Abstract Body (Do not enter title and authors here): Background: The NLRP3 inflammasome has been implicated in neutrophil polarization and neutrophil extracellular trap (NET) extrusion and facilitates secretion of interleukin-1β (IL-1β).
Research Questions: Evaluate the role of neutrophil NLRP3 inflammasome in IL-1β production, von Willebrand Factor (VWF) release, and NETosis following myocardial Infarction (MI).
Methods: 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 WT or NLRP3^−/− neutrophils. Echocardiography and triphenyltetrazolium chloride (TTC) staining were performed at 12 h to evaluate cardiac function and infarct size. NETs were quantified using immunofluorescence staining for Ly6G and H3Cit and tissue levels of IL-1β by ELISA. In-vivo recruitment of externally labeled donor neutrophils and cell counts were measured by Flow Cytometry (FC).
Results: NLRP3 deficiency reduced infarct size to roughly a third of WT heart injury (22.92±3.9% vs. 70.7±1.5 % of LV; n= 6 vs. 4; p<0.01) and preserved left ventricular (LV) function (42.3±1.9 % vs. 26.1±2.1% Ejection Fraction, EF; p=0.009) at 12 hours after MI as assessed by echocardiography and TTC staining. Transfusion of WT but not NLRP3^−/− neutrophils after MI increased infarct size in NLRP3^−/− mice (46.0±2.6% vs. 26.1±4.4%; n=7; p<0.01) and significantly reduced LV function (30.8±2.6 % vs. 40.8±1.5 % EF; n=7; p=0.006). The key features of myocardial tissue in WT neutrophil transfused recipients were increased H3Cit-positive deposits with NET-like morphology (6.03±1.6% vs. 0.79±0.39% H3Cit⁺, n=5; p=0.02), tissue levels of IL-1β (8.4±1.1 vs. 3.3±1.0 pg/mg; n=6 vs. 5; p<0.5), and plasma levels of VWF (830.4±62.6 vs. 472.4±63.4 ng/ml; n=7; p=0.002). FC analysis also revealed that neutrophil NLRP3 increased the number of labeled and transfused neutrophils in the bone marrow of recipient mice following MI (0.68±0.05 % vs. 0.40±0.04 %; n=6; p<0.01).
Conclusion: Our data suggest a key role for neutrophil NLRP3 in the production of IL-1β and 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.