Azithromycin and Chloramphenicol Diminish Neutrophil Extracellular Traps (Nets) Release

International Journal of Molecular Sciences

Article Azithromycin and Chloramphenicol Diminish Neutrophil Extracellular Traps (NETs) Release

Weronika Bystrzycka 1,2,3, Aneta Manda-Handzlik 1,3,*, Sandra Sieczkowska 2, Aneta Moskalik 2, Urszula Demkow 1 and Olga Ciepiela 1,* ID

1 Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, 02-091 Warsaw, Poland ; [email protected] (W.B.); [email protected] (U.D.) 2 Student’s Scientiﬁc Group at Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, 02-091 Warsaw, Poland; [email protected] (S.S.); [email protected] (A.M.) 3 Postgraduate School of Molecular Medicine, Medical University of Warsaw, 02-091 Warsaw, Poland * Correspondence: [email protected] (A.M.-H.); [email protected] (O.C.); Tel.: +48-2231-79503 (A.M.-H.); +48-2231-79503 (O.C.)

Received: 27 October 2017; Accepted: 5 December 2017; Published: 8 December 2017

Abstract: Neutrophils are one of the first cells to arrive at the site of infection, where they apply several strategies to kill pathogens: degranulation, respiratory burst, phagocytosis, and release of neutrophil extracellular traps (NETs). Antibiotics have an immunomodulating effect, and they can influence the properties of numerous immune cells, including neutrophils. The aim of this study was to investigate the effects of azithromycin and chloramphenicol on degranulation, apoptosis, respiratory burst, and the release of NETs by neutrophils. Neutrophils were isolated from healthy donors by density-gradient centrifugation method and incubated for 1 h with the studied antibiotics at different concentrations (0.5, 10 and 50 µg/mL—azithromycin and 10 and 50 µg/mL—chloramphenicol). Next, NET release was induced by a 3 h incubation with 100 nM phorbol 12-myristate 13-acetate (PMA). Amount of extracellular DNA was quantified by fluorometry, and NETs were visualized by immunofluorescent microscopy. Degranulation, apoptosis and respiratory burst were assessed by flow cytometry. We found that pretreatment of neutrophils with azithromycin and chloramphenicol decreases the release of NETs. Moreover, azithromycin showed a concentration-dependent effect on respiratory burst in neutrophils. Chloramphenicol did not affect degranulation, apoptosis nor respiratory burst. It can be concluded that antibiotics modulate the ability of neutrophils to release NETs influencing human innate immunity.

Keywords: azithromycin; chloramphenicol; neutrophil extracellular traps

1. Introduction Neutrophils are the most abundant immune cells in human peripheral blood, and play a crucial role in the innate immune response by defending the body against pathogens. They are major antimicrobial effector cells designed to kill microbes by several strategies: degranulation, phagocytosis, generation of reactive oxygen species (ROS), and by the—recently discovered—release of neutrophil extracellular traps (NETs) in a process called NETosis. NETs are fragile ﬁbers of decondensed chromatin decorated with antimicrobial proteins and histones [1]. These web-like structures are released from the cell to form a physical barrier for the pathogens that limits their spread throughout the organism, and to generate a high local concentration of antimicrobial factors. Despite their beneﬁcial role, NETs have been also reported to contribute to the development of several diseases, including rheumatoid arthritis, diabetes, and thrombosis [2]. Therefore, a substantial effort is being made to identify agents modulating

