The immune system is the body's mechanism of defense against harmful pathogens such as bacteria, viruses, and fungi, to abnormal cells and plays an adamant role in tissue repair after damage. It consists of a complex network of cells, tissues, and molecules working together to maintain homeostasis. Therefore, when developing a new drug or chemical compound, it is crucial to ensure that it does not interfere with this intricate system.
The immune system is divided into innate and adaptative systems that are complementary to each other. On one hand, the innate immune system acts rapidly through an unspecific response, for instance when an infection or tissue damage occurs, or a cell became tumoral. The adaptative immune system produces specific responses, much more effective but that takes longer to be produced.
Immunotoxicity is associated with any adverse effect on the structure or function of the immune system; this includes evaluations of other systems such as the nervous or endocrine, as the immune system works together with them.
This toxicity embraces a variety of serious adverse effects on the immune system and may result in either immunosuppression or enhancement of the immune response. Suppression of the immune response can lead to decreased host resistance to infectious agents or tumor cells, increasing susceptibility to infection and cancer, while enhancing the immune response is associated with the development of autoimmune reactivity such as delayed hypersensitivity, atopy, systemic or organ-specific immunopathology, and granuloma formation.
The immune system is extremely vulnerable to the action of xenobiotics and even mild disturbances could result in detrimental health effects. Therefore, it is extremely important to assess if new drugs under development have harmful effects on the immune system. Traditionally, the use of laboratory animals, usually rodents, has been necessary for toxicity evaluations of new drugs in the Early Discovery Phase, and toxicity tests of chemical compounds, being tremendously costly, time-consuming, and presenting ethical concerns.
Consequently, many New Alternative Models (NAMs) have been developed to overcome these problems and minimize laboratory animal use in line with the 3Rs Principles (Replacement, Reduction, and Refinement). In this sense, zebrafish embryos/larvae, are an ideal NAM that is highly reliable, genetically similar to humans, and enables the use of fish embryos for screening methods.
Zebrafish becoming each and every day more and more popular in toxicology and immunology studies due to their multiple benefits, such as their small size, rapid organogenesis, high fecundity, cost-effective maintenance, and transparent embryos. Their genome and immune systems are highly similar to humans, and several transgenic lines are available to visualize immune cells. Using zebrafish as a NAM for immunotoxicity assays may help to test the toxicity of pharmacological, agrochemical, industrial, or cosmetic compounds to the immune system in a High-Content manner and cost-efficient way. This is of special relevance in the Early Drug Discovery process where a high number of compounds have to be tested in a time and cost-efficient manner.
Zebrafish as Immunotoxicity Model for Early Drug Discovery
The immune system of zebrafish possesses various defense mechanisms and is very sensitive to environmental factors. They have a highly developed innate and adaptive immune system. Although the adaptive immune response is only operational in the juvenal stage, the innate immune system is functional at 48 hours post-fertilization (hpf), so it is possible to test on embryos of less than 6 days post-fertilization, with fewer ethical concerns and in line with the 3Rs Principles.
Xenobiotics can impair the immune defense through different mechanisms, including interference with signaling pathways in immune cells, and suppressing immune functions such as oxidative burst activity or induction of apoptosis. To assess immunotoxicity, zebrafish embryos are exposed to xenobiotics to evaluate different immune-related disturbances such as morphological changes, neutrophils and macrophage number and movement, oxidative stress, inflammation, and the expression of apoptosis-related genes.
In order to establish the immunotoxicity assays on zebrafish, different transgenic or mutant zebrafish have been developed as their genome is well-known. Regarding the applications of transgenic zebrafish for the evaluation of toxicity to the immune system, different immune cells such as neutrophils and macrophages can be fluorescently labeled for in vivo screening of immunotoxicity effects, allowing rapid characterization of the immune response. When an infection or tissue damage occurs, the inflammation process is activated as part of the innate immune response to activate the defense and repair mechanisms. This process is crucial for the correct activation of the immune system and organ repair process, and it is mediated by the production of cytokines, chemokines, and the infiltration of neutrophils and macrophages into the damaged tissue. It is possible to directly visualize zebrafish embryos under the fluorescent microscope for quantification of the amount and migration of neutrophils and macrophages.
