AbstractAcinetobacter baumannii is a common cause of health care associated infections worldwide. A. pittii is an opportunistic pathogen also frequently isolated from Acinetobacter infections other than those from A. baumannii. Knowledge of Acinetobacter virulence factors and their role in pathogenesis is scarce. Also, there are no detailed published reports on the interactions between A. pittii and human phagocytic cells. Using confocal laser and scanning electron microscopy, immunofluorescence, and live-cell imaging, our study shows that immediately after bacteria-cell contact, neutrophils rapidly and continuously engulf and kill bacteria during at least 4鈥塰ours of infection in vitro. After 3鈥塰 of infection, neutrophils start to release neutrophil extracellular traps (NETs) against Acinetobacter. DNA in NETs colocalizes well with human histone H3 and with the specific neutrophil elastase. We have observed that human neutrophils use large filopodia as cellular tentacles to sense local environment but also to detect and retain bacteria during phagocytosis. Furthermore, co-cultivation of neutrophils with human differentiated macrophages before infections shows that human neutrophils, but not macrophages, are key immune cells to control Acinetobacter. Although macrophages were largely activated by both bacterial species, they lack the phagocytic activity demonstrated by neutrophils. IntroductionAcinetobacter baumannii has been extensively studied because infections caused by this pathogen have been associated with high morbidity and mortality rates1, 2. Also, their ability to survive in dry conditions and their resistance to disinfectants allows these microorganisms to survive in the hospital environment3, 4. Furthermore, this organism frequently presents multidrug or pan-resistance5, 6. Due to those three attributes (survival in the hospital environment, antimicrobial resistance and virulence) it is likely that this organism will gain even increasing importance in the near future. Among Acinetobacter genus, A. pittii is another clinically relevant species. The significant role of A. pittii in human infections and the emergence of resistant strains have also become a great medical concern7,8,9.When Acinetobacter strains penetrate epithelial barriers and invade the host tissues, they first encounter the so-called 鈥減rofessional phagocytes鈥? macrophages and neutrophils. Professional phagocytes play a key role in host defence by engulfing and killing microorganisms. Little is known about the relative contribution of macrophages and neutrophils in the initial phase of encounter with Acinetobacter strains.Neutrophils (also known as polymorphonuclears, PMNs) are the most abundant leukocytes in the blood which are rapidly recruited to the inflammatory site upon inflammation. Neutrophils can eliminate microbes using three basic strategies: phagocytosis, degranulation, and by a recently discovered mechanism called NETosis, a specific type of cell death different from both necrosis and apoptosis10,11,12,13. Bacterial metabolites and inflammatory stimuli induce NETosis and the release of neutrophil extracellular traps (NETs). NETs are released to the extracellular space by activated neutrophils, but additional studies are required to establish under what conditions NETs play an important role in bacterial killing. Importantly, some pathogens are able to overcome these bactericidal mechanisms14,15,16,17,18.In this study, we investigated the interaction of A. baumannii and A. pittii clinical isolates with professional phagocytes. Understanding the mechanisms by which Acinetobacter interacts with immune cells is a prerequisite for the development of new prophylactic or therapeutic agents to treat the infections caused by these bacteria. Therefore, the aim of this work was to clarify the mechanisms of host-microbe interaction between neutrophils and Acinetobacter with focus on phagocytosis and neutrophil extracellular traps release.ResultsPhagocytosis and clearance of Acinetobacter strains by human neutrophilsHuman neutrophils are round cells that remain semi-attached and roll along the surfaces used in this study (glass or plastic). The presence of human (2%) or bovine serum (10%) in the protocol used to cultivate cells did not affect neutrophil behavior nor the outcome of the in vitro infections. The capability of neutrophils to bind and internalize A. baumannii and A. pittii is presented in Fig.聽1. In presence of Acinetobacter, neutrophils can flatten and become phagocytic. The transition to active phagocytosis is sudden, with extension of the cell-bacteria contact area followed by the emergence of pseudopods to form a phagocytic arm that progresses to complete engulfment of the bacteria. Bacteria were associated with neutrophils as early as 30鈥塵in post-infection (Fig.聽1a). Neutrophils were in contact with some of the surrounding bacteria through filopodia or pseudopods (arrow in Fig.聽1b), and multiple attempts at phagocytosis were observed at neutrophil surfaces. At this time, whole bacteria (indicated by red fluorescence) were observed inside human neutrophils (Fig.聽1c). After 2鈥塰, bacteria already remained largely inside neutrophils, but with different sizes and some loss of their characteristic red immunofluorescent pattern, indicating that the phagocytosed bacteria were probably being degraded (Fig.聽1d,e). Morphology in control neutrophils remains unchanged (Fig.