Original researchA critical role of foxp3a-positive regulatory T cells in maintaining immune homeostasis in zebrafish testis development
Introduction
Regulatory T cells (Treg cells) are essential mediators of immunological tolerance to self- and non-self-antigens by activating immunosuppression to control autoimmunity and inflammatory pathology. The differentiation and functions of Treg cells depend on a crucial transcription factor named Forkhead box protein P3 (FOXP3), the most reliable marker to recognize this type of T-cell subset (Fontenot et al., 2003; Hori et al., 2003; Huang et al., 2020). In humans, mutations of FOXP3 result in an autoimmune syndrome referring to immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome (Bennett et al., 2001; Wildin and Freitas, 2005). Besides, Foxp3 mutation in mice causes a fatal lymphoproliferative disorder and hypogonadal development (Brunkow et al., 2001; Jasurda et al., 2014). These results show a key role of Treg cells in the suppression of autoimmune diseases.
The orthologues of Foxp3 have been identified in several fish species, including zebrafish (Danio rerio). Probably owing to the whole-genome duplication event in teleost fishes (Glasauer and Neuhauss, 2014), there are two foxp3 genes in zebrafish, termed foxp3a and foxp3b, while foxp3a is more conserved to human Foxp3, with 31.5% identity (Mitra et al., 2010). With the labeling of foxp3a, Treg-like cells have been identified in puffer fish and zebrafish (Wen et al., 2011; Kasheta et al., 2017). Moreover, the expression of Foxp3 ortholog has been detected in zebrafish after lipopolysaccharide treatment (Mitra et al., 2010), suggesting the involvement of a FOXP3-dependent program in the progress of inflammation during infection. Accordingly, it has been reported that an immunosuppressive function of the zebrafish Foxp3 ortholog can be induced in mouse T cells in vitro (Quintana et al., 2010). A recent study showed that zebrafish Treg cells retain a certain plasticity to support immune homeostasis and regeneration of diverse tissues by producing tissue-specific growth factors (Hui et al., 2017). However, the function of foxp3a and Treg cells in gonadal development and sex differentiation has never been described in a fish system.
For zebrafish, a well-studied model organism of nonmammalian vertebrates, the sex differentiation and gonadal development seem to be fairly complicated processes in domesticated strains compared with wild populations due to the loss of differentiated sex chromosomes (Traut and Winking, 2001; Orban et al., 2009; Liew and Orban, 2014; Wilson et al., 2014). Immune-germ cell interaction has been involved in autoimmune inflammatory response against spermatic antigens (Hubert et al., 2009; Pelletier et al., 2009), and an increased influx of immune cells has been observed in the experimental autoimmune orchitis (EAO) of rats (Jacobo, 2018, Jacobo et al., 2011). Although plenty of studies have been focusing on the process of sex differentiation in zebrafish (Rodriguez-Mari et al., 2005; Lin et al., 2017), it remains unknown that how the immune function contributes to the gonadal development of zebrafish.
In this study, we generated zebrafish foxp3a mutants using clustered regularly-interspaced short palindromic repeat (CRISPR) /Cas9 technology and showed that foxp3a is essential for maintaining immune homeostasis in zebrafish testis development. We found that foxp3a was specifically expressed in a subset of T cells in zebrafish testis, and knockout of foxp3a led to deficiency of foxp3a-positive Treg cells, and the majority of foxp3a–/– mutants developed as subfertile males. Further study revealed that a proportion of foxp3a–/– females displayed sex reversal in early gonadal differentiation. In the developing testis of foxp3a–/– mutants, owing to the absence of foxp3a-positive Treg cells, which play immunosuppressive function, abundant effector T cells and macrophages expanded to disrupt the immunosuppressive milieu, resulting in defective germ cells and gonadal somatic cells. Therefore, our study provides the first line of evidence for the key role of foxp3a-mediated immune tolerance in the testis development of zebrafish.
Section snippets
Expression patterns of foxp3a and foxp3b in zebrafish
We first utilized semi-quantitative polymerase chain reaction (semi-qPCR) to analyze the expression profile of foxp3a and foxp3b in different tissues from wild-type (WT) zebrafish (Fig. 1A), and the results showed that except for heart and liver, foxp3a mRNA was detected in many organs such as brain, eye, gill, skin, muscle, spleen, kidney, gut and ovary, with the highest expression level in the testis. However, foxp3b, another ortholog of the mammalian Foxp3 gene in zebrafish, only shows
Discussion
Treg cells are critical for the maintenance of peripheral tolerance and preventing aggressive and harmful immune responses. Numerous studies on Treg cells have focused on mammals, such as humans and mice. Although the Treg cells in pufferfish and zebrafish have been identified recently (Wen et al., 2011; Kasheta et al., 2017), their function in vivo has not been well understood, especially in gonadal development. In this study, we explored the function of Treg cells during gonadal development
Zebrafish
The WT zebrafish (AB line) and the transgenic lines Tg(lck:EGFP)cz2Tg, Tg(mpeg1:EGFP)ihb20Tg and Tg(piwil1:EGFP-UTRnanos3)ihb327Tg used in this study were obtained from China Zebrafish Resource Center of National Aquatic Biological Resource Center (Wuhan, China; http://zfish.cn) and raised in the fish facilities of Institute of Hydrobiology, Chinese Academy of Sciences. All experimental and animal care procedures were approved by the Institutional Animal Care and Use Committee of the Institute
CRediT authorship contribution statement
Xianmei Li: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - Original Draft. Fenghua Zhang: Methodology, Validation. Nan Wu: Resources. Ding Ye: Resources. Yaqing Wang: Methodology. Xiaofan Zhang: Methodology. Yonghua Sun: Conceptualization, Writing - Review & Editing, Supervision, Project administration, Funding acquisition. Yong-An Zhang: Conceptualization, Supervision, Funding acquisition.
