Involvement of Activin and Follistatin in the Pathogenesis of Chronic Testicular Inflammation in Mice

Experimental autoimmune epididymo-orchitis (EAEO) is a rodent model of chronic testicular inflammation that reproduces the pathology observed in some types of human infertility, characterized by elevated levels of pro-inflammatory cytokines, immune cell recruitment, germ cell loss and ultimately sub- or infertility.
   Activins A and B are pro-inflammatory, pro-fibrotic cytokines, but also regulate spermatogenesis and steroidogenesis under normal conditions. The roles of activin A, B and the endogenous activin antagonists, inhibin and follistatin, were examined in EAEO. The disease was induced in adult mice by immunization with syngeneic testicular homogenate.
   Age-matched untreated mice and controls showed no pathology, with activin A localized to Sertoli cells and interstitial macrophages. Immunized mice developed EAEO by 50 days (induction rate of 100%), and were characterized by a >50% reduction in testis weight, complete loss of germ cells, and a marked peritubular fibrotic response. Similar changes were also observed in biopsies from human testes with inflammatory infiltrates. Moreover, changes were accompanied by increased expression of key inflammatory mediators such as tumor necrosis factor, monocyte chemoattractant protein-1 and interleukin-10. An increase of the total CD45+ leukocytes, comprising CD3+ T cells, CD4+CD8- and CD4+CD25+ T cells, and a novel population of CD4+CD8+ double positive T cells was also detected in EAEO testes. Activin A and B protein levels were significantly increased in EAEO testes at 50 days, compared with untreated controls but not at 80 days, whereas the inhibin subunit mRNA levels (Inha and Inhbb) decreased in EAEO testes, becoming significantly lower after 80 days compared with control animals. Activin A receptor acvr1b and acvr2b mRNA levels were also significantly decreased in EAEO testes. In contrast, testicular follistatin levels were significantly elevated at 50 and 80 days of EAEO.
   These data suggest that there is a direct association between the onset of EAEO and increased activin expression. Therefore, activin may play a role in promoting inflammation and fibrosis during EAEO in mouse testis.          
  Consequently, the development of EAEO in a mouse model with elevated circulating levels of the activin antagonist follistatin (FST), was examined. Follistatin levels were increased by a single injection of a non-replicative recombinant adenovirus-associated viral vector carrying a gene cassette of the circulating form of follistatin (FST315). Controls received an rAAV injection with an empty cassette. EAEO was induced and testes were collected 30 and 50 days after the first immunization. Serum follistatin levels were increased 5-fold in FST315-vector injected mice compared with the control group 30 days after vector injection and remained elevated until the end of the experiment. The EAEO induction rate was 40% for the FST315-vector injected group compared with 75% for the control group. Elevated levels of inflammatory mediators and activins, recruitment of immune cells, increased fibrosis, disruption of the blood-testis-barrier, and increased apoptosis were directly proportional to the observed testicular damage in EAEO.
   These data suggest that blocking activin activity alone, by increasing circulating follistatin levels before inducing EAEO, was not sufficient to inhibit the development of testicular inflammation and fibrosis.