聽1f).Figure 1Contact and phagocytosis of Acinetobacter by human neutrophils. Human neutrophils were infected for 30鈥塵in (a), 60鈥塵in (b,c) or 2鈥塰 (d鈥揻) with A. baumannii ATCC 19606T, fixed and processed for immunofluorescence labelling. Bacteria were detected with anti-A. baumannii rabbit antibody (red). Actin cytoskeleton was labelled with Atto 488 phalloidin (green) and nuclei are stained with DAPI (blue) (a鈥揷,f). (a) Single stack; (b and d鈥揻) maximal projections; (c) cross-sectional view. Arrow in (b) indicates a pseudopod in close contact with a bacterium. In (d,e) double-immunofluorescence images show extracellular bacteria (green), debris of intracellular bacteria (red) and bacterial and cellular DNA (blue). (f) As control, fresh untreated neutrophils were incubated in parallel during 4鈥塰. Micrographs were originally captured at 脳400 magnification (a,f) or 脳600 magnification (b鈥揺). Scale bars, (a,f) 5鈥壜祄; (b,d,e) 2鈥壜祄.Full size imageFurthermore, a Live/Dead staining was used to examine survival of Acinetobacter spp. after phagocytosis by unfixed primary human neutrophils. The dyes were added in the presence of 0.1% saponin, which sequesters cholesterol to preferentially permeabilize host cell plasma membranes, not Acinetobacter membranes. All acinetobacters stain with SYTO9, but only bacteria with compromised membranes stain with propidium iodide. The propidium iodide overcomes the SYTO9 fluorescence, so live bacteria appear green and dead bacteria appear red. Intracellular dead bacteria increased over time during the infection period (Fig.聽2). From 2 to 4鈥塰 post-infection bacteria attached to plastic or glass surfaces divided rapidly and neutrophils tried to contain the bacterial overgrowth by quickly and continuously engulfing these pathogens (Supplementary videos聽1 and 2).Figure 2Live/Dead staining in unfixed neutrophils. Neutrophils were infected with Acinetobacter strains, then exposed to components of the live/dead kit, propidium iodide and SYTO9. Upper panels: in merged images, live bacteria appear green, dead bacteria appear red and eukaryotic nuclei appear pink. Lower panels show selected z-stacks at high magnifications (red channel) of the boxed areas in the upper panel. Untreated similarly stained cells served as control (C). Original magnifications: upper panels 脳400; lower panels 脳1000. Scale bars, 5鈥壜祄.Full size imageInterestingly, using scanning electron microscopy and immunofluorescence, we observed that human neutrophils used very large filopodia (more than 50鈥壜祄) to not only sense the environment, but also to detect and retain bacteria (Fig.聽3). These large filopodia were also observed during experiments using live cell imaging on glass or plastic (Supplementary video聽3).Figure 3Capture and phagocytosis of Acinetobacter by human neutrophils. Pictures show SEM microphotographs (a,c,d,e) or immunofluorescence (b) images of infected neutrophils (3鈥塰, strain ATCC 19606T). Large filopodia were observed in infected cultures in close contact with bacteria (b,c). Some of these filopodia completely surround two bacteria (asterisks in c) while pseudopods are catching bacteria attached to the inert surface (arrows in c). In (b) bacteria were detected with anti-A. baumannii rabbit antibody (red), actin cytoskeleton was labelled with Atto 488 phalloidin (green) and nuclei were stained with DAPI (blue). Unstimulated neutrophils show round shapes (d). (e) Detail of the boxed area in (d) Micrographs were originally captured at 脳4000 (a), 脳600 (b), 脳10000 (c), 脳500 (d) or 脳9000 magnification (e). Scale bars, (a) 10鈥壜祄; (b,c,e) 5鈥壜祄; (d) 100鈥壜祄.Full size imageImportantly, preincubation of neutrophils with actin-cytoskeleton inhibitor cytochalasin D abrogated phagocytosis of Acinetobacter strains. This was demonstrated by the presence of neutrophils without bacteria 3鈥塰 after infection (Supplementary Figure聽1a,b). Of note, this cytoskeleton inhibitor reduces up to 90% of the number of neutrophils in the microscopic fields indicating that not only was phagocytosis affected, but also adherence of these cells to inert surfaces (the remained neutrophil morphology totally round).Gentamicin protection assays also demonstrated that intracellular bacteria had died because no live bacteria were recovered 3鈥塰 after infections following gentamicin treatment (Supplementary Figure聽1c). After performing quantitative CFUs counting experiments, difference in numbers between wells containing Acinetobacter and wells containing Acinetobacter plus neutrophils was not significative, despite neutrophils are able to eat at least 50 bacteria/cell (as observed by confocal microscopy) after 4鈥塰 of infection (Supplementary Figure聽1d).We incubated human neutrophils cells with extracellular products (ECPs) produced by all the Acinetobacter strains during growth in liquid medium, and no cytotoxicity was observed after 5鈥塰 of incubation with increasing volumes of bacterial ECPs (not shown).Production of neutrophil extracellular trapsNeutrophils that had become engorged with microbes (some neutrophils were shown to harbour more than 50 bacteria) started to die after 3鈥塰 post infection (Fig.聽4a,b). Neutrophils started to lose their individual nuclear lobules resulting in globular or horseshoe shape structures. During their final stage, nuclear and cytoplasmic integrity was lost, and most cells finally round up again and finally release NETs (Fig.聽4c,d). Very occasionally, NETs form large aggregates (up to 1 mm in length) (Fig.聽4e). In many cases, NETs clearly seems to entrap bacteria (Supplementary Figure聽2a and Supplementary video聽4). Immunofluorescence analyses confirmed the co-localization of histones (H3) and neutrophil elastase (NE) in extracellular traps released from human neutrophils (Supplementary Figure聽2b,c). These NETs appear to be flexible, and to emerge from the cell from which they originated (Supplementary Figure聽3a). The presence of NETs in infected cultures was highly variable. Non-infected neutrophils were used as controls for immunofluorescence staining in the nucleus (colocalization with histone H3) and cytoplasm (intracellular neutrophil elastase), and, as expected, Pseudomonas aeruginosa PAO1 infection used as positive control induced NET formation (Supplementary Figure聽3b鈥揹).Figure 4NETs production by human neutrophils infected with Acinetobacter. Pictures show a SEM microphotograph (a) or immunofluorescence images (b鈥揺) of neutrophils 4鈥塰 post-infection: (a,c鈥揺) strain ATCC 19606T; (b) strain HUMV 06-2790. From (b to e) bacteria were detected with anti-A. baumannii rabbit antibody (red), actin was labelled with Atto 488 phalloidin (green), and DNA was stained with DAPI (blue). Micrographs were originally captured at 脳15000 (a), 脳600 (b,d), 脳400 (c) or 脳200 magnification (e). Scale bars, (a鈥揹) 5鈥壜祄; (e) 100鈥壜祄.Full size imageTo quantify NETosis and NET release by in vitro-infected human neutrophils, neutrophil elastase and citrullinated histone H3 were measured by a NETosis assay and ELISA kit respectively. These assays demonstrated that Acinetobacter strains were able to induce the release of certain amounts of NETs by human neutrophils in vitro (Fig.