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1.
Nat Commun ; 11(1): 5258, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067458

RESUMO

Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identify 353 Toxoplasma genes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which are further confirmed. We show that one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages.


Assuntos
Interferon gama/imunologia , Macrófagos/imunologia , Toxoplasma/genética , Toxoplasmose/imunologia , Animais , Feminino , Genoma de Protozoário , Interações Hospedeiro-Parasita , Humanos , Interferon gama/genética , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/metabolismo , Toxoplasma/patogenicidade , Toxoplasmose/genética , Toxoplasmose/parasitologia , Virulência
2.
Exp Parasitol ; 218: 107989, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32941888

RESUMO

As the causative agent of hard-to-treat diffuse cutaneous leishmaniasis, Leishmania (L.) amazonensis persists in the host organism sheltered within large Parasitophorous Vacuoles (PVs) formed mainly in macrophages. In the present study, I present a simple and efficient method for L. amazonensis PV isolation. Isolated PVs are intact as demonstrated by the conservation of lysosomal probes loaded into PVs before the procedure. The method is useful for studies aiming at a complete and accurate molecular profile of these structures, to better understand the biogenesis of this pathogen-containing vacuole and its implication in parasite persistence and in leishmaniasis pathogenesis.


Assuntos
Leishmania mexicana/isolamento & purificação , Leishmaniose Tegumentar Difusa/parasitologia , Macrófagos/parasitologia , Animais , Humanos , Leishmania mexicana/crescimento & desenvolvimento , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Proteína 2 de Membrana Associada ao Lisossomo/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Especificidade da Espécie , Vacúolos/parasitologia
3.
PLoS Negl Trop Dis ; 14(9): e0008608, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32925918

RESUMO

The receptor Signaling Lymphocyte-Activation Molecule Family 1 (SLAMF1) controls susceptibility to Infection by the lethal Trypanosoma cruzi Y strain. To elucidate whether genetic diversity of the parasite was related with disease susceptibility, we further analyzed the role of SLAMF1 using 6 different Trypanosoma cruzi strains including Y. The interaction of SLAMF1 receptor with T. cruzi was evidenced by fluorescence microscopy, flow cytometry and quantitative PCR. All the strains, except VFRA, showed a decrease in parasite load in infected macrophages in Slamf1-/- compared to BALB/c. In macrophages gene expression NADPH oxidase (NOX2), and reactive oxygen species (ROS) production increased in Slamf1-/- compared to BALB/c in 5 out of 6 strains. However, Slamf1-/-macrophages infected with VFRA strain exhibited a divergent behavior, with higher parasite load, lower NOX2 expression and ROS production compared to BALB/c. Parasitological and immunological studies in vivo with Y strain showed that in the absence of SLAMF1 the immune response protected mice from the otherwise lethal Y infection favoring a proinflammatory response likely involving CD4, CD8, dendritic cells and classically activated macrophages. In the case of VFRA, no major changes were observed in the absence of SLAMF1. Thus, the results suggest that the T. cruzi affects SLAMF1-dependent ROS production, controlling parasite replication in macrophages and affecting survival in mice in a strain-dependent manner. Further studies will focus in the identification of parasite molecules involved in SLAMF1 interaction to explain the immunopathogenesis of the disease.


Assuntos
Macrófagos/parasitologia , Espécies Reativas de Oxigênio/metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Trypanosoma cruzi/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Doença de Chagas/imunologia , Chlorocebus aethiops , Células Dendríticas/imunologia , Suscetibilidade a Doenças/imunologia , Células HEK293 , Coração/parasitologia , Humanos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Miocárdio/patologia , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , Carga Parasitária , Células Vero
4.
PLoS Pathog ; 16(9): e1008799, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898164