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antimicrobial factors. Despite their beneficial role, NETs have been also reported to contribute to the development of several diseases, including rheumatoid arthritis, diabetes, and thrombosis [2]. Therefore, a substantial effort is being made to identify agents modulating the function of Int. J. Mol. Sci. 2017, 18, 2666 2 of 9 neutrophils, as this may help develop new therapeutic strategies for the treatment of patients suffering from NET-related diseases. the functionIt is proposed of neutrophils, that antibiotics as this may may help act develop as agents new therapeuticaffecting NET strategies release. for The the treatmentinfluence ofof patientsantibiotics suffering on the fromimmune NET-related system has diseases. been an object of investigation for nearly 70 years [3]. There are manyIt is proposed reports thatregarding antibiotics their may immunomodulati act as agents affectingng effect NET on release. granulocytic The influence and oflymphocytic antibiotics onfunctions the immune [4–6]. Hoeben system haset al. been have an demonstrated object of investigation that antibiotics for nearly affect 70the years respiratory [3]. There burst are activity many reportsand phagocytosis regarding their of immunomodulatingneutrophils [7]. Moreover, effect on Lai granulocytic et al. report and lymphocytic that azithromycin-loaded functions [4–6]. Hoebenneutrophils et al. are have more demonstrated effective in that bacterial antibiotics killing affect by thephagocytosis respiratory burst[8]. One activity of the and first phagocytosis groups to ofdescribe neutrophils the impact [7]. Moreover, of antibiot Lai etics al. on report the thatrelease azithromycin-loaded of NETs was Jerjomiceva neutrophils et are al. more, who effective observed in bacterialenhanced killing NETosis by phagocytosisin bovine granulocytes [8]. One of pretreated the first groups with toenrofloxacin describe the [9]. impact To date, of antibiotics there have on been the releasefew studies of NETs regarding was Jerjomiceva the impact et al.,of antibiotics who observed on NET enhanced release; NETosis thus, inwe bovine decided granulocytes to widen pretreatedknowledge withabout enrofloxacin this topic. There [9]. To are date, findings there concerning have been the few link studies between regarding antibiotic the impacttreatment of antibioticsand NETosis; on NEThowever, release; these thus, predominantly we decided to describe widen knowledge the induction about of thisNET topic. release There by the are addition findings concerningof bacteria theto the link cell between containing antibiotic the treatment antibiotic and [10,11]. NETosis; The however,aim of this these study predominantly was to investigate describe thechanges induction in the of functions NET release of neutrophils by the addition (NETosis, of bacteria degranulation, to the cell containingoxidative burst) the antibiotic caused [by10 ,11the]. Thepresence aim of of this antibiotics study was in tothe investigate cell medium. changes in the functions of neutrophils (NETosis, degranulation, oxidative burst) caused by the presence of antibiotics in the cell medium. 2. Results 2. Results 2.1. Degranulation 2.1. Degranulation Phorbol 12-myristate 13-acetate (PMA) was used as an inducer of degranulation during analysisPhorbol of the 12-myristate morphological 13-acetate complexity (PMA) of was neutro usedphils as an in inducer the side of degranulationscatter channel during (SSC) analysisby flow ofcytometry. the morphological We found complexitythat none of of the neutrophils studied antibiotics in the side alone scatter caused channel cell degranulation (SSC) by flow cytometry.(Figure 1). WeInstead, found we that found none that ofthe azithromycin studied antibiotics at doses alone of 10 caused and 50 cell μg/mL degranulation prevented (Figure cell degranulation1). Instead, weupon found PMA that treatment. azithromycin at doses of 10 and 50 µg/mL prevented cell degranulation upon PMA treatment.

Figure 1. Effect of azithromycin and chloramphenicol on neutrophil degranulation assessed by Figure 1. Effect of azithromycin and chloramphenicol on neutrophil degranulation assessed by measuring the granularity degree of neutrophils in the side scatter channel (SSC). 100 nM phorbol measuring the granularity degree of neutrophils in the side scatter channel (SSC). 100 nM phorbol 12-myristate 13-acetate (PMA) was used as a positive control of degranulation (n ≥ 3) (* p ≤ 0.05 vs. 12-myristate 13-acetate (PMA) was used as a positive control of degranulation (n ≥ 3) (* p ≤ 0.05 vs. unstimulated (Un) cells). unstimulated (Un) cells). 2.2. Apoptosis 2.2. Apoptosis Incubation of granulocytes with antibiotics did not induce cells apoptosis, although a slight, non-signiﬁcantIncubation increaseof granulocytes in the numberwith antibiotics of annexin did Vnot positive induce neutrophilscells apoptosis, compared although to a control, slight, untreatednon-significant cells wasincrease observed in the after number the incubation of annexi ofn the V cellspositive with 50neutrophilsµg/mL azithromycin compared to (Figure control,2). untreated cells was observed after the incubation of the cells with 50 μg/mL azithromycin (Figure 2).

