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2.
J Vet Diagn Invest ; 31(5): 726-731, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31272298

RESUMO

A 5-y-old male Poodle mix was presented with intermittent vomiting, anorexia, and weight loss. Physical examination revealed emaciation, lethargy, dehydration, hypothermia, respiratory distress, and splenomegaly. Based on clinicopathologic, serologic, and parasitologic findings, diagnoses of severe leishmaniosis and dirofilariasis were made. Extracellular, intraneutrophilic, and intramonocytic Leishmania amastigotes were observed on blood smear and buffy coat smear examination. In blood smears, 0.2% of neutrophils were observed to be infected; in buffy coat smears, 0.5% of neutrophils and 0.1% of monocytes were found to be infected. Leishmania amastigotes were also found engulfed by eosinophils and neutrophil precursors in bone marrow aspiration cytology. The detection of Leishmania amastigotes in blood smears is rare, and the clinical significance is uncertain. In circulating blood, Leishmania amastigotes are primarily found phagocytized by neutrophils. Although debatable, there is growing evidence that neutrophils are used as carriers enabling the "silent entry" of the protozoa into macrophages ("Trojan horse" theory). To date, cytologic screening of blood smears for the diagnosis of canine leishmaniosis is not a routine practice. Clinical pathologists and practitioners should be aware that Leishmania amastigotes may be present in neutrophils and less frequently monocytes during blood smear evaluation; neutrophil precursors and eosinophils may also be parasitized in bone marrow specimens.


Assuntos
Células da Medula Óssea/parasitologia , Doenças do Cão/parasitologia , Leishmania/isolamento & purificação , Leishmaniose/veterinária , Células Mieloides/parasitologia , Animais , Dirofilariose/complicações , Cães , Leishmaniose/complicações , Leishmaniose/parasitologia , Leishmaniose Visceral , Masculino , Neutrófilos , Baço/patologia
3.
Methods Mol Biol ; 1971: 237-247, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980307

RESUMO

Murine bone marrow-derived macrophages (BMMs) can be differentiated within 10 days from ex vivo bone marrow progenitor cells by supplementing the cell growth medium with colony stimulating factor-1 (CSF-1). Mature macrophages express specific myeloid markers which can be labeled and detected by flow cytometry (FACS).BMMs are a valuable tool to investigate the interactions between the Leishmania parasites and their host cell as well as to screen anti-Leishmania components. Options for the readout of in vitro infection experiments are diverse and may range from simple counting of intracellular parasites to the determination of metabolic changes of the intracellular parasite or the infected cell, thus providing the investigator with valuable results.


Assuntos
Células da Medula Óssea/parasitologia , Leishmania/crescimento & desenvolvimento , Leishmaniose/metabolismo , Macrófagos/parasitologia , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Diferenciação Celular , Células Cultivadas , Feminino , Leishmaniose/patologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C
4.
J Parasitol ; 105(2): 359-370, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31033389

RESUMO

Species of the genus Leishmania are the causal agents of leishmaniasis, a disease with diametrically different clinical manifestations that have been attributed to the species and host immune response. Some Leishmania species, including Leishmania mexicana, are capable of causing both localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). Therefore, it is possible that intraspecific differences may exist that contribute to the development of distinct clinical forms. Dendritic cells (DC) are important host cells of Leishmania spp. parasites, and cytokine production and phagocytosis upon infection with the parasite are significant for the outcome of the disease. In the present study we analyzed the production of IL-12, TNF-α, and IL-10 by DC infected with L. mexicana amastigotes isolated from a patient with LCL (amastigote = Lac) and from a patient with DCL (amastigote = Diact) by murine DC. Furthermore, we compared the frequency of phagocytosis of L. mexicana amastigotes of each isolate by fluorescence and optical microscopy and by flow cytometry. We show that the infection of DC with Diact amastigotes elicited the secretion of IL-10, TNF-α, and IL-12 by DC to a major extent as compared to the infection with Lac amastigotes. On the other hand, Lac and Diact amastigotes were similarly phagocytosed by DC, but interestingly there were more vacuoles in DC infected with Diact amastigotes. Our results suggest that isolates from a same species of Leishmania, such as L. mexicana, with different degrees of virulence according to the clinical manifestation they cause, differ in their capacity to elicit cytokine production and form vacuoles in DC.


