quantification-respiratory-burst-response-as-an-indicator-innate

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

using-zebrafish-models-human-influenza-virus-infections-to-screen

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

University of Maine

In mammals, Toll-IL-1R-containing adaptor molecule 1 (TICAM1)-dependent TLR pathways induce NF-kappaB and IFN-beta responses. TICAM1 activates NF-kappaB through two different pathways involving its interactions with TNFR-associated factor 6 and receptor-interacting protein 1. It also activates IFN regulatory factor 3/7 through its interaction with TANK-binding kinase-1, leading to the robust up-regulation of IFN-beta. In this study, we describe the role of zebrafish (Danio rerio) TICAM1 in activating NF-kappaB and zebrafish type I IFN. Zebrafish IFN is unique in that it cannot be categorized as being alpha- or beta-like. Through comprehensive sequence, phylogenetic, and syntenic analyses, we fully describe the identification of a zebrafish TICAM1 ortholog. Zebrafish TICAM1 exhibits sequence divergence from its mammalian orthologs and our data demonstrate that these sequence differences have functional consequences. Zebrafish TICAM1 activates zebrafish IFN; however, it does so in an apparently IFN regulatory factor 3/7-independent manner. Furthermore, zebrafish TICAM1 does not interact with zebrafish TNFR-associated factor 6, thus NF-kappaB activation is dependent upon its interaction with receptor-interacting protein 1. Comparative genome analysis suggests that TICAM1 and TICAM2 evolved from a common vertebrate TICAM ancestor following a gene duplication event and that TICAM2 was lost in teleosts following the divergence of the rayfin and lobefin fishes 450 million years ago. These studies provide evidence, for the first time, of the evolving function of a vertebrate TLR pathway.

Evidence for evolving Toll-IL-1 receptor-containing adaptor molecule function in vertebrates.

The zebrafish, Danio rerio, has come to the forefront of biomedical research as a powerful model for the study of development, neurobiology, and genetics of humans. In recent years, use of the zebrafish system has extended into studies in behaviour, immunology and toxicology, retaining the concept that it will serve as a model for human disease. As one of the most thoroughly studied teleosts, with a wealth of genetic and genomic information available, the zebrafish is now being considered as a model for pathogen studies in finfishes. Its genome is currently being sequenced and annotated, and gene microarrays and insertional mutants are commercially available. The use of gene-specific knockdown of translation through morpholino oligonucleotides is widespread. As a result, several laboratories have developed bacterial and viral disease models with the zebrafish to study immune responses to infection. Although many of the zebrafish pathogen models were developed to address human infectious disease, the results of these studies should provide important clues for the development of effective vaccines and prophylactic measures against bacterial and viral pathogens in economically important fishes. In this review, the capabilities and potential of the zebrafish model system will be discussed and an overview of information on zebrafish infectious disease models will be presented.

Zebrafish as a model for infectious disease and immune function.

Mammalian immune responses to LPS exposure are typified by the robust induction of NF-kappaB and IFN-beta responses largely mediated by TLR4 signal transduction pathways. In contrast to mammals, Tlr4 signal transduction pathways in nontetrapods are not well understood. Comprehensive syntenic and phylogenetic analyses support our hypothesis that zebrafish tlr4a and tlr4b genes are paralogous rather than orthologous to human TLR4. Furthermore, we provide evidence to support our assertion that the in vivo responsiveness of zebrafish to LPS exposure is not mediated by Tlr4a and Tlr4b paralogs because they fail to respond to LPS stimulation in vitro. Zebrafish Tlr4a and Tlr4b paralogs were also unresponsive to heat-killed Escherichia coli and Legionella pneumophila. Using chimeric molecules in which portions of the zebrafish Tlr4 proteins were fused to portions of the mouse TLR4 protein, we show that the lack of responsiveness to LPS was most likely due to the inability of the extracellular portions of zebrafish Tlr4a and Tlr4b to recognize the molecule, rather than to changes in their capacities to transduce signals through their Toll/IL-1 receptor (TIR) domains. Taken together, these findings strongly support the notion that zebrafish tlr4a and tlr4b paralogs have evolved to provide alternative ligand specificities to the Tlr immune defense system in this species. These data demonstrate that intensive examination of gene histories when describing the Tlr proteins of basally diverging vertebrates is required to obtain fuller appreciation of the evolution of their function. These studies provide the first evidence for the functional evolution of a novel Tlr.

The gene history of zebrafish tlr4a and tlr4b is predictive of their divergent functions.