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Figure 2. Effect of azithromycin and chloramphenicol on apoptosis assessed by annexin V-FITC and Figure 2. Effect of azithromycin and chloramphenicol on apoptosis assessed by annexin V-FITC and propodiumFigure 2. Effect iodide of azithromycinbinding; paraformaldehyde and chloramphenicol (PFA) constitutedon apoptosis positive assessed control by annexin (n = 6) V-FITC (** p ≤ 0.01and propodium iodide binding; paraformaldehyde (PFA) constituted positive control (n = 6) (** p ≤ 0.01 vs. vspropodium. unstimulated iodide cells binding; (Un)). paraformaldehyde (PFA) constituted positive control (n = 6) (** p ≤ 0.01 unstimulatedvs. unstimulated cells cells (Un)). (Un)). 2.3. Oxidative Burst 2.3. Oxidative Burst None ofof the the studied studied antibiotics antibiotics alone affectedalone affected oxidative oxidative burst in neutrophils. burst in However,neutrophils. pretreatment However, of None of the studied antibiotics alone affected oxidative burst in neutrophils. However, cellspretreatment with 50 µ g/mLof cells azithromycin with 50 μg/mL signiﬁcantly azithromycin inhibited significantly ROS production inhibited after ROS stimulation production with PMAafter pretreatment of cells with 50 μg/mL azithromycin significantly inhibited ROS production after (stimulationp ≤ 0.05) (Figure with 3PMA). (p ≤ 0.05) (Figure 3). stimulation with PMA (p ≤ 0.05) (Figure 3).

Figure 3. Effect of azithromycin and chloramphenicol on respiratory burst (n = 6), $$ p ≤ 0.01 vs. Figure 3. Effect of azithromycin and chloramphenicol on respiratory burst (n = 6), $$ p ≤ 0.01 vs. unstimulated cells (Un), * p ≤ 0.05 vs. phorbol 12-myristate 13-acetate (PMA). unstimulatedFigure 3. Effect cells of (Un),azithromycin * p ≤ 0.05 and vs. chloramphenicolphorbol 12-myristate on respiratory 13-acetate (PMA).burst ( n = 6), $$ p ≤ 0.01 vs. 2.4. NETosisunstimulated cells (Un), * p ≤ 0.05 vs. phorbol 12-myristate 13-acetate (PMA). 2.4. NETosis 2.4. NETosisOur studies revealed that pretreatment of the cells with 10 µg/mL chloramphenicol alone led to reducedOur studies spontaneous revealed release that pretreatment of NETs by cells of the (p ≤cells0.01). with Moreover, 10 μg/mL we chloramphenicol found that incubation alone led of Our studies revealed that pretreatment of the cells with 10 μg/mL chloramphenicol alone led theto reduced cells with spontaneous 10 µg/mL azithromycinrelease of NETs and by 10 cellsµg/mL (p ≤chloramphenicol 0.01). Moreover, causedwe found a signiﬁcant that incubation decrease of to reduced spontaneous release of NETs by cells (p ≤ 0.01). Moreover, we found that incubation of inthe NET cells release with 10 after μg/mL stimulation azithromycin with and PMA 10 ( pμ≤g/mL0.05 chloramphenicol for 10 µg/mL azithromycin caused a significant and p ≤ decrease0.01 for the cells with 10 μg/mL azithromycin and 10 μg/mL chloramphenicol caused a significant decrease 10in µNETg/mL release chloramphenicol after stimulation (Figure with4). FluorescentPMA (p ≤ 0.05 microscopy for 10 μg/mL conﬁrmed azithromycin the above and results p ≤ 0.01 (Figure for5 10). μing/mL NET chloramphenicol release after stimulation (Figure 4).with Fluorescent PMA (p ≤ micr 0.05oscopy for 10 confirmedμg/mL azithromycin the above resultsand p ≤(Figure 0.01 for 5). 10 μg/mL chloramphenicol (Figure 4). Fluorescent microscopy confirmed the above results (Figure 5).