Assuntos
Células da Medula Óssea/fisiologia , Citocinas/biossíntese , Células Dendríticas/fisiologia , Leishmania mexicana/fisiologia , Fagocitose , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/parasitologia , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Ensaio de Imunoadsorção Enzimática , Fêmur/citologia , Citometria de Fluxo , Leishmania mexicana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia , Microscopia de Fluorescência , Tíbia/citologia
5.
Exp Parasitol ; 196: 12-21, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30465732

RESUMO

Helminths and their products are strong candidates for the treatment of autoimmunological disorders and allergies. Being a key population of antigen-presenting cells, dendritic cells play a crucial role in the therapeutic potential of worms. The study compares the effects of live pre-male and pre-female L4 stage Heligmosomoides polygyrus administration on the maturation and activation of the JAWS II line of immature dendritic cells. On stimulation with L4 stage H. polygyrus, JAWS II cells acquire semi-mature status and induce Th2 and regulatory responses in vitro. The strongest immunosuppressive effect on JAWS II cells was observed following stimulation with both sexes of nematodes together; this was manifested as immature dendritic cell morphology, proliferation inhibition, cell cycle change, decreased translocation of NF-κB into the nucleus, and lower expression of surface cellular costimulatory molecules CD80, CD86 and MHC I. However, greater production of proinflammatory (IL-12p70, TNF-α, IL-6) and Th2 response-promoting cytokines (IL-4) was observed by JAWS II following exposure to both sexes compared to male or female larvae alone. Sex had no influence on the viability, apoptosis process or endocytosis abilities of the JAWS II cell line. The findings indicate that the presence of only a single sex of the parasite influences a developed response, resulting in reduced proinflammatory and an antiparasitic reaction.


Assuntos
Células Dendríticas/parasitologia , Nematospiroides dubius/fisiologia , Animais , Apoptose , Células da Medula Óssea/imunologia , Células da Medula Óssea/parasitologia , Células da Medula Óssea/fisiologia , Ciclo Celular , Linhagem Celular , Quimiocinas/análise , Citocinas/análise , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Endocitose , Feminino , Larva/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/análise , Nematospiroides dubius/crescimento & desenvolvimento , Fatores Sexuais , Organismos Livres de Patógenos Específicos
6.
Front Immunol ; 9: 2794, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30619242

RESUMO

After infection with Trypanosoma cruzi, the etiologic agent of Chagas disease, immunosuppression, and apoptosis of mature lymphocytes contribute to the establishment of the parasite in the host and thereby to persistence and pathology in the chronic stage of infection. In a systemic mouse model of experimental Chagas disease, we have demonstrated a strong depletion of mature B cells in the spleen during the first 2 weeks of infection. Remarkably, the decrease in this cell population commenced already in the bone marrow from infected mice and was a concomitant of an increased apoptosis in pro- and pre-B cell populations. Pro- and pre-B cells in the bone marrow showed a significant reduction accompanied by a functional disturbance of bone marrow-derived stromal cells resulting in diminished levels of IL-7, an essential factor for the development of B cell precursors. Ex vivo, stromal cells isolated from the bone marrow of infected mice had a strikingly impaired capacity to maintain the development of pro- and pre-B cells obtained from uninfected animals. Together, the reduction of an active humoral immune response during acute Chagas disease suggests to be an initial immune evasion mechanism of the parasite to establish persistent infection. Therefore, prevention of B cell depletion by rescuing the stromal cells during this early phase, could give rise to new therapeutic approaches.


Assuntos
Células da Medula Óssea/imunologia , Doença de Chagas/imunologia , Tolerância Imunológica , Trypanosoma cruzi/imunologia , Doença Aguda , Animais , Apoptose/imunologia , Células da Medula Óssea/parasitologia , Células da Medula Óssea/patologia , Doença de Chagas/parasitologia , Doença de Chagas/patologia , Doença Crônica , Interleucina-7/imunologia , Camundongos , Células Precursoras de Linfócitos B/imunologia , Células Precursoras de Linfócitos B/parasitologia , Células Precursoras de Linfócitos B/patologia , Baço/imunologia , Baço/parasitologia , Baço/patologia , Células Estromais/imunologia , Células Estromais/parasitologia , Células Estromais/patologia
7.
Cell Immunol ; 326: 77-85, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29248120

