Rainbow trout USP4 downregulates LPS-induced inflammation by removing the K63-linked ubiquitin chain on TAK1

Ubiquitin-specific protease 4 (USP4) is pivotal in negatively regulating the Toll-like receptor (TLR) signaling -mediated innate immune response. Although USP4 has been well studied in mammals, its role in TLR signaling pathways in fish remains largely unknown. In this study, we investigated the role of USP4 (OmUSP4) in regulating TLR response in rainbow trout Oncorhynchus mykiss. OmUSP4 contained the characteristic domains conserved in other USP4s: domain in USP (DUSP), ubiquitin-like (UBL), and the bi-part catalytic domain known as USP. OmUSP4 expression was increased in RTH-149 cells by stimulation with fish-pathogenic bacteria and bacterial ligands. Gain-and loss-of-function experiments revealed that OmUSP4 mitigated the activation of MAPKs and NF-kappa B, as well as the expression of pro-inflammatory cytokines in LPS-stimulated cells. OmUSP4 interacted with TAK1, a critical mediator in TLR-mediated NF-kappa B signaling pathways. LPS stimulation increased the K63-linked polyubiquitination of TAK1, which was significantly suppressed when OmUSP4 was compelled to be overexpressed. These results imply that OmUSP4 might function like mammals to downregulate LPS-induced inflammation in rainbow trout by removing the K63-linked ubiquitin chain on TAK1.