RESUMO

Professional antigen-presenting cells (APCs), like macrophages (Mϕs) and dendritic cells (DCs), are central players in the induction of natural and vaccine-induced immunity to malaria, yet very little is known about the interaction of SPZ with human APCs. Intradermal delivery of whole-sporozoite vaccines reduces their effectivity, possibly due to dermal immunoregulatory effects. Therefore, understanding these interactions could prove pivotal to malaria vaccination. We investigated human APC responses to recombinant circumsporozoite protein (recCSP), SPZ and anti-CSP opsonized SPZ both in monocyte derived MoDCs and MoMϕs. Both MoDCs and MoMϕs readily took up recCSP but did not change phenotype or function upon doing so. SPZ are preferentially phagocytosed by MoMϕs instead of DCs and phagocytosis greatly increased after opsonization. Subsequently MoMϕs show increased surface marker expression of activation markers as well as tolerogenic markers such as Programmed Death-Ligand 1 (PD-L1). Additionally they show reduced motility, produce interleukin 10 and suppressed interferon gamma (IFNγ) production by antigen specific CD8+ T cells. Importantly, we investigated phenotypic responses to SPZ in primary dermal APCs isolated from human skin explants, which respond similarly to their monocyte-derived counterparts. These findings are a first step in enhancing our understanding of pre-erythrocytic natural immunity and the pitfalls of intradermal vaccination-induced immunity.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Macrófagos/imunologia , Malária/imunologia , Plasmodium berghei/imunologia , Proteínas de Protozoários/imunologia , Pele/imunologia , Esporozoítos/imunologia , Animais , Células Cultivadas , Feminino , Humanos , Macrófagos/parasitologia , Malária/parasitologia , Camundongos , Pele/parasitologia
5.
Am J Trop Med Hyg ; 103(4): 1493-1495, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32748768

RESUMO

Mucosal leishmaniasis (ML) affects predominantly the nose and occurs usually weeks or months after the cure of the primary cutaneous lesion. The pathology of ML is characterized by an exaggerated inflammatory reaction with infiltration of lymphocytes, macrophages, and plasma cells. There is also a paucity of parasites and a strong delayed-type hypersensitivity reaction. Herein, we report a case of a young man who had a large ulcer in his left leg and complained of dysphagia. In nasofibrolaryngoscopy, there were nodular lesions in the oropharynx and rhinopharynx. The skin lesion biopsy showed a chronic inflammation with amastigotes inside macrophages, and DNA of Leishmania braziliensis confirmed the diagnosis of ML in tissue biopsied from the pharynx. The leishmaniasis skin test was negative. Cytokine evaluation showed lack of production of interferon (IFN)-γ, interleukin (IL)-1ß, and IL-17 with enhancement of these cytokine levels after cure.


Assuntos
Leishmania braziliensis/isolamento & purificação , Leishmaniose Mucocutânea , Antiprotozoários/uso terapêutico , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Transtornos de Deglutição/tratamento farmacológico , Transtornos de Deglutição/etiologia , Humanos , Leishmania braziliensis/efeitos dos fármacos , Leishmaniose Mucocutânea/diagnóstico , Leishmaniose Mucocutânea/tratamento farmacológico , Leishmaniose Mucocutânea/patologia , Macrófagos/parasitologia , Masculino , Antimoniato de Meglumina/uso terapêutico , Pessoa de Meia-Idade , Nasofaringe/parasitologia , Nasofaringe/patologia , Orofaringe/parasitologia , Orofaringe/patologia , Pele/parasitologia , Pele/patologia
6.
PLoS Pathog ; 16(8): e1008327, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32853276

RESUMO

Host resistance to Toxoplasma gondii relies on CD8 T cell IFNγ responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFNγ responses to a vacuolar resident antigen of T. gondii, TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 were independent of the parasite's protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including 'avirulent' ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFNγ responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFNγ differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFNγ production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFNγ responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFNγ production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFNγ responses to a vacuolar antigen.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Inflamassomos/imunologia , Interferon gama/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Animais , Linfócitos T CD8-Positivos/parasitologia , Feminino , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas de Protozoários/genética , Toxoplasmose Animal/parasitologia , Vacúolos/imunologia , Vacúolos/metabolismo , Vacúolos/parasitologia , Virulência/imunologia
7.
PLoS Negl Trop Dis ; 14(7): e0008470, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32644998

