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Immunomodulatory Role of Clarithromycin in Acinetobacter baumannii Infection via Formation of Neutrophil Extracellular Traps Theocharis Konstantinidis,a Konstantinos Kambas,a Alexandros Mitsios,a Maria Panopoulou,b Victoria Tsironidou,a Erminia Dellaporta,c Georgios Kouklakis,c Athanasios Arampatzioglou,a Iliana Angelidou,a Ioannis Mitroulis,d Panagiotis Skendros,a,e Konstantinos Ritisa,e Laboratory of Molecular Hematology, Department of Medicine, Democritus University of Thrace, Alexandroupolis, Greecea; Laboratory of Microbiology, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis, Greeceb; Gastrointestinal Endoscopy Unit, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis, Greecec; Department of Clinical Pathobiochemistry and Institute for Clinical Chemistry and Laboratory Medicine, Faculty of Medicine, Technische Universität Dresden, Dresden, Germanyd; First Department of Internal Medicine, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis, Greecee
Macrolide antibiotics have been shown to act as immunomodulatory molecules in various immune cells. However, their effect on neutrophils has not been extensively investigated. In this study, we investigated the role of macrolide antibiotics in the generation of neutrophil extracellular traps (NETs). By assessing ex vivo and in vivo NET formation, we demonstrated that clarithromycin is able to induce NET generation both in vitro and in vivo. Clarithromycin utilizes autophagy in order to form NETs, and these NETs are decorated with antimicrobial peptide LL-37. Clarithromycin-induced NETs are able to inhibit Acinetobacter baumannii growth and biofilm formation in an LL-37-dependent manner. Additionally, LL-37 antimicrobial function depends on NET scaffold integrity. Collectively, these data expand the knowledge on the immunomodulatory role of macrolide antibiotics via the generation of LL-37-bearing NETs, which demonstrate LL-37-dependent antimicrobial activity and biofilm inhibition against A. baumannii.
I
n the last 2 decades, macrolides have been demonstrated as potential immunomodulatory agents, due to their ability to induce the activity of various immune cells and their regulatory role in chemokine and cytokine production (1, 2). The addition of macrolide antibiotics (azithromycin, clarithromycin, and erythromycin) to the standard treatment of severe community-acquired pneumonia (CAP) due to macrolide-resistant microorganisms led to decreased mortality of patients, providing evidence for their effect on immunomodulation (3). Moreover, in ventilator-associated pneumonia (VAP) patients with clarithromycin-resistant bacterial infections, combined treatment with clarithromycin was positively correlated with the time of resolution of the infection (4). Although it was proposed that some macrolides could induce degranulation and bacterial killing in polymorphonuclear neutrophils (PMNs), knowledge regarding their role in these cells has been very limited (5, 6). Neutrophils are the most abundant circulating inflammatory cells and the first line of defense against pathogens. They employ three major strategies to fight against microbes: phagocytosis, degranulation, and the release of neutrophil extracellular traps (NETs) (7, 8). The discovery of the mechanism of NETs has redefined our perception of the role and functions of neutrophils. NETs are composed of chromatin which is decorated with neutrophilic proteins (cytoplasmic, granular, and nuclear). NETs not only elicit their antimicrobial effect through pathogen immobilization via entrapment (7) but also have a direct microbicidal effect that depends on antimicrobial peptides (7, 8), histones (7, 9), or DNA (10). On the other hand, it has been also reported that some microorganisms, such as Acinetobacter baumannii, escape NETs (11) or possess inhibitory strategies against this mechanism (12, 13). One of the strategies that some microbes utilize to protect their colonies is the formation of biofilms. Biofilms are constituted by
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an exopolymeric extracellular matrix comprised of lipids, proteins that frequently exhibit amyloid-like properties, extracellular DNA, and exopolysaccharides (14, 15). Biofilms facilitate microbial attachment to and growth on abiotic surfaces and also render bacterial cultures less susceptible to antibiotics (16). Recently, this mechanism was also found in a multidrug-resistant nosocomial Gram-negative bacterium, A. baumannii, and the formation of biofilm contributed to the resistance of this microorganism to multiple antibiotics (17). Considering the above, we investigated the role of macrolide antibiotics in the generation of NETs, how these NETs could affect pathogens that naturally are not able to trigger the mechanism of NETosis, and their effect on bacterial defense mechanisms, such as the formation of biofilm. MATERIALS AND METHODS Patients and sample collection. To examine the in vivo NET generation potential of clarithromycin, 10 patients suffering from Helicobacter pylori-
Received 24 August 2015 Returned for modification 7 October 2015 Accepted 23 November 2015 Accepted manuscript posted online 7 December 2015 Citation Konstantinidis T, Kambas K, Mitsios A, Panopoulou M, Tsironidou V, Dellaporta E, Kouklakis G, Arampatzioglou A, Angelidou I, Mitroulis I, Skendros P, Ritis K. 2016. Immunomodulatory role of clarithromycin in Acinetobacter baumannii infection via formation of neutrophil extracellular traps. Antimicrob Agents Chemother 60:1040 –1048. doi:10.1128/AAC.02063-15. Address correspondence to Konstantinos Ritis, [email protected]. T.K. and K.K. contributed equally to this article. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.02063-15. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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TABLE 1 Patient and control group characteristicsa Value for: Characteristic Gender, no. of males/no. of females Age, yr (mean ⫾ SD) No. on standard eradication regimen No. on sequential eradication regimen No. on omeprazole monotherapy
Patients with H. pylori-positive gastritis (n ⫽ 10)
Patients with H. pylori-negative gastritis (n ⫽ 5)
Controls(n ⫽ 10)
4/6 42.9 ⫾ 14.3 6 4
2/3 44.2 ⫾ 6.4
4/6 35.4 ⫾ 5.7
5
a
The standard eradication regimen consisted of amoxicillin at 1 g twice a day (b.i.d.), clarithromycin at 500 mg b.i.d., and omeprazole at 20 mg b.i.d., for 10 days. The sequential eradication regimen consisted of amoxicillin at 1 g b.i.d. plus omeprazole at 20 mg b.i.d. for the first 5 days, followed by clarithromycin at 500 mg b.i.d., metronidazole at 500 mg b.i.d., and omeprazole at 20 mg b.i.d. for the next 5 days.
positive gastritis were recruited, since these patients are administered clarithromycin as a standard treatment. Five patients with H. pylori-negative gastritis (omeprazole monotherapy) and 10 age- and sex-matched control individuals were also recruited (details in Table 1). Additionally, to investigate the ability of A. baumannii to induce in vivo NET generation, 4 septic patients with A. baumannii bacteremia were enrolled in this study. PMNs and sera were obtained from patients with H. pylori-positive gastritis 12 to 24 h before treatment (n ⫽ 10) and during the 5th day of the standard eradication regimen. Moreover, PMNs and sera were obtained from the patients with H. pylori-negative gastritis before and during the 5th day of omeprazole treatment, as well as from the patients with A. baumannii bacteremia at various time points during the disease course. The study protocol was in accordance with the Declaration of Helsinki and was approved by the ethics review board of the University Hospital of Alexandroupolis. Written informed consent was obtained from each individual. Reagents. Daptomycin (Novartis) was dissolved at a concentration of 50 mg/ml per stock, ciprofloxacin (Vianex AE) stock was used at 200 mg/100 ml or 2 mg/ml, amoxicillin (Cooper pharmaceuticals) was dissolved at a concentration of 1 g/vial per stock, azithromycin (Anfarm Hellas) was dissolved at a concentration of 100 mg/ml per stock, and clarithromycin (Anfarm Hellas) was dissolved at a concentration of 50 mg/ml per stock. Antibodies. As primary antibodies, a mouse anti-myeloperoxidase (anti-MPO) monoclonal antibody (MAb), rabbit anti-neutrophil elastase (anti-NE) MAb (Santa Cruz Biotechnology Inc.), mouse anti-cathelicidin antimicrobial peptide LL-37 MAb (Santa Cruz Biotechnology), antiLC3b Ab, and anti-p62 Ab were used for NET staining. 4=,6-Diamidino2-phenylindole (DAPI; Sigma-Aldrich) was used for DNA staining. Polyclonal rabbit anti-mouse Alexa Fluor 488 antibody and polyclonal goat anti-rabbit Alexa Fluor 647 (Invitrogen) were used as secondary antibodies. Anti-MPO MAb (Upstate, Millipore) was used as a capture antibody for MPO-DNA complex enzyme-linked immunosorbent assay (ELISA). MSU crystal preparation. Monosodium urate (MSU) crystals were prepared as previously described (18) under pyrogen-free conditions. In particular, urate acid sodium salt (Sigma-Aldrich) was dissolved in 1 M NaOH (25 mg/ml) and boiled for 2 h at 200°C prior to crystallization. The solution was left to cool at room temperature and filtered through a 0.2-m filter. It was then incubated at room temperature for 7 days. The resulting crystals were washed with ethanol and acetone and allowed to air dry under sterile conditions. PMN isolation, stimulation, and inhibition studies. PMNs were isolated from heparinized venous blood and cultured in 5% CO2 at 37°C in a total volume of 500 l of RPMI medium (Gibco BRL) in the presence of 2% heterologous healthy donor serum and different stimulatory agents. For in vitro studies, PMNs from control individuals were treated with antibiotics for 3 h. Furthermore, PMNs were treated with other inflammatory stimuli, i.e., MSU crystals (noninfectious) or phorbol 12-myristate 13-acetate (PMA), for determination of LL-37 expression on NETs.
