Disruption of Myd88 in a salmonid epithelioid cell line reveals its contribution to bacterial detection and immune response.

Abstract

Toll-like receptors (TLRs) are a class of pattern-recognition receptors that recognize pathogen- and damage-associated molecular patterns and initiate immune responses. TLRs selectively recruit distinct adapter molecules such as the myeloid differentiation primary response protein 88 (MyD88) that mediates signaling downstream of all TLRs, with the exception of TLR3. To investigate TLR signaling pathways in fish, we engineered a knockout clonal epitheliod fish cell line, named MYD88C2, using CRISPR/Cas9-mediated genome editing to disrupt the myd88 gene. We characterized the phenotype of this cell line alongside a wild-type cell line through gene-expression profiling and reporter-gene analyses in the context of stimulation with heat-killed Vibrio anguillarum, heat-killed Escherichia coli, flagellin, zymosan, and inoculation with a panel of viruses. We demonstrate that the proinflammatory response to zymosan and flagellin, as measured through the induction of proinflammatory genes, was greatly reduced in the MYD88C2 cell line. The responsiveness to zymosan was found to be partially restored by transfecting the MYD88C2 cell line with a myd88-expression plasmid. In contrast, the loss of the myd88 gene had no impact on the cytopathic effect associated with the replication of viral haemorrhagic septicemia virus (VHSV), infectious haematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), spring viraemia of carp virus (SVCV) or infectious salmon anaemia virus (ISAV). These findings highlight the critical role of MyD88 in mediating specific proinflammatory responses to bacterial and fungal stimuli, while its absence has no detectable impact on viral replication or cytopathogenicity in epithelioid fish cells under the conditions tested.