RESUMO

Immunosuppression is a well-established risk factor for Visceral Leishmaniasis. Post-immunosuppression leishmaniasis is characterized by an increase of parasite burden, hematopoietic disorders and unusual clinical manifestations. Although there are many reports on bone marrow findings in VL, less is known about the relationship between parasite dynamics in this organ and the function of either hematopoietic stem cells and progenitor cells themselves. In the present study, we tackle these issues using a new approach of infecting human stem cells derived from bone marrow with L. infantum. Using this strategy, we show that human hematopoietic stem cells (hHSC) are able to phagocytize L. infantum promastigotes and release modulatory and pro-inflammatory cytokines, mainly TNF-α. Our results demonstrated that L. infantum infection in vitro enhances hematopoiesis, favoring the development of erythrocitic lineage through a mechanism yet unknown. Moreover, we found that L. infantum infection alters the phenotypic profile of the hematopoietic progeny; modifying the surface markers expression of differentiated cells. Thus, our study represents a rare opportunity to monitor the in vitro differentiation of human stem cells experimentally infected by L. infantum to better understand the consequences of the infection on phenotypic and functional profile of the cell progeny.


Assuntos
Diferenciação Celular/imunologia , Eritropoese/imunologia , Células-Tronco Hematopoéticas/imunologia , Leishmania infantum/imunologia , Fagocitose/imunologia , Adulto , Idoso , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/parasitologia , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/parasitologia , Interações Hospedeiro-Parasita/imunologia , Humanos , Leishmania infantum/fisiologia , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
8.
J Biol Chem ; 292(33): 13615-13634, 2017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-28679535

RESUMO

Malaria infections cause several systemic and severe single- or multi-organ pathologies, killing hundreds of thousands of people annually. Considering the existing widespread resistance of malaria parasites to anti-parasitic drugs and their high propensity to develop drug resistance, alternative strategies are required to manage malaria infections. Because malaria is a host immune response-driven disease, one approach is based on gaining a detailed understanding of the molecular and cellular processes that modulate malaria-induced innate and adaptive immune responses. Here, using a mouse cerebral malaria model and small-molecule inhibitors, we demonstrate that inhibiting MEK1/2, the upstream kinases of ERK1/2 signaling, alters multifactorial components of the innate and adaptive immune responses, controls parasitemia, and blocks pathogenesis. Specifically, MEK1/2 inhibitor treatment up-regulated B1 cell expansion, IgM production, phagocytic receptor expression, and phagocytic activity, enhancing parasite clearance by macrophages and neutrophils. Further, the MEK1/2 inhibitor treatment down-regulated pathogenic pro-inflammatory and helper T cell 1 (Th1) responses and up-regulated beneficial anti-inflammatory cytokine responses and Th2 responses. These inhibitor effects resulted in reduced granzyme B expression by T cells, chemokine and intracellular cell adhesion molecule 1 (ICAM-1) expression in the brain, and chemokine receptor expression by both myeloid and T cells. These bimodal effects of the MEK1/2 inhibitor treatment on immune responses contributed to decreased parasite biomass, organ inflammation, and immune cell recruitment, preventing tissue damage and death. In summary, we have identified several previously unrecognized immune regulatory processes through which a MEK1/2 inhibitor approach controls malaria parasitemia and mitigates pathogenic effects on host organs.


Assuntos
Antimaláricos/uso terapêutico , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Malária Cerebral/tratamento farmacológico , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Imunidade Adaptativa/efeitos dos fármacos , Animais , Antimaláricos/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/parasitologia , Células da Medula Óssea/patologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Células Dendríticas/patologia , Feminino , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Imunidade Inata/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/parasitologia , Células Matadoras Naturais/patologia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Malária Cerebral/imunologia , Malária Cerebral/parasitologia , Malária Cerebral/patologia , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Malária Falciparum/patologia , Masculino , Camundongos Endogâmicos C57BL , Carga Parasitária , Parasitemia/prevenção & controle , Fagocitose/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Plasmodium falciparum/imunologia , Plasmodium falciparum/isolamento & purificação , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Piridonas/farmacologia , Piridonas/uso terapêutico , Pirimidinonas/farmacologia , Pirimidinonas/uso terapêutico , Análise de Sobrevida
9.
J Biol Chem ; 292(32): 13087-13096, 2017 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-28607148

