Resveratrol prevents the release of neutrophil extracellular traps (NETs) by controlling hydrogen peroxide levels and nuclear elastase migration.

Neutrophil extracellular traps (NETs) are defense mechanisms that trap and kill microorganisms and degrade cytokines. However, excessive production, dysregulation of suppression mechanisms, or inefficient removal of NETs can contribute to increased inflammatory response and the development of pathological conditions. Therefore, research has focused on identifying drugs that inhibit or delay the NET release process. Since reactive oxygen species (ROS) play a significant role in NET release, we aimed to investigate whether resveratrol (RSV), with a wide range of biological and pharmacological properties, could modulate NET release in response to different stimuli. Thus, human neutrophils were pretreated with RSV and subsequently stimulated with PMA, LPS, IL-8, or Leishmania. Our findings revealed that RSV reduced the release of NETs in response to all tested stimuli. RSV decreased hydrogen peroxide levels in PMA- and LPS-stimulated neutrophils, inhibited myeloperoxidase activity, and altered the localization of neutrophil elastase. RSV inhibition of NET generation was not mediated through A2A or A2B adenosine receptors or PKA. Based on the observed effectiveness of RSV in inhibiting NET release, our study suggests that this flavonoid holds potential as a candidate for treating NETs involving pathologies.

Chick heterophils release DNA extracellular traps (DETs) in vitro and in vivo upon Aspergillus fumigatus conidia exposure.

Aspergillosis is a common fungal disease in avian species, causing high mortality in young chicks in agricultural farms and yards. It is caused by fungi belonging to the genus Aspergillus. Aspergillosis occurs by inhalation of fungal conidia, and in chickens, effective infection control relies on a rapid and large influx of heterophils to the lungs. Heterophils, upon different stimuli, release to the extracellular milieu their chromatin associated with several proteins that ensnare and kill different pathogens similarly to neutrophil extracellular traps. Here, we showed that Aspergillus fumigatus conidia and the peptidogalactomannan (PGM), isolated from the fungus cell wall, induce the release of DNA extracellular traps (DETs) in chicks' blood and lung heterophils. We demonstrated that reactive oxygen species, elastase and peptidyl arginine deiminase (PAD) were involved in DETs extrusion, the occurrence of DETs in the lungs of A. fumigatus-exposed chicks in vivo, and its role in chick survival. These results may contribute to developing more efficient tools for the therapeutic and diagnosis of aspergillosis.

Human neutrophil extracellular traps do not impair in vitro Toxoplasma gondii infection.

Introduction: Toxoplasma gondii, responsible for causing toxoplasmosis, is a prevalent food and waterborne pathogen worldwide. It commonly infects warm-blooded animals and affects more than a third of the global human population. Once ingested, the parasite enters the host's small intestine and rapidly disseminates throughout the body via the bloodstream, infiltrating various tissues. Leukocyte-driven responses are vital against T. gondii, with neutrophils playing a dual role: swiftly recruited to infection sites, releasing inflammatory mediators, and serving as a replication hub and Trojan horses, aiding parasite spread. Neutrophils from various hosts release extracellular traps (NETs) against the protozoan. However, gaps persist regarding the mechanisms of NETs production to parasite and their significance in infection control. This study investigates the interplay between human neutrophils and T. gondii, exploring dynamics, key molecules, and signaling pathways involved in NETs production upon protozoan challenge. Methods and Results: Using confocal and electron microscopy, live cell imaging, pharmacological inhibitors, and DNA quantification assays, we find that human neutrophils promptly release both classical and rapid NETs upon pathogen stimulation. The NETs structure exhibits diverse phenotypes over time and is consistently associated with microorganisms. Mechanisms involve neutrophil elastase and peptidylarginine deiminase, along with intracellular calcium signaling and the PI3K pathway. Unexpectedly, human traps do not diminish viability or infectivity, but potentially aid in capturing parasites for subsequent neutrophil phagocytosis and elimination. Discussion: By revealing NETs formation mechanisms and their nuanced impact on T. gondii infection dynamics, our findings contribute to broader insights into host-pathogen relationships.

Acanthamoeba castellanii trophozoites escape killing by neutrophil extracellular traps using their 3'-nucleotidase/nuclease activity.

