Characterization of rag1 mutant zebrafish leukocytes

<p>Abstract</p> <p>Background</p> <p>Zebrafish 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 <it>recombination activation gene </it>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, <it>recombination activation gene 1</it>^-/-mutant zebrafish (<it>rag</it>1 mutants).</p> <p>Results</p> <p>Differential counts of peripheral blood leukocytes (PBL) showed that <it>rag</it>1 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 <it>rag</it>1 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.</p> <p>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 <it>rag</it>1 mutants and 17.6% in wild-types. Population B consisted of hematopoietic precursors, and comprised 50% of the gated cells for <it>rag</it>1 mutants and 53% for wild-types. Population C consisted of lymphocytes and lymphocyte-like cells and comprised 7% of the gated cells in the <it>rag</it>1 mutants and 26% in the wild-types.</p> <p>Reverse transcriptase polymerase chain reaction (RT-PCR) assays demonstrated <it>rag</it>1 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.</p> <p>Conclusion</p> <p>Our study demonstrates that in comparison to wild-type zebrafish, <it>rag</it>1 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 <it>rag </it>1 and/or 2 mutant mice, that possess macrophages, natural killer cells and neutrophils, but lack T and B lymphocytes. <it>Rag</it>1 mutant zebrafish will provide the platform for remarkable investigations in fish and innate immunology, as <it>rag </it>1 and 2 mutant mice did for mammalian immunology.</p

Zebrafish Kidney Phagocytes Utilize Macropinocytosis and Ca2+-Dependent Endocytic Mechanisms

Background: The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including active endocytosis by macrophages and granulocytes. Endocytosis can be used as a reliable measure of selective and non-selective mechanisms of antigen uptake in the early phase of an immune response. Numerous assays have been developed to measure this response in a variety of mammalian and fish species. The small size of the zebrafish has prevented the large-scale collection of monocytes/macrophages and granulocytes for these endocytic assays. Methodology/Principal Findings: Pooled zebrafish kidney hematopoietic tissues were used as a source of phagocytic cells for flow-cytometry based endocytic assays. FITC-Dextran, Lucifer Yellow and FITC-Edwardsiella ictaluri were used to evaluate selective and non-selective mechanisms of uptake in zebrafish phagocytes. Conclusions/Significance: Zebrafish kidney phagocytes characterized as monocytes/macrophages, neutrophils and lymphocytes utilize macropinocytosis and Ca 2+-dependant endocytosis mechanisms of antigen uptake. These cells do not appear to utilize a mannose receptor. Heat-killed Edwardsiella ictaluri induces cytoskeletal interactions for internalization in zebrafish kidney monocytes/macrophages and granulocytes. The proposed method is easy to implement and should prove especially useful in immunological, toxicological and epidemiological research

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

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 na?ve 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

Evaluation of visible implant elastomer tags in zebrafish (Danio rerio)

Summary The use of the visible implant elastomer (VIE) tagging system in zebrafish (Danio rerio) was examined. Two tag orientations (horizontal and vertical) at the dorsal fin base were tested for tag retention, tag fragmentation and whether VIE tags affected growth and survival of juvenile zebrafish (1–4 month post hatch). Six tag locations (abdomen, anal fin base, caudal peduncle, dorsal fin base, pectoral fin base, isthmus) and 5 tag colors (yellow, red, pink, orange, blue) were evaluated for ease of VIE tag application and tag visibility in adult zebrafish. Long-term retention (1 year) and multiple tagging sites (right and left of dorsal fin and pectoral fin base) were examined in adult zebrafish. Lastly, survival of recombination activation gene 1−/− (rag1−/−) zebrafish was evaluated after VIE tagging. The best tag location was the dorsal fin base, and the most visible tag color was pink. Growth rate of juvenile zebrafish was not affected by VIE tagging. Horizontal tagging is recommended in early stages of fish growth (1–2 months post hatch). VIE tags were retained for 1 year and tagging did not interfere with long-term growth and survival. There was no mortality associated with VIE tagging in rag1−/− zebrafish. The VIE tagging system is highly suitable for small-sized zebrafish. When familiar with the procedure, 120 adult zebrafish can be tagged in one hour. It does not increase mortality in adult zebrafish or interfere with growth in juvenile or adult zebrafish

