J Infect Chemother xxx (2014) 1e7
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Original article
Acinetobacter baumannii escape from neutrophil extracellular traps (NETs) Go Kamoshida*, Takane Kikuchi-Ueda, Shigeru Tansho-Nagakawa, Ryuichi Nakano, Akiyo Nakano, Hirotoshi Kikuchi, Tsuneyuki Ubagai, Yasuo Ono Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2014 Received in revised form 28 August 2014 Accepted 29 August 2014 Available online xxx
Acinetobacter baumannii and Pseudomonas aeruginosa are the same aerobic gram-negative bacillus and are usually harmless but cause infectious diseases in compromised hosts. Neutrophils play a critical role in infective protection against the extracellular growth of bacteria. Recently, a new biological defense mechanism called neutrophil extracellular traps (NETs) has been attracting attention. In present study, we investigated the responsiveness of neutrophils to A. baumannii and P. aeruginosa, focusing on NET formation. Neutrophils were co-cultured with A. baumannii or P. aeruginosa, and then DNA, histone and neutrophil elastase were stained, and the formation of NETs was evaluated. Neutrophils stimulated with A. baumannii had spread, but their shapes was maintained, and the nucleus was observed as clearly as that in non-stimulated neutrophils. However, neutrophils stimulated with P. aeruginosa did not maintain their cellular morphology, and the nucleus was disrupted with DNA, histones, and neutrophil elastase released into the extracellular space. These results suggest that A. baumannii does not induce NET formation, in contrast to P. aeruginosa. In addition, we measured expression of myeloperoxidase (MPO), reactive oxygen species (ROS) and superoxide in neutrophils, and we found that these expression in P. aeruginosa-stimulated neutrophils was stronger than that in A. baumannii-stimulated neutrophils. Furthermore, A. baumannii was not killed by neutrophils, in contrast to P. aeruginosa. In this study, we show that the reactivity of neutrophils and their biological defense mechanism are different between A. baumannii and P. aeruginosa, which is important for understanding the pathogenicity of these bacteria.
Keywords: Neutrophil Neutrophil extracellular traps (NETs) Acinetobacter baumannii Pseudomonas aeruginosa
© 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
1. Introduction Acinetobacter baumannii and Pseudomonas aeruginosa are the same aerobic gram-negative bacillus and are an environmental bacterium widely distributed in nature. Although these bacteria are usually harmless, they cause various infectious diseases in compromised hosts with immunodeficiency and in ventilated patients. Both types of bacteria easily acquire drug resistance, thereby inducing a natural transformation to order to capture the DNA fragments present in the extracellular space and to incorporate this DNA into the chromosomal DNA [1e3]. As a result, multidrugresistant strains of A. baumannii and P. aeruginosa have become an important problem in hospital-acquired infections [4,5].
* Corresponding author. Tel.: þ81 3 3964 1211; fax: þ81 3 5375 5284. E-mail address: [email protected] (G. Kamoshida).
Refractory infections such as ventilator-associated pneumonia (VAP) caused by multidrug-resistant A. baumannii (MDRA) have increased rapidly in Europe and the United States since the late 1990s [6]. MDRA infection an emerging infectious disease because the number of patients who have died from this infection in the world continue to increase [7,8]. The bacterial capsule, biofilm formation, and high iron-acquisition capacity are known virulence factors of A. baumannii [1,9]. However, specific virulence factor of A. baumannii remain unclear. Many studies of A. baumannii have evaluated drug-resistance mechanisms including drug-resistance genes [1,7]. Although neutrophils play a critical role in the infective protection against extracellular growing bacteria, studies on their interaction with A. baumannii and neutrophils are limited. Neutrophils migrate toward sites of infection and then combat bacteria by phagocytosis and producing toxic substances such as reactive oxygen species (ROS) [10]. Recently, in addition to the host defense mechanism, i.e., phagocytosis and ROS production, a new
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Please cite this article in press as: Kamoshida G, et al., Acinetobacter baumannii escape from neutrophil extracellular traps (NETs), J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.08.032
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biological defense mechanism called neutrophil extracellular traps (NETs) has been attracting attention [11e13]. Strongly activated neutrophils induce the formation of NETs in a new cell death pathway that is neither apoptosis nor necrosis [14,15]. NET formation is accompanied by destruction of the nucleus with the release of cellular DNA and chromatin into the extracellular space and the formation of a web-like structure. In general, it is considered that NETs efficiently catch pathogens and inactivate them using anti-microbial proteins such as neutrophil elastase that are localized on NETs [16,17]. Although NETs are effective for protection against infection, they induce inflammation, autoimmune diseases, and thrombosis, thereby releasing several factors present in cells; therefore, NETs appear to have paradoxical functions [18,19] and their function remains unclear. In this study, we investigated neutrophil responsiveness against A. baumannii and P. aeruginosa, focusing on NET formation. 