Acanthamoeba castellanii is a free-living amoeba that acts as an opportunistic pathogen for humans and is the pathogenic agent of Acanthamoeba keratitis (AK). A. castellanii may present as proliferative and infective trophozoites or as resistant cysts during their life cycle. The immune response against AK is still poorly explored; however, it is well established that macrophages and neutrophils play essential roles in controlling corneal infection during the disease outcome. The release of NETs is one of the innate immune strategies to prevent parasite infection, especially when neutrophils interact with microorganisms that are too large to be phagocytosed, which is the case for amoeba species. The present work demonstrated that A. castellanii trophozoites can trigger NET formation upon in vitro interaction with neutrophils. Using DNase as a control, we observed increased parasite survival after coinciding with neutrophils, which may be correlated with NET degradation. Indeed, A. castellanii trophozoites degrade the NET DNA scaffold. Molecular analysis confirmed the occurrence of a 3'-nucleotidase/nuclease (3'-NT/NU) in the A. castellanii genome. We also demonstrated that trophozoites exhibit significantly higher 3'-NT/NU activity than cysts, which cannot trigger NET release. Considering that previous studies indicated the pathological role of 3'-NT-/NU in parasite infection, we suggest that this enzyme may act as the mechanism of escape of A. castellanii trophozoites from NETs.

Peripheral nervous system is injured by neutrophil extracellular traps (NETs) elicited by nonstructural (NS) protein-1 from Zika virus.

The involvement of innate immune mediators to the Zika virus (ZIKV)-induced neuroinflammation is not yet well known. Here, we investigated whether neutrophil extracellular traps (NETs), which are scaffolds of DNA associated with proteins, have the potential to injure peripheral nervous. The tissue lesions were evaluated after adding NETs to dorsal root ganglia (DRG) explants and to DRG constituent cells or injecting them into mouse sciatic nerves. Identification of NET harmful components was achieved by pharmacological inhibition of NET constituents. We found that ZIKV inoculation into sciatic nerves recruited neutrophils and elicited the production of the cytokines CXCL1 and IL-1ß, classical NET inducers, but did not trigger NET formation. ZIKV blocked PMA- and CXCL8-induced NET release, but, in contrast, the ZIKV nonstructural protein (NS)-1 induced NET formation. NET-enriched supernatants were toxic to DRG explants, decreasing neurite area, length, and arborization. NETs were toxic to DRG constituent cells and affected myelinating cells. Myeloperoxidase (MPO) and histones were identified as the harmful component of NETs. NS1 injection into mouse sciatic nerves recruited neutrophils and triggered NET release and caspase-3 activation, events that were also elicited by the injection of purified MPO. In summary, we found that ZIKV NS1 protein induces NET formation, which causes nervous tissue damages. Our findings reveal new mechanisms leading to neuroinflammation by ZIKV.

Leishmania infantum Axenic Amastigotes Induce Human Neutrophil Extracellular Traps and Resist NET-Mediated Killing.

Neutrophils are multifaceted cells that, upon activation, release meshes of chromatin associated with different proteins, known as neutrophil extracellular traps (NETs). Leishmania amazonensis promastigotes and amastigotes induce NET release, and we have identified the signaling pathways involved in NET extrusion activated by promastigotes. Amastigotes maintain the infection in vertebrate hosts, and we have shown the association of NETs with amastigotes in human biopsies of cutaneous leishmaniasis. However, the interaction of amastigotes and neutrophils remains poorly understood. Our study aimed to characterize the pathways involved in the formation of NETs induced by axenic amastigotes from L. infantum, the causal agent of visceral leishmaniasis. Human neutrophils pretreated with signaling pathway inhibitors were incubated with amastigotes, and NET release was quantified in the culture supernatant. Amastigote viability was checked after incubation with NETs. We found that the release of NETs by neutrophils stimulated with these amastigotes requires the participation of elastase and peptidyl arginine deaminase and the involvement of PI3K, ROS, and calcium. Moreover, amastigotes are not susceptible to NET-mediated killing. Altogether, these findings improve our comprehension of the signaling pathways implicated in the interaction between amastigotes and human neutrophils.

Leishmania infantum Defective in Lipophosphoglycan Biosynthesis Interferes With Activation of Human Neutrophils.

