Zebrafish: A Relevant Genetic Model for Human Primary Immunodeficiency (PID) Disorders?

Primary immunodeficiency (PID) disorders, also commonly referred to as inborn errors of immunity, are a heterogenous group of human genetic diseases characterized by defects in immune cell development and/or function. Since these disorders are generally uncommon and occur on a variable background profile of potential genetic and environmental modifiers, animal models are critical to provide mechanistic insights as well as to create platforms to underpin therapeutic development. This review aims to review the relevance of zebrafish as an alternative genetic model for PIDs. It provides an overview of the conservation of the zebrafish immune system and details specific examples of zebrafish models for a multitude of specific human PIDs across a range of distinct categories, including severe combined immunodeficiency (SCID), combined immunodeficiency (CID), multi-system immunodeficiency, autoinflammatory disorders, neutropenia and defects in leucocyte mobility and respiratory burst. It also describes some of the diverse applications of these models, particularly in the fields of microbiology, immunology, regenerative biology and oncology.

Investigation of the causal etiology in a patient with T-B+NK+ immunodeficiency.

Newborn screening for severe combined immunodeficiency (SCID) has not only accelerated diagnosis and improved treatment for affected infants, but also led to identification of novel genes required for human T cell development. A male proband had SCID newborn screening showing very low T cell receptor excision circles (TRECs), a biomarker for thymic output of nascent T cells. He had persistent profound T lymphopenia, but normal numbers of B and natural killer (NK) cells. Despite an allogeneic hematopoietic stem cell transplant from his brother, he failed to develop normal T cells. Targeted resequencing excluded known SCID genes; however, whole exome sequencing (WES) of the proband and parents revealed a maternally inherited X-linked missense mutation in MED14 (MED14V763A), a component of the mediator complex. Morpholino (MO)-mediated loss of MED14 function attenuated T cell development in zebrafish. Moreover, this arrest was rescued by ectopic expression of cDNA encoding the wild type human MED14 ortholog, but not by MED14V763A , suggesting that the variant impaired MED14 function. Modeling of the equivalent mutation in mouse (Med14V769A) did not disrupt T cell development at baseline. However, repopulation of peripheral T cells upon competitive bone marrow transplantation was compromised, consistent with the incomplete T cell reconstitution experienced by the proband upon transplantation with bone marrow from his healthy male sibling, who was found to have the same MED14V763A variant. Suspecting that the variable phenotypic expression between the siblings was influenced by further mutation(s), we sought to identify genetic variants present only in the affected proband. Indeed, WES revealed a mutation in the L1 cell adhesion molecule (L1CAMQ498H); however, introducing that mutation in vivo in mice did not disrupt T cell development. Consequently, immunodeficiency in the proband may depend upon additional, unidentified gene variants.

Generation and Characterization of a Zebrafish IL-2Rγc SCID Model.

The IL-2 family of cytokines act via receptor complexes that share the interleukin-2 receptor gamma common (IL-2Rγc) chain to play key roles in lymphopoiesis. Inactivating IL-2Rγc mutations results in severe combined immunodeficiency (SCID) in humans and other species. This study sought to generate an equivalent zebrafish SCID model. The zebrafish il2rga gene was targeted for genome editing using TALENs and presumed loss-of-function alleles analyzed with respect to immune cell development and impacts on intestinal microbiota and tumor immunity. Knockout of zebrafish Il-2rγc.a resulted in a SCID phenotype, including a significant reduction in T cells, with NK cells also impacted. This resulted in dysregulated intestinal microbiota and defective immunity to tumor xenotransplants. Collectively, this establishes a useful zebrafish SCID model.

Loss of Ribosomal Protein Paralog Rpl22-like1 Blocks Lymphoid Development without Affecting Protein Synthesis.

Ribosomal proteins are thought to primarily facilitate biogenesis of the ribosome and its ability to synthesize protein. However, in this study, we show that Rpl22-like1 (Rpl22l1) regulates hematopoiesis without affecting ribosome biogenesis or bulk protein synthesis. Conditional loss of murine Rpl22l1 using stage or lineage-restricted Cre drivers impairs development of several hematopoietic lineages. Specifically, Tie2-Cre-mediated ablation of Rpl22l1 in hemogenic endothelium impairs the emergence of embryonic hematopoietic stem cells. Ablation of Rpl22l1 in late fetal liver progenitors impairs the development of B lineage progenitors at the pre-B stage and development of T cells at the CD44-CD25+ double-negative stage. In vivo labeling with O-propargyl-puromycin revealed that protein synthesis at the stages of arrest was not altered, indicating that the ribosome biogenesis and function were not generally compromised. The developmental arrest was associated with p53 activation, suggesting that the arrest may be p53-dependent. Indeed, development of both B and T lymphocytes was rescued by p53 deficiency. p53 induction was not accompanied by DNA damage as indicated by phospho-γH2AX induction or endoplasmic reticulum stress, as measured by phosphorylation of EIF2α, thereby excluding the known likely p53 inducers as causal. Finally, the developmental arrest of T cells was not rescued by elimination of the Rpl22l1 paralog, Rpl22, as we had previously found for the emergence of hematopoietic stem cells. This indicates that Rpl22 and Rpl22l1 play distinct and essential roles in supporting B and T cell development.

A Novel Germline Heterozygous BCL11B Variant Causing Severe Atopic Disease and Immune Dysregulation.

B-cell lymphoma/leukemia 11B (BCL11B) is a C2H2 zinc finger transcription factor that is critically important for regulating the development and function of a variety of systems including the central nervous system, the skin, and the immune system. Germline heterozygous variants are associated with a spectrum of clinical disorders, including severe combined immunodeficiency as well as neurological, craniofacial, and dermal defects. Of these individuals, ~50% present with severe allergic disease. Here, we report the detailed clinical and laboratory workup of one of the most severe BCL11B-dependent atopic cases to date. Leveraging a zebrafish model, we were able to confirm a strong T-cell defect in the patient. Based on these data, we classify germline BCL11B-dependent atopic disease as a novel primary atopic disorder.