RESUMO

BACKGROUND: Sm16, also known as SPO-1 and SmSLP, is a low molecular weight protein (~16kDa) secreted by the digenean trematode parasite Schistosoma mansoni, one of the main causative agents of human schistosomiasis. The molecule is secreted from the acetabular gland of the cercariae during skin invasion and is believed to perform an immune-suppressive function to protect the invading parasite from innate immune cell attack. METHODOLOGY/PRINCIPAL FINDINGS: We show that Sm16 homologues of the Schistosomatoidea family are phylogenetically related to the helminth defence molecule (HDM) family of immunomodulatory peptides first described in Fasciola hepatica. Interrogation of 69 helminths genomes demonstrates that HDMs are exclusive to trematode species. Structural analyses of Sm16 shows that it consists predominantly of an amphipathic alpha-helix, much like other HDMs. In S. mansoni, Sm16 is highly expressed in the cercariae and eggs but not in adult worms, suggesting that the molecule is of importance not only during skin invasion but also in the pro-inflammatory response to eggs in the liver tissues. Recombinant Sm16 and a synthetic form, Sm16 (34-117), bind to macrophages and are internalised into the endosomal/lysosomal system. Sm16 (34-117) elicited a weak pro-inflammatory response in macrophages in vitro but also suppressed the production of bacterial lipopolysaccharide (LPS)-induced inflammatory cytokines. Evaluation of the transcriptome of human macrophages treated with a synthetic Sm16 (34-117) demonstrates that the peptide exerts significant immunomodulatory effects alone, as well as in the presence of LPS. Pathways most significantly influenced by Sm16 (34-117) were those involving transcription factors peroxisome proliferator-activated receptor (PPAR) and liver X receptors/retinoid X receptor (LXR/RXR) which are intricately involved in regulating the cellular metabolism of macrophages (fatty acid, cholesterol and glucose homeostasis) and are central to inflammatory responses. CONCLUSIONS/SIGNIFICANCE: These results offer new insights into the structure and function of a well-known immunomodulatory molecule, Sm16, and places it within a wider family of trematode-specific small molecule HDM immune-modulators with immuno-biotherapeutic possibilities.


Assuntos
Antígenos de Helmintos/metabolismo , Proteínas de Helminto/metabolismo , Schistosoma mansoni/metabolismo , Animais , Anticorpos Anti-Helmínticos , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Células da Medula Óssea , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Proteínas de Helminto/genética , Humanos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Óvulo , Filogenia , Transporte Proteico
8.
PLoS Negl Trop Dis ; 14(7): e0008396, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32722702

RESUMO

The parasitophorous vacuoles (PVs) that insulate Leishmania spp. in host macrophages are vacuolar compartments wherein promastigote forms differentiate into amastigote that are the replicative form of the parasite and are also more resistant to host responses. We revisited the biogenesis of tight-fitting PVs that insulate L. infantum in promastigote-infected macrophage-like RAW 264.7 cells by time-dependent confocal laser multidimensional imaging analysis. Pharmacological disassembly of the cellular microtubule network and silencing of the dynein gene led to an impaired interaction of L. infantum-containing phagosomes with late endosomes and lysosomes, resulting in the tight-fitting parasite-containing phagosomes never transforming into mature PVs. Analysis of the shape of the L. infantum parasite within PVs, showed that factors that impair promastigote-amastigote differentiation can also result in PVs whose maturation is arrested. These findings highlight the importance of the MT-dependent interaction of L. infantum-containing phagosomes with the host macrophage endolysosomal pathway to secure the intracellular fate of the parasite.


Assuntos
Leishmania infantum/fisiologia , Leishmaniose Visceral/parasitologia , Macrófagos/parasitologia , Microtúbulos/parasitologia , Animais , Endossomos/metabolismo , Humanos , Leishmania infantum/crescimento & desenvolvimento , Leishmaniose Visceral/metabolismo , Camundongos , Microtúbulos/metabolismo , Fagossomos/metabolismo , Células RAW 264.7
9.
Exp Parasitol ; 216: 107939, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32535115

RESUMO

Gaucher disease is a lysosomal storage disease in which a genetic deficiency in ß-glucocerebrosidase leads to the accumulation of glycosphingolipids in lysosomes. Macrophages are amongst the cells most severely affected in Gaucher disease patients. One phenotype associated with Gaucher macrophages is the impaired capacity to fight bacterial infections. Here, we investigate whether inhibition of ß-glucocerebrosidase activity affects the capacity of macrophages to phagocytose and act on the early containment of human pathogens of the genus Leishmania. Towards our aim, we performed in vitro infection assays on macrophages derived from the bone marrow of C57BL/6 mice. To mimic Gaucher disease, macrophages were incubated with the ß-glucocerebrosidase inhibitor, conduritol B epoxide (CBE), prior to contact with Leishmania. This treatment guaranteed that ß-glucocerebrosidase was fully inhibited during the contact of macrophages with Leishmania, its enzymatic activity being progressively recovered along the 48 h that followed removal of the inhibitor. Infections were performed with L. amazonensis, L. infantum, or L. major, so as to explore potential species-specific responses in the context of ß-glucocerebrosidase inactivation. Parameters of infection, recorded immediately after phagocytosis, as well as 24 and 48 h later, revealed no noticeable differences in the infection parameters of CBE-treated macrophages relative to non-treated controls. We conclude that blocking ß-glucocerebrosidase activity during contact with Leishmania does not interfere with the phagocytic capacity of macrophages and the early onset of leishmanicidal responses.


