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Leishmania Amazonensis Promastigotes Induce and Are Killed by Neutrophil Extracellular Traps

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Leishmania Amazonensis Promastigotes Induce and Are Killed by Neutrophil Extracellular Traps Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps Anderson B. Guimara˜ es-Costaa, Michelle T. C. Nascimentoa, Giselle S. Fromenta, Rodrigo P. P. Soaresb, Fernanda N. Morgadoc,Fa´ tima Conceic¸a˜ o-Silvac, and Elvira M. Saraivaa,1 aDepartamento de Imunologia, Instituto de Microbiologia Prof. Paulo de Go´es, Universidade Federal do Rio de Janeiro, Bloco I, 21941-902, Rio de Janeiro, Brazil; bLaborato´rio de Entomologia Me´dica, Centro de Pesquisas Rene´Rachou, Fundac¸a˜ o Oswaldo Cruz, 30190-002, Belo Horizonte, Brazil; and cLaborato´rio de Imunoparasitologia, Fundac¸a˜ o Oswaldo Cruz, 21045-900, Rio de Janeiro, Brazil Edited by Philippa Marrack, National Jewish Medical and Research Center, Denver, CO, and approved March 9, 2009 (received for review January 9, 2009) Neutrophils are short-lived leukocytes that die by apoptosis, ne- are inoculated by the insect vector into the skin and local crosis, and NETosis. Upon death by NETosis, neutrophils release inflammation begins. Neutrophils are rapidly recruited to the fibrous traps of DNA, histones, and granule proteins named neu- bite site, are activated, and ingest and kill parasites (11, 12). trophil extracellular traps (NETs), which can kill bacteria and fungi. However, some phagocytosed parasites resist the killing mech- Inoculation of the protozoan Leishmania into the mammalian skin anisms triggered in the phagolysosomes, delaying neutrophil causes local inflammation with neutrophil recruitment. Here, we apoptosis long enough to allow macrophage arrival at the investigated the release of NETs by human neutrophils upon their infection site (11, 13). Interestingly, it has been shown that interaction with Leishmania parasites and NETs’ ability to kill this apoptotic neutrophils harboring intracellular parasites are then protozoan. The NET constituents DNA, elastase, and histones were phagocytosed by macrophages (13). detected in traps associated to promastigotes by immunofluores- Although an increasing number of studies have investigated cence. Electron microscopy revealed that Leishmania was ensnared the role of neutrophils in the immune response to Leishmania, by NETs released by neutrophils. Moreover, Leishmania and its the influence of NETs on Leishmania survival is still unknown. surface lipophosphoglycan induced NET release by neutrophils in Induction of NETs and release of antimicrobial components may a parasite number- and dose-dependent manner. Disruption of contribute to the killing of Leishmania parasites before they are NETs by DNase treatment during Leishmania–neutrophil interac- engulfed by professional phagocytes. In the present study, we MEDICAL SCIENCES tion increased parasite survival, evidencing NETs’ leishmanicidal report that L. amazonensis promastigotes and amastigotes are effect. Leishmania killing was also elicited by NET-rich superna- able to induce NET formation in human neutrophils and that tants from phorbol 12-myristate 13-acetate-activated neutrophils. these webs possess leishmanicidal activity. Additionally, Leish- Immunoneutralization of histone during Leishmania–neutrophil mania LPG induces NET formation in neutrophils. Released interaction partially reverted Leishmania killing, and purified his- NETs exert an extracellular leishmanicidal activity caused, at tone killed the parasites. Meshes composed of DNA and elastase least in part, by the histones present in the meshes. Immuno- were evidenced in biopsies of human cutaneous leishmaniasis. NET histochemical analysis confirmed the occurrence of these webs in is an innate response that might contribute to diminish parasite lesions from human cutaneous leishmaniasis. burden in the Leishmania inoculation site. Results histone ͉ protozoa ͉ trypanosomatid NETs from Phorbol 12-Myristate 13-Acetate (PMA)-Activated or Naïve Neutrophils Kill Leishmania. Initially, we compared the parasite eutrophils are the most abundant leukocytes in the blood killing abilities of untreated and PMA-activated neutrophils Nand are the first cells recruited to inflamed tissues. They (Fig. S1A). Exposure of promastigotes to PMA-activated neu- have a pivotal role in immunity to infection not only by ingesting trophils resulted in lower promastigote survival relative to and destroying microbes, but also by secreting various cytokines. exposure to untreated neutrophils. PMA-activated neutrophils Although their lifespan is increased in the inflammatory site, killed 53% more promastigotes than naïve cells (Fig. S1A). Next, they are short-lived cells that die by spontaneous apoptosis (1). to distinguish between phagocytic and NET-derived microbici- NETosis is a recently described