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• W2011491918 abstract "Neutrophils contribute to pathogen clearance by producing neutrophil extracellular traps (NETs), which are genomic DNA-based net-like structures that capture bacteria and fungi. Although NETs also express antiviral factors, such as myeloperoxidase and α-defensin, the involvement of NETs in antiviral responses remains unclear. We show that NETs capture human immunodeficiency virus (HIV)-1 and promote HIV-1 elimination through myeloperoxidase and α-defensin. Neutrophils detect HIV-1 by Toll-like receptors (TLRs) TLR7 and TLR8, which recognize viral nucleic acids. Engagement of TLR7 and TLR8 induces the generation of reactive oxygen species that trigger NET formation, leading to NET-dependent HIV-1 elimination. However, HIV-1 counteracts this response by inducing C-type lectin CD209-dependent production of interleukin (IL)-10 by dendritic cells to inhibit NET formation. IL-10 suppresses the reactive oxygen species-dependent generation of NETs induced upon TLR7 and TLR8 engagement, resulting in disrupted NET-dependent HIV-1 elimination. Therefore, NET formation is an antiviral response that is counteracted by HIV-1. Neutrophils contribute to pathogen clearance by producing neutrophil extracellular traps (NETs), which are genomic DNA-based net-like structures that capture bacteria and fungi. Although NETs also express antiviral factors, such as myeloperoxidase and α-defensin, the involvement of NETs in antiviral responses remains unclear. We show that NETs capture human immunodeficiency virus (HIV)-1 and promote HIV-1 elimination through myeloperoxidase and α-defensin. Neutrophils detect HIV-1 by Toll-like receptors (TLRs) TLR7 and TLR8, which recognize viral nucleic acids. Engagement of TLR7 and TLR8 induces the generation of reactive oxygen species that trigger NET formation, leading to NET-dependent HIV-1 elimination. However, HIV-1 counteracts this response by inducing C-type lectin CD209-dependent production of interleukin (IL)-10 by dendritic cells to inhibit NET formation. IL-10 suppresses the reactive oxygen species-dependent generation of NETs induced upon TLR7 and TLR8 engagement, resulting in disrupted NET-dependent HIV-1 elimination. Therefore, NET formation is an antiviral response that is counteracted by HIV-1. Neutrophils sense HIV-1 by TLR7 and TLR8 to produce NETs NETs capture HIV-1 to prevent HIV-1 from spreading NETs ensure high local concentrations of antiviral factors to eliminate HIV-1 HIV-1 induces CD209-dependent IL-10 production by DCs to suppress NET formation Neutrophils play a pivotal role in the elimination of pathogens invading a host (Papayannopoulos and Zychlinsky, 2009Papayannopoulos V. Zychlinsky A. NETs: a new strategy for using old weapons.Trends Immunol. 2009; 30: 513-521Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). Recent studies have revealed that neutrophils undergo suicide that is beneficial for host defense, releasing net-like structures into the extracellular space to prevent the spreading of bacteria and fungi (Brinkmann et al., 2004Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6178) Google Scholar; Urban et al., 2009Urban C.F. Ermert D. Schmid M. Abu-Abed U. Goosmann C. Nacken W. Brinkmann V. Jungblut P.R. Zychlinsky A. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans.PLoS Pathog. 2009; 5: e1000639https://doi.org/10.1371/journal.ppat.1000639Crossref PubMed Scopus (1114) Google Scholar). The extracellular structures are composed of genomic DNA and histones, and are designated neutrophil extracellular traps (NETs) (Brinkmann et al., 2004Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6178) Google Scholar). Mitochondrial DNA is also a component of NETs (Yousefi et al., 2009Yousefi S. Mihalache C. Kozlowski E. Schmid I. Simon H.U. Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps.Cell Death Differ. 2009; 16: 1438-1444Crossref PubMed Scopus (621) Google Scholar). Eosinophils and mast cells also produce DNA-based NET-like structures to prevent the spreading of bacteria (von Köckritz-Blickwede et al., 2008von Köckritz-Blickwede M. Goldmann O. Thulin P. Heinemann K. Norrby-Teglund A. Rohde M. Medina E. Phagocytosis-independent antimicrobial activity of mast cells by means of extracellular trap formation.Blood. 2008; 111: 3070-3080Crossref PubMed Scopus (422) Google Scholar; Yousefi et al., 2008Yousefi S. Gold J.A. Andina N. Lee J.J. Kelly A.M. Kozlowski E. Schmid I. Straumann A. Reichenbach J. Gleich G.J. et al.Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense.Nat. Med. 2008; 14: 949-953Crossref PubMed Scopus (702) Google Scholar). Reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) mediate NET formation induced by phorbol myristate acetate (PMA), a potent inducer of NET formation (Fuchs et al., 2007Fuchs T.A. Abed U. Goosmann C. Hurwitz R. Schulze I. Wahn V. Weinrauch Y. Brinkmann V. Zychlinsky A. Novel cell death program leads to neutrophil extracellular traps.J. Cell Biol. 2007; 176: 231-241Crossref PubMed Scopus (2181) Google Scholar; Papayannopoulos et al., 2010Papayannopoulos V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.J. Cell Biol. 2010; 191: 677-691Crossref PubMed Scopus (1232) Google Scholar; Hakkim et al., 2011Hakkim A. Fuchs T.A. Martinez N.E. Hess S. Prinz H. Zychlinsky A. Waldmann H. Activation of the Raf-MEK-ERK pathway is required for neutrophil extracellular trap formation.Nat. Chem. Biol. 2011; 7: 75-77Crossref PubMed Scopus (479) Google Scholar). MAPK activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to induce ROS generation in response to PMA. ROS damage the membrane of secretory granules and lysosomes, and induce the translocation of neutrophil elastase from these organelles to the nucleus. Neutrophil elastase then promotes the cleavage of histones, leading to chromatin release into the cytosol. NETs are highly sticky and capture extracellular microbes such as bacteria and fungi (Brinkmann et al., 2004Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6178) Google Scholar; Urban et al., 2009Urban C.F. Ermert D. Schmid M. Abu-Abed U. Goosmann C. Nacken W. Brinkmann V. Jungblut P.R. Zychlinsky A. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans.PLoS Pathog. 2009; 5: e1000639https://doi.org/10.1371/journal.ppat.1000639Crossref PubMed Scopus (1114) Google Scholar). Furthermore, various antimicrobial factors, such as cathelicidin, calprotectin, myeloperoxidase (MPO), and α-defensin, are expressed on NETs to mediate the NET-dependent elimination of bacteria and fungi (Brinkmann et al., 2004Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6178) Google Scholar; Urban et al., 2009Urban C.F. Ermert D. Schmid M. Abu-Abed U. Goosmann C. Nacken W. Brinkmann V. Jungblut P.R. Zychlinsky A. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans.PLoS Pathog. 2009; 5: e1000639https://doi.org/10.1371/journal.ppat.1000639Crossref PubMed Scopus (1114) Google Scholar). Although the pathological role of NETs remains to be elucidated, NET formation is potently induced in lungs of mice infected with influenza A virus and is affected in neutrophils isolated from cats infected with feline immunodeficiency virus (Wardini et al., 2010Wardini A.B. Guimarães-Costa A.B. Nascimento M.T. Nadaes N.R. Danelli M.G. Mazur C. Benjamim C.F. Saraiva E.M. Pinto-da-Silva L.H. Characterization of neutrophil extracellular traps in cats naturally infected with feline leukemia virus.J. Gen. Virol. 2010; 91: 259-264Crossref PubMed Scopus (90) Google Scholar; Hemmers et al., 2011Hemmers S. Teijaro J.R. Arandjelovic S. Mowen K.A. PAD4-mediated neutrophil extracellular trap formation is not required for immunity against influenza infection.PLoS ONE. 2011; 6: e22043https://doi.org/10.1371/journal.pone.0022043Crossref PubMed Scopus (186) Google Scholar; Narasaraju et al., 2011Narasaraju T. Yang E. Samy R.P. Ng H.H. Poh W.P. Liew A.A. Phoon M.C. van Rooijen N. Chow V.T. Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis.Am. J. Pathol. 2011; 179: 199-210Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar). Human immunodeficiency virus (HIV)-1 belongs to the lentivirus subfamily of retroviruses and infects human CD4+ T cells and human macrophages (Haseltine, 1988Haseltine W.A. Replication and pathogenesis of the AIDS virus.J. Acquir. Immune Defic. Syndr. 1988; 1: 217-240PubMed Google Scholar). HIV-1 infection disrupts the effectiveness of the human immune system and renders hosts susceptible to opportunistic infections, leading to the development of acquired immunodeficiency syndrome (AIDS). Thus, elucidation of the antiviral response against HIV-1 is clearly important. Although MPO and α-defensin which are expressed on NETs, have the ability to inactivate HIV-1 (Klebanoff and Coombs, 1992Klebanoff S.J. Coombs R.W. Viricidal effect of polymorphonuclear leukocytes on human immunodeficiency virus-1. Role of the myeloperoxidase system.J. Clin. Invest. 