Reg proteins direct accumulation of functionally distinct macrophage subsets after myocardial infarction

Myocardial infarction (MI) causes a massive increase of macrophages in the heart, which serve various non-redundant functions for cardiac repair. The identities of signals controlling recruitment of functionally distinct cardiac macrophages to sites of injury are only partially known. Previous work identified Regenerating islet-derived protein 3 beta (Reg3β) as a novel factor directing macrophages to sites of myocardial injury. Herein, we aim to characterize functionally distinct macrophage subsets and understand the impact of different members of the Reg protein family including Reg3β, Reg3γ, and Reg4 on their accumulation in the infarcted heart. We have determined dynamic changes of three phenotypically distinct tissue macrophage subpopulations in the mouse heart after MI by flow cytometry. RNA sequencing and bioinformatics analysis identified inflammatory gene expression patterns in MHC-IIhi/Ly6Clo and MHC-IIlo/Ly6Clo cardiac tissue macrophages while Ly6Chi cardiac tissue macrophages are characterized by gene activities associated with healing and revascularization of damaged tissue. Loss- and gain-of-function experiments revealed specific roles of Reg proteins for recruitment of cardiac tissue macrophage subpopulations to the site of myocardial injury. We found that expression of Reg3β, Reg3γ, and Reg4 is strongly increased after MI in mouse and human hearts with Reg3β providing the lead, followed by Reg3γ and Reg4. Inactivation of the Reg3β gene prevented the increase of all types of cardiac tissue macrophages shortly after MI whereas local delivery of Reg3β, Reg3γ, and Reg4 selectively stimulated recruitment of MHC-IIhi/Ly6Clo and MHC-IIlo/Ly6Clo but repressed accumulation of Ly6Chi cardiac tissue macrophages. We conclude that distinct cardiac macrophage subpopulations are characterized by substantially different gene expression patterns reflecting their pathophysiological role after MI. We argue that sequential, local production of Reg proteins orchestrates accumulation of macrophage subsets, which seem to act in a parallel or partially overlapping rather than in a successive manner.