Assuntos
Glucosilceramidase/antagonistas & inibidores , Leishmania/fisiologia , Macrófagos/parasitologia , Fagocitose , Animais , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Doença de Gaucher/complicações , Doença de Gaucher/fisiopatologia , Glucosilceramidase/efeitos dos fármacos , Glucosilceramidase/genética , Inositol/análogos & derivados , Inositol/farmacologia , Leishmania infantum/fisiologia , Leishmania major/fisiologia , Leishmania mexicana/fisiologia , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Fagocitose/efeitos dos fármacos
10.
PLoS Pathog ; 16(6): e1008291, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32479529

RESUMO

The protozoan parasite Leishmania donovani (L. donovani) causes visceral leishmaniasis, a chronic infection which is fatal when untreated. Herein, we investigated whether in addition to altering transcription, L. donovani modulates host mRNA translation to establish a successful infection. Polysome-profiling revealed that one third of protein-coding mRNAs expressed in primary mouse macrophages are differentially translated upon infection with L. donovani promastigotes or amastigotes. Gene ontology analysis identified key biological processes enriched for translationally regulated mRNAs and were predicted to be either activated (e.g. chromatin remodeling and RNA metabolism) or inhibited (e.g. intracellular trafficking and antigen presentation) upon infection. Mechanistic in silico and biochemical analyses showed selective activation mTOR- and eIF4A-dependent mRNA translation, including transcripts encoding central regulators of mRNA turnover and inflammation (i.e. PABPC1, EIF2AK2, and TGF-ß). L. donovani survival within macrophages was favored under mTOR inhibition but was dampened by pharmacological blockade of eIF4A. Overall, this study uncovers a vast yet selective reprogramming of the host cell translational landscape early during L. donovani infection, and suggests that some of these changes are involved in host defense mechanisms while others are part of parasite-driven survival strategies. Further in vitro and in vivo investigation will shed light on the contribution of mTOR- and eIF4A-dependent translational programs to the outcome of visceral leishmaniasis.


Assuntos
Fator de Iniciação 4A em Eucariotos/metabolismo , Leishmania donovani/metabolismo , Leishmaniose Visceral , Macrófagos , Biossíntese de Proteínas , RNA/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/patologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Macrófagos/patologia , Camundongos
11.
Nucleic Acids Res ; 48(11): 6081-6091, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32402089

RESUMO

Herein, we characterize the cellular uptake of a DNA structure generated by rolling circle DNA amplification. The structure, termed nanoflower, was fluorescently labeled by incorporation of ATTO488-dUTP allowing the intracellular localization to be followed. The nanoflower had a hydrodynamic diameter of approximately 300 nanometer and was non-toxic for all mammalian cell lines tested. It was internalized specifically by mammalian macrophages by phagocytosis within a few hours resulting in specific compartmentalization in phagolysosomes. Maximum uptake was observed after eight hours and the nanoflower remained stable in the phagolysosomes with a half-life of 12 h. Interestingly, the nanoflower co-localized with both Mycobacterium tuberculosis and Leishmania infantum within infected macrophages although these pathogens escape lysosomal degradation by affecting the phagocytotic pathway in very different manners. These results suggest an intriguing and overlooked potential application of DNA structures in targeted treatment of infectious diseases such as tuberculosis and leishmaniasis that are caused by pathogens that escape the human immune system by modifying macrophage biology.


Assuntos
DNA/química , DNA/metabolismo , Leishmania infantum/metabolismo , Macrófagos/microbiologia , Macrófagos/parasitologia , Mycobacterium tuberculosis/metabolismo , Fagossomos/metabolismo , DNA/análise , Replicação do DNA , Fluorescência , Meia-Vida , Humanos , Leishmaniose/terapia , Macrófagos/citologia , Macrófagos/imunologia , Nanoestruturas/análise , Nanoestruturas/química , Técnicas de Amplificação de Ácido Nucleico , Fagocitose , Fagossomos/química , Fagossomos/microbiologia , Fagossomos/parasitologia , Tuberculose/terapia
12.
PLoS Pathog ; 16(5): e1008572, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32413093

