Pathogenic Bacterium Acinetobacter baumannii Inhibits the Formation of Neutrophil Extracellular Traps by Suppressing Neutrophil Adhesion

Hospital-acquired infections caused by Acinetobacter baumannii have become problematic because of high rates of drug resistance. A. baumannii is usually harmless, but it may cause infectious diseases in an immunocompromised host. Although neutrophils are the key players of the initial immune response against bacterial infection, their interactions with A. baumannii remain largely unknown. A new biological defense mechanism, termed neutrophil extracellular traps (NETs), has been attracting attention. NETs play a critical role in bacterial killing by bacterial trapping and inactivation. Many pathogenic bacteria have been reported to induce NET formation, while an inhibitory effect on NET formation is rarely reported. In the present study, to assess the inhibition of NET formation by A. baumannii, bacteria and human neutrophils were cocultured in the presence of phorbol 12-myristate 13-acetate (PMA), and NET formation was evaluated. NETs were rarely observed during the coculture despite neutrophil PMA stimulation. Furthermore, A. baumannii prolonged the lifespan of neutrophils by inhibiting NET formation. The inhibition of NET formation by other bacteria was also investigated. The inhibitory effect was only apparent with live A. baumannii cells. Finally, to elucidate the mechanism of this inhibition, neutrophil adhesion was examined. A. baumannii suppressed the adhesion ability of neutrophils, thereby inhibiting PMA-induced NET formation. This suppression of cell adhesion was partly due to suppression of the surface expression of CD11a in neutrophils. The current study constitutes the first report on the inhibition of NET formation by a pathogenic bacterium, A. baumannii, and prolonging the neutrophil lifespan. This novel pathogenicity to inhibit NET formation, thereby escaping host immune responses might contribute to a development of new treatment strategies for A. baumannii infections.

type of cell death mechanism, termed NETosis (19,23), and it would be very interesting if bacteria could suppress the death of immune cells. Many pathogenic bacteria have been reported to induce NET formation, while an inhibitory effect on NET formation is rarely reported (20,24,25). A. baumannii may possess a number of escape mechanisms enabling it to evade several immune responses, as demonstrated above. In the current study, we investigated the inhibitory effect of A. baumannii on NET formation. We found that A. baumannii inhibits NET formation via the action of phorbol 12-myristate 13-acetate (PMA)-induced neutrophils. Furthermore, we provide evidence that suppression of neutrophil adhesion underpins this phenomenon.
Neutrophils were isolated from the human peripheral blood of healthy volunteers (n = 10), as described previously (26). Heparinized human blood was mixed with dextran 200,000/ saline (final concentration of 1%) to sediment most of the erythrocytes. The supernatant was then processed by density gradient centrifugation using lymphosepar I (Wako Pure Chemical Industries, Osaka, Japan). Neutrophils were purified from the pelleted cells after hypotonic conditions induced lysis of the remaining erythrocytes. Purity of the neutrophil preparation was greater than 95%, as assessed by Diff Quick staining.
Written informed consent was obtained from all study participants, in accordance with the Declaration of Helsinki. The study was approved by the Ethical Review Committee of the School of Medicine of Teikyo University.

cell-adhesion assay
Neutrophils (1 × 10 6 cells/mL, 0.1 mL) were placed in a 96-well culture plate, and incubated at 37°C for 30 min in RPMI 1640 supplemented with 2% human serum. In some experiments, neutrophils were stimulated with 200-nM PMA when cocultured with A. baumannii (MOI 50), before adding them to the wells. The non-adhering neutrophils were removed by washing the wells three times with PBS. The adherent cells remaining in the wells were lysed with 1% Nonidet P-40, and LDH from adherent neutrophils was detected using an MTX-LDH assay kit, as described above. The adhesion rate of neutrophils was calculated as a percentage of the lysed neutrophils. In some experiments, the assay was performed in the presence of 0.1-µg/mL CD11ablocking antibody or control IgG.

Flow-cytometry analysis
The surface expression of CD11a and CD11b on neutrophils was determined by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA), using specific antibodies [FITC-labeled anti-CD11a antibody and PE-labeled anti-CD11b antibody (BD Biosciences)].

statistical analysis
Data are presented as the mean ± SD. The values were compared using paired Student's t-test, and differences at p < 0.01 were considered statistically significant.