Visceral leishmaniasis (VL) is often associated with hematologic manifestations that may interfere with neutrophil response. Lipophosphoglycan (LPG) is a major molecule on the surface of Leishmania promastigotes, which has been associated with several aspects of the parasite-vector-host interplay. Here, we investigated how LPG from Leishmania (L.) infantum, the principal etiological agent of VL in the New World, influences the initial establishment of infection during interaction with human neutrophils in an experimental setting in vitro. Human neutrophils obtained from peripheral blood samples were infected with either the wild-type L. infantum (WT) strain or LPG-deficient mutant (∆lpg1). In this setting, ∆lpg1 parasites displayed reduced viability compared to WT L. infantum; such finding was reverted in the complemented ∆lpg1+LPG1 parasites at 3- and 6-h post-infection. Confocal microscopy experiments indicated that this decreased survival was related to enhanced lysosomal fusion. In fact, LPG-deficient L. infantum parasites more frequently died inside neutrophil acidic compartments, a phenomenon that was reverted when host cells were treated with Wortmannin. We also observed an increase in the secretion of the neutrophil collagenase matrix metalloproteinase-8 (MMP-8) by cells infected with ∆lpg1 L. infantum compared to those that were infected with WT parasites. Furthermore, collagen I matrix degradation was found to be significantly increased in ∆lpg1 parasite-infected cells but not in WT-infected controls. Flow cytometry analysis revealed a substantial boost in production of reactive oxygen species (ROS) during infection with either WT or ∆lpg1 L. infantum. In addition, killing of ∆lpg1 parasites was shown to be more dependent on the ROS production than that of WT L. infantum. Notably, inhibition of the oxidative stress with Apocynin potentially fueled ∆lpg1 L. infantum fitness as it increased the intracellular parasite viability. Thus, our observations demonstrate that LPG may be a critical molecule fostering parasite survival in human neutrophils through a mechanism that involves cellular activation and generation of free radicals.

Simvastatin Downregulates the SARS-CoV-2-Induced Inflammatory Response and Impairs Viral Infection Through Disruption of Lipid Rafts.

Coronavirus disease 2019 (COVID-19) is currently a worldwide emergency caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). In observational clinical studies, statins have been identified as beneficial to hospitalized patients with COVID-19. However, experimental evidence of underlying statins protection against SARS-CoV-2 remains elusive. Here we reported for the first-time experimental evidence of the protective effects of simvastatin treatment both in vitro and in vivo. We found that treatment with simvastatin significantly reduced the viral replication and lung damage in vivo, delaying SARS-CoV-2-associated physiopathology and mortality in the K18-hACE2-transgenic mice model. Moreover, simvastatin also downregulated the inflammation triggered by SARS-CoV-2 infection in pulmonary tissue and in human neutrophils, peripheral blood monocytes, and lung epithelial Calu-3 cells in vitro, showing its potential to modulate the inflammatory response both at the site of infection and systemically. Additionally, we also observed that simvastatin affected the course of SARS-CoV-2 infection through displacing ACE2 on cell membrane lipid rafts. In conclusion, our results show that simvastatin exhibits early protective effects on SARS-CoV-2 infection by inhibiting virus cell entry and inflammatory cytokine production, through mechanisms at least in part dependent on lipid rafts disruption.

Laminin Triggers Neutrophil Extracellular Traps (NETs) and Modulates NET Release Induced by Leishmania amazonensis.

Neutrophils are recruited from the blood and transmigrate through the endothelium to reach tissues, where they are prone to respond through different mechanisms, including the release of neutrophil extracellular traps (NETs). These responses occur in close contact with proteins from the basement membrane and extracellular matrix, where laminins are abundant. Thus, we investigated the interactions between neutrophils and different laminin (LM) isoforms and analyzed the induction of NETs. We showed that neutrophils stimulated with LM isoforms 111, 211, 332, 411, 421, and 511 released NETs. The same occurred when neutrophils interacted with polymerized LMs 111, 411, and 511. LM-induced NETs were partially inhibited by pretreatment of neutrophils with an anti-α6 integrin antibody. Furthermore, NETs triggered by laminins were dependent on elastase and peptidylarginine deiminase (PAD)-4, enzymes that participate in chromatin decondensation. We also found that LMs 411 and LM 511 potentiated the NET release promoted by promastigotes of the protozoan parasite Leishmania, and that NETs stimulated by LMs alone display leishmanicidal activity. The ability of LM to induce NET release may have potential implications for the course of inflammation or infection.

Protective effect of methyl gallate on murine antigen-induced arthritis by inhibiting inflammatory process and bone erosion.

