ORIGINAL ARTICLE
J Appl Biomater Funct Mater 2014 ; 12 (1): 21-26 DOI: 10.5301/jabfm.5000204
The Pseudomonas quinolone signal (PQS) stimulates chemotaxis of polymorphonuclear neutrophils Gertrud M. Hänsch1, Birgit Prior1, Gerald Brenner-Weiss2, Ursula Obst2, Joerg Overhage2 1 2
Institute of Immunology, University of Heidelberg, Heidelberg - Germany Institute of Functional Interfaces, Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen - Germany
ABSTRACT Cross-talk between bacteria and mammalian cells is increasingly recognized as an important factor, especially during chronic infections. In particular, the interaction of extracellular bacterial signaling molecules with cells of the innate immune response is of special interest. In this context, we investigated whether the Pseudomonas quinolone signal (PQS) which is a quorum sensing molecule produced by bacteria and participates in biofilm formation and virulence has any influence on polymorphonuclear neutrophils (PMN), the cells of the “first line defense” against bacterial infections. We found that PQS did not enhance the bactericidal activity of PMN and did not induce apoptosis at concentrations up to 100 µM. However, PQS stimulated chemotaxis of PMN in doses of 10-100 µM. This PQS-dependent chemotaxis could be inhibited with SB203580 which blocks MAPkinase p38, suggesting a signaling pathway similar to AHL-12 induction. Using bacterial cell culture supernantants of Pseudomonas aeruginosa wild-type cells and a PQS-deficient mutant strain support the in vivo relevance of these findings. Since PQS is produced in the early phase of biofilm formation, PMN infiltration could be timely enough to eradicate bacteria before biofilm formation is completed, which confers the bacteria with a relative resistance to host defense mechanisms. Key words: Chemotaxis, Quorum sensing molecule, Human neutrophils, Pseudomonas aeruginosa, Pseudomonas quinolone signal (PQS), Biofilm Accepted: November 12, 2013
INTRODUCTION Biofilm formation is the crucial factor in many chronic bacterial infections such as implant-associated osteomyelitis, endocarditis in patients with artificial heart valves, and vascular catheter or ventilator tube infections (1, 2). Moreover, pulmonary biofilm infections are the leading cause of morbidity and mortality in patients with cystic fibrosis due to persistent infections that eventually lead to lung damage and respiratory failure (3). Bacteria grown in biofilms are imbedded in a slimy, self-produced polymeric matrix composed of polysaccharides, nucleic acids, lipids and proteins (4-7). Once established, these sessile communities are protected from the host immune response and are difficult to eradicate due to their high inherent resistance against antibiotic agents (8-12). Bacterial cell-to-cell communication, also named quorum sensing (QS), is known to be involved in biofilm development and is mediated by a variety of different QS signaling molecules. One major group of QS molecules are the N-acylhomoserine lactones (AHL),
which are synthesized and secreted by many Gramnegative bacteria like the opportunistic human pathogen Pseudomonas aeruginosa. In P. aeruginosa, the two AHLs (3-oxododecanoyl)-L- (AHL-12) and N-(butanoyl)L-homoserine lactone (AHL-4) control the production of extracellular virulence factors and promote biofilm maturation by inducing expression of more than 300 genes (13). Moreover, it has been shown previously that AHL-12 strongly impacts mammalian cells, in particular host immune cells by inhibiting T-cell proliferation and antigen presentation or inducing apoptosis (14, 15). Recently, we and others have shown that AHL-12 also stimulates the host defense by increasing phagocytosis of bacteria by macrophages or neutrophils and induces chemotaxis of neutrophils through induction of p38 and leukocyte specific protein 1 (16-18). In addition to AHLs, P. aeruginosa uses the non-AHL signal 2-heptyl-3-hydroxy-4-quinolone (the Pseudomonas quinolone signal; PQS, Fig. 1) as an extra QS molecule. PQS is closely linked to the AHL signaling network and possesses numerous regulatory and physiological functions,
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
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PQS stimulates chemotaxis
Fig. 1 - The Pseudomonas quinolone signal (PQS).