RESUMO

The apicomplexan Toxoplasma gondii induces strong protective immunity dependent upon recognition by Toll-like receptors (TLR)11 and 12 operating in conjunction with MyD88 in the murine host. However, TLR11 and 12 proteins are not present in humans, inspiring us to investigate MyD88-independent pathways of resistance. Using bicistronic IL-12-YFP reporter mice on MyD88+/+ and MyD88-/- genetic backgrounds, we show that CD11c+MHCII+F4/80- dendritic cells, F4/80+ macrophages, and Ly6G+ neutrophils were the dominant cellular sources of IL-12 in both wild type and MyD88 deficient mice after parasite challenge. Parasite dense granule protein GRA24 induces p38 MAPK activation and subsequent IL-12 production in host macrophages. We show that Toxoplasma triggers an early and late p38 MAPK phosphorylation response in MyD88+/+ and MyD88-/- bone marrow-derived macrophages. Using the uracil auxotrophic Type I T. gondii strain cps1-1, we demonstrate that the late response does not require active parasite proliferation, but strictly depends upon GRA24. By i. p. inoculation with cps1-1 and cps1-1:Δgra24, we identified unique subsets of chemokines and cytokines that were up and downregulated by GRA24. Finally, we demonstrate that cps1-1 triggers a strong host-protective GRA24-dependent Th1 response in the absence of MyD88. Our data identify GRA24 as a major mediator of p38 MAPK activation, IL-12 induction and protective immunity that operates independently of the TLR/MyD88 cascade.


Assuntos
Interleucina-12/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Interleucina-12/genética , Sistema de Sinalização das MAP Quinases/genética , Macrófagos/parasitologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasmose/genética , Toxoplasmose/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética
13.
PLoS Pathog ; 16(5): e1008586, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453782

RESUMO

The murine innate immune response against Toxoplasma gondii is predominated by the interaction of TLR11/12 with Toxoplasma profilin. However, mice lacking Tlr11 or humans, who do not have functional TLR11 or TLR12, still elicit a strong innate immune response upon Toxoplasma infection. The parasite factors that determine this immune response are largely unknown. Herein, we investigated two dense granule proteins (GRAs) secreted by Toxoplasma, GRA15 and GRA24, for their role in stimulating the innate immune response in Tlr11-/- mice and in human cells, which naturally lack TLR11/TLR12. Our results show that GRA15 and GRA24 synergistically shape the early immune response and parasite virulence in Tlr11-/- mice, with GRA15 as the predominant effector. Nevertheless, acute virulence in Tlr11-/- mice is still dominated by allelic combinations of ROP18 and ROP5, which are effectors that determine evasion of the immunity-related GTPases. In human macrophages, GRA15 and GRA24 play a major role in the induction of IL12, IL18 and IL1ß secretion. We further show that GRA15/GRA24-mediated IL12, IL18 and IL1ß secretion activates IFNγ secretion by peripheral blood mononuclear cells (PBMCs), which controls Toxoplasma proliferation. Taken together, our study demonstrates the important role of GRA15 and GRA24 in activating the innate immune response in hosts lacking TLR11.


Assuntos
Imunidade Inata/imunologia , Macrófagos/imunologia , Proteínas de Protozoários/imunologia , Receptores Toll-Like/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Animais , Citocinas/genética , Citocinas/imunologia , Humanos , Imunidade Inata/genética , Macrófagos/parasitologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Células RAW 264.7 , Receptores Toll-Like/genética , Toxoplasmose/genética , Toxoplasmose/patologia
14.
Parasite Immunol ; 42(6): e12719, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32248547

RESUMO

AIMS: Visceral leishmaniasis (VL), caused by Leishmania donovani in India, is fatal if untreated, having serious concern of limited chemotherapeutic options. In this study, we evaluated antileishmanial efficacy of purified chlorogenic acid (CGA) against promastigotes and intracellular amastigotes infected into RAW264.7 macrophages. METHODS AND RESULTS: Chlorogenic acid was effective both on promastigotes (IC50  = 78.394 µmol/L, i.e. 27.75 µg/mL) and intracellular amastigotes (ED50  = 26.752 µmol/L, i.e. 9.47 µg/mL). In promastigotes, significant retardation in mitotic growth was caused both by cell-death and reduction of metabolic activity, evidenced by propidium-iodide uptake and MTT assay, respectively. Flow cytometric analysis revealed that retardation of mitotic growth was due to cell-cycle arrest at G1/S checkpoint. Complete clearance of amastigotes from infected RAW264.7 cells, assessed by microscopic counting, was achieved with 60 µmol/L (21.24 µg/mL) CGA for 24 hours, with negligible toxicity to host macrophages. This parasite clearing efficacy was comparable to 1.0 µg/mL (1.082 µmol/L) Amphotericin B, and 20 µmol/L Miltefosine, two standard antileishmanial drugs. Cytokine-ELISA revealed that elevated IL-10 production by infected macrophages was reduced after parasite clearance. Consequently, IL-12, TNF and NO (assayed by Griess test) production by macrophages were significantly increased after successful resolution of infection. CONCLUSION: Chlorogenic acid might emerge as a potential antileishmanial drug.