1992; 89: 2014-2017Crossref PubMed Scopus (76) Google Scholar; Moguilevsky et al., 1992Moguilevsky N. Steens M. Thiriart C. Prieels J.P. Thiry L. Bollen A. Lethal oxidative damage to human immunodeficiency virus by human recombinant myeloperoxidase.FEBS Lett. 1992; 302: 209-212Abstract Full Text PDF PubMed Scopus (13) Google Scholar; Ding et al., 2009Ding J. Chou Y.Y. Chang T.L. Defensins in viral infections.J. Innate Immun. 2009; 1: 413-420Crossref PubMed Scopus (67) Google Scholar), there are few reports describing a role of NETs in the elimination of HIV-1. Here, we examined the involvement of NETs in the anti-HIV-1 host defense response. Human neutrophils produced NETs consisting of DNA after PMA stimulation (see Figure S1A available online). The size and formation ratio of the NETs were defined by the strength of the PMA stimulation. Superresolution structured illumination microscopy (SR-SIM), a recently developed imaging technique (Schermelleh et al., 2010Schermelleh L. Heintzmann R. Leonhardt H. A guide to super-resolution fluorescence microscopy.J. Cell Biol. 2010; 190: 165-175Crossref PubMed Scopus (977) Google Scholar), enabled us to observe the structure of the NETs at high resolution, and revealed that the NETs formed multilobulated structures composed of twisted DNA-based fibers (Figure S1B). Scanning electron microscopy (SEM) further confirmed that net-like structures were produced by neutrophils in the extracellular space (Figure S1C). We used SR-SIM to detect HIV-1 virions, because conventional fluorescence microscopy is unable to detect a single HIV-1 virion with sizes of less than 200 nm, the theoretical resolution limit (Schermelleh et al., 2010Schermelleh L. Heintzmann R. Leonhardt H. A guide to super-resolution fluorescence microscopy.J. Cell Biol. 2010; 190: 165-175Crossref PubMed Scopus (977) Google Scholar). SR-SIM revealed that HIV-1 virions were captured on the outer face and in a ditch of the NETs (Figures 1A–1C). Consistently, SEM revealed that the NETs produced by neutrophils bound to HIV-1 virions as 120 nm spheres (Figure 1D). The HIV-1 Gag protein was detected on the culture plates after stimulation of neutrophils with PMA, but was not detected after DNase I treatment (Figure 1E), indicating that the capture and removal of HIV-1 from the culture supernatant were dependent on DNA on the culture plates. The HIV-1 envelope glycoprotein was not involved in the binding of HIV-1 virions to the NETs (Figures 1D and 1F). A moment of NET binding to HIV-1 virions was observed by the time-lapse imaging analysis (Movie S1). Since histones, major components of NETs, are rich in positively charged basic amino acids, extracellular histones could attract the negatively charged envelope of HIV-1 virions. Consistently, HIV-1 virions were detected on histone H3 of the NETs (Figure S1D). Therefore, NETs capture HIV-1 to prevent HIV-1 from spreading. Next, we assessed whether the infectivity of HIV-1 captured by the NETs was altered. The infectivity of HIV-1 in the culture supernatant was reduced after incubation with PMA-stimulated neutrophils, but was not reduced after DNase I treatment (Figures 2A–2C), indicating that DNA produced by PMA-stimulated neutrophils mediates inactivation of HIV-1. Immunofluorescence analyses revealed that MPO and α-defensin were abundantly expressed on the NETs produced by neutrophils (Figure 2D, Figures S2A and S2B). Inhibition of MPO activity and neutralization of α-defensin resulted in an impaired virucidal response to HIV-1 on the NETs (Figure 2E). However, MPO inhibitor and anti-α-defensin-neutralizing antibody did not directly affect infectivity of HIV-1 (Figure 2F). Although histones have an antimicrobial activity (Brinkmann et al., 2004Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6178) Google Scholar), HIV-1 inactivation by NETs was not affected by neutralizing antibody against histone H1 and H2 (data not shown). Histone H3 and H4 might be involved in HIV-1 inactivation by NETs. These findings indicate that NETs ensure high local concentrations of MPO and α-defensin to inactivate HIV-1. The finding of HIV-1 elimination by NETs prompted us to assess whether the innate immune system detects HIV-1 and induces NET formation. We found that neutrophils efficiently produced NETs, which were rich in MPO and α-defensin, after stimulation with HIV-1MN, a replication-competent CXCR4-tropic HIV-1 strain (Figure 3A and Figure S2C). Neutrophils also produced NETs after stimulation with the CCR5-tropic strain HIV-1JR-FL and the HIV-1 vector HIV-1CSII (Figure 3A). Neutrophils sensed HIV-1, resulting in the capture of HIV-1 by NETs (Figure 3B). When CD4+ T cells and neutrophils were cocultured, neutrophils reduced HIV-1 infection efficiency in an extracellular DNA-dependent manner, indicating that NETs inhibit HIV-1 infection of CD4+ T cells (Figure 3C). These findings suggest that pathogen-recognition receptors expressed on neutrophils sense HIV-1 and induce the NET-dependent HIV-1 elimination. Toll-like receptors (TLRs) are the sensors for viruses that induce the antiviral response (Kawai and Akira, 2009Kawai T. Akira S. The roles of TLRs, RLRs and NLRs in pathogen recognition.Int. Immunol. 