l-kynurenine, enhanced production of IL-10 in cultured splenic B cells. Finally, 3,4-DAA treatment reduced lesion formation and inflammation after collar-induced arterial injury in ApoE(-/-) mice, and reduced cytokine and chemokine production in ex vivo human atheroma cell cultures. Our data demonstrate that endogenous production of tryptophan metabolites via IDO is an essential feedback loop that controls atherogenesis and athero-inflammation. We show that the IDO pathway induces production of IL-10 in B cells in vivo and in vitro, suggesting that IDO may induce immunoregulatory functions of B cells in atherosclerosis. The favorable effects of anthranilic acid derivatives in atherosclerosis indicate a novel approach toward therapy of CVD.

Keywords: B cell; atherosclerosis; cardiovascular disease; indoleamine 2,3-dioxygenase; inflammation.

Fig. 1.

IDO deficiency accelerates early atherosclerotic lesion formation in the aortic root. ApoE^−/− and ApoE^−/−Indo^−/− mice fed a normal chow diet were killed at 15, 20, or 30 wk of age. (A) Representative photomicrographs of aortic roots from 15-, 20-, or 30-wk-old mice stained with Oil red O and Hematoxylin. (Scale bars, 500 µm.) (B) Cross-sectional aortic root lesion size (×10³ µm², Left) and the percentage aortic root lesion area (percent, Right). Data show the mean lesional area per individual mouse. Line represents the group mean (n = 9–12; *P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 2.

IDO deficiency promotes a vulnerable plaque phenotype. (A) Representative photomicrographs of aortic root sections from 15-wk-old ApoE^−/− and ApoE^−/−Indo^−/− mice stained with an antibody against CD68 (brown staining) and Hematoxylin. (B) Lesion area staining positive (×10³ µm², Left, and percentage, Right) for CD68. (C) Representative photomicrographs of aortic root sections from 15-wk-old mice stained with an antibody against CD4 (brown staining) and Hematoxylin. Arrows denote positive cells. (D) Lesion area staining positive (cells per section, Left, and cells per square millimeter, Right) for CD4. (E) Representative photomicrographs of aortic root sections from 30-wk-old mice stained with an antibody against α-smooth muscle actin (Cy3-red) and DAPI (blue). (F) Lesion area staining positive (×10³ µm², Left and percentage, Right) for SMC. (G) Representative photomicrographs of aortic root sections from 30-wk-old mice stained with H&E. Areas of necrosis denoted by dotted lines. (H) Necrotic lesion area (×10³ µm², Left, and percentage, Right). (A, C, E, and G) Dashed lines denote internal elastic lamina. (Scale bars, 100 µm.) (B, D, F, and H) Line represents the group mean (n = 8–12; *P < 0.05, **P < 0.01, ***P < 0.001).

Fig. S1.

IDO deficiency affects lesional macrophage content but not CD4, SMC, or necrotic core content at 20 wk. (A) Representative photomicrographs of aortic root sections from 20-wk-old ApoE^−/− and ApoE^−/−Indo^−/− mice stained with an antibody against CD68 (brown staining) and Hematoxylin. (B) Lesion area staining positive (×10³ µm², Left, and percentage, Right) for CD68. (C) Representative photomicrographs of aortic root sections from 20-wk-old ApoE^−/− and ApoE^−/−Indo^−/− mice stained with an antibody against CD4 (brown staining) and Hematoxylin. Arrows denote positive cells. (D) Lesion area staining positive (cells per section, Left, and cells per square millimeter, Right) for CD4. (E) Representative photomicrographs of aortic root sections from 20-wk-old ApoE^−/− and ApoE^−/−Indo^−/− mice stained with an antibody against α-smooth muscle actin (Cy3, red) and DAPI. (F) Lesion area staining positive (×10³ µm², Left, and percent, Right) for SMC. (G) Representative photomicrographs of aortic root sections from 20-wk-old ApoE^−/− and ApoE^−/−Indo^−/− mice stained with H&E. Areas of necrosis denoted by dotted lines. (H) Necrotic lesion area (×10³ µm², Left, and percent, Right). (A, C, E, and G) Dashed line denotes internal elastic lamina. (Scale bars, 100 µm.) (B, D, F, and H) Line represents the group mean (n = 9–10; *P < 0.05). SMC content and necrosis was not quantifiable at 15 wk because of the small nature of the lesions.

Fig. S2.

IDO deficiency does not affect lesional macrophage or T-cell content at 30 wk. (A) Representative photomicrographs of aortic root sections from 30-wk-old ApoE^−/− and ApoE^−/−Indo^−/− mice stained with an antibody against CD68 (brown staining) and Hematoxylin. (B) Lesion area staining positive (×10³ µm², Left, and percent, Right) for CD68. (C) Representative photomicrographs of aortic root sections from 30-wk-old ApoE^−/− and ApoE^−/−Indo^−/− mice stained with an antibody against CD4 (brown staining) and Hematoxylin. Arrows denote positive cells. (D) Lesion area staining positive (cells per section, Left, and cells per square millimeter, Right) for CD4. (A and C) Dashed line denotes internal elastic lamina. (Scale bars, 100 µm.) (B and D) Line represents the group mean (n = 9–12).

Fig. 3.

Aortas of ApoE^−/−Indo^−/− mice display increased myeloid-marker gene expression. Aortas from 15-wk-old ApoE^−/− and ApoE^−/−Indo^−/− mice were collected at killing and RNA extracted and gene expression of myeloid and lymphoid markers examined by RT-PCR. Stacked bars show mean + SEM. (n = 5–6; *P < 0.05).

