RESUMO
Hemocyanins are used as immunomodulators in clinical applications because they induce a strong Th1-biased cell-mediated immunity, which has beneficial effects. They are multiligand glycosylated molecules with abundant and complex mannose-rich structures. It remains unclear whether these structures influence hemocyanin-induced immunostimulatory processes in human APCs. We have previously shown that hemocyanin glycans from Concholepas concholepas (CCH), Fissurella latimarginata (FLH), and Megathura crenulata (KLH), participate in their immune recognition and immunogenicity in mice, interacting with murine C-type lectin receptors (CLRs). Here, we studied the interactions of these hemocyanins with two major mannose-binding CLRs on monocyte-derived human DCs: MR (mannose receptor) and DC-SIGN (DC-specific ICAM-3-grabbing nonintegrin). Diverse analyses showed that hemocyanins are internalized by a mannose-sensitive mechanism. This process was calcium dependent. Moreover, hemocyanins colocalized with MR and DC-SIGN, and were partly internalized through clathrin-mediated endocytosis. The hemocyanin-mediated proinflammatory cytokine response was impaired when using deglycosylated FLH and KLH compared to CCH. We further showed that hemocyanins bind to human MR and DC-SIGN in a carbohydrate-dependent manner with affinity constants in the physiological concentration range. Overall, we showed that these three clinically valuable hemocyanins interact with human mannose-sensitive CLRs, initiating an immune response and promoting a Th1 cell-driving potential.
Assuntos
Moléculas de Adesão Celular/imunologia , Células Dendríticas/imunologia , Hemocianinas/imunologia , Fatores Imunológicos/imunologia , Lectinas Tipo C/imunologia , Lectinas de Ligação a Manose/imunologia , Receptores de Superfície Celular/imunologia , Animais , Células CHO , Linhagem Celular Tumoral , Células Cultivadas , Cricetulus , Humanos , Imunidade Celular/imunologia , Imunização/métodos , Receptor de Manose , Monócitos/imunologia , Células U937RESUMO
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
Serum is an important source of proteins that interact with pathogens. Once bound to the cell surface, serum proteins can stimulate the innate immune system. The phagocytosis of Sporothrix schenckii conidia by human macrophages is activated through human serum opsonisation. In this study, we have attempted to characterise human blood serum proteins that bind to the cell wall of S. schenckii conidia. We systematically observed the same four proteins independent of the plasma donor: albumin, serum amyloid protein (SAP), α-1 antitrypsin (AAT), and transferrin were identified with the help of tandem mass spectrometry. Phagocytosis depended on the concentration of the SAP or α-1 antitrypsin that was used to opsonise the conidia; however, transferrin or albumin did not have any effect on conidia internalisation. The presence of mannose did not affect macrophage phagocytosis of the conidia opsonised with SAP or α-1 antitrypsin, which suggests that these proteins are not recognised by the mannose receptor.
Assuntos
Proteínas Sanguíneas/imunologia , Macrófagos/imunologia , Fagocitose , Esporos Fúngicos/imunologia , Sporothrix/imunologia , Esporotricose/imunologia , Proteínas Sanguíneas/química , Humanos , Lectinas Tipo C/imunologia , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Receptores de Superfície Celular/imunologia , Esporos Fúngicos/genética , Sporothrix/genética , Esporotricose/microbiologiaRESUMO
BACKGROUND: Lipopolysaccharide (LPS)-tolerant monocytes produce small amounts of inflammatory cytokines, which is one of the characteristics of the alternative activated macrophages (AAM). These cells exhibited an increased expression of CD206 and CD163. Given the functional similarities of AAMs with the modulation of monocytes' functions observed during sepsis and LPS-tolerance, we evaluated whether the inhibition of inflammatory cytokine production by LPS-tolerant monocytes is associated with the phenotype of cells expressing CD206 and CD163. METHODS: We investigated whether tolerant human monocytes would modulate their expression of CD206 and CD163, markers of alternative activation, and whether the level of their expression would be related to cytokines detection. Tolerance to LPS was induced in peripheral blood mononuclear cell by pre-incubating the cells with increasing concentrations of LPS. The expression of CD206 and CD163 and intracellular TNF-α and IL-6 was determined 24 h after LPS challenge by flow cytometry. RESULTS: No differences in CD163 expression were observed between tolerant and non-tolerant cells, while the expression of CD206, which was decreased following LPS stimulation in non-tolerized cells, was further reduced in tolerant cells. Decreased production of inflammatory cytokines was observed in the tolerized cells, regardless of the expression of CD163 and CD206, with the exception of IL-6 in CD206+ monocytes, which was similarly expressed in both tolerized and non-tolerized cells. CONCLUSIONS: The effect of LPS in the expression of CD163 and CD206 on monocytes is not reverted in LPS tolerant cells, and the inhibition of inflammatory cytokines in tolerant cells is not related with modulation of these receptors. © 2016 International Clinical Cytometry Society.
