Zebrafish Models for Immunology and Inflammation Research

Inflammation is at the core of some of the most prevalent and poorly understood diseases worldwide, from chronic inflammatory conditions to cancer. Yet modeling these processes in vivo, with the resolution and throughput needed for drug discovery, remains a challenge. 

Zebrafish have emerged as a powerful platform to address this gap. Their conserved immune system, optical transparency, and compatibility with high-throughput screening make them uniquely suited for immunology research, from dissecting inflammatory pathways to evaluating next-generation immunotherapies such as CAR-T cells.

Zebrafish as an Experimental Model for Immunology Research

The use of zebrafish to study inflammation started during the early 2000s, and the number of publications has grown substantially since 2011, reflecting growing recognition of this model's translational value. Their popularity in immunology stems from a combination of features that no other vertebrate model offers at the same scale: optical transparency of larvae for real-time visualization of immune cell behavior, a fully sequenced genome with 82% of human disease-related protein-coding genes conserved, and the possibility to produce hundreds of embryos per breeding pair per week.

Zebrafish possess both innate and adaptive immune systems that closely parallel those of mammals. Their hematopoietic stem cells give rise to the same major leukocyte lineages found in humans (neutrophils, macrophages, B and T lymphocytes, and innate lymphoid cells), which display comparable morphology and functional behavior. Key signaling pathways driving immune activation, including NF-κB, JAK/STAT, and MAPK, are conserved, as are major pattern recognition receptors such as Toll-like receptors (TLRs) and NOD-like receptors. The zebrafish cytokine repertoire, including orthologs of IL-1β, TNF-α, IL-6, IL-10, and TGF-β, is well characterized and functionally similar to that of mammals, supporting the translational validity of findings obtained in this model.

Innate and Adaptive Immune Responses in Zebrafish

The development of the zebrafish immune system occurs in two distinct phases. The first hematopoietic site is the intermediate cell mass (ICM), which produces primitive blood cells, including erythrocytes and myeloid progenitors such as macrophages and neutrophils. The organogenesis of the thymus and head kidney, the primary lymphoid organs, initiates in the middle to late embryonic period but remains rudimentary throughout early larval stages. Macrophages become phagocytically active around 24-26 hours post fertilization (hpf), and neutrophils appear at 48 hpf. The adaptive immune system starts to develop around 4-6 days post-fertilization, but only becomes fully functional later, during the juvenile stage, several weeks after fertilization (Figure 1). 

Figure 1. Hematopoiesis and development of the immune system in zebrafish. The development of the immune system starts with primitive hematopoiesis at 11 h post-fertilization (hpf). Myeloid and erythroid cells originate in the anterior lateral plate mesoderm (ALPM) and posterior lateral mesoderm (PLPM)). Specifically, myeloid cells develop in the rostral blood islands (RBI) and erythroid cells in the intermediate cell mass (ICM), respectively. At about 2 days post-fertilization (dpf), hematopoietic stem cells (HSCs) appear in the dorsal aorta (DA) and then transit into the caudal hematopoietic tissues (CHT). The terminal phase of hematopoiesis involves the migration of HSCs to the thymus and pronephros (i.e., the first stage of kidney development), where the full maturation of the blood cells occurs. Notably, at 3 dpf, zebrafish emerge from the chorion and make contact with the outside environment without fully developed CD4+/CD8+ lymphocytes, which appear at 3 weeks post-fertilization (wpf). Adapted from: Franza M. et al.  Zebrafish (Danio rerio) as a Model System to Investigate the Role of the Innate Immune Response in Human Infectious Diseases. Int J Mol Sci. 2024 Nov 8;25(22):12008. 

Zebrafish also possess antigen-presenting cells, B and T lymphocytes, and secondary lymphoid organs, including the spleen, which perform functions analogous to those in mammals. While the adaptive immune system develops later than in mammals, the transparency of zebrafish larvae enables real-time fluorescence imaging of immune cell behavior throughout development, a direct visualization in vivo that is not possible in other vertebrates. 

Zebrafish Inflammation Models for Drug Discovery and Immunotherapy

A broad toolkit of inflammation models has been validated in zebrafish, covering physical, chemical, and biological induction strategies. Physical injury models based on tail fin amputation or laser ablation trigger reproducible neutrophil and macrophage recruitment within minutes, enabling kinetic studies of cell migration and resolution. Chemical models using lipopolysaccharide (LPS), carrageenan, copper sulfate, or dextran sodium sulfate (DSS) allow dose-controlled induction of acute or chronic inflammation in larvae and adult fish. Validated genetic models of chronic inflammation, including Sirt1 knockout and hai1 mutant lines, further expand the scope of disease modeling possible in this species.

Critically, zebrafish inflammation models are pharmacologically responsive. Classical steroidal agents such as dexamethasone and beclomethasone reduce leukocyte infiltration in zebrafish in line with their known mechanisms of action. Nonsteroidal anti-inflammatory drugs (NSAIDs), including indomethacin, aspirin, and diclofenac, also produce expected anti-inflammatory and analgesic effects. These findings support the use of zebrafish inflammation models in early pharmacological screening.

Evaluating CAR-T Cell Therapies In Vivo with Zebrafish Xenografts

Perhaps one of the most interesting applications of zebrafish in this field is related to the fact that they lack a functional adaptive immune system at embryonic and larval stages (equivalent to the nude rodent model). It allows the generation of xenograft tumor models, since zebrafish do not reject engrafted human tumor cells, which means there is no need for immunosuppression. Xenograft tumor models can be generated either using immortalized cell lines or using patient-derived tumor cells to create patient-derived xenografts (zAvatar). They provide a controlled setting to co-inject human immune effector cells alongside tumor cells and observe their interactions in real time. 

