Neutrophil extracellular traps exhibit antibacterial activity against Burkholderia pseudomallei and

34 Pages·2012·2.87 MB·English

IAI Accepts, published online ahead of print on 27 August 2012
Infect. Immun. doi:10.1128/IAI.00806-12
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
1 Neutrophil extracellular traps exhibit antibacterial activity against Burkholderia
2 pseudomallei and are influenced by bacterial and host factors
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4 Donporn Riyapa , Surachat Buddhisa , Sunee Korbsrisate , Jon Cuccui , Brendan W. Wren ,
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5 Mark P. Stevens , Manabu Ato and Ganjana Lertmemongkolchai
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7 The Centre for Research & Development of Medical Diagnostic Laboratories, Faculty of lo
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8 Associated Medical Sciences, Khon Kaen University, Thailand, Department of Immunology, d

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9 Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand, London School of m

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10 Hygiene and Tropical Medicine, UK, Roslin Institute and Royal (Dick) School of Veterinary tp
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11 Studies, University of Edinburgh, UK, Department of Immunology, National Institute of i.
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12 Infectious Diseases, Tokyo, Japan. m
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14 Running title: NET formation induced by B. pseudomallei n

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16 Reprints or Correspondence to: 2
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17 Ganjana Lertmemongkolchai, PhD 9

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18 The Centre for Research and Development of Medical Diagnostic Laboratories,
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19 Faculty of Associated Medical Sciences, Khon Kaen University, e
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20 Khon Kaen 40002, Thailand. Tel: [phone], Fax: [phone]
21 E-mail: [email protected]; [email protected]
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23 Word count of the abstract: 188
24 Total word count of the text: 4,816
25 Potential conflict of interest: The authors have no commercial or other associations that
26 might pose a conflict of interest.
27 Presentation: This work was presented in part at the VI World Melioidosis Congress during
28 November 30 – December 2, 2010 in Townsville, Queensland, Australia. D
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30 Abstract
31 Burkholderia pseudomallei is the causative pathogen of melioidosis, of which a major
32 predisposing factor is diabetes mellitus. Polymorphonuclear neutrophils (PMNs) kill
33 microbes extracellularly by the release of neutrophil extracellular traps (NETs). PMNs play a
34 key role in the control of melioidosis but the involvement of NETs in killing of B.
35 pseudomallei remains obscure. Here, we showed that bacteriocidal NETs were released from D
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36 human PMNs in response to B. pseudomallei in a dose- and time- dependent manner. B.
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37 pseudomallei-induced NET formation required NADPH oxidase activation but not PI3K, a
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38 MAPKs, and Src family kinase signaling pathways. B. pseudomallei mutants defective in the
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39 virulence-associated Bsa Type III protein secretion system (T3SS) or capsular polysaccharide m

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40 I (CPS-I) induced elevated levels of NETs. NET induction by such mutants was associated tt
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41 with increased bacterial killing, phagocytosis and oxidative burst by PMNs. Taken together ia
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42 the data imply that T3SS and the capsule may play a role in evading the induction of NETs. s
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43 Importantly, PMNs from diabetic subjects released NETs at a lower level than PMNs from r
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44 healthy subjects. Modulation of NET formation may therefore be associated with the o
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45 pathogenesis and control of melioidosis. p
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48 KEYWORDS: y

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49 Neutrophil extracellular traps; NETs; diabetes; melioidosis; Burkholderia pseudomallei, e
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50 virulence
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52
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53 Introduction
54 Melioidosis is caused by the motile Gram-negative facultative intracellular pathogen
55 Burkholderia pseudomallei and is endemic in Southeast Asia and Northern Australia.
56 Melioidosis can establish with a variety of clinical features ranging from acute fulminant
57 septicemia to chronic localized infection. The case fatality rate of patients with severe
58 melioidosis is approximately 50% in Thailand (7, 16, 31, 39). B. pseudomallei infection often D
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59 affects individuals with one or more underlying predisposing conditions associated with
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60 impaired immune responses, the major risk factor being diabetes mellitus (DM) (18, 25). a
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61 There has been much scientific interest in understanding B. pseudomallei-host interactions at
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62 the cellular level as the organism is resistant to many antibiotics and no licensed vaccine m

