Functional identification of the zebrafish Interleukin-1 receptor in an embryonic model of Il-1β-induced systemic inflammation

Abstract

Interleukin-1β (IL-1β) is a potent proinflammatory cytokine that plays a vital role in the innate immune system. To observe the innate immune responsein vivo, several transgenic zebrafish lines have been developed to model IL-1β-induced inflammation and to visualize immune cell migration and proliferation in real time. However, our understanding of the IL-1β response in zebrafish is limited due to an incomplete genome annotation and a lack of functional data for the cytokine receptors involved in the inflammatory process. Here, we use a combination of database mining, genetic analyses, and functional assays to identify zebrafish Interleukin-1 receptor, type 1 (Il1r1). We identified putative zebrafishil1r1candidate genes that encode proteins with predicted structures similar to human IL1R1. To examine functionality of these candidates, we designed highly effective morpholinos to disrupt gene expression in a zebrafish model of embryonic Il-1β-induced systemic inflammation. In this double transgenic model,ubb:Gal4-EcR,uas:il1βmat, the zebrafishubiquitin b(ubb) promoter drives expression of the modified Gal4 transcription factor fused to the ecdysone receptor (EcR), which in turn drives the tightly-regulated expression and secretion of mature Il-1β only in the presence of the ecdysone analog tebufenozide (Teb). Application of Teb toubb:Gal4-EcR,uas:il1βmatembryos causes premature death, fin degradation, substantial neutrophil expansion, and generation of reactive oxygen species (ROS). To rescue these deleterious phenotypes, we injectedubb:Gal4-EcR,uas:il1βmatembryos with putativeil1r1morpholinos and found that knockdown of only one candidate gene prevented the adverse effects caused by Il-1β. Mosaic knockout ofil1r1using the CRISPR/Cas9 system phenocopied these results. Taken together, our study identifies the functional zebrafish Il1r1 utilizing a genetic model of Il-1β-induced inflammation and provides valuable new insights to study inflammatory conditions specifically driven by Il-1β or related to Il1r1 function in zebrafish.