Neutrophil NLRP3 promotes cardiac injury following acute myocardial infarction through IL-1β production, VWF release and NET deposition in the myocardium

Abstract

NLRP3 inflammasome has been implicated in neutrophil polarization and extrusion of neutrophil extracellular traps (NETs) in vitro and facilitates secretion of Il1-beta (IL-1β). Permanent ligation of the left anterior descending artery was used to induce MI in WT and NLRP3^-/- mice as well as in NLRP3^-/- recipient mice transfused with either WT or NLRP3^-/- neutrophils. NLRP3 deficiency reduced infarct size to roughly a third of WT heart injury and preserved left ventricular (LV) function at 12 h after MI as assessed by echocardiography and triphenyltetrazolium chloride staining of live tissue. Transfusion of WT but not NLRP3^-/- neutrophils after MI increased infarct size in NLRP3^-/- mice and significantly reduced LV function. The key features of myocardial tissue in WT neutrophil transfused recipients were increased H3Cit-positive deposits with NET-like morphology and increased tissue levels of IL-1β and plasma levels of von Willebrand Factor (VWF). Flow cytometry analysis also revealed that neutrophil NLRP3 increased the number of labeled and transfused neutrophils in the bone marrow of recipient mice following MI. Our data suggest a key role for neutrophil NLRP3 in the production of IL-1β and deposition of NETs in cardiac tissue exacerbating injury following MI. We provide evidence for a link between neutrophil NLRP3 and VWF release likely enhancing thromboinflammation in the heart. Neutrophil NLRP3 deficiency conferred similar cardioprotective effects to general NLRP3 deletion in MI rendering anti-neutrophil NLRP3 therapy a promising target for early cardioprotective treatment.

Figure 1

Nlrp3 deficiency alleviates infarct size in the early inflammatory phase. (A) Depiction of the experimental design: Ligation of the left anterior descending artery (LAD) in WT/NLRP3^+/+ and KO/NLRP3^−/− was used to induce MI. Mice were evaluated for heart function and euthanized ≈ 12 h post-MI. (B) Representative pictures of Triphenyl tetrazolium chloride (TTC) staining at 12 h post MI with viable myocardium staining red and infarcted/necrotic area white (*) (scale bar 2 mm). (C) Quantification of Infarct size defined as percent of non-TTC stained Left ventricular (LV) area calculated in ImageJ. (D) Representative M-Mode images, depicting end-systolic (Sys) and end-diastolic (Dia) left ventricular diameters, acquired using echocardiography, at indicated timepoints in WT/NLRP3^+/+ and NLRP3^−/− mice after MI. (E) Ejection fraction (EF), Fractional Shortening (FS), left ventricular end-diastolic volume (LVEDV), and end-systolic (LVESV) volume as assessed by echocardiography at 10 h following MI. (F) Plasma levels of Troponin-I as measured by ELISA in WT/NLRP3^+/+ and NLRP3^−/− mice with MI. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; Mann–Whitney U-test

Figure 2

NLRP3^+/+ neutrophils restore adverse infarct outcome in Nlrp3^−/− mice and provide a major source of IL-1β in infarcted myocardium. (A) Experimental design depicting the strategy for transfer of neutrophils isolated from WT and NLRP3^−/− (KO) mice (donors) into NLRP3^−/− mice (recipients) (n = 7). Purified neutrophils (≈ 99.4%) from the bone marrow (BM) of donors were pooled, labeled with CellTrace Carboxyfluorescein succinimidyl ester (CFSE), and injected in equal numbers into NLRP3^−/− recipients then subjected to MI (LAD-Ligation). Blood from the recipients was collected and echocardiographic analysis was performed at termination (≈ 12 h post-MI). (B) Representative pictures of TTC staining at 12 h post MI with viable myocardium staining red and infarcted/necrotic area staining white (*) (scale bar 2 mm). (C) Comparative analysis of infarct size in both groups defined as percent of non-TTC stained LV area calculated in ImageJ. (D) Quantitative comparison of plasma levels of Troponin-I determined using ELISA. (E) Representative M-Mode images, acquired using echocardiography. Systole (Sys), Diastole (Dia) and LV are marked with arrows. (F) Ejection fraction (EF), left ventricular end-diastolic (LVEDV) and end-systolic (LVESV) volume as assessed by echocardiography. (G) Quantitative comparison of heart tissue levels of IL-1β determined using ELISA. (H) Quantitative comparison of plasma levels of IL-1β determined using ELISA. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; Mann–Whitney U-test.

