Neutrophil Extracellular Trap (NET)-Mediated Killing of Pseudomonas aeruginosa: Evidence of Acquired Resistance within the CF Airway, Independent of CFTR
The inability of neutrophils to eradicate Pseudomonas aeruginosa within the cystic fibrosis (CF) airway eventually results in chronic infection by the bacteria in nearly 80 percent of patients. Phagocytic killing of P. aeruginosa by CF neutrophils is impaired due to decreased cystic fibrosis transmembrane conductance regulator (CFTR) function and virulence factors acquired by the bacteria. Recently, neutrophil extracellular traps (NETs), extracellular structures composed of neutrophil chromatin complexed with granule contents, were identified as an alternative mechanism of pathogen killing. The hypothesis that NET-mediated killing of P. aeruginosa is impaired in the context of the CF airway was tested. P. aeruginosa induced NET formation by neutrophils from healthy donors in a bacterial density dependent fashion. When maintained in suspension through continuous rotation, P. aeruginosa became physically associated with NETs. Under these conditions, NETs were the predominant mechanism of killing, across a wide range of bacterial densities. Peripheral blood neutrophils isolated from CF patients demonstrated no impairment in NET formation or function against P. aeruginosa. However, isogenic clinical isolates of P. aeruginosa obtained from CF patients early and later in the course of infection demonstrated an acquired capacity to withstand NET-mediated killing in 8 of 9 isolates tested. This resistance correlated with development of the mucoid phenotype, but was not a direct result of the excess alginate production that is characteristic of mucoidy. Together, these results demonstrate that neutrophils can kill P. aeruginosa via NETs, and in vitro this response is most effective under non-stationary conditions with a low ratio of bacteria to neutrophils. NET-mediated killing is independent of CFTR function or bacterial opsonization. Failure of this response in the context of the CF airway may occur, in part, due to an acquired resistance against NET-mediated killing by CF strains of P. aeruginosa.
References
[1]
Comeau AM, Parad RB, Dorkin HL, Dovey M, Gerstle R, et al. (2004) Population-based newborn screening for genetic disorders when multiple mutation DNA testing is incorporated: a cystic fibrosis newborn screening model demonstrating increased sensitivity but more carrier detections. Pediatrics 113: 1573–1581.
[2]
Sontag MK, Hammond KB, Zielenski J, Wagener JS, Accurso FJ (2005) Two-tiered immunoreactive trypsinogen-based newborn screening for cystic fibrosis in Colorado: screening efficacy and diagnostic outcomes. J Pediatr 147: S83–88.
Cystic Fibrosis Foundation (2009) Cystic Fibrosis Foundation Patient Registry 2008 Annual Data Report. Bethesda, Maryland.
[5]
Stick SM, Brennan S, Murray C, Douglas T, von Ungern-Sternberg BS, et al. (2009) Bronchiectasis in infants and preschool children diagnosed with cystic fibrosis after newborn screening. J Pediatr 155: 623–628 e621.
[6]
Dakin CJ, Numa AH, Wang H, Morton JR, Vertzyas CC, et al. (2002) Inflammation, infection, and pulmonary function in infants and young children with cystic fibrosis. Am J Respir Crit Care Med 165: 904–910.
[7]
Emerson J, Rosenfeld M, McNamara S, Ramsey B, Gibson RL (2002) Pseudomonas aeruginosa and other predictors of mortality and morbidity in young children with cystic fibrosis. Pediatr Pulmonol 34: 91–100.
[8]
Frederiksen B, Koch C, Hoiby N (1997) Antibiotic treatment of initial colonization with Pseudomonas aeruginosa postpones chronic infection and prevents deterioration of pulmonary function in cystic fibrosis. Pediatr Pulmonol 23: 330–335.
[9]
Kosorok MR, Zeng L, West SE, Rock MJ, Splaingard ML, et al. (2001) Acceleration of lung disease in children with cystic fibrosis after Pseudomonas aeruginosa acquisition. Pediatr Pulmonol 32: 277–287.