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63 exists. tt
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64 Polymorphonuclear neutrophils (PMNs) are highly specialized effector cells involved ia
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65 in host inflammatory responses and immune surveillance (22), and play a key role in control s
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66 of melioidosis in a murine model (10). A novel antimicrobial activity of PMNs was recently r
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67 reported in which the leukocytes generate neutrophil extracellular traps (NETs). Upon o
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68 activation, PMNs can release NETs composed of chromatin decorated with granular proteins. p
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69 NETs possess an antimicrobial activity that can entrap and damage microbes extracellularly, 3
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70 and it is believed they may result from activation of a novel cell death pathway termed 0
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71 NETosis (4, 5, 13, 34, 35). b
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72 The pathways leading to release of NETs are ill defined, but it has been reported to u
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73 require activation of one or more of the phagosomal membrane-bound NADPH oxidase t
74 enzyme complexes found in professional phagocytes and B lymphocytes (13). These
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75 enzymes catalyze the production of superoxide anions (O ), which in turn produce reactive
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76 oxygen species (ROS), including dioxygen and hydrogen peroxide (H O ), which cause
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77 oxidative damage to microorganisms in the phagosome (2, 13). PMNs from neonatal PMNs
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78 fail to form NETs when activated by inflammatory stimuli and consequently exhibit impaired
79 bacteriocidal activity against extracellular bacteria (13, 41). A variety of different pro-
80 inflammatory agonists have been shown to activate NET formation, including the mitogen
81 phorbolmyristate acetate (PMA), bacterial lipopolysaccharide (LPS), and the CXC family
82 chemokine interleukin 8 (IL-8) (4, 13). Such agonists have been reported to act in activated
83 PMNs via a chemokine receptor pathway (20), however the downstream signaling leading to D
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84 NETosis is not well understood. Src family kinases and p38 mitogen- activated protein
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85 kinases (MAPKs) are activated following stimulation with IL-8. There is also evidence that a
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86 IL-8 stimulates phosphatidylinositol-3 kinase (PI3K) activity in human PMNs (23, 24, 32).
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87 The interaction of B. pseudomallei with host cells is known to be influenced by a m

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88 bacterial type III secretion system (T3SS) encoded by the bsa locus. B. pseudomallei mutants tt
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89 lacking components of the Bsa secretion and translocation apparatus, including bsaZ, bsaQ, ia
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90 bipB and bipD, are impaired in invasion of epithelial cells, intracellular net replication, s
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91 survival in macrophages, escape from endocytic vacuoles, and virulence in mice (28-30). r
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92 Additionally, the Bsa T3SS influences the virulence of Burkholderia mallei (33). A o
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93 polysaccharide capsule encoded by the wcb operon also plays a pivotal role in the p
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94 pathogenesis of murine melioidosis (37). It has been previously reported that a 3
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95 polysaccharide capsule protects Streptococcus pneumoniae against entrapment in NETs (38); 0
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96 however, the role of capsule and of the Bsa T3SS in interactions with human PMN has b
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97 received little study. u
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98 Here, we investigated that role of NETs in the innate response of human PMN to B. t
99 pseudomallei, and of bacterial virulence factors in counteracting such responses. As we have
100 previously discovered that PMNs from diabetic subjects have impaired antibacterial functions
101 (6), we also explored the possibility that NET formation is altered, or impaired, in PMNs
102 from DM subjects.
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103 Materials and Methods
104 PMN isolation
105 Human PMNs were isolated from fresh heparinized venous blood from healthy and
106 diabetic subjects using the previously reported criteria and methods (6). Permission was
107 obtained from the Khon Kaen University Ethics Committee for Human Research, number
108 HE470506. Briefly, cells were isolated by 3.0% dextran T-500 sedimentation and separated D
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109 on a Ficoll-Hypaque density gradient centrifugation (Sigma), followed by hypotonic lysis to
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110 remove residual erythrocytes. Purity was >95%, as measured by differential count following a
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111 Giemsa staining and >99% viability as determined by trypan blue exclusion.
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112 Bacterial stains m