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Figure 4. Effect of azithromycin and chloramphenicol on NET release. 100 nM phorbol 12-myristate FigureFigure13-acetate 4. Effect (PMA) of azithromycin was added to and stimulate chloramphenicol the releas eon of NET NETs, release. neutrophils 100 nM treated phorbol with 12-myristate antibiotics 13-acetatewithout stimulation (PMA) was was served added added as to to the stimulate negative the the control releas release (en of= of 6). NETs, neutrophils treated with antibiotics without stimulation served as the negative control (n = 6).

Azithromycin Azithromycin Azithromycin Chloramphenicol Chloramphenicol Control Azithromycin Azithromycin Azithromycin Chloramphenicol Chloramphenicol 0.5 μg/mL 10 μg/mL 50 μg/mL 10 μg/mL 50 μg/mL Control 0.5 μg/mL 10 μg/mL 50 μg/mL 10 μg/mL 50 μg/mL

FigureFigure 5. 5.Visualization Visualization of theof releasethe release of NETs of performedNETs performed by fluorescence by fluorescence microscopy microscopy after incubation after Figurewithincubation azithromycin 5. Visualization with azithromycin and chloramphenicol, of the and release chloramphenicol, withof NETs or without performed with stimulationor without by fluorescence stimulation with 100 nM withmicroscopy PMA. 100 The nM green afterPMA. incubationcolorThe green represents with color azithromycin MPO, represents red DNA MPO, and (n chloramphenicol,red= 6). DNA (n = 6). with or without stimulation with 100 nM PMA. The green color represents MPO, red DNA (n = 6). 3. Discussion 3. Discussion 3. DiscussionIn this study, we were able to show that azithromycin and chloramphenicol can modify the In this study, we were able to show that azithromycin and chloramphenicol can modify the functions of neutrophils. Although these antibiotics at the tested concentrations do not affect neutrophil functionsIn this ofstudy, neutrophils. we were Althouable togh show these that antibiotics azithromycin at the and tested chloramphenicol concentrations can do modify not affect the degranulation and apoptosis, they may inhibit NETosis and affect production of ROS. functionsneutrophil of degranulation neutrophils. Althouand apoptosis,gh these they antibiotics may inhibit at theNETosis tested and concentrations affect production do notof ROS. affect Azithromycin belongs to the group of macrolides, which are able to accumulate in tissues, neutrophilAzithromycin degranulation belongs and to apoptosis, the group they of maymacrolides, inhibit NETosis which are and able affect to production accumulate of in ROS. tissues, most importantly in white blood cells [12]. Therefore, intracellular activity of macrolides against mostAzithromycin importantly belongsin white to blood the groupcells [12]. of macrolides, Therefore, intracellularwhich are able activity to accumulate of macrolides in tissues, against pathogens is much stronger than that of other antibiotics [13], as they can be easily transported to mostpathogens importantly is much in strongerwhite blood than cellsthat [12].of other Therefore, antibiotics intracellular [13], as they activity can beof easilymacrolides transported against to the site of infection [14]. In our study, azithromycin exerted a concentration-dependent effect on ROS pathogensthe site of is infection much stronger [14]. In than our thatstudy, of otherazithrom antiycinbiotics exerted [13], asa concentration-dependentthey can be easily transported effect toon production, and degranulation by PMA-stimulated neutrophils. Although this effect was significant theROS site production, of infection and [14]. degranulation In our study, byazithrom PMA-stimulatedycin exerted neutrophils. a concentration-dependent Although this effecteffect wason only at concentrations of 50 µg/mL for ROS release, we observed that increasing concentrations of ROSsignificant production, only andat concentrations degranulation of by 50 PMA-stimulated μg/mL for ROS neutrophils. release, we Although observed thisthat effect increasing was azithromycin tended to gradually decrease the ability of neutrophils to produce ROS. Regarding significantconcentrations only ofat azithromycinconcentrations tended of 50 to μ graduallyg/mL for decreaseROS release, the ability we observed of neutrophils that increasingto produce degranulation, azithromycin at high concentrations (10 and 50 µg/mL) did cause less effective concentrationsROS. Regarding of azithromycindegranulation, tended azithromycin to gradually at high decrease concentrations the ability (10 of and neutrophils 50 μg/mL) to didproduce cause degranulation of neutrophils after stimulation with PMA. Overall, this could suggest an inhibiting ROSless. effectiveRegarding degranulation degranulation, of neutrophilsazithromycin after at highstimulation concentrations with PMA. (10 Overall,and 50 μ thisg/mL) could did suggest cause or stabilizing effect of azithromycin on stimulated neutrophils. What is more, azithromycin had lessan effectiveinhibiting degranulation or stabilizing of neutrophilseffect of azithromycin after stimulation on stimulated with PMA. neutrophils.Overall, this Whatcould suggestis more, an inhibitory effect on NET release, with a significant decrease at a concentration of 10 µg/mL anazithromycin inhibiting orhad stabilizing an inhibitory effect effect of onazithromycin NET release, on with stimulated a significant neutrophils. decrease atWhat a concentration is more, azithromycin vs. PMA only. Surprisingly, this effect was no longer observed at a concentration azithromycinof 10 μg/mL had azithromycin an inhibitory vs. effect PMA on only. NET Surprisingly, release, with thisa significant effect was decrease no longer at a concentrationobserved at a of 50 µg/mL. As PMA is a ROS-dependent inducer of NETosis [15], and azithromycin showed an ofconcentration 10 μg/mL azithromycin of 50 μg/mL. vs.As PMA isonly. a ROS-dependent Surprisingly, induthis cereffect of NETosiswas no longer[15], and observed azithromycin at a inhibitory effect on ROS production, we hypothesize that azithromycin at least partially influences the concentrationshowed an inhibitory of 50 μg/mL. effect As on PMA ROS is production, a ROS-dependent we hypothesize inducer of that NETosis azithromycin [15], and at azithromycin least partially release of NETs by affecting ROS production. The fact that the highest concentration of azithromycin showedinfluences an inhibitorythe release effect of NETs on ROS by affecting production, ROS we pr oduction.hypothesize The that fact azithromycin that the highest at least concentration partially does not significantly inhibit NETosis despite a strong decrease in ROS production may be surprising. influencesof azithromycin the release does of not NETs significantly by affecting inhibit ROS NE prTosisoduction. despite The a factstrong that decrease the highest in ROS concentration production However, one should be aware that NET release is, without a doubt, a complex process, involving a ofmay azithromycin be surprising. does However, not significantly one should inhibit be NEawareTosis that despite NET releasea strong is, decrease without in a ROSdoubt, production a complex mayprocess, be surprising. involving However, a number oneof molecular should be events, aware nothatt only NET ROS release production. is, without Therefore, a doubt, ita cannotcomplex be process, involving a number of molecular events, not only ROS production. Therefore, it cannot be

