Encephalitozoon cuniculi takes advantage of efferocytosis to evade the immune response

Microsporidia are recognized as opportunistic pathogens in individuals with immunodeficiencies, especially related to T cells. Although the activity of CD8+ T lymphocytes is essential to eliminate these pathogens, earlier studies have shown significant participation of macrophages at the beginning of the infection. Macrophages and other innate immunity cells play a critical role in activating the acquired immunity. After programmed cell death, the cell fragments or apoptotic bodies are cleared by phagocytic cells, a phenomenon known as efferocytosis. This process has been recognized as a way of evading immunity by intracellular pathogens. The present study evaluated the impact of efferocytosis of apoptotic cells either infected or not on macrophages and subsequently challenged with Encephalitozoon cuniculi microsporidia. Macrophages were obtained from the bone marrow monocytes from C57BL mice, pre-incubated with apoptotic Jurkat cells (ACs), and were further challenged with E. cuniculi spores. The same procedures were performed using the previously infected Jurkat cells (IACs) and challenged with E. cuniculi spores before macrophage pre-incubation. The average number of spores internalized by macrophages in phagocytosis was counted. Macrophage expression of CD40, CD206, CD80, CD86, and MHCII, as well as the cytokines released in the culture supernatants, was measured by flow cytometry. The ultrastructural study was performed to analyze the multiplication types of pathogens. Macrophages pre-incubated with ACs and challenged with E. cuniculi showed a higher percentage of phagocytosis and an average number of internalized spores. Moreover, the presence of stages of multiplication of the pathogen inside the macrophages, particularly after efferocytosis of infected apoptotic bodies, was observed. In addition, pre-incubation with ACs or IACs and/or challenge with the pathogen decreased the viability of macrophages, reflected as high percentages of apoptosis. The marked expression of CD206 and the release of large amounts of IL-10 and IL-6 indicated the polarization of macrophages to an M2 profile, compatible with efferocytosis and favorable for pathogen development. We concluded that the pathogen favored efferocytosis and polarized the macrophages to an M2 profile, allowing the survival and multiplication of E. cuniculi inside the macrophages and explaining the possibility of macrophages acting as Trojan horses in microsporidiosis.