A. baumannii inhibiting neT Formation
Previously, we reported that A. baumannii does not induce NET formation after a 1-h coculture with neutrophils and that neutrophils are unable to kill this bacterium (21). Thus, A. baumannii appeared to possess escape mechanisms to evade immune responses. In the present study, we investigated the effect of A. baumannii on PMA-induced NET formation. Neutrophils and A. baumannii were cocultured for 3 h in the presence of PMA, DNA was stained with DAPI, and observed under a fluorescence microscope. NET formation was observed upon PMA stimulation. However, NETs were rarely observed when neutrophils were cocultured with A. baumannii, regardless of PMA stimulation ( Figure 1A). To quantify the amount of the released extracellular DNA, the signal of SYTOX green-stained DNA in the medium was measured. The amount of extracellular neutrophil DNA increased after PMA stimulation, but it was significantly reduced upon coculture with A. baumannii ( Figure 1B). Furthermore, neutrophil cell death was evaluated by determining the amount of released LDH. Extracellular LDH levels were elevated upon PMA stimulation, but, in comparison, they were significantly reduced upon coculture with A. baumannii ( Figure 1C). Next, the survival of PMA-stimulated neutrophils was evaluated in coculture with A. baumannii. Upon PMA stimulation, most neutrophils died; in contrast, many remained alive during coculture with A. baumannii ( Figure 1D). Furthermore, A. baumannii alone did not induce neutrophil cell death. Consequently, A. baumannii prolonged the lifespan of neutrophils by inhibiting NET formation, i.e., a cell death pathway, in PMA stimulation. Furthermore, to investigate the kinetics of the inhibitory effect of A. baumannii on NET formation, NET formation was monitored during a 5-h neutrophil culture with PMA, in the presence or absence of A. baumannii by time-lapse microscopy (Figure 2A; Videos S1 and S2 in Supplementary Material), and detection of extracellular DNA ( Figure 2B). As observed, PMA-induced NET formation was inhibited by coculture with A. baumannii for up to 5 h. Compared with PMA stimulation alone, the amount of extracellular neutrophil DNA was also inhibited by coculture with A. baumannii. Hence, A. baumannii inhibited PMA-induced NET formation, thereby inhibiting PMA-stimulated cell death.

inhibitory effect of Various Bacteria on neT Formation
To investigate the inhibitory effect of A. baumannii on NET formation in more detail, the formation of NETs was quantified in the presence of various bacteria. A. baumannii reference strain (ATCC 19606) suppressed PMA-induced NET formation in an MOI-dependent manner. MDRA clinical isolates (T14 and T2) exerted the same inhibitory effect ( Figure 3A). In another experiment, A. baumannii cells were heat-killed     or formalin-killed, and the NET quantification assay was performed. Upon killing, the bacteria lost the ability to inhibit NET formation ( Figure 3B). Next, the ability of bacteria other than A. baumannii to inhibit NET formation was examined. Other bacteria from the same genus, A. calcoaceticus and A. hemolyticus, and bacteria from a different genus, E. coli, did not inhibit NET formation (Figure 4). These observations suggested that only live A. baumannii cells are able to inhibit PMA-induced NET formation.

A. baumannii inhibiting neT Formation by suppressing neutrophil adhesion
Neutrophil adhesion to the scaffold is thought to be required for NET formation. However, we previously demonstrated that neutrophil mobility is enhanced upon stimulation with A. baumannii (15). Furthermore, we evaluated neutrophil morphology at 1 h post PMA stimulation. The cytoplasm in PMA-stimulated neutrophils was thin and roundly spread, and these neutrophils appeared to adhere strongly. In contrast, PMA and A. baumannii-stimulated neutrophils appeared to adhere weakly compared with only PMA-stimulated neutrophils ( Figure S1 in Supplementary Material). Therefore, we speculated that the adhesion ability of neutrophils was reduced upon A. baumannii stimulation. Cell-adhesion assay was performed to evaluate the adhesion ability of neutrophils upon PMA stimulation and coculture with A. baumannii. The adhesion ability was enhanced by PMA stimulation, but this enhancement was suppressed during coculture with A. baumannii ( Figure 5A). Next, NET quantification assay was performed using plates coated with a cell-adhesion inhibitor, poly-2-hydroxyethyl methacrylate (poly-HEMA), to examine whether inhibition of adhesion would affect NET formation. Indeed, PMA-induced NET formation was suppressed to the  A. baumannii coculture levels when the neutrophil adhesion was inhibited (Figure 5B). These observations suggested that the inhibition of neutrophil adhesion suppresses NET formation. Furthermore, we found that A. baumannii could suppress the adhesion of PMA-stimulated neutrophils.