Methyl gallate (MG) is a plant-derived phenolic compound known to present remarkable anti-inflammatory effect in different experimental models, such as paw oedema, pleurisy, zymosan-induced arthritis and colitis. Herein we investigated the effect of MG in the mice model of antigen-induced arthritis (AIA), a model with complex inflammatory response, driven primally by immune process and that cause bone and cartilage erosion similarly found in rheumatoid arthritis. Arthritis was induced by intra-articular injection of albumin methylated from bovine serum (mBSA) in C57BL/6 male mice previously immunized. The dose-response analysis of MG (0.7-70 mg/kg; p.o) showed that maximum inhibition was reached with the dose of 7 mg/kg on paw oedema and cell infiltration induced by AIA at 7 h. Treatment with MG (7 mg/kg; p.o) or with the positive control, dexamethasone (Dexa, 10 mg/kg, ip) reduced AIA oedema formation, leukocyte infiltration, release of extracellular DNA and cytokine production 7 and 24 h (acute response). Mice treated daily with MG for 7 days showed no significant weight loss or liver and kidney toxicity contrary to dexamethasone that induced some degree of toxicity. Prolonged treatment with MG inhibited the late inflammatory response (28 days) reducing oedema formation, cell infiltration, synovial hyperplasia, pannus formation and cartilage degradation as observed in histopathological analyses. Ultimately, MG reduced bone resorption as evidenced by a decrease in tartrate-resistant acid phosphate (TRAP)-positive cells number in femur histology. Altogether, we demonstrate that MG ameliorates the inflammatory reaction driven primarily by the immune process, suggesting a potential therapeutic application in arthritis treatment.

Leishmania Parasites Drive PD-L1 Expression in Mice and Human Neutrophils With Suppressor Capacity.

Neutrophils play an important role in the outcome of leishmaniasis, contributing either to exacerbating or controlling the progression of infection, a dual effect whose underlying mechanisms are not clear. We recently reported that CD4+ and CD8+ T cells, and dendritic cells of Leishmania amazonensis-infected mice present high expression of PD-1 and PD-L1, respectively. Given that the PD-1/PD-L1 interaction may promote cellular dysfunction, and that neutrophils could interact with T cells during infection, we investigated here the levels of PD-L1 in neutrophils exposed to Leishmania parasites. We found that both, promastigotes and amastigotes of L. amazonensis induced the expression of PD-L1 in the human and murine neutrophils that internalized these parasites in vitro. PD-L1-expressing neutrophils were also observed in the ear lesions and the draining lymph nodes of L. amazonensis-infected mice, assessed through cell cytometry and intravital microscopy. Moreover, expression of PD-L1 progressively increased in neutrophils from ear lesions as the disease evolved to the chronic phase. Co-culture of infected neutrophils with in vitro activated CD8+ T cells inhibits IFN-γ production by a mechanism dependent on PD-1 and PD-L1. Importantly, we demonstrated that in vitro infection of human neutrophils by L braziliensis induced PD-L1+ expression and also PD-L1+ neutrophils were detected in the lesions of patients with cutaneous leishmaniasis. Taken together, these findings suggest that the Leishmania parasite increases the expression of PD-L1 in neutrophils with suppressor capacity, which could favor the parasite survival through impairing the immune response.

Adenosine Diphosphate Improves Wound Healing in Diabetic Mice Through P2Y12 Receptor Activation.

