Diminazene aceturate inhibits the SARS-CoV-2 spike protein-induced inflammation involving leukocyte migration and DNA extracellular traps formation.

AIMS: To investigate the SARS-CoV-2 Spike protein (Spk)-induced inflammatory response and its downmodulation by diminazene aceturate (DIZE). MATERIALS AND METHODS: Through inducing Spk inflammation in murine models, leukocyte migration to the peritoneum, levels of myeloperoxidase (MPO), malondialdehyde (MDA), rolling and adhesion of mesenteric leukocytes, and vascular permeability were investigated. Extracellular DNA traps (DETs) induced by Spk and the production of IL-6 and TNF-α were analyzed using human neutrophils, monocytes, and macrophages. In silico assays assessed the molecular interaction between DIZE and molecules related to leukocyte migration and DETs induction. KEY FINDINGS: Spk triggered acute inflammation, demonstrated by increasing leukocyte migration. Oxidative stress was evidenced by elevated levels of MPO and MDA in the peritoneal liquid. DIZE attenuated cell migration, rolling, and leukocyte adhesion, improved vascular barrier function, mitigated DETs, and reduced the production of Spk-induced pro-inflammatory cytokines. Computational studies supported our findings, showing the molecular interaction of DIZE with targets such as ß2 integrin, PI3K, and PAD2 due to its intermolecular coupling. SIGNIFICANCE: Our results outline a novel role of DIZE as a potential therapeutic agent for mitigating Spk-induced inflammation.

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.

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.

Cloning, expression and purification of 3'-nucleotidase/nuclease, an enzyme responsible for the Leishmania escape from neutrophil extracellular traps.

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.

Tumor-Derived Exosomes Induce the Formation of Neutrophil Extracellular Traps: Implications For The Establishment of Cancer-Associated Thrombosis.

Cancer patients are at an increased risk of developing thromboembolic complications. Several mechanisms have been proposed to explain cancer-associated thrombosis including the release of tumor-derived extracellular vesicles and the activation of host vascular cells. It was proposed that neutrophil extracellular traps (NETs) contribute to the prothrombotic phenotype in cancer. In this study, we evaluated the possible cooperation between tumor-derived exosomes and NETs in cancer-associated thrombosis. Female BALB/c mice were orthotopically injected with 4T1 breast cancer cells. The tumor-bearing animals exhibited increased levels of plasma DNA and myeloperoxidase in addition to significantly increased numbers of circulating neutrophils. Mice were subjected to either Rose Bengal/laser-induced venous thrombosis or ferric chloride-induced arterial thrombosis models. The tumor-bearing mice exhibited accelerated thrombus formation in both models compared to tumor-free animals. Treatment with recombinant human DNase 1 reversed the prothrombotic phenotype of tumor-bearing mice in both models. Remarkably, 4T1-derived exosomes induced NET formation in neutrophils from mice treated with granulocyte colony-stimulating factor (G-CSF). In addition, tumor-derived exosomes interacted with NETs under static conditions. Accordingly, the intravenous administration of 4T1-derived exosomes into G-CSF-treated mice significantly accelerated venous thrombosis in vivo. Taken together, our observations suggest that tumor-derived exosomes and neutrophils may act cooperatively in the establishment of cancer-associated thrombosis.

Neutrophil Extracellular Traps Reprogram IL-4/GM-CSF-Induced Monocyte Differentiation to Anti-inflammatory Macrophages.

Monocyte-derived dendritic cells (mo-DCs) are essential for the development of a Th1 protective immune response against Leishmania parasites. It is well known that IL-4 and GM-CSF drive differentiation of human monocytes to dendritic cells (DCs). Here, we investigate if neutrophil extracellular traps (NETs) disrupt this process. NETs-enriched supernatants, generated after human neutrophil activation by Leishmania promastigotes, were added to monocytes and differentiation monitored by expression of molecules associated with macrophage and DCs phenotypes, cytokine production, and parasite killing. We found that NETs addition to IL-4/GM-CSF-treated monocytes prevented then to fully differentiate into DCs. No effect was observed if NETs were treated with DNase or by filtering the traps. Moreover, NETs closely interact with monocytes and downregulate the expression of the IL-4 receptor, which in turn disrupts fully differentiation of monocytes into DCs. Neutrophil elastase inhibition rescues the monocytes to DCs differentiation. Monocytes cultured with IL-4/GM-CSF and NETs differentiated into macrophages, as observed by the increased expression of CD68, CD32, and CD163, and decreased expression of CD80. Moreover, NET addition to IL-4/GM-CSF-treated monocytes rendered these cells less efficient to kill Leishmania parasites. Altogether, our results show that NETs interfere with IL-4/GM-CSF driven differentiation, reprogramming the generation of mo-DCs to an anti-inflammatory macrophage.

Neutrophil extracellular traps release induced by Leishmania: role of PI3Kγ, ERK, PI3Kσ, PKC, and [Ca2+].

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.

Classical ROS-dependent and early/rapid ROS-independent release of Neutrophil Extracellular Traps triggered by Leishmania parasites.

Neutrophil extracellular traps (NETs) extruded from neutrophils upon activation are composed of chromatin associated with cytosolic and granular proteins, which ensnare and kill microorganisms. This microbicidal mechanism named classical netosis has been shown to dependent on reactive oxygen species (ROS) generation by NADPH oxidase and also chromatin decondensation dependent upon the enzymes (PAD4), neutrophil elastase (NE) and myeloperoxidase (MPO). NET release also occurs through an early/rapid ROS-independent mechanism, named early/rapid vital netosis. Here we analyze the role of ROS, NE, MPO and PAD4 in the netosis stimulated by Leishmania amazonensis promastigotes in human neutrophils. We demonstrate that promastigotes induce a classical netosis, dependent on the cellular redox imbalance, as well as by a chloroamidine sensitive and elastase activity mechanism. Additionally, Leishmania also induces the early/rapid NET release occurring only 10 minutes after neutrophil-parasite interaction. We demonstrate here, that this early/rapid mechanism is dependent on elastase activity, but independent of ROS generation and chloroamidine. A better understanding of both mechanisms of NET release, and the NETs effects on the host immune system modulation, could support the development of new potential therapeutic strategies for leishmaniasis.

A Metabolic Shift toward Pentose Phosphate Pathway Is Necessary for Amyloid Fibril- and Phorbol 12-Myristate 13-Acetate-induced Neutrophil Extracellular Trap (NET) Formation.

Neutrophils are the main defense cells of the innate immune system. Upon stimulation, neutrophils release their chromosomal DNA to trap and kill microorganisms and inhibit their dissemination. These chromatin traps are termed neutrophil extracellular traps (NETs) and are decorated with granular and cytoplasm proteins. NET release can be induced by several microorganism membrane components, phorbol 12-myristate 13-acetate as well as by amyloid fibrils, insoluble proteinaceous molecules associated with more than 40 different pathologies among other stimuli. The intracellular signaling involved in NET formation is complex and remains unclear for most tested stimuli. Herein we demonstrate that a metabolic shift toward the pentose phosphate pathway (PPP) is necessary for NET release because glucose-6-phosphate dehydrogenase (G6PD), an important enzyme from PPP, fuels NADPH oxidase with NADPH to produce superoxide and thus induce NETs. In addition, we observed that mitochondrial reactive oxygen species, which are NADPH-independent, are not effective in producing NETs. These data shed new light on how the PPP and glucose metabolism contributes to NET formation.