mechanism of neutrophil death dal activity, PMA-activated neutrophils were treated with the that occurs upon neutrophil activation ultimately leading to the actin inhibitor cytochalasin D (CytD) to block phagocytosis, release of neutrophil extracellular traps (NETs). NETs are webs thereby allowing the exclusive measurement of extracellular composed of chromatin and granular proteins that have been killing. CytD addition increased by 3-fold the parasite survival shown not only to ensnare bacteria and fungi, but also to provide compared with PMA-treated control cells, indicating that a high local concentration of antimicrobial molecules (2–7). phagocytosis contributed to parasite killing by PMA-activated Leishmania protozoa are the etiological agents of leishmaniasis, neutrophils (Fig. 1A). Because NETs are degraded by treatment which include a wide spectrum of disease in humans characterized with DNase (2), this enzyme was added to PMA-treated neu- trophils followed by addition of promastigotes to the cell cul- by lesions in the skin, mucosal surfaces, and visceral organs. tures. DNase treatment protected promastigotes from killing, Worldwide, Ͼ10 million people are affected by this disease that doubling parasite survival compared with those exposed to causes significant morbidity and mortality (8). Leishmania ama- PMA-activated neutrophils (Fig. 1A). Importantly, NET release zonensis is the causative agent of human cutaneous leishmaniasis in the New World, with a high proportion of cases evolving to the severe anergic diffuse cutaneous leishmaniasis (9). Author contributions: F.C.-S. and E.M.S. designed research; A.B.G.-C., M.T.C.N., G.S.F., and Two major Leishmania developmental stages are recognized: F.N.M. performed research; R.P.P.S. contributed new reagents/analytic tools; A.B.G.-C., amastigotes that live intracellularly in macrophages, and pro- M.T.C.N., and E.M.S. analyzed data; and E.M.S. wrote the paper. mastigotes that multiply inside the insect vector. Promastigotes The authors declare no conflict of interest. of all Leishmania species synthesize lipophosphoglycan (LPG), a This article is a PNAS Direct Submission. glycoconjugate localized over the entire protozoan cell surface, 1To whom correspondence should be addressed. E-mail: [email protected]. which is one of the first parasite molecules to contact the host This article contains supporting information online at www.pnas.org/cgi/content/full/ cells (10). Leishmania infection is initiated when promastigotes 0900226106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900226106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 30, 2021 ** A ** B 300 400 * ** 300 200 ** 200 (% of control) 100 (% of control) 100 survival Leishmania Leishmania survival Leishmania 0 0 PMA PMA + CytD PMA + DNase Medium CYTD DNase CYTD + DNase Neutrophil treatment Neutrophil treatment Fig. 1. Killing of L. amazonensis by NETs from activated and naïve neutrophils. PMA-activated (A) or naïve (B) neutrophils were treated with CytD or DNase-1 and incubated with promastigotes (1 cell/0.1 parasite ratio) for2hat35°C.Schneider’s complete medium was added to the cultures, and live parasites were counted after 2 days of incubation at 26 °C. Results of at least 7 independent experiments are shown as mean ϩ SEM. PMA raw number: 8.2 ϫ 106 Ϯ 0.7 ϫ 106 promastigotes; medium raw number: 3.2 ϫ 106 Ϯ 0.7 ϫ 106 promastigotes. *, P Ͻ 0.002; **, P Ͻ 0.0001. was not affected by CytD treatment (Fig. S1B). The number of Because the presence of DNA released from killed parasites in promastigotes cultured in the absence of neutrophils, increased this assay cannot be excluded, we measured the amount of DNA between 70 and 272 times over the initial number. Of note, released by 107 Tween-lysed promastigotes and found 2.5 ␮g/mL promastigote growth was also unaffected by PMA, DNase, DNA released by this procedure. At the 10:1 promastigote/ CytD, or DMSO (CytD’s diluent) at the same concentrations neutrophil ratio, 22 ␮g/mL of DNA was detected (Fig. 4A). used for neutrophil treatments. Based on this value, even if all 107 promastigotes in the inter- We then tested the NET killing characteristics of naïve action medium of our original assay were killed, parasite-derived neutrophils activated only by contact with parasites. We ob- DNA would account for no more than 11% of the total DNA served that parasites were able to induce NETs, and that these content measured. In addition to L. amazonensis, promastigotes structures participated in promastigote killing (Fig. 1B). Parasite of Leishmania chagasi and Leishmania major induced NET survival increased 39% and 60% over controls when neutrophils release by naïve neutrophils (Fig. 4C). were treated with CytD and DNase, respectively. When we simultaneously treated neutrophils with CytD and DNase, pro- Promastigote LPG Induce NETs. To explore parasite
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