Assuntos
Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Tolerância Imunológica/genética , Lectinas Tipo C/genética , Macrófagos/imunologia , Lectinas de Ligação a Manose/genética , Receptores de Superfície Celular/genética , Adulto , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Citocinas/biossíntese , Citocinas/imunologia , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Humanos , Lectinas Tipo C/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/toxicidade , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Macrófagos/metabolismo , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Receptores de Superfície Celular/imunologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
The immunomodulatory properties of mannose-binding lectins ConBr (Canavalia brasiliensis) and CFL (Cratylia argentea) were investigated comparatively in a model of Salmonella infection. The lectins were intraperitoneally (i.p.) administered to mice daily for three days before the bacterial challenge with Salmonella enterica Ser. Typhimurium (0.2 mL i.p.; 10(7) CFU/mL). In vivo assays have shown that both lectins induced a significant leukocyte infiltration into the peritoneal cavity of uninfected mice, which was higher in the CFL group 3 days post-infection. Total and differential cell counts in the bloodstreams have shown uninfected animals pretreated with ConBr and CFL exhibited accentuated lymphopenia. Conversely, there was an increasing population of lymphocytes following 3 days post-infection in mice pretreated with both lectins. In addition, the bacterial burden was significantly reduced into the peritoneal cavity, bloodstreams, spleen and the liver in these mice. The lectins did not induce the release of pro- or anti-inflammatory cytokines into the peritoneal fluid of uninfected animals. However, following infection, the release of TNF-α and IL-10 in the peritoneal fluid were down-regulated in mice pretreated with both lectins whereas IL-1 was only reduced in mice pretreated with ConBr. Uninfected animals pretreated with CFL exhibited high nitric oxide (NO) content in the peritoneal fluid, which was decreased after infection in comparison to ConBr group. The lectins did not alter the serum levels of NO in uninfected mice but treatments with ConBr significantly reduced the NO content in infected animals in comparison to CFL group 24h after the bacterial challenge. Survival experiments have shown survival rates ranging from 70% to 100% in mice that received CFL or ConBr. On the other hand, untreated mice (PBS group) died 1-6 days after infection. We conclude that ConBr and CFL are prospective phytotherapeutics capable of modulate the cascade of pro-inflammatory plus regulatory cytokines and nitric oxide release derived from systemic infections.