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To study autophagy induction, neutrophils were treated with clarithromycin for 90 min and for 3 h. To inhibit the autophagic machinery, neutrophils were pretreated with bafilomycin A1 (30 nM; Sigma-Aldrich) for 30 min before clarithromycin stimulation. Immunofluorescence. NET generation, preparation, and visualization by immunofluorescence confocal microscopy were performed as previously described (19). Samples were stained using anti-MPO MAb, anti-LL-37 MAb, or anti-NE polyclonal Ab. A polyclonal rabbit antimouse Alexa Fluor 488 antibody or a polyclonal goat anti-rabbit Alexa Fluor 647 antibody (Invitrogen) was utilized as a secondary antibody. DAPI (Sigma-Aldrich) was used for DNA counterstaining. Visualization was performed in a confocal microscope (Revolution spinning disk confocal system; Andor, Ireland) with PLAPON 606O/TIRFM-SP (numerical aperture [NA], 1.45) and UPLSAPO 100XO (NA, 1.4) objectives (Olympus). The percentage of NET-releasing PMNs was calculated by counting 200 cells in total. To study autophagy induction, samples were stained with anti-LC3b polyclonal antibody, followed by a polyclonal goat anti-rabbit Alexa Fluor 488 antibody utilized as a secondary antibody. DNA was counterstained using DAPI (Sigma-Aldrich). NET isolation. A total of 1.5 ⫻ 106 ex vivo- or in vitro-stimulated neutrophils were cultured for 4 h. After medium removal, cells were washed with RPMI medium. RPMI medium was added to each well, and NETs were collected on supernatant medium after vigorous agitation. The medium was centrifuged at 20 ⫻ g for 5 min, and the supernatant phase, containing NETs, was collected and stored at ⫺20°C until use. MPO-DNA complex ELISA. To quantify NET release by ex vivo- or in vitro-stimulated PMNs, MPO-DNA complex was measured in NET structures isolated from 1.5 ⫻ 106 PMNs as previously described (19) or in plasma from patients and control individuals. More specifically, capture antibody, 5 g/ml of anti-MPO MAb (Upstate, Millipore), was used to coat 96-well plates (dilution, 1:500 in 50 l) overnight at 4°C. After three washes (300 l each), 20 l each of samples was added to the wells with 80 l of incubation buffer containing a peroxidase-labeled anti-DNA MAb (Cell Death ELISAPLUS; Roche; dilution, 1:25). The plate was incubated for 2 h with shaking at 300 rpm at room temperature. After three washes (300 l each), 100 l of peroxidase substrate (2,2=-azinobis[3-ethylbenzthiazolinesulfonic acid] [ABTS]) was added. Absorbance at a 405-nm wavelength was measured after 20 min of incubation at room temperature in the dark. NET release was calculated as percent increase compared to that in controls. Western blot analysis. Western blot analysis for p62/SQSTM1 determination was performed with cells treated with clarithromycin for 3 h, as previously described (20). Briefly, incubation of polyvinylidene difluoride (PVDF) membranes was carried out at 4°C using an anti-p62 antibody (stock concentration, 200 g/ml; 1/300 dilution). Membranes were probed with horseradish peroxidase (HRP)-conjugated secondary antibody (stock concentration, 400 g/ml; 1/1,000 dilution) for 45 min at room temperature. To verify equal loading, membranes were reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Moreover,
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FIG 1 Macrolide antibiotics induce NET generation in vitro. (A) NET generation in control PMNs treated with relevant concentrations of antibiotics, as assessed by immunofluorescence (confocal microscopy). Green, MPO; red, NE; blue, DAPI-DNA. Results of one representative out of six independent experiments are shown. Original magnification, ⫻600. Scale bar, 5 m. (B and C) Percentages of NET-releasing PMNs (B) and MPO-DNA complex in isolated NET structures from control PMNs (C) treated with antibiotics. In panels B and C, data from six independent experiments are presented as means ⫾ SDs (*, P ⬍ 0.05; n.s., not significant compared to control value).
LL37-specific mouse MAb (stock concentration, 200 g/ml; 1/500 dilution) was utilized for the measurement of LL-37 expressed in NETs, using the same protocol. Bacterial strains. A. baumannii strains, including a reference strain (ATCC 19606) and a clinical isolate, were cultured as previously described (21). Bacteria were preserved in glycerol broth at ⫺80°C. Bacteria from an overnight culture on MacConkey agar were suspended in saline to an optical density of 0.5 McFarland, corresponding to a concentration of approximately 108 CFU/ml. Bacterial strains of A. baumannii were cocultured with NETs isolated as described above (under “NET isolation”), generated by clarithromycin or generic inducers such as MSU or PMA in a 1/10 concentration. For LL-37 inhibition of NETs, NETs were incubated with anti-LL-37 antibody prior to their introduction to bacterial cultures. Mouse monoclonal IgG1 antibody was used as a control to anti-LL-37 and did not affect NET-induced bacterial killing. Biofilm assay. Biofilm assay was conducted by a microtiter plate method as described previously (22). Briefly, each microbial strain was grown overnight in Trypticase soy broth (TSB) at 37°C. Next, the overnight growth was diluted in a ratio of 1:40 in TSB. Two hundred microliters of cell suspension was inoculated to sterile 96-well polystyrene microtiter plates in the presence or absence of NETs in a 1/10 concentration, isolated as described above. After 24 h of incubation at 37°C, the wells were gently washed three times with 200 l of phosphate-buffered saline (PBS) and stained with 0.5% crystal violet for 15 min. The wells were rinsed again in 200 l of 95% ethanol to solubilize crystal violet. Each assay was performed in duplicate. Finally, the optical density at 550 nm (OD550) was determined using a microplate reader. OD550 values for each
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well were subtracted from those of the blank, which contained only TSB without inocula. Statistical analysis. Statistical analyses were performed using one-way analysis of variance (ANOVA) (Scheffé test in uniform sample size and least significant difference [LSD] test in nonuniform sample size for post hoc comparisons). All statistical analyses were performed with OriginPro 8. P values less than 0.05 were considered significant.