聽5a,b). However, NET release by infected neutrophils was always lower than neutrophils stimulated with the well known activator of full NETs release, PMA. To compare the induction of NETs by different strains, NETs formation was examined using the extracellular nucleic acid dye SYTOX Green by live-cell imaging during infections (Supplementary Figure聽4). Furthermore, using a quantitative fluorescence assay, NETs formation by several strains was compared with untreated neutrophils and with neutrophils treated with PMA. Fluorescence from NETs in infected cultures with several Acinetobacter strains was also higher than in untreated neutrophils and lower that in PMA stimulated neutrophils. By this method, one strain (HUMV 06-2790) failed to clearly demonstrate NETs release (Fig.聽5c).Figure 5Quantification of elastase, citrullinated histone H3 and extracellular DNA using SYTOX Green. (a) Measurement of released neutrophil elastase. Human neutrophils were infected with Acinetobacter strains or treated with PMA for four hours, washed, and treated with S7 nuclease for 15鈥塵in. The supernatant from each well was assayed. Samples were tested in triplicate. (b) Measurement of citrullinated histone H3 (CitH3). Human neutrophils were infected with Acinetobacter strains for 4鈥塰ours. Supernatants were centrifuged to remove cellular debris and then tested in the ELISA. Samples were tested in triplicate. The concentrations of total neutrophil elastase and CitH3 in the analyzed samples were estimated from standard curves obtained for each assay. (c) Quantification of fluorescence after infection experiments using SYTOX Green. Supernatants from unstained infected cultures were partially digested with DNAse I and stained with SYTOX Green. Each bar indicates the average of three independent experiments鈥壜扁€塖D. Asterisks indicate: *p鈥?鈥?.0004; **p鈥?lt;鈥?.00001; n.s., p鈥?鈥?.1610.Full size imageInfection of macrophage-neutrophil co-culturesTo test whether host cell type contributed mostly to clearance of this pathogen, we performed infections of mixed cultures containing human neutrophils and differentiated macrophages. Incubation of Acinetobacter with macrophages and neutrophils did not induce an important phagocytosis in macrophages, although produced remarkable important cell activation (compared with untreated macrophages), as demonstrated by the elongated cell shape. After 3鈥塰 of infection, 90% of macrophages were in contact with 5 or less bacteria despite that Acinetobacter was largely occupying the glass surface. On the other hand, neutrophils were full of bacteria (Supplementary Figure聽5).DiscussionNeutrophils and macrophages are the first lines of defence against invading microbes. Neutrophils are terminally differentiated, rapidly reach the infection site, and are equipped with antimicrobial proteins to kill bacteria19. However, little is known about the relative contribution of neutrophils during the initial phase after encountering Acinetobacter spp. in human infections. Moreover, although several animal infection models were used to study the infection by A. baumannii (sepsis and lung infections), neutrophils from mammals and fish differ from human neutrophils in many ways20,21,22. As the success of A. baumannii and A. pittii as pathogens depends on its ability to avoid killing by components of the innate immune system, the aim of the current study was to characterize the human neutrophil response to these microbes. When neutrophils were assessed for their inherent abilities to neutralize Acinetobacter strains, both bacterial species were recognized within 20鈥?0鈥塵in of co-incubation with cells. Immunofluorescence staining and double-immunofluorescence performed from 30鈥塵in to 4鈥塰 demonstrated that neutrophils catch bacteria continuously. This was also confirmed by time-lapse microscopy. Moreover, we examined live and dead acinetobacters inside neutrophils by using confocal microscopy. The primary goal of these experiments was to confirm whether the bacteria were located physically inside or outside the host cells and that dead bacteria inside cells lost their immunogenic surface (stained with a polyclonal antibody) because, although neutrophils kill the vast majority of bacteria, some microbes circumvent killing by these cells14,15,16. Using anti-Acinetobacter antibodies, whole bacteria were seen as red at the glass surface and associated with cells, but dead or damaged bacteria inside cells lost their characteristic red fluorescence. To unequivocally demonstrate that human neutrophils kill Acinetobacter, and therefore bacterial survival is compromised in presence of these cells, we performed an in situ Live/Dead staining on unfixed cells. This staining demonstrated that, once inside neutrophils, Acinetobacter die. This was observed along the experiments demonstrating that human neutrophils can easily and effectively kill both A. baumannii and A. pittii in vitro. In accordance with several authors, uptake of bacteria could lead to full activation of the anti-microbial arsenal of the neutrophil killing the ingested bacteria23. In conclusion, our findings strongly indicate that all the strains tested were phagocytosed and killed by human neutrophils. This is clearly in contrast to reports by others24, 25. Based on the experimental methods described in these previous publications, there is no obvious indication for the discrepancies in the reported results, apart from bacteria-cell contact time 1鈥塰26 vs 4鈥塰. According to our immunofluorescence, SEM, CFUs counting and live-cell and live/dead imaging experiments, neutrophils are in contact with Acinetobacter at 1鈥塰, but further