Infection and inflammation of the male reproductive tract have been estimated to be responsible for ~15% of all cases of infertility. Experimental autoimmune orchitis (EAO) in mice is a model of immunologic male infertility, pathologically characterised by lymphocytic inflammation causing breakdown of the testicular immune privilege, spermatogenesis impairment and fibrosis. EAO in mice was induced by immunisation with testicular homogenate (TH) + adjuvant + Bordetella pertussis toxin.Activin A, a member of the transforming growth factor-beta superfamily of immunoregulatory cytokines, is a regulator of fibrosis and inflammation in various tissues, highlighting an emerging potential as a target for novel diagnostic and therapeutic strategies. It was found to be increased in EAO mouse testes. However, it is not known whether activin A is directly involved in testicular inflammation and fibrosis observed in EAO.Results of this study show that activin A subunit (INHBA) gene expression levels and the key fibrotic proteins, collagen and fibronectin, are elevated in human testicular biopsies with impaired spermatogenesis and leukocytic infiltrates, and that activin A is positively correlated with collagen and the extent of leukocytic infiltration in these biopsies. Collagen and fibronectin were also found significantly increased in murine testes with EAO, whereby fibronectin expression positively correlated with the severity of the disease. Moreover, pre-treatment of mice with a vector carrying a gene cassette of the activin antagonist follistatin 315 (rAAV-FST315) prior to immunisation with TH reduces testicular fibronectin expression, and hence, possibly positively influences EAO. Treatment of mouse primary peritubular cells (PTC) and NIH 3T3 cells with activin A resulted in increased levels of fibronectin mRNA and elevated production of collagen type I and fibronectin. Activin A treatment also increased collagen type IV mRNA levels in PTC, while in NIH 3T3 cells alphaSMA mRNA and protein expression were elevated by activin A. Moreover, the existence of cells co-expressing collagen type I and CD45 or F4/80 suggests the involvement of macrophages in the fibrotic response in the EAO testis. These data indicate that multiple cell types may contribute to fibrosis development in EAO testis including PTC, resident fibroblasts and macrophages, and implicate activin A as a key mediator of this process, at least in fibroblasts and PTC. Results of this work also showed that TNF stimulated activin A production in Sertoli cells (SC). The role of SC-derived activin A was investigated in mice lacking activin A expression in SC (InhbaSCKO). Preliminary results showed no differences in the EAO induction rate, amount of leukocytic infiltration and fibrosis severity between TH-immunised InhbaSCKO and InhbaFLOX mice, while activin A and the EAO damage score still were positively correlated indicating that SC-derived activin A does not play a role in regulating progression to EAO.
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