Rapid Natural Killer Cell Gene Responses, Generated by TLR Ligand-Induced Trained Immunity, Provide Protection to Bacterial Infection in rag1−/− Mutant Zebrafish (Danio rerio)

T and B cell-deficient rag1−/− mutant zebrafish develop protective immunity mediated by trained immunity. In mammals, trained immune responses can be induced by Toll-like receptor (TLR) ligands. This study evaluated protective trained immunity in rag1−/− zebrafish through exposure to TLR ligands (beta glucan, R848, poly I:C), RE33® (a live-attenuated Edwardsiella ictaluri vaccine), or combinations thereof, followed by wild-type E. ictaluri challenge one month later. Survival analyses revealed that all TLR ligands and vaccine treatments provided significantly higher protection than the control, with beta glucan inducing significantly greater protection than RE33®, while R848 and poly I:C were equivalent to the vaccine. Survivals for the treatments were beta glucan 70%, beta glucan + RE33® 60%, R848 + RE33® 54%, poly I:C + RE33® 50%, R848 49%, poly I:C 32%, RE33® 24%, and control 0%. Gene expression analysis of kidney and liver tissues post challenge revealed that beta glucan training elicited early and strong increased expressions of nklb (5536 fold @ 6 hpi), nkld (147 fold @ 12 hpi), and ifng (575 fold @ 12 hpi) in the kidney, and ifng (1369 fold @ 6 hpi), nkla (250 fold @ 6 hpi), nklb (734 fold @ 6 hpi), nklc (2135 fold @ 6 hpi) and nkld (589 fold @ 6 hpi) in the liver. Principal component analysis (PCA) revealed that early kidney gene expressions at 6–12 h post secondary infection (nkla @ 12 hpi, nklb @ 6 and 12 hpi, nklc @ 6 and 12 hpi, nkld @ 6 and 12 hpi, ifng @ 6 and 12 hpi, t-bet @ 6, 12 and 48 hpi, and nitr9 @24 hpi) in the kidney and liver (nkla, nklb, nklc, nkld, ifng, t-bet and nitr9 @ 6 hpi) were associated with the highest survival. This study highlights that TLR ligand-induced trained immunity boosts innate immunity and survival, with NK cell subpopulations in kidney and liver tissues responding differently to mediate protective responses.