Single-cell RNA Sequencing Revealed the Immunophenotypic Features of Macrophages in Cardiac Transplants and Uncovered Lgals9 Promoted Their Polarization towards The M2b Subtype

Abstract Macrophages play a crucial role in the immune response during allograft rejection in organ transplantation. Therefore, our study aimed to explore the genomic features of macrophages in mouse heart transplants and use single-cell RNA sequencing to investigate Galectin-9 (Gal-9, Lgals9), a lectin that can mediate the activation and differentiation of immune cells through ligand-receptor interactions, and the effects of its regulation in transplantation. We discovered a new subset of macrophages called "Myoz2+ macrophages", which specifically expressed genes related to myocardial contraction. We identified a distinct differentiation trajectory and process for the Saa3+ macrophage population, representing anti-inflammatory functionality. Also, we observed a significant downregulation of Lgals9 expression in the macrophages after mouse heart transplantation. Then, we validated our findings using RT-qPCR and Western blotting and also investigated the impact of Lgals9 on macrophage function through flow cytometry and ELISA. Furthermore, in vitro, we found that rLgals9 (Recombinant Mouse Galectin-9 Protein) treated macrophages polarized towards the M2b phenotype at appropriate concentrations.