A critical role of foxp3a-positive regulatory T cells in maintaining immune homeostasis in zebrafish testis development

Abstract

Suppressive regulatory T cells (Treg cells) play a vital role in preventing autoimmunity and restraining excessive immune response to both self- and non-self-antigens. Studies on humans and mice show that the Forkhead box p3 (Foxp3) is a key regulatory gene for the development and function of Treg cells. In zebrafish, Treg cells have been identified by using foxp3a as a reliable marker. However, little is known about the function of foxp3a and Treg cells in gonadal development and sex differentiation. Here, we show 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, while knockout of foxp3a led to deficiency of foxp3a-positive Treg cells in the testis. More than 80% of foxp3a^–/– mutants developed as subfertile males, and the rest of the mutants developed as fertile females with decreased ovulation. Further study revealed that foxp3a^–/– mutants had a delayed juvenile ovary-to-testis transition in definite males and sex reversal in about half of the definite females, which led to a dominance of later male development. Owing to the absence of foxp3a-positive Treg cells in the differentiating testis of foxp3a^–/– mutants, abundant T cells and macrophages expand to disrupt an immunosuppressive milieu, resulting in defective development of germ cells and gonadal somatic cells and leading to development of infertile males. Therefore, our study reveals that foxp3a-positive Treg cells play an essential role in the orchestration of gonadal development and sex differentiation in zebrafish.

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.