These xenografts offer a fast and cost-effective complement to mouse models for CAR-T cell evaluation. CAR-T has proven highly effective for B-cell malignancies; however, expanding this success to solid tumors and optimizing CAR designs for safety, specificity, and efficacy in immunosuppressive environments demands better preclinical tools. Mouse xenograft models, the current gold standard, are expensive, slow, and provide limited imaging access to the tumor-immune cell interface.

Several practical advantages make this platform particularly attractive for iterative CAR design testing:

• The small size of zebrafish embryos allows experiments in 96-well format, enabling automated image acquisition and higher-throughput analysis. 
• Novel CAR designs incorporating small-molecule ON/OFF switches can be conveniently tested in this system, as small compounds dissolved in the surrounding water are directly absorbed by the embryo. 
• The number of tumor cells required per zebrafish embryo is very low — typically between 50 and 300 — meaning that a single tumor sample can yield a large number of independent xenografts. 

Zebrafish have already proven to be a fast and cost-effective model to complement the mouse for CAR-T cell evaluation. Pascoal et al. injected GFP-expressing Nalm-6 human leukemia cells into the circulation of 48 hpf zebrafish embryos, followed two hours later by co-injection of CD19-specific CAR-T cells labeled with a fluorescent dye. Fluorescence-based quantification of the tail region (where both cell populations accumulate and interact) showed a mean reduction of approximately 70% in leukemia cell numbers within 24 hours in embryos receiving CAR-T cells, compared to no significant change in controls receiving T cells without a CAR (Figure 2). 

Figure 2. Zebrafish embryos were injected with Nalm-6 cells (green) at approximately 48 hpf. Around 2 hours later, either CD19 CAR-T cells (red cells in (A)) or T cells without a CAR (red cells in (B)) were injected, and a time-lapse movie was recorded, starting approximately 4 hours post-injection of T cells. Several time points of one embryo co-injected with CD19 CAR-T cells (A) or mock T cells (B) are shown as single frames. Adapted from: Pascoal S. et al. A Preclinical Embryonic Zebrafish Xenograft Model to Investigate CAR-T Cells In Vivo. Cancers (Basel). 2020 Feb 29;12(3):567. 

At ZeClinics, we have the infrastructure and expertise to generate and characterize zebrafish xenograft models at scale. We have validated human xenografts in zebrafish using several immortalized cell lines (i.e. MDA-MB-231)

Our automated phenotyping platforms are designed to maximize throughput while delivering quantitative, high-content data: the VAST BioImager automates the handling, positioning, and high-resolution imaging of individual larvae, enabling fluorescent visualization at the organ level across large cohorts (Figure 3). 

Figure 3.  A) High-resolution imaging of a zebrafish larva acquired by ZeClinics shortly after the xenotransplantations (timepoint 1), and the same larva after a few days of vehicle (DMSO) treatment (timepoint 2).  B) Quantification of tumor progression as the ratio of tumor volume at time-point 2 divided by tumor volume at time-point 1.

If you are exploring zebrafish xenograft models to evaluate tumor behavior, screen compounds with potential anti-tumoral efficacy, or the efficacy of cellular immunotherapies such as CAR T cells, we can support you from model generation and candidate screening to data reporting.

ZeClinics can help you

Sources

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Belo MAA, Oliveira MF, Oliveira SL, Aracati MF, Rodrigues LF, Costa CC, Conde G, Gomes JMM, Prata MNL, Barra A, Valverde TM, de Melo DC, Eto SF, Fernandes DC, Romero MGMC, Corrêa Júnior JD, Silva JO, Barros ALB, Perez AC, Charlie-Silva I. Zebrafish as a model to study inflammation: A tool for drug discovery. Biomed Pharmacother. 2021 Dec;144:112310. doi: 10.1016/j.biopha.2021.112310.

Franza M, Varricchio R, Alloisio G, De Simone G, Di Bella S, Ascenzi P, di Masi A. Zebrafish (Danio rerio) as a Model System to Investigate the Role of the Innate Immune Response in Human Infectious Diseases. Int J Mol Sci. 2024 Nov 8;25(22):12008. doi: 10.3390/ijms252212008.

Grissenberger S, Salzer B, Pascoal S, Wenninger-Weinzierl A, Lehner M, Distel M. Preclinical testing of CAR T cells in zebrafish xenografts. Methods Cell Biol. 2022;167:133-147. doi: 10.1016/bs.mcb.2021.07.002.

Pascoal S, Salzer B, Scheuringer E, Wenninger-Weinzierl A, Sturtzel C, Holter W, Taschner-Mandl S, Lehner M, Distel M. A Preclinical Embryonic Zebrafish Xenograft Model to Investigate CAR T Cells In Vivo. Cancers (Basel). 2020 Feb 29;12(3):567. doi: 10.3390/cancers12030567.

Zanandrea R, Bonan CD, Campos MM. Zebrafish as a model for inflammation and drug discovery. Drug Discov Today. 2020 Dec;25(12):2201–2211. doi: 10.1016/j.drudis.2020.09.036.

By Javier Terriente

Javier is the co-founder of ZeClinics and ZeCardio Therapeutics, two biotech firms specializing in zebrafish-based preclinical drug discovery for cardiovascular, neural, and toxicology applications. He combines scientific leadership with business acumen, having successfully driven fundraising efforts and strategic partnerships.

Currently leading scientific efforts at ZeCardioTx (and formerly CSO at ZeClinics), Javier also serves on the Board of Directors of AseBio, where he advocates for industry collaboration. His academic background includes a PhD in Molecular Biology and a Marie Curie Fellowship. Recognized as an expert in zebrafish models, he has published extensively and has supervised five industrial PhD theses.