RESUMO
Transforming growth factor beta (TGF-ß) is a cytokine with important involvement in biological processes related to the pathogenesis of sickle cell disease (SCD), including endothelial and vascular dysfunction, inflammation, and hematopoietic homeostasis. This study is aimed at investigating associations between levels of TGF-ß1 and classical laboratory biomarkers and inflammatory mediators, as well as the tissue inhibitor of metalloproteases-1 (TIMP-1) and matrix metalloproteinase-9 (MMP-9), in pediatric patients (n = 123) with SCD in steady state: 84 with sickle cell anemia (HbSS) and 39 with hemoglobin SC disease (HbSC). A healthy control (HC) group of 59 individuals was also included. Hematological and biochemical analyses were carried out using electronic methods. TGF-ß1, TIMP-1, and MMP-9 plasma quantifications were performed by ELISA. TGF-ß1 plasma levels were higher in HbSS individuals than in HbSC and HC. In individuals with HbSS, TGF-ß1 levels were positively correlated with red blood cells, hemoglobin, hematocrit, platelets, and TIMP-1. In addition, HbSS individuals with TGF-ß1 levels above the median (≥72.29 ng/mL) also presented increased monocyte counts and decreased albumin levels. In patients with HbSC, TGF-ß1 levels were positively correlated with leukocytes, eosinophils, lymphocytes, monocytes, and platelets, as well as levels of TIMP-1, VLDL-C, triglycerides, heme, and AST. Additionally, HbSC individuals with TGF-ß1 levels above the median (≥47.80 ng/mL) presented increased leukocyte and platelet counts, as well as increased levels of triglycerides, VLDL-C, MMP-9, and TIMP-1, and decreased HDL-C. Our findings suggest that TGF-ß1 may play important roles in vascular remodeling, vasculopathy, angiogenesis, and inflammation in pediatric patients with SCD.
Assuntos
Anemia Falciforme , Hemólise , Fator de Crescimento Transformador beta1 , Anemia Falciforme/diagnóstico , Biomarcadores/sangue , Criança , Humanos , Inflamação , Metaloproteinase 9 da Matriz , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta1/sangueRESUMO
Sickle cell anemia (SCA) is a hemolytic disease in which vaso-occlusion is an important pathophysiological mechanism. The treatment is based on hydroxyurea (HU), which decreases leukocyte counts and increases fetal hemoglobin synthesis. Different cell types are thought to contribute to vaso-occlusion. Nevertheless, the role of monocytes subsets remains unclear. We investigated frequencies of monocytes subsets in blood and their response to HU therapy, testing their ability to express pro-inflammatory molecules and tissue factor (TF). We identified major changes in monocyte subsets, with classical monocytes (CD14++CD16-) appearing highly frequent in who were not taking HU, whereas those with patrolling phenotype (CD14dimCD16+) were enriched in individuals undergoing therapy. Additionally, HU decreased the production of TNF-α, IL1-ß, IL-6, IL-8 as well as TF by the LPS-activated monocytes. Likewise, frequency of TF-expressing monocytes is increased in patients with previous vaso-occlusion. Moreover, activated monocytes expressing TF produced several pro-inflammatory cytokines simultaneously. Such polyfunctional capacity was dramatically dampened by HU therapy. The frequency of classical monocytes subset was positively correlated with percentage cytokine producing cells upon LPS stimulation. These findings suggest that classical monocytes are the subset responsible for multiple pro-inflammatory cytokine production and possibly drive inflammation and vaso-occlusion in SCA which is damped by HU.
Assuntos
Anemia Falciforme/tratamento farmacológico , Hidroxiureia/farmacologia , Mediadores da Inflamação/metabolismo , Monócitos/efeitos dos fármacos , Adolescente , Anemia Falciforme/sangue , Anemia Falciforme/imunologia , Criança , Feminino , Humanos , Hidroxiureia/uso terapêutico , Contagem de Leucócitos , Masculino , Monócitos/imunologia , Monócitos/metabolismoRESUMO
Sand flies bite mammalian hosts to obtain a blood meal, driving changes in the host inflammatory response that support the establishment of Leishmania infection. This effect is partially attributed to components of sand fly saliva, which are able to recruit and activate leukocytes. Our group has shown that heme oxygenase-1 (HO-1) favors Leishmania survival in infected cells by reducing inflammatory responses. Here, we show that exposure to sand fly bites is associated with induction of HO-1 in vivo. Histopathological analyses of skin specimens from human volunteers experimentally exposed to sand fly bites revealed that HO-1 and Nrf2 are produced at bite sites in the skin. These results were recapitulated in mice ears injected with a salivary gland sonicate (SGS) or exposed to sand fly bites, indicating that vector saliva may be a key factor in triggering HO-1 expression. Resident skin macrophages were the main source HO-1 at 24-48 h after bites. Additionally, assays in vivo after bites and in vitro after stimulation with saliva both demonstrated that HO-1 production by macrophages was Nrf2-dependent. Collectively, our data demonstrates that vector saliva induces early HO-1 production at the bite sites, representing a major event associated with establishment of naturally-transmitted Leishmania infections.
