Identification and Characterization of Zebrafish Tlr4 Coreceptor Md-2.

The zebrafish () 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 TLR4 and myeloid differentiation factor 2 (Tlr4/Md-2) recognizes the bacterial molecule 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, whereas the zebrafish genes appear to be paralogs, not orthologs, of amniote We revisited these conclusions. We identified a zebrafish gene encoding Md-2, Using single-cell RNA sequencing, we found that is transcribed in cells that also transcribe genes diagnostic for innate immune cells, including the zebrafish -like genes. In larval zebrafish, is expressed in a small number of macrophage-like cells. In a functional assay, zebrafish Md-2 and Tlr4ba form a complex that activates NF-κB signaling in response to LPS. In larval zebrafish loss-of-function mutations perturbed LPS-induced cytokine production but gave little protection against LPS toxicity. Finally, by analyzing the genomic context of genes in 11 jawed vertebrates, we found that arose 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 a de novo invention 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.