2. Materials and methods 2.1. Reagents and antibodies Heparin was purchased from Novo Nordisk (Copenhagen, Denmark). Dextran 200,000 and Lymphosepar I were obtained from Wako Pure Chemical Industries (Osaka, Japan) and ImmunoBiological Laboratries (Takasaki, Japan), respectively. Diff-Quik, and Dapi Fluoromount-G were purchased from Sysmex (Kobe, Japan) and SouthernBiotech (Birmingham, AL, USA), respectively. Recombinant DNase I was obtained from TaKaRa (Osaka, Japan). Total ROS/superoxide detection kit was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Antibodies directed to histone H3 and neutrophil elastase were purchased from Cell Signaling Technology (Beverly, MA, USA) and Abcam (Cambridge, MA, USA), respectively. FITC-conjugated antiMPO antibody was obtained from Dako Cytomation (Glostrup, Denmark). Anti-rabbit IgG (Alexa Fluor 555 conjugate) and anti-rabbit IgG (HRP conjugate) were purchased from Cell Signaling Technology. 2.2. Bacteria and neutrophils A. baumannii (ATCC19606) and P. aeruginosa (PAO1) were used as a reference strain. ATCC19606 and PAO1 were grown for 16 h at 37 C in LuriaeBertani broth (Wako Pure Chemical Industries) [11]. These bacteria were then washed three times with phosphatebuffered saline (PBS), and used to suspend fresh RPMI 1640 medium (Life Technologies, Gaithersburg, MD, USA). Neutrophils were isolated from the venous blood of healthy volunteers as described previously [20]. Heparinized human blood was mixed with dextran 200,000/saline (final concentration of 1%) to sediment most of the erythrocytes. After the samples were left to stand for 30 min, the supernatant was then subjected to Lymphosepar I density gradient centrifugation at 1600 rpm for 30 min. Neutrophils were purified from the pelleted cells by hypotonic conditions to lyse the remaining erythrocytes. The purity of the neutrophils was higher than 95% as assessed by Diff-Quik staining, which is a rapid and easy method for May-Grünwald-Giemsa staining. The significance of this study was explained to all participants to obtain informed consent. The protocol was approved by the Ethical Review Committee at Teikyo University School of Medicine (No. 07104). 2.3. Visualization and quantification of NETs Neutrophils (1 106 cells/ml) and bacteria (5 107 CFU) were co-cultured on slides for 1 h at 37 C under a 5% CO2 atmosphere in
RPMI 1640/2% human serum. These cells were then fixed with 5% formaldehyde at room temperature for 10 min and washed three times with PBS. These cells were stained with Diff-Quik and observed with microscopy. DNA was stained with DAPI and observed with fluorescence microscopy (BX53, Olympus, Tokyo, Japan). Quantification of NETs was performed to measure the extent of DNA stained by DAPI per cell using ImageJ (US National Institutes of Health, Bethesda, MD, USA). 2.4. Immunofluorescence microscopy Neutrophils were stimulated with bacteria for 1 h and fixed, and then treated with 2.5% skim milk (Wako Pure Chemical Industries) for 30 min, followed by treatment with anti-histone H3 antibody (1:100 dilution) or anti-neutrophil elastase antibody (1:200 dilution) for 1 h. The cells were then stained with Alexa Fluor 555conjugated anti-rabbit IgG (1:1000 dilution) secondary antibody for 30 min. After each reaction step, the cells were washed five times with PBS. Finally, the DNA was stained with DAPI. The fluorescently labeled cells were observed with fluorescence microscopy as described previously [21]. 2.5. Immunoblotting For detection of histone H3 that was released and remained attached to the extracellular DNA, immunoblotting was performed as described previously [22]. Neutrophils and bacteria were cocultured on 24-well plates for 1 h, and the supernatant was removed and supplemented with DNase I (50 U/ml) in RPMI 1640/ 10% fetal calf serum (FCS). After incubation for 15 min at 37 C, the supernatant was recovered and gently removed from the cells by centrifugation. Specimens were subjected to SDS-polyacrylamide gel (5e20%) electrophoresis (AE-6530, ATTO, Tokyo, Japan), and separated proteins were electroblotted onto a nitrocellulose membrane (Hybound-ECL, GE Healthcare, Buckinghamshire, UK) by semi-dry blotting (BE-330, Bio-Craft, Tokyo, Japan). The blotted membrane was blocked with 2.5% skim milk for 30 min, and the membrane was treated with anti-histone H3 antibody (1:2000 dilution) for 20 h and then with HRP-conjugated anti-rabbit IgG antibody (1:3000 dilution) for 30 min. The membrane was washed with Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-T) three times after each reaction step. Proteins were detected using an enhanced chemiluminescence (ECL) detection system (Bio-Rad Laboratories, Hercules, CA, USA and ImageQuant LAS 500, GE). 2.6. Flow cytometric analysis The expression of myeloperoxidase (MPO) was measured with a flow cytometer (FACSCanto II, BD Biosciences, San Diego, CA, USA) using an FITC-labeled antibody against MPO, essentially as described previously [23]. Neutrophils were stimulated with bacteria for 1 h, fixed with 5% formaldehyde, and stained with an antiMPO antibody (1:100 dilution) for 30 min. After each step, the cells were washed three times with PBS containing 0.5% bovine serum albumin (BSA). Expression of reactive oxygen species (ROS) and superoxide in neutrophils was detected by total ROS/superoxide detection kit. 2.7. Survival analysis of bacteria Bacteria (5 107 CFU) and neutrophils (1 106 cells/ml) were co-cultured on 24-well plates for 1 h, and then lysed with 1% Triton X-100 (Wako). Appropriate dilutions were spread onto LuriaeBertani agar plates, incubated at 37 C overnight, colony were counted, and calculated the survival rate.