Assuntos
Regulação Enzimológica da Expressão Gênica , Heme Oxigenase-1/biossíntese , Mordeduras e Picadas de Insetos/enzimologia , Insetos Vetores , Proteínas de Membrana/biossíntese , Psychodidae , Saliva , Pele/enzimologia , Animais , Feminino , Humanos , Mordeduras e Picadas de Insetos/patologia , Leishmania/metabolismo , Masculino , Camundongos , Camundongos Knockout , Células RAW 264.7 , Pele/patologia , Células THP-1RESUMO
Leishmania braziliensis infection causes skin ulcers, typically found in localized cutaneous leishmaniasis (LCL). This tissue pathology associates with different modalities of cell necrosis, which are subverted by the parasite as a survival strategy. Herein we examined the participation of necroptosis, a specific form of programmed necrosis, in LCL lesions and found reduced RIPK3 and PGAM5 gene expression compared to normal skin. Assays using infected macrophages demonstrated that the parasite deactivates both RIPK3 and MLKL expression and that these molecules are important to control the intracellular L. braziliensis replication. Thus, LCL-related necroptosis may be targeted to control infection and disease immunopathology.
RESUMO
Necroptosis is a pro-inflammatory cell death, which happens in the context of caspase-8 inhibition, allowing activation of the receptor interacting protein kinase 1-receptor interacting protein kinase 3-mixed lineage kinase domain-like (RIPK1-RIPK3-MLKL) axis. Recently, necroptosis has emerged as a key component of resistance against pathogens including infected macrophage by Leishmania infantum, the ethiologic agent of Visceral leishmaniasis (VL). VL is the most severe form of Leishmaniasis, characterized by systemic inflammation and neutropenia. However, the role of neutrophil cell death in VL has not been characterized. Here, we showed that VL patients exhibited increased lactate dehydrogenase levels in the serum, a hallmark of cell death and tissue damage. We investigated the effect of necroptosis in neutrophil infection in vitro. Human neutrophils pretreated with zVAD-fmk (pan-caspase inhibitor) and zIETD-fmk (caspase-8 inhibitor) increased reactive oxygen species (ROS) level in response to Leishmania infection, which is associated with necroptotic cell death. MLKL, an important effector molecule downstream of necroptosis pathway, was also required for Leishmania killing. Moreover, in absence of caspases-8, murine neutrophils displayed loss of membrane integrity, higher levels of ROS, and decreased L. infantum viability. Pharmacological inhibition of RIPK1 or RIPK3 increased parasite survival when caspase-8 was blocked. Electron microscopy assays revealed morphological features associated with necroptotic death in L. infantum infected-neutrophils pretreated with caspase inhibitor, whereas infected cells pretreated with RIPK1 and RIPK3 inhibitors did not show ultra-structural alterations in membrane integrity and presented viable Leishmania within parasitophorous vacuoles. Taken together, these findings suggest that inhibition of caspase-8 contributes to elimination of L. infantum in neutrophils by triggering necroptosis. Thus, targeting necroptosis may represent a new strategy to control Leishmania replication.
Assuntos
Leishmania infantum/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose , Biomarcadores , Caspase 8/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/patologia , Masculino , Camundongos , Necrose , Neutrófilos/parasitologia , Neutrófilos/ultraestruturaAssuntos
Anemia Falciforme/patologia , Mediadores da Inflamação/sangue , Síndrome Torácica Aguda/sangue , Síndrome Torácica Aguda/diagnóstico , Anemia Falciforme/diagnóstico , Biomarcadores/sangue , Brasil , Estudos de Casos e Controles , Criança , Pré-Escolar , Citocinas/sangue , Feminino , Humanos , MasculinoRESUMO
Free heme is an inflammatory molecule capable of inducing migration and activation of neutrophils. Here, we examine the heme-driven oxidative stress-associated cell death mechanisms in human neutrophils infected with Leishmania infantum, an etiologic agent of visceral leishmaniasis (VL). We first performed exploratory analyses in a population of well characterized treatment-naïve VL patients as well as uninfected controls, who were part of previously reported studies. We noted a positive correlation between serum concentrations of heme with heme oxygenase-1 (HO-1) and lactate deydrogenase, as well as, a negative correlation between heme values and peripheral blood neutrophils counts. Moreover, in vitro infection with L. infantum in the presence of heme enhanced parasite burden in neutrophils, while increasing the production of reactive oxygen species and release of neutrophilic enzymes. Additional experiments demonstrated that treatment of infected neutrophils with ferrous iron (Fe+2), a key component of the heme molecule, resulted in increased parasite survival without affecting neutrophil activation status. Furthermore, stimulation of infected neutrophils with heme triggered substantial increases in HO-1 mRNA expression as well as in superoxide dismutase-1 enzymatic activity. Heme, but not Fe+2, induced oxidative stress-associated cell death. These findings indicate that heme promotes intracellular L. infantum survival via activation of neutrophil function and oxidative stress. This study opens new perspectives for the understanding of immunopathogenic mechanisms involving neutrophils in VL.