RESULTS
Macrolide antibiotics induce generation of NETs. Considering that macrolide antibiotics can act as immunomodulatory agents (1, 2), we sought to investigate whether this effect can be mediated through neutrophils and, more specifically, through the generation of NETs. We initially studied the ability of different antibiotic groups to generate NETs. In vitro stimulation of PMNs from healthy individuals with clinically relevant concentrations of commonly used classes of antibiotics has shown that macrolide antibiotics (azithromycin and clarithromycin) induced NET formation, whereas ampicillin (semisynthetic penicillin), penicillin, daptomycin, and ciprofloxacin did not, as observed by immunofluorescence (Fig. 1A and B) and MPO-DNA complex ELISA (Fig. 1C). We next investigated the ability of clarithromycin to induce NETs in vivo. To assess this, patients suffering from H. pyloripositive gastritis were enrolled as a study group, since clarithro-
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FIG 2 Clarithromycin induces NET generation in vivo in patients with H. pylori gastritis. (A) NET generation in ex vivo PMNs from patients with H. pylori-positive gastritis before and after treatment with clarithromycin, amoxicillin, and omeprazole and from control individuals, as assessed by immunofluorescence (confocal microscopy). Green, MPO; red, NE; blue, DAPI-DNA. Results of one representative out of six independent experiments are shown. Original magnification, ⫻600. Scale bar, 5 m. (B and C) Percentages of NET-releasing PMNs (B) and MPO-DNA complex (in isolated NET structures from ex vivo PMNs (C) isolated from patients with H. pylori-positive gastritis before and after standard eradication treatment with clarithromycin, amoxicillin, and omeprazole (n ⫽ 6), patients with sequential eradication (amoxicillin and omeprazole) (n ⫽ 4), patients with H. pylori-negative gastritis (omeprazole monotherapy) (n ⫽ 5), and control individuals (n ⫽ 10). Data are presented as medians ⫾ 90th percentile (*, P ⬍ 0.05; n.s., not significant compared to control value).
mycin is used for standard eradication therapy and these patients do not demonstrate clinical and laboratory evidence of systemic inflammation (23) that could potentially influence in vivo NET formation. Thus, PMNs were isolated before and during the 5th day of standard eradication therapy with clarithromycin, amoxicillin, and omeprazole. The collected PMNs during the 5th day of eradication therapy demonstrated increased ex vivo NET formation compared to that of PMNs before clarithromycin initiation, PMNs from healthy individuals, or PMNs from patients under sequential eradication treatment, which comprises only amoxicillin plus omeprazole for the first 5 days of eradication (details in Table 1), as determined by immunofluorescence (Fig. 2A and B) and MPO-DNA complex ELISA (Fig. 2C). Patients with H. pylori-negative gastritis that were treated with omeprazole alone did not demonstrate any ex vivo NET generation (Fig. 2). These findings demonstrate the ability of macrolides to induce NET formation. Clarithromycin utilizes autophagy to induce NET generation. Several lines of evidence support the involvement of autophagy in neutrophil functions, including NET release (18, 24, 25). Thus, we assessed endogenous LC3B cellular distribution and p62/SQSTM1 degradation in PMNs from control subjects treated with clarithromycin. Formation of LC3B puncta and increased p62/SQSTM1 degradation were observed in PMNs treated with
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clarithromycin compared to controls, as determined by immunofluorescence and immunoblotting, respectively, suggesting the induction of the autophagic machinery (Fig. 3A and B). In addition, PMNs pretreated with bafilomycin A1 prior to stimulation with clarithromycin demonstrated reduced NET generation compared to that of PMNs treated with clarithromycin alone (Fig. 3C), further verifying the involvement of autophagy in clarithromycin induction of NET formation. These findings demonstrate that clarithromycin induces NET formation in an autophagy-dependent manner. NETs generated by clarithromycin are decorated with LL-37. Considering that LL-37 is a potent antimicrobial peptide (26, 27) and that it is released through NETs under certain inflammatory conditions (28, 29), we examined the presence of LL-37 on clarithromycin-induced NETs. NETs released by PMNs from control subjects treated with clarithromycin were decorated with LL-37, as determined by immunofluorescence and immunoblotting (Fig. 4). We then assessed LL-37 expression on NETs released from PMNs derived from patients treated with clarithromycin. Indeed, PMNs derived from these patients demonstrated the presence of LL-37 on NETs (Fig. 4A). Furthermore, to assess whether the presence of LL-37 is a generic finding of NETs, we investigated its presence on NETs using
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FIG 3 Clarithromycin-induced NET generation in PMNs by utilizing autophagy. Autophagy induction was determined with LC3B staining as assessed by confocal microscopy (A) or p62/SQSTM1 immunoblotting in control PMNs treated with clarithromycin (B). Red, LC3B; blue, DAPI-DNA. (C) NET generation in control PMNs treated with clarithromycin in the presence or absence of bafilomycin A1 (autophagy inhibitor), as assessed by immunofluorescence (confocal microscopy). Green, MPO; red, NE; blue, DAPI-DNA. (D) MPO-DNA complex in isolated NET structures from control PMNs treated with clarithromycin in the presence or absence of autophagy inhibitor. In panels A to C, results for one representative out of four independent experiments are shown. Original magnifications: ⫻1,000 (A) and ⫻600 (C). Scale bar, 5 m. In panel D, data from four independent experiments are presented as means ⫾ SDs (*, P ⬍ 0.05; n.s., not significant).
other alternative inducers of NETosis, such as MSU and PMA. LL-37 was absent from NETs induced by MSU (Fig. 4), as assessed by immunofluorescence and immunoblotting of NET proteins. Similar to MSU, PMA is not able to induce LL-37-bearing NETs (Fig. 4A). These findings suggest that clarithromycin has the ability to induce NETs containing LL-37.
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Clarithromycin-dependent LL-37 expression on NETs modulates antimicrobial activity against Acinetobacter baumannii. Since neutrophils during infection produce NETs as a defense mechanism against pathogens (7, 8), we investigated if clarithromycin-driven NETs conserve this ability. To test the microbicidal activity of clarithromycin-driven NETs, our model was based on A. baumannii, since this microorganism is not able to trigger the
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FIG 4 LL-37 is present on NETs induced by clarithromycin. (A) Localization of LL-37 on NETs in in vitro control PMNs stimulations with clarithromycin or ex vivo patients treated with clarithromycin, amoxicillin, and omeprazole, as observed by confocal microscopy. MSU and PMA were used as generic NET inducers. Green, LL-37; red, NE; blue, DAPI-DNA. Original magnification, ⫻600. Scale bar, 5 m. (B) Expression of LL-37 on NET proteins derived from in vitro stimulations of control PMNs with clarithromycin or MSU, as assessed by immunoblotting. Each panel shows results of one representative out of four independent experiments.
mechanism of NET by itself in vitro (11) or ex vivo (see Fig. S1 in the supplemental material) and is also resistant to treatment with clarithromycin (30). Clinical and ATCC 19606 strains of A. baumannii were cultured in the presence or absence of clarithromycin-induced NETs. NETs were obtained either by in vitro stimulation or ex vivo from H. pylori-positive patients treated with clarithromycin. We observed that NETs induced by clarithromycin both in vitro (Fig. 5A) and ex vivo (Fig. 5B) significantly reduced bacterial growth compared to that in control cultures. This activity was attributed to the presence of LL-37, since LL-37 neutralization with anti-LL-37 antibody abolished the antimicrobial action of clarithromycin-induced NETs (Fig. 5A and B). To further verify the specific activity of clarithromycin-driven NETs, we used NETs obtained after stimulation from inducers that are not able to generate LL-37-bearing NETs, such as MSU or PMA. These generic NETs, in contrast to clarithromycin-induced NETs, indicated an absence of LL-37 (Fig. 4A and B) and significantly reduced capacity to inhibit bacterial growth (Fig. 5A). We further sought to investigate the effect of clarithromycininduced NETs on the generation of biofilm by A. baumannii. Interestingly, clarithromycin-induced NETs significantly reduced
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the formation of biofilms in an LL-37-dependent manner, as demonstrated by LL-37 inhibition (Fig. 5C). However, generic NETs induced by MSU crystals or PMA did not affect biofilm generation by A. baumannii (Fig. 5C). Treatment of NET structures with DNase attenuated the inhibitory effect on biofilms (Fig. 5C). Since the NET scaffold affects the functionality of various NET-bound components (7, 19), we wanted to test whether LL-37 functionality is affected by NET integrity. DNase treatment of clarithromycin-induced NETs abolished LL-37 antimicrobial function on A. baumannii cultures (Fig. 5D). These findings suggest an indirect antimicrobial role of clarithromycin against strains of A. baumannii through NET formation in an LL-37-dependent manner. DISCUSSION
The present study demonstrates for the first time the in vitro and in vivo ability of macrolide antibiotics to induce NET formation, expanding our understanding of their immunomodulatory role. We show that clarithromycin-induced NETs are decorated by the antimicrobial peptide LL-37, which is in its active form and is able to act against multidrug-resistant A. baumannii in vitro.