[10]
Nixon GM, Armstrong DS, Carzino R, Carlin JB, Olinsky A, et al. (2001) Clinical outcome after early Pseudomonas aeruginosa infection in cystic fibrosis. J Pediatr 138: 699–704.
[11]
Parad RB, Gerard CJ, Zurakowski D, Nichols DP, Pier GB (1999) Pulmonary outcome in cystic fibrosis is influenced primarily by mucoid Pseudomonas aeruginosa infection and immune status and only modestly by genotype. Infect Immun 67: 4744–4750.
[12]
Schaedel C, de Monestrol I, Hjelte L, Johannesson M, Kornfalt R, et al. (2002) Predictors of deterioration of lung function in cystic fibrosis. Pediatr Pulmonol 33: 483–491.
[13]
Burns JL, Gibson RL, McNamara S, Yim D, Emerson J, et al. (2001) Longitudinal assessment of Pseudomonas aeruginosa in young children with cystic fibrosis. J Infect Dis 183: 444–452.
[14]
Khan TZ, Wegner JS, Bost T, Martinez J, Accurso FJ, et al. (1995) Early Pulmonary Inflammation in Infants with Cystic Fibrosis. Am J Respir Crit Care Med 151: 1075–1082.
[15]
Hayes E, Pohl K, McElvaney NG, Reeves EP (2011) The cystic fibrosis neutrophil: a specialized yet potentially defective cell. Arch Immunol Ther Exp (Warsz) 59: 97–112.
[16]
Painter RG, Valentine VG, Lanson NA Jr, Leidal K, Zhang Q, et al. (2006) CFTR Expression in human neutrophils and the phagolysosomal chlorination defect in cystic fibrosis. Biochemistry 45: 10260–10269.
[17]
Adib-Conquy M, Pedron T, Petit-Bertron AF, Tabary O, Corvol H, et al. (2008) Neutrophils in cystic fibrosis display a distinct gene expression pattern. Mol Med 14: 36–44.
[18]
Makam M, Diaz D, Laval J, Gernez Y, Conrad CK, et al. (2009) Activation of critical, host-induced, metabolic and stress pathways marks neutrophil entry into cystic fibrosis lungs. Proc Natl Acad Sci U S A 106: 5779–5783.
[19]
Tirouvanziam R, Gernez Y, Conrad CK, Moss RB, Schrijver I, et al. (2008) Profound functional and signaling changes in viable inflammatory neutrophils homing to cystic fibrosis airways. Proc Natl Acad Sci U S A 105: 4335–4339.
[20]
Dai Y, Dean TP, Church MK, Warner JO, Shute JK (1994) Desensitisation of neutrophil responses by systemic interleukin 8 in cystic fibrosis. Thorax 49: 867–871.
Moriceau S, Lenoir G, Witko-Sarsat V (2010) In cystic fibrosis homozygotes and heterozygotes, neutrophil apoptosis is delayed and modulated by diamide or roscovitine: evidence for an innate neutrophil disturbance. J Innate Immun 2: 260–266.
[23]
Painter RG, Marrero L, Lombard GA, Valentine VG, Nauseef WM, et al. (2010) CFTR-mediated halide transport in phagosomes of human neutrophils. J Leukoc Biol 87: 933–942.
[24]
Bonvillain RW, Painter RG, Adams DE, Viswanathan A, Lanson NA Jr, et al. (2010) RNA interference against CFTR affects HL60-derived neutrophil microbicidal function. Free Radic Biol Med 49: 1872–1880.
[25]
McKeon DJ, Cadwallader KA, Idris S, Cowburn AS, Pasteur MC, et al. (2010) Cystic fibrosis neutrophils have normal intrinsic reactive oxygen species generation. Eur Respir J 35: 1264–1272.
[26]
Oliver A, Canton R, Campo P, Baquero F, Blazquez J (2000) High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science 288: 1251–1254.
[27]
Mena A, Smith EE, Burns JL, Speert DP, Moskowitz SM, et al. (2008) Genetic adaptation of Pseudomonas aeruginosa to the airways of cystic fibrosis patients is catalyzed by hypermutation. J Bacteriol 190: 7910–7917.