RESUMO

Leishmaniasis is caused by protozoan parasites of the genus Leishmania In mammalians, these parasites survive and replicate in macrophages and parasite elimination by macrophages is critical for host resistance. Endosomal Toll-like receptors (TLRs) have been shown to be crucial for resistance to Leishmania major in vivo For example, mice in the resistant C57BL/6 genetic background that are triple-deficient for TLR3, -7, and -9 (Tlr3/7/9-/-) are highly susceptible to L. major infection. Tlr3/7/9-/- mice are as susceptible as mice deficient in MyD88 or UNC93B1, a chaperone required for appropriate localization of endosomal TLRs, but the mechanisms are unknown. Here we found that macrophages infected with L. major undergo autophagy, which effectively accounted for restriction of parasite replication. Signaling via endosomal TLRs was required for autophagy because macrophages deficient for TLR3, -7, and 9, UNC93B1, or MyD88 failed to undergo L. major-induced autophagy. We also confirmed that Myd88-/-, Tlr3/7/9-/-, and Unc93b1-/- cells were highly permissive to L. major replication. Accordingly, shRNA-mediated suppression of Atg5, an E3 ubiquitin ligase essential for autophagosome elongation, in macrophages impaired the restriction of L. major replication in C57BL/6, but did not affect parasite replication in Myd88-/- or Unc93b1-/- macrophages. Rapamycin treatment reduced inflammatory lesions formed in the ears of Leishmania-infected C57BL/6 and Tlr3/7/9-/- mice, indicating that autophagy operates downstream of TLR signaling and is relevant for disease development in vivo Collectively, our results indicate that autophagy contributes to macrophage resistance to L. major replication, and mechanistically explain the previously described endosomal TLR-mediated resistance to L. major infection.


Assuntos
Autofagia , Endossomos/parasitologia , Leishmania major/imunologia , Macrófagos/parasitologia , Proteínas de Membrana Transportadoras/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 3 Toll-Like/metabolismo , Animais , Proteína 5 Relacionada à Autofagia/antagonistas & inibidores , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/parasitologia , Células da Medula Óssea/patologia , Células Cultivadas , Resistência à Doença , Endossomos/imunologia , Endossomos/metabolismo , Endossomos/patologia , Feminino , Leishmania major/crescimento & desenvolvimento , Leishmania major/fisiologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Interferência de RNA , Transdução de Sinais , Receptor 3 Toll-Like/genética , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo
10.
J Biol Chem ; 292(29): 12324-12338, 2017 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-28550086

RESUMO

Leishmaniasis is one of the leading globally neglected diseases, affecting millions of people worldwide. Leishmania infection depends on the ability of insect-transmitted metacyclic promastigotes to invade mammalian hosts, differentiate into amastigotes, and replicate inside macrophages. To counter the hostile oxidative environment inside macrophages, these protozoans contain anti-oxidant systems that include iron-dependent superoxide dismutases (SODs) in mitochondria and glycosomes. Increasing evidence suggests that in addition to this protective role, Leishmania mitochondrial SOD may also initiate H2O2-mediated redox signaling that regulates gene expression and metabolic changes associated with differentiation into virulent forms. To investigate this hypothesis, we examined the specific role of SODA, the mitochondrial SOD isoform in Leishmania amazonensis Our inability to generate L. amazonensis SODA null mutants and the lethal phenotype observed following RNAi-mediated silencing of the Trypanosoma brucei SODA ortholog suggests that SODA is essential for trypanosomatid survival. L. amazonensis metacyclic promastigotes lacking one SODA allele failed to replicate in macrophages and were severely attenuated in their ability to generate cutaneous lesions in mice. Reduced expression of SODA also resulted in mitochondrial oxidative damage and failure of SODA/ΔsodA promastigotes to differentiate into axenic amastigotes. SODA expression above a critical threshold was also required for the development of metacyclic promastigotes, as SODA/ΔsodA cultures were strongly depleted in this infective form and more susceptible to reactive oxygen species (ROS)-induced stress. Collectively, our data suggest that SODA promotes Leishmania virulence by protecting the parasites against mitochondrion-generated oxidative stress and by initiating ROS-mediated signaling mechanisms required for the differentiation of infective forms.


Assuntos
Ferro/metabolismo , Leishmania mexicana/enzimologia , Mitocôndrias/enzimologia , Proteínas de Protozoários/metabolismo , Superóxido Dismutase/metabolismo , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/parasitologia , Células da Medula Óssea/patologia , Linhagem Celular , Células Cultivadas , Células Clonais , Feminino , Técnicas de Inativação de Genes , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/patogenicidade , Leishmania mexicana/ultraestrutura , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Carga Parasitária , Transporte Proteico , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Interferência de RNA , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/genética , Virulência
11.
Trends Parasitol ; 33(7): 492-495, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28366603

RESUMO

Accumulation of erythrocytic parasites in bone marrow and the spleen has been reported in cases of Plasmodium vivax malaria. If this occurs commonly, these stages represent a possible source of early, relapse-like homologous recurrences. Moreover, they might hinder the elimination of malaria from human populations. Pertinent research suggestions have been made.