Please cite this article in press as: Kamoshida G, et al., Acinetobacter baumannii escape from neutrophil extracellular traps (NETs), J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.08.032
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Fig. 1. Condition for neutrophil interaction with bacteria. Neutrophils (1 106 cells/ml) and A. baumannii or P. aeruginosa (5 107 CFU) were co-cultured for 1 h in RPMI 1640/2% human serum. These cells were then fixed with 5% formaldehyde and stained Diff-Quik (A). DNA was stained with DAPI and observed with fluorescence microscopy (B). Scale bar ¼ 50 mm. (C) Quantification of NETs was performed to measure the extent of DNA stained with DAPI. The data are shown as the mean with SD of 4e6 fields selected at random. The experiment was conducted in triplicate. ***p < 0.005 vs. control neutrophils.
Please cite this article in press as: Kamoshida G, et al., Acinetobacter baumannii escape from neutrophil extracellular traps (NETs), J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.08.032
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3. Results 3.1. Observation and quantification of NETs with bacterial stimulation Neutrophils were co-cultured with A. baumannii (ATCC19606) or P. aeruginosa (PAO1) for 1 h in RPMI 1640/2% human serum, and the cells were then stained with Diff-Quick. Neutrophils stimulated with A. baumannii had spread, but their shapes were clearly the same as those of non-stimulated neutrophils. However, P. aeruginosa-stimulated neutrophils did not maintain their cell shape (Fig. 1A). When viewed on an enlarged scale, A. baumannii was observed to be adherent around neutrophils, and thus not phagocytosed. In contrast, P. aeruginosa had aggregated on ruptured neutrophils (Fig. 1A lower panels). We next stained the nuclear DNA with DAPI. The nucleus of A. baumannii-stimulated neutrophils was observed as clearly as the nucleus of nonstimulated cells, whereas stimulation with P. aeruginosa disrupted the nucleus and DNA was released into the extracellular space (Fig. 1B). Thus, A. baumannii does not induce the formation of neutrophil extracellular traps (NETs), whereas P. aeruginosa induces NET formation. Furthermore, we measured the spread of DNA per cell for quantification of NETs and observed an increase of
approximately 3 fold in the amount of DNA in P. aeruginosa-stimulated neutrophils compared to A. baumannii-stimulation cells and control cells (Fig. 1C). In addition, NET formation was not induced in neutrophils in the range of 1e100 MOI (multiplicity of infection) A. baumannii (data not shown) or with 3 h of incubation time (data not shown). Additionally, even when we performed these experiments using clinical isolate strains of multidrug-resistant A. baumannii rather than the ATCC19606 strain, NET formation was not induced (data not shown). In NET formation, it is known that histone and neutrophil elastase are released by neutrophils and then localized on extracellular DNA [11,13]. Therefore, we performed immunofluorescence microscopy to investigate the co-localization of histones, neutrophil elastase, and NETs. Neutrophils were stimulated by A. baumannii or P. aeruginosa for 1 h, and the cells were stained with an anti-histone H3 antibody or anti-neutrophil elastase antibody, and then DNA (NETs) was stained with DAPI. The P. aeruginosa stimulation induce NET formation and released histone and neutrophil elastase, which were co-localized with NETs. However, A. baumannii stimulation did not induce NET formation; therefore, the release of histone and neutrophil elastase were hardly detected (Fig. 2A). We then performed an immunoblot analysis against histone H3, which is released and remains attached to the
Fig. 2. Detection of extracellular histone H3 and neutrophil elastase upon NET induction. (A) Neutrophils and A. baumannii or P. aeruginosa were co-cultured for 1 h in RPMI 1640/2% human serum, and the cells were fixed with 5% formaldehyde. The fixed cells were then stained with anti-histone H3 (upper panels) or anti-neutrophil elastase antibodies (lower panels), followed by treatment with a fluorescent secondary antibody. DNA was stained with DAPI and observed with fluorescence microscopy. Scale bar ¼ 50 mm. (B) Detection of histone H3 that was released and remained on extracellular DNA was performed with immunoblotting. The co-cultured supernatant was removed and supplemented with DNase I in RPMI 1640/10% FCS. After incubation for 15 min at 37 C, the supernatant was recovered and analyzed by immunoblotting using an anti-histone H3 antibody.
Please cite this article in press as: Kamoshida G, et al., Acinetobacter baumannii escape from neutrophil extracellular traps (NETs), J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.08.032
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cultured on 24-well plates for 1 h, the survived bacteria was counted, and calculated the survival rate. As a results, the survival rate of P. aeruginosa was less than 50% in the presence of neutrophils. However, A. baumannii was not affected by neutrophils and most bacteria survived (Fig. 4). We found that A. baumannii was not killed by neutrophils, in contrast to P. aeruginosa.
extracellular DNA, for quantification of NETs. The amount of histones that was released and remained on NETs increased when neutrophils were stimulated with P. aeruginosa but not with A. baumannii stimulation (Fig. 2B). 3.2. Expression of MPO, ROS and superoxide in neutrophils induced by bacterial stimulation
4. Discussion We measured expression of MPO, ROS and superoxide in neutrophils using a flow cytometer. The flow cytometric analysis showed that MPO, ROS and superoxide expression in P. aeruginosa-stimulated neutrophils was stronger than that in cells stimulated by A. baumannii (Fig. 3). These expression in P. aeruginosa-stimulation was comparable to the PMA stimulation. We found that the increase in expression of MPO, ROS and superoxide in neutrophils with P. aeruginosa stimulation was higher than that in cells stimulated by A. baumannii.