including control of biofilm development, secondary metabolite and virulence factor production as well as iron acquisition, among others (19). Interestingly, PQS is present at sites of infection, e.g. in sputum samples of cystic fibrosis patients (20, 21), and is capable of modulating the host immune response by modulating activity in T-cell proliferation and cytokine (IL-2) release, suppressing interleukin 12 (IL-12) and tumor necrosis factor alpha (TNF-α) secretion by LPS-stimulated macrophages and repressing HIF-1α protein levels (19, 22-25). In this study we investigated the potential of PQS to activate human neutrophils and analyzed signaling pathways related to migration. We found an induction of chemotaxis and up-regulation of adherence proteins and cellular functions that enhance the local neutrophil response. MATERIAL AND METHODS Bacterials strains and materials Pseudomonas aeruginosa PA14 wild-type and the corresponding pqsA transposon insertion mutant ID23621 (mutant) were obtained from the P. aeruginosa PA14 mutant library (26). Cells of wild-type and mutant strain were grown for 20 h in 30 mL BHI medium at 37°C under shaking conditions. The culture medium was centrifuged at 5800×g for 10 min and the supernatant was subsequently sterilized by filtration and used for functional assays (see below). A FITC-labeled Escherichia coli was used in the phagocytosis assay. PQS, calcein AM and f-MLP were purchased from Sigma-Aldrich (Seelze, Germany) and SB 203580 from Calbiochem (La Jolla, USA). PQS measurement by liquid chromatography/tandem mass spectrometry (LC-MS/MS) Sterilized cell-free supernatants of bacterial overnight cultures were mixed with two volumes of dichloromethane and the lower organic layer containing PQS was transferred into a new reaction tube following evaporation under nitrogen gas at room temperature. The pellet was resuspended in pure methanol following quantification by LC-MS/MS using a Zorbax Eclipse XCB-C8 5 µm, 150×3.6 mm HPLC 22
column (Agilent, Santa Clara, CA, USA) and a mobile phase consisting of acetonitrile - water 80:20 (v/v) with 100 µM EDTA and 0.1 % acetic acid at a flow rate of 0.40 ml/min. Electron spray ionization (ESI)-MS was performed on an API 365 triple quadrupole mass spectrometer (PE Sciex, Toronto, Canada) using a turbo ion spray interface used in positive mode. Single MS experiments (Q1 scan), MS/MS experiments (product ion scan, PIC), and multiple reaction monitoring (MRM) were performed using nitrogen as curtain gas, nebulizer gas, heater gas and collision gas. Instrumental parameters were optimized by infusion experiments with PQS standard solution (10 µg/ml; Sigma-Aldrich, Seelze, Germany) infused into the mass spectrometer using a syringe pump (Harvard Apparatus, South Natick, MA, USA) at a flow rate of 10 µl/min. To quantify PQS with high selectivity and sensitivity, MRM experiments were performed using the transitions from precursor ion to fragment ion: 260/175 (quantifier), 260/146, 260/147 and 260/188 (qualifier). An external calibration was performed using PQS standard solutions with concentrations ranging from 10 ng/ml up to 1000 ng/ml. Isolation of PMN from peripheral blood Heparin blood from volunteers (mainly laboratory personnel and students) was layered on PolymorphPrep (Nycomed, Oslo, Norway) after receiving approval for the procedure from the local ethics committee. After centrifugation, the PMN fraction was harvested and washed repeatedly in phosphate-buffered saline (PBS, pH 7.4). For the analyses of chemotaxis, the cells were suspended in Hank’s balanced salt solution (HBSS) containing 0.5% bovine serum albumin (BSA), at a final concentration of 1 x 106 cells per milliliter HBSS. Because of the wide intraindividual variation of the cell response, all assays were performed with cells of at least three different donors. Determination of cell death and apoptosis Intercalation of propidium iodide into DNA was measured by cytofluorometry as described by Belloc et al (1994) (27). In parallel, binding of Annexin V FITC which binds to the membrane phospholipid phosphatidylserine (PS) in the presence of Ca2+ was determined. Isolated PMN were incubated for 30 min with PQS in doses ranging from 1 µM to 100 µM; formalydehyde (3%) was used as a “death” control. Phagocytosis A commercially available kit (Glycotope Biotechnology, Heidelberg, Germany) was used. In brief, FITC-labeled Escherichia coli were incubated with whole heparinized blood at either 4ºC or 37ºC for 15 min. Uptake into PMN of bacteria was quantified by cytofluorometry.