Chronic myeloid leukemia (CML) is induced by the BCR-ABL oncogene, a product of Philadelphia (Ph) chromosome. The BCR-ABL kinase inhibitor imatinib is a standard treatment for Ph+ leukemia, and has been shown to induce a complete hematologic and cytogenetic response in most chronic phrase CML patients. However, imatinib does not cure CML, and one of the reasons is that imatinib does not kill leukemia stem cells (LSCs) in CML both in vitro and in vivo. Recently, several new targets or drugs have been reported to inhibit LSCs in cultured human CD34+ CML cells or in mouse model of BCR-ABL induced CML, including an Alox5 pathway inhibitor, Hsp90 inhibitors, omacetaxine, hedgehog inhibitor and BMS-214662. Specific targeting of LSCs but not normal stem cell is a correct strategy for developing new anti-cancer therapies in the future.

Novel therapeutic agents against cancer stem cells of chronic myeloid leukemia.

Inhibition of BCR-ABL with kinase inhibitors has become a well-accepted strategy for targeted therapy of Philadelphia-positive (Ph(+)) chronic myeloid leukemia (CML) and has been shown to be highly effective in controlling the disease. However, BCR-ABL kinase inhibitors do not efficiently kill leukemic stem cells (LSCs), indicating that this therapeutic strategy does not lead to a cure of CML. Development of curative therapies of CML require the identification of genes/pathways that play critical roles in survival and self-renewal of LSCs. Targeting of these key BCR-ABL downstream genes provides an opportunity to eradicate LSCs, as shown in our work that identifies the Alox5 gene as a key regulator of the function of CML LSCs. Immediate clinical trials are necessary to test the effectiveness of targeting a key BCR-ABL downstream gene in eradicating LSCs in CML patients. In this review, we will discuss current targeted therapies of CML using BCR-ABL kinase inhibitors, with a focus on the importance of developing a targeted therapy of CML through identification of target genes in CML LSCs.

Targeted therapy of chronic myeloid leukemia.

Arsenic trioxide (ATO) is a first-line anti-cancer agent for acute promyelocytic leukemia, and induces apoptosis in other solid cancer cell lines including breast cancer cells. However, as with arsenites found in drinking water and used as raw materials for wood preservatives, insecticides, and herbicides, low doses of ATO can induce carcinogenesis after long-term exposure. At 24 h after exposure, ATO (0.01-1 µM) significantly increased cell proliferation and promoted cell cycle progression from the G1 to S/G2 phases in the non-tumorigenic MCF10A breast epithelial cell line. The expression of 14 out of 96 cell-cycle-associated genes significantly increased, and seven of these genes including cell division cycle 6 (CDC6) and cyclin D1 (CCND1) were closely related to cell cycle progression from G1 to S phase. Low-dose ATO steadily increased gene transcript and protein levels of both CDC6 and cyclin D1 in a dose- and time-dependent manner. Low-dose ATO produced reactive oxygen species (ROS), and activated the p38 MAPK, Akt, and ERK1/2 pathways at different time points within 60 min. Small molecular inhibitors and siRNAs inhibiting the activation of p38 MAPK, Akt, and ERK1/2 decreased the ATO-increased expression of CDC6 protein. Inhibiting the activation of Akt and ERK1/2, but not p38 MAPK, decreased the ATO-induced expression of cyclin D1 protein. This study reports for the first time that p38 MAPK/Akt/ERK1/2 activation is required for the protein stabilization of CDC6 in addition to cyclin D1 in ATO-induced cell proliferation and cell cycle modulation from G1 to S phase.

Activation of the p38 MAPK/Akt/ERK1/2 signal pathways is required for the protein stabilization of CDC6 and cyclin D1 in low-dose arsenite-induced cell proliferation.

We have shown that Alox5 is a critical regulator of leukemia stem cells (LSCs) in a BCR-ABL-induced chronic myeloid leukemia (CML) mouse model, and we hypothesize that the Alox5 pathway represents a major molecular network that regulates LSC function. Therefore, we sought to dissect this pathway by comparing the gene expression profiles of wild type and Alox5(-/-) LSCs. DNA microarray analysis revealed a small group of candidate genes that exhibited changes in the levels of transcription in the absence of Alox5 expression. In particular, we noted that the expression of the Msr1 gene was upregulated in Alox5(-/-) LSCs, suggesting that Msr1 suppresses the proliferation of LSCs. Using CML mouse model, we show that Msr1 is downregulated by BCR-ABL and this down-regulation is partially restored by Alox5 deletion, and that Msr1 deletion causes acceleration of CML development. Moreover, Msr1 deletion markedly increases LSC function through its effects on cell cycle progression and apoptosis. We also show that Msr1 affects CML development by regulating the PI3K-AKT pathway and β-Catenin. Together, these results demonstrate that Msr1 suppresses LSCs and CML development. The enhancement of the tumor suppressor function of Msr1 may be of significance in the development of novel therapeutic strategies for CML.

