CYLD suppresses LPS-induced inflammation through RIP1 deubiquitination in rainbow trout

Abstract

The deubiquitinase cylindromatosis (CYLD) negatively regulates the MAPK and NF-κB signaling pathways by removing ubiquitin from upstream regulatory elements in the TLR pathway. Although the regulatory mechanisms of mammalian CYLD are well-characterized, its function in TLR signaling pathways in fish is still largely unexplored. Herein, we investigated the function of CYLD in modulating the TLR response in rainbow trout (Oncorhynchus mykiss). LPS stimulation induced the expression of OmCYLD in RTH-149 cells. Using approaches to increase or decrease gene function, we demonstrated that OmCYLD inhibits MAPK and NF-κB activation and reduces the production of proinflammatory cytokines in LPS-stimulated RTH-149 cells. OmCYLD interacted with RIP1, a critical regulator of TLR-mediated NF-κB signaling, via its third CAP-Gly domain and USP domain, independently of its deubiquitinating activity. LPS stimulation led to increased polyubiquitination of RIP1 in RTH-149 cells, which was suppressed by OmCYLD overexpression. Moreover, mutation of the USP domain impaired the deubiquitination of polyubiquitinated RIP1, confirming that the USP domain is essential for its deubiquitinating activity. These results suggest that, similar to mammals, OmCYLD regulates LPS-induced inflammation in rainbow trout, probably by modulating the ubiquitination status of RIP1.