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
Hänsch et al
Fig. 2 - PQS does not induce apoptosis of PMN. PMN were incubated with (left panel) or without PQS (100 µM) for 30 min. Apoptosis and cell death were determined by staining with AnnexinV FITC and intercalation of propidium iodide, respectively. On the right panel, the “death control” is shown.
Production of oxygen radicals To determine the production of oxygen radicals, we used the method described by Babior et al (28). In brief, PMN (1×106) were suspended in cytochrome c solution (Sigma-Aldrich, Seelze, Germany, 1 mg/ml HBSS,) and stimulated with phorbol ester (PMA) (Sigma-Aldrich), or with PQS in the concentrations as indicated in the text. Reduction of cytochrome c was measured photometrically after 30 min and calculated as ΔOD (550 nm). Cytofluorometry To determine the expression of surface receptors on PMN, a FITC-labeled antibody to CD66b (Coulter Immunotech, Marseille, France) was used to identify PMN, and phycoerythrin (PE)-labeled antibodies to either anti-CD11b (BD Biosciences, Heidelberg, Germany) or anti-CD18 (Serotec GmbH, Düsseldorf, Germany). One hundred microliters of whole blood was incubated with anti-CD66b-FITC and the respective PE-labeled antibodies (0.1-5 µg) for comparison with mouse IgG (either PE- or FITC labelled; Beckman Coulter Marseille, France) for 20 min. Then the erythrocytes were lysed using BD Facs Lysing solution (BD Biosciences) and cells were subjected to cytofluorometry using FACSCalibur and CellQuest Pro software (Becton Dickinson, Heidelberg, Germany). Ten thousand events were counted, and expression of CD11b or CD18 on CD66b positive cells was shown as a histogram. Expression of CD11b or CD18 was quantified as mean fluorescence intensity (MFI). Chemotaxis Chemotaxis was determined using the BD Falcon HTS Fluoroblok 96-Multiwell Insert System with a polyethylene terephthalate (PET) membrane insert plate with 3 µm pore size. Isolated PMN were labeled with calcein (106 cells/ml were incubated for 15 min at 37°C with calcein (1 µg/ml), then washed three times with HBSS and seeded in a concentration of 2×105/50 µL into the insert
plate. PQS in concentrations of 1 µM, 10 µM, 100 µM, and for comparison HBSS or fMLP (10-7 M) was added to lower wells. After 2 h at 37°C the insert plate was removed and fluorescence was determined by a PerkinElmer VICTOR2™ microplate reader using Wallac Workstation software (Waltham, MA, USA). RESULTS AND DISCUSSION PQS does not induce apoptosis or bactericidal activity of PMN Polymorphonuclear neutrophils were isolated from at least three different donors and subsequently incubated with PQS to identify any toxic effect. After 30 min of incubation, PQS did not show any cytotoxicity towards PMN when used in doses up to 100 µM (Fig. 2). To answer the question whether PQS would modulate PMN functions, in a first set of experiments an impact on the phagocytosis of bacteria was studied. PMN efficiently took up opsonized, FITC-labeled E. coli demonstrating that PQS did not enhance phagocytosis when used in doses of 1 µM to 100 µM (example in Fig. 3). In this regard, PQS exhibited functional differences in comparison to the AHL-12 quorum sensing molecule, which was shown to significantly enhance phagocytosis (17, 29) and induce apoptosis (14). In a further set of experiments, the possible effect of PQS on the production of reactive oxygen species was tested. In concentrations of 1 to 100 µM, PQS did not induce reactive oxygen species and did not modulate the reactive oxygen production induced by f-MLP (107 or 108 M) (data not shown). In that respect, PQS does not directly affect the bactericidal activity of PMN. Furthermore, the effect of PQS on the constitutively expressed adhesion proteins CD11b/CD18 was analyzed. As demonstrated in the example shown in Figure 4, PQS only marginally enhanced their expression; on average an increase of 22% for CD11b and 17% for CD18 (mean
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PQS stimulates chemotaxis
Fig. 3 - PQS does not affect phagocytosis of bacteria by PMN. PMN in whole blood were incubated either with PQS (100 µM; thick line) or without (thin line) with opsonized, FITC-labeled E. coli at either 4ºC or 37ºC for 15 min. Thereafter, green fluorescence associated with the cells was determined by cytofluorometry.