Acknowledgments
This work was supported by the National Key Research and Development Program of China (2018YFD0900505 to Y.-A.Z. and 2018YFA0801000 to Y.S.) and the National Natural Science Foundation of China (32025037 and 31721005 to Y.S.). We would like to thank Kuoyu Li at the China Zebrafish Resource Center for zebrafish rearing.
References (60)
- et al.
A critical role of follicle-stimulating hormone (Fsh) in mediating the effect of clotrimazole on testicular steroidogenesis in adult zebrafish
Toxicology
(2012) - et al.
nanos3 maintains germline stem cells and expression of the conserved germline stem cell gene nanos2 in the zebrafish ovary
Dev. Biol.
(2013) - et al.
Estrogen regulation of gene expression in the teleost fish immune system
Fish Shellfish Immunol.
(2016) - et al.
Chemokines: key players in innate and adaptive immunity
J. Invest. Dermatol.
(2005) Chapter three - macrophages: their untold story in T cell activation and function
- et al.
Characterization of testicular expression of P450 17alpha-hydroxylase, 17,20-lyase in zebrafish and its perturbation by the pharmaceutical fungicide clotrimazole
Gen. Comp. Endocrinol.
(2011) - et al.
Molecular feature and therapeutic perspectives of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome
J. Genet. Genomics
(2020) - et al.
Testicular autoimmunity
Autoimmun. Rev.
(2011) - et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method
Methods
(2001) - et al.
Long and winding roads: testis differentiation in zebrafish
Mol. Cell. Endocrinol.
(2009)
Characterization and expression pattern of zebrafish anti-Mullerian hormone (Amh) relative to sox9a, sox9b, and cyp19a1a, during gonad development
Gene Expr. Patterns
Spermatogenesis in fish
Gen. Comp. Endocrinol.
Alteration of immune function endpoints and differential expression of estrogen receptor isoforms in leukocytes from 17β-estradiol exposed rainbow trout (Oncorhynchus mykiss)
Gen. Comp. Endocrinol.
Zebrafish FOXP3 is required for the maintenance of immune tolerance
Dev. Comp. Immunol.
Adverse effects of bisphenol A on Sertoli cell blood-testis barrier in rare minnow Gobiocypris rarus
Ecotoxicol. Environ. Saf.
IPEX and FOXP3: clinical and research perspectives
J. Autoimmun.
The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3
Nat. Genet.
Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse
Nat. Genet.
Vertebrate sex determination: evolutionary plasticity of a fundamental switch
Nat. Rev. Genet.
Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos
Cell Res.
Short technical reports. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources
Biotechniques
Bmp15 is an oocyte-produced signal required for maintenance of the adult female sexual phenotype in zebrafish
PLoS Genet.
Identification of germ-line stem cells in zebrafish
Methods Mol. Biol.
Foxp3 programs the development and function of CD4+CD25+ regulatory T cells
Nat. Immunol.
Whole-genome duplication in teleost fishes and its evolutionary consequences
Mol. Genet. Genom.
Macrophage clearance of apoptotic cells: a critical assessment
Front. Immunol.
Control of regulatory T cell development by the transcription factor Foxp3
Science
Aire-deficient C57BL/6 mice mimicking the common human 13-base pair deletion mutation present with only a mild autoimmune phenotype
J. immunol.
Zebrafish regulatory T cells mediate organ-specific regenerative programs
Dev. Cell
The role of regulatory T Cells in autoimmune orchitis
Andrologia
Cited by (9)
Expression analysis of grass carp Foxp3 and its biologic effects on CXCL-8 transcription in non-lymphoid cells
2022, Developmental and Comparative ImmunologyCitation Excerpt :Genes encoding Foxp3 orthologs and Foxp3+ Treg-like cells have been found in several fish species (Kasheta et al., 2017; Kikuchi, 2020; Wen et al., 2011). By retroviral transduction with zebrafish foxp3 gene in mouse non-Treg cells or generating foxp3 mutant zebrafish demonstrated conserved immunosuppressive function of fish Foxp3 (Li et al., 2020; Quintana et al., 2010; Sugimoto et al., 2017). Nevertheless, previous study indicated that expression of Nile tilapia Foxp3 mRNA and protein were detected both in lymphocytes and epithelial-like cells of stomach (Jia et al., 2015).
A landscape of differentiated biological processes involved in the initiation of sex differentiation in zebrafish
2022, Water Biology and SecurityCitation Excerpt :The cytokine-mediated and chemokine-mediated signaling pathways may be related immunoregulation which is discussed below. In previous studies, we found that oocytes were undergoing apoptosis in the juvenile testis at 30 dpf, resulting in an immune response and inflammation (Li et al., 2020; Ye et al., 2019). Excessive inflammation in the testis would disrupt both germ cells and gonadal somatic cells leading to male infertility.
CKLF instigates a “cold” microenvironment to promote MYCN-mediated tumor aggressiveness
2024, Science AdvancesDirect male development in chromosomally ZZ zebrafish
2024, Frontiers in Cell and Developmental BiologyInduced formation of primordial germ cells from zebrafish blastomeres by germplasm factors
2023, Nature CommunicationsVISUAL DETECTION OF POLY-L-LYSINE ON THE COVER GLASS
2023, Acta Hydrobiologica Sinica