The State of Art of Extracellular Traps in Protozoan Infections (Review).

Protozoan parasite infection causes severe diseases in humans and animals, leading to tremendous economic and medical pressure. Natural immunity is the first line of defence against parasitic infection. Currently, the role of natural host immunity in combatting parasitic infection is unclear, so further research on natural host immunity against parasites will provide a theoretical basis for the prevention and treatment of related parasitic diseases. Extracellular traps (ETs) are an important natural mechanism of immunity involving resistance to pathogens. When immune cells such as neutrophils and macrophages are stimulated by external pathogens, they release a fibrous network structure, consisting mainly of DNA and protein, that can capture and kill a variety of extracellular pathogenic microorganisms. In this review, we discuss the relevant recently reported data on ET formation induced by protozoan parasite infection, including the molecular mechanisms involved, and discuss the role of ETs in the occurrence and development of parasitic diseases.

Eosinophils and Neutrophils Eliminate Migrating Strongyloides ratti Larvae at the Site of Infection in the Context of Extracellular DNA Trap Formation.

Parasitic nematodes such as hookworms actively penetrate the skin of their hosts, encountering skin-resident innate immune cells that represent the host´s first line of defense. Here we use Strongyloides ratti as a model for an intestinal helminth parasite with tissue migrating stages. We show that interception and killing of migrating larvae in mice during a 1st infection occurred predominantly in skin and muscle tissue before larvae migrated via lung and head tissue to the intestine. Inhibition of larval migration was even more efficient in immune mice during a 2nd infection where larvae barely left the site of entry i.e. the foot. Using cell-deficient mice we show that interception in the tissue was predominantly mediated by neutrophils and eosinophils while basophils and mast cells were dispensable in vivo. Likewise, neutrophils and eosinophils inhibited S. ratti L3 motility in vitro in the context of ETosis. Thereby eosinophils were strictly dependent on the presence of anti-S. ratti antibodies while neutrophils inhibited L3 motility as such. Also, MPO and MMP-9 were released by neutrophils in response to L3 alone, but immune plasma further stimulated MPO release in an antibody-dependent manner. In summary, our findings highlight the central role of the skin as first line of defense against helminth parasites in both, innate and adaptive immunity.

TatD DNases of African trypanosomes confer resistance to host neutrophil extracellular traps.

African trypanosomatid parasites escape host acquired immune responses through periodic antigenic variation of their surface coat. In this study, we describe a mechanism by which the parasites counteract innate immune responses. Two TatD DNases were identified in each of Trypanosoma evansi and Trypanosoma brucei. These DNases are bivalent metal-dependent endonucleases localized in the cytoplasm and flagella of the parasites that can also be secreted by the parasites. These enzymes possess conserved functional domains and have efficient DNA hydrolysis activity. Host neutrophil extracellular traps (NETs) induced by the parasites could be hydrolyzed by native and recombinant TatD DNases. NET disruption was prevented, and the survival rate of parasites was decreased, in the presence of the DNase inhibitor aurintricarboxylic acid. These data suggest that trypanosomes can counteract host innate immune responses by active secretion of TatD DNases to degrade NETs.

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.

Neutrophil properties in healthy and Leishmania infantum-naturally infected dogs.

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.

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.

The state of art of neutrophil extracellular traps in protozoan and helminthic infections.

Neutrophil extracellular traps (NETs) are DNA fibers associated with histones, enzymes from neutrophil granules and anti-microbial peptides. NETs are released in a process denominated NETosis, which involves sequential steps that culminate with the DNA extrusion. NETosis has been described as a new mechanism of innate immunity related to defense against different pathogens. The initial studies of NETs were carried out with bacteria and fungi, but currently a large variety of microorganisms capable of inducing NETs have been described including protozoan and helminth parasites. Nevertheless, we have little knowledge about how NETosis process is carried out in response to the parasites, and about its implication in the resolution of this kind of disease. In the best case, the NETs entrap and kill parasites in vitro, but in others, immobilize the parasites without affecting their viability. Moreover, insufficient studies on the NETs in animal models of infections that would help to define their role, and the association of NETs with chronic inflammatory pathologies such as those occurring in several parasitic infections have left open the possibility of NETs contributing to pathology instead of protection. In this review, we focus on the reported mechanisms that lead to NET release by protozoan and helminth parasites and the evidence that support the role of NETosis in the resolution or pathogenesis of parasitic diseases.