Assuntos
Antiprotozoários/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Ácido Clorogênico/uso terapêutico , Citocinas/metabolismo , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Óxido Nítrico/metabolismo , Animais , Linhagem Celular , Índia , Leishmaniose Visceral/mortalidade , Leishmaniose Visceral/parasitologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Fosforilcolina/análogos & derivados , Fosforilcolina/uso terapêutico , Células RAW 264.7
15.
Parasit Vectors ; 13(1): 183, 2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32268913

RESUMO

BACKGROUND: Angiostrongylus cantonensis can cause severe symptoms of central nervous system infections. In the host, this parasite localizes in the blood and cerebrospinal fluid, and its secreted components can impact immune responses. Our previous study demonstrated that immune responses were inhibited in A. cantonensis-infected mice immunized with Ac-Galectin-1 (AcGal-1). However, the mechanisms by which AcGal-1 regulates the immune responses remain unclear. Macrophages are innate immune cells that rapidly respond to infection. The direct impact of AcGal-1 on macrophages may affect the immune responses. METHODS: AcGal-1 protein was purified by nickel ion affinity chromatography. The effect of AcGal-1 on the apoptosis of macrophages was detected using CCK-8 assay, flow cytometry and western blot. Macrophage membrane proteins bound to AcGal-1 were obtained using the His-tag-based pull-down assay and identified via mass spectrometry. Co-localization of AcGal-1 and the macrophage membrane protein Annexin A2 was observed by immunofluorescence microscopy, and their interaction was validated by co-immunoprecipitation experiments. SiRNA-mediated knockdown of Annexin A2 was used to determine if AcGal-1-induced macrophage apoptosis required interaction with Annexin A2. The phosphorylation level of apoptotic signal pathway protein was detected by phospho-antibody microarray and western blot. RESULTS: Our study showed that AcGal-1 caused apoptosis of the macrophages. AcGal-1 increased the expression of apoptosis proteins caspase-3, caspase-9, Bax, but reduced the expression of anti-apoptosis protein Bcl-2. AcGal-1 interacted with the membrane protein Annexin A2, and knockdown of Annexin A2 expression increased Bcl-2 but decreased Bax levels in AcGal-1-treated cells. Moreover, AcGal-1 increased JNK phosphorylation and the inhibition of JNK phosphorylation in AcGal-1-treated cells decreased the expression of caspase-3, -9, Bax and almost restored Bcl-2 to the level observed in control cells. CONCLUSIONS: AcGal-1 can induce the apoptosis of macrophages by binding to Annexin A2 and activating JNK downstream the apoptotic signaling pathway.


Assuntos
Anexina A2/imunologia , Apoptose , Galectina 1/imunologia , Sistema de Sinalização das MAP Quinases , Macrófagos/parasitologia , Angiostrongylus cantonensis , Animais , Proliferação de Células , Técnicas de Silenciamento de Genes , Humanos , Macrófagos/imunologia , Ligação Proteica , RNA Interferente Pequeno , Células THP-1
16.
PLoS Negl Trop Dis ; 14(4): e0008167, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32275661

RESUMO

Leishmania donovani, an intracellular protozoan parasite upon infection, encounters a range of antimicrobial factors within the host cells. Consequently, the parasite has evolved mechanisms to evade this hostile defense system through inhibition of macrophage activation that, in turn, enables parasite replication and survival. There is growing evidence that epigenetic down-regulation of the host genome by intracellular pathogens leads to acute infection. Epigenetic modification is mediated by chromatin remodeling, histone modifications, or DNA methylation. Histone deacetylases (HDACs) removes acetyl groups from lysine residues on histones, thereby leading to chromatin remodeling and gene silencing. Here, using L. donovani infected macrophages differentiated from THP-1 human monocytic cells, we report a link between host chromatin modifications, transcription of defense genes and intracellular infection with L. donovani. Infection with L. donovani led to the silencing of host defense gene expression. Histone deacetylase 1 (HDAC1) transcript levels, protein expression, and enzyme activity showed a significant increase upon infection. HDAC1 occupancy at the promoters of the defense genes significantly increased upon infection, which in turn resulted in decreased histone H3 acetylation in infected cells, resulting in the down-regulation of mRNA expression of host defense genes. Small molecule mediated inhibition and siRNA mediated down-regulation of HDAC1 increased the expression levels of host defense genes. Interestingly, in this study, we demonstrate that the silencing of HDAC1 by both siRNA and pharmacological inhibitors resulted in decreased intracellular parasite survival. The present data not only demonstrate that up-regulation of HDAC1 and epigenetic silencing of host cell defense genes is essential for L. donovani infection but also provides novel therapeutic strategies against leishmaniasis.


