RESUMO
Neutrophil extracellular traps (NETs) evolved as a unique effector mechanism contributing to resistance against infection that can also promote tissue damage in inflammatory conditions. Malaria infection can trigger NET release, but the mechanisms and consequences of NET formation in this context remain poorly characterized. Here we show that patients suffering from severe malaria had increased amounts of circulating DNA and increased neutrophil elastase (NE) levels in plasma. We used cultured erythrocytes and isolated human neutrophils to show that Plasmodium-infected red blood cells release macrophage migration inhibitory factor (MIF), which in turn caused NET formation by neutrophils in a mechanism dependent on the C-X-C chemokine receptor type 4 (CXCR4). NET production was dependent on histone citrullination by peptidyl arginine deiminase-4 (PAD4) and independent of reactive oxygen species (ROS), myeloperoxidase (MPO) or NE. In vitro, NETs functioned to restrain parasite dissemination in a mechanism dependent on MPO and NE activities. Finally, C57/B6 mice infected with P. berghei ANKA, a well-established model of cerebral malaria, presented high amounts of circulating DNA, while treatment with DNAse increased parasitemia and accelerated mortality, indicating a role for NETs in resistance against Plasmodium infection.
Assuntos
Eritrócitos/imunologia , Armadilhas Extracelulares/imunologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Malária/imunologia , Neutrófilos/imunologia , Plasmodium/imunologia , Receptores CXCR4/metabolismo , Animais , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/parasitologia , Humanos , Malária/metabolismo , Malária/parasitologia , Malária/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Neutrófilos/parasitologia , Parasitemia/imunologia , Parasitemia/metabolismo , Parasitemia/parasitologia , Parasitemia/patologiaRESUMO
Visceral leishmaniasis is a chronic disease that affects humans and dogs as well. Dogs, the domestic reservoir of Leishmania, play a central role in the transmission of visceral leishmaniasis, the most severe form of this disease. Neutrophils are the most abundant leukocytes in blood and interact with the parasite after infection. Here, we evaluate the effector properties of neutrophils from healthy and naturally Leishmania infantum-infected dogs. Our results showed that the parasite induced neutrophil extracellular trap (NET) release from neutrophils in both groups. Additionally, phagocytosis and NETs contributed differently to parasite killing by neutrophils from healthy and infected animals, and IFN-γ, IL-8, IL-4 and TNF-α production by neutrophils from both groups were differentially modulated by the parasite. Our results contribute to a better understanding of the complex role played by neutrophils in canine visceral leishmaniasis, which may favor the development of more effective therapies.
Assuntos
Leishmania infantum/patogenicidade , Leishmaniose Visceral/veterinária , Neutrófilos/parasitologia , Animais , Doenças do Cão/sangue , Doenças do Cão/parasitologia , Cães , Armadilhas Extracelulares/parasitologia , Feminino , Interferon gama/metabolismo , Interleucina-4/metabolismo , Interleucina-8/metabolismo , Leishmaniose Visceral/sangue , Masculino , Neutrófilos/metabolismo , Fagocitose , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Leishmaniasis is one of the most significant of the neglected tropical diseases, with 350 million people in 98 countries worldwide living at risk of developing one of the many forms of the disease. During the transmission of the parasite from its vector to the vertebrate host, neutrophils are rapidly recruited to the site of the sandfly bite. Using different strategies, neutrophils can often kill a large number of parasites. However, some parasites can resist neutrophil-killing mechanisms and survive until macrophage arrival at the infection site. One of the strategies for neutrophil-mediated killing is the production of neutrophil extracellular traps (NETs). Because of its ecto-localized nuclease activity, the enzyme 3'-nucleotidase/nuclease (3'NT/NU), present in different Leishmania species, was recently identified as part of a possible parasite escape mechanism from NET-mediated death. Previous studies showed that 3'NT/NU also plays an important role in the establishment of Leishmania infection by generating extracellular adenosine that favors the parasite and macrophage interaction. This study aims to deepen the knowledge about 3'NT/NU, mainly with respect to its nuclease activity that is little studied in the current literature. For this, we cloned, expressed and purified the recombinant La3'NT/NU and have confirmed its contribution to the parasite escape from NET-mediated killing.