incubation time renders active phagocytosis. Our findings also correlate with current in vivo studies in mice and fish reporting the significance of neutrophils on Acinetobacter infections26,27,28. Moreover, our results correlate well with in vitro models using human neutrophils against other microbes, where phagocytosis seems to be the main mechanism to clear bacteria10, 29. Filopodia are abundant in macrophages30, but little is known about their role during phagocytosis or chemotaxis in neutrophils. An unexpected finding of the study was the presence of very large filopodia emerging from the neutrophil body to sense the environment and even to catch bacteria in vitro. Although quantitation of the filopodial dynamics or the cytoskeletal reorganization during neutrophil chemotaxis or phagocytosis is beyond the scope of this paper, new knowledge through a deeper study on the modulation and regulation of these filopodia may prove helpful in understanding the pathogenesis of this and other bacteria.After 3鈥?鈥塰 post-infection, neutrophils started to die in presence of growing acinetobacters. In our assays, both Acinetobacter species grow actively in cell culture media and large numbers of bacteria were achieved 4鈥塰 after infections. Despite these in vitro assays did not allow new neutrophils recruitment, cells are full of dead bacteria 4鈥塰 after infections as demonstrate by confocal microscopy and gentamycin protection assays. This could mean that neutrophils play an important role against Acinetobacter in vivo.Neutrophil cell death is fundamentally divided into necrosis, apoptosis, autophagy and the newly recognized NETosis. NETosis is a complex process that occurs with dramatic changes in the morphology of the neutrophil that finally lead to cell death10. The release of NETs against Acinetobacter was identical when human neutrophils were seeded on glass or plastic, as well as when using human or bovine serum. NETs are able to trap bacteria, fungi, and parasites31, but the possibility that the microbes ensnared in NETs are alive is controversial32. In our hands, A. baumannii and A. pittii induce a moderate cell death during the first 2鈥塰 of infection and NETs release by human neutrophils started after 3鈥塰, similar to those induced by P. aeruginosa.One of the most widely used techniques to observe NET induction is confocal microscopy. This approach is very informative as to the presence or absence of NETs, but microscopy images did not allow quantification of NETs. In this work, quantification of neutrophil elastase and citrullinated histone H3 demonstrated a strain-dependent variation in the NETs induction. Using SYTOX Green to stain and to quantify extracellular DNA, one strain failed to induce significant amounts of DNA release as compared with untreated controls. However, neutrophil extracellular traps release after Acinetobacter infections correlates with the presence of specific NETosis markers such as neutrophil elastase and histone H333. Therefore, and in agreement with Naccache and Fernandes33 the experimental approaches to investigate NET formation underscore the need for consensus on standardized experimental approaches in the NET field.Our results show that some bacteria were entrapped by NETs, and therefore this neutrophil response to these pathogens could partially prevent dissemination during the infection. A recent study shows that there are no ex vivo NETs production in neutrophils isolated from Acinetobacter baumannii bacteremia34. However, neither the presence of NETs in vivo was studied nor the neutrophil-Acinetobacter interactions in vitro.Finally, using differentiated human macrophages in co-culture with neutrophils to study Acinetobacter host-microbe interactions, we show that neutrophils play a key role in controlling the infections caused by these bacteria. This is important because neutrophils make also an essential contribution in the recruitment and activation of macrophages during infections35. Our results also correlate with those of others showing that neutrophils, but not macrophages, are crucially to control early steps during bacterial and fungal infections35, 36.In this work, our first objective was to demonstrate phagocytosis and killing of these two important pathogens by human neutrophils as a defence mechanism, but the induction of NETs in a small number of human neutrophils could be also important to fight infection. As neutrophils are also responsible for tissue damage and inflammation during certain circumstances, an overactivation of these cells (i.e. excessive NETs release) could be detrimental to the host. Therefore, future detailed studies at the molecular level will help to decipher the mechanisms involved in the regulation of neutrophils in presence of Acinetobacter or other pathogens, both alone or in combination with other immune cells.MethodsBacterial strains and growth conditionsThe nine Acinetobacter clinical isolates (A. baumannii n鈥?鈥?; A. pittii n鈥?鈥?) used in this work were all previously described37. Reference strains A. baumannii ATCC 19606T and A. pittii LMG 10559 were also included (Table聽1). The strains were routinely cultured on blood agar (BA) plates, brain hearth infusion broth (BHIB) or Luria Bertani broth (LB) at 37鈥壜癈, and frozen at 鈭?0鈥壜癈 with 20% glycerol. As control for NETs induction, Pseudomonas aeruginosa strain PAO1 was used38. P. aeruginosa was cultured in LB at 37鈥壜癈.Table 1 Acinetobacter strains used in this study.Full size tableNeutrophil isolation from whole human bloodAll studies involving human samples were in accordance with international standards for research ethics and were approved by the local institutional review board (Hospital Universitario Marqu茅s de Valdecilla). Neutrophils were isolated from whole venous blood obtained from healthy human volunteers after informed consent. The EasySepTM Direct Human Neutrophil enrichment kit (StemCell) was used, following the manufacturer鈥檚 instructions. Briefly, 50鈥壩糒 of EasySep庐 neutrophil enrichment cocktail, containing a mix of tetrameric antibody complexes