Fig. S3.

Inflammatory gene expression in spleen and LN of ApoE^−/−Indo^−/− vs. ApoE^−/− mice. Spleens and LN from 15-wk-old ApoE^−/− and ApoE^−/−Indo^−/− mice were collected at killing and RNA extracted. Gene expression of myeloid and lymphoid markers in spleen (A) and LN (B) of ApoE^−/− and ApoE^−/−Indo^−/− mice were then examined by RT-PCR. Stacked bars show mean + SEM. (n = 5–6; *P < 0.05).

Fig. S4.

Serum Kyn but not Trp levels are altered in ApoE^−/−Indo^−/− mice. (A) Serum Kyn levels (µM) in ApoE^−/− vs. ApoE^−/−Indo^−/− mice aged 15, 20, and 30 wk. (B) Serum Trp levels (mM) in ApoE^−/− vs. ApoE^−/−Indo^−/− mice aged 15, 20, and 30 wk. (C) Serum Kyn:Trp ratio levels in ApoE^−/− vs. ApoE^−/−Indo^−/− mice aged 15, 20, and 30 wk. Bars show mean + SEM, (n = 9–11; **P < 0.01, ***P < 0.001; ****P < 0.0001).

Fig. 4.

ApoE^−/−Indo^−/− mice display reduced serum IL-10 and IL-10–expressing B cells. (A) IL-10 levels (pg/mL) in serum of 15-wk-old ApoE^−/− vs. ApoE^−/−Indo^−/− mice. (B and C) Percentage of CD19⁺IL-10⁺ cells in the spleen (B) and LN (C) of 15-wk-old ApoE^−/− versus ApoE^−/−Indo^−/− mice. (D and E) CD19⁺IL-10⁺ cells (as percentage of vehicle) in splenocytes from ApoE^−/− mice cultured in the presence or absence of 3,4-DAA (D) or l-Kyn (E) at various concentrations for 48 h. Bars show mean + SEM, (n = 7–12; *P < 0.05, **P < 0.01, ***P < 0.001).

Fig. S5.

IDO deficiency affects serum IL-10 but not IL-12 levels. IL-10 (A) and IL-12p40 (B) levels (pg/mL) in serum of 15-, 20-, and 30-wk-old ApoE^−/− vs. ApoE^−/−Indo^−/− mice as measured by MSD. Bars show mean + SEM, (n = 9–11; *P < 0.05).

Fig. S6.

IDO-deficiency does not affect splenic or LN regulatory T-cell content. (A) Percentage of CD4⁺FoxP3⁺ cells in the spleen (Left) and LN (Right) of 15-wk-old ApoE^−/− vs. ApoE^−/−Indo^−/− mice. (B) Percentage of Tr1 (IL-10–expressing CD4⁺ cells) cells in the spleen (Left) and LN (Right) of 15-wk-old ApoE^−/− vs. ApoE^−/−Indo^−/− mice. Bars show mean + SEM, (n = 7–16).

Fig. 5.

3,4-DAA inhibits cytokine production by human atheroma cells. Production of IL-6 (A), TNF-α (B), GMCSF (C), IL-10 (D), and CXCL1 (E) (as percent untreated) in the supernatants of human atheroma cells cultured in the presence or absence of 3,4-DAA at various concentrations for 48 h as measured by luminex. (F) MTT assay to assess cell viability following 48-h treatment with 3,4-DAA. (n = 5 donors) Bars show group mean + SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.

3,4-DAA treatment inhibits accelerated atherosclerosis development induced by carotid collar injury. ApoE^−/− mice with a perivascular collar on the carotid artery were treated with 3,4-DAA (400 mg/kg) or vehicle. Mice were culled 21 d after collar placement. (A) Representative photomicrographs of injured carotid arteries from ApoE^−/− mice treated with 3,4-DAA or vehicle stained for elastin. (Scale bars, 100 µm). (B) Intima/media ratio (IMR) of carotid arteries 21 d after injury. Data show values for individual mice. Line represents group mean, (n = 7; *P < 0.05).

Fig. S7.

3,4-DAA treatment inhibits macrophage accumulation induced by carotid collar injury. ApoE^−/− mice with a perivascular collar on the carotid artery were treated with 3,4-DAA (400 mg/kg) or vehicle. Mice were culled 21 d after collar placement. (A) Representative photomicrographs of injured carotid arteries from ApoE^−/− mice treated with 3,4-DAA or vehicle stained with an antibody against the macrophage marker CD68 and counterstained with Hematoxylin. (Scale bars, 100 µm). (B) Lesion area staining positive for CD68 (×10³ µm², Left, percent, Right) in 3,4-DAA and vehicle-treated ApoE^−/− mice. Data show values for individual mice. Line represents group mean, (n = 7; *P < 0.05).

Fig. S8.

3,4-DAA-treatment does not affect serum Kyn and Trp levels in ApoE^−/− mice. (A) Serum Kyn levels (µM) in 3,4-DAA vs. Vehicle-treated ApoE^−/− mice that underwent collar-induced injury. (B) Serum Trp levels (mM) in 3,4-DAA vs. Vehicle-treated ApoE^−/− mice that underwent collar-induced injury. (C) Serum Kyn:Trp ratio levels in 3,4-DAA vs. Vehicle-treated ApoE^−/− mice that underwent collar-induced injury. Bars show mean + SEM, (n = 6–9).