Heat-killed Malassezia pachydermatis suspension modulates the activity of macrophages challenged with Encephalitozoon cuniculi.

Phagocytic responses are critical for effective host defense against opportunistic fungal pathogens, such as Encephalitozoon cuniculi, an obligate intracellular fungus that causes emerging encephalitozoonosis in humans and other animals. Malassezia has immunomodulatory effects and can modulate the production of pro- and anti-inflammatory cytokines via keratinocytes and human monocytes. In this study, we evaluated the modulatory effects of heat-killed Malassezia pachydermatis suspension on macrophages challenged with Encephalitozoon cuniculi. Macrophages were treated with heat-killed M. pachydermatis suspension before being infected with spores of E. cuniculi. The cultures were stained with calcofluor, and the spores, internalized or not, were counted to determine their phagocytic capacity and index (PC and PI, respectively). Microbicidal and phagocytic activities were evaluated by transmission electron microscopy (TEM). The untreated macrophages had higher PC and PI and number of phagocytosed spores than treated macrophages. However, TEM revealed that treated macrophages had higher microbicidal activity because there were few spores in different degrees of degeneration and amorphous materials in the phagocytic vacuoles. Macrophages treated with heat-killed M. pachydermatis suspension had lower PC and PI and incipient presence of E. cuniculi in phagosomes. Treated macrophages had a mixed pattern of cytokine release with Th1, Th2, and Th17 profiles, with emphasis on interleukin (IL)-10, IL-4, IL-17, IL-6, and interferon (IFN)-γ secretion, and particularly high production of anti-inflammatory cytokines. Our results suggest that treatment with heat-killed M. pachydermatis suspension increases the release of cytokines and decreases the phagocytic activity of macrophages challenged with E. cuniculi.

Heat-killed Malassezia pachydermatis suspension modulates the activity of macrophages challenged with Encephalitozoon cuniculi

Phagocytic responses are critical for effective host defense against opportunistic fungal pathogens, such as Encephalitozoon cuniculi, an obligate intracellular fungus that causes emerging ncephalitozoonosis in humans and other animals. Malassezia has immunomodulatory effects and can modulate the production of pro- and anti-inflammatory cytokines via keratinocytes and human monocytes. In this study, we evaluated the modulatory effects of heat-killed Malassezia pachydermatis suspension on macrophages challenged with Encephalitozoon cuniculi. Macrophages were treated with heat-killed M. pachydermatis suspension before being infected with spores of E. cuniculi. The cultures were stained with calcofluor, and the spores, internal- ized or not, were counted to determine their phagocytic capacity and index (PC and PI, respectively). Micro-bicidal and phagocytic activities were evaluated by transmission electron microscopy (TEM). The untreated macrophages had higher PC and PI and number of phagocytosed spores than treated macrophages. However, TEM revealed that treated macrophages had higher microbicidal activity because there were few spores in different degrees of degeneration and amorphous materials in the phagocytic vacuoles. Macrophages treated with heat-killed M. pachydermatis suspension had lower PC and PI and incipient presence of E. cuniculi in phagosomes. Treated macrophages had a mixed pattern of cytokine release with Th1, Th2, and Th17 profiles, with emphasis on interleukin (IL)-10, IL-4, IL-17, IL-6, and interferon (IFN)-g secretion, and particularly high production of anti-inflammatory cytokines. Our results suggest that treatment with heat-killed M. pachyder-matis suspension increases the release of cytokines and decreases the phagocytic activity of macrophages challenged with E. cuniculi.

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.

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.