Int. J. Mol. Sci. 2017, 18, 2666 5 of 9 number of molecular events, not only ROS production. Therefore, it cannot be excluded that at the highest concentration of azithromycin, other processes that promote NETosis may be upregulated. Chloramphenicol was introduced for use in 1949; but after only 48 years, it was banned from use, as it had been reported to cause, among other things, aplastic anaemia. Nowadays, it is mostly used as a drug of last resort, but is still a first line drug for bacterial conjuctivitis [16]. To our knowledge, the impact of chloramphenicol on the process of NETosis has not been investigated, yet. In our study, chloramphenicol at a concentration of 10 µg/mL decreased the release of NETs after incubation with PMA. Moreover, pretreatment of cells with 10 µg/mL chloramphenicol reduced the number of cells undergoing spontaneous NETosis in unstimulated samples. Hoeben et al. reported that chloramphenicol may reduce myeloperoxidase (MPO) activity in bovine granulocytes [7]. Considering the fact that, during NETosis, chromatin decondensation occurs due to the synergistic work of MPO and neutrophil elastase [17], it seems feasible that chloramphenicol inhibits NETosis via the inhibition of MPO. Antibiotics, aside from their antimicrobial properties, can induce an immunomodulating effect on cells [18–21], which varies depending on the antibiotic used, its concentration, and the target cells. In our previous studies, we have already demonstrated the impact of clindamycin, amoxicillin, gentamicine and cefotaxime on the release of NETs [22,23]. We found that amoxicillin induces NET release, and that gentamicine inhibits NETosis; meanwhile, we found that cefotaxime and clindamycin have no effect of NET release. Based on our current investigation, it can be concluded that both azithromycin and chloramphenicol may influence innate immunity by reducing the ability of neutrophils to release NETs. Overall, as the effects of different antibiotics on neutrophils are quite distinct, it cannot be excluded that the effect of a given drug may depend on its chemical composition and mechanism of action. When interpreting data of our study, one should bear in mind the limitation that we analyzed the influence of antibiotics solely on the basis of NET formation induced with an artificial stimulus, PMA. Even though PMA is considered to mimic signals derived from microbes or immune products [24], the availability of data using physiological stimuli, e.g., lipopolysaccharide, would further support our conclusions, and it would be beneficial to perform such studies in the future. What is more, we would like to point out that the influence of antibiotics on neutrophils in vivo may be influenced by extracellular milieu, such as the changes in local pH during infection or availability of plasma proteins [25,26], whilst our studies were performed in protein-free medium, and the pH was stabilized with HEPES buffer. NETs play an important role in the innate immune system, as they form a physical barrier for the pathogens that limits their spread throughout the organism, and generate a high local concentration of antimicrobial proteins, increasing their efficacy [1]. Disorders in NET formation have been shown to cause increased susceptibility to opportunistic infections. Despite their beneficial role, NETs have also been reported to damage cells located near the web-like structures, and contribute to the development of several diseases. As NETs constitute a considerable source of autoantigens, most often they are described as contributors to autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis [2]. Identification of agents modulating the function of neutrophils has become an important goal for the scientific community, as it may help develop new therapeutic strategies for the treatment of patients suffering from NET-related diseases. When it comes to antimicrobial therapy, the choice of an antibiotic that decreases NETosis may impair natural mechanisms of innate immunity, while at the same time diminishing the release of autoantigens stimulating autoimmune processes. It would be thus advantageous for patients suffering from autoimmune diseases if doctors, whenever possible, chose antibiotics that negatively influenced NET release. Int. J. Mol. Sci. 2017, 18, 2666 6 of 9