Assuntos
Canavalia/imunologia , Fatores Imunológicos/uso terapêutico , Leucócitos/efeitos dos fármacos , Lectinas de Ligação a Manose/uso terapêutico , Infecções por Salmonella/tratamento farmacológico , Salmonella typhi/imunologia , Animais , Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Mediadores da Inflamação/metabolismo , Injeções Intraperitoneais , Leucócitos/imunologia , Leucócitos/microbiologia , Lectinas de Ligação a Manose/imunologia , Camundongos , Óxido Nítrico/metabolismo , Infecções por Salmonella/imunologiaRESUMO
Leishmania species are dimorphic protozoan parasites that live and replicate in the gut of sand flies as promastigotes or in mammalian hosts as amastigotes. Different immune cells, including DCs, and receptors differ in their involvement in phagocytosis of promastigotes and amastigotes and in recognition of different Leishmania species. In the case of L. mexicana, differences in phagocytosis of promastigotes and amastigotes by DCs and participation of C-type lectin receptors (CLRs) have not been established. In the present study, flow cytometry and confocal microscopy were used to investigate the phagocytosis by monocyte-derived dendritic cells (moDCs) of L. mexicana promastigotes and amastigotes in the presence or absence of immune serum during various periods of time. Blocking antibodies against mannose receptors and DC-SIGN were used to explore the participation of these receptors in the phagocytosis of L. mexicana by moDC. The major differences in interactions of L. mexicana promastigotes and amastigotes with moDC were found to occur within the first 3 hr, during which phagocytosis of promastigotes predominated as compared with opsonization of promastigotes and amastigotes. However, after 6 hr of incubation, opsonized promastigotes were preferentially phagocytosed as compared with unopsonized promastigotes and amastigotes and after 24 hr of incubation there were no differences in the phagocytosis of promastigotes and amastigotes. Finally, after 3 hr incubation, DC-SIGN was involved in the phagocytosis of promastigotes, but not of amastigotes.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Leishmania mexicana/imunologia , Monócitos/imunologia , Monócitos/parasitologia , Fagocitose/fisiologia , Animais , Moléculas de Adesão Celular/imunologia , Células Cultivadas , Células Dendríticas/citologia , Citometria de Fluxo/métodos , Interações Hospedeiro-Parasita , Humanos , Lectinas Tipo C/imunologia , Leishmaniose/sangue , Leishmaniose/imunologia , Leishmaniose/parasitologia , Macrófagos/imunologia , Macrófagos/parasitologia , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Camundongos Endogâmicos BALB C , Microscopia Confocal/métodos , Monócitos/citologia , Receptores de Superfície Celular/imunologiaRESUMO
The clinical course of infection with Mycobacterium leprae varies widely and depends on the pattern of the host immune response. Dendritic cells play an important role in the activation of the innate and adaptive immune system and seem to be essential for the development of the disease. To analyze the presence of epidermal dendritic cells (CD1a and CD207), plasmacytoid dendritic cells (CD123) and dermal dendrocytes (factor XIIIa) in lesion fragments of leprosy patients, skin samples from 30 patients were studied. These samples were submitted to immunohistochemistry against CD1a, CD207, FXIIIa, and CD123. The results showed a larger number of Langerhans cells, detected with the CD1a or CD207 marker, dermal dendrocytes and plasmacytoid dendritic cells in patients with the tuberculoid form. A positive correlation was observed between the Langerhans cell markers CD1a and CD207 in both the tuberculoid and lepromatous forms, and between Langerhans cells and dermal dendrocytes in samples with the tuberculoid form. The present results indicate the existence of a larger number of dendritic cells in patients at the resistant pole of the disease (tuberculoid) and suggest that the different dendritic cells studied play a role, favoring an efficient immune response against infection with M. leprae.
Assuntos
Antígenos CD1/imunologia , Antígenos CD/imunologia , Células Dendríticas/imunologia , Fator XIIIa/imunologia , Subunidade alfa de Receptor de Interleucina-3/imunologia , Células de Langerhans/imunologia , Lectinas Tipo C/imunologia , Hanseníase/imunologia , Lectinas de Ligação a Manose/imunologia , Pele/imunologia , Derme/citologia , Derme/imunologia , Humanos , Hanseníase/microbiologia , Hanseníase/patologia , Mycobacterium leprae/fisiologia , Pele/patologiaRESUMO
BACKGROUND: Jorge Lobo's disease (JLD) is a cutaneous chronic mycosis caused by Lacazia loboi. We studied Factor XIIIa + dermal dendrocytes (FXIIIa + DD), Langerhans cells (LC) through the expression of langerin and the expression of S100 protein. METHODS: A total of 41 biopsies and 10 normal skins (control) were developed with a polymer-based immunohistochemical method. RESULTS: Lesions presented infiltrate comprising macrophages, some asteroid corpuscles, lymphocytes, multinucleated giant cells and a large number of fungi. LCs presented short dendrites and were scarcely distributed. Dermal langerin + cells were detected in nine JLD lesions. FXIIIa + DD were hypertrophic, visualized in the inflammatory infiltrate of JLD lesions. Cells S100+ were present in JLD and control group with a similar number of cells. A total of 14 specimens did not express FXIIIa, and this considerable number probably contributed to the statistical similarity with the control group. CONCLUSIONS: The results indicate that LCs are present in the immune response against Lacazia loboi. Some dermal langerin + cells could be another subset of dendritic cells. Our data indicate changes of LCs in JLD cutaneous lesions and present, for the first time, results that show langerin + cells in the dermis and corroborate previous observations on the participation of FXIIIa + DD in the in situ immune response in JLD.
