A cell reporter system to examine toll-like receptor ligands in channel catfish

Abstract Toll-like receptors (TLR) are a family of innate immune receptors that recognize microbe- or danger-associated molecular patterns (MAMPs or DAMPs). TLR ligand binding results in the recruitment of adaptor proteins, which initiate signaling cascades that lead to the activation of NF-κB and interferon regulatory factors (IRFs). Importantly, this signaling initiates both innate and adaptive immune responses. Channel catfish is an important aquacultural species, and the catfish genome encodes 20 TLRs. In contrast, mammalian genomes encode from 10 to 13 TLRs. Our hypothesis is that fish, due to their aquatic environment, express additional TLRs that provide protection from pathogens and pollutants. Our goal is to gain a functional understanding of catfish TLRs by identifying their ligands. To screen potential ligands, we use the HEK Dual-null cell line, which is engineered to not express endogenous TLRs. This cell line is stably transfected with a SEAP reporter gene under the control of NF-κB. Using this model, it was confirmed that TLR7 is activated by imiquimod, a synthetic ssRNA analog. In addition, we have identified the full repertoire of IFN genes in the catfish genome. This allows us to assess products downstream of TLR activation by qPCR. Taken together, this system permits us to study the TLR signaling pathways that drive catfish immune responses. In the future, once TLR signaling is better understood, specific ligands can be incorporated into vaccines as adjuvants to direct favorable immune responses and protect farmed and domesticated animal species. Supported by pre-doctoral fellowship USDA-NIFA-AFRI 2022-67011-36580 and grant NSF-IOS 1656419