Transcript analysis of natural killer (NK) cell specific genes in the liver, kidney and spleen tissues of rag1 −/− mutant zebrafish in response to in vivo administration of TLR ligands

Abstract

Abstract T and B cells mediate specific protective immunity. NK cells can also mediate a memory response to murine cytomegalovirus in T and B cell deficient rag2−/− mice. T and B cell deficient rag1−/− mutant zebrafish mediate protective immunity to intracellular bacteria. NK cells have not been characterized in zebrafish due to a lack of tools to target specific markers. The aim of this study was to evaluate NK cell tissue distribution and transcript response to TLR ligands in rag1−/− mutant zebrafish. We accomplished this by determining the level of expression of ifnγ, t-bet, nitr9, and NK lysin (nkl) a, b, c and d transcripts following exposure to β glucan, Poly I:C, R-848 and VTX-2337. Expression levels were normalized to the basal level of expression and the fold changes were compared to that of endotoxin free PBS (carrier control) injected group. R848 and VTX-2337 exposure changed the gene expression profile in a tissue specific manner. Nitr9 protein expression was analyzed by western blot and detectable levels were found in the liver. Nitr9 expression correlated with the expression of ifnγ in liver and kidney. In another experiment, fish were injected with TLR ligand when vaccinated, were only vaccinated, or not vaccinated. After bacteria challenge, fish that received β glucan or R848 when vaccinated had the highest survival. This suggests the TLR ligands have an adjuvant activity on NK cell protective responses. Our results of NK cell stimulation indirectly demonstrate the presence of NK cells in zebrafish and suggest a possible means of enhancing NK cell based immunity. These findings demonstrate the clinical significance of the rag1−/− mutant zebrafish model.