Identification and characterization of zebrafish Tlr4 co-receptor Md-2

Abstract

ABSTRACTThe zebrafish (Danio rerio) is a powerful model organism for studies of the innate immune system. One apparent difference between human and zebrafish innate immunity is the cellular machinery for LPS-sensing. In amniotes, the protein complex formed by Toll-like receptor 4 and myeloid differentiation factor 2 (Tlr4/Md-2) recognizes the bacterial molecule lipopolysaccharide (LPS) and triggers an inflammatory response. It is believed that zebrafish have neither Md-2 nor Tlr4: Md-2 has not been identified outside of amniotes, while the zebrafishtlr4genes appear to be paralogs, not orthologs, of amnioteTLR4s. We revisited these conclusions. We identified a zebrafish gene encoding Md-2,ly96. Using single-cell RNA-Seq, we found thatly96is transcribed in cells that also transcribe genes diagnostic for innate immune cells, including the zebrafishtlr4-like genes. Unlike amnioteLY96, zebrafishly96expression is restricted to a small number of macrophage-like cells. In a functional assay, zebrafish Md-2 and Tlr4a form a complex that activates NF-κB signaling in response to LPS, butly96loss-of-function mutations gave little protection against LPS-toxicity in larval zebrafish. Finally, by analyzing the genomic context oftlr4genes in eleven jawed vertebrates, we found thattlr4arose prior to the divergence of teleosts and tetrapods. Thus, an LPS-sensitive Tlr4/Md-2 complex is likely an ancestral feature shared by mammals and zebrafish, rather than ade novoinvention on the tetrapod lineage. We hypothesize that zebrafish retain an ancestral, low-sensitivity Tlr4/Md-2 complex that confers LPS-responsiveness to a specific subset of innate immune cells.