REG3β limits lymphatic drainage of cardiac leukocytes and improves resolution of myocardial inflammation

Abstract

The initiation and resolution of immune-inflammatory processes through precise regulation of leukocyte accumulation from the blood and lymphatic drainage, respectively, are critical for efficient wound healing and tissue remodeling after myocardial infarction. Previous work from my group identified regenerating islet-derived protein 3 beta (REG3β) as a novel cardiomyocyte-derived secretory protein that restricts neutrophil accumulation in the injured myocardium. In the present work, I investigated the role of the cardiac lymphatic network in REG3β-mediated resolution of cardiac inflammation. Flow cytometry-based kinetic immune cell profiling in combination with light sheet fluorescence microscopy imaging of the lymphatic network of infarcted hearts revealed a persistent accumulation of innate immune cells and increased lymphatic vessel formation in REG3β-deficient (Reg3b-/-) mice. The Reg3b mutants showed an accumulation of Ly6Chi macrophages, which stimulated lymphangiogenesis by secretion of VEGF-C/-D. In addition, the levels of chemokines CCL19 and CCL21 were higher in the walls of the lymphatic vessels in the heart of infarcted Reg3b-/- mice. The enhanced expression of CCL19 and CCL21 correlated with an increased emigration of CCR7+ pHrodo dye-labelled cardiac neutrophil granulocytes and macrophages towards mediastinal lymph nodes and bone marrow. AAV9-mediated overexpression of sVEGFR3 blocked this process. RNAseq analysis of heart-derived leukocytes in the bone marrow showed that such leukocytes are able to promote granulopoiesis. Indeed, more neutrophil granulocytes were produced in the bone marrow of Reg3b-/- mice after myocardial infarction, which explains the continuous infiltration of newly formed BrdU+ neutrophils into infarcted Reg3b-/- hearts. Additionally, an increased proliferation of macrophages was found in the infarcted hearts of Reg3b mutants, which is probably the cause for the significantly increased amount of myeloid leukocytes during myocardial healing. Overall, my findings suggest an increased inflammatory circuit upon myocardial infarction in which REG3β restricts lymphatic drainage and homing of cardiac leukocytes to the bone marrow, thereby attenuating signals that stimulate the production of new immune cells and cause excessive cardiac inflammation.