RESUMO
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.
Assuntos
Apoptose/genética , Encephalitozoon cuniculi/imunologia , Evasão da Resposta Imune , Macrófagos/microbiologia , Esporos Fúngicos/imunologia , Animais , Medula Óssea/imunologia , Medula Óssea/microbiologia , Diferenciação Celular , Técnicas de Cocultura , Encephalitozoon cuniculi/genética , Encephalitozoon cuniculi/crescimento & desenvolvimento , Feminino , Expressão Gênica , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Células Jurkat , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Macrófagos/imunologia , Receptor de Manose , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose , Cultura Primária de Células , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
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.
RESUMO
Here, we have investigated the possible effect of B-1 cells on the activity of peritoneal macrophages in E. cuniculi infection. In the presence of B-1 cells, peritoneal macrophages had an M1 profile with showed increased phagocytic capacity and index, associated with the intense microbicidal activity and a higher percentage of apoptotic death. The absence of B-1 cells was associated with a predominance of the M2 macrophages, reduced phagocytic capacity and index and microbicidal activity, increased pro-inflammatory and anti-inflammatory cytokines production, and higher percentual of necrosis death. In addition, in the M2 macrophages, spore of phagocytic E. cuniculi with polar tubular extrusion was observed, which is an important mechanism of evasion of the immune response. The results showed the importance of B-1 cells in the modulation of macrophage function against E. cuniculi infection, increasing microbicidal activity, and reducing the fungal mechanisms involved in the evasion of the immune response.
Assuntos
Subpopulações de Linfócitos B , Encephalitozoon cuniculi/imunologia , Encefalitozoonose/imunologia , Encefalitozoonose/patologia , Macrófagos Peritoneais/imunologia , Animais , Apoptose , Células Cultivadas , Feminino , Macrófagos Peritoneais/microbiologia , Camundongos Endogâmicos BALB C , Fagocitose/imunologia , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/imunologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genéticaRESUMO
Malassezia pachydermatis and Malassezia furfur are lipophilic yeasts of the cutaneous microbiome, although these organisms are occasionally responsible for serious invasive infections in neonates. Since phagocytosis is an important mechanism mediating the adaptive immune response, here we evaluated the phagocytosis capacity and production of nitric oxide and cytokine by macrophages after challenged with M. furfur CBS-1878 and M. pachydermatis CBS-1696. The phagocytic indexes was determined using RAW 264.7 cultivated or not with M. furfur or M. pachydermatis in the concentrations of 5:1 or 2:1 (yeasts:macrophages ratio) for 6 h, 24 h, and 48 h following the challenges. Evaluation of nitric oxide and pro- and anti-inflammatory cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17A, interferon (IFN)-γ and tumor necrosis factor [TNF]-α) by Griess method and flow cytometry, respectively, were performed in the different intervals by collecting the cell culture supernatant. Results showed a higher phagocytic index in the 5:1 ratio in 24 h for both species. Malassezia pachydermatis-infected macrophages had superior phagocytic indexes than M. furfur-infected macrophages. Phagocytosis evaluation at 48 h showed significant microorganisms proliferation and macrophages death, particularly in macrophages infected with M. pachydermatis, suggesting yeast evasion mechanism. Significant variations in the nitric oxide production were observed in macrophages infected with both species. Levels of TNF-α and IL-4 cytokines have increased in M. furfur and M. pachydermatis macrophage-infected cultures, respectively. The low microbicidal activity and the presence of pro- and anti-inflammatory cytokines reinforce the dichotomous character of the relation of these yeasts with the host, acting as a commensal in the cutaneous microbiome or causing infection.