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113 B. pseudomallei wild-type (WT) strain K96243 is the prototype genome-sequenced tt
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114 strain (15) and WT strain 10276 was isolated from a fatal case of human melioidosis in ia
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115 Bangladesh (29). The 10276 bsaZ and K96243 bsaQ mutant strains lacking the function of s
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116 the Bsa T3SS have been described elsewhere (21, 29). We also used K96243 wcbB and wcbN r
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117 mutants lacking enzymes required for capsule synthesis as described previously (8). B. o
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118 pseudomallei WT strains K96243 and 10276 were grown in Luria-Bertani (LB) broth, p
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119 whereas type III secretion and capsule mutants were grown in LB broth containing 3
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120 chloramphenicol and kanamycin, respectively. The number of viable bacteria used was 0
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121 determined by retrospective plating of serial ten-fold dilutions on LB agar plates. The details b
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122 of bacteria used in this study are summarized in Table 1. u
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123 Quantification of NET release t
124 PMNs were incubated for 90 min with B. pseudomallei WT, mutant strains or killed
125 B. pseudomallei at a multiplicity of infection (MOI) of 10. Typically the number of bacteria
126 used for inoculation of 7 log10 PMN cells was 8 log10 colony-forming units (CFU). As a
127 positive control, PMNs were separately treated with 100 nM of PMA (Sigma, St Louise,
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128 USA). Twenty units/ml each of restriction enzymes, EcoRI and HindIII (Invitrogen, Paisly,
129 UK) were added to cultures for NET digestion for 2 h at 37°C. The activity of restriction
130 enzymes was stopped with 5 mM EDTA for 15 min at 65°C. Extracellular DNA was then
131 quantified by using the Picogreen dsDNA kit (Invitrogen), in accordance with the
132 manufacturer’s instructions.
133 NET-mediated bacterial killing D
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134 Purified PMNs were placed in 24-well tissue culture plates and incubated for 30 min
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135 at 37°C in the presence and absence of the phagocytosis inhibitor, cytochalasin D (10 μg/ml; a
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136 Sigma). To inhibit NET-mediated bacterial killing, PMNs were incubated with DNaseI (100
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137 units/ml; Invitrogen) for 15 min prior to addition of bacteria. B. pseudomallei WT K96243 m

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138 was then added to the PMNs at a MOI of 10, followed by continued incubation in the tt
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139 presence or absence of DNaseI for 90 min. Infected PMNs were then lysed with 1% Triton-X ia
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140 100 for 10 min and each well was scraped to recover all lysed cells. Serial dilutions of the s
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141 lysates were plated to LB agar plates and bacterial colonies counted after 48 h incubation. r
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142 Phagocytosis assay by using flow cytometry o
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143 B. pseudomallei WT and mutant strains were cultured overnight cultures in LB broth p
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144 were adjusted to 1×10 CFU/ml in phosphate-buffered saline (PBS). Bacteria were collected ,

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145 by centrifugation, incubated with 1 μg/ml fluorescein isothiocyanate (FITC; Sigma) for 60 1
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146 min at room temperature, then washed three times in 1 ml of PBS. The intensity of the FITC y

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147 signal due to labeled bacteria was determined prior to use. FITC-labeled bacteria at a MOI of e
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148 10 were incubated at 37°C for 90 min in 5x10 cells/ml of PMNs purified from healthy
149 donors. Cells were washed twice in ice-cold PBS and then fixed in 2% (v/v)
150 paraformaldehyde in PBS prior to flow cytometric analysis.
151 Oxidative burst assay by using flow cytometry
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152 Bacteria at a MOI of 10 were added into 50 μl diluted heparinized whole blood at
153 37°C for 90 min and 800 ng/ml PMA was used as positive control. Hydroethidine (Sigma)
154 was added to a final concentration of 2,800 ng/ml of blood at 37°C for 5 min. Cells were
155 incubated in 2 ml FACS lysing solution (BD Bioscience) to lyse red blood cells, washed
156 twice with PBS, and fixed with 2% (v/v) paraformaldehyde in PBS. Following lysis and
157 fixation, flow cytometric analysis was performed with a FACScan flow cytometer by using D
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158 Cellquest software (BD Bioscience). n
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159 Immunofluorescence assay ad
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160 PMNs from healthy subjects were incubated at a concentration of 2.5×106 cells/ml d f
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161 with medium control or B. pseudomallei WT K96243 at a MOI of 10 (unless specified
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162 elsewhere) at 37°C for 90 min in the absence or presence of 100 units/ml of DNaseI p
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163 (Invitrogen). Samples were fixed with 4% (v/v) paraformaldehyde and blocked with 3% i.
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164 (w/v) bovine serum albumin (BSA; Sigma) in PBS for 30 min at room temperature. Bacteria m
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165 were stained with a rabbit polyclonal anti-B. pseudomallei antibody or mouse polyclonal anti- rg
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166 LPS antibody and primary antibodies were detected by a mouse anti-rabbit Ig-Alexa-Flour n