In this study, we investigated neutrophil responsiveness to A. baumannii and P. aeruginosa, focusing on NET formation. A. baumannii (ATCC19606) did not induce NET formation, whereas P. aeruginosa (PAO1) induced NET formation (Figs. 1 and 2). In bacterial infection, neutrophils phagocytose bacteria and eradicate them by producing reactive oxygen species (ROS) [10,24]. Myeloperoxidase (MPO), which is a ROS producing enzyme, is abundant in the azurophilic granules of neutrophils [25]. Neutrophils combat bacteria using MPO; therefore, expression of MPO is increased by bacterial infection. Additionally, it has been reported that MPO and ROS are involved in the induction of NETs [15,26,27]. We showed that the increased expression of MPO, ROS and superoxide in
3.3. Survival of bacteria by co-culture with neutrophils We then analyze survival rate of bacteria by co-culture with neutrophils. A. baumannii or P. aeruginosa and neutrophils were co-
A Fluorescence intensity mean
x103 7 6 5 4 3 2
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Fluorescence intensity mean
B
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C
x102 9 8 7
6 5
4 3
***
MPO
x103 8 ROS
***
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Superoxide
***
6 5 4
3
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2
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1
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Fig. 3. Expression of MPO, ROS and superoxide in neutrophils following bacterial stimulation. Neutrophils that had been co-cultured with A. baumannii or P. aeruginosa for 1 h were analyzed by flow cytometry after being stained with MPO (A), ROS (B) and superoxide (C). The mean fluorescence intensity was measured and shown. () indicates without antiMPO antibody. The experiment were conducted in triplicate. ***p < 0.005 vs. A. baumannii.
Please cite this article in press as: Kamoshida G, et al., Acinetobacter baumannii escape from neutrophil extracellular traps (NETs), J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.08.032
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G. Kamoshida et al. / J Infect Chemother xxx (2014) 1e7
Survival rate (%)
150
100
*** 50
0 Neutrophils
-
+
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Fig. 4. Survival rate of bacteria by co-culture with neutrophils. A. baumannii or P. aeruginosa and neutrophils were co-cultured for 1 h, the survived bacteria was counted, and calculated the survival rate. The experiment were conducted in triplicate. ***p < 0.005 vs. neutrophils () P. aeruginosa.
formalin, NET induction was completely inhibited (data not shown). Consequently, soluble factors produced by P. aeruginosa appear to be important for NET induction. Indeed, Rada et al. reported that pyocyanin enhanced NET formation via ROS [35]. Several reports have indicated that MPO and ROS are involved in NET induction [15,26,27]. Thus, we measured expression of MPO, ROS and superoxide in neutrophils and found that these expression in P. aeruginosa-stimulated neutrophils was stronger than that in A. baumannii-stimulated cells (Fig. 3). As expected, this finding raises the possibility that differences in MPO and ROS production may be involved in the differences in NET induction. Future studies should address the detailed mechanism of NET induction, why NETs are not induced in A. baumannii, and what factor of P. aeruginosa induces NET formation. In this study, the in vitro NET induction system provided a useful model to investigate the NET formation and interaction with neutrophils and bacteria. The findings of the present study indicate a difference in neutrophil reactivity to A. baumannii and P. aeruginosa, which is important for understanding the pathogenicity of these bacteria. Conflict of interest None.
neutrophils with P. aeruginosa stimulation was higher than that with A. baumannii stimulation (Fig. 3). Furthermore, A. baumannii was not killed by neutrophils, in contrast to P. aeruginosa (Fig. 4). These results suggested that reactivity of neutrophils and biological defense mechanisms are different between A. baumannii infection and P. aeruginosa infection. It is known that A. baumannii and P. aeruginosa, which are important opportunistic pathogens, have similar characteristics such as drug resistance and biofilm formation [3,28,29]. However, the neutrophil responses to A. baumannii and P. aeruginosa seem different. Neutrophils play a critical role in protection against infection by extracellular growing bacteria including A. baumannii and P. aeruginosa; therefore the results of this study are important. Recent studies have demonstrated that NET formation was induced by P. aeruginosa, and it is thus considered that neutrophils combat bacteria using NETs [30]. The finding that A. baumannii did not induce NET formation was somewhat surprising to us because we assumed that A. baumannii would induce NET formation similarly to P. aeruginosa. Furthermore, we observed that A. baumannii does not seem to be phagocytosed by neutrophils (Fig. 1A). Neutrophils combat P. aeruginosa by phagocytosis and NET formation, but A. baumannii appears to be different. These results show that the neutrophil response to A. baumannii is different from the to P. aeruginosa. In A. baumannii infection, because ventilator-associated pneumonia (VAP) is an important problem, interactions with lung and airway epithelial cells have been studied [31,32]. Gaddy et al. reported that A. baumannii adheres to epithelial cells via outer membrane protein A (OmpA) [33]. Moreover, Choi et al. reported that in addition to adhesion, A. baumannii can invade epithelial cells [34]. Thus, A. baumannii adheres to neutrophils using these mechanism described above and may thus avoid phagocytosis and NET formation. We assumed that P. aeruginosa induces NET formation through phagocytosis, but A. baumannii does not escape phagocytosis from neutrophils. Therefore, we then performed a NET analysis by suppressing the phagocytosis of neutrophils using cyochalasin B, which is an inhibitor of phagocytosis. NET induction by P. aeruginosa was induced even under suppression of phagocytosis (data not shown). We found that NET formation and phagocytosis are thus likely unrelated. However, by fixing P. aeruginosa with
