| Background and Objectives:The mammalian testis is an immunoprivileged organ where male germ cell autoantigens are immunologically ignored. However, some pathological conditions can disrupt the immunoprivileged status and induce autoimmune orchitis, an etiological factor of male infertility. Mechanisms underlying autoimmune orchitis induction are largely unknown. Both systemic immune tolerance to autoantigens and local immunosuppressive milieu contribute to the testicular immune privilege. Testicular immunosuppression has been intensively studied, but information on systemic immune tolerance to autoantigens is lacking. Toll-like receptor (TLR) plays an important role in initiating immune response and involving in autoimmune disease development. TLR signaling pathway suppressed by TAM (Tyro3, Axl, Mer) receptor tyrosine kinases. In the present study, we aimed to determine the role of TLR and TAM receptors in maintaining the systemic tolerance to male germ cell antigens using experimental autoimmune orchitis (EAO) model.Materials and Methods:Gene knockout and WT mice were immunized with syngeneic testicular homogenates emulsified in complete Freund’s adjuvant (CFA) to induce EAO. Histopathology of testis were checked by HE stain and immunochemistry; Expression of proinflammatory cytokines were analyzed by real time qRT-PCR and ELISA; Macrophages and Lymphocytes were determined by flow cytometry; Autoantibody generation in the sera was determined by Western Blot; Biotin tracing method was used to evaluate the permeability of testicular blood-testis barrier (BTB).Results:Axl and Mer double knockout (Axl-/-Mer-/-) mice developed evident EAO after a single immunization. EAO was characterized by the enlargement of lymph nodes and accumulation of macrophages and T lymphocytes in the testis. Damage to the seminiferous epithelium was also observed. EAO induction was associated with pro-inflammatory cytokine upregulation in the testis, impaired permeability of BTB, and generation of autoantibodies against germ cell antigens in Axl-/-Mer-/- mice. Immunization also induced mild EAO in Axl or Mer single gene knockout mice. By contrast, a single immunization failed to induce EAO in WT mice. WT mice developed severe EAO after three immunizations, TLR2 or TLR4 deficient mice showed relatively low susceptibility to EAO induction compared with WT mice. Notably, TLR2 and TLR4 double knockout mice were almost completely protected from EAO induction. Moreover, we demonstrated that TLR2 was crucial in mediating autoantibody production in response to immunization.Conclusions:The results imply that TLR2 and TLR4 cooperatively mediate EAO induction, and we speculate that Axl and Mer receptors play a critical role in inhibiting EAO induction by suppressing TLR signaling pathway. In this way, Axl and Mer receptors cooperatively regulate the systemic immune tolerance to male germ cell antigens. |