4. Materials and Methods

4.1. Antibiotics Azithromycin and chloramphenicol were purchased from Sigma-Aldrich and diluted in protein-free RPMI medium supplemented with 10 mM HEPES (ThermoFisher Scientiﬁc, Waltham, MA, USA) to give a ﬁnal concentration of 0.5, 10 and 50 µg/mL (azithromycin) or 10 and 50 µg/mL (chloramphenicol) in each experiment. The concentrations used in the study for chloramphenicol are the same as its therapeutic serum concentration, which is 2–50 µg/mL [27]. For azithromycin, we used concentrations similar to those achieved in white blood cells during therapy (0–40 µg/mL; serum concentrations: up to 0.4 µg/mL) [28].

4.2. Neutrophil Isolation and Preparation Peripheral venous blood was collected from healthy donors into citrate tubes. All experiments were approved by Ethics Committee at Medical University of Warsaw. Written informed consent was obtained from each volunteer. Neutrophils were isolated by the density-gradient centrifugation method, as described previously [22]. In all the performed experiments, neutrophils were preincubated with the studied antibiotics for ◦ 1 h at 37 C, 5% CO2.

4.3. Degranulation Degranulation was analyzed using ﬂow cytometry [9] (Cytomics FC500 Beckman Coulter, Beckman Coulter Inc., Brea, CA, USA) by assessing the granularity degree of neutrophils in the side scatter channel (SSC). For this purpose, 2.5 × 105/mL neutrophils were incubated with the studied ◦ antibiotics for 1 h at 37 C, 5% CO2. Positive control with 100 nM phorbol 12-myristate 13-acetate (PMA, Merck Millipore, Burlington, MA, USA) was used, whereas granulocytes with RPMI alone constituted negative control.

4.4. Apoptosis Apoptosis was assessed with Annexin V Apoptosis Detection Kit FITC (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) by flow cytometry. After incubation with antibiotics for ◦ 5 1 h at 37 C, 5% CO2, 2.5 × 10 /mL of granulocytes were washed, centrifuged, suspended in binding buffer and double-stained with annexin V-FITC and propidium iodide according to the manufacturer’s instructions. Positive control (apoptotic cells) was obtained by adding 4% final concentration (f.c.) paraformaldehyde (PFA) to the cell suspension. Negative control contained cells resuspended in RPMI.

