Interleukin-27 receptor limits atherosclerosis in Ldlr-/- mice

Abstract

Rationale: Atherosclerosis is a chronic inflammatory disease of the arterial wall. Several proinflammatory cytokines are known to promote atherosclerosis, but less is known about the physiological role of anti-inflammatory cytokines. Interleukin (IL)-27 is a recently discovered member of the IL-6/IL-12 family. The IL-27 receptor is composed of IL-27 receptor A (WSX-1) and gp130 and is required for all established IL-27 signaling pathways. The expression of the IL-27 subunit Ebi3 is elevated in human atheromas, yet its function in atherosclerosis remains unknown.

Objective: The aim of this study was to test the role of IL-27 receptor signaling in immune cells in atherosclerosis development.

Methods and results: Atherosclerosis-prone Ldlr(-/-) mice transplanted with Il27ra(-/-) bone marrow and fed Western diet for 16 weeks developed significantly larger atherosclerotic lesions in aortic roots, aortic arches, and abdominal aortas. Augmented disease correlated with increased accumulation of CD45(+) leukocytes and CD4(+) T cells in the aorta, which produced increased amounts of IL-17A and tumor necrosis factor. Several chemokines, including CCL2, were upregulated in the aortas of Ldlr(-/-) mice receiving Il27ra(-/-) bone marrow, resulting in accumulation of CD11b(+) and CD11c(+) macrophages and dendritic cells in atherosclerotic aortas.

Conclusions: The absence of anti-inflammatory IL-27 signaling skews immune responses toward T-helper 17, resulting in increased production of IL-17A and tumor necrosis factor, which in turn enhances chemokine expression and drives the accumulation of proatherogenic myeloid cells in atherosclerotic aortas. These findings establish a novel antiatherogenic role for IL-27 receptor signaling, which acts to suppress the production of proinflammatory cytokines and chemokines and to curb the recruitment of inflammatory myeloid cells into atherosclerotic aortas.

Figure 1. Increased atherosclerotic plaque area inIl27ra^−/−bone marrow–transplanted mice

Ldlr^−/− mice were lethally irradiated and reconstituted with 5×10⁶ unfractioned bone marrow cells from C57BL/6 (wild-type [wt]) or Il27ra^−/− mice. After 4 weeks of reconstitution, mice were fed with western diet for 16 weeks. A, Images of aortic arch of Ldlr^−/− mice receiving Il27ra^−/− bone marrow or C57BL/6 (wt) control. B, Representative en face images of Sudan IV–stained whole aortas of Ldlr^−/− mice receiving Il27ra^−/− or wt bone marrow. B, right, Quantification of plaque area as percentage of aortic surface in Ldlr^−/− mice receiving Il27ra^−/− or wt bone marrow (n=6–7 females or males per group, respectively). C, left, Representative images of aortic roots (top) and single valve (bottom) of Ldlr^−/− mice receiving Il27ra^−/− or C57BL/6 (wt) bone marrow. C, right, Aortic root lesions were quantified on frozen sections stained with Oil-red-O in the 300 µm following the aortic valve (wt [n=8–9 males or females] mice and Il27ra^−/− [n=8–9 males or females] mice from 3 experiments).

Figure 2. Leukocyte composition of mouse aorta

A, Aortas from bone marrow–transplanted mice were made into single-cell suspensions by enzymatic digestion and stained. Live CD45⁺ leukocytes were counted in aortas of 16-week Western diet–fed Ldlr^−/− mice transplanted with Il27ra^−/− (n=7) or C57BL/6 (wild-type [wt]; n=7) bone marrow. Left panels show representative dot plots. Right panel shows mean±SEM, *P<0.05. B, left, Live CD45⁺ cells from aortas were stained for TCRβ and CD4. Numbers indicate percentage in indicated gates. B, right, Absolute number of live CD45⁺, TCRβ⁺, CD4⁺TCRβ⁺, and CD8⁺TCRβ⁺ cells in aortas from Ldlr^−/− mice transplanted with Il27ra^−/− or wt bone marrow (n=7) each. Mean±SEM *P<0.05. Localization of CD3⁺ cells in aortic roots (C) and abdominal aortas (D) of Ldlr^−/− mice transplanted with Il27ra^−/− or C57BL/6 (wt) bone marrow as seen by immunofluorescence. L indicates lumen; P, plaque; A, adventitia. Representative images from 1 of 3 independent experiments. Mean±SEM number of CD3⁺ cells per section in aortic roots (C, right) and abdominal aortas (D, right) of Ldlr^−/− mice transplanted with Il27ra^−/− (n=6) or C57BL/6 (wt; n=6) bone marrow are shown.

Figure 3. Enhanced production of T-helper 17 cytokines and transcription factors in mice receivingIl27ra^−/− bone marrow

A, Relative gene expression in Il27ra^−/−→Ldlr^−/− (n=6) mice normalized to β-actin and then normalized to gene expression in wild-type (wt)→Ldlr^−/− (n=6) mice in aortic arches, abdominal aortas (Ab. aorta), paraaortic lymph nodes (paLNs), and spleens after 16 weeks of Western diet. B and C, Interleukin (IL)-17A, tumor necrosis factor (TNF), IL-6, and interferon (IFN) γ were measured by bead array in supernatants of aortic (n=8; B) and splenic (n=8; C) cell suspensions, stimulated with anti-CD3/anti-CD28 for 48 h. Mean±SEM; *P<0.05.