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FIG 5 In vitro- and ex vivo-generated clarithromycin-induced NETs act against Acinetobacter baumannii in an LL-37-dependent manner. (A) Cultures of clinical and ATCC 19606 strains of A. baumannii in the presence of clarithromycin-induced NETs generated in vitro before and after pretreatment with anti-LL-37 antibody. MSU and PMA were used as controls (LL-37 negative NETs). (B) Cultures of clinical and ATCC 19606 strains of A. baumannii in the presence of NETs derived from H. pylori-positive patients treated with clarithromycin, amoxicillin, and omeprazole, before and after pretreatment with anti-LL-37 antibody. Data from six independent experiments are presented as means ⫾ SDs (*, P ⬍ 0.05; n.s., not significant). (C) Determination of biofilm generation in clinical and ATCC 19606 strains of A. baumannii in the presence of clarithromycin-induced NETs generated in vitro before and after pretreatment with anti-LL-37 antibody or DNase I, as assessed by crystal violet staining. MSU and PMA were used as controls (LL-37 negative NETs). (D) Cultures of clinical and ATCC 19606 strains of A. baumannii in the presence of clarithromycin-induced NETs generated in vitro before and after pretreatment with DNase I. In panels A to C and panel D, data from 6 and 4 independent experiments, respectively, are presented as means ⫾ SDs (*, P ⬍ 0.05; n.s., not significant).
Clarithromycin-induced NETs exerted their antimicrobial function against both clinical and ATCC 19606 strains of multidrug-resistant A. baumannii, a pathogen that is not able to trigger the mechanism of NET by itself. However, specific NETs (such as clarithromycin-driven NETs) restrict A. baumannii in cultures. Additionally, clarithromycin-induced NETs were able to inhibit A. baumannii biofilm formation. This is an interesting finding and gives more information on the ongoing debate on the cross talk of biofilms and NETs, since neutrophils constitute indispensable biofilm controllers but can also contribute to biofilm stability (31). These findings suggest an indirect antimicrobial action of macrolide antibiotics, through the induction of NET generation. As a side note, we also provide evidence that A. baumannii does not induce NET generation in vivo, as observed with ex vivo-isolated PMNs from patients with A. baumannii bacteremia. Since neutrophils have been shown to express LL-37 under
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certain inflammatory conditions (28, 29), we investigated the presence of this antimicrobial peptide on clarithromycin-induced NETs. We demonstrated for the first time that clarithromycin induces both in vitro and ex vivo NETs decorated with LL-37, while other generic inducers of NETs, such as MSU or PMA, were not able to generate amounts of active LL-37-bearing NETs. Recent studies showed that PMA-induced NETs did not express active LL-37. Although traces of LL-37 have been detected mainly by electron microscopy (32), the absence of LL-37 activity is in accordance with our findings. Furthermore, considering the susceptibility of A. baumannii to LL-37 (27), we further demonstrate that LL-37 mediated the microbicidal activity of NETs and attenuated the formation of biofilms by this pathogen. Additionally, digestion of the chromatin scaffold with DNase abolished completely the bactericidal activity of clarithromycin-induced NETs, similarly to anti-LL37 inhibition. This indicates the importance of the
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NET chromatin scaffold in LL-37 functionality, previously shown also with other NET-bound proteins (7, 19). Moreover, since autophagy plays an important role in NET generation, we investigated whether clarithromycin regulates this active mechanism to generate NETs. Indeed, clarithromycin was involved in the in vitro upregulation of autophagy leading to generation of NETs, as demonstrated by the end-stage autophagy inhibition with bafilomycin A1. These findings suggest that clarithromycin can act additionally as an autophagy inducer in PMNs, thus expanding its role by regulating autophagy, a mechanism necessary for NET formation. A previous clinical trial has demonstrated that administration of clarithromycin in patients with VAP accelerated the resolution of pneumonia and decreased the risk of death from septic shock, suggesting a potential immunomodulatory effect of clarithromycin on the host (4). It should be noted that most of the patients (54.5%) were suffering from A. baumannii infection. Besides the limitations related to the design of the study, including the short period of clarithromycin administration (only 3 days), these results support our in vitro findings and encourage the investigation of the in vivo synergistic role of macrolide-induced NETs in severe infections, such as those caused by multidrug-resistant species of A. baumannii. Additionally, whether NETs are involved in the adjunctive potential of clarithromycin when administered in combination with other antibiotics is a query that needs to be investigated in future studies. In conclusion, this is the first report suggesting the formation of LL-37-bearing NETs by clarithromycin as a part of its immunomodulatory role. The role of clarithromycin in inducing NETs is dependent on autophagy, and the LL-37 present in NETs is important for both microbicidal activity and biofilm inhibition. Of note, the functionality of LL-37 is highly dependent on NET chromatin scaffold integrity. Our study supports the use of clarithromycin as a synergic agent in clinical trials with patients with A. baumannii infection in order to determine whether the observed in vitro microbicidal function through NETs is repeated in vivo. ACKNOWLEDGMENT We declare no conflict of interest.
FUNDING INFORMATION This study was supported by the Scientific Committee of Democritus University of Thrace, grant number 80895.
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5. Schultz MJ, Speelman P, Hack CE, Buurman WA, van Deventer SJ, van Der Poll T. 2000. Intravenous infusion of erythromycin inhibits CXC chemokine production, but augments neutrophil degranulation in whole blood stimulated with Streptococcus pneumoniae. J Antimicrob Chemother 46:235–240. http://dx.doi.org/10.1093/jac/46.2.235. 6. Iskandar I, Walters JD. 2011. Clarithromycin accumulation by phagocytes and its effect on killing of Aggregatibacter actinomycetemcomitans. J Periodontol 82:497–504. http://dx.doi.org/10.1902/jop.2010.100221. 7. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A. 2004. Neutrophil extracellular traps kill bacteria. Science 303:1532–1535. http://dx.doi.org/10.1126/science.1092385. 8. Yipp BG, Petri B, Salina D, Jenne CN, Scott BN, Zbytnuik LD, Pittman K, Asaduzzaman M, Wu K, Meijndert HC, Malawista SE, de Boisfleury Chevance A, Zhang K, Conly J, Kubes P. 2012. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat Med 18:1386 –1393. http://dx.doi.org/10.1038/nm.2847. 9. Wang Y, Chen Y, Xin L, Beverley SM, Carlsen ED, Popov V, Chang KP, Wang M, Soong L. 2011. Differential microbicidal effects of human histone proteins H2A and H2B on Leishmania promastigotes and amastigotes. Infect Immun 79:1124 –1133. http://dx.doi.org/10.1128/IAI .00658-10. 10. Halverson TW, Wilton M, Poon KKH, Petri B, Lewenza S. 2015. DNA is an antimicrobial component of neutrophil extracellular traps. PLoS Pathog 11(1):e1004593. http://dx.doi.org/10.1371/journal.ppat.1004593. 11. Kamoshida G, Kikuchi-Ueda T, Tansho-Nagakawa S, Nakano R, Nakano A, Kikuchi H, Ubagai T, Ono Y. 2015. Acinetobacter baumannii escape from neutrophil extracellular traps (NETs). J Infect Chemother 21:43– 49. http://dx.doi.org/10.1016/j.jiac.2014.08.032. 12. Berends ET, Horswill AR, Haste NM, Monestier M, Nizet V, von Kockritz-Blickwede M. 2010. Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps. J Innate Immun 2:576 –586. http://dx.doi.org/10.1159/000319909. 13. Lauth X, von Kockritz-Blickwede M, McNamara CW, Myskowski S, Zinkernagel AS, Beall B, Ghosh P, Gallo RL, Nizet V. 2009. M1 protein allows group A streptococcal survival in phagocyte extracellular traps through cathelicidin inhibition. J Innate Immun 1:202–214. http://dx.doi .org/10.1159/000203645. 14. Hall-Stoodley L, Costerton JW, Stoodley P. 2004. Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2:95–108. http://dx.doi.org/10.1038/nrmicro821. 15. Fuxman Bass JI, Russo DM, Gabelloni ML, Geffner JR, Giordano M, Catalano M, Zorreguieta A, Trevani AS. 2010. Extracellular DNA: a major proinflammatory component of Pseudomonas aeruginosa biofilms. J Immunol 184:6386 – 6395. http://dx.doi.org/10.4049/jimmunol .0901640. 