zDHHC20b-mediated TRIF palmitoylation drives infection-induced necroptosis through the TRIF-RIPK3 axis in teleost monocytes/macrophages

Palmitoylation is a reversible post-translational modification that regulates protein trafficking, stability, and function, yet its contribution to immune cell death in teleosts remains largely unknown. Using large yellow croaker (Larimichthys crocea) as a teleost model, this study identified the palmitoyltransferase zDHHC20b (LczDHHC20b) as the infection-responsive zDHHC20 paralog that promoted necroptosis in monocytes/macrophages (MO/MΦ) during Pseudomonas plecoglossicida infection. LczDHHC20b knockdown disrupted inflammatory cytokine regulation and markedly reduced necroptotic cell death. Acyl-biotin exchange assays combined with liquid chromatography-tandem mass spectrometry identified TIR-domain-containing adapter-inducing interferon-β (TRIF), a central adaptor in innate immune signaling, as a cell death-related palmitoylation substrate of LczDHHC20b. Infection progressively increased TRIF palmitoylation at Cys92 and Cys142, which enhanced recruitment of receptor-interacting serine/threonine kinase 3 (RIPK3), promoted phosphorylation of RIPK3 and mixed lineage kinase domain-like protein (MLKL), and activated necroptosis. LczDHHC20b knockdown or pharmacological inhibition of palmitoylation with 2-bromopalmitate (2-BP) impaired TRIF-RIPK3 complex assembly and suppressed downstream necroptotic signaling. Palmitoylation-deficient TRIF also exhibited reduced localization to endoplasmic reticulum- and trans-Golgi network-associated compartments, suggesting that palmitoylation contributes to the spatial organization of TRIF-dependent signaling. These findings provide the first evidence of TRIF palmitoylation and establish this modification as a previously unrecognized post-translational mechanism regulating TRIF-mediated immune cell death. The identified LczDHHC20b-TRIF-RIPK3 axis further defines a lipid-dependent pathway that controls necroptosis in teleost macrophages and shapes host-pathogen interactions during bacterial infection.