produced from monoclonal antibodies directed against the cell surface antigens CD2, CD3, CD9, CD19, CD36, CD56 and magnetic particles were added per 1鈥塵L of blood. The blood/antibody/bead solution was adjusted to a total volume of 50鈥塵L with recommended media and placed into an Easy 50 magnet for 10鈥塵in at room temperature (RT). Unbound neutrophils were pipetted into a new tube and placed in the Easy 50 magnet before addition of new magnetic particles. This step was repeated once. Highly-pure unbound neutrophils were briefly centrifuged and resuspended in RPMI 1640 media plus 10% fetal bovine serum (FBS) or 2% human serum. Neutrophils were also separated from other leukocytes using dextran density gradient centrifugation and red blood cells lysis as described elsewhere39. Neutrophils were isolated from samples from at least 14 donors and purity of neutrophil preparations was determined by morphology after staining of nuclei with NucBlue (Molecular Probes).Phagocytosis experimentsAcinetobacter strains were cultured overnight in 10鈥塵l BHIB or LB at 37鈥壜癈 with shaking at 175鈥塺pm. Neutrophils were infected with bacteria at a multiplicity of infection (MOI, bacterium: eukaryotic cell ratio) of ~100:1. The number of colony forming units (CFUs) inoculated per well was determined by serial dilution in phosphate buffered saline (PBS) and plating on BA and incubated for 24鈥塰. The infected plates were centrifuged for 4鈥塵in at 200鈥壝椻€?i>g prior to the incubation to promote adherence of bacteria to cells and to synchronize infections. Infected cells were then incubated at 37鈥壜癈 with 5% CO2 for different times. For quantification of live bacteria (extracellular and intracellular), external non-adherent bacteria were removed by washing four times with PBS, and human cells were then disrupted by addition of 100鈥壜祃 Triton X-100 (1% in PBS) per well. To determine if A. baumannii is able to survive inside neutrophils after phagocytosis, strain A. baumannii ATCC 19606T was selected. The MIC of gentamicin for this strain was previously determined37. Cells were infected for 2鈥塰, washed with PBS, and the culture medium was replaced by medium containing 200鈥壜礸鈥塵l鈭? of gentamicin (Gibco). Cells were incubated for a further 2鈥塰, and lysed as described before. After this time, number of putative viable intracellular bacteria was counted. To do this, serial dilutions of the disrupted mixture were plated onto BA and incubated for 48鈥塰 at 37鈥壜癈. Growth of 3 Acinetobacter strains in presence or absence of neutrophils was monitored during 4鈥塰. Viability/growth of Acinetobacter was calculated as the average of the total number of CFUs per total initial inoculum and expressed as a percentage. Quantitative phagocytosis experiments and growth experiments were repeated at least four times.Incubation with cytochalasin DNeutrophils were incubated with the actin-cytoskeleton inhibitor cytochalasin D (5鈥壜礸鈥塵l鈭?) (Sigma) for 30鈥塵in before the bacteria were added. Neutrophils were then infected for 3鈥塰 as described for the immunofluorescence assays.Immunofluorescence assaysCells were placed in 24-well tissue culture plates containing round glass coverslips. Bacteria were cultured as described above. Infected monolayers were incubated at 37鈥壜癈 with 5% CO2 for different times (from 30鈥塵in up to 4鈥塰). Cells were washed four times and fixed with cold paraformaldehyde (3.2% in PBS) for 20鈥塵in at room temperature. Then, cells were permeabilized with Triton X-100 (0.1% in PBS) for 5鈥塵in at RT and washed five times with PBS. Atto-488 phalloidin (Sigma), which binds polymerized F-actin, was used to identify actin filaments and fibers. Differential double immunofluorescent labelling of Acinetobacter allowed extracellular bacteria to be differentiated from intracellular bacteria. For double immunofluorescence assays, strains A. baumannii ATCC 19606T and A. pittii LMG 10559 were used to produce polyclonal sera as previously described40. Antiserum was collected 8 weeks after the first boost, processed and stored using standard protocols40. Histones in NETs were stained with a rabbit polyclonal anti-histone H3 antibody (Abcam). Specific human neutrophil elastase was stained with an anti-neutrophil elastase rabbit monoclonal antibody (Abcam). Secondary antibodies conjugated to Alexa Fluor 594 or Alexa Fluor 488 goat anti-rabbit IgG were purchased from Invitrogen. After infections, coverslips were mounted on glass slides with Fluoroshield mounting medium containing DAPI (Sigma Aldrich) to stain double-stranded DNA. All preparations were examined with a Nikon A1R confocal scanning laser microscope equipped with 403鈥塶m, 488鈥塶m and 561鈥塶m lasers. Images were captured at random with a 脳20 Plan-Apo 0.75 NA, 脳40 Plan-Fluor 1.3 NA or 脳100 Apo-TIRF 1.49 NA objectives, and processed using the NIS-Elements 3.2 software. All immunofluorescence experiments for each strain were repeated with neutrophils from at least three different blood samples.Assessing Bacterial Viability inside neutrophils with Live/Dead stainingBacterial viability inside neutrophils was determined by using the BacLight Live/Dead bacterial viability kit (Molecular Probes Inc.). Live/Dead Staining was performed in presence of 0.1% saponin for 20鈥塵in at 1鈥塰, 2鈥塰, 3鈥塰 and 4鈥塰 post-infection. A series of optical sections was obtained with a Nikon A1R confocal scanning laser microscope (CLSM); the excitation wavelengths were 488鈥塶m (green) and 561鈥塶m (red), and 500- to 550-nm and 570- to 620鈥塶m emission filters were used, respectively. Images were captured at random with a 100脳 Apo TIRF (numerical aperture [NA], 1.49) objective. Reconstructions of confocal sections were assembled using NIS-Elements software, version 3.2.Time-lapse fluorescence microscopyTime-lapse microscopy was carried out on a Nikon Eclipse Ti-E microscope (Nikon), equipped with a PlanFluor 20鈥?0鈥壝椻€?.6NA objective (Nikon) and a CO2 incubator. Neutrophils cells were seeded in 6-well plates (Nunc), in coated 4-well 碌-slides (Ibidi, Martinsried, Germany) or in 24-well plates containing coverslips and infected as described