TLR4 Induces PANoptosis in Annulus Fibrosus Cells by Activating NLRP12 in Intervertebral Disc Degeneration.

Purpose: Intervertebral disc degeneration (IVDD), a common degenerative disorder, is characterized by chronic inflammation and progressive cell death. PANoptosis, an integrated form of programmed cell death that merges pyroptosis, apoptosis, and necroptosis, has recently emerged as a key contributor to degenerative diseases. Although Toll-like receptor 4 (TLR4) and NLRP12 are known to regulate inflammatory signaling and cell death, their roles in mediating annulus fibrosus cells (AFCs). This study investigated how the TLR4-NLRP12 axis regulates AFC PANoptosis and contributes to IVDD progression. Methods: Single-cell RNA sequencing (scRNA-seq) profiled cell heterogeneity and PANoptosis-related signaling in healthy/degenerative annulus fibrosus (AF) tissues. An in vitro IVDD model was established by treating AFCs with TNF-α to mimic inflammation. NLRP12 knockdown was performed via lentiviral transduction. PANoptosis was assessed by Western blot (cleaved-Caspase-1, cleaved-Caspase-3, p-MLKL, c-GSDMD), TUNEL staining, and flow cytometry. In vivo, a rat IVDD model was induced by acupuncture puncture, followed by TLR4 knockdown via lentiviral injection. X-ray imaging was used to assess intervertebral space height, histology to evaluate disc morphology, and TUNEL assay to detect annulus fibrosus cell death. Results: scRNA-seq revealed an increased abundance of Fibro-AFCs and Adh-AFCs in IVDD, alongside enrichment of cell death-related pathways. PANoptosis-related gene expression and cell death scores were elevated in degenerative samples. In vitro, TLR4 activation induced PANoptosis in AFCs, which was attenuated by either TLR4 inhibition or NLRP12 knockdown. Formation of the PANoptosome complex-characterized by colocalization of Caspase-8, ASC, and RIPK3-was observed in degenerative AFCs. In vivo, TLR4 silencing ameliorated IVDD pathology, as evidenced by improved disc height, and reduced cell death. To our knowledge, this is the first study to identify the occurrence of PANoptosis in AFCs. Conclusion: TLR4 promotes PANoptosis in AFCs through NLRP12 activation, driving IVDD pathogenesis. The TLR4-NLRP12-PANoptosis presents a potential therapeutic target for IVDD.