Title: The Role of Extracellular Histones in Influenza Virus Pathogenesis
Abstract: Although exaggerated host immune responses have been implicated in influenza-induced lung pathogenesis, the etiologic factors that contribute to these events are not completely understood. We previously demonstrated that neutrophil extracellular traps exacerbate pulmonary injury during influenza pneumonia. Histones are the major protein components of neutrophil extracellular traps and are known to have cytotoxic effects. Here, we examined the role of extracellular histones in lung pathogenesis during influenza. Mice infected with influenza virus displayed high accumulation of extracellular histones, with widespread pulmonary microvascular thrombosis. Occluded pulmonary blood vessels with vascular thrombi often exhibited endothelial necrosis surrounded by hemorrhagic effusions and pulmonary edema. Histones released during influenza induced cytotoxicity and showed strong binding to platelets within thrombi in infected mouse lungs. Nasal wash samples from influenza-infected patients also showed increased accumulation of extracellular histones, suggesting a possible clinical relevance of elevated histones in pulmonary injury. Although histones inhibited influenza growth in vitro, in vivo treatment with histones did not yield antiviral effects and instead exacerbated lung pathology. Blocking with antihistone antibodies caused a marked decrease in lung pathology in lethal influenza–challenged mice and improved protection when administered in combination with the antiviral agent oseltamivir. These findings support the pathogenic effects of extracellular histones in that pulmonary injury during influenza was exacerbated. Targeting histones provides a novel therapeutic approach to influenza pneumonia. Although exaggerated host immune responses have been implicated in influenza-induced lung pathogenesis, the etiologic factors that contribute to these events are not completely understood. We previously demonstrated that neutrophil extracellular traps exacerbate pulmonary injury during influenza pneumonia. Histones are the major protein components of neutrophil extracellular traps and are known to have cytotoxic effects. Here, we examined the role of extracellular histones in lung pathogenesis during influenza. Mice infected with influenza virus displayed high accumulation of extracellular histones, with widespread pulmonary microvascular thrombosis. Occluded pulmonary blood vessels with vascular thrombi often exhibited endothelial necrosis surrounded by hemorrhagic effusions and pulmonary edema. Histones released during influenza induced cytotoxicity and showed strong binding to platelets within thrombi in infected mouse lungs. Nasal wash samples from influenza-infected patients also showed increased accumulation of extracellular histones, suggesting a possible clinical relevance of elevated histones in pulmonary injury. Although histones inhibited influenza growth in vitro, in vivo treatment with histones did not yield antiviral effects and instead exacerbated lung pathology. Blocking with antihistone antibodies caused a marked decrease in lung pathology in lethal influenza–challenged mice and improved protection when administered in combination with the antiviral agent oseltamivir. These findings support the pathogenic effects of extracellular histones in that pulmonary injury during influenza was exacerbated. Targeting histones provides a novel therapeutic approach to influenza pneumonia. Influenza pneumonia, caused by highly pathogenic influenza viral infection, has remained a top global health concern for more than a century. The majority of patients who succumb to severe influenza pneumonia develop complications of acute respiratory distress syndrome (ARDS), which is characterized by alveolar capillary damage, edema, parenchymal hemorrhage, pulmonary microvascular thrombosis (MT), and hyperinflammatory cytokine responses.1Short K.R. Kroeze E.J. Fouchier R.A. Kuiken