of three experiments with cells of different donors) was detectable. PQS induces chemotaxis of PMN In addition, we investigated the chemotaxis of PMN in response to different PQS concentrations and observed that PQS was chemotactic for isolated PMN. In doses of 10 µM to 100 µM PQS significantly induced chemotaxis (examples and summary of data are shown in Figs. 5A and B). Compared to bona fide chemokines such as f-MLP or IL-8, the molar concentrations required for induction of chemotaxis were much higher, a phenomenon that was also observed for AHL-12, and which raises the question of the in vivo relevance. To this end, we investigated the effect of bacterial supernatants from P. aeruginosa PA14 wild-type cells as well as the corresponding PQS-deficient mutant strain ID23621 (26) which is not able to synthesize PQS due to a transposon mutation in the PQS synthesis gene pqsA. Bacteria were grown for 20 h at 37°C in BHI medium under shaking conditions and supernatants of the wild-type and mutant strain were sterilized and subsequently analyzed for PQS using LC–MS/MS. The amount of PQS in supernatants was quantified according the settings given above using the specific transitions from m/z 298 (molecule ion) to m/z 197 and m/z 102. As expected, PQS was only detectable in the wild-type supernatant (1240 ng/ml supernatant). Using these supernatants in our chemotaxis assay, we observed that supernatants of P. aeruginosa induced a chemotactic response, which was in accordance to our previous studies on AHL-12 (16). Culture supernatant of the mutant strain induced a lower response in comparison to the wild-type, which, however, could be reconstituted by PQS (Fig. 5C). 24
Fig. 4 - PQS upregulates expression of CD11b/CD18. PMN in whole blood were cultivated with (thick line) or without (thin line) PQS (100 µM) for 30 min at 37ºC. Shown is the surface expression on PMN (identified as CD66b positive cells).
These results indicate that sufficient PQS is produced by wild-type bacterial cells to induce chemotaxis of PMN. It is not the only chemokine, however, a likely candidate for an additional molecule is AHL-12. This notion is supported by our previous observation that AHL-12-deficient supernatants still induced chemotaxis of PMN, though to a lesser extent (16). PQS-induced chemotaxis was inhibited when cells were pre-incubated with SB203580, which blocks the MAPkinase p38 (Fig. 5D). This enzyme is required for the induction of cellular migration by so-called “end-point” chemokines, and suggests a signaling pathway similar to AHL-12. How PQS is recognized by PMN is not yet clear. As a lipid, it may enter the cell freely; chemotaxis, however, supposedly requires a polarization of the cells, which is accomplished by a clustering of appropriate surface receptors, a phenomenon that needs further investigation. CONCLUSIONS The results of our studies demonstrate that the Pseudomonas quinolone signal does not exhibit cytotoxic effects on PMN nor does it have an impact on apoptosis. However, we found that PQS stimulates chemotaxis of PMN, likely via a signaling pathway similar to AHL-12. Regardless of the activation mechanisms, the fact that PQS induces chemotaxis might have an implication for the local host defense. Since PQS is produced in a similar way to AHL-12, before and in the early state of biofilm formation, PMN as a first-line defense could be recruited in a phase of infection where bacteria might still be vulnerable and can be eradicated (30, 31). On the other hand, infiltration of PMN into sites of infection can also enhance the local inflammatory response, thus leading to a progression of tissue damage. A better understanding of the dynamics of host-pathogen interactions, in particular in cell signaling events is increasingly needed for the development of new
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
Hänsch et al
A
B
Fig. 5 - PQS induces chemotaxis of PMN. A. The migration of PMN toward the bona fide chemokine IL-8 and in response to PQS. On the very left, random migration is shown (data from one donor). B. The compiled data of 4 independent experiments with cells of 4 individuals (PQS 10 µM) (A, A, BB. According to the U-test, induction of chemotaxis by PQS is different from random migration). C. Supernatants of P. aeruginosa-induced chemotaxis of PMN; supernatants of the PQS mutant strain which is deficient in PQS induced less chemotaxis. Addition of PQS (10 µM) reconstituted the chemotactic activity. D. Preincubation of PMN with the MAP-kinase inhibitor SB203580 reduced the chemotaxis towards PQS (data compiled from two experiments using cells of different donors).