before. NucBlue (one drop/well, Molecular Probes) or 10鈥壜礛 SYTOX Green were added to each well to stain nuclei. Cells were infected as described before, and images were collected from 30鈥塵in up to 120鈥塵in post-infection every 2鈥塵in (NucBlue) or from 40鈥塵in up to 190鈥塵in post-infection every 1.5鈥塵in (SYTOX Green) with an ORCA- R2 CCD camera (Hamamatsu) powered by Nis Elements 3.2 software. For NucBlue, a 375鈥?90鈥塶m excitation, 420鈥?90鈥塶m emission filter was used and for SYTOX Green, a 485鈥?20鈥塶m excitation, 521/25鈥塶m emission filter was used. Individual time-lapse frames were imported to the open source image analysis software, ImageJ (http://rsbweb.nih.gov/ij).NETosis assayIn separate experiments, we used a NETosis assay kit (Cayman Chemical) to determine the activity of NET-bound neutrophil elastase, according to manufacturer鈥檚 instructions. The assay is based on the enzymatic activity of neutrophil elastase in the culture medium that has been released from NETs through the action of S7 Nuclease. A colorimetric assay employing a specific elastase substrate (N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide) was used after washing away non-NET associated elastase, as to measure only NET-associated elastase activity. The 5 substrate is selectively cleaved by elastase to give a 4-nitroaniline product that absorbs light at 405鈥塶m. The concentration of neutrophil elastase was measured by optical densitometry in a Multiskan FC microplate reader (Thermo Fisher).Citrullinated Histone H3 assayQuantitative determination of citrullinated histone was made using an ELISA Kit (citrullinated histone H3 ELISA kit, Cayman Chemical) according to manufacturer鈥檚 instructions. The concentration of citrullinated H3 was measured by optical densitometry at 450鈥塶m in a Multiskan FC microplate reader (Thermo Fisher).Quantification of NET-DNANeutrophils were left untreated, treated with PMA (100鈥塶M) or infected with Acinetobacter strains for 4鈥塰. Wells containing infected cultures and controls were then treated with DNAse I (Sigma Aldrich) for 15鈥塵in at RT. The reaction was stopped with 0.5鈥塎 EDTA and cultures were centrifuged for 10鈥塵in at 8,000鈥壝椻€?i>g. 150鈥壜祃 supernatants from each well were transferred in triplicate into black 96-well plates (Thermo Scientific鈩?. SYTOX Green was added (10鈥壜礛) to each well for 15鈥塵in and then fluorescence was quantified with excitation/emission wavelengths of 485/535鈥塶m using a Synergy鈩?HTX Multi-Mode Microplate Reader (Biotek). All data were derived from three independent experiments. Statistical analysis of the data was carried out with the paired two-tailed Student t-test. A p-value less than 0.05 was considered statistically significant.Cytotoxicity of bacterial extracellular productsTo determine the cytotoxic potential of the ECPs present in Acinetobacter culture supernatants, bacteria were grown on LB or BHIB for 24鈥塰 and collected by centrifugation at 3,000鈥塺pm for 15鈥塵in at RT. The supernatants were sterilized via membrane filtration (0.22鈥壜祄, Millipore) and used immediately to challenge human neutrophils plated at density of 2鈥壝椻€?04 cells/well. ECPs were added directly to the cell culture medium at different volumes (100鈥?00鈥壩糽, each in duplicate) and cells were incubated for periods up to 24鈥塰 and processed for immunofluorescence. Control cultures were incubated with the same volumes using fresh bacterial culture medium.Scanning Electron MicroscopyCoverslips containing infected neutrophils were fixed in ice-cold 3% glutaraldehyde for 20鈥塵in at 4鈥壜癈. Samples were dehydrated with a graded ethanol series, dried by the critical point method, coated with gold in a Fine coat ion sputter JFC-1100 226 (JEOL, Ltd), and observed with an Inspect S microscope (FEI Company) working at 25鈥塳V.Isolation and differentiation of macrophages from human bloodHuman monocyte-derived macrophages (HMDM) were isolated from the peripheral blood of healthy donors as previously described. Briefly, blood was layered at a ratio of 2:1 (blood/Ficoll medium) on Ficoll Histopaque-1077 (Sigma) in 15鈥塵l centrifuge tubes and spun for 30鈥塵in at 2000 rpm in an Allegra X-22R centrifuge (Beckman Coulter). The layer containing the peripheral blood mononuclear cells was collected and then resuspended in 15鈥塵l of PBS, and recentrifuged for 10鈥塵in at 1000鈥塺pm. After two washes in PBS, cells were resuspended in DMEM containing 10% FBS, L-Glutamine and 100 units ml鈭? penicillin and 100鈥塵g鈥塵l鈭? streptomycin on 12 mm diameter coverslips in 24-well plates. Non-adherent cells were removed after 4鈥塰. The cells were subsequently cultured in cell culture medium containing 50 ng ml鈭? granulocyte macrophage colony stimulating factor (GM-CSF) (Sigma Aldrich) in an atmosphere containing 5% CO2. Cultures were fed daily, and infection experiments were performed 10 days after the peripheral blood was collected. Infections were performed with MOI of 100:1:1 (bacteria/neutrophil/macrophage) ratio. Change history11 March 2020An amendment to this paper has been published and can be accessed via a link at the top of the paper.References1.Clark, N. M., Zhanel, G. G. Lynch, J. P. 3rd. Emergence of antimicrobial resistance among Acinetobacter species: a global threat. Curr Opin Crit Care 22, 491鈥?99 (2016).Article聽Google Scholar聽 2.Lahmer, T. et al. Acinetobacter baumannii sepsis is fatal in medical intensive care unit patients: six cases and review of literature. Anaesth Intensive Care 42, 666鈥?68 (2014).CAS聽 PubMed聽Google Scholar聽 3.Greene, C., Vadlamudi, G., Newton, D., Foxman, B. Xi, C. The influence of biofilm formation and multidrug resistance on environmental survival of clinical and environmental isolates of Acinetobacter baumannii. Am J Infect Control 44, e65鈥?1 (2016).CAS聽 Article聽Google Scholar聽 4.Espinal, P., Marti, S. Vila, J. Effect of biofilm formation on the survival of Acinetobacter baumannii on dry surfaces. J Hosp Infect 80, 56鈥?0 (2012).CAS聽 Article聽Google Scholar聽 5.Vila-Farres, X. et al. In vitro activity of several antimicrobial peptides against colistin-susceptible and colistin-resistant Acinetobacter