Abstract
In the absence of lymphocytes, rag1-/- mutant zebrafish develop protective immunity to bacteria. In mammals, induction of protection by innate immunity can be mediated by macrophages or natural killer (NK) cells. To elucidate potential responsive cell populations, we morphologically characterized lymphocyte-like cells (LLCs) from liver, spleen and kidney hematopoietic tissues. In fish, these cells include NK cells and Non-specific cytotoxic cells (NCCs). We also evaluated the transcriptional expression response of select genes that are important indicators of NK and macrophage activation after exposure to specific TLR ligands. The LLC cell populations could be discriminated by size and further discriminated by the presence of cytoplasmic granules. Expression levels of mx, tnfα, ifnγ, t-bet and nitr9 demonstrated dynamic changes in response to intra-coelomically administered β glucan (a TLR2/6 ligand), Poly I:C (a TLR3 ligand) and resiquimod (R848) (a TLR7/8 ligand). Following TLR 2/6 stimulation, there was a greater than 100 fold increase in ifnγ in liver, kidney and spleen and moderate increases in tnfα in liver and kidney. TLR3 stimulation caused broad up regulation of mx, down-regulation of tnfα in kidney and spleen tissues and up regulation of nitr9 in the kidney. Following TLR 7/8 stimulation, there was a greater than 100 fold increase in ifnγ in liver and kidney and t-bet in liver. Our gene expression findings suggest that LLCs and macrophages are stimulated following β glucan exposure. Poly I:C causes type I interferon response and mild induction of LLC in the kidney and R-848 exposure causes the strongest LLC stimulation. Overall, the strongest NK like gene expression occurred in the liver. These differential effects of TLR ligands in rag1-/- mutant zebrafish shows strong NK cell-like gene expression responses, especially in the liver, and provides tools to evaluate the basis for protective immunity mediated by the innate immune cells of fish.
Highlights
Innate immunity provides essential protection against pathogens during early life stages of fish because adaptive immunity is absent [1,2,3]
natural killer (NK) lysin is expressed by NK cells [51], and NCCRP-1 is expressed by non-specific cytotoxic cells (NCCs) cells [52], suggesting that NCCs and NK cells are included in the LCC populations we observed
We feel the significant increases seen in t-bet following Toll like receptors (TLRs) ligand treatments suggests that R848 likely resulted in an increased numbers of non-specific cytotoxic cells (NCCs) and NK cells, and changes in nitr9 expression may reflect changes in the NK cell population
Summary
Innate immunity provides essential protection against pathogens during early life stages of fish because adaptive immunity is absent [1,2,3]. Rag1-/- mutant zebrafish lack T cell receptor (TCR) and immunoglobulin (Ig) but have non-specific cytotoxic cells (NCCs), NK cells, monocytes/macrophages and neutrophils [5]. Using these fish as a model for studying immune responses in young fish, we found that fish lacking effector lymphocytes can develop protective immunity to bacteria after vaccination [6]. Increased ifnγ expression was associated with both primary and secondary immune responses [11] These findings suggested functional responses of cell division and enhanced phagocytosis, but did not indicate a specific cell population was associated with the protective secondary response
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