C
D
and more efficient antibiotic treatment options for biofilm infections.
Future” of the Karlsruhe Institute of Technology (KIT) as part of the German Excellence Initiative.
ACKNOWLEDGEMENTS
Conflict of interest: The authors declare that there are no conflicts of interest.
We thank Anke Neidig and Michael Nusser for technical assistance. Financial support: This study was in part funded by the BioInterfaces (BIF) Program of the Karlsruhe Institute of Technology (KIT) in the Helmholtz Association and by the “Concept for the
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Corresponding Author: Prof. Dr. Gertrud M. Hänsch Institute of Immunology University of Heidelberg Im Neuenheimer Feld 305 69120 Heidelberg, Germany [email protected]
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Ehrlich GD, Arciola CR. From Koch’s postulates to biofilm theory. The lesson of Bill Costerton. Int J Artif Organs. 2012; 35(10): 695-699. 7. Montanaro L, Poggi A, Visai L, et al. Extracellular DNA in biofilms. Int J Artif Organs. 2011; 34(9): 824-831. 8. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet. 2001; 358(9276): 135-138. 9. Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev. 2002; 15(2): 167-193. 10. Van Staden AD, Dicks LM. Calcium orthophosphate-based bone cements (CPCs): Applications, antibiotic release and alternatives to antibiotics. J Appl Biomater Funct Mater. 2012 Jun 26; 10(1): 2-11. 11. Baino F, Fiorilli S, Mortera R et al. Mesoporous bioactive glass as a multifunctional system for bone regeneration and controlled drug release. J Appl Biomater Funct Mater. 2012; 26; 10(1): 12-21. 12. Drenkard E, Ausubel FM. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature. 2002; 416(6882): 740-743. 13. Schuster M, Lostroh CP, Ogi T, Greenberg EP. Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J Bacteriol. 2003; 185(7): 2066-2079. 14. Cooley M, Chhabra SR, Williams P. N-Acylhomoserine lactone-mediated quorum sensing: a twist in the tail and a blow for host immunity. Chem Biol. 24 2008; 15(11): 1141-1147. 15. Ritchie AJ, Yam AO, Tanabe KM, Rice SA, Cooley MA. Modification of in vivo and in vitro T- and B-cell-mediated immune responses by the Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone. Infect Immun. 2003; 71(8): 4421-4431. 16. Kahle NA, Brenner-Weiss G, Overhage J, Obst U, Hansch GM. Bacterial quorum sensing molecule induces chemotaxis of human neutrophils via induction of p38 and leukocyte specific protein 1 (LSP1). Immunobiology. 2013; 218(2): 145-151. 17. Vikstrom E, Magnusson KE, Pivoriunas A. The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)L-homoserine lactone stimulates phagocytic activity in human macrophages through the p38 MAPK pathway. Microbes Infect. 2005; 7(15): 1512-1518. 18. Zimmermann S, Wagner C, Muller W, et al. Induction of neutrophil chemotaxis by the quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone. Infect Immun. 2006; 74(10): 5687-5692. 19. Dubern JF, Diggle SP. Quorum sensing by 2-alkyl-4-quinolones in Pseudomonas aeruginosa and other bacterial species. Mol Biosyst. 2008; 4(9): 882-888.
