The Role of Macrophages in Implementing the Effects of Secretome of Mesenchymal Stromal Cells in the Spermatogonial Stem Cell Niche.

Postnatal stem cells surrounded by a niche support the renewal and regeneration of tissues and organs throughout life. The dysfunction of niche components can lead to the development of diseases, which are hard to cure. We previously showed that the subtunical injection of mesenchymal stromal cell (MSC) secretome induced the spermatogonial stem cell niche restoration, although the mechanisms of the process are not fully revealed. The study aimed at analyzing the effect of the MSC secretome on resident macrophages in animal models of male infertility and on peritoneal macrophages of intact animals.

Materials and methods: To study the effect of the MSC secretome on resident macrophages, doxorubicin-induced damage of murine spermatogenesis was modeled by intraperitoneal injections of 1 mg/kg of doxorubicin once in two days to reach a cumulative dose of 10 mg/kg. The second animal model of spermatogenesis injury was the abdominal cryptorchidism in rats. The MSC secretome was injected under the tunica albuginea. The animals were divided into the following groups: "intact", "damage", "MSC secretome". After isolating the testes, the number of macrophages was estimated using the immunohistochemical analysis. To investigate the phagocytic activity of macrophages mice were intramuscularly injected into the thigh with the MSC secretome with the following isolation of peritoneal macrophages. The ability of peritoneal macrophages to absorb FITC latex particles was analyzed.

Results: In cryptorchidism model the number of CD163⁺ M2 macrophages in the interstitium of testes increased significantly. The MSC secretome injection under the tunica albuginea of the testicle led to decreasing the number of CD163⁺ M2 macrophages. In the model of the toxic damage of spermatogenesis with doxorubicin the number of CD163⁺ M2 macrophages in the interstitium increased, however, there were no effects in the group with the MSC secretome injection. The number of M2 macrophages in this model, positive for another classical marker CD206, also increased, but the administration of MSC secretome reduced their number neither during the early nor in the late periods after damage. The study of MSC secretome effects on peritoneal macrophages demonstrated that a single intramuscular injection of MSC secretome in doses lower and higher than the therapeutic dose didn't reduce, but conversely increased the phagocytic activity of macrophages.

Conclusion: Our findings indicate the impact of the damage etiology and pathogenesis on the involvement of M2 macrophages in the implementation of the MSC secretome effects and the absence of its systemic immunotoxicity.