T. Pathogenesis of influenza-induced acute respiratory distress syndrome.Lancet Infect Dis. 2014; 14: 57-69Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 2Kumar A. Zarychanski R. Pinto R. Cook D.J. Marshall J. Lacroix J. Stelfox T. Bagshaw S. Choong K. Lamontagne F. Turgeon A.F. Lapinsky S. Ahern S.P. Smith O. Siddiqui F. Jouvet P. Khwaja K. McIntyre L. Menon K. Hutchison J. Hornstein D. Joffe A. Lauzier F. Singh J. Karachi T. Wiebe K. Olafson K. Ramsey C. Sharma S. Dodek P. Meade M. Hall R. Fowler R.A. Canadian Critical Care Trials Group H1N1 CollaborativeCritically ill patients with 2009 influenza A (H1N1) infection in Canada.JAMA. 2009; 302: 1872-1879Crossref PubMed Scopus (1127) Google Scholar, 3Harms P.W. Schmidt L.A. Smith L.B. Newton D.W. Pletneva M.A. Walters L.L. Tomlins S.A. Fisher-Hubbard A. Napolitano L.M. Park P.K. Blaivas M. Fantone J. Myers J.L. Jentzen J.M. Autopsy findings in eight patients with fatal H1N1 influenza.Am J Clin Pathol. 2010; 134: 27-35Crossref PubMed Scopus (103) Google Scholar, 4Wang H. Xiao X. Lu J. Chen Z. Li K. Liu H. Luo L. Wang M. Yang Z. Factors associated with clinical outcome in 25 patients with avian influenza A (H7N9) infection in Guangzhou, China.BMC Infect Dis. 2016; 16: 534Crossref PubMed Scopus (30) Google Scholar, 5Nie Q. Zhang D.Y. Wu W.J. Huang C.L. Ni Z.Y. Extracorporeal membrane oxygenation for avian influenza A (H7N9) patient with acute respiratory distress syndrome: a case report and short literature review.BMC Pulm Med. 2017; : 17-38Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar The pathologic and clinical manifestations of ARDS are well characterized in animal models of influenza.6Xu T. Qiao J. Zhao L. Wang G. He G. Li K. Tian Y. Gao M. Wang J. Wang H. Dong C. Acute respiratory distress syndrome induced by avian influenza A (H5N1) virus in mice.Am J Respir Crit Care Med. 2006; 174: 1011-1017Crossref PubMed Scopus (109) Google Scholar, 7Narasaraju 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 (605) Google Scholar, 8Traylor Z.P. Aeffner F. Davis I.C. Influenza A H1N1 induces declines in alveolar gas exchange in mice consistent with rapid post-infection progression from acute lung injury to ARDS.Influenza Other Respir Viruses. 2013; 7: 472-479Crossref PubMed Scopus (42) Google Scholar In addition to virus-inflicted damage, host-mediated factors are linked to lung pathology and respiratory failure9Cheung C.Y. Leung C.Y. Nicholls J.M. Innate immune responses to influenza A H5N1: friend or foe?.Trends Immunol. 2009; 30: 574-584Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 10Kash J.C. Taubenberger J.K. The role of viral, host, and secondary bacterial factors in influenza pathogenesis.Am J Pathol. 2015; 185: 1528-1536Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 11Ramos I. Fernandez-Sesma A. Modulating the innate immune response to influenza A virus: potential therapeutic use of anti-inflammatory drugs.Front Immunol. 2015; 6: 361Crossref PubMed Scopus (88) Google Scholar in severe influenza pneumonia. Our previous studies have demonstrated that excessive neutrophils and neutrophil extracellular traps (NETs) contribute to acute lung damage in influenza infection, as well as during Streptococcus pneumoniae superinfection after influenza infection, in mice.7Narasaraju 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 (605) Google Scholar, 12Narayana Moorthy A. Narasaraju T. Rai P. Perumalsamy R. Tan K.B. Wang S. Engelward B. Chow V.T. In vivo and in vitro studies on the roles of neutrophil extracellular traps during secondary pneumococcal pneumonia after primary pulmonary influenza infection.Front Immunol. 2013; 4: 56Crossref PubMed Scopus (95) Google Scholar, 13Moorthy A.N. Rai P. Jiao H. Wang S. Tan K.B. Qin L. Watanabe H. Zhang Y. Teluguakula N. Chow V.T. Capsules of virulent pneumococcal serotypes enhance formation of neutrophil extracellular traps during in vivo pathogenesis of pneumonia.Oncotarget. 