Figure 3

Neutrophil NLRP3 promotes NETosis in ischemic myocardium and homing to the BM in the early inflammatory phase. (A) Representative image of LV section of recipients transfused with NLRP3^+/+ donor neutrophils immunostained for DAPI (blue), H3Cit (green) and Ly6G (red) at 100X magnification (left). Representative images at 50X magnification of LV sections of recipients transfused with either NLRP3^−/− (top right) or NLRP3^+/+ neutrophils (down right) immunostained for DAPI (blue) and H3Cit (green). Extracellular H3Cit (*) next to Ly6G⁺ cells are considered NETs. Intranuclear staining of H3Cit (^x) is considered citrullination of Histone 3 without NET release (scale bar 20 µm). (B) Comparative analysis of percent of total extracellular H3Cit⁺ (green) in LV heart sections quantified by ImageJ. (C) Comparative analysis of total Ly6G⁺ cell counts (per mm²) of LV heart sections counted manually by two independent operators in ImageJ. (D) Flow cytometry gating strategy used for isolated bone marrow cells. CD11b⁺ and CD45⁺ double-positive events were considered myeloid cells and Ly6G⁺ (out of CD11b⁺ and CD45⁺/myeloid cells) were considered neutrophils. Neutrophils (Ly6G⁺CD11b⁺CD45⁺) staining positive for CellTrace CFSE were considered originally labeled and transfused bone marrow neutrophils (BMN). (E) Quantitative comparison of originally labeled and transfused bone marrow neutrophils (% of CellTrace⁺ events out of Ly6G⁺ [CD11b⁺CD45⁺] events). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; Mann–Whitney U-test.

Figure 4

Neutrophil NLRP3 exacerbates endothelial activation with increased VWF deposition and ICAM-1 expression. (A) Representative images of LV sections of recipients transfused with either NLRP3^−/−- or NLRP3^+/+-neutrophils immunostained for DAPI (blue), von Willebrand Factor (VWF) (green), an endothelial adhesive protein deposited in the vasculature, and CD31 (pink), an endothelial cell marker (scale bar 20 µm). VWF deposits (*) in the Vessel lumen (V) (B) Comparative analysis of VWF deposition in % of LV sections quantified by ImageJ. (C) Quantitative comparison of plasma levels of VWF (ng/ml) determined using ELISA. (D) Representative images of LV sections of recipients transfused with either NLRP3^−/−- or NLRP3^+/+-neutrophils immunostained for DAPI (blue), CD31 (green) and ICAM-1 (intercellular adhesion molecule-1) (red), an endothelial adhesion molecule upregulated upon activation (scale bar 50 µm). Vessel lumen (V), ICAM-1 (*). (E) Comparative analysis of ICAM-1 and CD31 positive area in % of LV quantified by ImageJ. (F) Representative images of LV sections of recipients transfused with either NLRP3^−/−- or NLRP3^+/+-neutrophils immunostained for DAPI (blue), tumor necrosis factor α (TNF-α) (green)(^x), a proinflammatory and cytotoxic mediator secreted by macrophages and CD68 (red) (*), a surface marker mainly expressed by monocytes and macrophages (scale bar 20 µm). (G) Comparative analysis of CD68⁺ cells relative to all cells (DAPI⁺) counted manually by two independent operators in ImageJ. (H) Comparative analysis of TNF-α positive area in % of LV quantified by ImageJ. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; Mann–Whitney U-test.