Assuntos
Células de Langerhans/imunologia , Lobomicose/patologia , Antígenos CD/imunologia , Humanos , Imuno-Histoquímica , Lacazia/isolamento & purificação , Lacazia/fisiologia , Células de Langerhans/química , Lectinas Tipo C/imunologia , Lobomicose/imunologia , Lectinas de Ligação a Manose/imunologia , Proteínas S100/imunologia , Pele/química , Pele/imunologia , Pele/patologia , Coloração e RotulagemRESUMO
The functions of phagocytic cells against pathogens are initiated by the interaction between membrane receptors and molecular structures which compose the cell wall of these microorganisms. Thus our study aimed to identify the neutrophil receptors involved in the recognition of different strains of Paracoccidioides brasiliensis and the consequent modulation of immune response through the production of cytokines and inflammatory mediators. Neutrophils did not produce TNF-alfa in response to both strains. However, these cells produce IL-12, mainly in response to Pb 265, with participation of TLR2 and dectin-1. These cells also produce L-10, whose levels were higher for Pb 18 with involvement of TLR2 and MR and only TLR2 for Pb 265. The production of PGE2 and LTB4 was detected similarly for the two strains. For PGE2, MR and dectin-1 were involved, while in relation to LTB4, none of them. In summary, we demonstrated that neutrophils have a dynamic role during host immune response to P. brasiliensis, since in addition to their role as effector cells of innate immunity; they have the capacity to modulate innate and adaptative immune response against this fungus by producing cytokines and lipidic mediators. This modulation may be toward a pró- or anti-inflammatory pattern in a dependence of P. brasiliensis strains and PRR involved in fungus recognition by these cells.
Assuntos
Lectinas Tipo C/imunologia , Lectinas de Ligação a Manose/imunologia , Neutrófilos/imunologia , Paracoccidioides/imunologia , Receptores de Superfície Celular/imunologia , Receptor 2 Toll-Like/imunologia , Células Cultivadas , Dinoprostona/biossíntese , Dinoprostona/imunologia , Humanos , Interleucina-10/biossíntese , Interleucina-10/imunologia , Interleucina-12/biossíntese , Interleucina-12/imunologia , Subunidade p35 da Interleucina-12 , Leucotrieno B4/biossíntese , Leucotrieno B4/imunologia , Receptor de Manose , Paracoccidioides/classificação , Paracoccidioidomicose/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
De novo ectopic lymphoid tissue formation is known to occur in certain disease and inflammatory settings. After an effective vaccination with dendritic cells (DC) charged with melanoma apoptotic/necrotic cells (Apo/Nec), a subcutaneous tertiary lymphoid structure was organized, where retained vaccine cells interacted with recruited inflammatory and T cells. In this work we report for the first time the recruitment of two morphologically different CD207(+) cells to vaccination site. The time-course behavior of CD207(+) cells was reciprocal between vaccination site and draining lymph nodes (DLNs). After 6-10 days, CD207(+) cells localized at the paracortical region of DLNs, in close contact with T cell population. DLNs were enriched in a peculiar MHCII(+) CD11c((-)) CD207(+) population, whose role remains to be determined. Whether CD207(+) cells migration to the vaccination site can be associated with a differential anti-tumoral response remains as an open and exciting question.