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Original article
Acinetobacter baumannii escape from neutrophil extracellular traps (NETs) Go Kamoshida*, Takane Kikuchi-Ueda, Shigeru Tansho-Nagakawa, Ryuichi Nakano, Akiyo Nakano, Hirotoshi Kikuchi, Tsuneyuki Ubagai, Yasuo Ono Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2014 Received in revised form 28 August 2014 Accepted 29 August 2014 Available online xxx
Acinetobacter baumannii and Pseudomonas aeruginosa are the same aerobic gram-negative bacillus and are usually harmless but cause infectious diseases in compromised hosts. Neutrophils play a critical role in infective protection against the extracellular growth of bacteria. Recently, a new biological defense mechanism called neutrophil extracellular traps (NETs) has been attracting attention. In present study, we investigated the responsiveness of neutrophils to A. baumannii and P. aeruginosa, focusing on NET formation. Neutrophils were co-cultured with A. baumannii or P. aeruginosa, and then DNA, histone and neutrophil elastase were stained, and the formation of NETs was evaluated. Neutrophils stimulated with A. baumannii had spread, but their shapes was maintained, and the nucleus was observed as clearly as that in non-stimulated neutrophils. However, neutrophils stimulated with P. aeruginosa did not maintain their cellular morphology, and the nucleus was disrupted with DNA, histones, and neutrophil elastase released into the extracellular space. These results suggest that A. baumannii does not induce NET formation, in contrast to P. aeruginosa. In addition, we measured expression of myeloperoxidase (MPO), reactive oxygen species (ROS) and superoxide in neutrophils, and we found that these expression in P. aeruginosa-stimulated neutrophils was stronger than that in A. baumannii-stimulated neutrophils. Furthermore, A. baumannii was not killed by neutrophils, in contrast to P. aeruginosa. In this study, we show that the reactivity of neutrophils and their biological defense mechanism are different between A. baumannii and P. aeruginosa, which is important for understanding the pathogenicity of these bacteria.
Keywords: Neutrophil Neutrophil extracellular traps (NETs) Acinetobacter baumannii Pseudomonas aeruginosa
© 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
1. Introduction Acinetobacter baumannii and Pseudomonas aeruginosa are the same aerobic gram-negative bacillus and are an environmental bacterium widely distributed in nature. Although these bacteria are usually harmless, they cause various infectious diseases in compromised hosts with immunodeficiency and in ventilated patients. Both types of bacteria easily acquire drug resistance, thereby inducing a natural transformation to order to capture the DNA fragments present in the extracellular space and to incorporate this DNA into the chromosomal DNA [1e3]. As a result, multidrugresistant strains of A. baumannii and P. aeruginosa have become an important problem in hospital-acquired infections [4,5].
* Corresponding author. Tel.: þ81 3 3964 1211; fax: þ81 3 5375 5284. E-mail address: [email protected] (G. Kamoshida).
Refractory infections such as ventilator-associated pneumonia (VAP) caused by multidrug-resistant A. baumannii (MDRA) have increased rapidly in Europe and the United States since the late 1990s [6]. MDRA infection an emerging infectious disease because the number of patients who have died from this infection in the world continue to increase [7,8]. The bacterial capsule, biofilm formation, and high iron-acquisition capacity are known virulence factors of A. baumannii [1,9]. However, specific virulence factor of A. baumannii remain unclear. Many studies of A. baumannii have evaluated drug-resistance mechanisms including drug-resistance genes [1,7]. Although neutrophils play a critical role in the infective protection against extracellular growing bacteria, studies on their interaction with A. baumannii and neutrophils are limited. Neutrophils migrate toward sites of infection and then combat bacteria by phagocytosis and producing toxic substances such as reactive oxygen species (ROS) [10]. Recently, in addition to the host defense mechanism, i.e., phagocytosis and ROS production, a new
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Please cite this article in press as: Kamoshida G, et al., Acinetobacter baumannii escape from neutrophil extracellular traps (NETs), J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.08.032
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biological defense mechanism called neutrophil extracellular traps (NETs) has been attracting attention [11e13]. Strongly activated neutrophils induce the formation of NETs in a new cell death pathway that is neither apoptosis nor necrosis [14,15]. NET formation is accompanied by destruction of the nucleus with the release of cellular DNA and chromatin into the extracellular space and the formation of a web-like structure. In general, it is considered that NETs efficiently catch pathogens and inactivate them using anti-microbial proteins such as neutrophil elastase that are localized on NETs [16,17]. Although NETs are effective for protection against infection, they induce inflammation, autoimmune diseases, and thrombosis, thereby releasing several factors present in cells; therefore, NETs appear to have paradoxical functions [18,19] and their function remains unclear. In this study, we investigated neutrophil responsiveness against A. baumannii and P. aeruginosa, focusing on NET formation. 