Trichomonas vaginalis triggers the release of THP-1 extracellular traps.

Trichomonas vaginalis is responsible for the prevalence of trichomoniasis, which may be one of the most epidemic nonviral sexually transmitted pathogens. Extracellular traps (ET) are a unique form of innate immunity against infection; they bind to and kill microorganisms. However, the effect of T. vaginalis on ET release in the human monocytic cell line THP-1 remains unclear. In the present study, the morphology of ET derived from THP-1 in response to T. vaginalis was observed by scanning electron microscopy (SEM). The results demonstrated ET entangling T. vaginalis. Then, the colocalization of histone (H3) and myeloperoxidase (MPO) with DNA was observed via fluorescence confocal microscopy. Colocalization revealed the classic characteristics of DNA decorated with H3 and MPO. T. vaginalis significantly increased reactive oxygen species (ROS) and THP-1-derived ET. In addition, we measured the levels of lactic dehydrogenase (LDH) and the phosphorylation of the P38 and ERK1/2 MAPK signaling pathways. The results indicated that the formation of ET induced by T. vaginalis was related to phosphorylation of the P38 and ERK1/2 MAPK signaling pathways but not to LDH levels. These data confirmed the phenomenon of THP-1-derived ET being triggered by T. vaginalis in vitro; this process may play a pivotal role in innate immunity during defense against T. vaginalis infection.

Bovine macrophage-derived extracellular traps act as early effectors against the abortive parasite Neospora caninum.

Macrophages are multipurpose phagocytes and are considered to be irreplaceable during the early host innate immune response against microbial and parasitic pathogens. However, no report has investigated the novel anti-parasitic mechanism of macrophage-derived extracellular traps (ETs) against the abortive apicomplexan parasite Neospora caninum (N. caninum) in cattle. Scanning electron microscopy (SEM) was used to visualize and characterize N. caninum tachyzoite-induced macrophage-triggered ETs in exposed bovine macrophages. Fluorescence confocal microscopy was used to confirm the classical backbone structure of DNA embedded with histone 3 (H3) and myeloperoxidase (MPO) in N. caninum tachyzoite-induced macrophage-derived ETs. Furthermore, the lactate dehydrogenase (LDH) levels in the supernatants of parasite-exposed macrophages were detected by a LDH Cytotoxicity Assay® kit. The results clearly demonstrated that N. caninum tachyzoites triggered bovine macrophage-derived ET-like structures. Inhibiting assays revealed that N. caninum tachyzoite-induced macrophage-mediated ET formation may be an ERK 1/2- and p38 MAPK-dependent cell death process. In conclusion, the present study is the first report on the formation of ETs in bovine macrophages against N. caninum tachyzoites and adds new data on the possible role of macrophages in vivo infection by capturing invasive stages and exposing them to other leukocytes.

Ovine vital neutrophil extracellular traps bind and impair Haemonchus contortus L3 in a breed-dependent manner.

This study aimed to characterize neutrophil response to Haemonchus contortus (Hc) in vitro using cells from parasite-resistant St. Croix (STC) and parasite-susceptible Suffolk (SUF) sheep. Neutrophils from Hc-primed and naive STC and SUF sheep were incubated with Hc larval antigen (HcLA), Hc worm antigen (HcWA) or complete media (CM). After HcLA exposure, neutrophils from STC and SUF formed extracellular traps composed of DNA. Stimulation with HcLA induced a 35-fold increase in extracellular DNA compared to CM controls. However, extracellular DNA was not found when neutrophils were cultured with HcWA. The formation of neutrophil extracellular traps (NET) in response to HcLA yields a low percentage of necrotic cells indicating a form of vital NETosis. Neutrophils from primed and naïve STC bound Hc L3 greater (93% and 68%) than SUF (78% and 45%; P < 0.001). Furthermore, STC neutrophils significantly reduced larval ATP levels compared to SUF neutrophils (0.05 µmol/L vs 0.1 µmol/L ATP, P < 0.001). These data indicate that ovine neutrophils bind, form vital NET and reduce ATP to Hc L3 in a breed and infection status-dependent manner.

Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps.