16. Donlan RM, Costerton JW. 2002. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15:167–193. http://dx .doi.org/10.1128/CMR.15.2.167-193.2002. 17. Espinal P, Martí S, Vila J. 2012. Effect of biofilm formation on the survival of Acinetobacter baumannii on dry surfaces. J Hosp Infect 80:56 – 60. http://dx.doi.org/10.1016/j.jhin.2011.08.013. 18. Mitroulis I, Kambas K, Chrysanthopoulou A, Skendros P, Apostolidou E, Kourtzelis I, Drosos GI, Boumpas DT, Ritis K. 2011. Neutrophil extracellular trap formation is associated with IL-1 and autophagyrelated signaling in gout. PLoS One 6(12):e29318. http://dx.doi.org/10 .1371/journal.pone.0029318. 19. Stakos DA, Kambas K, Konstantinidis T, Mitroulis I, Apostolidou E, Arelaki Tsironidou SV, Giatromanolaki A, Skendros P, Konstantinides S, Ritis K. 2015. Expression of functional tissue factor by neutrophil extracellular traps in culprit artery of acute myocardial infarction. Eur Heart J 36:1405–1414. http://dx.doi.org/10.1093/eurheartj/ehv007. 20. Chrysanthopoulou A, Mitroulis I, Kambas K, Skendros P, Kourtzelis I, Vradelis S, Kolios G, Aslanidis S, Doumas M, Ritis K. 2011. Tissue factor-thrombin signaling enhances the fibrotic activity of myofibroblasts in systemic sclerosis through up-regulation of endothelin receptor A. Arthritis Rheum 63:3586 –3597. http://dx.doi.org/10.1002/art.30586. 21. Bouvet PJ, Grimont PA. 1986. Taxonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii sp. nov., Acinetobacter haemolyticus sp. nov., Acinetobacter johnsonii sp. nov., and Acinetobacter junii sp. nov. and emended descriptions of Acinetobacter calcoaceticus and Acinetobacter lwoffii. Int J Syst Bacteriol 36:228 –240. 22. O’Toole GA. 2011. Microtiter dish biofilm formation assay. J Vis Exp 2011(47):e2437. http://dx.doi.org/10.3791/2437.
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Immunomodulatory Role of Clarithromycin in Acinetobacter baumannii Infection via Formation of Neutrophil Extracellular Traps Theocharis Konstantinidis,a Konstantinos Kambas,a Alexandros Mitsios,a Maria Panopoulou,b Victoria Tsironidou,a Erminia Dellaporta,c Georgios Kouklakis,c Athanasios Arampatzioglou,a Iliana Angelidou,a Ioannis Mitroulis,d Panagiotis Skendros,a,e Konstantinos Ritisa,e Laboratory of Molecular Hematology, Department of Medicine, Democritus University of Thrace, Alexandroupolis, Greecea; Laboratory of Microbiology, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis, Greeceb; Gastrointestinal Endoscopy Unit, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis, Greecec; Department of Clinical Pathobiochemistry and Institute for Clinical Chemistry and Laboratory Medicine, Faculty of Medicine, Technische Universität Dresden, Dresden, Germanyd; First Department of Internal Medicine, Democritus University of Thrace, University General Hospital of Alexandroupolis, Alexandroupolis, Greecee
Macrolide antibiotics have been shown to act as immunomodulatory molecules in various immune cells. However, their effect on neutrophils has not been extensively investigated. In this study, we investigated the role of macrolide antibiotics in the generation of neutrophil extracellular traps (NETs). By assessing ex vivo and in vivo NET formation, we demonstrated that clarithromycin is able to induce NET generation both in vitro and in vivo. Clarithromycin utilizes autophagy in order to form NETs, and these NETs are decorated with antimicrobial peptide LL-37. Clarithromycin-induced NETs are able to inhibit Acinetobacter baumannii growth and biofilm formation in an LL-37-dependent manner. Additionally, LL-37 antimicrobial function depends on NET scaffold integrity. Collectively, these data expand the knowledge on the immunomodulatory role of macrolide antibiotics via the generation of LL-37-bearing NETs, which demonstrate LL-37-dependent antimicrobial activity and biofilm inhibition against A. baumannii.
I
n the last 2 decades, macrolides have been demonstrated as potential immunomodulatory agents, due to their ability to induce the activity of various immune cells and their regulatory role in chemokine and cytokine production (1, 2). The addition of macrolide antibiotics (azithromycin, clarithromycin, and erythromycin) to the standard treatment of severe community-acquired pneumonia (CAP) due to macrolide-resistant microorganisms led to decreased mortality of patients, providing evidence for their effect on immunomodulation (3). Moreover, in ventilator-associated pneumonia (VAP) patients with clarithromycin-resistant bacterial infections, combined treatment with clarithromycin was positively correlated with the time of resolution of the infection (4). Although it was proposed that some macrolides could induce degranulation and bacterial killing in polymorphonuclear neutrophils (PMNs), knowledge regarding their role in these cells has been very limited (5, 6). Neutrophils are the most abundant circulating inflammatory cells and the first line of defense against pathogens. They employ three major strategies to fight against microbes: phagocytosis, degranulation, and the release of neutrophil extracellular traps (NETs) (7, 8). The discovery of the mechanism of NETs has redefined our perception of the role and functions of neutrophils. NETs are composed of chromatin which is decorated with neutrophilic proteins (cytoplasmic, granular, and nuclear). NETs not only elicit their antimicrobial effect through pathogen immobilization via entrapment (7) but also have a direct microbicidal effect that depends on antimicrobial peptides (7, 8), histones (7, 9), or DNA (10). On the other hand, it has been also reported that some microorganisms, such as Acinetobacter baumannii, escape NETs (11) or possess inhibitory strategies against this mechanism (12, 13). One of the strategies that some microbes utilize to protect their colonies is the formation of biofilms. Biofilms are constituted by
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an exopolymeric extracellular matrix comprised of lipids, proteins that frequently exhibit amyloid-like properties, extracellular DNA, and exopolysaccharides (14, 15). Biofilms facilitate microbial attachment to and growth on abiotic surfaces and also render bacterial cultures less susceptible to antibiotics (16). Recently, this mechanism was also found in a multidrug-resistant nosocomial Gram-negative bacterium, A. baumannii, and the formation of biofilm contributed to the resistance of this microorganism to multiple antibiotics (17). Considering the above, we investigated the role of macrolide antibiotics in the generation of NETs, how these NETs could affect pathogens that naturally are not able to trigger the mechanism of NETosis, and their effect on bacterial defense mechanisms, such as the formation of biofilm. MATERIALS AND METHODS Patients and sample collection. To examine the in vivo NET generation potential of clarithromycin, 10 patients suffering from Helicobacter pylori-
Received 24 August 2015 Returned for modification 7 October 2015 Accepted 23 November 2015 Accepted manuscript posted online 7 December 2015 Citation Konstantinidis T, Kambas K, Mitsios A, Panopoulou M, Tsironidou V, Dellaporta E, Kouklakis G, Arampatzioglou A, Angelidou I, Mitroulis I, Skendros P, Ritis K. 2016. Immunomodulatory role of clarithromycin in Acinetobacter baumannii infection via formation of neutrophil extracellular traps. Antimicrob Agents Chemother 60:1040 –1048. doi:10.1128/AAC.02063-15. Address correspondence to Konstantinos Ritis, [email protected]. T.K. and K.K. contributed equally to this article. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.02063-15. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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TABLE 1 Patient and control group characteristicsa Value for: Characteristic Gender, no. of males/no. of females Age, yr (mean ⫾ SD) No. on standard eradication regimen No. on sequential eradication regimen No. on omeprazole monotherapy
Patients with H. pylori-positive gastritis (n ⫽ 10)
Patients with H. pylori-negative gastritis (n ⫽ 5)
Controls(n ⫽ 10)
4/6 42.9 ⫾ 14.3 6 4
2/3 44.2 ⫾ 6.4
4/6 35.4 ⫾ 5.7
5
a
The standard eradication regimen consisted of amoxicillin at 1 g twice a day (b.i.d.), clarithromycin at 500 mg b.i.d., and omeprazole at 20 mg b.i.d., for 10 days. The sequential eradication regimen consisted of amoxicillin at 1 g b.i.d. plus omeprazole at 20 mg b.i.d. for the first 5 days, followed by clarithromycin at 500 mg b.i.d., metronidazole at 500 mg b.i.d., and omeprazole at 20 mg b.i.d. for the next 5 days.