RESUMO
Here, we have investigated the possible effect of B-1 cells on the activity of peritoneal macrophages in E. cuniculi infection. In the presence of B-1 cells, peritoneal macrophages had an M1 profile with showed increased phagocytic capacity and index, associated with the intense microbicidal activity and a higher percentage of apoptotic death. The absence of B-1 cells was associated with a predominance of the M2 macrophages, reduced phagocytic capacity and index and microbicidal activity, increased pro-inflammatory and anti-inflammatory cytokines production, and higher percentual of necrosis death. In addition, in the M2 macrophages, spore of phagocytic E. cuniculi with polar tubular extrusion was observed, which is an important mechanism of evasion of the immune response. The results showed the importance of B-1 cells in the modulation of macrophage function against E. cuniculi infection, increasing microbicidal activity, and reducing the fungal mechanisms involved in the evasion of the immune response
RESUMO
Malassezia pachydermatis and Malassezia furfur are lipophilic yeasts of the cutaneous microbiome, although these organisms are occasionally responsible for serious invasive infections in neonates. Since phagocytosis is an important mechanism mediating the adaptive immune response, here we evaluated the phagocytosis capacity and production of nitric oxide and cytokine by macrophages after challenged with M. furfur CBS-1878 and M. pachydermatis CBS-1696. The phagocytic indexes was determined using RAW 264.7 cultivated or not with M. furfur or M. pachydermatis in the concentrations of 5:1 or 2:1 (yeasts:macrophages ratio) for 6 h, 24 h, and 48 h following the challenges. Evaluation of nitric oxide and pro- and anti-inflammatory cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17A, interferon (IFN)-gama and tumor necrosis factor [TNF]-a) by Griess method and flow cytometry, respectively, were performed in the different intervals by collecting the cell culture supernatant. Results showed a higher phagocytic index in the 5:1 ratio in 24 h for both species. Malassezia pachydermatis-infected macrophages had superior phagocytic indexes than M. furfur-infected macrophages. Phagocytosis evaluation at 48 h showed significant microorganisms proliferation and macrophages death, particularly in macrophages infected with M. pachydermatis, suggesting yeast evasion mechanism. Significant variations in the nitric oxide production were observed in macrophages infected with both species. Levels of TNF-a and IL-4 cytokines have increased in M. furfur and M. pachydermatis macrophage-infected cultures, respectively. The low microbicidal activity and the presence of pro- and anti-inflammatory cytokines reinforce the dichotomous character of the relation of these yeasts with the host, acting as a commensal in the cutaneous microbiome or causing infection.
RESUMO
BACKGROUND: In previous results mice treated with high dilutions of antimony presented reduction of monocyte migration to the site of infection with increase in B lymphocytes population in the local lymph node. AIMS: To know the mechanisms involved, a series of in vitro studies was done, using co-cultures of macrophages (RAW 264.7) and Leishmania (L.) amazonensis treated with different dilutions of antimony (Antimonium crudum or AC), in different times. METHODOLOGY: Spreading, phagocytosis, the oxidative activity of macrophages, the viability of free promastigotes and the cytokines/chemokines concentration in the supernatant were evaluated. The assays were performed in quadruplicate. RESULTS: Cells treated with AC 30cH (10-58M) and AC 200cH (10-398M) presented a temporary reduction of the spreading after 02h of incubation, followed by increase after 48h, being the most significant increase observed after the AC 200cH treatment. However, the percentage of internalized parasites at 48, 96 and 120h of incubation was also higher in cells treated with AC 200cH. It is suggested that the AC 200cH improves the ability of phagocytes to internalize the parasites, but not to digest them. The cytokines-chemokines panel corroborated these results. Both dilutions potentiated the parasite-induced reduction of cytokines production, especially IL-6, IL 12 p40 and γ-IFN, after 48h of incubation. In addition, the production of MIP-1 beta (CCL4), a chemokine involved in chronic inflammation, was also reduced after 120h. A specific effect of AC 30cH was seen by the inhibition of two peaks of CCL2 (MCP-1) observed in infected macrophages, at 24 and 120h. Since this cytokine is an important chemokine for monocytes, it explains the results obtained formerly in vivo. The morphology of macrophages after acridine orange staining revealed that the treatment with AC 30cH reduced substantially the acid vacuoles in the cytoplasm, indicating a certain inability of these cells to digest the parasites. On the other hand, a large peak of VEGF-A, associated with increase of internalized parasites was observed after 120h of treatment with AC 200cH, which could be associated to the regulation of the chronic inflammation events by M1-M2 polarization. There was no statistical difference among groups regarding the production of TNF, NO and H2O2, showing that the drugs do not alter macrophage cytotoxic activity. A clear quantitative and qualitative variation of the modulatory effects of AC 30cH and 200cH was seen, in function of time. CONCLUSIONS: Both dilutions were able to potentiate the decrease of most of cytokines and chemokines induced by the parasite infection in vitro, which explains the clinical improvement seen previously in vivo, however, the mechanisms involved and the epidemiological significance of these findings are still under discussion.