Trained Immunity Provides Long-Term Protection against Bacterial Infections in Channel Catfish

Beta glucan exposure induced trained immunity in channel catfish that conferred long-term protection against Edwardsiella ictaluri and Edwardsiella piscicida infections one month post exposure. Flow cytometric analyses demonstrated that isolated macrophages and neutrophils phagocytosed higher amounts of E. ictaluri and E. piscicida. Beta glucan induced changes in the distribution of histone modifications in the monomethylation and trimethylation of H3K4 and modifications in the acetylation and trimethylation of H3K27. KEGG pathway analyses revealed that these modifications affected expressions of genes controlling phagocytosis, phagosome functions and enhanced immune cell signaling. These analyses correlate the histone modifications with gene functions and to the observed enhanced phagocytosis and to the increased survival following bacterial challenge in channel catfish. These data suggest the chromatin reconfiguration that directs trained immunity as demonstrated in mammals also occurs in channel catfish. Understanding the mechanisms underlying trained immunity can help us design prophylactic and non-antibiotic based therapies and develop broad-based vaccines to limit bacterial disease outbreaks in catfish production

Comparison of survival by day (left panel) and cumulative mortality (right panel) between naïve and vaccinated rag1^−/− mutant zebrafish in Trial 1.

<p>Asterisk indicates a significantly lower mortality rate in vaccinated rag1^−/− mutant zebrafish when compared to naïve rag1^−/− mutant zebrafish. Fish were vaccinated with RE33® an attenuated strain of <i>E. ictaluri</i> and 4 weeks later challenged by a secondary injection of virulent <i>E. ictaluri</i>. DPI = days post secondary exposure. Error bars indicate standard error of the mean, SEM, between tanks (n = 9).</p

Flow cytometry data of CFSE stained leukocytes re-isolated from recipient rag1^−/− mutant kidneys.

<p><b>A.</b> Scatter plot of kidney leukocytes demonstrating the gating of cell populations (gate 1 is phagocytes, gate 2 is lymphocyte like cells). <b>B.</b> Histogram of positive control: CFSE stained cells injected into the recipient. <b>C.</b> Histogram of negative control: cells isolated from a PBS injected fish. <b>D.</b> Histograms CSFE stained cell counts from gate 1 (phagocytes) and gate 2 (lymphocyte like) from kidney tissue of recipient rag1^−/− mutants at 24 h post infusion. <b>E.</b> Graph indicating the CFSE stained cell counts in the kidney of transfused fish at 1 h, then 1, 2, 3, 4, 7 and 18 days post transfusion. Error bars indicate standard deviation (n = 2).</p

Comparison of survival by day (left panel) and cumulative mortality (right panel) between naïve and vaccinated rag1^−/− mutant zebrafish fed antibiotic feed in Trial 3.

<p>Asterisk indicates a significantly lower mortality rate in vaccinated rag1^−/− mutant zebrafish when compared to naïve rag1^−/− mutant zebrafish. Fish were vaccinated with RE33® an attenuated strain of <i>E. ictaluri</i> and 4 weeks later challenged by a secondary injection of virulent <i>E. ictaluri</i>. Ten days after the vaccination, fish received feed supplemented with oxolinic acid for 7 days. DPI = days post secondary exposure. Error bars indicate standard error of the mean, SEM, between tanks (n = 16).</p

Homologous and heterologous protection trial in Trial 4.

<p>Percent survival, A, and relative percent survival [(1– mean mortality of vaccinated/mean mortality of sham vaccinated) × 100] B, of <i>E. ictaluri</i> RE33® vaccinated rag1^−/− mutant zebrafish after secondary challenge with <i>E</i>. <i>ictaluri</i>, <i>Y. ruckeri</i> or <i>E. tarda.</i> Error bars indicate standard deviation between tanks (n = 4).</p