Figure 4. Enhanced production of T-helper 17 cytokines in aortas of mice receiving Il27ra^−/− bone marrow

Percent of CD4 T cells expressing tumor necrosis factor (TNF), interleukin (IL)-17A, and interferon (IFN) γ by intracellular staining in aorta (A, left), spleen (B, left), or paraaortic lymph nodes (paLNs) (C, left) of Ldlr^−/− mice transplanted with Il27ra^−/− (n=6) or wild-type (wt; n=6) bone marrow fed 16 weeks with Western diet. A–C, right, Representative fluorescence-activated cell sorter plot of CD4⁺TCRβ⁺ T cells stained for IL-17A and TNF in aortas (A), spleens (B), and paLNs (C) of Ldlr^−/− mice transplanted with Il27ra^−/− or wt bone marrow fed 16 weeks with Western diet. Mean±SEM; *P<0.05.

Figure 5. Regulatory T cells in spleen and lymph nodes inIl27ra^−/− bone marrow–transplanted mice

A, Percentage of live CD4⁺TCRβ⁺Foxp3⁺ T regulatory cells (Treg) in spleens and paraaortic lymph nodes (paLNs) from Ldlr^−/− mice transplanted with Il27ra^−/− (n=10) or wild-type (wt) bone marrow (n=10). B, Percentage of live CD4⁺TCRβ⁺, Foxp3⁻IL10⁺ Tr1 cells in spleens and paLNs of Ldlr^−/− mice transplanted with Il27ra^−/− (n=10) or wt bone marrow (n=10). Mean±SEM; *P<0.05. LN indicates lymph node.

Figure 6. Analysis of interleukin (IL)-17–dependent chemokine expression in mouse aortas and lymphoid organs

A, Relative chemokine gene expression in Il27ra^−/−→Ldlr^−/− (n=6) normalized to β-actin and then normalized to gene expression in wild-type (wt)→Ldlr^−/− (n=6) mice in aortic arches, abdominal aortas (Ab. aortas), paraaortic lymph nodes (LNs), and spleens after 16 weeks of Western diet. CCL2 protein in supernatants of aortic cell suspensions unstimulated (n=6; B) or stimulated with anti-CD3/anti-CD28 for 48 h (n=8; C). Mean±SEM; *P<0.05.

Figure 7. Interleukin-27 receptor (IL-27R) controls the recruitment of myeloid cells to atherosclerotic aortas

A, left, Live CD45⁺ cells from aortas of Ldlr^−/− mice transplanted with bone marrow from Il27ra^−/− or wild-type (wt) mice fed with Western diet for 16 weeks were stained for CD11b and CD11c. Numbers indicate percentage in each quadrant. A, right, Absolute number of live CD45⁺, CD11b⁺CD11c⁻, CD11b⁺CD11c⁺, and CD11b⁻CD11c⁺ cells per aorta of Ldlr^−/− transplanted with Il27ra^−/− (n=8) or wt (n=7) bone marrow was determined based on flow cytometry data. Mean±SEM; *P<0.05. B and C, Localization and abundance of CD11b⁺CD11c⁻(green), CD11b⁺CD11c⁺ (yellow), CD11b⁻CD11c⁺(red), and MOMA-2⁺ (blue) cells in aortic roots of Ldlr^−/− mice transplanted with Il27ra^−/− (B) or C57BL/6 (wt; C) bone marrow characterized by immunofluorescence. Dotted white lines indicate border of lamina muscularis. L indicates lumen; P, plaque; A, adventitia. Representative images from 1 of 6 independent experiments. D, Quantification of CD11b⁺CD11c⁻, CD11b⁺CD11c⁺, CD11b⁻CD11c⁺, and MOMA-2⁺ cells in the aortic roots of Ldlr^−/− mice transplanted with Il27ra^−/− (n=6) or C57BL/6 (wt) (n=6) bone marrow as in B and C. Mean±SEM; *P<0.05.

Figure 8. Interleukin-27 receptor (IL-27R) controls the recruitment of myeloid cells to abdominal aortas of atherosclerotic mice

Localization and abundance of CD11b⁺CD11c⁻(green), CD11b⁺CD11c⁺ (yellow), CD11b⁻CD11c⁺(red), and MOMA-2⁺ (blue) cells in abdominal aortas of Ldlr^−/− mice transplanted with Il27ra^−/− (A) or C57BL/6 (wild-type [wt]) (B) bone marrow characterized by immunofluorescence. Dotted white lines indicate border of lamina muscularis. L indicates lumen; P, plaque; A, adventitia. Representative images from 1 of 6 independent experiments. C, Quantification of immunofluoresecent staining for CD11b⁺CD11c⁻, CD11b⁺CD11c⁺, CD11b⁻CD11c⁺, and MOMA-2⁺ cells in the abdominal aortas of Ldlr^−/− mice transplanted with Il27ra^−/− (n=6) or C57BL/6 (wt; n=6) bone marrow as shown in A and B. Mean±SEM; *P<0.05.