baumannii. Clin Microbiol Infect 18, 383鈥?87 (2012).CAS聽 Article聽Google Scholar聽 6.Antunes, L. C., Imperi, F., Minandri, F. Visca, P. In vitro and in vivo antimicrobial activities of gallium nitrate against multidrug-resistant Acinetobacter baumannii. Antimicrobial agents and chemotherapy 56, 5961鈥?970 (2012).CAS聽 Article聽Google Scholar聽 7.Yamamoto, M. et al. Regional dissemination of Acinetobacter species harbouring metallo-beta-lactamase genes in Japan. Clin Microbiol Infect 19, 729鈥?36 (2013).CAS聽 Article聽Google Scholar聽 8.Pagano, M. et al. Emergence of NDM-1-producing Acinetobacter pittii in Brazil. Int J Antimicrob Agents 45, 444鈥?45 (2015).CAS聽 Article聽Google Scholar聽 9.Kamolvit, W., Derrington, P., Paterson, D. L. Sidjabat, H. E. A case of IMP-4-, OXA-421-, OXA-96-, and CARB-2-producing Acinetobacter pittii sequence type 119 in Australia. J Clin Microbiol 53, 727鈥?30 (2015).CAS聽 Article聽Google Scholar聽 10.Fuchs, T. A. et al. Novel cell death program leads to neutrophil extracellular traps. J Cell Biol 176, 231鈥?41 (2007).CAS聽 Article聽Google Scholar聽 11.Brinkmann, V. Zychlinsky, A. Beneficial suicide: why neutrophils die to make NETs. Nat Rev Microbiol 5, 577鈥?82 (2007).CAS聽 Article聽Google Scholar聽 12.Standish, A. J. Weiser, J. N. Human neutrophils kill Streptococcus pneumoniae via serine proteases. J Immunol 183, 2602鈥?609 (2009).CAS聽 Article聽Google Scholar聽 13.Kumar, V. Sharma, A. Neutrophils: Cinderella of innate immune system. Int Immunopharmacol 10, 1325鈥?334 (2010).CAS聽 Article聽Google Scholar聽 14.Greenlee-Wacker, M., DeLeo, F. R. Nauseef, W. M. How methicillin-resistant Staphylococcus aureus evade neutrophil killing. Curr Opin Hematol 22, 30鈥?5 (2015).CAS聽 Article聽Google Scholar聽 15.Voyich, J. M. et al. Insights into mechanisms used by Staphylococcus aureus to avoid destruction by human neutrophils. J Immunol 175, 3907鈥?919 (2005).CAS聽 Article聽Google Scholar聽 16.Johnson, M. B. Criss, A. K. Resistance of Neisseria gonorrhoeae to neutrophils. Front Microbiol 2, 77 (2011).Article聽Google Scholar聽 17.Kobayashi, S. D. et al. Phagocytosis and Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils. J Infect Dis 213, 1615鈥?622 (2016).CAS聽 Article聽Google Scholar聽 18.Silva, M. T. When two is better than one: macrophages and neutrophils work in concert in innate immunity as complementary and cooperative partners of a myeloid phagocyte system. J Leukoc Biol 87, 93鈥?06 (2010).CAS聽 Article聽Google Scholar聽 19.Kaufmann, S. H. Dorhoi, A. Molecular Determinants in Phagocyte-Bacteria Interactions. Immunity 44, 476鈥?91 (2016).CAS聽 Article聽Google Scholar聽 20.Rosen, H. Editorial: of mice and men鈥搚et again. J Leukoc Biol 94, 210鈥?12 (2013).CAS聽 Article聽Google Scholar聽 21.Mestas, J. Hughes, C. C. Of mice and not men: differences between mouse and human immunology. J Immunol 172, 2731鈥?738 (2004).CAS聽 Article聽Google Scholar聽 22.Bhuiyan, M. S. et al. Acinetobacter baumannii phenylacetic acid metabolism influences infection outcome through a direct effect on neutrophil chemotaxis. Proc Natl Acad Sci USA 113, 9599鈥?604 (2016).CAS聽 Article聽Google Scholar聽 23.Nordenfelt, P. Tapper, H. Phagosome dynamics during phagocytosis by neutrophils. J Leukoc Biol 90, 271鈥?84 (2011).CAS聽 Article聽Google Scholar聽 24.Kamoshida, G. et al. Acinetobacter baumannii escape from neutrophil extracellular traps (NETs). J Infect Chemother 21, 43鈥?9 (2015).CAS聽 Article聽Google Scholar聽 25.Kamoshida, G. et al. A novel bacterial transport mechanism of Acinetobacter baumannii via activated human neutrophils through interleukin-8. J Leukoc Biol 100, 1405鈥?412 (2016).CAS聽 Article聽Google Scholar聽 26.Breslow, J. M. et al. Innate immune responses to systemic Acinetobacter baumannii infection in mice: neutrophils, but not interleukin-17, mediate host resistance. Infect Immun 79, 3317鈥?327 (2011).CAS聽 Article聽Google Scholar聽 27.van Faassen, H. et al. Neutrophils play an important role in host resistance to respiratory infection with Acinetobacter baumannii in mice. Infect Immun 75, 5597鈥?608 (2007).Article聽Google Scholar聽 28.Guo, B. et al. Quantitative impact of neutrophils on bacterial clearance in a murine pneumonia model. Antimicrobial agents and chemotherapy 55, 4601鈥?605 (2011).CAS聽 Article聽Google Scholar聽 29.Surewaard, B. G. et al. Staphylococcal alpha-phenol soluble modulins contribute to neutrophil lysis after phagocytosis. Cell Microbiol 15, 1427鈥?437 (2013).CAS聽 Article聽Google Scholar聽 30.Kress, H. et al. Filopodia act as phagocytic tentacles and pull with discrete steps and a load-dependent velocity. Proc Natl Acad Sci USA 104, 11633鈥?1638 (2007).ADS聽 CAS聽 Article聽Google Scholar聽 31.Lu, T., Kobayashi, S. D., Quinn, M. T. Deleo, F. R. A NET Outcome. Front Immunol 3, 365 (2012).Article聽Google Scholar聽 32.Menegazzi, R., Decleva, E. Dri, P. Killing by neutrophil extracellular traps: fact or folklore? Blood 119, 1214鈥?216 (2016).Article聽Google Scholar聽 33.Naccache, P. H. Fernandes, M. J. Challenges in the characterization of neutrophil extracellular traps: The truth is in the details. European journal of immunology 46, 52鈥?5 (2016).CAS聽 Article聽Google Scholar聽 34.Konstantinidis, T. et al. Immunomodulatory Role of Clarithromycin in Acinetobacter baumannii Infection via Formation of Neutrophil Extracellular Traps. Antimicrobial agents and chemotherapy 60, 1040鈥?048 (2015).Article聽Google Scholar聽 35.Chertov, O. et al. Identification of human neutrophil-derived cathepsin G and azurocidin/CAP37 as chemoattractants for mononuclear cells and neutrophils. J Exp Med 186, 739鈥?47 (1997).CAS聽 Article聽Google Scholar聽 36.Mircescu, M. M., Lipuma, L., van Rooijen, N., Pamer, E. G. Hohl, T. M. Essential role for neutrophils but not alveolar macrophages at early time points following Aspergillus fumigatus infection. J Infect Dis 200, 647鈥?56 (2009).CAS聽 Article聽Google Scholar聽 37.Lazaro-Diez, M. et al. Acinetobacter baumannii and A. pittii clinical isolates lack adherence and cytotoxicity to lung epithelial cells in vitro. Microbes Infect 18, 559鈥?64 (2016).CAS聽 Article聽Google Scholar聽 38.Ocampo-Sosa, A. A. et al. Alterations of OprD in carbapenem-intermediate and -susceptible strains of Pseudomonas aeruginosa isolated from patients with bacteremia in a Spanish multicenter study. Antimicrobial agents and chemotherapy 56, 1703鈥?713 (2012).CAS聽 Article聽Google Scholar聽 39.Kuhns, D. B., Long Priel, D. A., Chu, J. Zarember, K. A. Isolation and Functional Analysis of Human Neutrophils. Curr Protoc Immunol 111(7.23), 21鈥?6 (2015). Google Scholar聽 40.Ramos-Vivas, J. et al. Rhodococcus equi human clinical isolates enter and survive within human alveolar epithelial cells. Microbes Infect 13, 438鈥?46 (2011).CAS聽 Article聽Google Scholar聽 Download referencesAcknowledgementsM.L.