Chronic wounds are a public health problem worldwide, especially those related to diabetes. Besides being an enormous burden to patients, it challenges wound care professionals and causes a great financial cost to health system. Considering the absence of effective treatments for chronic wounds, our aim was to better understand the pathophysiology of tissue repair in diabetes in order to find alternative strategies to accelerate wound healing. Nucleotides have been described as extracellular signaling molecules in different inflammatory processes, including tissue repair. Adenosine-5'-diphosphate (ADP) plays important roles in vascular and cellular response and is immediately released after tissue injury, mainly from platelets. However, despite the well described effect on platelet aggregation during inflammation and injury, little is known about the role of ADP on the multiple steps of tissue repair, particularly in skin wounds. Therefore, we used the full-thickness excisional wound model to evaluate the effect of local ADP application in wounds of diabetic mice. ADP accelerated cutaneous wound healing, improved new tissue formation, and increased both collagen deposition and transforming growth factor-ß (TGF-ß) production in the wound. These effects were mediated by P2Y12 receptor activation since they were inhibited by Clopidogrel (Clop) treatment, a P2Y12 receptor antagonist. Furthermore, P2Y1 receptor antagonist also blocked ADP-induced wound closure until day 7, suggesting its involvement early in repair process. Interestingly, ADP treatment increased the expression of P2Y12 and P2Y1 receptors in the wound. In parallel, ADP reduced reactive oxygen species (ROS) formation and tumor necrosis factor-α (TNF-α) levels, while increased IL-13 levels in the skin. Also, ADP increased the counts of neutrophils, eosinophils, mast cells, and gamma delta (γδ) T cells (Vγ4+ and Vγ5+ cells subtypes of γδ+ T cells), although reduced regulatory T (Tregs) cells in the lesion. In accordance, ADP increased fibroblast proliferation and migration, myofibroblast differentiation, and keratinocyte proliferation. In conclusion, we provide strong evidence that ADP acts as a pro-resolution mediator in diabetes-associated skin wounds and is a promising intervention target for this worldwide problem.

Leishmanicidal effect of 1,3,4-thiadiazolium mesoionic salts on Leishmania amazonensis in vitro.

Leishmaniasis is a neglected broad clinical spectrum disease caused by protozoa of the genus Leishmania, which affect millions of people annually in the world and the treatment has severe side effects and resistant strains have been reported. Mesoionic salts are a subclass of the betaine group with extensive biological activity such as microbicide and anti-inflammatory In this work, we analyze the cytotoxic effects of mesoionic salts, 4-phenyl-5-(X-phenyl)-1,3,4-thiadiazolium-2-phenylamine chloride (X = 4 Cl; 3,4 diCl and 3,4 diF), on Leishmania amazonensis in vitro. Initially, Mesoionic salts toxicity were evaluated by XTT assay on L. amazonensis promastigotes. Our results show that the mesoionic salts MI-3,4 diCl, MI-4 Cl and MI-3,4 diF were toxic to the promastigote parasite with IC50 values of 14.3, 40.1 and 61.8 µM, respectively. The amastigote survival was evaluated in treated infected-macrophages, and the results demonstrate that MI-4 Cl (IC50 = 33 µM) and MI-3,4 diCl (IC50 = 43 µM) have a toxic effect against these forms. None of the mesoionic compounds tested present host cell toxicity up to the tested concentration of 100 µM. The selectivity index for MI-3,4 diCl and MI-4 Cl were 3.94 and 6.97, respectively. Nitric oxide (NO) production assayed by Griess reagent, in LPS-activated macrophages or not, in the presence of the salts showed that only the MI-3,4 diCl compound reduced NO levels. Lipid profile analysis of treated-promastigotes showed no alteration of neutral lipids. Evaluation of mitochondrial membrane potential (∆Ψm) showed that the MI-4Cl compound was able to reduce (∆Ψm) by 50%. Therefore, our results suggest that the chlorinated compounds are promising biomolecules, which cause inhibition of L.amazonensis promastigotes, amastigotes, leading to mitochondrial damage.

Neutrophil extracellular traps from healthy donors and HIV-1-infected individuals restrict HIV-1 production in macrophages.

Neutrophils release extracellular traps (NETs) after interaction with microorganisms and physiological or synthetic products. NETs consist of decondensed chromatin complexed with proteins, some of them with microbicidal properties. Because NETs can modulate the functioning of HIV-1 target cells, we aimed to verify whether they modify HIV-1 replication in macrophages. We found that exposure of HIV-1-infected macrophages to NETs resulted in significant inhibition of viral replication. The NET anti-HIV-1 action was independent of other soluble factors released by the activated neutrophils, but otherwise dependent on the molecular integrity of NETs, since NET-treatment with protease or DNase abolished this effect. NETs induced macrophage production of the anti-HIV-1 ß-chemokines Rantes and MIP-1ß, and reduced the levels of integrated HIV-1 DNA in the macrophage genome, which may explain the decreased virus production by infected macrophages. Moreover, the residual virions released by NET-treated HIV-1-infected macrophages lost infectivity. In addition, elevated levels of DNA-elastase complexes were detected in the plasma from HIV-1-infected individuals, and neutrophils from these patients released NETs, which also inhibited HIV-1 replication in in vitro infected macrophages. Our results reveal that NETs may function as an innate immunity mechanism able to restrain HIV-1 production in macrophages.

