Neutrophil extracellular traps promote macrophage inflammation and impair atherosclerosis resolution in diabetic mice

Abstract

Neutrophil extracellular traps (NETs) promote inflammation and atherosclerosis progression. NETs are increased in diabetes and impair the resolution of inflammation during wound healing. Atherosclerosis resolution, a process resembling wound healing, is also impaired in diabetes. Thus, we hypothesized that NETs impede atherosclerosis resolution in diabetes by increasing plaque inflammation. Indeed, transcriptomic profiling of plaque macrophages from NET+ and NET- areas in low-density lipoprotein receptor-deficient (Ldlr-/-) mice revealed inflammasome and glycolysis pathway upregulation, indicating a heightened inflammatory phenotype. We found that NETs declined during atherosclerosis resolution, which was induced by reducing hyperlipidemia in nondiabetic mice, but they persisted in diabetes, exacerbating macrophage inflammation and impairing resolution. In diabetic mice, deoxyribonuclease 1 treatment reduced plaque NET content and macrophage inflammation, promoting atherosclerosis resolution after lipid lowering. Given that humans with diabetes also exhibit impaired atherosclerosis resolution with lipid lowering, these data suggest that NETs contribute to the increased cardiovascular disease risk in this population and are a potential therapeutic target.

Keywords: Atherosclerosis; Cardiology; Diabetes; Inflammation.

Figure 1. Neutrophil extracellular traps in atherosclerotic plaques skew macrophages to a proinflammatory phenotype.

(A) Representative atherosclerotic plaque section stained for neutrophil extracellular traps (NETs), as determined by colocalization of myeloperoxidase (MPO), the lymphocyte antigen 6 complex locus G6D (Ly6G), citrullinated histone H3 (H3Cit), and macrophages (CD68). CD68⁺ cells were collected via laser capture microdissection (LCM) in NET⁺ and NET^– areas and sequenced. Scale bar: 100 μm. (B) Heatmap and (C) volcano plot showing differential gene expression in NET⁺ versus NET^– macrophages. (D) Upregulation of the glycolysis and (E) inflammasome pathways in NET⁺ compared with NET^– macrophages, as determined using Ingenuity Pathway Analysis. (F) Immunofluorescence staining and (G) quantification confirming the inflammasome pathway activation (nod-like receptor family pyrin domain-containing 3 [NLRP3]) in NETs⁺ area. Scale bar: 50 μm. (H) Quantification of iNOS immunofluorescence staining showing an increase in iNOS in NET⁺ versus NET^– areas (%DAPI). n = 3–5/group, *P < 0.05 using unpaired t test.

Figure 2. DNase1 treatment promotes atherosclerosis resolution in diabetic mice.

(A) Male Ldlr^–/– mice were fed a Western diet for 16 weeks to develop baseline plaques. At week 15, a subset of mice received streptozocin injections to induce diabetes. At week 16, a subset of mice were harvested for baseline measures, and all other mice were switched to a chow diet to induce resolution of plaques. These mice were then split into 2 groups and over a 4-week period received either DNase1 (62.5 μg/mouse) or vehicle injections every other day. At week 20, mouse tissues were harvested for plaque analyses. (B) NET staining and (C) quantification in aortic roots, as determined by composite staining of myeloperoxidase (MPO), the lymphocyte antigen 6 complex locus G6D (Ly6G), and citrullinated histone H3 (H3Cit). Scale bar: 100 μm. (D) Representative images and (E) quantification of CD68 staining as a marker of plaque macrophage content. Scale bar: 100 μm. Data are shown as mean ± SEM. n = 7–12/group, *P < 0.05, 1-way ANOVA with Tukey’s multiple comparison test.

Figure 3. Effects of DNase1 treatment on plaque size, collagen content, and necrotic area in atherosclerotic plaques.

More analyses of mice described in Figure 2. Quantification of (A) plaque size, (B) Picrosirius red staining to determine collagen content, and (C) plaque necrotic area. Data are shown as mean ± SEM. n = 4–12/group, *P < 0.05, ** P < 0.005, ***P < 0.001 1-way ANOVA with Dunnett’s multiple comparison test compared with the baseline (A and B) or diabetes (C) group.

Figure 4. DNase1 treatment promotes atherosclerosis resolution in diabetic mice by reducing NETs-induced plaque macrophage inflammation.

More analyses of mice described in Figure 2. (A) Overlapping NET (left) and CD68 staining (right) in diabetic mice versus diabetic mice treated with DNase1. Scale bar: 50 μm. (B) Percentage of macrophages colocalized with NETs. (C) Quantification of NLRP3⁺ and caspase-1⁺ cells and (D) iNos⁺ cells, all as the percentage of DAPI staining of plaques. Data are shown as mean ± SEM. n = 3–5/group, *P < 0.05, 1-way ANOVA with Tukey’s multiple comparison test.

Figure 5. Effects of diabetes and DNAse1 treatment on cholesterol crystal content in resolving plaques.

More analyses of mice described in Figure 2. (A) Cholesterol crystal visualization (white) and (B) quantification (as % plaque area) in atherosclerotic plaques with DAPI (blue) used as a counterstain. Scale bar: 100 μm. Data are shown as mean ± SEM. n = 5–8/group, *P < 0.05, 1-way ANOVA with Tukey’s multiple comparison test.