Lymphatic-macrophage crosstalk during neonatal mouse heart regeneration and transition to fibrotic repair

Abstract Background In adult mice, myocardial infarction (MI) activates the cardiac lymphatics, which undergo sprouting angiogenesis (lymphangiogenesis) and function to drain interstitial fluid and traffic macrophages to mediastinal lymph nodes (MLNs). This prevents oedema and reduces inflammatory immune cell content to improve cardiac function. Purpose Given the importance of the adult cardiac lymphatics in macrophage clearance after injury, we investigated their role across the neonatal "regenerative window". At post-natal day 1 (P1) neonatal mice fully regenerate their heart following MI, in a macrophage-dependent manner, whereas equivalent injury at day 7 (P7) leads to scarring driven by pro-fibrotic macrophages. We hypothesised that lymphatics respond and function differently during this "window," to clear macrophage-specific subtypes depending upon their requirement for regeneration versus fibrotic repair. Methods & Results Extensive lymphatic growth and sprouting was evident in intact neonatal hearts until P16, with strain-dependent developmental differences. The response to injury revealed limited lymphangiogenesis and minimal clearance of macrophages from P1 compared to P7 infarcted hearts, as determined by adoptive transfer experiments and monitoring of cleared macrophages to MLNs. This is coincident with maturation of lymphatic endothelial cell junctions across the neonatal period via transition from "zipper" (impermeable) to "button" (permeable) -type junctions. To gain molecular insight into the mechanisms underpinning lymphatic endothelium macrophage interactions at P1 versus P7, we generated unbiased single cell RNA sequencing datasets from neonatal heart samples after MI and observed altered signalling between lymphatic endothelial cells and macrophages across the neonatal period, including altered expression of the lymphangiocrine factor Reelin (RELN). Finally, in mice lacking the lymphatic endothelial receptor-1 (LYVE1), that exhibit impaired transmigration of interstitial macrophages to lymphatic vessels, magnetic resonance imaging (MRI) revealed a surprising impaired functional outcome in P1 mice 28 days post-MI. Given our observations that pro-regenerative macrophages at P1 are not trafficked, this suggested a distinct role for LYVE1 in tissue-resident (TR) macrophages, consistent with its expression in developing and post-natal hearts. Macrophage-specific deletion of Lyve1 during neonatal heart injury revealed impaired heart regeneration, characterised by reduced neovascular response and function, suggesting a hitherto unappreciated role for LYVE1 in regulating the pro-regenerative function of TR macrophages. Conclusions Collectively, we reveal that cardiac lymphatics are developmentally compromised for immune cell clearance in early neonates, which enables retention of pro-regenerative TR macrophages, and that LYVE1 plays an essential role in TR macrophages enabling heart regeneration via the induction of coronary angiogenesis.