Increased NETosis in Patients With Heart Failure Is Associated With Macrophage Activation Towards a Proinflammatory Phenotype

Figures

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  FIGURE 1. Neutrophils are increased in HF patients. Representative confocal micrographs of CD66b‐positive cells (green, white arrows) in a control (A), DCM (B) and in a patient with infCM (C). Notice a remarkable increase in CD66b‐positive cells in HF patients. Nuclei are shown in blue after staining with DAPI, and F‐Actin is shown in red after labelling with Alexa647‐phalloidin.

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  FIGURE 2. NETosis is increased in HF. Typical immunofluorescent examples of citH3‐positive cells (arrows) in a control patient (A) and in a patient with ICM (B). The insert in (B) is a higher magnification of the boxed region and shows NETS (arrowheads), which are positive for citH3 and DAPI. F‐actin is shown in green and nuclei in blue. MPO‐positive cells in a control (C) and in a patient with infCM (D). Notice a marked increase of the MPO‐positive cells in the latter patient. F‐actin is shown in red and nuclei in blue.

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  FIGURE 3. Representative confocal images of CD66b/citH3− (A, B) and CD66b/MPO/citH3‐positive cells (C, D), as well as quantifications of these cells in the groups studied (E, F).

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  FIGURE 4. In HF patients, the number of macrophages is increased and correlates positively with the number of neutrophils. Representative immuno‐fluorescence images of CD68‐positive cells (red, arrows) in control groups (A), DCM (B), ICM (C) and infCM (D) as well as the quantitative analysis of macrophages in the examined groups (E). The total number of macrophages per myocardial tissue area correlates significantly with the number of neutrophils (F).

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  FIGURE 5. The phenotype of macrophages in the control group. Representative confocal images of double labeling of CD68 (green) and CD206 (arrowhead in B) in a control myocardium (A, B). F‐actin is labelled in red (A) and grey (B). Triple labeling for CD68 (C) with CD206 (arrowhead in E and F) and TNF‐α (arrow in E and F) in a control patient. Note that of seven CD68‐positive cells (C), only one cell is CD206‐positive (D) and another cell is TNF‐α‐positive (E). Panel (F) is the superimposition of all three markers; the cell nuclei are coloured pseudo‐magenta, and F‐actin is shown in grey.

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  FIGURE 6. The phenotype of macrophages in a patient with DCM. Representative confocal image of triple labeling for CD68 (A), TNF‐α (B) and CD206 (C). Panel D is the overlay of all three labels and shows that the majority of yellow‐labelled macrophages are TNF‐α‐positive cells that are mixed with some CD206‐positive cells (arrows). In panel (D), the cell nuclei are coloured pseudo‐magenta, and F‐Actin is shown in grey.

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  FIGURE 7. The M1‐like macrophages predominate over M2‐like macrophages in a patient with infCM. Triple labeling of CD68 (grey in A), IL‐6 (green in B) and arginase 1 (red in B). Note that IL‐6‐positive cells (white arrows) are more numerous than arginase 1‐positive cells (yellow arrows). F‐actin is marked red in (A) and grey in (B). Cell nuclei are stained blue with DAPI.

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  FIGURE 8. Boxplots of the percentage of CD66b+/TNF‐α+ M1‐ and CD66b+/CD206+ M2‐like macrophages in the examined groups (A, B) and the correlation analysis between these cells and the number of citH3/MPO/CD66b‐positive cells (C, D).

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  FIGURE 9. NETosis is more strongly associated with the number of M1‐like rather than with M2‐like macrophages. Representative confocal images of triple labeling for citH3 (purple colour resulting from co‐localization of the red signal of citH3 with the blue colour of DAPI), TNF‐α (white arrows) and CD206 (yellow arrows) in a patient with ICM. Note that citH3 positivity is accompanied by the higher number of TNF‐α‐positive cells rather than with some CD206‐positive cells. Asterisks denote nuclear citH3 and arrowheads indicate typical long NET structures.