positive gastritis were recruited, since these patients are administered clarithromycin as a standard treatment. Five patients with H. pylori-negative gastritis (omeprazole monotherapy) and 10 age- and sex-matched control individuals were also recruited (details in Table 1). Additionally, to investigate the ability of A. baumannii to induce in vivo NET generation, 4 septic patients with A. baumannii bacteremia were enrolled in this study. PMNs and sera were obtained from patients with H. pylori-positive gastritis 12 to 24 h before treatment (n ⫽ 10) and during the 5th day of the standard eradication regimen. Moreover, PMNs and sera were obtained from the patients with H. pylori-negative gastritis before and during the 5th day of omeprazole treatment, as well as from the patients with A. baumannii bacteremia at various time points during the disease course. The study protocol was in accordance with the Declaration of Helsinki and was approved by the ethics review board of the University Hospital of Alexandroupolis. Written informed consent was obtained from each individual. Reagents. Daptomycin (Novartis) was dissolved at a concentration of 50 mg/ml per stock, ciprofloxacin (Vianex AE) stock was used at 200 mg/100 ml or 2 mg/ml, amoxicillin (Cooper pharmaceuticals) was dissolved at a concentration of 1 g/vial per stock, azithromycin (Anfarm Hellas) was dissolved at a concentration of 100 mg/ml per stock, and clarithromycin (Anfarm Hellas) was dissolved at a concentration of 50 mg/ml per stock. Antibodies. As primary antibodies, a mouse anti-myeloperoxidase (anti-MPO) monoclonal antibody (MAb), rabbit anti-neutrophil elastase (anti-NE) MAb (Santa Cruz Biotechnology Inc.), mouse anti-cathelicidin antimicrobial peptide LL-37 MAb (Santa Cruz Biotechnology), antiLC3b Ab, and anti-p62 Ab were used for NET staining. 4=,6-Diamidino2-phenylindole (DAPI; Sigma-Aldrich) was used for DNA staining. Polyclonal rabbit anti-mouse Alexa Fluor 488 antibody and polyclonal goat anti-rabbit Alexa Fluor 647 (Invitrogen) were used as secondary antibodies. Anti-MPO MAb (Upstate, Millipore) was used as a capture antibody for MPO-DNA complex enzyme-linked immunosorbent assay (ELISA). MSU crystal preparation. Monosodium urate (MSU) crystals were prepared as previously described (18) under pyrogen-free conditions. In particular, urate acid sodium salt (Sigma-Aldrich) was dissolved in 1 M NaOH (25 mg/ml) and boiled for 2 h at 200°C prior to crystallization. The solution was left to cool at room temperature and filtered through a 0.2-m filter. It was then incubated at room temperature for 7 days. The resulting crystals were washed with ethanol and acetone and allowed to air dry under sterile conditions. PMN isolation, stimulation, and inhibition studies. PMNs were isolated from heparinized venous blood and cultured in 5% CO2 at 37°C in a total volume of 500 l of RPMI medium (Gibco BRL) in the presence of 2% heterologous healthy donor serum and different stimulatory agents. For in vitro studies, PMNs from control individuals were treated with antibiotics for 3 h. Furthermore, PMNs were treated with other inflammatory stimuli, i.e., MSU crystals (noninfectious) or phorbol 12-myristate 13-acetate (PMA), for determination of LL-37 expression on NETs.
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To study autophagy induction, neutrophils were treated with clarithromycin for 90 min and for 3 h. To inhibit the autophagic machinery, neutrophils were pretreated with bafilomycin A1 (30 nM; Sigma-Aldrich) for 30 min before clarithromycin stimulation. Immunofluorescence. NET generation, preparation, and visualization by immunofluorescence confocal microscopy were performed as previously described (19). Samples were stained using anti-MPO MAb, anti-LL-37 MAb, or anti-NE polyclonal Ab. A polyclonal rabbit antimouse Alexa Fluor 488 antibody or a polyclonal goat anti-rabbit Alexa Fluor 647 antibody (Invitrogen) was utilized as a secondary antibody. DAPI (Sigma-Aldrich) was used for DNA counterstaining. Visualization was performed in a confocal microscope (Revolution spinning disk confocal system; Andor, Ireland) with PLAPON 606O/TIRFM-SP (numerical aperture [NA], 1.45) and UPLSAPO 100XO (NA, 1.4) objectives (Olympus). The percentage of NET-releasing PMNs was calculated by counting 200 cells in total. To study autophagy induction, samples were stained with anti-LC3b polyclonal antibody, followed by a polyclonal goat anti-rabbit Alexa Fluor 488 antibody utilized as a secondary antibody. DNA was counterstained using DAPI (Sigma-Aldrich). NET isolation. A total of 1.5 ⫻ 106 ex vivo- or in vitro-stimulated neutrophils were cultured for 4 h. After medium removal, cells were washed with RPMI medium. RPMI medium was added to each well, and NETs were collected on supernatant medium after vigorous agitation. The medium was centrifuged at 20 ⫻ g for 5 min, and the supernatant phase, containing NETs, was collected and stored at ⫺20°C until use. MPO-DNA complex ELISA. To quantify NET release by ex vivo- or in vitro-stimulated PMNs, MPO-DNA complex was measured in NET structures isolated from 1.5 ⫻ 106 PMNs as previously described (19) or in plasma from patients and control individuals. More specifically, capture antibody, 5 g/ml of anti-MPO MAb (Upstate, Millipore), was used to coat 96-well plates (dilution, 1:500 in 50 l) overnight at 4°C. After three washes (300 l each), 20 l each of samples was added to the wells with 80 l of incubation buffer containing a peroxidase-labeled anti-DNA MAb (Cell Death ELISAPLUS; Roche; dilution, 1:25). The plate was incubated for 2 h with shaking at 300 rpm at room temperature. After three washes (300 l each), 100 l of peroxidase substrate (2,2=-azinobis[3-ethylbenzthiazolinesulfonic acid] [ABTS]) was added. Absorbance at a 405-nm wavelength was measured after 20 min of incubation at room temperature in the dark. NET release was calculated as percent increase compared to that in controls. Western blot analysis. Western blot analysis for p62/SQSTM1 determination was performed with cells treated with clarithromycin for 3 h, as previously described (20). Briefly, incubation of polyvinylidene difluoride (PVDF) membranes was carried out at 4°C using an anti-p62 antibody (stock concentration, 200 g/ml; 1/300 dilution). Membranes were probed with horseradish peroxidase (HRP)-conjugated secondary antibody (stock concentration, 400 g/ml; 1/1,000 dilution) for 45 min at room temperature. To verify equal loading, membranes were reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Moreover,
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FIG 1 Macrolide antibiotics induce NET generation in vitro. (A) NET generation in control PMNs treated with relevant concentrations of antibiotics, as assessed by immunofluorescence (confocal microscopy). Green, MPO; red, NE; blue, DAPI-DNA. Results of one representative out of six independent experiments are shown. Original magnification, ⫻600. Scale bar, 5 m. (B and C) Percentages of NET-releasing PMNs (B) and MPO-DNA complex in isolated NET structures from control PMNs (C) treated with antibiotics. In panels B and C, data from six independent experiments are presented as means ⫾ SDs (*, P ⬍ 0.05; n.s., not significant compared to control value).
