Modulation of the lipid metabolism in macrophages through chaperone-mediated autophagy in primary immune thrombocytopenia Free

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by thrombocytopenia and bleeding episodes. Macrophages play a central role in platelet clearance and inflammatory responses in ITP. Our previous clinical studies have identified a significant correlation between dyslipidemia and more severe ITP bleeding symptoms, as well as reduced responsiveness to standard treatments. Adiponectin, a key regulator of lipid metabolism known to enhance insulin sensitivity and fatty acid oxidation, also exerts anti-inflammatory effects on macrophages by suppressing NLRP3 inflammasome activation and inhibiting M1 polarization. Based on these properties, we hypothesized that adiponectin signaling may serve as a novel target of both lipid metabolism and immune dysregulation in ITP. We employed AdipoRon, an orally bioavailable adiponectin receptor agonist that overcomes the pharmacokinetic limitations of native adiponectin, to investigate the regulatory effect of adiponectin signaling in ITP. Mechanistically, we focused on chaperone-mediated autophagy (CMA), a selective lysosomal degradation process that regulates both lipid metabolism and macrophage functional plasticity. Peripheral blood samples from 56 patients with ITP and 31 sex- and age-matched healthy controls were collected with EDTA anticoagulation. Macrophages were generated from THP-1 cells or peripheral blood mononuclear cells (PBMC) from patients with ITP and were treated with AdipoRon (20 μM) or vehicle control. Macrophage polarization, activation, and phagocytic functions were assessed using flow cytometry, quantitative PCR (qPCR), and immunofluorescence microscopy. Plasma levels of adiponectin and inflammatory cytokines were quantified by enzyme-linked immunosorbent assay (ELISA). Intracellular accumulation of lipid was evaluated using BODIPY and Oil Red O staining. Blocking of the CMA pathway was performed using siRNA against the Lamp2a gene and myeloid cell-specific Lamp2a knock-out mice (Lamp2a^fl/flLysM-Cre) in vitro and in vivo, respectively. Fatty acid oxidation and oxidative phosphorylation of macrophages were detected by a Seahorse substrate oxidation stress kit. Active and passive ITP murine models were established to evaluate the therapeutic efficacy and underlying mechanisms of AdipoRon in vivo. Our results show that plasma adiponectin levels were significantly elevated in patients with ITP (median 7.18 [IQR 2.77–13.50]) compared to healthy controls (median 5.57 [IQR 2.74–9.177], P < 0.001). In patients with ITP, adiponectin levels were positively correlated with platelet counts (P = 0.003, R² = 0.2332) and negatively correlated with plasma low-density lipoprotein (P = 0.048), total cholesterol (P = 0.045), and bleeding severity (P = 0.003, R² = 0.2646). AdipoRon reduced lipid accumulation and enhanced the fatty acid oxidation in macrophages, accompanied by decreased M1 polarization and diminished secretion of pro-inflammatory cytokines, including IL-1β, TNF-α, and IL-6. Additionally, AdipoRon attenuated antibody-mediated platelet phagocytosis by macrophages in vitro. Using both passive and active ITP murine models, AdipoRon ameliorated thrombocytopenia and suppressed the inflammatory responses and phagocytic capacity of macrophages in the spleen, liver, and bone marrow. Transcriptomic analysis revealed that AdipoRon treatment induced an enrichment of lysosomal and autophagy-related gene expression profiles. Specifically, the expression of LAMP2A—a key mediator of CMA—was both upregulated in macrophages derived from patients with ITP, and ITP mice. Blocking of CMA via LAMP2A silencing or using Lamp2a^fl/flLysM-Cre mice showed completely abolished effects of AdipoRon on lipid metabolism, inflammation suppression, and its therapeutic efficacy in ITP murine models. In conclusion, our study demonstrates that AdipoRon ameliorates ITP through CMA-dependent metabolic reprogramming of macrophages. Thus, AdipoRon maybe a novel therapeutic target in ITP by modulating lipid metabolism and platelet phagocytic functions in a CMA-dependent pathway.