2016; 7: 19327-19340Crossref PubMed Scopus (30) Google Scholar, 14Narasaraju T. Harshini A. Neutrophils as possible therapeutic targets in severe influenza pneumonia.J Infect Pulm Dis. 2016; 2https://doi.org/10.16966/2470-3176.115PubMed Google Scholar, 15Ivan F.X. Rajapakse J.C. Welsch R.E. Rozen S.G. Narasaraju T. Xiong G.M. Engelward B.P. Chow V.T. Differential pulmonary transcriptomic profiles in murine lungs infected with low and highly virulent influenza H3N2 viruses reveal dysregulation of TREM1 signaling, cytokines, and chemokines.Funct Integr Genomics. 2012; 12: 105-117Crossref PubMed Scopus (24) Google Scholar, 16Audrey-Ann L. Narasaraju T. Tan K.B. Wang S. Phoon M.C. Chow V.T. Caspase-1-deficient mice are more susceptible to influenza pneumonitis.Eur J Inflamm. 2014; 12: 117-130Crossref Scopus (2) Google Scholar Chromatin strands in the NETs disrupt the alveolar–capillary barrier, resulting in epithelial injury, vascular leakage, and hemorrhage.7Narasaraju 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 (605) Google Scholar Although the bactericidal effects of NETs have been well characterized, influenza-induced NETs lack bactericidal effects.7Narasaraju 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 (605) Google Scholar, 12Narayana Moorthy A. Narasaraju T. Rai P. Perumalsamy R. Tan K.B. Wang S. Engelward B. Chow V.T. In vivo and in vitro studies on the roles of neutrophil extracellular traps during secondary pneumococcal pneumonia after primary pulmonary influenza infection.Front Immunol. 2013; 4: 56Crossref PubMed Scopus (95) Google Scholar Histones are the major protein components of NETs and are essential components in chromatin organization in the intact cell. However, accumulating evidence indicates that histone proteins released from NETs or dying cells contribute to tissue injury and death in several clinical conditions, including trauma, sepsis, aggravated kidney injury, and acid aspiration–induced ARDS.17Xu J. Zhang X. Pelayo R. Monestier M. Ammollo C.T. Semeraro F. Taylor F.B. Esmon N.L. Lupu F. Esmon C.T. Extracellular histones are major mediators of death in sepsis.Nat Med. 2009; 15: 1318-1321Crossref PubMed Scopus (1051) Google Scholar, 18Abrams S.T. Zhang N. Manson J. Liu T. Dart C. Baluwa F. Wang S.S. Brohi K. Kipar A. Yu W. Wang G. Toh C.H. Circulating histones are mediators of trauma-associated lung injury.Am J Respir Crit Care Med. 2013; 187: 160-169Crossref PubMed Scopus (371) Google Scholar, 19Allam R. Scherbaum C.R. Darisipudi M.N. Mulay S.R. Hägele H. Lichtnekert J. Hagemann J.H. Rupanagudi K.V. Ryu M. Schwarzenberger C. Hohenstein B. Hugo C. Uhl B. Reichel C.A. Krombach F. Monestier M. Liapis H. Moreth K. Schaefer L. Anders H.J. Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4.J Am Soc Nephrol. 2012; 23: 1375-1388Crossref PubMed Scopus (306) Google Scholar, 20Wildhagen K.C. Wiewel M.A. Schultz M.J. Horn J. Schrijver R. Reutelingsperger C.P. van der Poll T. Nicolaes G.A. Extracellular histone H3 levels are inversely correlated with antithrombin levels and platelet counts and are associated with mortality in sepsis patients.Thromb Res. 2015; 136: 542-547Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 21Zhang Y. Wen Z. Guan L. Jiang P. Gu T. Zhao J. Lv X. Wen T. Extracellular histones play an inflammatory role in acid aspiration-induced acute respiratory distress syndrome.Anesthesiology. 2015; 122: 127-139Crossref PubMed Scopus (49) Google Scholar, 22Lee D.Y. Huang C.M. Nakatsuji T. Thiboutot D. Kang S.A. Monestier M. Gallo R.L. Histone H4 is a major component of the antimicrobial action of human sebocytes.J Invest Dermatol. 2009; 129: 2489-2496Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar Histone proteins interact with membrane phospholipids and enhance intracellular calcium influx, which triggers cytotoxic responses.19Allam R. Scherbaum C.R. Darisipudi M.N. Mulay S.R. Hägele H. Lichtnekert J. Hagemann J.H. Rupanagudi K.V. Ryu M. Schwarzenberger C. Hohenstein B. Hugo C. Uhl B. Reichel C.A. Krombach F. Monestier M. Liapis H. Moreth K. Schaefer L. Anders H.J. Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4.J Am Soc Nephrol. 2012; 23: 1375-1388Crossref PubMed Scopus (306) Google Scholar Histone proteins also promote platelet aggregation by interacting with fibrinogen receptors.23Fuchs T.A. Bhandari A.A. Wagner D.D. Histones induce rapid and profound thrombocytopenia in mice.Blood. 2011; 118: 3708-3714Crossref PubMed Scopus (312) Google Scholar Increased pulmonary capillary thrombosis, platelet activation and aggregation, and endothelial damage have been linked to inflammation and severe lung pneumonia during influenza.24Taubenberger J.K. Morens D.M. The pathology of influenza virus infections.Annu Rev Pathol. 2008; 3: 499-522Crossref PubMed Scopus (743) Google Scholar, 25Lê V.B. Schneider J.G. Boergeling Y. Berri F. Ducatez M. Guerin J.L. Adrian I. Errazuriz-Cerda E. Frasquilho S. Antunes L. Lina B. Bordet J.C. Jandrot-Perrus M. Ludwig S. Riteau B. Platelet activation and aggregation promote lung inflammation and influenza virus pathogenesis.Am J Respir Crit Care Med. 2015; 191: 804-819Crossref PubMed Scopus (118) Google Scholar, 26Yang Y. Tang H. Aberrant coagulation causes a hyper-inflammatory response in severe influenza pneumonia.Cell Mol Immunol. 2016; 13: 432-442Crossref PubMed Scopus (97) Google Scholar Among various inflammatory mediators that contribute to the development of thrombosis, CD40 ligand (L), a tumor necrosis family protein cytokine, is well characterized in its potential role in platelet aggregation and thrombosis.27Yacoub D. Hachem A. Théorêt J.F. Gillis M.A. Mourad W. Merhi Y. Enhanced levels of soluble CD40 ligand exacerbate platelet aggregation and thrombus formation through a CD40-dependent tumor necrosis factor receptor-associated factor-2/Rac1/p38 mitogen-activated protein kinase signaling pathway.Arterioscler Thromb Vasc Biol. 2010; 30: 2424-2433Crossref PubMed Scopus (62) Google Scholar CD40L is abundantly expressed in platelets and other leukocytes.28Schönbeck U. Libby P. The CD40/CD154 receptor/ligand dyad.Cell Mol Life Sci. 2001; 58: 4-43Crossref PubMed Google Scholar Soluble (s) CD40L, a cleavage product of CD40L, is a strong inducer of platelet aggregation and induces B-cell proliferation and isotope switching during immune responses.27Yacoub D. Hachem A. Théorêt J.F. Gillis M.A. Mourad W. Merhi Y. Enhanced levels of soluble CD40 ligand exacerbate platelet aggregation and thrombus formation through a CD40-dependent tumor necrosis factor receptor-associated factor-2/Rac1/p38 mitogen-activated protein kinase signaling pathway.Arterioscler Thromb Vasc Biol. 2010; 30: 2424-2433Crossref PubMed Scopus (62) Google Scholar, 29Andre P. Nannizzi-Alaimo L. Prasad S.K. Phillips D.R. Platelet-derived CD40L: the switch-hitting player of cardiovascular disease.Circulation. 2002; 106: 896-899Crossref PubMed Scopus (490) Google Scholar It has been reported that 95% of circulating sCD40L is secreted from platelets, suggesting that histone–platelet interaction may be crucial in the development of thrombosis.30Zarnegar B. He J.Q. Oganesyan G. Hoffmann A. Baltimore D. Cheng G. Unique CD40-mediated biological program in B cell activation requires both type 1 and type 2 NF-κB activation pathways.Proc Natl Acad Sci U S A. 2004; 101: 8108-8113Crossref PubMed Scopus (103) Google Scholar Due to the difficulty in predicting emerging new influenza strains, the preparation of an effective influenza vaccine is very challenging. Because severe lung pathology in influenza is mediated by virus and host factors, treatment with antiviral drugs does not always effectively control mortality rates.31Suzuki H. Saito R. Masuda H. Oshitani H. Sato M. Sato I. Emergence of amantadine-resistant influenza A viruses: epidemiological study.J Infect Chemother. 