Assuntos
Portador Sadio/parasitologia , Erradicação de Doenças , Eritrócitos/parasitologia , Malária/parasitologia , Plasmodium vivax/fisiologia , Animais , Células da Medula Óssea/parasitologia , Humanos , Estágios do Ciclo de Vida/fisiologia , Malária/prevenção & controle , Recidiva , Baço/parasitologia
12.
EMBO Rep ; 17(12): 1753-1765, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27861124

RESUMO

Cross-presentation by MHC class I molecules allows the detection of exogenous antigens by CD8+ T lymphocytes. This process is crucial to initiate cytotoxic immune responses against many pathogens (i.e., Toxoplasma gondii) and tumors. To achieve efficient cross-presentation, dendritic cells (DCs) have specialized endocytic pathways; however, the molecular effectors involved are poorly understood. In this work, we identify the small GTPase Rab22a as a key regulator of MHC-I trafficking and antigen cross-presentation by DCs. Our results demonstrate that Rab22a is recruited to DC endosomes and phagosomes, as well as to the vacuole containing T. gondii parasites. The silencing of Rab22a expression did not affect the uptake of exogenous antigens or parasite invasion, but it drastically reduced the intracellular pool and the recycling of MHC-I molecules. The knockdown of Rab22a also hampered the cross-presentation of soluble, particulate and T. gondii-associated antigens, but not the endogenous MHC-I antigen presentation through the classical secretory pathway. Our findings provide compelling evidence that Rab22a plays a central role in the MHC-I endocytic trafficking, which is crucial for efficient cross-presentation by DCs.


Assuntos
Apresentação de Antígeno , Proteínas de Transporte/metabolismo , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas Nucleares/metabolismo , Toxoplasma/imunologia , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/fisiologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/parasitologia , Linfócitos T CD8-Positivos/imunologia , Proteínas de Transporte/genética , Apresentação Cruzada , Proteínas de Ligação a DNA , Células Dendríticas/parasitologia , Endocitose , Endossomos/metabolismo , Endossomos/parasitologia , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Camundongos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fagossomos/metabolismo , Fagossomos/parasitologia , Transporte Proteico , Proteínas de Ligação a RNA , Toxoplasma/fisiologia , Vacúolos/metabolismo , Vacúolos/parasitologia
13.
PLoS Negl Trop Dis ; 10(10): e0005061, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27736880

RESUMO

Accumulating evidences have assigned a central role to parasite-derived proteins in immunomodulation. Here, we report on the proteomic identification and characterization of immunomodulatory excretory-secretory (ES) products from the metacestode larva (tetrathyridium) of the tapeworm Mesocestoides corti (syn. M. vogae). We demonstrate that ES products but not larval homogenates inhibit the stimuli-driven release of the pro-inflammatory, Th1-inducing cytokine IL-12p70 by murine bone marrow-derived dendritic cells (BMDCs). Within the ES fraction, we biochemically narrowed down the immunosuppressive activity to glycoproteins since active components were lipid-free, but sensitive to heat- and carbohydrate-treatment. Finally, using bioassay-guided chromatographic analyses assisted by comparative proteomics of active and inactive fractions of the ES products, we defined a comprehensive list of candidate proteins released by M. corti tetrathyridia as potential suppressors of DC functions. Our study provides a comprehensive library of somatic and ES products and highlight some candidate parasite factors that might drive the subversion of DC functions to facilitate the persistence of M. corti tetrathyridia in their hosts.


