Differential gene expression following stimulation of TLRs 2, 3, 7 and 8 in rag1-/- mutant zebrafish (IRC4P.613)

Abstract Natural Killer (NK) cells have shown to mediate adaptive immune responses to chemical haptens and cytomegalovirus in T and B cell deficient mice. T and B cell deficient zebrafish mount specific protection to intracellular bacteria. We hypothesize that a sub-population of zebrafish NK cells mediates this protection, and these cells have a mechanism for enhanced discrimination of a bacterial target. However, zebrafish NK cells have not been characterized, and their response to TLR agonists has not been investigated. We utilized rag1-/- mutant zebrafish as an in vivo fish model to determine if TLR agonists (TLR2: βglucan, TLR3: Poly I:C and TLR 7/8: R-848) stimulate NK cells in liver, kidney marrow and spleen tissues. We used IFNγ expression as an indicator of NK cell activation, TNFα expression as an indicator of macrophage activation, and MxA expression as an indicator of IFNa and IFNβ production. With RT-qPCR we quantified transcript levels at 1h, 6h, 12h and 24h after injecting rag1-/- mutant zebrafish with either βglucan, POLY I:C or R-848. Our results show significant increases of IFNγ, MxA and TNFα transcripts in the three tissues with βglucan, POLY I:C or R-848 compared to PBS injected control fish. Liver had the highest expression of IFNγ in response to R-848 and βglucan. The data presented are the initial findings in an investigation to help define mechanisms involved in NK cell mediated protective immunity in rag1-/- mutant zebrafish.