Heat shock protein A4L is a potent autoantigen for testicular autoimmunity in mice

Highlights

• Immunization with testicular germ cells (TGC) induces experimental autoimmune orchitis (EAO).

• HSPA4L, a purified TGC protein, was found to be a strong EAO inducer.

• HSPA4L could be a target and a useful biomarker of EAO.

Abstract

Experimental autoimmune orchitis (EAO) may be used as a model to investigate immunological infertility in men. Murine EAO is induced via immunization with auto-immunogenic antigens (AIAgs) from testicular germ cells (TGCs). CD4 + T cells play a crucial role in EAO induction. However, whether AIAgs induce an immune response remains unclear. We aimed to identify self-antigens that induce EAO by screening a phage display library of random TGC peptides using IgG from EAO-induced A/J mice. Twenty TGC-specific AIAgs were detected, and G protein-coupled receptor kinase 2 interacting protein-1 (GIT1) and heat shock protein A4L (HSPA4L) were identified as candidate AIAgs that induce EAO. Immunization with GIT1 or HSPA4L, emulsified in complete Freund’s adjuvant, resulted in 66 % or 100 % incidence of EAO, respectively, indicating that HSPA4L is a most potent AIAg that induces EAO in mice. These findings may expectedly help improve the diagnostic procedures and treatment of immunological infertility in men.

Introduction

Infertility affects one in six couples in developed nations, and in 40–50 % of cases, infertility manifests in the male partner (Ilacqua et al., 2018). A significant number of male infertility cases may be caused by immunity-related disorders. Several idiopathic inflammatory diseases of the testes are known to cause male infertility in humans (Tung and Lu, 1991). Furthermore, testicular biopsies of infertile individuals are characterized by lymphocytic inflammation and immune deposits (Taylor et al., 1978; Hatakeyama, 1965; Hassanin and Ayad, 2016). However, the detailed molecular mechanism underlying orchitis remains unclear.

The testis is a critical target for autoimmune damage. Haploid germ cells, such as spermatids and spermatozoa, are not found in the seminiferous epithelium until puberty, the stage at which immune tolerance is established (Tung et al., 1981). Therefore, these contain various autoimmunogenic antigens (AIAgs) that are recognized as foreign by the self-immune system. The blood-testis barrier (BTB), formed by Sertoli cells, protects autoimmunogenic spermatozoa from the self-immune system (Setchell et al., 1969; Dym and Fawcett, 1970). However, if the BTB is functionally damaged, AIAgs leakage causes continuous immune stimulation, which results in prolonged testicular inflammation. Indeed, damage to the BTB in one testis, following an infection, biopsy, or surgery in the scrotal area, induces orchitis in the contralateral testis (Suominen and Söderström, 1982; Rodriguez et al., 2006).

Animal experimental autoimmune orchitis (EAO) models have provided important insights into testicular inflammation-based pathology and organ-specific autoimmunity. EAO is induced in mice, rats, and guinea pigs via immunization with a testicular antigen (Tung et al., 1971). Evidently, both cellular (Itoh et al., 1992; Yule and Tung, 1993) and humoral (Tung et al., 1981) immunity play roles in EAO models. However, it remains unclear which testicular autoantigens are recognized by these inflammatory cells.

Previous studies using various rodent EAO models have proposed multiple substances that may trigger a testicular autoimmune response. For example, western blot analysis of germ cell lysates revealed the presence of autoantibodies that recognize antigens with molecular weights between 20 and 250 kDa in the sera of EAO mice (Pelletier et al., 2011; Qu et al., 2010). Immunizing male guinea pigs with sperm adhesion molecule 1, a sperm surface protein, resulted in severe spermatogenesis disturbances (Tung et al., 1997). A study conducted by Fijak et al. (2005) indicated that the 70-kDa heat shock protein (HSP) 5, heterogeneous nuclear ribonucleoprotein H1 and sperm outer dense fiber major protein 2 are testis-specific antigens associated with EAO in rats. Furthermore, Terayama et al. (2016) identified new candidate antigens, HSP1-like (HSC70 T), and DAZ-associated protein 1 (DAZAP1) that were related to the testicular autoimmune response. However, the EAO induction rate of these proteins was low (25–30 %) and no reports have indicated that 100 % EAO incidence can be induced using purified proteins.

In this study, we aimed to detect TGC-specific AIAgs in a mouse EAO model. We analyzed reactivity between phage libraries constructed using TGC genes and EAO serum autoantibodies and purified recombinant antigens that efficiently and accurately induced EAO. We expect that these findings may help improve diagnostic procedures as well as treatment methods targeting male infertility in humans.