Multi-omics and experimental evidence in human chondrocytes identify caspase-8 as a non-apoptotic regulator of inflammatory, senescent, and fibrotic signaling in osteoarthritis

<h4>Background</h4>Osteoarthritis (OA) is characterized by chronic inflammation, cellular senescence, and progressive cartilage remodeling, yet effective disease-modifying therapeutic targets remain limited. Caspase-8 has classically been regarded as a key initiator of the extrinsic apoptotic pathway; however, accumulating evidence suggests that it exerts broad non-apoptotic regulatory functions in a highly context- and cellular state-dependent manner. Here, we systematically investigated the role of caspase-8 in OA pathogenesis and its therapeutic potential.<h4>Methods</h4>We integrated bulk RNAseq, single-cell RNAseq, and spatial transcriptomics databases, in vitro genetic knockdown, inhibitor titration, caspase activity assays and functional assays, quantitative proteomics, in silico protein-protein docking analysis and population-level causal inference using two-sample Mendelian randomization (MR) and SMR/HEIDI analyses to construct and validate a caspase-8-centered regulatory network in human OA chondrocytes.<h4>Results</h4>Across two independent transcriptomic cohorts (GSE168505 and GSE246425), the Death-Inducing Signaling Complex (DISC)-caspase-8 axis and a caspase-8 activation signature were significantly upregulated in osteoarthritic cartilage and in senescent (late passage) OA chondrocytes, and were strongly associated with inflammatory and senescence-related programs. Additional analyses of aging, murine destabilization of the medial meniscus (DMM), and spatial transcriptomic datasets (GSE287861, GSE26475, and GSE254844) highlighted context-dependent CASP8 patterns, including senescence- but not inflammation-associated CASP8 elevation in naturally aged cartilage, no consistent CASP8 expression or activation-score increase after DMM surgery, and regionally heterogeneous CASP8 distribution across cartilage zones. Single-cell RNA sequencing (GSE255460) localized these signatures to OA-expanded inflammatory and fibrocartilage-like chondrocyte subpopulations, in which high CASP8 gene expression defined a distinct transcriptional state characterized by suppression of hyaline cartilage matrix and metabolic programs, together with enrichment of TNFα/interferon and TGF-β-associated inflammatory-fibrotic signaling. CASP8-high chondrocytes also displayed altered inferred ligand-receptor communication, particularly involving extracellular matrix-, adhesion-, and growth factor-related interactions. Stratification by caspase-8 activation potential recapitulated these transcriptional features and revealed induction of senescence-associated secretory phenotype (SASP) programs across multiple chondrocyte lineages. Functionally, pharmacological inhibition of caspase-8 with Z-IETD-FMK improved metabolic activity, reduced cellular senescence and MMP-13 secretion without inducing apoptosis, and partially restored proliferation and migration in OA chondrocytes under inflammatory conditions, with less effects in non-OA cells and no effects in siRNA mediated CASP8 knocked down cells. Quantitative proteomics demonstrated that caspase-8 inhibition attenuated inflammatory, senescence, and canonical NF-κB signaling without suppressing apoptosis, while reshaping proteostasis-chromatin regulatory networks and dampening fibrocartilage-like matrix remodeling. In silico protein-protein docking further showed favorable predicted binding affinities between caspase-8 and selected proteomically altered candidates, such as TGF-β3 and MMP13. Finally, MR and SMR/HEIDI analyses supported CASP8 as a causal risk regulator for knee OA, influenced by multi-tissue expression and epigenetic regulation.<h4>Conclusions</h4>Collectively, our findings identify caspase-8 as a central non-apoptotic signaling hub that couples inflammatory-senescence circuits with fibrotic remodeling in chondrocytes. Consequently, caspase-8 represents a promising genetic and pharmacological therapeutic target, warranting further drug development and translational investigation.