Extracellular Traps Released by Neutrophils from Cats are Detrimental to Toxoplasma gondii Infectivity.

Toxoplasma gondii is the causative agent of toxoplasmosis, an infectious disease that affects over 30% of the human world population, causing fatal infections in immunocompromised individuals and neonates. The life cycle of T. gondii is complex, and involves intermediate hosts (birds and mammals) and definitive hosts (felines, including domestic cats). The innate immune repertoire against the parasite involves the production of neutrophil extracellular traps (NET), and neutrophils from several intermediate hosts produce NET induced by T. gondii. However, the mechanisms underlying NET release in response to the parasite have been poorly explored. Therefore, the aims of this study were to investigate whether neutrophils from cats produce NET triggered by T. gondii and to understand the mechanisms thereby involved. Neutrophils from cats were stimulated with T. gondii tachyzoites and NET-derived DNA in the supernatant was quantified during the time. The presence of histone H1 and myeloperoxidase was detected by immunofluorescence. We observed that cat neutrophils produce both classical and rapid/early NET stimulated by T. gondii. Inhibition of elastase, intracellular calcium, and phosphatidylinositol 3-kinase (PI3K)-δ partially blocked classical NET release in response to the parasite. Electron microscopy revealed strands and networks of DNA in close contact or completely entrapping parasites. Live imaging showed that tachyzoites are killed by NET. We conclude that the production of NET is a conserved strategy to control infection by T. gondii amongst intermediate and definitive hosts.

CXCR4 and MIF are required for neutrophil extracellular trap release triggered by Plasmodium-infected erythrocytes.

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.

DH82 Canine and RAW264.7 Murine Macrophage Cell Lines Display Distinct Activation Profiles Upon Interaction With Leishmania infantum and Leishmania amazonensis.

Leishmaniasis is an anthropozoonotic disease, and dogs are considered the main urban reservoir of the parasite. Macrophages, the target cells of Leishmania sp., play an important role during infection. Although dogs have a major importance in the epidemiology of the disease, the majority of the current knowledge about Leishmania-macrophage interaction comes from murine experimental models. To assess whether the canine macrophage strain DH82 is an accurate model for the study of Leishmania interaction, we compared its infection by two species of Leishmania (Leishmania infantum and L. amazonensis) with the murine macrophage cell line (RAW264.7). Our results demonstrated that L. amazonensis survival was around 40% at 24 h of infection inside both macrophage cell lines; however, a reduction of 4.3 times in L. amazonensis infection at 48 h post-infection in RAW 264.7 macrophages was observed. The survival index of L. infantum in DH82 canine macrophages was around 3 times higher than that in RAW264.7 murine cells at 24 and 48 h post-infection; however, at 48 h a reduction in both macrophages was observed. Surprisingly at 24 h post-infection, NO and ROS production by DH82 canine cells stimulated with LPS or menadione or during Leishmania infection was minor compared to murine RAW264.7. However, basal arginase activity was higher in DH82 cells when compared to murine RAW264.7 cells. Analysis of the cytokines showed that these macrophages present a different response profile. L. infantum induced IL-12, and L. amazonensis induced IL-10 in both cell lines. However, L. infantum and L. amazonensis also induced TGF-ß in RAW 264.7. CD86 and MHC expression showed that L. amazonensis modulated them in both cell lines. Conversely, the parasite load profile did not show significant difference between both macrophage cell lines after 48 h of infection, which suggests that other mechanisms of Leishmania control could be involved in DH82 cells.

In vitro leishmanicidal activity of monoterpenes present in two species of Protium (Burseraceae) on Leishmania amazonensis.

