PROJECT SUMMARY/ABSTRACT Reperfusion of the ischemic heart is lifesaving in myocardial infarction, but the physiologic processes triggered by reperfusion can cause subsequent damage. The innate immune system plays a prominent role in reperfusion injury. Neutrophils release multiple toxic mediators and the appreciation of the role of neutrophil extracellular traps (NETs) in MI/R pathophysiology is gaining importance. Adenosine is known to mitigate many pro-inflammatory immune processes while ATP released from dying and necrotic cells, activates immune cells. ATP is metabolized to adenosine by ectonucleotidases expressed on many cells including mesenchymal stromal cells (MSCs). MSCs are known to highly express ecto-nucleoside triphosphate diphosphohydrolase-1 (NTDPase1, CD39) and ecto-5’-nucleotidase (CD73). Modulation of purinergic metabolism may be a powerful mechanism used by MSCs to inhibit inflammation in their native bone marrow as well as when transplanted for therapeutic purpose. Currently little is known about endogenous regulation of NET production and we have hypothesize that adenosine may be a key endogenous regulator. Our preliminary data shows that MSC transplanted to the heart encapsulated in alginate can modulate the innate inflammatory response to MI/R. Furthermore, encapsulated MSCs increase myocardial adenosine levels, reduce reactive oxygen species, and decrease the number of leukocytes and neutrophils in the heart after 24 hours. We will use human neutrophils as well as neutrophils isolated from mice lacking key adenosine receptors to understand the role of adenosine signaling in MI/R. We will also use a rodent model of MI/R to test our novel NET-regulatory strategy in vivo. The results of these studies will identify new therapeutic strategies for treatment of innate immune MI/R injury. It is our aims to 1) delineate the importance of adenosine receptor 2A (A2A) signaling in the regulation of NET formation; 2) evaluate the intracellular signaling pathways involved in NET regulation; 3) test the feasibility of MSC modulation of purinergic metabolism to prevent NETs in MI/R.