2003; 9: 195-200Abstract Full Text PDF PubMed Scopus (157) Google Scholar, 32Moscona A. Oseltamivir resistance-disabling our influenza defenses.N Engl J Med. 2005; 353: 2633-2636Crossref PubMed Scopus (294) Google Scholar, 33Dharan N.J. Gubareva L.V. Meyer J.J. Okomo-Adhiambo M. McClinton R.C. Marshall S.A. St George K. Epperson S. Brammer L. Klimov A.I. Bresee J.S. Fry A.M. Infections with oseltamivir-resistant influenza A (H1N1) virus in the United States.JAMA. 2009; 301: 1034-1041Crossref PubMed Scopus (455) Google Scholar The use of antiviral drugs is also limited by adverse effects on the host and the rapid development of resistance in several strains of influenza.32Moscona A. Oseltamivir resistance-disabling our influenza defenses.N Engl J Med. 2005; 353: 2633-2636Crossref PubMed Scopus (294) Google Scholar, 33Dharan N.J. Gubareva L.V. Meyer J.J. Okomo-Adhiambo M. McClinton R.C. Marshall S.A. St George K. Epperson S. Brammer L. Klimov A.I. Bresee J.S. Fry A.M. Infections with oseltamivir-resistant influenza A (H1N1) virus in the United States.JAMA. 2009; 301: 1034-1041Crossref PubMed Scopus (455) Google Scholar Similarly, we found that targeting a host-mediated factor alone does not protect from lethal influenza challenge.34Ng H.H. Narasaraju T.A. Sim M.K. Chow V.T. Doxycyline treatment attenuates acute lung injury in mice infected with highly virulent influenza H3N2 virus: involvement of matrix metalloproteases.Exp Mol Pathol. 2012; 92: 287-295Crossref PubMed Scopus (61) Google Scholar Targeting both the virus and host factors may be a better choice for influenza treatment.35Smee D.F. Hurst B.L. Wong M.H. Bailey K.W. Tarbet E.B. Morrey J.D. Furuta Y. Effects of the combination of favipiravir (T-705) and oseltamivir on influenza A virus infections in mice.Antimicrob Agents Chemother. 2010; 54: 126-133Crossref PubMed Scopus (92) Google Scholar, 36Ilyushina N.A. Bovin N.V. Webster R.G. Govorkova E.A. Combination chemotherapy, a potential strategy for reducing the emergence of drug-resistant influenza A variants.Antivir Res. 2006; 70: 121-131Crossref PubMed Scopus (156) Google Scholar, 37Sharma G. Champalal Sharma D. Hwei Fen L. Pathak M. Bethur N. Pendharkar V. Peiris M. Altmeyer R. Reduction of influenza virus-induced lung inflammation and mortality in animals treated with a phosophodisestrase-4 inhibitor and a selective serotonin reuptake inhibitor.Emerg Microbes Infect. 2013; 2: e54Crossref PubMed Scopus (27) Google Scholar In support of this hypothesis, our previous findings showed that treatment with hepatocyte growth factor, a mitogen for alveolar epithelial cells, together with the antiviral agent oseltamivir, decreases lung pathology in mice challenged with lethal influenza virus.38Narasaraju T.A. Edwin Y.Y. Ng H.H. Audrey A. Phoon M.C. Chow V.T. Therapeutic effect of combining hepatocyte growth factor and oseltamivir in amelioration of influenza pneumonitis.Curr Mol Med. 2014; 14: 1-13Crossref PubMed Scopus (7) Google Scholar We hypothesized that extracellular histones released during influenza infection contribute to alveolitis and endothelial damage and trigger MT; these events eventually lead to the pathologic manifestations of ARDS. Here, we examined the role of extracellular histones in influenza virus pathogenesis using an animal model of lethal influenza. We also evaluated the release of extracellular histones in nasal wash and plasma samples collected from influenza-infected patients. Our results show that lethal influenza viral infection promotes the extensive release of extracellular histones into the lung air space. The released extracellular histones induce cytotoxicity and MT, which exacerbate tissue damage. Finally, treatment with antihistone antibodies in combination with oseltamivir ameliorates lung pathology in lethal influenza–challenged mice. Influenza A/Puerto Rico/8/34, H1N1 virus was obtained from ATCC (Manassas, VA). Virus levels were determined by tissue culture infectivity dose (TCID50) assay via infection of Madin-Darby canine kidney cells. Female BALB/c mice (6 to 8 weeks old) were used in this study. The animals were housed in microisolator cages in a Biosafety Level 2 animal facility. The protocol of the animal experiments was approved by the