Assuntos
Infecções por Cestoides/imunologia , Células Dendríticas/imunologia , Proteínas de Helminto/imunologia , Mesocestoides/química , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/parasitologia , Infecções por Cestoides/parasitologia , Células Dendríticas/parasitologia , Proteínas de Helminto/química , Proteínas de Helminto/genética , Humanos , Larva/química , Larva/genética , Larva/imunologia , Mesocestoides/genética , Mesocestoides/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteômica
14.
J Biol Chem ; 291(7): 3496-507, 2016 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-26670606

RESUMO

Apoptosis is one of the mechanisms used by host cells to remove unwanted intracellular organisms, and often found to be subverted by pathogens through use of host anti-apoptotic proteins. In the present study, with the help of in vitro and in vivo approaches, we documented that the macrophage anti-apoptotic protein myeloid cell leukemia 1 (MCL-1) is exploited by the intra-macrophage parasite Leishmania donovani to protect their "home" from actinomycin D-induced mitochondria-dependent apoptosis. Among all the anti-apoptotic BCL-2 family members, infection preferentially up-regulated expression of MCL-1 at both the mRNA and protein levels and compared with infected control, MCL-1-silenced infected macrophages documented enhanced caspase activity and increased apoptosis when subjected to actinomycin D treatment. Phosphorylation kinetics and ChIP assay demonstrated that infection-induced MCL-1 expression was regulated by transcription factor CREB (cAMP-response element-binding protein) and silencing of CREB resulted in reduced expression of MCL-1 and increased apoptosis. During infection, MCL-1 was found to be localized in mitochondria and this was significantly reduced in Tom70-silenced macrophages, suggesting the active role of TOM70 in MCL-1 transport. In the mitochondria, MCL-1 interacts with the major pro-apoptotic protein BAK and prevents BAK-BAK homo-oligomer formation thereby preventing cytochrome c release-mediated mitochondrial dysfunction. Silencing of MCL-1 in the spleen of infected mice showed decreased parasite burden and increased induction of splenocyte apoptosis. Collectively our results showed that L. donovani exploited the macrophage anti-apoptotic protein MCL-1 to prevent BAK-mediated mitochondria-dependent apoptosis thereby protecting its niche, which is essential for disease progression.


Assuntos
Antiparasitários/farmacologia , Apoptose/efeitos dos fármacos , Interações Hospedeiro-Parasita/efeitos dos fármacos , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Animais , Antiparasitários/uso terapêutico , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/parasitologia , Células da Medula Óssea/patologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dactinomicina/farmacologia , Dactinomicina/uso terapêutico , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/fisiologia , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/patologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Células RAW 264.7 , Interferência de RNA , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/parasitologia , Baço/patologia
15.
Immunity ; 42(6): 1130-42, 2015 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-26070484

RESUMO

Tissue-infiltrating Ly6C(hi) monocytes play diverse roles in immunity, ranging from pathogen killing to immune regulation. How and where this diversity of function is imposed remains poorly understood. Here we show that during acute gastrointestinal infection, priming of monocytes for regulatory function preceded systemic inflammation and was initiated prior to bone marrow egress. Notably, natural killer (NK) cell-derived IFN-γ promoted a regulatory program in monocyte progenitors during development. Early bone marrow NK cell activation was controlled by systemic interleukin-12 (IL-12) produced by Batf3-dependent dendritic cells (DCs) in the mucosal-associated lymphoid tissue (MALT). This work challenges the paradigm that monocyte function is dominantly imposed by local signals after tissue recruitment, and instead proposes a sequential model of differentiation in which monocytes are pre-emptively educated during development in the bone marrow to promote their tissue-specific function.


Assuntos
Células da Medula Óssea/imunologia , Células Dendríticas/imunologia , Mucosa Intestinal/imunologia , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Animais , Antígenos Ly/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Células da Medula Óssea/parasitologia , Diferenciação Celular , Células Cultivadas , Interferon gama/metabolismo , Interleucina-12/genética , Interleucina-12/metabolismo , Mucosa Intestinal/parasitologia , Células Matadoras Naturais/parasitologia , Leucócitos Mononucleares/parasitologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Imunológicos , Especificidade de Órgãos/imunologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
16.
J Exp Med ; 212(6): 875-82, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25964370

RESUMO

Group 2 innate lymphoid cells (ILC2s) are often found associated with mucosal surfaces where they contribute to protective immunity, inappropriate allergic responses, and tissue repair. Although we know they develop from a common lymphoid progenitor in the bone marrow (BM), the specific lineage path and transcriptional regulators that are involved are only starting to emerge. After ILC2 gene expression analysis we investigated the role of Bcl11b, a factor previously linked to T cell commitment, in ILC2 development. Using combined Bcl11b-tom and Id2-gfp reporter mice, we show that Bcl11b is expressed in ILC2 precursors in the BM and maintained in mature ILC2s. In vivo deletion of Bcl11b, by conditional tamoxifen-induced depletion or by Bcl11b(-/-) fetal liver chimera reconstitution, demonstrates that ILC2s are wholly dependent on Bcl11b for their development. Notably, in the absence of Bcl11b there is a concomitant expansion of the RORγt(+) ILC3 population, suggesting that Bcl11b may negatively regulate this lineage. Using Nippostrongylus brasiliensis infection, we reveal that the absence of Bcl11b leads to impaired worm expulsion, caused by a deficit in ILC2s, whereas Citrobacter rodentium infection is cleared efficiently. These data clearly establish Bcl11b as a new factor in the differentiation of ILC2s.


