Programmed cell death protein 1 (PD1) deficiency induces cardiac inflammation during baseline conditions and ischemia/reperfusion injury

Abstract

Abstract Background The Programmed cell death protein 1 (PD1) immune checkpoint is densely expressed on cardiac endothelial cells. The increasing clinical application of immune checkpoint inhibitor therapy targeting PD1 for the treatment of advanced malignancies has revealed profound cardiovascular side effects that have recently paralleled in a preclinical model. However, the effects of PD1 deficiency during baseline conditions and myocardial injury are so far unknown. Purpose This study aims to assess the impact of PD1 deficiency on myocardia immunity, and to evaluate the relevance of PD1 signalling in cardiac disease. Methods C57BL/6J wild-type mice and C57BL/6J Pdcd1−/− mice were purchased and bred at the animal facility. In-vivo ischaemia/reperfusion (I/R) injury was applied to assess the response in cardiac injury. In brief, mice were anesthetised followed by lateral thoracotomy and ligation of the left coronary artery for 45 min. For flow cytometry, hearts were removed and subjected to an enzymatic digestion. Single cell solutions were stained with different antibody panels to assess cardiac immune cells and the expression of programmed cell death protein 1 ligand 1 (PDL1). Western blot was conducted after homogenization of snap-frozen heart tissue using specific primary and corresponding secondary antibodies. Immunofluorescence and conventional haematoxylin and eosin stain from 4 μm sections were used to visualize the distribution of PDL1 in cardiac tissue. Results While no overt cardiac phenotype was observed in Pdcd1−/− mice, a profound upregulation of pro-inflammatory cytokines was determined during baseline conditions, including interleukin (IL) 1α IL4, and extracellular-signal regulated kinase (ERK) 1/2. NADPH oxidase 1 (NOX1) which is involved in production of reactive oxygen species and endothelial injury response was downregulated in Pdcd1−/− mice. Following I/R injury, a significant decline in endothelial PDL1 expression was observed, which was attributed to changes in the area at risk, as shown by immunofluorescence staining. The infarct size following in-vivo I/R injury was not altered upon PD1-deficiency as determined by TTC staining. However, flow cytometry determined increased cell numbers of distinct leukocyte subsets during reperfusion-related inflammation. Conclusion Deficiency of PD1/PDL1 signalling shows distinct inflammatory changes in cardiac tissue at baseline and I/R injury. The results indicate that relevant PD1-related detrimental effects are not limited to complications from cancer therapy but can be expected in various forms of cardiovascular disease, hence requiring further investigations. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Department of Cardiology and Vascular Medicine, West German Heart and Vascular Center, University Hospital Essen, Germany