2. Materials and methods 2.1. Reagents and antibodies Heparin was purchased from Novo Nordisk (Copenhagen, Denmark). Dextran 200,000 and Lymphosepar I were obtained from Wako Pure Chemical Industries (Osaka, Japan) and ImmunoBiological Laboratries (Takasaki, Japan), respectively. Diff-Quik, and Dapi Fluoromount-G were purchased from Sysmex (Kobe, Japan) and SouthernBiotech (Birmingham, AL, USA), respectively. Recombinant DNase I was obtained from TaKaRa (Osaka, Japan). Total ROS/superoxide detection kit was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Antibodies directed to histone H3 and neutrophil elastase were purchased from Cell Signaling Technology (Beverly, MA, USA) and Abcam (Cambridge, MA, USA), respectively. FITC-conjugated antiMPO antibody was obtained from Dako Cytomation (Glostrup, Denmark). Anti-rabbit IgG (Alexa Fluor 555 conjugate) and anti-rabbit IgG (HRP conjugate) were purchased from Cell Signaling Technology. 2.2. Bacteria and neutrophils A. baumannii (ATCC19606) and P. aeruginosa (PAO1) were used as a reference strain. ATCC19606 and PAO1 were grown for 16 h at 37 C in LuriaeBertani broth (Wako Pure Chemical Industries) [11]. These bacteria were then washed three times with phosphatebuffered saline (PBS), and used to suspend fresh RPMI 1640 medium (Life Technologies, Gaithersburg, MD, USA). Neutrophils were isolated from the venous blood of healthy volunteers as described previously [20]. Heparinized human blood was mixed with dextran 200,000/saline (final concentration of 1%) to sediment most of the erythrocytes. After the samples were left to stand for 30 min, the supernatant was then subjected to Lymphosepar I density gradient centrifugation at 1600 rpm for 30 min. Neutrophils were purified from the pelleted cells by hypotonic conditions to lyse the remaining erythrocytes. The purity of the neutrophils was higher than 95% as assessed by Diff-Quik staining, which is a rapid and easy method for May-Grünwald-Giemsa staining. The significance of this study was explained to all participants to obtain informed consent. The protocol was approved by the Ethical Review Committee at Teikyo University School of Medicine (No. 07104). 2.3. Visualization and quantification of NETs Neutrophils (1 106 cells/ml) and bacteria (5 107 CFU) were co-cultured on slides for 1 h at 37 C under a 5% CO2 atmosphere in
RPMI 1640/2% human serum. These cells were then fixed with 5% formaldehyde at room temperature for 10 min and washed three times with PBS. These cells were stained with Diff-Quik and observed with microscopy. DNA was stained with DAPI and observed with fluorescence microscopy (BX53, Olympus, Tokyo, Japan). Quantification of NETs was performed to measure the extent of DNA stained by DAPI per cell using ImageJ (US National Institutes of Health, Bethesda, MD, USA). 2.4. Immunofluorescence microscopy Neutrophils were stimulated with bacteria for 1 h and fixed, and then treated with 2.5% skim milk (Wako Pure Chemical Industries) for 30 min, followed by treatment with anti-histone H3 antibody (1:100 dilution) or anti-neutrophil elastase antibody (1:200 dilution) for 1 h. The cells were then stained with Alexa Fluor 555conjugated anti-rabbit IgG (1:1000 dilution) secondary antibody for 30 min. After each reaction step, the cells were washed five times with PBS. Finally, the DNA was stained with DAPI. The fluorescently labeled cells were observed with fluorescence microscopy as described previously [21]. 2.5. Immunoblotting For detection of histone H3 that was released and remained attached to the extracellular DNA, immunoblotting was performed as described previously [22]. Neutrophils and bacteria were cocultured on 24-well plates for 1 h, and the supernatant was removed and supplemented with DNase I (50 U/ml) in RPMI 1640/ 10% fetal calf serum (FCS). After incubation for 15 min at 37 C, the supernatant was recovered and gently removed from the cells by centrifugation. Specimens were subjected to SDS-polyacrylamide gel (5e20%) electrophoresis (AE-6530, ATTO, Tokyo, Japan), and separated proteins were electroblotted onto a nitrocellulose membrane (Hybound-ECL, GE Healthcare, Buckinghamshire, UK) by semi-dry blotting (BE-330, Bio-Craft, Tokyo, Japan). The blotted membrane was blocked with 2.5% skim milk for 30 min, and the membrane was treated with anti-histone H3 antibody (1:2000 dilution) for 20 h and then with HRP-conjugated anti-rabbit IgG antibody (1:3000 dilution) for 30 min. The membrane was washed with Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-T) three times after each reaction step. Proteins were detected using an enhanced chemiluminescence (ECL) detection system (Bio-Rad Laboratories, Hercules, CA, USA and ImageQuant LAS 500, GE). 2.6. Flow cytometric analysis The expression of myeloperoxidase (MPO) was measured with a flow cytometer (FACSCanto II, BD Biosciences, San Diego, CA, USA) using an FITC-labeled antibody against MPO, essentially as described previously [23]. Neutrophils were stimulated with bacteria for 1 h, fixed with 5% formaldehyde, and stained with an antiMPO antibody (1:100 dilution) for 30 min. After each step, the cells were washed three times with PBS containing 0.5% bovine serum albumin (BSA). Expression of reactive oxygen species (ROS) and superoxide in neutrophils was detected by total ROS/superoxide detection kit. 2.7. Survival analysis of bacteria Bacteria (5 107 CFU) and neutrophils (1 106 cells/ml) were co-cultured on 24-well plates for 1 h, and then lysed with 1% Triton X-100 (Wako). Appropriate dilutions were spread onto LuriaeBertani agar plates, incubated at 37 C overnight, colony were counted, and calculated the survival rate.