Institutional Animal Care and Use Committee at Oklahoma State University (protocol number VM-11-43), and the experiments were performed in strict accordance with their recommendations. For infection studies, animals were anesthetized with a mixture of ketamine (7.5 mg/kg) and xylazine (0.1 mg/kg) by i.p. injection. The mice were then intranasally challenged with a lethal dose of 2500 TCID50 of influenza virus in 50 μL phosphate buffered saline. Equal volumes of phosphate-buffered saline were inoculated into control mice. Mice were anesthetized with ketamine/xylazine by i.p. injection. Tissues for histopathology were collected as described previously.34Ng H.H. Narasaraju T.A. Sim M.K. Chow V.T. Doxycyline treatment attenuates acute lung injury in mice infected with highly virulent influenza H3N2 virus: involvement of matrix metalloproteases.Exp Mol Pathol. 2012; 92: 287-295Crossref PubMed Scopus (61) Google Scholar Lungs from control and infected animals were fixed by the intratracheal instillation of 4% neutral-buffered formalin at 25 cm of water gauge pressure and embedded in paraffin. Other major organs, including heart, spleen, intestine, liver, and brain, were also fixed in formalin. Pathologic ARDS lesions were evaluated using a previously described semiquantitative histologic examination scoring method in a blinded manner.39Narasaraju T. Ng H.H. Phoon M.C. Chow V.T. MCP-1 antibody treatment enhances damage and impedes repair of the alveolar epithelium in influenza pneumonitis.Am J Respir Cell Mol Biol. 2010; 42: 732-743Crossref PubMed Scopus (80) Google Scholar The lung histopathology was scored based on necrotizing bronchiolitis (damage to airway epithelial cells, necrotic bodies, or denudation of airway epithelial lining), inflammation in the bronchioles (bronchioles filled with inflammatory cells, eg, macrophages, neutrophils, and lymphocytes), alveolitis (damaged alveolar epithelial cells or denuded epithelial lining), interstitial inflammation (inflammation in the alveoli or thickening of the alveolar interstitium), hemorrhage (presence of erythrocytes in the alveolar airspace, damaged capillaries, and hemorrhagic effusions in the damaged areas), edema (presence of proteinaceous material in the alveolar airspace), MT (presence of platelet aggregates within the capillaries or occlusions in the small blood vessels of the pulmonary vasculature), and endothelial damage (necrotic endothelium present with in small blood vessels or capillaries). Damage severity was scored on a 5-point scale: 0 indicates none or very minor; 1 indicates mild; 2 indicates intermediate; 3 indicates moderately severe; and 4 indicates severe and widespread. The total lung surface was scored at 400× magnification, and semiquantitative scores were expressed as means ± SEM as described previously.7Narasaraju 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 (605) Google Scholar Histopathology scores were represented as mean scores of major lesions, including bronchiolitis, alveolitis, pulmonary edema, hemorrhage, and MT. For bronchoalveolar lavage (BAL) fluid collection, the lungs were washed by two intratracheal administrations of 0.5 mL of phosphate-buffered saline.7Narasaraju 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 (605) Google Scholar The recovery of BAL fluid was >90% in all animals. The BAL fluids were centrifuged at 200 × g for 10 minutes, and the supernatants were stored at −80°C. The BAL cells were concentrated using the CytoFuge 2 cytocentrifuge (Stat Spin, Westwood, MA), and differential cell counts were performed using modified Giemsa staining. Protein leakage was measured by the determination of the total BAL protein content using a DC Protein Assay Kit (Bio-Rad, Hercules, CA). The BAL fluids were analyzed for the release of extracellular histones and modified histones (citrullinated histones, which are indicative of NET formation), as described previously.7Narasaraju 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 (605) Google Scholar