2009; 21: 317-337Crossref PubMed Scopus (1191) Google Scholar). In particular, TLR7 and TLR8 detect single-stranded RNA of the HIV-1 genome on plasmacytoid dendritic cells to induce the production of type I interferon, cytokines, and chemokines (Beignon et al., 2005Beignon A.S. McKenna K. Skoberne M. Manches O. DaSilva I. Kavanagh D.G. Larsson M. Gorelick R.J. Lifson J.D. Bhardwaj N. Endocytosis of HIV-1 activates plasmacytoid dendritic cells via Toll-like receptor-viral RNA interactions.J. Clin. Invest. 2005; 115: 3265-3275Crossref PubMed Scopus (539) Google Scholar; Heil et al., 2004Heil F. Hemmi H. Hochrein H. Ampenberger F. Kirschning C. Akira S. Lipford G. Wagner H. Bauer S. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8.Science. 2004; 303: 1526-1529Crossref PubMed Scopus (3054) Google Scholar; Gringhuis et al., 2010Gringhuis S.I. van der Vlist M. van den Berg L.M. den Dunnen J. Litjens M. Geijtenbeek T.B. HIV-1 exploits innate signaling by TLR8 and DC-SIGN for productive infection of dendritic cells.Nat. Immunol. 2010; 11: 419-426Crossref PubMed Scopus (215) Google Scholar). Because multiple TLRs are expressed on neutrophils and mediate ROS production (Hayashi et al., 2003Hayashi F. Means T.K. Luster A.D. Blood. 2003; 102: 2660-2669Crossref PubMed Scopus (719) Google Scholar; Hoarau et al., 2007Hoarau C. Gérard B. Lescanne E. Henry D. François S. Lacapère J.J. El Benna J. Dang P.M. Grandchamp B. Lebranchu Y. et al.TLR9 activation induces normal neutrophil responses in a child with IRAK-4 deficiency: involvement of the direct PI3K pathway.J. Immunol. 2007; 179: 4754-4765Crossref PubMed Scopus (53) Google Scholar; Wang et al., 2008Wang J.P. Bowen G.N. Padden C. Cerny A. Finberg R.W. Newburger P.E. Kurt-Jones E.A. Toll-like receptor-mediated activation of neutrophils by influenza A virus.Blood. 2008; 112: 2028-2034Crossref PubMed Scopus (118) Google Scholar), we examined an involvement of the TLR family members in NET formation induced by HIV-1. Inhibition of the vacuolar type H+-ATPase by bafilomycin A1 resulted in the suppression of HIV-1-induced NET formation (Figure 3D), suggesting that endolysosomal TLRs such as TLR3, TLR7, TLR8, and TLR9 mediate NET formation. Chemical inhibition of interleukin-1 receptor-associated kinase 4 (IRAK4), a critical regulator of all TLRs except TLR3 (Kawai and Akira, 2009Kawai T. Akira S. The roles of TLRs, RLRs and NLRs in pathogen recognition.Int. Immunol. 2009; 21: 317-337Crossref PubMed Scopus (1191) Google Scholar), also resulted in the suppression of HIV-1-induced NET formation (Figure 3D), suggesting that TLR7, TLR8, and TLR9 are involved in NET formation. Neither bafilomycin A1 nor the IRAK4 inhibitor suppressed NET formation by PMA, and these compounds did not affect cell viability (Figures S3A and S3B). Dual-iODN and iODN2088, antagonists for TLR7, TLR8, and TLR9, inhibited NET formation by HIV-1, whereas G-type iODN, a selective antagonist for TLR9, did not alter NET formation efficiency (Figure 3E). These inhibitory oligonucleotides did not affect NET formation by PMA and cell viability (Figures S3C and S3D). When CD4+ T cells and neutrophils were cocultured, neutrophils reduced HIV-1 infection efficiency in a TLR7- and TLR8-dependent manner (Figure 3F). These findings indicate that TLR7 and TLR8 are responsible for the NET-dependent HIV-1 elimination. We next examined the molecular mechanism of TLR7- and TLR8-mediated NET formation. We focused on a role of ROS, because NADPH oxidase-dependent ROS generation was involved in the NET-dependent elimination of HIV-1 by PMA (Figures S3E–S3G). Neutrophils produced NETs, which were rich in MPO and α-defensin, in response to R848 and CL075, synthetic ligands for TLR7 and TLR8 (Figure 3G and Figure S2D). Both HIV-1 and R848 triggered the generation of ROS, and R848-induced ROS generation was mediated by NADPH oxidase (Figures 3H and 3I). Butylated hydroxyanisole (BHA), a ROS scavenger, and apocynin, an inhibitor of NADPH oxidase, inhibited the NET formation induced by R848, but did not alter cell viability (Figure 3J and Figure S3H). Consistently, R848 induced NET-dependent HIV-1 elimination through ROS generated by NADPH oxidase (Figure 3K). Type I IFN, which is often slightly induced by R848 (Kawai and Akira, 2009Kawai T. Akira S. The roles of TLRs, RLRs and NLRs in pathogen recognition.Int. Immunol. 