Colchicine Impacts Leukocyte Trafficking in Atherosclerosis and Reduces Vascular Inflammation

Abstract

Background: Inflammation strongly contributes to atherosclerosis initiation and progression. Consequently, recent clinical trials pharmacologically targeted vascular inflammation to decrease the incidence of atherosclerosis-related complications. Colchicine, a microtubule inhibitor with anti-inflammatory properties, reduced cardiovascular events in patients with recent acute coronary syndrome and chronic coronary disease. However, the biological basis of these observations remains elusive. We sought to explore the mechanism by which colchicine beneficially alters the course of atherosclerosis.

Methods and results: In mice with early atherosclerosis (Apoe^-/- mice on a high cholesterol diet for 8 weeks), we found that colchicine treatment (0.25 mg/kg bodyweight once daily over four weeks) reduced numbers of neutrophils, inflammatory monocytes and macrophages inside atherosclerotic aortas using flow cytometry and immunohistochemistry. Consequently, colchicine treatment resulted in a less inflammatory plaque composition and reduced plaque size. We next investigated how colchicine prevented plaque leukocyte expansion and found that colchicine treatment mitigated recruitment of blood neutrophils and inflammatory monocytes to plaques as revealed by adoptive transfer experiments. Causally, we found that colchicine reduced levels of both leukocyte adhesion molecules and receptors for leukocyte chemoattractants on blood neutrophils and monocytes. Further experiments showed that colchicine treatment reduced vascular inflammation also in post-myocardial infarction accelerated atherosclerosis through similar mechanisms as documented in early atherosclerosis. When we examined whether colchicine also decreased numbers of macrophages inside atherosclerotic plaques by impacting monocyte/macrophage transitioning or in-situ proliferation of macrophages, we report that colchicine treatment did not influence macrophage precursor differentiation or macrophage proliferation using cell culture experiments with bone marrow derived macrophages.

Conclusions: Our data reveal that colchicine prevents expansion of plaque inflammatory leukocytes through lowering recruitment of blood myeloid cells to plaques. These data provide novel mechanistic clues on the beneficial effects of colchicine in the treatment of atherosclerosis and may inform future anti-inflammatory interventions in patients at risk.

Keywords: anti-inflammatory treatment; atherosclerosis; colchicine; innate immunity; leukocyte recruitment; monocytes/macrophages; neutrophils; vascular inflammation.

Figure 1

Colchicine treatment reduces plaque inflammation. (A) Flow cytometric gating and (B) quantification of myeloid cells in atherosclerotic aortas in vehicle- vs. colchicine-treated Apoe^-/- mice (n=18-21 per group, 62-67% female, Welch’s t-test for macrophages (Mø), Mann-Whitney U-test for monocytes (Mono) and neutrophils as appropriate). Numbers next to gates indicate population frequencies (%). Representative immunohistochemical staining for (C) myeloid cells (CD11b) and (D) neutrophils (Ly6G) of sectioned aortic roots from vehicle- vs. colchicine-treated Apoe^-/- mice (n=8-10 per group, 60-75% female, Mann-Whitney U-test for CD11b, Student’s t-test for Ly6G). Bar graphs show quantification of positive CD11b- and Ly6G-area, respectively. Scale bars represent 500 µm. (E) Quantitative real-time PCR for gene expression quantification of fibrotic, inflammatory and cytokine genes in aortas of vehicle- vs. colchicine-treated Apoe^-/- mice (n=5-12 per group, 55-88% female, Student’s/Welch’s t-test or Mann-Whitney U-test as appropriate). Mmp3/Mmp9/Mmp10 (matrix metalloproteinase-3/9/10), Col1/Col3 (collagen-1/3), Csf1 (colony stimulating factor 1), Il1β (interleukin 1 beta), Il6 (interleukin 6), Tnf (tumor necrosis factor), Tgfb1 (transforming growth factor beta 1), Ccl2 (C-C Motif Chemokine Ligand 2), Cxcl1 (C-X-C Motif Chemokine Ligand 1), Cxcl2 (C-X-C Motif Chemokine Ligand 2), Cxcl12 (C-X-C Motif Chemokine Ligand 12), Cx3cl1 (C-X3-C Motif Chemokine Ligand 1). Data are presented as mean+s.e.m. (F) Representative Masson Trichrome staining and quantification of total plaque area (n=8-11 per group, 64-75% female, Welch’s t-test). Scale bars represent 500 µm. Dots within bar plots show the gender of the mice with a color code: purple (male) and red (female).

