rFgl2-treated CD32b⁺M2 macrophages effectively inhibits cardiac transplant rejection in mice

The search for additional therapies against acute rejection is warranted. Alternatively activated macrophages (M2 macrophages) have immunomodulatory functions, and enhancement of their immunomodulatory capacity is a feasible complementary therapy. Fcγ receptor II b (FcγRIIb, CD32b), which is highly expressed by M2 macrophages, and its important ligand, fibrinogen-like protein 2 (Fgl2), are immunosuppressive molecules, and it is necessary to explore the potential of both to enhance the immunomodulatory capacity of M2 macrophages.

In vitro, CD32b⁺M2 macrophages obtained by magnetic bead sorting were treated with recombinant Fgl2 (rFgl2) to explore their immunomodulatory capacity under simulated inflammatory (lipopolysaccharide+interferon γ) stimuli and the possible mechanisms using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and western blotting (WB), and they were co-cultured with CD4⁺T cells to explore their modulation of the polarization of regulatory T cells (Tregs). In vivo, rFgl2-treated CD32b⁺M2 macrophages were used to treat abdominal heart transplantation model mice, and systemic inflammation, changes in systemic proportions of macrophages, CD4⁺, CD8⁺T cells, and Tregs, and infiltration in grafts were examined using ELISA, hematoxylin-eosin staining, immunohistochemistry, and flow cytometry.

In vitro CD32b⁺M2 macrophages treated with rFgl2 further expressed IL-10 and TGF-β1 and had increased levels of phosphorylation of SHP2 and STAT3, which effectively increased the proportion of Tregs after co-culture with CD4⁺T cells. In vivo rFgl2-treated CD32b⁺M2 macrophages increased the proportion of M2 macrophages and Tregs, improved systemic inflammation, attenuated graft injury, reduced inflammatory cell infiltration, and prolonged graft survival.

rFgl2 promotes the differentiation of Tregs by activating SHP2 and thus promoting STAT3 activation, which in turn promotes the secretion of the inflammation-suppressing cytokine IL-10 as well as TGF-β1. rFgl2-treated CD32b⁺M2 macrophages alleviate the acute rejection of cardiac transplants in mice by promoting differentiation of M2 macrophages as well as Tregs, which can be used as a complementary treatment to reduce the adverse effects of immunosuppression and prolong the survival time of allogeneic transplants.