2009; 21: 317-337Crossref PubMed Scopus (1191) Google Scholar), was not involved in NET-dependent HIV-1 elimination (Figure 3L). Thus, TLR7 and TLR8 induce NADPH oxidase-dependent ROS generation to eliminate HIV-1 by NETs. Accumulating evidence has revealed that HIV-1 evolves to manage immune cells, especially dendritic cells, for its efficient propagation. For example, HIV-1 binds to the dendritic cell-specific lectin CD209, also called DC-SIGN, to move along an infectious synapse generated between CD209+ dendritic cells and CD4+ T cells, resulting in efficient infection of CD4+ T cells (Geijtenbeek et al., 2000Geijtenbeek T.B. Kwon D.S. Torensma R. van Vliet S.J. van Duijnhoven G.C. Middel J. Cornelissen I.L. Nottet H.S. KewalRamani V.N. Littman D.R. et al.DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells.Cell. 2000; 100: 587-597Abstract Full Text Full Text PDF PubMed Scopus (2041) Google Scholar; Arrighi et al., 2004Arrighi J.F. Pion M. Garcia E. Escola J.M. van Kooyk Y. Geijtenbeek T.B. Piguet V. DC-SIGN-mediated infectious synapse formation enhances X4 HIV-1 transmission from dendritic cells to T cells.J. Exp. Med. 2004; 200: 1279-1288Crossref PubMed Scopus (199) Google Scholar). Since mucosal tissues are rich in CD209+ dendritic cells and CD4+ T cells, HIV-1 uses these tissues as major HIV-1 entry routes (Geijtenbeek et al., 2000Geijtenbeek T.B. Kwon D.S. Torensma R. van Vliet S.J. van Duijnhoven G.C. Middel J. Cornelissen I.L. Nottet H.S. KewalRamani V.N. Littman D.R. et al.DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells.Cell. 2000; 100: 587-597Abstract Full Text Full Text PDF PubMed Scopus (2041) Google Scholar; Jameson et al., 2002Jameson B. Baribaud F. Pöhlmann S. Ghavimi D. Mortari F. Doms R.W. Iwasaki A. Expression of DC-SIGN by dendritic cells of intestinal and genital mucosae in humans and rhesus macaques.J. Virol. 2002; 76: 1866-1875Crossref PubMed Scopus (228) Google Scholar). Importantly, mucosal tissues are also rich in neutrophils, and NETs are suggested to be involved in the mucosal inflammation (Mumy and McCormick, 2009Mumy K.L. McCormick B.A. The role of neutrophils in the event of intestinal inflammation.Curr. Opin. Pharmacol. 2009; 9: 697-701Crossref PubMed Scopus (36) Google Scholar; Savchenko et al., 2011Savchenko A.S. Inoue A. Ohashi R. Jiang S. Hasegawa G. Tanaka T. Hamakubo T. Kodama T. Aoyagi Y. Ushiki T. et al.Long pentraxin 3 (PTX3) expression and release by neutrophils in vitro and in ulcerative colitis.Pathol. Int. 2011; 61: 290-297Crossref PubMed Scopus (59) Google Scholar). Therefore, we assessed whether HIV-1 makes use of CD209+ dendritic cells to escape the NET-dependent antiviral response. Neutrophils produced NETs less efficiently in response to R848 after exposure to the culture supernatant of monocyte-derived dendritic cells incubated with HIV-1 (Figure 4A). HIV-1 with the envelope glycoprotein, but not HIV-1 lacking the envelope glycoprotein, induced the production of a NET-suppressive factor by dendritic cells (Figure 4A). Consistently, recombinant gp120 induced the production of the NET-suppressive factor by dendritic cells (Figure 4A). CD4, CXCR4, and CD209 are known to bind to the envelope glycoprotein of CXCR4-tropic HIV-1 and to induce an immune response (Lee et al., 2003Lee C. Liu Q.H. Tomkowicz B. Yi Y. Freedman B.D. Collman R.G. Macrophage activation through CCR5- and CXCR4-mediated gp120-elicited signaling pathways.J. Leukoc. Biol. 2003; 74: 676-682Crossref PubMed Scopus (110) Google Scholar; Shan et al., 2007Shan M. Klasse P.J. Banerjee K. Dey A.K. Iyer S.P. Dionisio R. Charles D. Campbell-Gardener L. Olson W.C. Sanders R.W. et al.HIV-1 gp120 mannoses induce immunosuppressive responses from dendritic cells.PLoS Pathog. 