A. baumannii inhibiting neT Formation by inhibiting neutrophil cD11a expression
Finally, to investigate the mechanism underlying the suppression of neutrophil adhesion upon stimulation with A. baumannii, flow cytometry was used to analyze the expression of neutrophil cell surface adhesion molecules. The levels of CD11a (integrin αL) and CD11b (integrin αM) were increased upon PMA stimulation. The enhancement of CD11a expression by PMA was suppressed upon coculture with A. baumannii (Figures 6A,C). In contrast, the enhancement of CD11b expression was further increased in the presence of A. baumannii (Figures 6B,D). Because the expression of CD11a in neutrophils was suppressed by A. baumannii, the effect of CD11a on the neutrophil adhesion ability and NET formation was next assessed. The enhancement of neutrophil adhesion by PMA was reduced in the presence of a CD11a-blocking antibody ( Figure 7A). Furthermore, PMA-induced NET formation was also reduced when a CD11a-blocking antibody was present ( Figure 7B). Collectively, these observations suggested that A. baumannii suppressed the neutrophil expression of CD11a, thereby inhibiting PMA-induced NET formation.

DiscUssiOn
A previous study reported that the probiotic bacterium L. rhamnosus inhibits NET formation (22). The immunomodulatory effect of probiotic bacteria, including lactic acid bacteria such as L. rhamnosus, on host immune cells is well known (27). Furthermore, it was reported that sugar constituents of the Cryptococcus neoformans capsule and of the Candida albicans or Streptococcus suis biofilms modulate NET formation (28)(29)(30). However, many common pathogenic bacteria were reported to induce NET formation, while the inhibition of NET formation is rarely reported (20,24,25). The current study constitutes the first-ever report that A. baumannii, a common pathogenic gram-negative bacterium, exerts such an inhibitory effect. Furthermore, it is surprising that the pathogenic bacterium A. baumannii prolongs the lifespan of neutrophils by inhibiting NET formation. Previous studies reported that L. rhamnosus and microbial sugars suppress ROS production in neutrophils, thereby inhibiting NET formation. Therefore, ROS production by A. baumannii-stimulated neutrophils was also investigated. The enhancement of ROS production by PMA was not suppressed by A. baumannii stimulation ( Figure S2 in Supplementary Material). Thus, A. baumannii did not appear to possess an antioxidative activity. Moreover, no capsule or biofilm formation by A. baumannii was observed under the culture conditions employed in the current study. Furthermore, L. rhamnosus was shown to inhibit bacterium-induced NET formation (22). The effect of A. baumannii on E. coli-induced NET formation was hence investigated. However, A. baumannii did not inhibit this process ( Figure S3 in Supplementary Material). Various pathways of NET formation are known (25,31,32). Based on data presented in the current study, we suggest that A. baumannii inhibits the formation of NETs that require long (3 h or more) and strong neutrophil activation, e.g., PMA stimulation, independently of antioxidative activity.
Cell adhesion is important for neutrophil activation (16,33). We thought that NETs are also similar, with the neutrophils  shapes (a,B). The determined mean fluorescence intensity of samples is shown in (c,D). The data are shown as the mean ± SD; n ≥ 3 per group. ***p < 0.001. The results are representative of at least three experiments. (a) Neutrophils were stimulated with 200-nM phorbol 12-myristate 13-acetate (PMA) for 30 min, the non-adherent neutrophils were removed, the adhering cells were lysed, and LDH levels were determined. In some experiments, the assay was performed in the presence of 0.1-µg/mL CD11a-blocking antibody or control IgG, and Acinetobacter baumannii (MOI 50). The adhesion rate was calculated as a percentage compared with lysed neutrophils. (B) Neutrophils were cultured for 3 h, and the extracellular DNA was stained with SYTOX green and the signal quantified. Some experiments were performed in the presence of 0.1-µg/mL CD11a-blocking antibody or control IgG, and A. baumannii (MOI 50). The data are shown as the mean ± SD; n ≥ 3 per group. ***p < 0.001 and **p < 0.01. The results are representative of at least three experiments. unable to release their DNA into the extracellular space without adhesion, but this was clearly not the case. In the current study, we showed that PMA-induced neutrophil NET formation was suppressed when the assay was performed using plates treated with a cell-adhesion inhibitor. Furthermore, we showed that A. baumannii inhibits neutrophil adhesion by suppressing the PMA-induced CD11a expression, thereby inhibiting NET formation. However, CD11a inhibition did not completely suppress the enhancement in adhesion stimulated by PMA. CD11b inhibition also suppressed adhesion and NET formation of neutrophils induced by PMA ( Figure S4 in Supplementary Material). Thus, cell adhesion is important for NET formation, and other cell-adhesion molecules may also be partially involved in this effect. In the current study, we demonstrated that CD11a is an important player in neutrophil adhesion and NET formation after PMA stimulation, and its levels are modulated by A. baumannii stimulation.