Assuntos
Linfócitos/citologia , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/microbiologia , Células da Medula Óssea/parasitologia , Linhagem da Célula , Citrobacter rodentium , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Humanos , Fígado/embriologia , Linfócitos/microbiologia , Linfócitos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nippostrongylus
17.
J Immunol ; 195(1): 194-202, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26026056

RESUMO

We previously showed that LIGHT and its receptor herpes virus entry mediator (HVEM) are important for development of optimal CD4(+) Th1 cell immunity and resistance to primary Leishmania major infection in mice. In this study, we further characterized the contributions of this molecule in dendritic cell (DC) maturation, initiation, and maintenance of primary immunity and secondary anti-Leishmania immunity. Flow-cytometric studies showed that CD8α(+) DC subset was mostly affected by HVEM-Ig and lymphotoxin ß receptor-Ig treatment. LIGHT signaling is required at both the priming and the maintenance stages of primary anti-Leishmania immunity but is completely dispensable during secondary immunity in wild type mice. However, LIGHT blockade led to impaired IL-12 and IFN-γ responses and loss of resistance in healed CD40-deficient mice after L. major challenge. The protective effect of LIGHT was mediated primarily via its interaction with lymphotoxin ß receptor on CD8α(+) DCs. Collectively, our results show that although LIGHT is critical for maintenance of primary Th1 response, it is dispensable during secondary anti-Leishmania immunity in the presence of functional CD40 signaling as seen in wild type mice.


Assuntos
Antígenos CD40/deficiência , Interações Hospedeiro-Patógeno/imunologia , Leishmaniose Cutânea/imunologia , Células Th1/imunologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/parasitologia , Células da Medula Óssea/patologia , Antígenos CD40/genética , Antígenos CD40/imunologia , Antígenos CD8/genética , Antígenos CD8/imunologia , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Células Dendríticas/patologia , Feminino , Regulação da Expressão Gênica , Interferon gama/genética , Interferon gama/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Leishmania major/imunologia , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Receptor beta de Linfotoxina/genética , Receptor beta de Linfotoxina/imunologia , Camundongos , Transdução de Sinais , Células Th1/parasitologia , Células Th1/patologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética
18.
PLoS One ; 9(10): e109803, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25299045

RESUMO

The obligate intracellular parasite, Toxoplasma gondii, disseminates through its host inside infected immune cells. We hypothesize that parasite nutrient requirements lead to manipulation of migratory properties of the immune cell. We demonstrate that 1) T. gondii relies on glutamine for optimal infection, replication and viability, and 2) T. gondii-infected bone marrow-derived dendritic cells (DCs) display both "hypermotility" and "enhanced migration" to an elevated glutamine gradient in vitro. We show that glutamine uptake by the sodium-dependent neutral amino acid transporter 2 (SNAT2) is required for this enhanced migration. SNAT2 transport of glutamine is also a significant factor in the induction of migration by the small cytokine stromal cell-derived factor-1 (SDF-1) in uninfected DCs. Blocking both SNAT2 and C-X-C chemokine receptor 4 (CXCR4; the unique receptor for SDF-1) blocks hypermotility and the enhanced migration in T. gondii-infected DCs. Changes in host cell protein expression following T. gondii infection may explain the altered migratory phenotype; we observed an increase of CD80 and unchanged protein level of CXCR4 in both T. gondii-infected and lipopolysaccharide (LPS)-stimulated DCs. However, unlike activated DCs, SNAT2 expression in the cytosol of infected cells was also unchanged. Thus, our results suggest an important role of glutamine transport via SNAT2 in immune cell migration and a possible interaction between SNAT2 and CXCR4, by which T. gondii manipulates host cell motility.