Studies with transgenic zebrafish lines are often complemented with other techniques that allow further characterization of the immune response, such as the analysis of genes involved in macrophage migration such as mpk8b, mmp13a, cdc42I, rac1, and rhoA-B, in the inflammatory process or evaluation of oxidative stress and cell death.
Oxidative stress, which is characterized by an imbalance of reactive oxygen species (ROS), is one of the most important processes triggering the immune response and responsible for cellular damage associated with aging, cancer, or metabolic syndrome. ROS activates many pro-inflammatory substances such as cytokines, neutrophils, and macrophages, directly or through the activation of programmed cell death (apoptosis). To assess the oxidative-stress level in zebrafish, the accumulation of ROS and expression changes in marker genes (il6, il8, il-1beta, tnf-alpha, tlr, etc) or apoptosis-related genes (p53, caspases, bcl2, etc.) can be evaluated. Apoptosis itself can also be visualized in zebrafish embryos under the fluorescence microscope.
Biobide’s Immunotox Assay
Biobide has developed a High-Content Screening (HCS) Assay for the identification of potential immunotoxic/immunosuppressant substances using zebrafish embryos as NAM. This assay is composed of four consecutive steps:
- Maximum Tolerated Concentration (MTC) Assay
An initial MTC assay is performed to characterize the systemic toxicity profile. Zebrafish embryos are exposed to the test compound and at 120 hpf, several systemic and developmental toxicity-related endpoints are assessed by visual observation under a stereomicroscope. Two doses with no toxic effect are selected for the following assays.
- Quantification of leukocyte population
Transgenic zebrafish embryos expressing green neutrophils and red macrophages are used to quantify the number of leukocytes by fluorescent intensity after the test substance exposure. The decrease in the number of leukocytes is related to an immuno-toxic effect of the test compound.
- Neutrophil migration Assay
Sterile inflammation is induced by tail injury of transgenic embryos to evaluate neutrophil response to inflammation in the presence of test compounds. The decrease in the number of neutrophils migrated to the tail wound is related to a disruption of the immune response to the sterile inflammation caused by the test compound.
- Gene expression analysis.
Gene expression of the pro-inflammatory cytokines il-1beta and tnf-alpha is analyzed to determine if the inflammatory response is correctly activated.
Assessment of immunotoxicity of new potential drugs, especially in the Early Discovery Phase or chemical compounds used in industry, pharmacology, or for cosmetic purposes is extremely important for claiming their safety. Thus, the use of NAMs such as zebrafish contributes to fastening and reducing the colossal cost of toxicology assays in animal models.
Specifically in immunologic toxicity assessments, this model provides imperative benefits such as the possibility of easily testing different parameters using transgenic animal models in HCS platforms that offer a fast and cost-effective alternative and is in line with the 3Rs Principles.
In this sense, Biobide has developed Immunotox Assays in zebrafish embryos able to test a large number of compounds in a time-compelling manner and with highly valuable qualitative and quantitative information. In consequence, the zebrafish embryo model offers a time- and cost-effective NAM to assess the potential immuno-toxic and/or immunosuppressant effect of drugs or chemicals, providing a tool for HCS and reducing the number of experimental animals used.
- Immunotoxicity Testing Guidance | FDA
- Effects of cyhalofop-butyl on the developmental toxicity and immunotoxicity in zebrafish (Danio rerio) - PubMed (nih.gov)
- Immunotoxicity responses to polystyrene nanoplastics and their related mechanisms in the liver of zebrafish (Danio rerio) larvae - PubMed (nih.gov)
- Transgenic zebrafish (Danio rerio) as an emerging model system in ecotoxicology and toxicology: Historical review, recent advances, and trends - PubMed (nih.gov)
- Exposure to pyrazosulfuron-ethyl induces immunotoxicity and behavioral abnormalities in zebrafish embryos - PubMed (nih.gov)
- Effects of haloxyfop-p-methyl on the developmental toxicity, neurotoxicity, and immunotoxicity in zebrafish - PubMed (nih.gov)
- Cyhalofop-butyl exposure induces the severe hepatotoxicity and immunotoxicity in zebrafish embryos - PubMed (nih.gov)
- Immunotoxicity induced by triclocarban exposure in zebrafish triggering the risk of pancreatic cancer - PubMed (nih.gov)
- Sulfoxaflor induces immunotoxicity in zebrafish (Danio rerio) by activating TLR4/NF-κB signaling pathway - PubMed (nih.gov)