Interleukin-35 plays a pivotal role in maintaining the testicular immune privilege

Abstract

Background: Testis is known as one of the immunologically privileged organs. In particular, blood-testis barrier formed by Sertoli cells protects autoimmunogeneic spermatozoa and spermatid from attack by the self-immune system. Moreover, it was demonstrated that Sertoli cells, Leydig cells and a few population of testicular macrophages exhibit immunosuppressive activity. Recent studies also suggest a possibility that some cytokines in the testis contribute to maintaining the immune privilege. Interleukin (IL)-35 is a heterodimeric cytokine composed of Epstein-Barr virus-induced gene 3 (EBI3) and the p35 subunit of IL-12. Although it is IL-35 has an important role in immunosuppression, its role in the testis remains unknown. Methods and Findings: In the present study, we investigated the role of intra-testicular IL-35 by histochemistry, immunohistochemistry and real-time RT-PCR using wild-type C57BL/6 mice and EBI3- and p35-deficient mice. EBI3 expression was detected in a part of CD163-positive macrophages and acrosomal regions of spermatids in testis of wild-type mice. Intriguingly, p35 expression was coincidently detected in a part of EBI3- and F4/80-positive macrophages, and also in basal lamina of seminiferous tubules, endothelial cells and acrosomal region of spermatids. A significant increase in the number of seminiferous tubules with spermatogenic disturbance was observed in both EBI3- and p35-deficient mice, compared with that in wild-type mice. Especially, p35-deficient mice showed severe spermatogenic disturbance. Moreover, CD4-, CD8- and B220-positive infiltrating cells were detected in the testicular interstitium of EBI3- and p35-deficient mice, but not of wild-type mice. Intra-testicular mRNA expression of interferon-gamma was significantly increased in EBI3- and p35-deficient mice. A similar increase in the expression of IL-10 was observed only in p35-deficient mice. Finally, autoantibodies to spermatids were detected in sera obtained from EBI3- and p35-deficient mice, but not from wild-type mice. Conclusions: In testis, there are EBI3- and p35-double positive macrophages, possibly producing immunosuppressive IL-35. And, lack of either EBI3 or p35 causes infiltration of lymphocytes into testis and spermtatogenic disturbance. These results indicate that IL-35 plays an important role in maintaining the testicular immune privilege.