Please cite this article in press as: Kamoshida G, et al., Acinetobacter baumannii escape from neutrophil extracellular traps (NETs), J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.08.032
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Fig. 1. Condition for neutrophil interaction with bacteria. Neutrophils (1 106 cells/ml) and A. baumannii or P. aeruginosa (5 107 CFU) were co-cultured for 1 h in RPMI 1640/2% human serum. These cells were then fixed with 5% formaldehyde and stained Diff-Quik (A). DNA was stained with DAPI and observed with fluorescence microscopy (B). Scale bar ¼ 50 mm. (C) Quantification of NETs was performed to measure the extent of DNA stained with DAPI. The data are shown as the mean with SD of 4e6 fields selected at random. The experiment was conducted in triplicate. ***p < 0.005 vs. control neutrophils.
Please cite this article in press as: Kamoshida G, et al., Acinetobacter baumannii escape from neutrophil extracellular traps (NETs), J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.08.032
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3. Results 3.1. Observation and quantification of NETs with bacterial stimulation Neutrophils were co-cultured with A. baumannii (ATCC19606) or P. aeruginosa (PAO1) for 1 h in RPMI 1640/2% human serum, and the cells were then stained with Diff-Quick. Neutrophils stimulated with A. baumannii had spread, but their shapes were clearly the same as those of non-stimulated neutrophils. However, P. aeruginosa-stimulated neutrophils did not maintain their cell shape (Fig. 1A). When viewed on an enlarged scale, A. baumannii was observed to be adherent around neutrophils, and thus not phagocytosed. In contrast, P. aeruginosa had aggregated on ruptured neutrophils (Fig. 1A lower panels). We next stained the nuclear DNA with DAPI. The nucleus of A. baumannii-stimulated neutrophils was observed as clearly as the nucleus of nonstimulated cells, whereas stimulation with P. aeruginosa disrupted the nucleus and DNA was released into the extracellular space (Fig. 1B). Thus, A. baumannii does not induce the formation of neutrophil extracellular traps (NETs), whereas P. aeruginosa induces NET formation. Furthermore, we measured the spread of DNA per cell for quantification of NETs and observed an increase of
approximately 3 fold in the amount of DNA in P. aeruginosa-stimulated neutrophils compared to A. baumannii-stimulation cells and control cells (Fig. 1C). In addition, NET formation was not induced in neutrophils in the range of 1e100 MOI (multiplicity of infection) A. baumannii (data not shown) or with 3 h of incubation time (data not shown). Additionally, even when we performed these experiments using clinical isolate strains of multidrug-resistant A. baumannii rather than the ATCC19606 strain, NET formation was not induced (data not shown). In NET formation, it is known that histone and neutrophil elastase are released by neutrophils and then localized on extracellular DNA [11,13]. Therefore, we performed immunofluorescence microscopy to investigate the co-localization of histones, neutrophil elastase, and NETs. Neutrophils were stimulated by A. baumannii or P. aeruginosa for 1 h, and the cells were stained with an anti-histone H3 antibody or anti-neutrophil elastase antibody, and then DNA (NETs) was stained with DAPI. The P. aeruginosa stimulation induce NET formation and released histone and neutrophil elastase, which were co-localized with NETs. However, A. baumannii stimulation did not induce NET formation; therefore, the release of histone and neutrophil elastase were hardly detected (Fig. 2A). We then performed an immunoblot analysis against histone H3, which is released and remains attached to the
Fig. 2. Detection of extracellular histone H3 and neutrophil elastase upon NET induction. (A) Neutrophils and A. baumannii or P. aeruginosa were co-cultured for 1 h in RPMI 1640/2% human serum, and the cells were fixed with 5% formaldehyde. The fixed cells were then stained with anti-histone H3 (upper panels) or anti-neutrophil elastase antibodies (lower panels), followed by treatment with a fluorescent secondary antibody. DNA was stained with DAPI and observed with fluorescence microscopy. Scale bar ¼ 50 mm. (B) Detection of histone H3 that was released and remained on extracellular DNA was performed with immunoblotting. The co-cultured supernatant was removed and supplemented with DNase I in RPMI 1640/10% FCS. After incubation for 15 min at 37 C, the supernatant was recovered and analyzed by immunoblotting using an anti-histone H3 antibody.
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cultured on 24-well plates for 1 h, the survived bacteria was counted, and calculated the survival rate. As a results, the survival rate of P. aeruginosa was less than 50% in the presence of neutrophils. However, A. baumannii was not affected by neutrophils and most bacteria survived (Fig. 4). We found that A. baumannii was not killed by neutrophils, in contrast to P. aeruginosa.
extracellular DNA, for quantification of NETs. The amount of histones that was released and remained on NETs increased when neutrophils were stimulated with P. aeruginosa but not with A. baumannii stimulation (Fig. 2B). 3.2. Expression of MPO, ROS and superoxide in neutrophils induced by bacterial stimulation
4. Discussion We measured expression of MPO, ROS and superoxide in neutrophils using a flow cytometer. The flow cytometric analysis showed that MPO, ROS and superoxide expression in P. aeruginosa-stimulated neutrophils was stronger than that in cells stimulated by A. baumannii (Fig. 3). These expression in P. aeruginosa-stimulation was comparable to the PMA stimulation. We found that the increase in expression of MPO, ROS and superoxide in neutrophils with P. aeruginosa stimulation was higher than that in cells stimulated by A. baumannii.