Assuntos
Antimônio/farmacologia , Leishmania/imunologia , Leishmaniose/imunologia , Macrófagos/imunologia , Monocinas/imunologia , Animais , Leishmaniose/patologia , Macrófagos/parasitologia , Camundongos , Células RAW 264.7RESUMO
ABSTRACT Pro-inflammatory cytokines and glial cells, especially microglial cells, have been implicated in persistent pain sensitization. Less is known about the role of astrocytes in pain regulation. This study aimed to observe the expression of the astrocytic biomarker glial fibrillary acidic protein (GFAP) and the serum levels of interleukin 1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) after short-term administration of central pain relievers in rats not submitted to noxious stimuli. Male Wistar rats were divided into five groups, receiving for nine days- (1) amitriptyline (Amt-10 mg/kg/day, by gavage); (2) gabapentin (Gb-60 mg/kg/day, by gavage; (3) methadone (Me-4.5 mg/kg/day, intraperitoneal route [IP]); (4) morphine (Mo-10 mg/kg/day, IP); or (5) 0.9% saline solution, IP. Brain samples were collected for immunohistochemical study of GFAP expression in the mesencephalon and nucleus accumbens (NAc). The area of GFAP-positive cells was calculated using MetaMorphï software and serum levels of IL-1ß and TNF-α were measured by enzyme-linked immunosorbent assay. Serum TNF-α levels were decreased in the groups treated with Mo, Me and Gb, but not in the Amt-treated group. IL-1ß decreased only in rats treated with Me. The astrocytic expression of GFAP was decreased in the brainstem with all drugs, while it was increased in the NAc with Amt, Me and Mo
Assuntos
Animais , Masculino , Ratos , Proteína Glial Fibrilar Ácida/análise , Analgésicos/farmacologia , Dor/tratamento farmacológico , Astrócitos/imunologia , Citocinas/classificaçãoRESUMO
Microsporidia are obligate intracellular mitochondria-lacking pathogens that rely on host cells to grow and multiply. Microsporidia, currently classified as fungi, are ubiquitous in nature and are found worldwide. They infect a large number of mammals and are recognized as opportunistic infection agents in HIV-AIDS patients. Its importance for veterinary medicine has been unveiled in recent years through the description of clinical and subclinical forms of infection in domestic and wild animals. Domestic and wild birds may be infected by the same human microsporidia, reinforcing their zoonotic potential. Microsporidiosis in fish is prevalent and causes significant economic losses for fish farming. Some species of microsporidia have been propagated in cell cultures, which may provide conditions for the development of diagnostic techniques, understanding of pathogenesis and immune responses and for the discovery of potential therapies. Unfortunately, the cultivation of these parasites is not fully standardized in most research laboratories, especially in the veterinary field. The aim of this review is to relate the most important microsporidia of veterinary interest and demonstrate how these pathogens can be grown and propagated in cell culture for diagnostic purposes or for pathogenesis studies. Cultivation of microsporidia allowed the study of its life cycle, metabolism, pathogenesis and diagnosis, and may also serve as a repository for these pathogens for molecular, biochemical, antigenic and epidemiological studies.