Assuntos
Citoplasma/metabolismo , Epigênese Genética , Histona Desacetilase 1/genética , Leishmania donovani/patogenicidade , Leishmaniose/genética , Macrófagos/parasitologia , Linhagem Celular , Montagem e Desmontagem da Cromatina , Citoplasma/parasitologia , Metilação de DNA , Regulação para Baixo , Regulação da Expressão Gênica , Inativação Gênica , Histona Desacetilase 1/metabolismo , Histonas/genética , Histonas/metabolismo , Interações Hospedeiro-Parasita/genética , Humanos , Monócitos/metabolismo , Monócitos/parasitologia , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Células THP-1
17.
PLoS Negl Trop Dis ; 14(4): e0008188, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32275665

RESUMO

Leishmaniasis is one of the Neglected Tropical Diseases (NTDs) which is closely associated with poverty and has gained much relevance recently due to its opportunistic coinfection with HIV. It is a protozoan zoonotic disease transmitted by a dipteran Phlebotomus, Lutzomyia/ Sergentomyia sandfly; during blood meals on its vertebrate intermediate hosts. It is a four-faceted disease with its visceral form being more deadly if left untreated. It is endemic across the tropics and sub-tropical regions of the world. It can be considered the third most important NTD after malaria and lymphatic filariasis. Currently, there are numerous drawbacks on the fight against leishmaniasis which includes: non-availability of vaccines, limited availability of drugs, high cost of mainstay drugs and parasite resistance to current treatments. In this study, we screened the antileishmanial activity, selectivity, morphological alterations, cell cycle progression and apoptotic potentials of six Pathogen box compounds from Medicine for Malaria Venture (MMV) against Leishmania donovani promastigotes and amastigotes. From this study, five of the compounds showed great promise as lead chemotherapeutics based on their high selectivity against the Leishmania donovani parasite when tested against the murine mammalian macrophage RAW 264.7 cell line (with a therapeutic index ranging between 19-914 (promastigotes) and 1-453 (amastigotes)). The cell cycle progression showed growth arrest at the G0-G1 phase of mitotic division, with an indication of apoptosis induced by two (2) of the pathogen box compounds tested. Our findings present useful information on the therapeutic potential of these compounds in leishmaniasis. We recommend further in vivo studies on these compounds to substantiate observations made in the in vitro study.


Assuntos
Antiprotozoários/farmacologia , Desenvolvimento de Medicamentos , Leishmania donovani/efeitos dos fármacos , Anfotericina B/farmacologia , Animais , Apoptose/efeitos dos fármacos , Concentração Inibidora 50 , Cinética , Leishmania donovani/crescimento & desenvolvimento , Macrófagos/parasitologia , Camundongos , Microscopia de Fluorescência , Células RAW 264.7
18.
Parasite Immunol ; 42(9): e12718, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32249437

RESUMO

AIM: To characterize several anti-Leishmania tropica nanobodies and to investigate their effect on Leishmania infection. METHODS: Several immunological tests were implied to characterize five different (as confirmed by sequencing) anti-L tropica nanobodies (NbLt05, NbLt06, NbLt14, NbLt24 and NbLt36) against parasite lysates or intact cells from different stages, promastigotes and amastigotes. Direct inhibitory effect of these nanobodies on parasite infection cycle on macrophages was tested in cell culture. RESULTS: All the five nanobodies (with distinguished characteristics) were more specific to L tropica than to L major, but could equally recognize the lysate and the outer surface of the intact cells from the two main stages of the parasite. Nanobodies recognized several leishmania antigens (majorly between 75 and 63 kDa), and their proteinaceous nature was confirmed. Because of its role in leishmania life cycle, gp63 was considered a potential antigen candidate for nanobodies, and bioinformatics predicted such interaction. All nanobodies have a negative effect on the infectivity of L tropica, as they decreased the number of infected macrophages and the amastigotes inside those macrophages. CONCLUSION: Such anti-leishmania nanobodies, with outstanding characteristics and important target, can be of great use in the development of promising treatment strategies against leishmaniasis.


