Novel mechanism in cardiac injury: immune checkpoints

Abstract

Abstract Background Immune checkpoint inhibitors (ICI), specifically directed against CTLA-4 and PD-1, have revolutionized cancer therapy but are associated with immune-related adverse events, including fulminant myocarditis. The mechanisms are unknown, but one possibility is that CTLA-4 and PD-1 play a critical role in cardiovascular homeostasis. Purpose The purpose of this study is to investigate the role of these immune checkpoints in cardiac injury. We hypothesize that cardiomyocytes can express immune checkpoint ligands in response to stress and that CTLA-4 and/or PD-1 play a key role in cardiac response to injury. Methods We measured expression levels of CTLA-4 ligands (Cd80, Cd86) and PD-1 ligands (Pdcdl1, Pdcdl2) in in vitro and in vivo models of cardiac injury, including iPSC-derived cardiomyocytes (iPSC-CM) and diseased human cardiac samples. Immunofluorescent staining and multiplex immunohistochemistry were used to derive more granular data on cell type expressing specific immune checkpoint associated proteins. To determine the functional role of CTLA-4 and PD-1 in cardiac injury, myocardial infarction (MI) was induced in C57Bl/6 mice treated with anti-CTLA-4 or in mice with a genetic knock-out of CTLA-4 and PD-1 (Pdcd1−/−Ctla4+/+ and Pdcd1−/−Ctla4+/−). Flow cytometry was performed 2-days post-MI to determine immune cell infiltration, echocardiography was performed 7-days and 28-days post-MI and plasma samples were analyzed for ANP and Troponin I. Results Doxorubicin or hypoxia increased expression of Cd80, Cd86, Pdcdl1 and Pdcdl2 in iPSC-CM. After MI, isolated cardiomyocytes from the ischemic/border zone area yielded significant increased expression of both Cd80 and Cd86, which was confirmed at the protein level. However, pharmacologic inhibition of CTLA-4 during MI resulted in better survival compared to no treatment (p<0.007). No differences were seen in immune cell infiltration, troponin I and ANP levels and echocardiography. Pdcd1−/-Ctla4+/+ and Pdcd1−/−Ctla4+/− mice showed a decrease in immune cell infiltration. Conclusions Whole hearts, isolated cardiomyocytes and iPSC-CM from both mice and humans express immune checkpoint ligands in response to cardiac injury. Pharmacologic or genetic inhibition of CTLA-4 and PD-1 in MI did not result in adverse effects regarding survival, cardiac function, immune cell infiltration and heart enzyme levels in mice. These data support the hypothesis that immune checkpoint pathways play a role in cardiac injury and in these preliminary studies immune checkpoint inhibition during cardiac ischemic injury did not result in adverse effects. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): National Institutes of Health grants R56 HL141466 and R01 HL141466