The Inflammatory Landscape during Myocardial Reperfusion Injury Depends on Distinct Neutrophil Phenotypes

Objectives: A hallmark of acute phase reperfusion injury following myocardial infarction (MI) is a leukocyte driven inflammatory response. During the first hours neutrophils are the most abundant cells that are recruited toward the heart and start migrating into the affected tissue within the first day after MI. It is acknowledged that neutrophils can shift between a pro- and anti-inflammatory state depending on their maturation state under disease conditions. However, in depth evaluation of the physiological impact of different neutrophil subsets is currently incomplete and presents a major research focus today. We reasoned that neutrophils could display different phenotypes in the course of reperfusion injury which might be associated with healing and scar formation of the heart. Methods: Mice were divided in 4 groups and subjected to 40 minutes of myocardial ischemia followed by reperfusion. Heart function was monitored over time using echocardiography and mice were sacrificed at day 1, 3, or 7 to quantify neutrophil count in the heart, blood, spleen and bone marrow. Expression of adhesion molecules (CD206, PSGL-1, CD62L, LFA-1, CD11b, CD177, CD49b) and activation state in each compartment was validated using flow cytometry. Results: Neutrophil count within the area at risk (AAR) reached a peak at day 3 (7887 cells/mg tissue) after induction of experimental MI and was relatively low at day 7 (167 cells/mg tissue). The proportion of pro- (CD206-) and anti-inflammatory (CD206+) neutrophils in the AAR shifted over the course of 7 days from > 99% CD206- at day 1, 80–95% at day 3, and 55–75% at day 7. Circulating neutrophils reflected a similar distribution with an average of 70% CD206- at day 1, 58% at day 3, and 38% at day 7. In line, the activation state was altered in blood neutrophils with CD11b showing peak expression at day 3 (40% MFI increase compared with d1) and reaching normal levels around day 7. Interestingly, LFA-1 was similarly increased at day 3 (70% MFI increase) but no decline was observed at day 7. Conclusions: Here we show for the first time that neutrophils change from a pro- to an anti-inflammatory phenotype during the first 7 days after MI. This is reflected in different expression profiles of the most relevant adhesion molecules which also represent alterations in the maturation state of neutrophils. It remains to be elucidated how neutrophils trigger recruitment and activation of other immune cells like monocytes, macrophages or platelets in the course of myocardial reperfusion. This preliminary study underlines the predominant role of neutrophils in acute phase cardiovascular inflammation and manipulating the switch between different states could become a promising approach to improve the outcome of patients suffering from MI.