β2-Adrenergic Receptor Agonist Terbutaline Regulates Macrophage Polarization Via HMGB1 in Immune Thrombocytopenia

Introduction: Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by decreased platelet counts and increased risk of hemorrhage. Autoantibody-mediated excessive platelet clearance by macrophages in the spleen is an important mechanism of ITP. Macrophages, heterogeneous cells with high functional plasticity, can polarize into the M1 type which is crucial for antigen presentation, and the M2 type which has an immunosuppressive function. An imbalance in M1/M2 polarization is involved in the pathogenesis of ITP. High Mobility Group Box 1 (HMGB1), a nuclear protein, regulates macrophage polarization and function. Our previous studies demonstrated that terbutaline, a β2-adrenergic receptor (β2-AR) agonist, significantly restored the percentage of Tregs in ITP patients. However, whether terbutaline can regulate M1/M2 polarization in ITP is unknown.

Methods: Peripheral blood (PB)-derived CD14⁺ monocytes were isolated from ITP patients and healthy donors and cultured in vitro. Spleen-derived macrophages were isolated from ITP model mice and WT mice. The percentages of M1 and M2 macrophages, the expression of macrophage markers, and the results of in vitro phagocytosis assays were analyzed via flow cytometry. Quantitative analysis of nontargeted and targeted metabolomics of macrophages was performed. ELISA was used to measure the levels of cytokines and chemokines in the plasma and supernatants. Terbutaline-treated PB-derived ITP-CD14⁺ monocytes and ITP murine models were used to explore their effects on macrophage polarization and function.

Results: An imbalance in M1/M2 polarization was observed in macrophages derived from CD14⁺ monocytes in the peripheral blood of ITP patients, which is consistent with previous reports. Flow cytometry revealed that the addition of terbutaline to ITP-derived macrophages inhibited the expression of M1 markers and promoted the expression of the M2 marker CD163 in vitro. ELISAs demonstrated that terbutaline-treated ITP-derived macrophages had significantly increased levels of IL-10 and decreased levels of TNF-α in the supernatant. Furthermore, metabolomics revealed alterations in metabolic pathways, particularly arginine, after terbutaline addition in the ITP group.

Compared with those in wild-type (WT) mice, the number of splenic sympathetic nerve fibers in ITP model mice is lower, the M1/M2 ratio is greater, phagocytosis toward platelets is greater, and platelet counts are lower. After chemical sympathectomy with 6-hydroxydopamine in WT mice, decreased platelet counts and increased polarization of splenic macrophages toward the M1 phenotype were observed, indicating that the loss of sympathetic nerves was associated with aberrant macrophage polarization. The expression of HMGB1 in splenic macrophages from ITP model mice was significantly greater than that in WT mice.

For in vitro experiments, splenic macrophages from ITP model mice were extracted and cultured. Compared with the vehicle group, the HMGB1 siRNA group exhibited decreased macrophage polarization toward M1, decreased phagocytosis toward platelets, and alterations in amino acid metabolites, which suggested that HMGB1 may regulate macrophage polarization and phagocytosis in ITP. In vitro, terbutaline inhibited ITP murine model-derived splenic macrophage polarization toward M1 macrophages and changed the metabolic pathways of amino acids, which could be reversed by recombinant mouse HMGB1.

In vivo, terbutaline ameliorated thrombocytopenia in an ITP murine model. Consistent with the in vitro results, terbutaline-treated ITP mice presented a decreased M1/M2 ratio and increased platelet count, without a difference in 7-day mortality.

Furthermore, our preliminary data demonstrated that eight ITP patients treated with terbutaline had a greater overall response and sustained response compared with the control group, which warrants further exploration. The Research Ethics Committee approved this study, which was performed in compliance with the provisions of the Declaration of Helsinki.

Conclusions: Terbutaline could modulate abnormal macrophage polarization and inhibit macrophage phagocytosis toward platelets via HMGB1. To our knowledge, this is the first report indicating that terbutaline can effectively ameliorate thrombocytopenia by regulating macrophage polarization via HMGB1 in ITP.