LL37-specific mouse MAb (stock concentration, 200 g/ml; 1/500 dilution) was utilized for the measurement of LL-37 expressed in NETs, using the same protocol. Bacterial strains. A. baumannii strains, including a reference strain (ATCC 19606) and a clinical isolate, were cultured as previously described (21). Bacteria were preserved in glycerol broth at ⫺80°C. Bacteria from an overnight culture on MacConkey agar were suspended in saline to an optical density of 0.5 McFarland, corresponding to a concentration of approximately 108 CFU/ml. Bacterial strains of A. baumannii were cocultured with NETs isolated as described above (under “NET isolation”), generated by clarithromycin or generic inducers such as MSU or PMA in a 1/10 concentration. For LL-37 inhibition of NETs, NETs were incubated with anti-LL-37 antibody prior to their introduction to bacterial cultures. Mouse monoclonal IgG1 antibody was used as a control to anti-LL-37 and did not affect NET-induced bacterial killing. Biofilm assay. Biofilm assay was conducted by a microtiter plate method as described previously (22). Briefly, each microbial strain was grown overnight in Trypticase soy broth (TSB) at 37°C. Next, the overnight growth was diluted in a ratio of 1:40 in TSB. Two hundred microliters of cell suspension was inoculated to sterile 96-well polystyrene microtiter plates in the presence or absence of NETs in a 1/10 concentration, isolated as described above. After 24 h of incubation at 37°C, the wells were gently washed three times with 200 l of phosphate-buffered saline (PBS) and stained with 0.5% crystal violet for 15 min. The wells were rinsed again in 200 l of 95% ethanol to solubilize crystal violet. Each assay was performed in duplicate. Finally, the optical density at 550 nm (OD550) was determined using a microplate reader. OD550 values for each
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well were subtracted from those of the blank, which contained only TSB without inocula. Statistical analysis. Statistical analyses were performed using one-way analysis of variance (ANOVA) (Scheffé test in uniform sample size and least significant difference [LSD] test in nonuniform sample size for post hoc comparisons). All statistical analyses were performed with OriginPro 8. P values less than 0.05 were considered significant.
RESULTS
Macrolide antibiotics induce generation of NETs. Considering that macrolide antibiotics can act as immunomodulatory agents (1, 2), we sought to investigate whether this effect can be mediated through neutrophils and, more specifically, through the generation of NETs. We initially studied the ability of different antibiotic groups to generate NETs. In vitro stimulation of PMNs from healthy individuals with clinically relevant concentrations of commonly used classes of antibiotics has shown that macrolide antibiotics (azithromycin and clarithromycin) induced NET formation, whereas ampicillin (semisynthetic penicillin), penicillin, daptomycin, and ciprofloxacin did not, as observed by immunofluorescence (Fig. 1A and B) and MPO-DNA complex ELISA (Fig. 1C). We next investigated the ability of clarithromycin to induce NETs in vivo. To assess this, patients suffering from H. pyloripositive gastritis were enrolled as a study group, since clarithro-
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FIG 2 Clarithromycin induces NET generation in vivo in patients with H. pylori gastritis. (A) NET generation in ex vivo PMNs from patients with H. pylori-positive gastritis before and after treatment with clarithromycin, amoxicillin, and omeprazole and from control individuals, as assessed by immunofluorescence (confocal microscopy). Green, MPO; red, NE; blue, DAPI-DNA. Results of one representative out of six independent experiments are shown. Original magnification, ⫻600. Scale bar, 5 m. (B and C) Percentages of NET-releasing PMNs (B) and MPO-DNA complex (in isolated NET structures from ex vivo PMNs (C) isolated from patients with H. pylori-positive gastritis before and after standard eradication treatment with clarithromycin, amoxicillin, and omeprazole (n ⫽ 6), patients with sequential eradication (amoxicillin and omeprazole) (n ⫽ 4), patients with H. pylori-negative gastritis (omeprazole monotherapy) (n ⫽ 5), and control individuals (n ⫽ 10). Data are presented as medians ⫾ 90th percentile (*, P ⬍ 0.05; n.s., not significant compared to control value).
mycin is used for standard eradication therapy and these patients do not demonstrate clinical and laboratory evidence of systemic inflammation (23) that could potentially influence in vivo NET formation. Thus, PMNs were isolated before and during the 5th day of standard eradication therapy with clarithromycin, amoxicillin, and omeprazole. The collected PMNs during the 5th day of eradication therapy demonstrated increased ex vivo NET formation compared to that of PMNs before clarithromycin initiation, PMNs from healthy individuals, or PMNs from patients under sequential eradication treatment, which comprises only amoxicillin plus omeprazole for the first 5 days of eradication (details in Table 1), as determined by immunofluorescence (Fig. 2A and B) and MPO-DNA complex ELISA (Fig. 2C). Patients with H. pylori-negative gastritis that were treated with omeprazole alone did not demonstrate any ex vivo NET generation (Fig. 2). These findings demonstrate the ability of macrolides to induce NET formation. Clarithromycin utilizes autophagy to induce NET generation. Several lines of evidence support the involvement of autophagy in neutrophil functions, including NET release (18, 24, 25). Thus, we assessed endogenous LC3B cellular distribution and p62/SQSTM1 degradation in PMNs from control subjects treated with clarithromycin. Formation of LC3B puncta and increased p62/SQSTM1 degradation were observed in PMNs treated with
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clarithromycin compared to controls, as determined by immunofluorescence and immunoblotting, respectively, suggesting the induction of the autophagic machinery (Fig. 3A and B). In addition, PMNs pretreated with bafilomycin A1 prior to stimulation with clarithromycin demonstrated reduced NET generation compared to that of PMNs treated with clarithromycin alone (Fig. 3C), further verifying the involvement of autophagy in clarithromycin induction of NET formation. These findings demonstrate that clarithromycin induces NET formation in an autophagy-dependent manner. NETs generated by clarithromycin are decorated with LL-37. Considering that LL-37 is a potent antimicrobial peptide (26, 27) and that it is released through NETs under certain inflammatory conditions (28, 29), we examined the presence of LL-37 on clarithromycin-induced NETs. NETs released by PMNs from control subjects treated with clarithromycin were decorated with LL-37, as determined by immunofluorescence and immunoblotting (Fig. 4). We then assessed LL-37 expression on NETs released from PMNs derived from patients treated with clarithromycin. Indeed, PMNs derived from these patients demonstrated the presence of LL-37 on NETs (Fig. 4A). Furthermore, to assess whether the presence of LL-37 is a generic finding of NETs, we investigated its presence on NETs using
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FIG 3 Clarithromycin-induced NET generation in PMNs by utilizing autophagy. Autophagy induction was determined with LC3B staining as assessed by confocal microscopy (A) or p62/SQSTM1 immunoblotting in control PMNs treated with clarithromycin (B). Red, LC3B; blue, DAPI-DNA. (C) NET generation in control PMNs treated with clarithromycin in the presence or absence of bafilomycin A1 (autophagy inhibitor), as assessed by immunofluorescence (confocal microscopy). Green, MPO; red, NE; blue, DAPI-DNA. (D) MPO-DNA complex in isolated NET structures from control PMNs treated with clarithromycin in the presence or absence of autophagy inhibitor. In panels A to C, results for one representative out of four independent experiments are shown. Original magnifications: ⫻1,000 (A) and ⫻600 (C). Scale bar, 5 m. In panel D, data from four independent experiments are presented as means ⫾ SDs (*, P ⬍ 0.05; n.s., not significant).
other alternative inducers of NETosis, such as MSU and PMA. LL-37 was absent from NETs induced by MSU (Fig. 4), as assessed by immunofluorescence and immunoblotting of NET proteins. Similar to MSU, PMA is not able to induce LL-37-bearing NETs (Fig. 4A). These findings suggest that clarithromycin has the ability to induce NETs containing LL-37.