Ernst RK, Yi EC, Guo L, Lim KB, Burns JL, et al. (1999) Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa. Science 286: 1561–1565.
[30]
Smith EE, Buckley DG, Wu Z, Saenphimmachak C, Hoffman LR, et al. (2006) Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc Natl Acad Sci U S A 103: 8487–8492.
[31]
Cabral DA, Loh BA, Speert DP (1987) Mucoid Pseudomonas aeruginosa resists nonopsonic phagocytosis by human neutrophils and macrophages. Pediatr Res 22: 429–431.
[32]
Worlitzsch D, Tarran R, Ulrich M, Schwab U, Cekici A, et al. (2002) Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients. J Clin Invest 109: 317–325.
[33]
Hassett DJ, Cuppoletti J, Trapnell B, Lymar SV, Rowe JJ, et al. (2002) Anaerobic metabolism and quorum sensing by Pseudomonas aeruginosa biofilms in chronically infected cystic fibrosis airways: rethinking antibiotic treatment strategies and drug targets. Adv Drug Deliv Rev 54: 1425–1443.
[34]
Hill D, Rose B, Pajkos A, Robinson M, Bye P, et al. (2005) Antibiotic susceptabilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions. J Clin Microbiol 43: 5085–5090.
[35]
Jesaitis AJ, Franklin MJ, Berglund D, Sasaki M, Lord CI, et al. (2003) Compromised host defense on Pseudomonas aeruginosa biofilms: characterization of neutrophil and biofilm interactions. J Immunol 171: 4329–4339.
[36]
Bayer AS, Speert DP, Park S, Tu J, Witt M, et al. (1991) Functional role of mucoid exopolysaccharide (alginate) in antibiotic-induced and polymorphonuclear leukocyte-mediated killing of Pseudomonas aeruginosa. Infect Immun 59: 302–308.
[37]
Govan JR, Deretic V (1996) Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 60: 539–574.
[38]
Rau MH, Hansen SK, Johansen HK, Thomsen LE, Workman CT, et al. (2010) Early adaptive developments of Pseudomonas aeruginosa after the transition from life in the environment to persistent colonization in the airways of human cystic fibrosis hosts. Environ Microbiol.
[39]
Henry RL, Mellis CM, Petrovic L (1992) Mucoid Pseudomonas aeruginosa is a marker of poor survival in cystic fibrosis. Pediatr Pulmonol 12: 158–161.
[40]
Konstan MW, Morgan WJ, Butler SM, Pasta DJ, Craib ML, et al. (2007) Risk factors for rate of decline in forced expiratory volume in one second in children and adolescents with cystic fibrosis. J Pediatr 151: 134–139, 139 e131.
[41]
Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, et al. (2004) Neutrophil extracellular traps kill bacteria. Science 303: 1532–1535.
[42]
Hirsch JG (1958) Bactericidal action of histone. J Exp Med 108: 925–944.
[43]
Fuchs TA, Abed U, Goosmann C, Hurwitz R, Schulze I, et al. (2007) Novel cell death program leads to neutrophil extracellular traps. J Cell Biol 176: 231–241.
[44]
Yousefi S, Mihalache C, Kozlowski E, Schmid I, Simon HU (2009) Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps. Cell Death Differ 16: 1438–1444.
[45]
Pilsczek FH, Salina D, Poon KK, Fahey C, Yipp BG, et al. (2010) A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J Immunol 185: 7413–7425.
[46]
Metzler KD, Fuchs TA, Nauseef WM, Reumaux D, Roesler J, et al. (2011) Myeloperoxidase is required for neutrophil extracellular trap formation: implications for innate immunity. Blood 117: 953–959.
[47]
Papayannopoulos V, Metzler KD, Hakkim A, Zychlinsky A (2010) Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J Cell Biol 191: 677–691.
[48]
Beiter K, Wartha F, Albiger B, Normark S, Zychlinsky A, et al. (2006) An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps. Curr Biol 16: 401–407.