Antibodies against histone proteins H1, H2A, H2B, H3, H4, citrullinated H3 (citH3), and citH4 (Millipore, MA) were used. Densitometry analysis was performed on all Western blots for histone proteins using ImageJ software version 1.51 (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Flow cytometry analysis was performed on platelets collected from mock- or influenza-infected mouse lungs at 3 days postinfection (dpi). Lungs were lavaged with Tyrode buffer pH 7.4 (150 mmol/L sodium chloride, 12 mmol/L sodium bicarbonate, 0.34 mmol/L sodium phosphate monobasic, 1 mmol/L magnesium chloride, 5 mmol/L HEPES, 5 mmol/L glucose, and 1% bovine serum albumin) containing 1% formaldehyde. Formalin was added to prevent any activation of platelets during isolation and staining. Leukocytes were removed by centrifuging BAL at 80 × g for 10 minutes. Cell-free BAL supernatants were further centrifuged at 400 × g for 10 minutes to recover platelet pellets. Platelets were then resuspended in Tyrode buffer and stained with antibodies including anti-CD40 PerCP-Cy5.5 or anti-CD41 phycoerythrin (eBioscience, San Diego, CA) or anti-CD40L fluorescein isothiocyanate or anti-CD62p (P-selectin) APC (BioLegend, San Diego, CA). Flow cytometry analysis was performed using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Immunohistochemistry analysis was performed for the detection of NET release using anti-citH3 + anti-myeloperoxidase (MPO) or anti-citH4 + anti-MPO antibodies. Anti-citH3 + anti–P-selectin, anti-fibrin + anti–P-selectin, or anti-citH3 + anti-sCD40L were used for the detection of MT as described previously.7Narasaraju 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 (605) Google Scholar Extracellular histones released into the cell-free BAL were measured by a cell death enzyme-linked immunosorbent assay (ELISA) kit (Roche, Pleasanton, CA) according to the manufacturer's instructions. To determine whether extracellular histones released during influenza infection were functionally active and produced cytotoxic effects, we performed a lactate dehydrogenase release assay using a cytotoxicity detection kit (Roche). The cell-free BAL fluids collected from the control and influenza-infected mice were incubated with alveolar epithelial cells (A549) in the presence or absence of antihistone antibodies (10 μg/mL; antihistones H1, H2A, H2B, H3, H4, citH3, and citH4). About 1.5 × 104 TCID50/mL virus levels in BAL fluids were found in influenza-infected mice at 3 dpi. To exclude any cytotoxic effects of the virus, antiserum raised against the influenza virus (100 μg/mL) was also added. To assess the toxic effects of individual histones, recombinant human histones H1, H2A, H2B, H3, H4 (Cayman Chemical Company, Ann Arbor, MI) or total histones (from calf thymus) 5 μg/mL were added to A549 cells or endothelial cells (EA.hy926), and the lactate dehydrogenase release assay was performed. Human nasal wash and plasma samples were a gift from Dr. Paul Thomas, St. Jude Children's Research Hospital (Memphis, TN). The inclusion criteria required that participants meet the clinical case definition of influenza virus infection at the time of enrollment as described by Oshansky et al.40Oshansky C.M. Gartland A.J. Wong S.S. Jeevan T. Wang D. Roddam P.L. Caniza M.A. Hertz T. Devincenzo J.P. Webby R.J. Thomas P.G. Mucosal immune responses predict clinical outcomes during influenza infection independently of age and viral load.Am J Respir Crit Care Med. 2014; 189: 449-462Crossref PubMed Scopus (117) Google Scholar The clinical criteria for the inclusion of subjects with influenza included fever or feverishness accompanied by cough or sore throat for 96 hours or less. Children and adults with suspected influenza viral infections receiving medical counsel were approached by study personnel to participate. Index cases, defined as those individuals who tested positive for influenza A/B infection, and their respective household contacts were enrolled in the study. The race/ethnicity clas