ETHNOPHARMACOLOGICAL RELEVANCE: Leishmaniasis is a neglected disease that affects millions of people around the world. Parasite resistance and the toxicity to the current treatments lead to the search for new effective molecules. Plants are widely used in traditional and indigenous medicine to treat different diseases. The oleoresin of the genus Protium, which is rich in volatile compounds active against different microorganisms, is among these plants. AIM: The aim of this study was to evaluate the leishmanicidal potential of Protium altsonii (PaEO) and P. hebetatum (PhEO) (Burseraceae) oleoresins, as well as of three representative monoterpenes in their constitution: α-pinene, p-cymene and 1,8-cineole. MATERIALS AND METHODS: Protium altsonii (PaEO) and P. hebetatum (PhEO) oleoresins and three of their constituents were tested in vitro on promastigotes and amastigotes-infected macrophages in different concentrations. Their toxicity for macrophages was analyzed by XTT assay and phagocytic ability. It was evaluated the ability of the compounds to induce NO production on treated-macrophages using Griess reaction and the effect of them in lipid profile on treated-parasite through Thin Layer Chromatography. RESULTS: Our data showed that both essential oils have toxic effect on promastigotes and amastigotes of L. amazonensis in vitro in a dose-dependent manner. PaEO IC50 were 14.8 µg/mL and 7.8 µg/mL and PhEO IC50s were 0.46 µg/mL and 30.5 µg/m for promastigotes and amastigotes, respectively. Toxicity to macrophages was not observed at 50 µg/mL with both EOs. The compounds 1,8- cineole, α-pinene, and p-cymene inhibited amastigotes survival in a dose-dependent manner with IC50s of 48.4 µg/mL, 37 µg/mL, 46 µg/mL, respectively. Macrophage viability was around 90% even at 200 µg/mL and the phagocytic capacity was not altered in the treated-macrophages to up 50 µg/mL. The compounds were not able to modulate the nitric oxide production either at rest or LPS-activated macrophages. In addition, treated promastigote revealed an important change in their lipid profile after 48 h at 50 µg/mL in the presence of the compounds. CONCLUSIONS: The results indicate that oleoresins of Protium genus are potent against Leishmania and α-pinene, p-cymene and 1,8-cineole have anti-Leishmania properties that could be explored in synergistic assays in order to develop new drug candidates.

Endocytosis and Exocytosis in Leishmania amazonensis Are Modulated by Bromoenol Lactone.

In the protozoan pathogen Leishmania, endocytosis, and exocytosis occur mainly in the small area of the flagellar pocket membrane, which makes this parasite an interesting model of strikingly polarized internalization and secretion. Moreover, little is known about vesicle recognition and fusion mechanisms, which are essential for both endo/exocytosis in this parasite. In other cell types, vesicle fusion events require the activity of phospholipase A2 (PLA2), including Ca2+-independent iPLA2 and soluble, Ca2+-dependent sPLA2. Here, we studied the role of bromoenol lactone (BEL) inhibition of endo/exocytosis in promastigotes of Leishmania amazonensis. PLA2 activities were assayed in intact parasites, in whole conditioned media, and in soluble and extracellular vesicles (EVs) conditioned media fractions. BEL did not affect the viability of promastigotes, but reduced the differentiation into metacyclic forms. Intact parasites and EVs had BEL-sensitive iPLA2 activity. BEL treatment reduced total EVs secretion, as evidenced by reduced total protein concentration, as well as its size distribution and vesicles in the flagellar pocket of treated parasites as observed by TEM. Membrane proteins, such as acid phosphatases and GP63, became concentrated in the cytoplasm, mainly in multivesicular tubules of the endocytic pathway. BEL also prevented the endocytosis of BSA, transferrin and ConA, with the accumulation of these markers in the flagellar pocket. These results suggested that the activity inhibited by BEL, which is one of the irreversible inhibitors of iPLA2, is required for both endocytosis and exocytosis in promastigotes of L. amazonensis.

Neutrophil extracellular trap-enriched supernatants carry microRNAs able to modulate TNF-α production by macrophages.

Neutrophil extracellular traps (NETs) emerge from the cell as a DNA scaffold associated with cytoplasmic and granular proteins, able to immobilize and kill pathogens. This association occurs following nuclear and granular membrane disintegration, allowing contact with the decondensed chromatin. Thus, it is reasonable to speculate that the DNA can also mix with miRNAs and carry them in NETs. Here, we report for the first time the presence of the miRNA carriers associated with NETs and miRNAs present in NET-enriched supernatants (NET-miRs), thus adding a novel class of molecules and new proteins that can be released and transported in the NET platform. We observed that the majority of NET-miRs were common to all four stimuli used (PMA, interleukin-8, amyloid fibrils and Leishmania), and that miRNA-142-3p carried by NETs down-modulates protein kinase Cα and regulates TNF-α production in macrophages upon NET interaction with these cells. Our findings unveil a novel role for NETs in the cell communication processes, allowing the conveyance of miRNA from neutrophils to neighboring cells.