[49]
Sumby P, Barbian KD, Gardner DJ, Whitney AR, Welty DM, et al. (2005) Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response. Proc Natl Acad Sci U S A 102: 1679–1684.
Guimaraes-Costa AB, Nascimento MT, Froment GS, Soares RP, Morgado FN, et al. (2009) Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps. Proc Natl Acad Sci U S A 106: 6748–6753.
[52]
Lippolis JD, Reinhardt TA, Goff JP, Horst RL (2006) Neutrophil extracellular trap formation by bovine neutrophils is not inhibited by milk. Vet Immunol Immunopathol 113: 248–255.
[53]
Martinelli S, Urosevic M, Daryadel A, Oberholzer PA, Baumann C, et al. (2004) Induction of genes mediating interferon-dependent extracellular trap formation during neutrophil differentiation. J Biol Chem 279: 44123–44132.
[54]
Buchanan JT, Simpson AJ, Aziz RK, Liu GY, Kristian SA, et al. (2006) DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps. Curr Biol 16: 396–400.
[55]
Jaillon S, Peri G, Delneste Y, Fremaux I, Doni A, et al. (2007) The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps. J Exp Med 204: 793–804.
[56]
Urban CF, Reichard U, Brinkmann V, Zychlinsky A (2006) Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms. Cell Microbiol 8: 668–676.
[57]
Clark SR, Ma AC, Tavener SA, McDonald B, Goodarzi Z, et al. (2007) Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med 13: 463–469.
[58]
Haslett C, Guthrie LA, Kopaniak M, Johnston RB Jr, Henson PM (1985) Modulation of multiple neutrophil functions by trace amounts of bacterial LPS and by preparative methods. Am J Pathol 119: 101–110.
[59]
von Kockritz-Blickwede M, Chow OA, Nizet V (2009) Fetal calf serum contains heat-stable nucleases that degrade neutrophil extracellular traps. Blood 114: 5245–5246.
[60]
Ogle JW, Janda JM, Woods DE, Vasil ML (1987) Characterization and use of a DNA probe as an epidemiological marker for Pseudomonas aeruginosa. J Infect Dis 155: 119–126.
[61]
Parks QM, Young RL, Poch KR, Malcolm KC, Vasil ML, et al. (2009) Neutrophil enhancement of Pseudomonas aeruginosa biofilm development: human F-actin and DNA as targets for therapy. J Med Microbiol 58: 492–502.
[62]
Vasil ML, Ogle JW, Grant CC, Vasil AI (1987) Recombinant DNA approaches to the study of the regulation of virulence factors and epidemiology of Pseudomonas aeruginosa. Antibiot Chemother 39: 264–278.
[63]
Jacobs MA, Alwood A, Thaipisuttikul I, Spencer D, Haugen E, et al. (2003) Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 100: 14339–14344.
[64]
Fuxman Bass JI, Russo DM, Gabelloni ML, Geffner JR, Giordano M, et al. (2010) Extracellular DNA: a major proinflammatory component of Pseudomonas aeruginosa biofilms. J Immunol 184: 6386–6395.
[65]
Bianchi M, Hakkim A, Brinkmann V, Siler U, Seger RA, et al. (2009) Restoration of NET formation by gene therapy in CGD controls aspergillosis. Blood 114: 2619–2622.
[66]
Van Ziffle JA, Lowell CA (2009) Neutrophil-specific deletion of Syk kinase results in reduced host defense to bacterial infection. Blood 114: 4871–4882.
[67]
Holloway BW (1955) Genetic recombination in Pseudomonas aeruginosa. J Gen Microbiol 13: 572–581.
[68]
Avdi NJ, Nick JA, Whitlock BB, Billstrom MA, Henson PM, et al. (2001) TNFa activation of the Jun NH2-terminal Kinase (JNK) pathway in human neutrophils: Integrin involvement in a pathway leading from cytoplasmic tyrosine kinase to apoptosis. J Biol Chem 276: 2189–2199.
[69]
Hammer MC, Baltch AL, Sutphen NT, Smith RP, Conroy JV (1981) Pseudomonas aeruginosa: quantitation of maximum phagocytic and bactericidal capabilities of normal human granulocytes. J Lab Clin Med 98: 938–948.