-D. holds a contract from the Instituto de Investigaci贸n Sanitaria Valdecilla IDIVAL and Universidad de Cantabria (PREVAL16/05). S.R.-S. holds a contract from the Instituto de Investigaci贸n Valdecilla IDIVAL. J.R.-V. holds a Miguel Servet II contract for Young Researchers from the Instituto de Salud Carlos III, Spain. The authors thank Dr. Fidel Madrazo (Electron Microscopy Unit, Technology Support Services, IDIVAL) for helping with confocal microscopy and live cell imaging. J.R.-V. acknowledges the receipt of a Sociedad Espa帽ola de Enfermedades Infecciosas y Microbiolog谋虂a Cl谋虂nica (SEIMC) fellowship. J.R.-V. thanks In茅s Montes and Adri谩n Fern谩ndez for technical assistance. J.R.-V. was supported by the Spanish Instituto de Salud Carlos III, Spain (grants PI13/01310 and PI16/01103). Research in our laboratories is supported by Plan Nacional de I鈥?鈥塂鈥?鈥塱 2008鈥?011 and Instituto de Salud Carlos III, Subdirecci贸n General de Redes y Centros de Investigaci贸n Cooperativa, Ministerio de Econom铆a y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD12/0015) - co-financed by European Development Regional Fund 鈥淎 way to achieve Europe鈥?ERDF. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.Author informationAffiliationsInstituto de Investigaci贸n Valdecilla IDIVAL, Santander, 39011, SpainMar铆a L谩zaro-D铆ez,聽Itziar Chapartegui-Gonz谩lez,聽Santiago Redondo-Salvo,聽David Merino,聽David San Segundo,聽Adri谩n Fern谩ndez,聽Jes煤s Navas,聽Alain Ocampo-Sosa聽 聽Jos茅 Ramos-VivasServicio de Microbiolog铆a, Hospital Universitario Marqu茅s de Valdecilla, Santander, 39008, SpainMar铆a L谩zaro-D铆ez,聽Itziar Chapartegui-Gonz谩lez,聽Alain Ocampo-Sosa聽 聽Jos茅 Ramos-VivasNew York University School of Medicine, New York, 10003, USAChike LeighServicio de Inmunolog铆a, Hospital Universitario Marqu茅s de Valdecilla, Santander, 39008, SpainDavid Merino聽 聽David San SegundoDepartamento de Biolog铆a Molecular, Universidad de Cantabria, Santander, 39011, SpainJes煤s NavasDepartamento de Anatom铆a y Biolog铆a Celular, Universidad de Cantabria, Santander, 39011, SpainJos茅 Manuel IcardoGrupo de Investigaci贸n en Acuicultura, Universidad de Las Palmas de Gran Canaria, Gran Canaria, 35214, SpainF茅lix AcostaRed Espa帽ola de Investigaci贸n en Patolog铆a Infecciosa (REIPI), Instituto de Salud Carlos III, Madrid, 28029, SpainMar铆a L谩zaro-D铆ez,聽Alain Ocampo-Sosa,聽Luis Mart铆nez-Mart铆nez聽 聽Jos茅 Ramos-VivasUnidad de Gesti贸n Cl铆nica de Microbiolog铆a, Hospital Universitario Reina Sof铆a, C贸rdoba, 14004, SpainLuis Mart铆nez-Mart铆nezInstituto Maim贸nides de Investigaci贸n Biom茅dica de C贸rdoba (IMIBIC), C贸rdoba, 14004, SpainLuis Mart铆nez-Mart铆nezAuthorsMar铆a L谩zaro-D铆ezView author publicationsYou can also search for this author in PubMed聽Google ScholarItziar Chapartegui-Gonz谩lezView author publicationsYou can also search for this author in PubMed聽Google ScholarSantiago Redondo-SalvoView author publicationsYou can also search for this author in PubMed聽Google ScholarChike LeighView author publicationsYou can also search for this author in PubMed聽Google ScholarDavid MerinoView author publicationsYou can also search for this author in PubMed聽Google ScholarDavid San SegundoView author publicationsYou can also search for this author in PubMed聽Google ScholarAdri谩n Fern谩ndezView author publicationsYou can also search for this author in PubMed聽Google ScholarJes煤s NavasView author publicationsYou can also search for this author in PubMed聽Google ScholarJos茅 Manuel IcardoView author publicationsYou can also search for this author in PubMed聽Google ScholarF茅lix AcostaView author publicationsYou can also search for this author in PubMed聽Google ScholarAlain Ocampo-SosaView author publicationsYou can also search for this author in PubMed聽Google ScholarLuis Mart铆nez-Mart铆nezView author publicationsYou can also search for this author in PubMed聽Google ScholarJos茅 Ramos-VivasView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsJ.R.V. conceived the experiments, J.R.V. and D.S.S. designed the experiments, M.L.D., I.C.G., S.R.S., C.L., D.M., A.F., F.A., A.O.S., J.M.I. and J.R.V. performed the experiments, M.L.D., I.C.G., J.N., F.A., J.M.I. L.M.M. and J.R.V. analyzed the data, A.O.S., D.S.S., J.N., F.A., J.M.I. contributed with reagents/materials/analysis tools, J.R.V. wrote the paper. All authors reviewed the manuscript.Corresponding authorCorrespondence to Jos茅 Ramos-Vivas.Ethics declarations Competing Interests The authors declare that they have no competing interests. Additional informationPublisher\'s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Electronic supplementary materialSupplementary InfoSupplementary video 1Supplementary video 2Supplementary video 3Supplementary video 4Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article鈥檚 Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article鈥檚 Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and PermissionsAbout this articleCite this articleL谩zaro-D铆ez, M., Chapartegui-Gonz谩lez, I., Redondo-Salvo, S. et al. Human neutrophils phagocytose and kill Acinetobacter baumannii and A. pittii. Sci Rep 7, 4571 (2017). https://doi.org/10.1038/s41598-017-04870-8Download citationReceived: 16 December 2016Accepted: 22 May 2017Published: 04 July 2017DOI: https://doi.org/10.1038/s41598-017-04870-8 Rohana P. Dassanayake, Taylor L. T. Wherry, Shollie M. Falkenberg, Timothy A. Reinhardt, Eduardo Casas Judith R. Stabel Veterinary Research (2021) CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.

>>> 更多资讯详情请访问蚂蚁淘商城