2007; 3: e169Crossref PubMed Scopus (124) Google Scholar). An anti-CD209-neutralizing antibody inhibited the production of the NET-suppressive factor by dendritic cells stimulated with HIV-1, but did not alter viability of dendritic cells and neutrophils (Figures 4B and 4C), indicating that HIV-1 stimulated CD209 on dendritic cells to suppress TLR-mediated NET formation. However, neither an anti-CD4-neutralizing antibody nor the CXCR4 antagonist T22 affected the production of the NET-suppressive factor by dendritic cells incubated with HIV-1 (Figure 4B). Thus, HIV-1 takes advantage of the functions of CD209 on dendritic cells not only for efficient infection of the target cells but also to escape the NET-mediated antiviral system. Previous studies have revealed that the immunosuppressive cytokine interleukin (IL)-10 is produced by dendritic cells after engagement of CD209, and that the expression level of plasma IL-10 correlates with the progression of AIDS (Stylianou et al., 1999Stylianou E. Aukrust P. Kvale D. Müller F. Frøland S.S. IL-10 in HIV infection: increasing serum IL-10 levels with disease progression—down-regulatory effect of potent anti-retroviral therapy.Clin. Exp. Immunol. 1999; 116: 115-120Crossref PubMed Scopus (161) Google Scholar; Geijtenbeek et al., 2003Geijtenbeek T.B. Van Vliet S.J. Koppel E.A. Sanchez-Hernandez M. Vandenbroucke-Grauls C.M. Appelmelk B. Van Kooyk Y. Mycobacteria target DC-SIGN to suppress dendritic cell function.J. Exp. Med. 2003; 197: 7-17Crossref PubMed Scopus (897) Google Scholar; Shan et al., 2007Shan M. Klasse P.J. Banerjee K. Dey A.K. Iyer S.P. Dionisio R. Charles D. Campbell-Gardener L. Olson W.C. Sanders R.W. et al.HIV-1 gp120 mannoses induce immunosuppressive responses from dendritic cells.PLoS Pathog. 2007; 3: e169Crossref PubMed Scopus (124) Google Scholar; Naicker et al., 2009Naicker D.D. Werner L. Kormuth E. Passmore J.A. Mlisana K. Karim S.A. Ndung'u T. Interleukin-10 promoter polymorphisms influence HIV-1 susceptibility and primary HIV-1 pathogenesis.J. Infect. Dis. 2009; 200: 448-452Crossref PubMed Scopus (58) Google Scholar). IL-33 is also produced by dendritic cells after engagement of CD209 and suppresses inflammation through a unique TH2 pathway (Anthony et al., 2011Anthony R.M. Kobayashi T. Wermeling F. Ravetch J.V. Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway.Nature. 2011; 475: 110-113Crossref PubMed Scopus (483) Google Scholar). Next, we attempted to identify the NET-suppressive factor produced by dendritic cells with CD209 stimulation. An anti-IL-10-neutralizing antibody, but not an anti-IL-33-neutralizing antibody, inhibited the suppression of NET formation by the culture supernatant of dendritic cells with CD209 stimulation (Figures 4D and 4E). When CD209+ dendritic cells and neutrophils were cocultured, dendritic cells inhibited HIV-1-induced NET formation in a CD209- and IL-10-dependent manner (Figure 4F), strongly suggesting that HIV-1 escapes from NETs under physiological conditions. Consistently, dendritic cells produced IL-10 after sensing HIV-1 with the envelope glycoprotein, but not HIV-1 lacking the envelope glycoprotein (Figures 4G and 4H). Dendritic cells also produced IL-10 after stimulation with recombinant gp120 (Figure 4H), indicating that CD209 senses HIV-1 envelope glycoprotein to induce IL-10 production. IL-10 inhibited the NET formation and NET-dependent HIV-1 elimination induced by R848, but did not alter cell viability (Figures 4I and 4J and Figure S3I). However, IL-10 failed to inhibit the NET formation and HIV-1 elimination induced by PMA (Figures S3J and S3K). Therefore, HIV-1 induces CD209-dependent production of IL-10 by dendritic cells to suppress NET formation. IL-10 is known to suppress the inflammatory immune response by blocking activation of the TLR signaling pathway (Chang et al., 2009Chang J. Kunkel S.L. Chang C.H. Negative regulation of MyD88-dependent signaling by IL-10 in dendritic cells.Proc. Natl. Acad. Sci. USA. 2009; 106: 18327-18332Crossref PubMed Scopus (28) Google Scholar). Phosphorylation of NCF1 is a key step in activation of the NADPH oxidase complex (Johnson et al., 1998Johnson J.L. Park J.W. Benna J.E. Faust L.P. Inanami O. Babior B.M. Activation of p47(PHOX), a cytosolic subunit of the leukocyte NADPH oxidase. Phosphorylation of ser-359 or ser-370 precedes phosphorylation at other sites and is required for activity.J. Biol. Chem. 1998; 273: 35147-35152Crossref PubMed Scopus (124) Google Scholar; Dang et al., 2006Dang P.M. Elbim C. Marie J.C. Chiandotto M. Gougerot-Pocidalo M.A. El-Benna J. Anti-inflammatory effect of interleukin-10 on human neutrophil respiratory burst involves inhibition of GM-CSF-induced p47PHOX phosphorylation through a decrease in ERK1/2 activity.FASEB J. 2006; 20: 1504-1506Crossref PubMed Scopus (56) Google Scholar). IL-10 inhibited R848-induced assembly of p38 MAPK and NCF1 (Figure S4A) and phosphorylation of NCF1 (Figure 4K). Consistently, IL-10 