In this study, we investigated neutrophil responsiveness to A. baumannii and P. aeruginosa, focusing on NET formation. A. baumannii (ATCC19606) did not induce NET formation, whereas P. aeruginosa (PAO1) induced NET formation (Figs. 1 and 2). In bacterial infection, neutrophils phagocytose bacteria and eradicate them by producing reactive oxygen species (ROS) [10,24]. Myeloperoxidase (MPO), which is a ROS producing enzyme, is abundant in the azurophilic granules of neutrophils [25]. Neutrophils combat bacteria using MPO; therefore, expression of MPO is increased by bacterial infection. Additionally, it has been reported that MPO and ROS are involved in the induction of NETs [15,26,27]. We showed that the increased expression of MPO, ROS and superoxide in
3.3. Survival of bacteria by co-culture with neutrophils We then analyze survival rate of bacteria by co-culture with neutrophils. A. baumannii or P. aeruginosa and neutrophils were co-
A Fluorescence intensity mean
x103 7 6 5 4 3 2
1 0
Fluorescence intensity mean
B
(-)
C
x102 9 8 7
6 5
4 3
***
MPO
x103 8 ROS
***
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Superoxide
***
6 5 4
3
2
2
1
1
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0
Fig. 3. Expression of MPO, ROS and superoxide in neutrophils following bacterial stimulation. Neutrophils that had been co-cultured with A. baumannii or P. aeruginosa for 1 h were analyzed by flow cytometry after being stained with MPO (A), ROS (B) and superoxide (C). The mean fluorescence intensity was measured and shown. () indicates without antiMPO antibody. The experiment were conducted in triplicate. ***p < 0.005 vs. A. baumannii.
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G. Kamoshida et al. / J Infect Chemother xxx (2014) 1e7
Survival rate (%)
150
100
*** 50
0 Neutrophils
-
+
A. baumannii
-
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Fig. 4. Survival rate of bacteria by co-culture with neutrophils. A. baumannii or P. aeruginosa and neutrophils were co-cultured for 1 h, the survived bacteria was counted, and calculated the survival rate. The experiment were conducted in triplicate. ***p < 0.005 vs. neutrophils () P. aeruginosa.
formalin, NET induction was completely inhibited (data not shown). Consequently, soluble factors produced by P. aeruginosa appear to be important for NET induction. Indeed, Rada et al. reported that pyocyanin enhanced NET formation via ROS [35]. Several reports have indicated that MPO and ROS are involved in NET induction [15,26,27]. Thus, we measured expression of MPO, ROS and superoxide in neutrophils and found that these expression in P. aeruginosa-stimulated neutrophils was stronger than that in A. baumannii-stimulated cells (Fig. 3). As expected, this finding raises the possibility that differences in MPO and ROS production may be involved in the differences in NET induction. Future studies should address the detailed mechanism of NET induction, why NETs are not induced in A. baumannii, and what factor of P. aeruginosa induces NET formation. In this study, the in vitro NET induction system provided a useful model to investigate the NET formation and interaction with neutrophils and bacteria. The findings of the present study indicate a difference in neutrophil reactivity to A. baumannii and P. aeruginosa, which is important for understanding the pathogenicity of these bacteria. Conflict of interest None.
neutrophils with P. aeruginosa stimulation was higher than that with A. baumannii stimulation (Fig. 3). Furthermore, A. baumannii was not killed by neutrophils, in contrast to P. aeruginosa (Fig. 4). These results suggested that reactivity of neutrophils and biological defense mechanisms are different between A. baumannii infection and P. aeruginosa infection. It is known that A. baumannii and P. aeruginosa, which are important opportunistic pathogens, have similar characteristics such as drug resistance and biofilm formation [3,28,29]. However, the neutrophil responses to A. baumannii and P. aeruginosa seem different. Neutrophils play a critical role in protection against infection by extracellular growing bacteria including A. baumannii and P. aeruginosa; therefore the results of this study are important. Recent studies have demonstrated that NET formation was induced by P. aeruginosa, and it is thus considered that neutrophils combat bacteria using NETs [30]. The finding that A. baumannii did not induce NET formation was somewhat surprising to us because we assumed that A. baumannii would induce NET formation similarly to P. aeruginosa. Furthermore, we observed that A. baumannii does not seem to be phagocytosed by neutrophils (Fig. 1A). Neutrophils combat P. aeruginosa by phagocytosis and NET formation, but A. baumannii appears to be different. These results show that the neutrophil response to A. baumannii is different from the to P. aeruginosa. In A. baumannii infection, because ventilator-associated pneumonia (VAP) is an important problem, interactions with lung and airway epithelial cells have been studied [31,32]. Gaddy et al. reported that A. baumannii adheres to epithelial cells via outer membrane protein A (OmpA) [33]. Moreover, Choi et al. reported that in addition to adhesion, A. baumannii can invade epithelial cells [34]. Thus, A. baumannii adheres to neutrophils using these mechanism described above and may thus avoid phagocytosis and NET formation. We assumed that P. aeruginosa induces NET formation through phagocytosis, but A. baumannii does not escape phagocytosis from neutrophils. Therefore, we then performed a NET analysis by suppressing the phagocytosis of neutrophils using cyochalasin B, which is an inhibitor of phagocytosis. NET induction by P. aeruginosa was induced even under suppression of phagocytosis (data not shown). We found that NET formation and phagocytosis are thus likely unrelated. However, by fixing P. aeruginosa with
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