Dataset Information

Rag1-/- mutant zebrafish demonstrate specific protection following bacterial re-exposure.

ABSTRACT:

Background

Recombination activation gene 1 deficient (rag1(-/-)) mutant zebrafish have a reduced lymphocyte-like cell population that lacks functional B and T lymphocytes of the acquired immune system, but includes Natural Killer (NK)-like cells and Non-specific cytotoxic cells (NCC) of the innate immune system. The innate immune system is thought to lack the adaptive characteristics of an acquired immune system that provide enhanced protection to a second exposure of the same pathogen. It has been shown that NK cells have the ability to mediate adaptive immunity to chemical haptens and cytomegalovirus in murine models. In this study we evaluated the ability of rag1(-/-) mutant zebrafish to mount a protective response to the facultative intracellular fish bacterium Edwardsiella ictaluri.

Methodology/principal findings

Following secondary challenge with a lethal dose of homologous bacteria 4 and 8 weeks after a primary vaccination, rag1(-/-) mutant zebrafish demonstrated protective immunity. Heterologous bacterial exposures did not provide protection. Adoptive leukocyte transfers from previously exposed mutants conferred protective immunity to naïve mutants when exposed to homologous bacteria.

Conclusions/significance

Our findings show that a component of the innate immune system mounted a response that provided significantly increased survival when rag1(-/-) mutant zebrafish were re-exposed to the same bacteria. Further, adoptive cell transfers demonstrated that kidney interstitial leukocytes from previously exposed rag1(-/-) mutant zebrafish transferred this protective immunity. This is the first report of any rag1(-/-) mutant vertebrate mounting a protective secondary immune response to a bacterial pathogen, and demonstrates that a type of zebrafish innate immune cell can mediate adaptive immunity in the absence of T and B cells.

SUBMITTER: Hohn C

PROVIDER: S-EPMC3435260 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Publications

Rag1-/- mutant zebrafish demonstrate specific protection following bacterial re-exposure.

Hohn Claudia C Petrie-Hanson Lora L

PloS one 20120906 9

<h4>Background</h4>Recombination activation gene 1 deficient (rag1(-/-)) mutant zebrafish have a reduced lymphocyte-like cell population that lacks functional B and T lymphocytes of the acquired immune system, but includes Natural Killer (NK)-like cells and Non-specific cytotoxic cells (NCC) of the innate immune system. The innate immune system is thought to lack the adaptive characteristics of an acquired immune system that provide enhanced protection to a second exposure of the same pathogen. ...[more]

PMID: 22970222

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Project description:BackgroundZebrafish may prove to be one of the best vertebrate models for innate immunology. These fish have sophisticated immune components, yet rely heavily on innate immune mechanisms. Thus, the development and characterization of mutant and/or knock out zebrafish are critical to help define immune cell and immune gene functions in the zebrafish model. The use of Severe Combined Immunodeficient (SCID) and recombination activation gene 1 and 2 mutant mice has allowed the investigation of the specific contribution of innate defenses in many infectious diseases. Similar zebrafish mutants are now being used in biomedical and fish immunology related research. This report describes the leukocyte populations in a unique model, recombination activation gene 1-/- mutant zebrafish (rag1 mutants).ResultsDifferential counts of peripheral blood leukocytes (PBL) showed that rag1 mutants had significantly decreased lymphocyte-like cell populations (34.7%) compared to wild-types (70.5%), and significantly increased granulocyte populations (52.7%) compared to wild-types (17.6%). Monocyte/macrophage populations were similar between mutants and wild-types, 12.6% and 11.3%, respectively. Differential leukocyte counts of rag1 mutant kidney hematopoietic tissue showed a significantly reduced lymphocyte-like cell population (8%), a significantly increased myelomonocyte population (57%), 34.8% precursor cells, and 0.2% thrombocytes, while wild-type hematopoietic kidney tissue showed 29.4% lymphocytes/lymphocyte-like cells, 36.4% myelomonocytes, 33.8% precursors and 0.5% thrombocytes. Flow cytometric analyses of kidney hematopoietic tissue revealed three leukocyte populations. Population A was monocytes and granulocytes and comprised 34.7% of the gated cells in rag1 mutants and 17.6% in wild-types. Population B consisted of hematopoietic precursors, and comprised 50% of the gated cells for rag1 mutants and 53% for wild-types. Population C consisted of lymphocytes and lymphocyte-like cells and comprised 7% of the gated cells in the rag1 mutants and 26% in the wild-types. Reverse transcriptase polymerase chain reaction (RT-PCR) assays demonstrated rag1 mutant kidney hematopoietic tissue expressed mRNA encoding Non-specific Cytotoxic cell receptor protein-1 (NCCRP-1) and Natural Killer (NK) cell lysin but lacked T cell receptor (TCR) and immunoglobulin (Ig) transcript expression, while wild-type kidney hematopoietic tissue expressed NCCRP-1, NK lysin, TCR and Ig transcript expression.ConclusionOur study demonstrates that in comparison to wild-type zebrafish, rag1 mutants have a significantly reduced lymphocyte-like cell population that likely includes Non-specific cytotoxic cells (NCC) and NK cells (and lacks functional T and B lymphocytes), a similar macrophage/monocyte population, and a significantly increased neutrophil population. These zebrafish have comparable leukocyte populations to SCID and rag 1 and/or 2 mutant mice, that possess macrophages, natural killer cells and neutrophils, but lack T and B lymphocytes. Rag1 mutant zebrafish will provide the platform for remarkable investigations in fish and innate immunology, as rag 1 and 2 mutant mice did for mammalian immunology.

Project description: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.

Dataset Information

Rag1-/- mutant zebrafish demonstrate specific protection following bacterial re-exposure.

Background

Methodology/principal findings

Conclusions/significance

Publications

Rag1-/- mutant zebrafish demonstrate specific protection following bacterial re-exposure.

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