Accumulating evidence suggests that CD4+ T cells mediate neutrophil recruitment and organ injury during ischemia and reperfusion (I/R). However, the precise mechanisms by which CD4+ T cell populations, circulating or tissue resident CD4+ T cells, contribute to myocardial I/R injury are not fully understood. A role of the spleen in mediating I/R injury has been reported in the kidney and brain. But, the role of spleen in mediating inflammatory response during acute phase of myocardial reperfusion injury remains unknown. Previous studies related to the spleen are concentrated on role of the spleen, especially the splenic mononuclear cells, on post-MI remodeling and later phase (>24 hours of reperfusion) of I/R injury. Different from post-MI remodeling, we found that during acute phase of post-ischemic reperfusion, circulating lymphocytes and monocytes are decreased. The myocardial infarct size finalized within one hour of reperfusion versus 24 hours of reperfusion. Thus, inflammatory response within one-hour reperfusion is more critical in causing myocardial tissue necrosis. Our preliminary study has shown that substances, HMGB1 and mitochondrial DNA (mtDNA), were released from the ischemic myocardium and activated splenocytes upon reperfusion by acting on RAGE and/or possible TLR9 receptors. Splenectomy before I/R reduced myocardial infarct size. Acute adoptive transfer of normal splenocytes into the bloodstream of the splenectomized mice restored infarct size to that of control mice. These results indicate that the spleen plays a central role in myocardial I/R injury via HMGB1/mtDNA?RAGE/TLR9 pathway. We therefore hypothesize that splenic CD4+ T cells are activated via a cardio-splenic axis during reperfusion and contribute importantly to myocardial I/R injury. To test these hypotheses, we will first determine the role of splenic CD4+ T cells in myocardial I/R injury by using WT mice with or without splenectomy in combination with adoptive transfer of splenocytes from WT, RAGE-/- and CD4-/- mice. Following these experiments, we will test that infarct-sparing effect of an A2AR agonist is due to its action on A2ARs of splenic CD4+ T cells and will develop a liposome system to deliver the A2AR agonist to the spleen to inhibit the splenic leukocytes. Secondly, we will determine that a cardio-splenic axis causes I/R injury via a HMGB1/mtDNA (from ischemic myocardium) - RAGE/TLR9 (on splenic CD4+ T cells) pathway. We will employ a mouse model with shorter I/R, 20-min ischemia and 60-min reperfusion and treat mice with ischemic heart homogenates (IHH) or coronary perfusate (CP) from mice with 40-min ischemia and plasma acquired at 5-min reperfusion. The HMGB1 and/or mtDNA in IHH, CP or plasma will activate splenic CD4+ T cells by either binding to RAGEs or to TLR9 receptors and exacerbate infarct size. Finally, we will determine if the therapeutic blockade of HMGB1 or mtDNA in the blood will suffice to block the cardio-splenic axis, reduce myocardial infarct size and preserve cardiac function.

Public Health Relevance

Splenic CD4+ T cells mediate myocardial ischemia-reperfusion injury Ischemic heart disease remains the single leading cause of death in the United States, accounting for one out of every five deaths. Myocardial infarction (heart attack) and heart failure account for the vast majority of the morbidity and mortality associated with ischemic heart disease. Over the last twenty years, genetically modified mice have greatly expedited biomedical research aimed at unraveling the role of individual genes in the progression of heart attack. Our previous studies have demonstrated CD4+ T lymphocytes play a pivotal role in mediating heart attack. This research project will further determine that the splenic CD4+ T cells, not the circulating CD4+ T cells, mediate the process of heart attack, explore how substances released from damaged heart activate splenic CD4+ T cells and identify new treatment strategies targeting the spleen, specifically use targeted delivery of adenosine 2A receptor agonist to the spleen during the procedures of restoring coronary blood flow, to reduce myocardial infarction.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Surgery, Anesthesiology and Trauma Study Section (SAT)
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Schwartz, Lisa
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University of Virginia
Schools of Medicine
United States
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