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Clarithromycin-dependent LL-37 expression on NETs modulates antimicrobial activity against Acinetobacter baumannii. Since neutrophils during infection produce NETs as a defense mechanism against pathogens (7, 8), we investigated if clarithromycin-driven NETs conserve this ability. To test the microbicidal activity of clarithromycin-driven NETs, our model was based on A. baumannii, since this microorganism is not able to trigger the
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FIG 4 LL-37 is present on NETs induced by clarithromycin. (A) Localization of LL-37 on NETs in in vitro control PMNs stimulations with clarithromycin or ex vivo patients treated with clarithromycin, amoxicillin, and omeprazole, as observed by confocal microscopy. MSU and PMA were used as generic NET inducers. Green, LL-37; red, NE; blue, DAPI-DNA. Original magnification, ⫻600. Scale bar, 5 m. (B) Expression of LL-37 on NET proteins derived from in vitro stimulations of control PMNs with clarithromycin or MSU, as assessed by immunoblotting. Each panel shows results of one representative out of four independent experiments.
mechanism of NET by itself in vitro (11) or ex vivo (see Fig. S1 in the supplemental material) and is also resistant to treatment with clarithromycin (30). Clinical and ATCC 19606 strains of A. baumannii were cultured in the presence or absence of clarithromycin-induced NETs. NETs were obtained either by in vitro stimulation or ex vivo from H. pylori-positive patients treated with clarithromycin. We observed that NETs induced by clarithromycin both in vitro (Fig. 5A) and ex vivo (Fig. 5B) significantly reduced bacterial growth compared to that in control cultures. This activity was attributed to the presence of LL-37, since LL-37 neutralization with anti-LL-37 antibody abolished the antimicrobial action of clarithromycin-induced NETs (Fig. 5A and B). To further verify the specific activity of clarithromycin-driven NETs, we used NETs obtained after stimulation from inducers that are not able to generate LL-37-bearing NETs, such as MSU or PMA. These generic NETs, in contrast to clarithromycin-induced NETs, indicated an absence of LL-37 (Fig. 4A and B) and significantly reduced capacity to inhibit bacterial growth (Fig. 5A). We further sought to investigate the effect of clarithromycininduced NETs on the generation of biofilm by A. baumannii. Interestingly, clarithromycin-induced NETs significantly reduced
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the formation of biofilms in an LL-37-dependent manner, as demonstrated by LL-37 inhibition (Fig. 5C). However, generic NETs induced by MSU crystals or PMA did not affect biofilm generation by A. baumannii (Fig. 5C). Treatment of NET structures with DNase attenuated the inhibitory effect on biofilms (Fig. 5C). Since the NET scaffold affects the functionality of various NET-bound components (7, 19), we wanted to test whether LL-37 functionality is affected by NET integrity. DNase treatment of clarithromycin-induced NETs abolished LL-37 antimicrobial function on A. baumannii cultures (Fig. 5D). These findings suggest an indirect antimicrobial role of clarithromycin against strains of A. baumannii through NET formation in an LL-37-dependent manner. DISCUSSION
The present study demonstrates for the first time the in vitro and in vivo ability of macrolide antibiotics to induce NET formation, expanding our understanding of their immunomodulatory role. We show that clarithromycin-induced NETs are decorated by the antimicrobial peptide LL-37, which is in its active form and is able to act against multidrug-resistant A. baumannii in vitro.
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FIG 5 In vitro- and ex vivo-generated clarithromycin-induced NETs act against Acinetobacter baumannii in an LL-37-dependent manner. (A) Cultures of clinical and ATCC 19606 strains of A. baumannii in the presence of clarithromycin-induced NETs generated in vitro before and after pretreatment with anti-LL-37 antibody. MSU and PMA were used as controls (LL-37 negative NETs). (B) Cultures of clinical and ATCC 19606 strains of A. baumannii in the presence of NETs derived from H. pylori-positive patients treated with clarithromycin, amoxicillin, and omeprazole, before and after pretreatment with anti-LL-37 antibody. Data from six independent experiments are presented as means ⫾ SDs (*, P ⬍ 0.05; n.s., not significant). (C) Determination of biofilm generation in clinical and ATCC 19606 strains of A. baumannii in the presence of clarithromycin-induced NETs generated in vitro before and after pretreatment with anti-LL-37 antibody or DNase I, as assessed by crystal violet staining. MSU and PMA were used as controls (LL-37 negative NETs). (D) Cultures of clinical and ATCC 19606 strains of A. baumannii in the presence of clarithromycin-induced NETs generated in vitro before and after pretreatment with DNase I. In panels A to C and panel D, data from 6 and 4 independent experiments, respectively, are presented as means ⫾ SDs (*, P ⬍ 0.05; n.s., not significant).
Clarithromycin-induced NETs exerted their antimicrobial function against both clinical and ATCC 19606 strains of multidrug-resistant A. baumannii, a pathogen that is not able to trigger the mechanism of NET by itself. However, specific NETs (such as clarithromycin-driven NETs) restrict A. baumannii in cultures. Additionally, clarithromycin-induced NETs were able to inhibit A. baumannii biofilm formation. This is an interesting finding and gives more information on the ongoing debate on the cross talk of biofilms and NETs, since neutrophils constitute indispensable biofilm controllers but can also contribute to biofilm stability (31). These findings suggest an indirect antimicrobial action of macrolide antibiotics, through the induction of NET generation. As a side note, we also provide evidence that A. baumannii does not induce NET generation in vivo, as observed with ex vivo-isolated PMNs from patients with A. baumannii bacteremia. Since neutrophils have been shown to express LL-37 under
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certain inflammatory conditions (28, 29), we investigated the presence of this antimicrobial peptide on clarithromycin-induced NETs. We demonstrated for the first time that clarithromycin induces both in vitro and ex vivo NETs decorated with LL-37, while other generic inducers of NETs, such as MSU or PMA, were not able to generate amounts of active LL-37-bearing NETs. Recent studies showed that PMA-induced NETs did not express active LL-37. Although traces of LL-37 have been detected mainly by electron microscopy (32), the absence of LL-37 activity is in accordance with our findings. Furthermore, considering the susceptibility of A. baumannii to LL-37 (27), we further demonstrate that LL-37 mediated the microbicidal activity of NETs and attenuated the formation of biofilms by this pathogen. Additionally, digestion of the chromatin scaffold with DNase abolished completely the bactericidal activity of clarithromycin-induced NETs, similarly to anti-LL37 inhibition. This indicates the importance of the
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NET chromatin scaffold in LL-37 functionality, previously shown also with other NET-bound proteins (7, 19). Moreover, since autophagy plays an important role in NET generation, we investigated whether clarithromycin regulates this active mechanism to generate NETs. Indeed, clarithromycin was involved in the in vitro upregulation of autophagy leading to generation of NETs, as demonstrated by the end-stage autophagy inhibition with bafilomycin A1. These findings suggest that clarithromycin can act additionally as an autophagy inducer in PMNs, thus expanding its role by regulating autophagy, a mechanism necessary for NET formation. A previous clinical trial has demonstrated that administration of clarithromycin in patients with VAP accelerated the resolution of pneumonia and decreased the risk of death from septic shock, suggesting a potential immunomodulatory effect of clarithromycin on the host (4). It should be noted that most of the patients (54.5%) were suffering from A. baumannii infection. Besides the limitations related to the design of the study, including the short period of clarithromycin administration (only 3 days), these results support our in vitro findings and encourage the investigation of the in vivo synergistic role of macrolide-induced NETs in severe infections, such as those caused by multidrug-resistant species of A. baumannii. Additionally, whether NETs are involved in the adjunctive potential of clarithromycin when administered in combination with other antibiotics is a query that needs to be investigated in future studies. In conclusion, this is the first report suggesting the formation of LL-37-bearing NETs by clarithromycin as a part of its immunomodulatory role. The role of clarithromycin in inducing NETs is dependent on autophagy, and the LL-37 present in NETs is important for both microbicidal activity and biofilm inhibition. Of note, the functionality of LL-37 is highly dependent on NET chromatin scaffold integrity. Our study supports the use of clarithromycin as a synergic agent in clinical trials with patients with A. baumannii infection in order to determine whether the observed in vitro microbicidal function through NETs is repeated in vivo. ACKNOWLEDGMENT We declare no conflict of interest.
FUNDING INFORMATION This study was supported by the Scientific Committee of Democritus University of Thrace, grant number 80895.
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