Heartworm disease is a zoonotic vector-borne disease caused by Dirofilaria immitis mainly affecting canids. Infectious third-stage larvae (L3) are transmitted to the definitive hosts via culicid mosquitoes; adult nematodes reside in the pulmonary arteries and in the right heart releasing unsheathed first-stage larvae (microfilariae) into the bloodstream leading to chronic and sometimes fatal disease. So far, early innate immune reactions triggered by these different D. immitis stages in the canine host have scarcely been investigated. Therefore, D. immitis microfilariae and L3 were analyzed for their capacity to induce neutrophil extracellular traps (NETs) in canine polymorphonuclear neutrophils (PMN). Overall, scanning electron microscopy analysis revealed both larval stages as strong inducers of canine NETosis. Co-localization of PMN-derived extracellular DNA with granulocytic histones, neutrophil elastase, or myeloperoxidase in parasite-entrapping structures confirmed the classical characteristics of NETosis. Quantitative analyses showed that both larval stages triggered canine NETs in a time-dependent but dose-independent manner. Moreover, parasite-induced NET formation was not influenced by the parasites viability since heat-inactivated microfilariae and L3 also induced NETs. In addition, parasite/PMN confrontation promoted significant entrapment but not killing of microfilariae and L3. Both, NETosis and larval entrapment was significantly reversed via DNase I treatments while treatments with the NADPH oxidase inhibitor diphenyleneiodonium failed to significantly influence these reactions. Interestingly, different types of NETs were induced by microfilariae and L3 since microfilarial stages merely induced spread and diffuse NETs while the larger L3 additionally triggered aggregated NET formation.

Entamoeba histolytica Trophozoites Induce a Rapid Non-classical NETosis Mechanism Independent of NOX2-Derived Reactive Oxygen Species and PAD4 Activity.

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.

Neutrophils and Malaria.

Neutrophils are abundant in the circulation and are one of the immune system's first lines of defense against infection. There has been substantial work carried out investigating the role of neutrophils in malaria and it is clear that during infection neutrophils are activated and are capable of clearing malaria parasites by a number of mechanisms. This review focuses on neutrophil responses to human malarias, summarizing evidence which helps us understand where neutrophils are, what they are doing, how they interact with parasites as well as their potential role in vaccine mediated immunity. We also outline future research priorities for these, the most abundant of leukocytes.

Resistance of Leishmania (Viannia) Panamensis to Meglumine Antimoniate or Miltefosine Modulates Neutrophil Effector Functions.

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.

Comparison of Picogreen and Sytox Orange Stains in Quantitative Analysis of Extracellular Trap Formed against Toxoplasma gondii in Polymorphonuclear Leukocytes from Sheep.

OBJECTIVE: The present study aimed to compare the effectiveness of two extracellular DNA dyes (Sytox Orange and PicoGreen) in quantitative measurement of the DNA that forms the backbone of the extracellular trap structures in vitro. Toward this aim, the co-culture of polymorphonuclear leucocytes (PMNs) and Toxoplasma gondii tachyzoites isolated from sheep was selected as model. METHODS: T. gondii tachyzoites and PMNs isolated from the sheep were incubated for varied durations (30, 60, 90 and 120 min); the extracellular DNA released following this incubation was stained using two different dyes. RESULTS: In the present study, no statistical significant difference was observed between the two extracellular DNA dyes with regard to the measurement of extracellular DNA released for all the incubation durations (p>0.05), except for the 30-min incubation (p=0.014). CONCLUSION: There was a statistically significant difference at 30 min incubation time. This difference may be attributed to the staining properties of the dye. Researchers studying in vitro netosis are recommended to use two different extracellular DNA dyes for the quantitative analysis of extracellular DNA formed during short-term incubation.

Role of NETs in the difference in host susceptibility to Toxoplasma gondii between sheep and cattle.

The main aim of this study was to compare extracellular traps (NETs) formation by polymorphonuclear neutrophils (PMNs) of cattle and sheep when exposed to T. gondii tachyzoites in vitro. The effects of parasite concentrations and different incubation periods on NETs development in cattle and sheep PMNs were studied. The effect of NET structures on host cell invasion by tachyzoites was also studied. This is the first report of NETs development by sheep and cattle PMNs against T. gondii in vitro. T. gondii-induced extracellular DNA production from PMNs was dependent on tachyzoite concentrations and incubation time in both sheep and cattle. Many nuclear and cytoplasmic changes were observed in sheep and cattle PMNs after exposure to T. gondii tachyzoites. The typical appearance of NETs, with MPO, NE and histone (H3) attached to extracellular DNA, was observed. Tachyzoites were entrapped within this structure. Myeloperoxidase (MPO) activity was higher in the cattle PMN-tachyzoite co-cultures than sheep. NETs structures released from sheep PMNs caused mechanical immobilisation of T. gondii tachyzoites, however, NET structures released from cattle PMNs may be lethal to tachyzoites. Bovine MPO may have a lethal effect on T. gondii tachyzoites in vitro during a 3h incubation. Besides other mechanisms that effect on host susceptibility to T. gondii in sheep and cattle, extracellular traps formation as a part of immunological reactions may be play a role in host susceptibility to T. gondii.