inhibited R848-induced generation of ROS and subsequent nuclear translocation of neutrophil elastase (Figure 4L and Figure S4B). However IL-10 did not inhibit NADPH oxidase-dependent ROS generation and nuclear translocation of neutrophil elastase induced by PMA (Figures S3L, S4A, and S4B). These findings indicated that IL-10 inhibits TLR7- and TLR8-signaling pathway, resulting in the suppression of NADPH oxidase-dependent NET formation. In the present study, we have shown NET induction by the innate immune system and HIV-1 elimination by NETs. Previous studies have revealed that NETs also eliminate Staphylococcus aureus and Candida albicans, causative agents of opportunistic infections in AIDS patients (Haseltine, 1988Haseltine W.A. Replication and pathogenesis of the AIDS virus.J. Acquir. Immune Defic. Syndr. 1988; 1: 217-240PubMed Google Scholar; Brinkmann et al., 2004Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6178) Google Scholar; Urban et al., 2009Urban C.F. Ermert D. Schmid M. Abu-Abed U. Goosmann C. Nacken W. Brinkmann V. Jungblut P.R. Zychlinsky A. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans.PLoS Pathog. 2009; 5: e1000639https://doi.org/10.1371/journal.ppat.1000639Crossref PubMed Scopus (1114) Google Scholar). Thus, induction of NET formation seems to be a promising immunotherapeutic strategy for AIDS patients. However, there are difficulties associated with eliminating HIV-1 by NETs. This is because a progressive decrease in the number of neutrophils occurs in AIDS patients, and the host defense systems are disrupted by HIV-1 (Scadden, 1992Scadden D.T. The use of GM-CSF in AIDS.Infection. 1992; 20: S103-S106Crossref PubMed Scopus (9) Google Scholar; Shan et al., 2007Shan M. Klasse P.J. Banerjee K. Dey A.K. Iyer S.P. Dionisio R. Charles D. Campbell-Gardener L. Olson W.C. Sanders R.W. et al.HIV-1 gp120 mannoses induce immunosuppressive responses from dendritic cells.PLoS Pathog. 2007; 3: e169Crossref PubMed Scopus (124) Google Scholar; D'Souza et al., 2008D'Souza A. Jaiyesimi I. Trainor L. Venuturumili P. Granulocyte colony-stimulating factor administration: adverse events.Transfus. Med. Rev. 2008; 22: 280-290Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The severe neutropenia and immune disruption by HIV-1 might complicate HIV-1 elimination by the NET antiviral system and lead to the progression of AIDS. To recover well from neutropenia, administration of granulocyte monocyte colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF) is effective (Scadden, 1992Scadden D.T. The use of GM-CSF in AIDS.Infection. 1992; 20: S103-S106Crossref PubMed Scopus (9) Google Scholar; D'Souza et al., 2008D'Souza A. Jaiyesimi I. Trainor L. Venuturumili P. Granulocyte colony-stimulating factor administration: adverse events.Transfus. Med. Rev. 2008; 22: 280-290Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). These growth factors stimulate the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. In addition, GM-CSF has the ability to assist NET formation (Martinelli et al., 2004Martinelli S. Urosevic M. Daryadel A. Oberholzer P.A. Baumann C. Fey M.F. Dummer R. Simon H.U. Yousefi S. Induction of genes mediating interferon-dependent extracellular trap formation during neutrophil differentiation.J. Biol. Chem. 2004; 279: 44123-44132Crossref PubMed Scopus (218) Google Scholar). Inhibition of HIV-1 interactions with CD209 may also support induction of the NET antiviral system because IL-10 inhibits not only the formation of NETs but also the activation of the G-CSF and GM-CSF signaling pathways (Dang et al., 2006Dang P.M. Elbim C. Marie J.C. Chiandotto M. Gougerot-Pocidalo M.A. El-Benna J. Anti-inflammatory effect of interleukin-10 on human neutrophil respiratory burst involves inhibition of GM-CSF-induced p47PHOX phosphorylation through a decrease in ERK1/2 activity.FASEB J. 2006; 20: 1504-1506Crossref PubMed Scopus (56) Google Scholar; Yoshimura et al., 2007Yoshimura A. Naka T. Kubo M. SOCS proteins, cytokine signalling and immune regulation.Nat. Rev. Immunol. 