Assuntos
Desoxirribonucleases/imunologia , Armadilhas Extracelulares/imunologia , Leishmania/enzimologia , Leishmaniose/imunologia , Neutrófilos/imunologia , Nucleotidases/imunologia , Proteínas de Protozoários/imunologia , Clonagem Molecular , Desoxirribonucleases/genética , Armadilhas Extracelulares/parasitologia , Humanos , Leishmania/genética , Leishmania/imunologia , Leishmaniose/parasitologia , Nucleotidases/genética , Proteínas de Protozoários/genéticaRESUMO
Neutrophil extracellular traps (NETs) are DNA fibers decorated with histones and antimicrobial proteins from cytoplasmic granules released into the extracellular space in a process denominated NETosis. The molecular pathways involved in NETosis have not been completely understood. Classical NETosis mechanisms involve the neutrophil elastase (NE) translocation to nucleus due to the generation of reactive oxygen species (ROS) by NADPH oxidase (NOX2) or the peptidyl arginine deiminase 4 (PAD4) activation in response to an increase in extracellular calcium influx; both mechanisms result in DNA decondensation. Previously, we reported that trophozoites and lipopeptidophosphoglycan from Entamoeba histolytica trigger NET release in human neutrophils. Here, we demonstrated in a quantitative manner that NETs were rapidly form upon treatment with amoebic trophozoites and involved both nuclear and mitochondrial DNA (mtDNA). NETs formation depended on amoeba viability as heat-inactivated or paraformaldehyde-fixed amoebas were not able to induce NETs. Interestingly, ROS were not detected in neutrophils during their interaction with amoebas, which could explain why NOX2 inhibition using apocynin did not affect this NETosis. Surprisingly, whereas calcium chelation reduced NET release induced by amoebas, PAD4 inhibition by GSK484 failed to block DNA extrusion but, as expected, abolished NETosis induced by the calcium ionophore A23187. Additionally, NE translocation to the nucleus and serine-protease activity were necessary for NET release caused by amoeba. These data support the idea that E. histolytica trophozoites trigger NETosis by a rapid non-classical mechanism and that different mechanisms of NETs release exist depending on the stimuli used.
Assuntos
Entamoeba histolytica/metabolismo , Entamebíase/metabolismo , Armadilhas Extracelulares/metabolismo , NADPH Oxidases/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trofozoítos/metabolismo , Acetofenonas/antagonistas & inibidores , Apoptose , Cálcio/metabolismo , DNA/efeitos dos fármacos , DNA/metabolismo , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Entamebíase/parasitologia , Armadilhas Extracelulares/parasitologia , Humanos , Elastase de Leucócito/metabolismo , Viabilidade Microbiana , Mitocôndrias/genética , Mitocôndrias/metabolismo , NADPH Oxidases/efeitos dos fármacos , Necrose , Neutrófilos/metabolismo , Neutrófilos/parasitologia , Oxirredução/efeitos dos fármacos , Peptidoglicano/metabolismo , Fosfolipídeos/metabolismo , Proteína-Arginina Desiminase do Tipo 4 , Inibidores de Serina Proteinase/metabolismo , Trofozoítos/genéticaRESUMO
Leishmania (Viannia) panamensis (L. (V.) p.) is the main causative agent of cutaneous leishmaniasis in Colombia and is usually treated with either meglumine antimoniate (MA) or miltefosine (MIL). In recent years, there has been increasing evidence of the emergence of drug-resistance against these compounds. Neutrophils are known to play an important role in immunity against Leishmania. These cells are rapidly recruited upon infection and are also present in chronic lesions. However, their involvement in the outcome of infection with drug-resistant Leishmania has not been examined. In this study, human and murine neutrophils were infected in vitro with MA or MIL drug-resistant L. (V.) p. lines derived from a parental L. (V.) p. drug-susceptible strain. Neutrophil effector functions were assessed analyzing the production of reactive oxygen species (ROS), the formation of neutrophil extracellular trap (NET) and the expression of cell surface activation markers. Parasite killing by neutrophils was assessed using L. (V.) p. transfected with a luciferase reporter. We show here that MA and MIL-resistant L. (V.) p. lines elicited significantly increased NET formation and MA-resistant L. (V.) p. induced significantly increased ROS production in both murine and human neutrophils, compared to infections with the parental MIL and MA susceptible strain. Furthermore, neutrophils exposed to drug-resistant lines showed increased activation, as revealed by decreased expression of CD62L and increased expression of CD66b in human neutrophils yet presented higher survival within neutrophils than the drug-susceptible strain. These results provide evidence that parasite drug-susceptibility may influences neutrophil activation and function as well as parasite survival within neutrophils. Further investigaton of the inter-relationship of drug susceptibility and neutrophil effector function should contribute to better understanding of the factors involved in susceptibility to anti-Leishmania drugs.