[70]
Leijh PC, van den Barselaar MT, van Zwet TL, Dubbeldeman-Rempt I, van Furth R (1979) Kinetics of phagocytosis of Staphylococcus aureus and Escherichia coli by human granulocytes. Immunology 37: 453–465.
[71]
Painter RG, Bonvillain RW, Valentine VG, Lombard GA, LaPlace SG, et al. (2008) The role of chloride anion and CFTR in killing of Pseudomonas aeruginosa by normal and CF neutrophils. J Leukoc Biol 83: 1345–1353.
[72]
Heale JP, Pollard AJ, Stokes RW, Simpson D, Tsang A, et al. (2001) Two distinct receptors mediate nonopsonic phagocytosis of different strains of Pseudomonas aeruginosa. J Infect Dis 183: 1214–1220.
[73]
Fuchs HJ, Borowitz DS, Christiansen DH, Morris EM, Nash ML, et al. (1994) Effect of aerosolized recombinant human DNase on exacerbations of respiratory symptoms and on pulmonary function in patients with cystic fibrosis. The Pulmozyme Study Group. N Engl J Med 331: 637–642.
[74]
Quan JM, Tiddens HA, Sy JP, McKenzie SG, Montgomery MD, et al. (2001) A two-year randomized, placebo-controlled trial of dornase alfa in young patients with cystic fibrosis with mild lung function abnormalities. J Pediatr 139: 813–820.
[75]
Walker TS, Tomlin KL, Worthen GS, Poch KR, Lieber JG, et al. (2005) Enhanced Pseudomonas aeruginosa biofilm development mediated by human neutrophils. Infect Immun 73: 3693–3701.
[76]
Wartha F, Beiter K, Albiger B, Fernebro J, Zychlinsky A, et al. (2007) Capsule and D-alanylated lipoteichoic acids protect Streptococcus pneumoniae against neutrophil extracellular traps. Cell Microbiol 9: 1162–1171.
[77]
Lauth X, von Kockritz-Blickwede M, McNamara CW, Myskowski S, Zinkernagel AS, et al. (2009) M1 protein allows Group A streptococcal survival in phagocyte extracellular traps through cathelicidin inhibition. J Innate Immun 1: 202–214.
[78]
Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, et al. (2000) Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406: 959–964.
[79]
Mulcahy H, Charron-Mazenod L, Lewenza S (2010) Pseudomonas aeruginosa produces an extracellular deoxyribonuclease that is required for utilization of DNA as a nutrient source. Environ Microbiol 12: 1621–9.
[80]
Bergsson G, Reeves EP, McNally P, Chotirmall SH, Greene CM, et al. (2009) LL-37 complexation with glycosaminoglycans in cystic fibrosis lungs inhibits antimicrobial activity, which can be restored by hypertonic saline. J Immunol 183: 543–551.
[81]
Chmiel JF, Davis PB (2003) State of the art: why do the lungs of patients with cystic fibrosis become infected and why can't they clear the infection? Respir Res 4: 8.
[82]
Moraes TJ, Plumb J, Martin R, Vachon E, Cherepanov V, et al. (2006) Abnormalities in the pulmonary innate immune system in cystic fibrosis. Am J Respir Cell Mol Biol 34: 364–374.
[83]
Rubin BK (2007) Mucus structure and properties in cystic fibrosis. Paediatr Respir Rev 8: 4–7.
[84]
Sheils CA, Kas J, Travassos W, Allen PG, Janmey PA, et al. (1996) Actin filaments mediate DNA fiber formation in chronic inflammatory airway disease. Am J Pathol 148: 919–927.
[85]
O'Donnell AE, Barker AF, Ilowite JS, Fick RB (1998) Treatment of idiopathic bronchiectasis with aerosolized recombinant human DNase I. rhDNase Study Group. Chest 113: 1329–1334.
[86]
Clinical Trials.gov (2010) Effectiveness of Pulmozyme in Infants with Cystic Fibrosis.