RESUMO
Visceral leishmaniasis (VL) remains a major public health problem worldwide. Cytokine balance is thought to play a critical role in the development of this disease. Here, we perform a prospective exploratory study addressing whether simultaneous assessment of circulating levels of different lipid mediators and cytokines could highlight specific pathways involved with VL pathogenesis. VL patients displayed substantial increases in serum levels of Prostaglandin F2α (PGF2α), Leukotriene B4 (LTB4), Resolvin D1 (RvD1), IL-1ß, IL-6, IL-8, IL-10, IL-12p70 and TNF-α compared with uninfected endemic control group, while exhibiting decreased levels of TGF-ß1. Hierarchical cluster analysis of the prospective changes in the expression level of theses parameters upon anti-Leishmania treatment initiation revealed that the inflammatory profile observed in active disease gradually changed over time and was generally reversed at day 30 of therapy. Furthermore, not only the individual concentrations of most of the inflammatory biomarkers changed upon treatment, but the correlations between those and several biochemical parameters used to characterize VL disease activity were also modified over time. These results demonstrate that an inflammatory imbalance hallmarks active VL disease and open perspective for manipulation of these pathways in future studies examining a potential host-directed therapy against VL.
Assuntos
Mediadores da Inflamação/metabolismo , Leishmania donovani , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/parasitologia , Adolescente , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Biomarcadores , Criança , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Mediadores da Inflamação/sangue , Leishmaniose Visceral/tratamento farmacológico , Masculino , Adulto JovemRESUMO
BACKGROUND: Eicosanoids and sand fly saliva have a critical role in the Leishmania infection. Here, we evaluated the effect of Lutzomyia longipalpis salivary gland sonicate (SGS) on neutrophil and monocyte recruitment and activation of eicosanoid production in a murine model of inflammation. METHODS: C57BL/6 mice were inoculated intraperitonealy with Lutzomyia longipalpis SGS or Leishmania infantum or both, followed by analyses of cell recruitment, parasite load and eicosanoid production. RESULTS: Intraperitoneal injection of Lutzomyia longipalpis SGS together with Leishmania infantum induced an early increased parasite viability in monocytes and neutrophils. L. longipalpis SGS increased prostaglandin E2 (PGE2), but reduced leukotriene B4 (LTB4) production ex vivo in peritoneal leukocytes. In addition, the pharmacological inhibition of cyclooxygenase 2 (COX-2) with NS-398 decreased parasite viability inside macrophages during Leishmania infection in the presence of L. longipalpis SGS arguing that PGE2 production is associated with diminished parasite killing. CONCLUSIONS: These findings indicate that L. longipalpis SGS is a critical factor driving immune evasion of Leishmania through modulation of PGE2/LTB4 axis, which may represent an important mechanism on establishment of the infection.
Assuntos
Dinoprostona/imunologia , Leishmania infantum/imunologia , Leishmaniose Visceral/imunologia , Leucotrieno B4/imunologia , Psychodidae/imunologia , Animais , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Modelos Animais de Doenças , Feminino , Humanos , Leishmaniose Visceral/parasitologia , Leucócitos/parasitologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nitrobenzenos/farmacologia , Psychodidae/parasitologia , Glândulas Salivares/imunologia , Sulfonamidas/farmacologiaRESUMO
We recently demonstrated that immunization with polyester poly(lactide-co-glycolide acid) (PLGA) nanoparticles loaded with the 11-kDa Leishmania vaccine candidate kinetoplastid membrane protein 11 (KMP-11) significantly reduced parasite load in vivo. Presently, we explored the ability of the recombinant PLGA nanoparticles to stimulate innate responses in macrophages and the outcome of infection with Leishmania braziliensis in vitro. Incubation of macrophages with KMP-11-loaded PLGA nanoparticles significantly decreased parasite load. In parallel, we observed the augmented production of nitric oxide, superoxide, TNF-α and IL-6. An increased release of CCL2/MCP-1 and CXCL1/KC was also observed, resulting in macrophage and neutrophil recruitment in vitro. Lastly, the incubation of macrophages with KMP-11-loaded PLGA nanoparticles triggered the activation of caspase-1 and the secretion of IL-1ß and IL-18, suggesting inflammasome participation. Inhibition of caspase-1 significantly increased the parasite load. We conclude that KMP-11-loaded PLGA nanoparticles promote the killing of intracellular Leishmania parasites through the induction of potent innate responses. FROM THE CLINICAL EDITOR: In this novel study, KMP-11-loaded PLGA nanoparticles are demonstrated to promote the killing of intracellular Leishmania parasites through enhanced innate immune responses by multiple mechanisms. Future clinical applications would have a major effect on our efforts to address parasitic infections.