Lgals3high Macrophages Orchestrate Neuroinflammation and Extracellular Matrix Remodelling via NF-κB Pathway in Acute Spinal Cord Injury.
TL;DR
Lgals3high macrophages are core drivers of ECM remodelling in ASCI via a Lgals3/NF-κB/p65/Ccl2 signalling axis that couples M1 polarization with monocyte recruitment and exerts therapeutic effects by regulating macrophage polarization and ECM remodelling, providing a novel "immune-matrix" dual-targeting strategy.
Abstract
Background
Acute spinal cord injury (ASCI) causes severe dysfunction, with secondary ECM remodelling being more destructive than primary injury. Macrophages regulate neuroinflammation and ECM remodelling in ASCI. Studies shown ECM related gene Lgals3 regulate macrophage polarization across various diseases. However, the role of Lgals3 in ASCI-related ECM remodelling and neuroinflammation remains unclear.
Methods
We integrated Single-cell RNA Sequencing (ScRNA-seq) analysis with in vitro and in vivo experiments. An ECM-related gene set was constructed for bioinformatic analyses of cell clustering, pseudotime trajectory, cell-cell communication, and transcription factor activity. LPS+IFN-γ-induced RAW264.7 macrophages were treated with the Lgals3 inhibitor GB1107, and the Lgals3/NF-κB/p65/Ccl2 axis was further dissected by siRNA-mediated Lgals3 knockdown, pharmacological NF-κB inhibition with QNZ, and TNF-α-mediated NF-κB reactivation rescue. ASCI models in SD rats were assessed via multiplex immunohistochemistry (mIHC), histology, TUNEL, western blot, and qRT-PCR.
Results
Seven cell types were identified in the ASCI microenvironment, with macrophages showing the most upregulated ECM-related genes. Four ECM-associated macrophage subtypes were defined; Lgals3high macrophages (Mac-1), characterized by high Lgals3 and Ccl2 expression, dominated early-phase ASCI. Mac-1 exhibited the strongest intercellular communication with prominent CCL signalling pathway activation. Lgals3, Ccl2, and p65 were specifically upregulated in M1 macrophages post-ASCI, with p65 nuclear translocation. GB1107 inhibited NF-κB, promoted M1-to-M2 polarization, reduced inflammatory infiltration and ECM remodelling, and decreased apoptosis. Mechanistically, siRNA-mediated Lgals3 silencing suppressed NF-κB/p65 activation and Ccl2 expression while inducing Arg1; this phenotype was recapitulated by NF-κB inhibition and reversed by TNF-α-driven NF-κB reactivation, validating the Lgals3/NF-κB/p65/Ccl2 axis as the obligate regulatory backbone of macrophage polarization.
Conclusion
Lgals3high macrophages are core drivers of ECM remodelling in ASCI via a Lgals3/NF-κB/p65/Ccl2 signalling axis that couples M1 polarization with monocyte recruitment. GB1107 disrupts this axis, reprograms macrophages from M1 to M2, attenuates inflammatory infiltration and ECM remodelling, and reduces apoptosis. GB1107 exerts therapeutic effects by regulating macrophage polarization and ECM remodelling, providing a novel "immune-matrix" dual-targeting strategy.