Figure 2

Colchicine treatment dampens leukocyte recruitment to atherosclerotic aortas by silencing neutrophil and monocyte activation. (A) Flow cytometric gating and quantification of GFP^high (GFP⁺) myeloid cells in atherosclerotic aortas 24h after adoptive transfer of GFP^high monocytes and neutrophils into vehicle- vs. colchicine-treated Apoe^-/- mice (n=8 per group, 38-50% female, Student’s t-test). (B) Gating strategy and histograms of leukocyte adhesion molecules on aortic endothelial cells (EC) from vehicle- vs. colchicine-treated Apoe^-/- mice. (C) Quantification of mean fluorescent intensities (MFI, representing relative protein levels) of adhesion molecules expressed by aortic endothelial cells from vehicle- vs. colchicine-treated Apoe^-/- mice (n=9-11 per group, 44-55% female, Student’s t-test or Mann-Whitney U-test as appropriate). Bar graphs indicate relative change of MFI standardized to controls. FMO (fluorescence minus one (respective antibody omitted)) control. Quantitative real-time PCR for gene expression quantification in fluorescence-activated cell sorting (FACS)-purified (D) aortic endothelial cells, (E) blood neutrophils and (F) blood Ly6C^high monocytes of vehicle- vs. colchicine-treated Apoe^-/- mice (n=3-12 per group, 33-100% female, Student’s/Welch’s t-test or Mann-Whitney U-test as appropriate). ICAM-1/Icam1 (intercellular adhesion molecule 1), ICAM-2/Icam2 (intercellular adhesion molecule 2), VCAM-1/Vcam1 (vascular cell adhesion protein 1), E-selectin/Sele, P-selectin/Selp, L-selectin/Sell, selectin P ligand/Selplg, platelet endothelial cell adhesion molecule-1/Pecam1, integrin subunit alpha L/Itgal, integrin subunit alpha M/Itgam, integrin subunit alpha 4/Itga4, integrin subunit beta 1/Itgb1, integrin subunit beta 2/Itgb2, C-C motif chemokine ligand 2/Ccl2, C-X-C motif chemokine ligands 1 + 2+12/Cxcl1/Cxcl2/Cxcl12, C-X3-C motif ligand 1/Cx3cl1, C-C chemokine receptor types 1 + 5/Ccr1/Ccr5, C-X-C Motif chemokine receptor types 2 + 4/Cxcr2/Cxcr4 and C-X3-C Motif Chemokine Receptor 1/Cx3cr1. Data are presented as mean+s.e.m. Numbers next to gates indicate population frequencies (%). Dots within bar plots show the gender of the mice with a color code: purple (male) and red (female).

Figure 3

Colchicine exposed neutrophils and monocytes show reduced recruitment capacities. (A) Experimental scheme. (B) Flow cytometric gating and (C) quantification of GFP^high myeloid cells (GFP⁺) in atherosclerotic aortas 24h after adoptive transfer of either vehicle- or colchicine-exposed GFP^high monocytes and neutrophils into Apoe^-/- mice (n=10 per group, 60% female, Mann-Whitney U-test). Data are presented as mean+s.e.m. Numbers next to gates indicate population frequencies (%). Dots within bar plots show the gender of the mice with a color code: purple (male) and red (female).

