Sustained elevation of NLRP3 expression primes neutrophils for NET release following ST-segment myocardial infarction

Abstract Background Neutrophils play a pivotal role in myocardial inflammatory injury through release of NETs. The NLRP3 inflammasome has a proximal role in auto-inflammatory diseases and NET release. Aim To evaluate neutrophil NLRP3 expression and function after reperfusion therapy in myocardial infarction (MI). Methods 47 patients with first manifestation of coronary artery disease (CAD) were included in this prospective single center trial. 27 patients with ST-Segment myocardial infarction (STEMI) and 20 with stable CAD. Neutrophils were isolated from peripheral blood at reperfusion therapy and follow-up visit after 30 days. NETosis of isolated neutrophils was visualized using immunofluorescence imaging and quantified using fluorescent readings of Sytox Orange intercalation to measure DNA release associated with NETosis as fold increase to baseline following stimulation. In parallel, following cell lysis and total RNA extraction using Qiagen RNeasy kit absolute quantification of neutrophil mRNA was performed using digital droplet PCR (ddPCR) for inflammasome receptor (NLRP3) and cellular levels of NLRP3 protein was measured by enzyme-linked immunosorbent assay (ELISA). Results Compared with stable CAD patients, NLRP3 expression in neutrophils from patients with STEMI was significantly upregulated following reperfusion (0.025 vs 0.012; p=0.009) and remained elevated after 30 days (0.020 vs. 0.011): p=0.010) Neutrophils from patients with STEMI had higher NLRP3 protein expression both after reperfusion (2.01 ±0.70 pg/ml vs 0.67 ±0.20 pg/ml: p=0.005) and at 30 days following the index event (0.95 ±0.17 pg/ml vs. 0.43 0.23 pg/ml): p=0.03) compared to stable CAD patients. Immunostaining of fresh isolated neutrophils of patients with STEMI showed that they were significantly primed for NET release when compared to CAD patients at both time points (1,81 vs. 1.32; p=0.03 and 2.44 vs. 1.39; p=0,02). Conclusion In the immediate aftermath post MI and revascularization in humans, neutrophil express higher levels of NLRP3 and are primed for NETosis. These inflammatory markers remain elevated at 30 days post MI compared to patients with chronic coronary syndrome pointing towards neutrophil trained immunity in MI patients and potential benefits of anti-inflammatory treatment.