Assuntos
Camelus/imunologia , Cadeias Pesadas de Imunoglobulinas/uso terapêutico , Leishmania tropica , Leishmaniose/terapia , Anticorpos de Domínio Único/uso terapêutico , Animais , Células Cultivadas , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Leishmania tropica/efeitos dos fármacos , Leishmania tropica/crescimento & desenvolvimento , Leishmania tropica/imunologia , Leishmaniose/imunologia , Estágios do Ciclo de Vida , Macrófagos/imunologia , Macrófagos/parasitologia , Anticorpos de Domínio Único/imunologia
19.
J Vis Exp ; (156)2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32150165

RESUMO

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to visceral lesions. Currently, 12 million people are estimated to be infected with Leishmania worldwide and over 1 billion people live at the risk of infection. Leishmania amazonensis is endemic in Central and South America and usually leads to the cutaneous form of the disease, which can be directly visualized in an animal model. Therefore, L. amazonensis strains are good models for cutaneous leishmaniasis studies because they are also easily cultivated in vitro. C57BL/6 mice mimic the L. amazonensis-driven disease progression observed in humans and are considered one of the best mice strains model for cutaneous leishmaniasis. In the vertebrate host, these parasites inhabit macrophages despite the defense mechanisms of these cells. Several studies use in vitro macrophage infection assays to evaluate the parasite infectivity under different conditions. However, the in vitro approach is limited to an isolated cell system that disregards the organism's response. Here, we compile an in vivo murine infection method that provides a systemic physiological overview of the host-parasite interaction. The detailed protocol for the in vivo infection of C57BL/6 mice with L. amazonensis comprises parasite differentiation into infective amastigotes, mice footpad cutaneous inoculation, lesion development, and parasite load determination. We propose this well-established method as the most adequate method for physiological studies of the host immune and metabolic responses to cutaneous leishmaniasis.


Assuntos
Modelos Animais de Doenças , Interações Hospedeiro-Parasita/imunologia , Leishmania/imunologia , Leishmania/patogenicidade , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Virulência , Animais , Feminino , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL
20.
Parasit Vectors ; 13(1): 94, 2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32085719

RESUMO

BACKGROUND: New therapeutic drugs are urgently needed against visceral leishmaniasis because current drugs, such as pentavalent antimonials and miltefosine, produce severe side effects and development of resistance. Whether cyclosporine A (CsA) and its derivatives can be used as therapeutic drugs for visceral leishmaniasis has been controversial for many years. METHODS: In this study, we evaluated the efficacy of CsA and its derivative, dihydrocyclosporin A (DHCsA-d), against promastigotes and intracellular amastigotes of Leishmania donovani. Sodium stibogluconate (SSG) was used as a positive control. RESULTS: Our results showed that DHCsA-d was able to inhibit the proliferation of L. donovani promastigotes (IC50: 21.24 µM and 12.14 µM at 24 h and 48 h, respectively) and intracellular amastigotes (IC50: 5.23 µM and 4.84 µM at 24 and 48 h, respectively) in vitro, but CsA treatment increased the number of amastigotes in host cells. Both DHCsA-d and CsA caused several alterations in the morphology and ultrastructure of L. donovani, especially in the mitochondria. However, DHCsA-d showed high cytotoxicity towards cells of the mouse macrophage cell line RAW264.7, with CC50 values of 7.98 µM (24 h) and 6.65 µM (48 h). Moreover, DHCsA-d could increase IL-12, TNF-α and IFN-γ production and decrease the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. On the contrary, CsA decreased IL-12, TNF-α, and IFN-γ production and increased the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. The expression of L. donovani cyclophilin A (LdCyPA) in promastigotes and intracellular amastigotes and the expression of cyclophilin A (CyPA) in RAW 264.7 cells were found to be significantly downregulated in the CsA-treated group compared to those in the untreated group. However, no significant changes in LdCyPA and CyPA levels were found after DHCsA-d or SSG treatment. CONCLUSIONS: Our findings initially resolved the dispute regarding the efficacy of CsA and DHCsA-d for visceral leishmaniasis treatment. CsA showed no significant inhibitory effect on intracellular amastigotes. DHCsA-d significantly inhibited promastigotes and intracellular amastigotes, but it was highly cytotoxic. Therefore, CsA and DHCsA-d are not recommended as antileishmanial drugs.


Assuntos
Antiprotozoários/farmacologia , Ciclosporina/farmacologia , Ciclosporinas/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/parasitologia , Animais , Avaliação Pré-Clínica de Medicamentos , Humanos , Interferon gama/imunologia , Interleucina-10/imunologia , Interleucina-2/imunologia , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/fisiologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/imunologia , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Células RAW 264.7
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