Myeloid cell-specific Irf5 deficiency stabilizes atherosclerotic plaques in Apoe-/- mice

Abstract

Objective: Interferon regulatory factor (IRF) 5 is a transcription factor known for promoting M1 type macrophage polarization in vitro. Given the central role of inflammatory macrophages in promoting atherosclerotic plaque progression, we hypothesize that myeloid cell-specific deletion of IRF5 is protective against atherosclerosis.

Methods: Female Apoe^-/-Lysm^Cre/+Irf5^fl/fl and Apoe^-/-Irf5^fl/fl mice were fed a high-cholesterol diet for three months. Atherosclerotic plaque size and compositions as well as inflammatory gene expression were analyzed. Mechanistically, IRF5-dependent bone marrow-derived macrophage cytokine profiles were tested under M1 and M2 polarizing conditions. Mixed bone marrow chimeras were generated to determine intrinsic IRF5-dependent effects on macrophage accumulation in atherosclerotic plaques.

Results: Myeloid cell-specific Irf5 deficiency blunted LPS/IFNγ-induced inflammatory gene expression in vitro and in the atherosclerotic aorta in vivo. While atherosclerotic lesion size was not reduced in myeloid cell-specific Irf5-deficient Apoe^-/- mice, plaque composition was favorably altered, resembling a stable plaque phenotype with reduced macrophage and lipid contents, reduced inflammatory gene expression and increased collagen deposition alongside elevated Mertk and Tgfβ expression. Irf5-deficient macrophages, when directly competing with wild type macrophages in the same mouse, were less prone to accumulate in atherosclerotic lesion, independent of monocyte recruitment. Irf5-deficient monocytes, when exposed to oxidized low density lipoprotein, were less likely to differentiate into macrophage foam cells, and Irf5-deficient macrophages proliferated less in the plaque.

Conclusion: Our study provides genetic evidence that selectively altering macrophage polarization induces a stable plaque phenotype in mice.

Keywords: Anti-inflammation; Aortic macrophages; Atherosclerosis; Interferon regulatory factor 5; Macrophage polarization (M1, M2); Plaque stabilization.

Figure 1

scRNA-Seq datasets from murine (Winkels et al., 2018 + Wirka et al., 2019, data integrated) and human (Wirka et al., 2019) atherosclerotic lesions were analyzed for Irf5 expression. (A, C) tSNE plots of mouse and human atheroma associated cell types and Irf5 expression herein (violin plots). (B, D) tSNE plots depicting foamy, inflammatory, and resident-like subtypes of murine (Winkels et al., 2018 + Wirka et al., 2019, data integrated) and human macrophages (Wirka et al., 2019) in atherosclerotic lesions. Violin plots showing expression levels of Irf5 and subtype defining genes in the respective macrophage subtypes. ∗p < 0.05 denotes statistically significant differences between macrophage subsets, adjusted p-value, Wilcoxon Rank–Sum test.

Figure 2

(A) Confirmation of myeloid cell-specific Irf5 deficiency in Bone-marrow derived macrophages (BMDM) isolated from Apoe^–/–Lysm^Cre/+Irf5^fl/flmice (KO) and WT controls (Apoe^−/−Irf5^fl/fl mice) by gel-electrophoresis of Irf5 PCR products. (B) BMDM generated from WT and KO bone marrow cells (M0) were polarized to M1 and M2 subsets in vitro through stimulation with LPS and IFNy or IL-4 and IL-13 for 12 h. Results of expressions for key macrophage-associated genes are presented as mean ± SEM. Gene expressions were analyzed using the ΔΔCq method and normalized to the expression of β-actin. ∗p < 0.05 denotes statistically significant differences between macrophage subtypes, Mann–Whitney, n = 4 per group. IFNγ = interferon, IL = interleukin, LPS = lipopolysaccharide, WT = wild type.