Many neutrophil surface molecules also change upon stimulation and regulate neutrophil activity (34,35). Recently, Zawrotniak et al. reported neutrophil surface molecules and signals that are important for promoting NET formation stimulated by C. albicans components such as mannan (36). Also, glucuronoxylomannan from the C. neoformans capsule (28) and matrix mannan from the C. albicans biofilm (29) inhibit NET formation. Thus, NET formation appears to be regulated via a complex mechanism involving the microorganism component and its neutrophil surface receptors. Indeed, in this study, CD11b, CD14, CD16, TLR4, and TLR2 expression was also affected by PMA stimulation and A. baumannii cocultivation (Figures 6B,D and Figure S5 in Supplementary Material). Investigating the bacterial factor(s), associated receptor(s), and signals is thus a challenge for future studies.
Acinetobacter baumannii appears to inhibit the activation of neutrophils by suppressing their adhesion, and extends their survival after PMA stimulation. Furthermore, when the neutrophils were cocultured with A. baumannii, the extracellular LDH levels were low and the majority of neutrophils remained alive. In contrast, many neutrophils died upon coculture with E. coli or P. aeruginosa ( Figure S6 and Video S3 in Supplementary Material). Although it is generally known that many common bacteria kill neutrophils by inducing apoptosis or exerting cytotoxicity (37,38), A. baumannii did not exert such a killing effect. Furthermore, our previous studies suggested that A. baumannii avoids neutrophil defense mechanisms, such as phagocytosis, at an initial point (at 1 h) (21). It appears that A. baumannii is not recognized as a foreign body that stimulates a strong inflammatory response by neutrophils. However, this is clearly in contrast to the findings of other (39). Lazaro-Diez et al. showed that A. baumannii is indeed phagocytosed by neutrophils and induces NET formation. These discrepancies may be associated with the difference in the experimental conditions, such the duration of coculture, MOI, and culture conditions. In another report, macrophages rather than neutrophils were shown to contribute to phagocytosis during the initial stages of A. baumannii infection (40 neutrophil depletion in mouse models (41)(42)(43). The recruitment of neutrophils to the A. baumannii infection site via cytokines and chemokines has been evidenced in many studies, including our own (15,41,44). Immune strategies to combat A. baumannii may involve the triggering of oxidative burst and cytokine/ chemokine production to amplify the immune response against the pathogen by the accumulated neutrophils (45). However, a part of A. baumannii can escape the immune response, including NETs, as demonstrated in the current study. In this case, A. baumannii would contribute to the dissemination of bacteria by utilizing the migratory capacity of neutrophils, as reported previously (15). Indeed, bacteremia and severe sepsis are caused by A. baumannii with high frequency in a compromised host (12)(13)(14). Furthermore, the bacteria are also detected in other organs, including the blood, in several mouse models of respiratory A. baumannii infection (42). The data reported in the current study suggest that A. baumannii inhibits NET formation, thereby escaping host immune responses and causing infection. This novel mechanism to inhibit NET formation might contribute to a development of new treatment strategies for A. baumannii infections.
In summary, we discovered that neutrophil adhesion is important for PMA-induced NET formation and that A. baumannii inhibits NET formation by suppressing neutrophil cell adhesion. Future studies should investigate the involved molecule(s) in detail and the mechanisms that control the expression of neutrophil CD11a, including bacterial factor(s) and intracellular signals.

eThics sTaTeMenT
Written informed consent was obtained from all study participants, in accordance with the Declaration of Helsinki. The study was approved by the Ethical Review Committee of the School of Medicine of Teikyo University.    ViDeO s2 | Time-lapse observation of the inhibition of PMA-induced NET formation by A. baumannii. Fluorescently-labeled neutrophils (red) and A. baumannii (MOI 50) were co-cultured for 0-5 h in the presence of 200 nM PMA; SYTOX green (green) was used to visualize the extracellular DNA. The images were captured every minute by a confocal microscope.
ViDeO s3 | Time-lapse observation of neutrophils co-cultured with A. baumannii. Fluorescently-labeled neutrophils (red) and A. baumannii (MOI 50) were co-cultured for 0-5 h; SYTOX green (green) was used to visualize the extracellular DNA. The images were captured every minute by a confocal microscope. reFerences