Figure 4

Colchicine treatment did not alter numbers of blood leukocyte subsets. Flow cytometric gating and quantification of myeloid cells in the blood (A, B) (n=18-21 per group, 62-67% female, Student’s t-test for total leukocytes (Leuko) and Ly6C^high monocytes (Ly6C^high), Mann-Whitney U-test for neutrophils (Neutro) and Ly6C^low monocytes (Ly6C^low) and bone marrow (C, D) in vehicle- vs. colchicine-treated Apoe^-/- mice (n=10-13 per group, 77-80% female, Mann-Whitney U-test). Data are presented as mean+s.e.m. Numbers inside/next to gates indicate population frequencies (%). Dots within bar plots show the gender of the mice with a color code: purple (male) and red (female).

Figure 5

Colchicine treatment impacts neither macrophage precursor differentiation nor macrophage proliferation. (A) Experimental scheme for precursor differentiation into macrophages (Fig. 6B+C). In brief, bone marrow cells were retrieved from one femur and cultured with M-CSF (macrophage colony-stimulating factor) for 6 days to generate bone marrow-derived macrophages (BMDM). Either vehicle or colchicine was added once every 24h starting immediately after seeding. (B) Flow cytometric gating and (C) quantification of macrophage (Mø) numbers 6 days after either vehicle or colchicine exposure (n=5 per group, each n represents one donor animal; repeated measures one-way ANOVA with Dunnett’s multiple comparisons test). (D) Experimental scheme for macrophage proliferation (Fig. 6E+F). In brief, bone marrow cells were retrieved from one femur and cultured with M-CSF (macrophage colony-stimulating factor) for 6 days to generate bone marrow-derived macrophages (BMDM). Either vehicle or colchicine was added once every 24h starting 4d after seeding. BrdU (bromodeoxyuridine) was administered 2h before the harvest (day 6 after seeding). (E) Flow cytometric gating and (F) quantification of BrdU⁺ macrophage (BrdU⁺ Mø) frequencies (Friedman test followed by Dunn’s multiple comparisons test) and total macrophage numbers 48h after either vehicle or colchicine exposure (repeated measures one-way ANOVA with Dunnett’s multiple comparisons test). n=5 per group (each n represents one donor animal). Data are presented as mean+s.e.m. Numbers next to gates indicate population frequencies (%).

Figure 6

Colchicine treatment reduces vascular inflammation in post-myocardial infarction accelerated atherosclerosis. (A) Experimental scheme for Figures 6B-E . In brief, Apoe^-/- mice on a high cholesterol diet (HCD) were subjected to myocardial infarction (MI) and treated with either vehicle or colchicine for four weeks starting one week after induction of MI. (B) Flow cytometric gating and (C) quantification of leukocyte subsets in atherosclerotic aortas in vehicle- vs. colchicine-treated Apoe^-/- mice which were infarcted five weeks prior (n=9-11 per group, 64-67% female, Student’s t-test for macrophages and neutrophils, Welch’s t-test for monocytes). Neutro: neutrophils, Mø: macrophages and Mono: monocytes (D) Flow cytometric gating and quantification of GFP^high myeloid cells (GFP⁺) in atherosclerotic aortas 24h after adoptive transfer of GFP^high monocytes and neutrophils into vehicle- vs. colchicine-treated Apoe^-/- mice which were infarcted five weeks prior (n=9-11 per group, 64-67% female, Welch’s t-test). (E) Quantification of blood leukocyte subsets in vehicle- vs. colchicine-treated Apoe^-/- mice which were infarcted five weeks prior (n=9-11 per group, 64-67% female, Welch’s t-test for neutrophils, Mann-Whitney U-test for Ly6C^high monocytes, Student’s t-test for Ly6C^low monocytes). Data are presented as mean+s.e.m. Numbers next to gates indicate population frequencies (%). Dots within bar plots show the gender of the mice with a color code: purple (male) and red (female).