2007; 7: 454-465Crossref PubMed Scopus (1192) Google Scholar). Therefore, a combination therapy, with recovery from neutropenia and inhibition of HIV-1 immune escape, may be effective in AIDS patients. Neutrophils (2 × 105 cells) were treated as indicated and then incubated for 6 hr with an HIV-1 vector capable of expressing firefly luciferase after integration into the host genome. Alternatively, the HIV-1 vector incubated for 6 hr in the absence of neutrophils was used as input control. Molt-4 T cells (2 × 105 cells) were incubated with the culture supernatant for 48 hr to determine the infectivity of the HIV-1 vector in the culture supernatant. Separately, Molt-4 T cells were seeded into a culture plate after harvesting of the culture supernatant and were incubated for 48 hr to determine the infectivity of the HIV-1 vector on the culture plate. After the incubation, the Molt-4 T cells were subjected to a luciferase reporter assay. The luciferase activity in Molt-4 T cells infected with the treated HIV-1 vector was divided by the luciferase activity in Molt-4 T cells infected with the input HIV-1 vector to determine the infectivity of the treated HIV-1 vector. The experiments were performed in accordance with the guidelines of the ethics committee of Osaka University. Immunofluorescence staining was performed as previously described (Saitoh et al., 2011Saitoh T. Satoh T. Yamamoto N. Uematsu S. Takeuchi O. Kawai T. Akira S. Antiviral protein Viperin promotes Toll-like receptor 7- and Toll-like receptor 9-mediated type I interferon production in plasmacytoid dendritic cells.Immunity. 2011; 34: 352-363Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The samples were examined under an IX81-DSU spinning disc confocal microscope (Olympus). For measurement of the efficiency of NET formation, 20 fields of view in each sample (each 100 × 100 μm) were randomly chosen for analysis. The number of views with NETs was counted to measure the rate of NET appearance. Superresolution images of the samples were obtained using an Elyra SR-SIM (Zeiss). For measurement of the efficiency of HIV-1 capture by NETs, six fields of view in each sample (each 5 × 5 μm) were randomly chosen for analysis. Rendering of z stack SR-SIM images by the software Imaris (Bitplane AG) was used to construct the three-dimensional SR-SIM image. Samples on plastic coverslips were fixed with 2% formaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), postfixed with 1% osmium tetroxide and 0.5% potassium ferrocyanide in the same buffer, dehydrated through a graded series of ethanol, substituted with t-butyl alcohol, and freeze-dried. After freeze-drying, the samples were coated with osmium tetroxide and observed with a S-4800 field emission scanning electron microscope (Hitachi High-Technologies). Immunoblotting was performed as previously described (Saitoh et al., 2011Saitoh T. Satoh T. Yamamoto N. Uematsu S. Takeuchi O. Kawai T. Akira S. Antiviral protein Viperin promotes Toll-like receptor 7- and Toll-like receptor 9-mediated type I interferon production in plasmacytoid dendritic cells.Immunity. 2011; 34: 352-363Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The levels of HIV-1 p24 and IL-10 were measured by ELISA according to the manufacturer's instructions. Fluorescence-activated cell sorting (FACS) was performed as previously described (Saitoh et al., 2011Saitoh T. Satoh T. Yamamoto N. Uematsu S. Takeuchi O. Kawai T. Akira S. Antiviral protein Viperin promotes Toll-like receptor 7- and Toll-like receptor 9-mediated type I interferon production in plasmacytoid dendritic cells.Immunity. 2011; 34: 352-363Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). We are grateful to Drs. D. Trono, H. Miyoshi, G. Melikian, and Y. Koyanagi for providing invaluable materials. We also thank the members of the Laboratory of Host Defense for their assistance. This work was supported by JSPS KAKENHI Grant Number 23659231 (to T.S.), JSPS KAKENHI Grant Number 20002008 (to S.A.), JSPS Funding Program for World-Leading Innovative R&D on Science and Technology (to S.A.), and National Institutes of Health Grant P01 AI070167 (to S.A.). Download .pdf (2.48 MB) Help with pdf files Document S1. Figure S1, Figure S2, Figure S3, Figure S4, Supplemental Experimental Procedures, and Supplemental References Download .avi (.87 MB) Help with avi files Movie S1. Time-Lapse Imaging of Genomic DNA Binding to HIV-1 Virion-like ParticlesHuman neutrophils were stimulated with PMA (100 nM) for 1 hr in the presence of Hoechst 33342. The cells were subsequently incubated with Gag-EGFP (HIV-1 virion-like particles). The fluorescence images of Gag-EGFP (Green) and DNA (Magenta) were obtained every 90 s using LSM780 confocal laser scanning microscopy (Zeiss). Movie S1 is related to Figure 1." @default.
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• W2011491918 title "Neutrophil Extracellular Traps Mediate a Host Defense Response to Human Immunodeficiency Virus-1" @default.
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