A tumor suppressor function of the Msr1 gene in leukemia stem cells of chronic myeloid leukemia.

Hematopoiesis is a tightly regulated biological process that relies upon complicated interactions between blood cells and their microenvironment to preserve the homeostatic balance of long-term hematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs), multipotent progenitors (MPPs), and differentiated cells. Adhesion molecules like P-selectin (encoded by the Selp gene) are essential to hematopoiesis, and their dysregulation has been linked to leukemogenesis. Like HSCs, leukemic stem cells (LSCs) depend upon their microenvironments for survival and propagation. P-selectin plays a crucial role in Philadelphia chromosome-positive (Ph(+)) chronic myeloid leukemia (CML). In this paper, we show that cells deficient in P-selectin expression can repopulate the marrow more efficiently than wild type controls. This results from an increase in HSC self-renewal rather than alternative possibilities like increased homing velocity or cell cycle defects. We also show that P-selectin expression on LT-HSCs, but not ST-HSCs and MPPs, increases with aging. In the absence of P-selectin expression, mice at 6 months of age possess increased levels of short-term HSCs and multipotent progenitors. By 11 months of age, there is a shift towards increased levels of long-term HSCs. Recipients of BCR-ABL-transduced bone marrow cells from P-selectin-deficient donors develop a more aggressive CML, with increased percentages of LSCs and progenitors. Taken together, our data reveal that P-selectin expression on HSCs and LSCs has important functional ramifications for both hematopoiesis and leukemogenesis, which is most likely attributable to an intrinsic effect on stem cell self-renewal.

Functional ramifications for the loss of P-selectin expression on hematopoietic and leukemic stem cells.

A therapeutic strategy for treating cancer is to target and eradicate cancer stem cells (CSCs) without harming their normal stem cell counterparts. The success of this approach relies on the identification of molecular pathways that selectively regulate CSC function. Using BCR-ABL-induced chronic myeloid leukemia (CML) as a disease model for CSCs, we show that BCR-ABL downregulates the Blk gene (encoding B-lymphoid kinase) through c-Myc in leukemic stem cells (LSCs) in CML mice and that Blk functions as a tumor suppressor in LSCs but does not affect normal hematopoietic stem cells (HSCs) or hematopoiesis. Blk suppresses LSC function through a pathway involving an upstream regulator, Pax5, and a downstream effector, p27. Inhibition of this Blk pathway accelerates CML development, whereas increased activity of the Blk pathway delays CML development. Blk also suppresses the proliferation of human CML stem cells. Our results show the feasibility of selectively targeting LSCs, an approach that should be applicable to other cancers.

The Blk pathway functions as a tumor suppressor in chronic myeloid leukemia stem cells.

FOXM1 is a critical regulator of the G1/S and G2/M cell cycle transitions, as well as of the mitotic spindle assembly. Previous studies have suggested that FOXM1 regulates CDC25A gene transcription, but the mechanism remains unknown. Here, we provide evidence that FOXM1 directly regulates CDC25A gene transcription via direct promoter binding and indirect activation of E2F-dependent pathways. Prior literature reported that CDC25B and CDC25C activate CDK1/cyclinB complexes in order to enable phosphorylation of FOXM1. It was unknown if CDC25A functions in a similar manner. We report that FOXM1 transcriptional activity is synergistically enhanced when co-expressed with CDC25A. The increase is dependent upon CDK1 phosphorylation of FOXM1 at T600, T611 and T620 residues. We also report a novel protein interaction between FOXM1 and CDC25A via the C-terminus of FOXM1. We demonstrate that the phosphorylation of Thr 600 and Thr 611 residues of FOXM1 enhanced this interaction, and that the interaction is dependent upon CDC25A phosphatase activity. Our work provides novel insight into the underlying mechanisms by which FOXM1 controls the cell cycle through its association with CDC25A.

Novel interactions between FOXM1 and CDC25A regulate the cell cycle.

Humans and viruses have a long co-evolutionary history. Viral illnesses have and will continue to shape human history: from smallpox, to influenza, to HIV, and beyond. Animal models of human viral illnesses are needed in order to generate safe and effective antiviral medicines, adjuvant therapies, and vaccines. These animal models must support the replication of human viruses, recapitulate aspects of human viral illnesses, and respond with conserved immune signaling cascades. The zebrafish is perhaps the simplest, most commonly used laboratory model organism in which innate and/or adaptive immunity can be studied. Herein, we will discuss the current zebrafish models of human viral illnesses and the insights they have provided. We will highlight advantages of early life stage zebrafish and the importance of innate immunity in human viral illnesses. We will also discuss viral characteristics to consider before infecting zebrafish with human viruses as well as predict other human viruses that may be able to infect zebrafish.