Assuntos
Antiprotozoários/farmacologia , Resistência a Medicamentos/imunologia , Leishmania guyanensis/imunologia , Leishmaniose Cutânea/tratamento farmacológico , Neutrófilos/imunologia , Animais , Antiprotozoários/uso terapêutico , Células Cultivadas , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/parasitologia , Humanos , Leishmania guyanensis/efeitos dos fármacos , Leishmania guyanensis/isolamento & purificação , Leishmaniose Cutânea/sangue , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Antimoniato de Meglumina/farmacologia , Antimoniato de Meglumina/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Neutrófilos/parasitologia , Testes de Sensibilidade Parasitária , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêutico , Cultura Primária de Células , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Upon in vitro stimulation, neutrophils undergo a cell death named netosis. This process is characterized by extracellular release of chromatin scaffold associated with granular and cytoplasmic proteins, which together, ensnare and kill microbes. We have previously described that interaction of Leishmania amazonensis with human neutrophils leads to the release of neutrophil extracellular traps, which trap and kill the parasite. However, the signaling leading to Leishmania induced netosis is still unknown. Thus, we sought to evaluate signaling events that drive L. amazonensis induced neutrophil extracellular trap release from human neutrophils. Here, we found that PI3K, independently of protein kinase B, has a role in parasite-induced netosis. We also described that the main isoforms involved are PI3Kγ and PI3Kδ, which work in reactive oxygen species-dependent and -independent ways, respectively. We demonstrated that activation of ERK downstream of PI3Kγ is important to trigger reactive oxygen species-dependent, parasite-induced netosis. Pharmacological inhibition of protein kinase C also significantly decreased parasite-induced neutrophil extracellular trap release. Intracellular calcium, regulated by PI3Kδ, represents an alternative reactive oxygen species-independent pathway of netosis stimulated by L. amazonensis Finally, intracellular calcium mobilization and reactive oxygen species generation are the major regulators of parasite-induced netosis. Our results contribute to a better understanding of the signaling behind netosis induced by interactions between Leishmania and neutrophils.
Assuntos
Sinalização do Cálcio/fisiologia , Classe I de Fosfatidilinositol 3-Quinases/fisiologia , Classe Ib de Fosfatidilinositol 3-Quinase/fisiologia , Armadilhas Extracelulares/parasitologia , Leishmania mexicana/imunologia , Sistema de Sinalização das MAP Quinases , Neutrófilos/imunologia , Proteína Quinase C/fisiologia , Cromatina/ultraestrutura , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Humanos , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Neutrophil extracellular traps (NETs) have been described as a network of extracellular fibers composed by DNA, histones and various proteins/enzymes. Studies have demonstrated that NETs could be responsible for the trapping and elimination of a variety of infectious agents. In order to verify the presence of NETs in American tegumentary leishmaniasis (ATL) and their relationship with the presence of amastigotes we evaluated active cutaneous lesions of 35 patients before treatment by the detection of parasites, neutrophils (neutrophil elastase) and histones through immunohistochemistry and confocal immunofluorescence. Intact neutrophils could be detected in all ATL lesions. NETs were present in 27 patients (median 1.1; range from 0.1 to 23.5/mm2) with lesion duration ranging from one to seven months. NETs were in close proximity with neutrophils (r = 0.586; p = 0.0001) and amastigotes (r = 0.710; p = 0.0001). Two patterns of NET formation were detected: small homogeneously distributed networks observed in all lesions; and large structures that could be visualized at a lower magnification in lesions presenting at least 20% of neutrophils. Lesions presenting the larger NET formation showed high parasite detection. A correlation between NET size and the number of intact amastigotes was observed (p=0.02). As we detected an association between NET and amastigotes, our results suggest that neutrophil migration and NET formation could be stimulated and maintained by stimuli derived from the parasite burden/parasite antigen in the extracellular environment. The observation of areas containing only antigens not intermingled with NETs (elastase and histone) suggests that the involvement of these structures in the control of parasite burden is a dynamic process in which the formation of NETs is exhausted with the destruction of the parasites. Since NETs were also associated with granulomas, this trapping would favor the activity of macrophages in order to control the parasite burden.