Figure 3

(A) Myeloid cell-specific Irf5 deficient Apoe^–/– mice (KO) and WT controls were fed a high cholesterol diet for 12 weeks. Body weight and plasma cholesterol levels are presented as mean ± SEM, n = 8 per group. ns = non-significant, t-test. (B) Representative dot plots and cell counts for leukocytes, neutrophils, monocytes, Ly6C^high-monocytes in particular, T-cells and B-cells in the blood are presented as mean ± SEM, n = 8 per group. ns = non-significant, t-test. (C) Representative histologic images and quantification of plaque area, lipid- (Oil Red O), macrophage- (CD68), collagen-contents and necrotic core (Masson Trichrome), and necrotic core (NC) in aortic root sections. The plaque area represents the intimal lesion area as a fraction of the total wall area (encompassing intima and media). Areas within lesions filled with lipids, macrophages, collagen and the necrotic core are related to the intimal lesion area. Results are presented as mean percentage ± SEM, n = 8 per group, ∗p < 0.05 denotes statistically significant differences between WT and KO mice, t-test. (D) Absolute aortic root lesion size measured in 6 serial sections at 50 μm intervals starting from valve initiation. Results are presented as mean ± SEM, n = 8 per group. ns = non-significant, t-test. (E) Change in gene expressions in aortas from myeloid cell-specific Irf5 KO-mice relative to expression levels in WT mice. Gene expressions were analyzed using the ΔΔCq method and normalized to the expression of β-actin. Results are presented as mean ± SEM, n = 8 per group. ∗p < 0.05 denotes statistically significant differences between WT and KO, t-test and Mann–Whitney. ORO=Oil red O, TWA = total wall area, NC = necrotic core.

Figure 4

(A, B) Lethaly irradiated Apoe^–/– mice were reconstituted with a mixture of Apoe^–/– CD45.1⁺ bone marrow cells (WT) and CD45.2⁺Apoe^–/–Lysm^Cre/+Irf5^fl/fl bone marrow cells (KO) to create mixed chimeric mice. CD45.1 and CD45.2 chimerism was analyzed in monocytes and macrophages in the blood and aortic tissue cell suspension in mixed chimeras fed a high cholesterol diet for 4 weeks. Results are presented as mean ± SEM, n = 4 mixed chimeras. (C) Relative change in CD45.1 and CD45.2 chimerism (%) between monocytes in the blood and aorta, and macrophages in the aorta is presented following 4 and 12 weeks of HCD feeding, Results are presented as mean ± SEM, n = 4–5 mixed chimeras, ∗p < 0.05 denotes statistically significant differences between cell populations, Kruskal–Wallis and Dunn's multiple comparisons test. (D) Quantification of proliferation (Ki67) and apoptosis (Casp3) among aortic macrophages in mixed chimeras following 4 and 12 weeks of HCD. Results are presented as mean ± SEM, n = 4–5 mixed chimeras, ∗p < 0.05 denotes statistically significant intraindividual differences between CD45.1 (blue) and CD45.2 (red) macrophages, Mann Whitney. (E) Quantification of CD36 surface expression on aortic macrophages in mixed chimeras following 12 weeks of HCD based on mean fluorescent intensities (MFI) as illustrated in the representative dot plot. CD45.1⁺Irf5^+/+ macrophages are shown in blue and CD45.2⁺Irf5^−/− macrophages are shown in red. Results are presented as mean ± SEM, n = 4 mixed chimeras, ∗p < 0.05 denotes statistically significant intraindividual differences between CD45.1 (blue) and CD45.2 (red) macrophages, Mann Whitney. (F) Monocytes isolated from WT and KO mice were cultured with M-CSF for 16 h to induce macrophage differentiation, subsequently stimulated with Dil-labeled oxLDL for 4 h generating macrophage foam cells. Quantification of the fraction of differentiated F4/80^high foam cells and oxLDL uptake by flow cytometry (representative dot plots and histogram with dashed line representing unstimulated control). Results are presented as mean ± SEM, n = 8 per group. ∗p < 0.05 denotes statistically significant differences between WT and KO, t-test. Dil-oxLDL = dil-labeled oxidized low density lipoprotein. HCD = high-cholesterol diet.