NLK facilitates Caspase‐8 activation to drive macrophage PANoptosis in sepsis

Mounting evidence indicates that macrophage PANoptosis-an integrated inflammatory cell-death program comprising pyroptosis, apoptosis, and necroptosis-plays a pivotal role in sepsis pathogenesis. However, its upstream regulation remains poorly understood. Here, we identify Nemo-like kinase (NLK) as a novel regulator of Caspase-8-mediated PANoptotic signalling in sepsis. Integrated analyses of bulk (GSE65682) and single-cell (GSE167363) transcriptomic datasets from patients with sepsis revealed elevated NLK expression in monocytes, strongly associated with PANoptotic effectors and adverse outcomes. Functional studies using NLK conditional knockout mice driven by Csf1r-iCre and lipopolysaccharide-stimulated bone-marrow-derived macrophages showed that NLK deficiency attenuated Caspase-8 cleavage, suppressed pyroptotic (cleaved Caspase-1, GSDMD-N) and apoptotic (cleaved Caspase-3/7) activation, and redirected cell-death execution towards a necroptosis-dominant program (p-RIPK1/3, p-MLKL). This death-mode redistribution was associated with attenuated cytokine release, reduced multiorgan injury, and improved survival. Mechanistically, NLK associated with the N-terminal death effector domains (DEDs; amino acids 1-216) of Caspase-8, thereby enhancing the efficiency of Caspase-8 recruitment and proximity-induced activation within FADD-RIPK1/3-containing PANoptosome complexes. NLK deletion impaired Caspase-8 activation within these complexes and promoted RIPK1-RIPK3 necrosome assembly. Moreover, Caspase-8 overexpression in NKO macrophages partially restored GSDMD and Caspase-3 cleavage and reduced p-MLKL, confirming that NLK is required for efficient Caspase-8 activation and optimal PANoptotic signalling. Collectively, these findings identify NLK as a regulatory rheostat of Caspase-8-associated PANoptosis in sepsis and highlight the NLK-Caspase-8 axis as a potential therapeutic target for fine-tuning sepsis-associated inflammatory cell death.