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<p>Neutrophil extracellular traps (NETs) exacerbate fibroblast inflammatory injury in hyperglycemic conditions, yet the role of glucose metabolism and O-linked N-acetylglucosamine (O-GlcNAc) glycosylation in this process remains unclear. Here, we investigate how glucose transporter protein 1 (GLUT1)-dependent glucose uptake regulates O-GlcNAcylation of high-mobility group box 1 (HMGB1) to drive NET formation and fibroblast inflammation. Mouse peripheral blood neutrophils (MPBN) were treated with high glucose (25 mM) and phorbol ester (PMA) to induce NETs. Co-culture of NETs with mouse fibroblasts (L929) reduced fibroblast viability by 1.1 fold and migration by 1.2 fold within 24&#xa0;h, while upregulating pro-inflammatory cytokines (Tumor Necrosis Factor-α (TNF-α): +1.3-fold; Interleukin-1β (IL-1β): +1.1-fold; Interleukin-6 (IL-6): +1.1-fold) and suppressing collagen synthesis (Collagen I (COL-I): − 1.7-fold; Collagen III (COL-III): −2.5-fold). Critically, high glucose elevated GLUT1 expression in MPBN (+ 1.2-fold), further amplified under co-culture conditions(+ 1.2-fold). Functional assays using GLUT1 knockdown confirmed that GLUT1 activity was essential for glucose uptake and subsequent O-GlcNAc modification of HMGB1, stabilizing its expression. Enhanced O-GlcNAcylation of high-mobility group box 1 (HMGB1) directly promoted NET formation, evidenced by elevated markers (Citrullinated histone H3 (Cit-H3): +1.6-fold; Myeloperoxidase (MPO): +1.2-fold; Circulating free DNA (cfDNA): +2-fold) and activation of c-Jun N-terminal kinase (JNK)/p38 phosphorylation. These effects were abolished by toll-like receptor 4 (TLR4) inhibition, linking HMGB1-TLR4 signaling to NET-driven inflammation. Mechanistically, GLUT1 knockdown reduced HMGB1 O-GlcNAcylation and reversed NET-induced fibroblast dysfunction. Our findings provide direct evidence that hyperglycemia enhances GLUT1 expression and activity, driving HMGB1 O-GlcNAcylation to maintain NETs formation through TLR4, which promotes fibroblast inflammatory injury. This pathway highlights a metabolic-inflammation axis relevant to diabetic complications.</p>

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GLUT1-mediated HMGB1 O-GlcNAcylation drives hyperglycemia-Induced neutrophil extracellular trap networks formation via TLR4 signaling and exacerbates fibroblast inflammation

  • 期刊论文

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  • 发表日期：

    2025年5月29日

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  • Weijing Sun,
  • Jinlong Xu,
  • Shijie Li,
  • Yue Zhao,
  • Jiachen Fu,
  • Lixia Di,
  • Dezhi Han

摘要

Neutrophil extracellular traps (NETs) exacerbate fibroblast inflammatory injury in hyperglycemic conditions, yet the role of glucose metabolism and O-linked N-acetylglucosamine (O-GlcNAc) glycosylation in this process remains unclear. Here, we investigate how glucose transporter protein 1 (GLUT1)-dependent glucose uptake regulates O-GlcNAcylation of high-mobility group box 1 (HMGB1) to drive NET formation and fibroblast inflammation. Mouse peripheral blood neutrophils (MPBN) were treated with high glucose (25 mM) and phorbol ester (PMA) to induce NETs. Co-culture of NETs with mouse fibroblasts (L929) reduced fibroblast viability by 1.1 fold and migration by 1.2 fold within 24 h, while upregulating pro-inflammatory cytokines (Tumor Necrosis Factor-α (TNF-α): +1.3-fold; Interleukin-1β (IL-1β): +1.1-fold; Interleukin-6 (IL-6): +1.1-fold) and suppressing collagen synthesis (Collagen I (COL-I): − 1.7-fold; Collagen III (COL-III): −2.5-fold). Critically, high glucose elevated GLUT1 expression in MPBN (+ 1.2-fold), further amplified under co-culture conditions(+ 1.2-fold). Functional assays using GLUT1 knockdown confirmed that GLUT1 activity was essential for glucose uptake and subsequent O-GlcNAc modification of HMGB1, stabilizing its expression. Enhanced O-GlcNAcylation of high-mobility group box 1 (HMGB1) directly promoted NET formation, evidenced by elevated markers (Citrullinated histone H3 (Cit-H3): +1.6-fold; Myeloperoxidase (MPO): +1.2-fold; Circulating free DNA (cfDNA): +2-fold) and activation of c-Jun N-terminal kinase (JNK)/p38 phosphorylation. These effects were abolished by toll-like receptor 4 (TLR4) inhibition, linking HMGB1-TLR4 signaling to NET-driven inflammation. Mechanistically, GLUT1 knockdown reduced HMGB1 O-GlcNAcylation and reversed NET-induced fibroblast dysfunction. Our findings provide direct evidence that hyperglycemia enhances GLUT1 expression and activity, driving HMGB1 O-GlcNAcylation to maintain NETs formation through TLR4, which promotes fibroblast inflammatory injury. This pathway highlights a metabolic-inflammation axis relevant to diabetic complications.