EZH2 Inhibition Alleviates Chronic Graft-Versus-Host Disease By Modulating Lipid Metabolic Reprogramming and Senescence of Neutrophils

【Background】Enhancer of zeste homolog 2 (EZH2)-mediated trimethylation of histone 3 lysine 27 (H3K27Me3) plays a critical role in immune regulation. Previous studies have shown that pharmacological inhibition of EZH2 can effectively reduce chronic graft-versus-host disease (cGvHD) by targeting follicular helper T cells (TFH) and germinal center (GC) B cells following allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the effects of EZH2 on neutrophils during cGvHD progression remain largely unexplored. This study investigates the impact of EZH2 inhibition (EZH2i) on neutrophil function in preclinical cGVHD models.

【Methods】Using the miHA-mismatched cGvHD (B10D2 to BALB/c) model, we found that EZH2i effectively alleviates cGvHD. Histopathological analyses, including H&E and Trichrome staining, revealed reduced IgG deposition and fibrosis in the skin and lungs following EZH2i treatment. Flow cytometry analysis demonstrated that EZH2i suppressed Tcon cells, plasma B cells, and neutrophil infiltration while expanding regulatory T cells (Tregs) in the skin and lung. Single-cell transcriptome profiling identified five distinct neutrophil clusters (Neu1-Neu5) in cGVHD lung tissues. Neu1 is enriched for granule-associated genes (Retnlg, Mmp8, S100a8, Ngp, Lcn2, and Ly6g) representing developed neutrophils, while Neu2 has genes associated with immature neutrophils and PMN-MDSCs. Neu3 consists of classical, mature neutrophils with interferon-stimulated genes, including Ifit3 and Isg15. Neu4 shows increased expression of chemokines and inflammation-related genes, indicating the activation status, and Neu5 is associated with phagocytic activity. Trajectory analysis indicates a developmental progression from Neu1 to Neu2, then to Neu3, Neu4, and ultimately Neu5. Activated (Neu4) and phagocytic neutrophils (Neu5) display the highest aging scores and upregulate several pathways that are also enhanced during neutrophil activation, including integrin and leukocyte adhesion, Toll-like receptor (TLR), NOD-like receptor (NLR), and NF-kB signaling pathways.

【Results】Metabolic profiling indicated that neutrophils from cGVHD-affected lungs upregulate genes involved in lipid metabolism, such as cholesterol biosynthesis and cellular lipid catabolic processes. Mechanistically, we discovered that EZH2-mediated H3K27Me3 suppresses PGC1a expression in neutrophils. Consequently, lung-infiltrating neutrophils in cGVHD display lower levels of the membrane fatty acid transporter CD36 and the rate-limiting enzyme of fatty acid oxidation (FAO), CPT1A. This FAO defect is associated with their senescent phenotype; senescent neutrophils produce more neutrophil extracellular traps (NETs) and reactive oxygen species (ROS). In contrast, EZH2 inhibition enhances the expression of CD36 and CPT1A, promoting a metabolic shift towards FAO and reversing neutrophil senescence. This metabolic reprogramming reduces NETs formation and ROS production. Additionally, the shift towards FAO also promotes the generation of Tregs, thereby alleviating cGVHD.

【Conclusion】In conclusion, pharmacological inhibition of EZH2 mitigates cGVHD by modulating neutrophil senescence and functional states. This study provides novel insights into the metabolic and functional plasticity of neutrophils in response to EZH2 inhibition and underscores the therapeutic potential of modulating epigenetic pathways in cGVHD therapy.