Testicular immune cell populations and macrophage polarisation in adult male mice and the influence of altered activin A levels

Detailed morphological characterization of testicular leukocytes in the adult CX₃CR₁^gfp/+ transgenic mouse identified two distinct CX₃CR₁⁺ mononuclear phagocyte (macrophage and dendritic cell) populations: stellate/dendriform cells opposed to the seminiferous tubules (peritubular), and polygonal cells associated with Leydig cells (interstitial). Using confocal microscopy combined with stereological enumeration of CX₃CR₁^gfp/+ cells established that there were twice as many interstitial cells (68%) as peritubular cells (32%). Flow cytometric analyses of interstitial cells from mechanically-dissociated testes identified multiple mononuclear phagocyte subsets based on surface marker expression (CX₃CR₁, F4/80, CD11c). These cells comprised 80% of total intratesticular leukocytes, as identified by CD45 expression. The remaining leukocytes were CD3⁺ (T lymphocytes) and NK1.1⁺ (natural killer cells). Functional phenotype assessment using CD206 (an anti-inflammatory/M2 marker) and MHC class II (an activation marker) identified a potentially tolerogenic CD206⁺MHCII⁺ sub-population (12% of total CD45⁺ cells). Rare testicular subsets of CX₃CR₁⁺CD11c⁺F4/80⁺ (4.3%) mononuclear phagocytes and CD3⁺NK1.1⁺ (3.1%) lymphocytes were also identified for the first time. In order to examine the potential for the immunoregulatory cytokine, activin A to modulate testicular immune cell populations, testes from adult mice with reduced activin A (Inhba^+/−) or elevated activin A (Inha^+/−) were assessed using flow cytometry. Although the proportion of F4/80⁺CD11b⁺ leukocytes (macrophages) was not affected, the frequency of CD206⁺MHCII⁺cells was significantly lower and CD206⁺MHCII⁻ correspondingly higher in Inha^+/− testes. This shift in expression of MHCII in CD206⁺ macrophages indicates that changes in circulating and/or local activin A influence resident macrophage activation and phenotype and, therefore, the immunological environment of the testis.