Gastropod-derived haemocyte extracellular traps entrap metastrongyloid larval stages of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior.

BACKGROUND: Phagocyte-derived extracellular traps (ETs) were recently demonstrated mainly in vertebrate hosts as an important effector mechanism against invading parasites. In the present study we aimed to characterize gastropod-derived invertebrate extracellular phagocyte trap (InEPT) formation in response to larval stages of important canine and feline metastrongyloid lungworms. Gastropod haemocytes were isolated from the slug species Arion lusitanicus and Limax maximus, and the snail Achatina fulica, and exposed to larval stages of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior and investigated for gastropod-derived InEPT formation. RESULTS: Phase contrast as well as scanning electron microscopy (SEM) analyses of lungworm larvae-exposed haemocytes revealed ET-like structures to be extruded by haemocytes thereby contacting and ensnaring the parasites. Co-localization studies of haemocyte-derived extracellular DNA with histones and myeloperoxidase in larvae-entrapping structures confirmed classical characteristics of ETs. In vivo exposure of slugs to A. vasorum larvae resulted in InEPTs being extruded from haemocytes in the slug mucous extrapallial space emphasizing the pivotal role of this effector mechanism against invasive larvae. Functional larval entrapment assays demonstrated that almost half of the haemocyte-exposed larvae were contacted or even immobilized by released InEPTs. Overall, as reported for mammalian-derived ETs, different types of InEPTs were here observed, i.e. aggregated, spread and diffused InEPTs. CONCLUSIONS: To our knowledge, this study represents the first report on metastrongyloid lungworm-triggered ETosis in gastropods thereby providing evidence of early mollusc host innate immune reactions against invading larvae. These findings will contribute to the better understanding on complex parasite-intermediate host interactions since different gastropod species bear different transmitting capacities for metastrongyloid infections.

Human Leukocytes Kill Brugia malayi Microfilariae Independently of DNA-Based Extracellular Trap Release.

BACKGROUND: Wuchereria bancrofti, Brugia malayi and Brugia timori infect over 100 million people worldwide and are the causative agents of lymphatic filariasis. Some parasite carriers are amicrofilaremic whilst others facilitate mosquito-based disease transmission through blood-circulating microfilariae (Mf). Recent findings, obtained largely from animal model systems, suggest that polymorphonuclear leukocytes (PMNs) contribute to parasitic nematode-directed type 2 immune responses. When exposed to certain pathogens PMNs release extracellular traps (NETs) in the form of chromatin loaded with various antimicrobial molecules and proteases. PRINCIPAL FINDINGS: In vitro, PMNs expel large amounts of NETs that capture but do not kill B. malayi Mf. NET morphology was confirmed by fluorescence imaging of worm-NET aggregates labelled with DAPI and antibodies to human neutrophil elastase, myeloperoxidase and citrullinated histone H4. A fluorescent, extracellular DNA release assay was used to quantify and observe Mf induced NETosis over time. Blinded video analyses of PMN-to-worm attachment and worm survival during Mf-leukocyte co-culture demonstrated that DNase treatment eliminates PMN attachment in the absence of serum, autologous serum bolsters both PMN attachment and PMN plus peripheral blood mononuclear cell (PBMC) mediated Mf killing, and serum heat inactivation inhibits both PMN attachment and Mf killing. Despite the effects of heat inactivation, the complement inhibitor compstatin did not impede Mf killing and had little effect on PMN attachment. Both human PMNs and monocytes, but not lymphocytes, are able to kill B. malayi Mf in vitro and NETosis does not significantly contribute to this killing. Leukocytes derived from presumably parasite-naïve U.S. resident donors vary in their ability to kill Mf in vitro, which may reflect the pathological heterogeneity associated with filarial parasitic infections. CONCLUSIONS/SIGNIFICANCE: Human innate immune cells are able to recognize, attach to and kill B. malayi microfilariae in an in vitro system. This suggests that, in vivo, the parasites can evade this ability, or that only some human hosts support an infection with circulating Mf.

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.