Assuntos
Sistemas de Transporte de Aminoácidos/genética , Células Dendríticas/parasitologia , Glutamina/metabolismo , Receptores CXCR4/genética , Toxoplasma/genética , Sistema A de Transporte de Aminoácidos , Sistemas de Transporte de Aminoácidos/imunologia , Animais , Animais Recém-Nascidos , Células da Medula Óssea/imunologia , Células da Medula Óssea/parasitologia , Células da Medula Óssea/patologia , Diferenciação Celular , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/genética , Quimiocina CXCL12/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/patologia , Fibroblastos/imunologia , Fibroblastos/parasitologia , Fibroblastos/patologia , Regulação da Expressão Gênica , Genes Reporter , Glutamina/farmacologia , Interações Hospedeiro-Patógeno , Humanos , Lipopolissacarídeos/farmacologia , Luciferases/genética , Luciferases/metabolismo , Organismos Geneticamente Modificados , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Receptores CXCR4/imunologia , Transdução de Sinais , Toxoplasma/imunologia , Toxoplasma/metabolismo
19.
Cytometry A ; 85(7): 588-600, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24804957

RESUMO

Previously, we showed that treating macrophages with ATP impairs the intracellular growth of Leishmania amazonensis, and that the P2X7 purinergic receptor is overexpressed during leishmaniasis. In the present study, we directly evaluated the effect of periodate-oxidized ATP (oATP) on parasite control in Leishmania-infected macrophages. We found that oATP impaired the attachment/entrance of L. amazonensis promastigotes to C57BL/6 mouse macrophages in a P2X7 receptor-independent manner, as macrophages from P2X7(-/-) mice were similarly affected. Although oATP directly inhibited the growth of axenic promastigotes in culture, promoted rapid ultrastructural alterations, and impaired Leishmania internalization by macrophages, it did not affect intracellular parasite multiplication. Upon infection, phagosomal acidification was diminished in oATP-treated macrophages, accompanied by reduced endosomal proteolysis. Likewise, MHC class II molecules expression and ectoATPase activity was decreased by oATP added to macrophages at the time of parasite infection. These inhibitory effects were not due to a cytotoxic effect, as no additional release of lactate dehydrogenase was detected in culture supernatants. Moreover, the capacity of macrophages to produce nitric oxide and reactive oxygen species was not affected by the presence of oATP during infection. We conclude that oATP directly affects extracellular parasite integrity and macrophage functioning.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Leishmaniose/tratamento farmacológico , Leishmaniose/imunologia , Macrófagos/imunologia , Receptores Purinérgicos P2X7/genética , Trifosfato de Adenosina/farmacologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/parasitologia , Antígenos de Histocompatibilidade Classe II/biossíntese , L-Lactato Desidrogenase/metabolismo , Leishmania/imunologia , Leishmaniose/parasitologia , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/biossíntese , Espécies Reativas de Oxigênio/metabolismo
20.
Pathog Dis ; 70(1): 17-27, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23913680

RESUMO

Dendritic cells (DCs) are the antigen-presenting cells capable of activating naïve T cells. Although CD4+ T cells are crucial for Cryptosporidium parvum clearance, little is known about the role of DCs in the immune response to this parasite. In this study, the interaction between mouse DCs and C. parvum was investigated both in vitro and in vivo. For in vitro experiments, mouse bone marrow-derived dendritic cells (BMDCs) derived from wild-type C57B1/6 or MyD88-/- or C3H/HeJ mice and DC cell line DC2.4 were pulsed with C. parvum. Active invasion of parasites was demonstrated by parasite colocalization with host cell membranes and actin-plaque formation at the site of attachment. DC activation induced by the parasite invasion was demonstrated by upregulation of costimulatory molecules CD40, CD80, and CD86, as well as inflammatory cytokines IL-12, TNF-α, and IL-6. BMDCs derived from MyD88-/- and C3H/HeJ mice failed to produce IL-12 in response to C. parvum, suggesting the importance of TLR-dependent signaling pathway specially presence of a functional TLR4 pathway, for C. parvum-induced cytokine production. In vivo experiments showed that both parasite antigens and live parasites were transported to mice mesenteric lymph nodes. All together, these data suggest that DCs play a key role in host immune responses to C. parvum and pathogenesis of the disease.


Assuntos
Criptosporidiose/imunologia , Cryptosporidium parvum/imunologia , Células Dendríticas/imunologia , Linfonodos/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/parasitologia , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/parasitologia , Criptosporidiose/parasitologia , Células Dendríticas/parasitologia , Interleucina-12/imunologia , Interleucina-6/imunologia , Linfonodos/parasitologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/imunologia
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