Studying the immune response to human viral infections using zebrafish.

In genome-wide studies, hundreds of thousands of hypothesis tests are performed simultaneously. Bonferroni correction and False Discovery Rate (FDR) can effectively control type I error but often yield a high false negative rate. We aim to develop a more powerful method to detect differentially expressed genes. We present a Weighted False Discovery Rate (WFDR) method that incorporate biological knowledge from genetic networks. We first identify weights using Integrative Multi-species Prediction (IMP) and then apply the weights in WFDR to identify differentially expressed genes through an IMP-WFDR algorithm. We performed a gene expression experiment to identify zebrafish genes that change expression in the presence of arsenic during a systemic Pseudomonas aeruginosa infection. Zebrafish were exposed to arsenic at 10 parts per billion and/or infected with P. aeruginosa. Appropriate controls were included. We then applied IMP-WFDR during the analysis of differentially expressed genes. We compared the mRNA expression for each group and found over 200 differentially expressed genes and several enriched pathways including defense response pathways, arsenic response pathways, and the Notch signaling pathway.

Testing multiple hypotheses through IMP weighted FDR based on a genetic functional network with application to a new zebrafish transcriptome study.

Small freshwater fish models, especially zebrafish, offer advantages over traditional rodent models, including low maintenance and husbandry costs, high fecundity, genetic diversity, physiology similar to that of traditional biomedical models, and reduced animal welfare concerns. The Collaborative Workshop on Aquatic Models and 21st Century Toxicology was held at North Carolina State University on May 5-6, 2014, in Raleigh, North Carolina, USA. Participants discussed the ways in which small fish are being used as models to screen toxicants and understand mechanisms of toxicity. Workshop participants agreed that the lack of standardized protocols is an impediment to broader acceptance of these models, whereas development of standardized protocols, validation, and subsequent regulatory acceptance would facilitate greater usage. Given the advantages and increasing application of small fish models, there was widespread interest in follow-up workshops to review and discuss developments in their use. In this article, we summarize the recommendations formulated by workshop participants to enhance the utility of small fish species in toxicology studies, as well as many of the advances in the field of toxicology that resulted from using small fish species, including advances in developmental toxicology, cardiovascular toxicology, neurotoxicology, and immunotoxicology. We alsoreview many emerging issues that will benefit from using small fish species, especially zebrafish, and new technologies that will enable using these organisms to yield results unprecedented in their information content to better understand how toxicants affect development and health.

ArticleTotal : 2	Year
Publication title Cited by 16	2013
Publication title Cited by 16	2017

Article	Year
Evidence for evolving Toll-IL-1 receptor-containing adaptor molecule function in vertebrates. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 17372010	2007
Zebrafish as a model for infectious disease and immune function. Fish & shellfish immunology\| PubMed ID: 18640057	2008
The gene history of zebrafish tlr4a and tlr4b is predictive of their divergent functions. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 19812203	2009
Novel therapeutic agents against cancer stem cells of chronic myeloid leukemia. Anti-cancer agents in medicinal chemistry\| PubMed ID: 20184539	2010
Targeted therapy of chronic myeloid leukemia. Biochemical pharmacology\| PubMed ID: 20470758	2010
Activation of the p38 MAPK/Akt/ERK1/2 signal pathways is required for the protein stabilization of CDC6 and cyclin D1 in low-dose arsenite-induced cell proliferation. Journal of cellular biochemistry\| PubMed ID: 20862710	2010
A tumor suppressor function of the Msr1 gene in leukemia stem cells of chronic myeloid leukemia. Blood\| PubMed ID: 21596859	2011
Functional ramifications for the loss of P-selectin expression on hematopoietic and leukemic stem cells. PloS one\| PubMed ID: 22039451	2011
The Blk pathway functions as a tumor suppressor in chronic myeloid leukemia stem cells. Nature genetics\| PubMed ID: 22797726	2012
Novel interactions between FOXM1 and CDC25A regulate the cell cycle. PloS one\| PubMed ID: 23240008	2012
Studying the immune response to human viral infections using zebrafish. Developmental and comparative immunology\| PubMed ID: 24718256	2014
Testing multiple hypotheses through IMP weighted FDR based on a genetic functional network with application to a new zebrafish transcriptome study. BioData mining\| PubMed ID: 26097506	2015
Advancing toxicology research using in vivo high throughput toxicology with small fish models. ALTEX\| PubMed ID: 27328013	2016

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