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167 488 or a goat anti-mouse IgG Alexa-Flour 568 (Molecular Probes, Leiden, Netherlands). r
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168 Histones were stained with a rabbit polyclonal anti-histone H3 antibody and a mouse anti- ,

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169 rabbit Ig-Alexa-Flour 488 (Molecular Probes) (17). Double-stranded DNA was stained with 1
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170 propidium iodide (PI) or 4'-6-Diamidino-2-phenylindole (DAPI) (Molecular Probes). Stained y

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171 cells were washed and analyzed by confocal laser scanning microscopy (LSM 510 META; e
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172 Carl Zeiss, Germany).
173 Treatment of PMNs with inhibitors
174 The NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI), the PI3K
175 inhibitor wortmannin, the p38-MAPK inhibitor SB203580, and the Src family kinase
176 inhibitor PP2 were purchased from Sigma. The chemokine CXCL8 (IL-8) was purchased
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177 from Peprotech (Rocky Hill, NJ). Briefly, PMNs were pretreated with DPI, wortmannin,
178 SB203580, or PP2 at doses indicated in the legend of Figure 3 at 37°C for 90 min prior to
179 stimulation with 100 nM PMA, 100 nM recombinant human CXCL8 (IL-8), or B.
180 pseudomallei WT K96243 at a MOI of 10 at 37°C for 90 min and NET release was then
181 quantified as described above.
182 Statistical analysis D
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183 Statistical analysis was performed using tests appropriate to the dataset, as specified
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184 in figure legends, using GraphPad Prism 5 software (GraphPad, San Diego, USA). P values a
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185 of ≤ 0.05 were considered statistically significant.
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186 m

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187 RESULTS
188 B. pseudomallei induces NET formation in time- and dose-dependent manner
189 We first studied whether NET formation was induced by B. pseudomallei. The
190 composition of extracellular material released by B. pseudomallei-infected human PMNs was
191 analyzed by confocal microscopy. We observed that B. pseudomallei co-localized with DNA
192 ejected from PMN which stained with antibody specific for histone H3 (Fig. 1A-D). The D
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193 morphology and composition of the ejected material is typical of NETs. To determine
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194 whether B. pseudomallei-induced NETs were degraded by DNase treatment, PMNs from a
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195 healthy human subjects were infected with B. pseudomallei in the absence or presence of
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196 DNase I enzyme. Compared to uninfected cells (Fig. 1E); propidium iodide staining of DNA m

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197 revealed the release of webs of extracellular DNA containing trapped bacteria upon B. tt
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198 pseudomallei infection (Fig. 1F). Treatment of such infected PMNs with DNase I resulted in ia
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199 loss of such extracellular DNA networks and the bacteria no longer appeared to be entrapped s
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200 (Fig. 1G), suggesting released nucleotides were composed of DNA but not RNA. r
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201 The kinetics of NET formation were next studied in B. pseudomallei-infected PMNs o
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202 over time and a range of MOIs. Extracellular DNA in culture supernatants was quantified as p
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203 an indicator of NET formation as described in Materials and Methods. Although the extent of 3
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204 DNA release was comparable to control levels at a MOI of 0.1 or 0.3, the amount of 0
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205 extracellular DNA released by infected PMN was significantly increased at MOIs from 1 to b
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206 10, and at 60 min post-infection and time intervals thereafter (Fig. 1H). u
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207 NETs kill B. pseudomallei t
208 We next examined the consequences of NET release for B. pseudomallei by
209 quantifying viable bacteria after infection of PMNs from healthy subjects. Under the assay
210 conditions, stimulation of the cells with PMA elicited the release of extracellular DNA
211 relative to PMNs in medium alone as expected (Fig. 2A). Cytochalasin D did not affect the
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