4.5. Respiratory Burst To analyze the respiratory burst in granulocytes, dihydrorhodamine 123 (DHR 123, (Thermo Fisher Scientific) was used as a fluorescent marker of the intracellular production of reactive oxygen species (ROS). For this purpose, 2.5 × 105/mL of neutrophils were incubated with 4 µg/mL DHR123 for ◦ ◦ 30 min in 37 C, 5% CO2. Subsequently, cells were incubated for 1 h at 37 C, 5% CO2, with the studied antibiotics. 100 nM PMA, which is an efficient stimulator of ROS production, was used as a positive control. Fluorescence intensity of rhodamine 123 formed during oxygen burst was measured at the first fluorescence channel with flow cytometry.

4.6. NET Quantification Isolated cells were seeded into 96-well black plates at density of 1 × 105 cells/well (5 × 105 cells/mL), ◦ treated with antibiotics or medium alone and left for 1 h at 37 C, 5% CO2. Then, 100 nM PMA was ◦ added to stimulate NETs formation for 3 h at 37 C, 5% CO2. Unstimulated granulocytes were used as control cells. Post stimulation, 500 mIU/mL of micrococcal nuclease (ThermoFisher Scientific) was Int. J. Mol. Sci. 2017, 18, 2666 7 of 9 added for 20 min at 37 ◦C to detach DNA from the bottom of the wells. Reaction was stopped with 5 mM EDTA, and then the plate was centrifuged at 415× g for 10 min. Then, supernatant was collected to black titer plates and 100 nM Sytox green fluorescent dye (Life Technologies, Waltham, MA, USA) was added to each well to measure the amount of extracellular DNA by fluorometry.

4.7. NET Visualization NET formation was visualized using fluorescent microscopy. Briefly, neutrophils (2.5 × 104 cells/well; 6.25 × 104 cells/well) were seeded onto 8-well Lab-Tek Chamber Slides (ThermoFisher Scientiﬁc) ◦ and incubated for 1 h at 37 C at 5% CO2 with antibiotics. Subsequently, 100 nM PMA was added to stimulate NETs release. After 3 h, samples were ﬁxed with 4% f.c. PFA and washed 3 times with PBS. Cells were permeabilized with 0.1% Triton X (Sigma-Aldrich, St. Louis, MO, USA) and again washed with PBS. Samples were stained overnight with anti-myeloperoxidase-FITC monoclonal antibody (1:500, 4 ◦C, Abcam ab11729, Cambridge, UK). DNA was then counterstained with nucleic acid dye Sytox Orange (Life Technologies, Waltham, MA, USA). NETs were visualized with a Leica DMi8 microscope (Wetzlar, Germany).

4.8. Statistical Analysis Statistical analysis was performed using GraphPad Prism v.6.0 (GraphPad Software, La Jolla, CA, USA). All values were analyzed with one-way ANOVA followed by a post-hoc test for paired data. All the results have been presented as mean ± standard error of the mean. Results were considered statistically signiﬁcant at p < 0.05.

Acknowledgments: A project was financed from the Grant for Young Scientists No. 1WW/PM11/15 funded by the Dean of the First Faculty of Medicine, Medical University of Warsaw. Grant holder: Olga Ciepiela. Author Contributions: Weronika Bystrzycka, Aneta Manda-Handzlik and Olga Ciepiela conceived and designed the experiments; Weronika Bystrzycka, Sandra Sieczkowska, Aneta Manda-Handzlik, Aneta Moskalik and Olga Ciepiela performed the experiments; Weronika Bystrzycka, Sandra Sieczkowska, Aneta Manda-Handzlik, Aneta Moskalik and Olga Ciepiela analyzed the data; Olga Ciepiela and Urszula Demkow contributed reagents/materials/analysis tools; Weronika Bystrzycka, Aneta Manda-Handzlik and Olga Ciepiela wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

ROS Reactive oxygen species NETs Neutrophil extracellular traps MPO Myeloperoxidase PMA Phorbol 12-myristate 13-acetate PFA Paraformaldehyde SSC Side scatter channel f.c. Final concentration

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