Journal: Circulation Research
Article Title: Macrophage DNases Limit Neutrophil Extracellular Trap–Mediated Defective Efferocytosis in Atherosclerosis
doi: 10.1161/CIRCRESAHA.125.326353
Figure Lengend Snippet: DNase deficiency in hematopoietic cells impairs neutrophil extracellular trap (NET) clearance in atherosclerotic plaques. A , The relative levels of DNase1 and DNase1L3 in lesional macrophages (Mac2+), smooth muscle cells (sm-actin [smooth muscle actin]+), and endothelial cells (CD31+) were quantified by measuring DNase1 and DNase1L3 fluorescence intensity in antibody-labeled aortic root sections of 16-week Western diet–fed Ldlr −/− mice. B , Bone marrow–derived macrophages (BMDMs) from wild-type (WT) and Dnase1 −/− Dnase1l3 −/− (double knockout [DKO]) mice were exposed to NETs (250 ng/mL) for either 2 or 6 hours. The quantity of remaining NETs in the supernatant was measured by Picogreen assay, and the NET clearance efficiency was determined relative to input NET concentration. C , BMDMs from WT and DKO mice were incubated for 2 hours with pHrodo-red labeled NETs. After washes, fluorescence microscopy was performed, and the percentage of macrophages that showed engulfment of fluorescent NETs was quantified. D , Eight-week-old female Ldlr −/− mice were lethally irradiated followed by administration of bone marrow cells from either WT or DKO mice. Six weeks postbone marrow reconstitution, WT and hematopoietic cell–specific DNase1/DNase1L3 knockout (DNase-HCKO) mice were fed a Western-type diet for 16 weeks and then euthanized for analysis. E , Brachiocephalic artery from WT and DNase-HCKO mice were homogenized, and the tissue extract was analyzed for DNase activity by single radial enzyme diffusion assay. n=10 mice per group. F , Aortic tree from both groups of mice (n=5) were harvested, fragmented, and maintained in culture as explants, followed by exposure to vehicle or NETs for 4 hours. The supernatant was collected for measurement of ( G ) DNase activity and ( H ) NET clearance efficiency. I , Lesional NET level was quantified by immunostaining for MPO (myeloperoxidase) and citrullinated histone H3 (Cit-H3) in DAPI (4′,6-diamidino-2-phenylindole)–stained aortic root sections of WT and DNase-HCKO mice. n=10 mice per group. Scale bar, 50 µm. J , Hematoxylin and eosin–stained aortic root sections were analyzed for total atherosclerotic lesion area, ( K ) necrotic area, and ( L ) plaque necrosis as a percentage of total lesion area. The regions of plaque necrosis are demarcated by the black dashed line. n=10 to 11 mice per group. Scale bar, 50 µm. M , In situ efferocytosis assay in aortic root sections labeled with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) reagent to detect apoptotic cells (ACs; red) followed by Mac2 immunolabelling (green) to identify lesional macrophages. Lesional efferocytosis efficiency was calculated as the ratio of macrophage-associated ACs to free-lying ACs. White arrows indicate ACs associated with macrophages, whereas white arrowheads show free-lying TUNEL+ cells. n=10 mice per group. Scale bar, 25 µm. N , Quantification of lesional collagen content in Mason’s trichrome-stained aortic root sections. n=10 mice per group. Scale bar, 50 µm. All data are represented as mean±SEM. Test for normality was conducted by the Shapiro-Wilk test. P value was determined by the Mann-Whitney U test ( B , C , E , G , and H ), or unpaired t test ( I through N ). AU indicates arbitrary units; Mφ, macrophage; ND, not detected; and S.N., supernatant.
Article Snippet: After TUNEL labeling, the sections were immunostained with anti-Mac2 (galectin-3) antibody as described above and counterstained with DAPI.
Techniques: Fluorescence, Labeling, Western Blot, Derivative Assay, Double Knockout, Picogreen Assay, Concentration Assay, Incubation, Microscopy, Irradiation, Knock-Out, Activity Assay, Diffusion-based Assay, Immunostaining, Staining, In Situ, TUNEL Assay, MANN-WHITNEY
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![DNase deficiency in hematopoietic cells impairs neutrophil extracellular trap (NET) clearance in atherosclerotic plaques. A , The relative levels of DNase1 and DNase1L3 in lesional macrophages <t>(Mac2+),</t> smooth muscle cells (sm-actin [smooth muscle actin]+), and endothelial cells (CD31+) were quantified by measuring DNase1 and DNase1L3 fluorescence intensity in antibody-labeled aortic root sections of 16-week Western diet–fed Ldlr −/− mice. B , Bone marrow–derived macrophages (BMDMs) from wild-type (WT) and Dnase1 −/− Dnase1l3 −/− (double knockout [DKO]) mice were exposed to NETs (250 ng/mL) for either 2 or 6 hours. The quantity of remaining NETs in the supernatant was measured by Picogreen assay, and the NET clearance efficiency was determined relative to input NET concentration. C , BMDMs from WT and DKO mice were incubated for 2 hours with pHrodo-red labeled NETs. After washes, fluorescence microscopy was performed, and the percentage of macrophages that showed engulfment of fluorescent NETs was quantified. D , Eight-week-old female Ldlr −/− mice were lethally irradiated followed by administration of bone marrow cells from either WT or DKO mice. Six weeks postbone marrow reconstitution, WT and hematopoietic cell–specific DNase1/DNase1L3 knockout (DNase-HCKO) mice were fed a Western-type diet for 16 weeks and then euthanized for analysis. E , Brachiocephalic artery from WT and DNase-HCKO mice were homogenized, and the tissue extract was analyzed for DNase activity by single radial enzyme diffusion assay. n=10 mice per group. F , Aortic tree from both groups of mice (n=5) were harvested, fragmented, and maintained in culture as explants, followed by exposure to vehicle or NETs for 4 hours. The supernatant was collected for measurement of ( G ) DNase activity and ( H ) NET clearance efficiency. I , Lesional NET level was quantified by immunostaining for MPO (myeloperoxidase) and citrullinated histone H3 (Cit-H3) in DAPI (4′,6-diamidino-2-phenylindole)–stained aortic root sections of WT and DNase-HCKO mice. n=10 mice per group. Scale bar, 50 µm. J , Hematoxylin and eosin–stained aortic root sections were analyzed for total atherosclerotic lesion area, ( K ) necrotic area, and ( L ) plaque necrosis as a percentage of total lesion area. The regions of plaque necrosis are demarcated by the black dashed line. n=10 to 11 mice per group. Scale bar, 50 µm. M , In situ efferocytosis assay in aortic root sections labeled with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) reagent to detect apoptotic cells (ACs; red) followed by Mac2 immunolabelling (green) to identify lesional macrophages. Lesional efferocytosis efficiency was calculated as the ratio of macrophage-associated ACs to free-lying ACs. White arrows indicate ACs associated with macrophages, whereas white arrowheads show free-lying TUNEL+ cells. n=10 mice per group. Scale bar, 25 µm. N , Quantification of lesional collagen content in Mason’s trichrome-stained aortic root sections. n=10 mice per group. Scale bar, 50 µm. All data are represented as mean±SEM. Test for normality was conducted by the Shapiro-Wilk test. P value was determined by the Mann-Whitney U test ( B , C , E , G , and H ), or unpaired t test ( I through N ). AU indicates arbitrary units; Mφ, macrophage; ND, not detected; and S.N., supernatant.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2999/pmc12542999/pmc12542999__res-137-1255-g001.jpg)
