1Universitätsklinikum Essen Klinik für Kardiologie und Angiologie Essen, Deutschland; 2Universitätsklinikum Essen Cardio Science Labs Essen, Deutschland
Background After reperfused acute myocardial infarction (repAMI), clearance of cellular debris is vital to cardiac healing. CD47 represents an immune cell checkpoint, modulating migration of and preventing phagocytosis by myeloid cells. Inhibition of CD47 signalling was shown to enhance tumor cell phagocytosis by macrophages and to reduce infarction size following repAMI, by increasing macrophage activity. Whether this mechanism is also present in neutrophils, as the first and most abundant infiltrating immune cells during repAMI, is incompletely understood.
Methods & Results We subjected mice to an in vivo ischemia/reperfusion (I/R) protocol with 45 min of ischemia and 24 h of reperfusion, with and without CD47 blockade. I/R injury size was determined by TTC/EB staining and was reduced in anti-CD47 treated mice. We hypothesized this reduction to be linked to immune cells, as both ex vivo Langendorff I/R and in vitro human cardiomyocyte (CM) hypoxia/reoxygenation models did not benefit from disrupting CD47 signalling. We could show that CD47 is expressed on endothelial cells, fibroblasts and CMs using flow cytometry and that this expression is increased after repAMI inside the infarction zone with immunostaining of histological slices. Total immune cell numbers did not differ between control and treatment groups in flow cytometry analysis, but using light sheet fluorescence microscopy, we were able to delineate higher neutrophil numbers per damaged area in anti-CD47 treated mice. We then induced apoptosis in isolated, primary mouse CMs labelled with Calcein by adding 2 µM staurosporine to culture medium for 2 h. Apoptotic CMs were co-cultured with mouse neutrophils in a 5:1 ratio with either CD47 or IgG antibody present. CD47-inhibited neutrophils showed higher phagocytosis than IgG controls.
Conclusion Our findings demonstrate that infarction size reduction by CD47 blockade following repAMI is associated with altered infiltration and most likely activity of neutrophils, as shown by the phagocytosis assay. Whether this modulation of neutrophils plays a grander role for the benefits of CD47 inhibition after repAMI needs to be investigated using long-term healing models.