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20. Collier DN, Anderson L, McKnight SL, et al. A bacterial cell to cell signal in the lungs of cystic fibrosis patients. FEMS Microbiol Lett. 2002; 215(1): 41-46. 21. Guina T, Purvine SO, Yi EC, et al. Quantitative proteomic analysis indicates increased synthesis of a quinolone by Pseudomonas aeruginosa isolates from cystic fibrosis airways. Proc Natl Acad Sci U S A. 2003; 100(5): 2771-2776. 22. Kim K, Kim SH, Lepine F, Cho YH, Lee GR. Global gene expression analysis on the target genes of PQS and HHQ in J774A.1 monocyte/macrophage cells. Microb Pathog. 2010; 49(4): 174-180. 23. Hooi DS, Bycroft BW, Chhabra SR, Williams P, Pritchard DI. Differential immune modulatory activity of Pseudomonas aeruginosa quorum-sensing signal molecules. Infect Immun. 2004; 72(11): 6463-6470. 24. Skindersoe ME, Zeuthen LH, Brix S, et al. Pseudomonas aeruginosa quorum-sensing signal molecules interfere with dendritic cell-induced T-cell proliferation. FEMS Immunol Med Microbiol. 2009; 55(3): 335-345. 25. Legendre C, Reen FJ, Mooij MJ, McGlacken GP, Adams C, O'Gara F. Pseudomonas aeruginosa Alkyl quinolones repress hypoxia-inducible factor 1 (HIF-1) signaling through HIF-1alpha degradation. Infect Immun. 2012; 80(11): 3985-3992. 26. Liberati NT, Urbach JM, Miyata S, et al. An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. Proc Natl Acad Sci USA. 2006; 103(8): 2833-2838. 27. Belloc F, Dumain P, Boisseau MR, et al. A flow cytometric method using Hoechst 33342 and propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells. Cytometry. 1994; 17(1): 59-65. 28. Babior BM, Kipnes RS, Curnutte JT. Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest. 1973; 52(3): 741-744. 29. Wagner C, Zimmermann S, Brenner-Weiss G, et al. The quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) enhances the host defence by activating human polymorphonuclear neutrophils (PMN). Anal Bioanal Chem. 2007; 387(2): 481-487. 30. Mathew S, Roch T, Frentsch M, Lendlein A, Wischke C. Adjuvant effect of microencapsulated NOD ligands studied in a human phagocytic cell line. J Appl Biomater Funct Mater. 2012; 10(3): 229-36. 31. Kotsougiani D, Pioch M, Prior B, Heppert V, Hänsch GM, Wagner C. Activation of T Lymphocytes in Response to Persistent Bacterial Infection: Induction of CD11b and of Toll-Like Receptors on T Cells. Int J Inflam. 2010; 2010: 526740.
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J Appl Biomater Funct Mater 2014 ; 12 (1): 21-26 DOI: 10.5301/jabfm.5000204
The Pseudomonas quinolone signal (PQS) stimulates chemotaxis of polymorphonuclear neutrophils Gertrud M. Hänsch1, Birgit Prior1, Gerald Brenner-Weiss2, Ursula Obst2, Joerg Overhage2 1 2
Institute of Immunology, University of Heidelberg, Heidelberg - Germany Institute of Functional Interfaces, Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen - Germany
ABSTRACT Cross-talk between bacteria and mammalian cells is increasingly recognized as an important factor, especially during chronic infections. In particular, the interaction of extracellular bacterial signaling molecules with cells of the innate immune response is of special interest. In this context, we investigated whether the Pseudomonas quinolone signal (PQS) which is a quorum sensing molecule produced by bacteria and participates in biofilm formation and virulence has any influence on polymorphonuclear neutrophils (PMN), the cells of the “first line defense” against bacterial infections. We found that PQS did not enhance the bactericidal activity of PMN and did not induce apoptosis at concentrations up to 100 µM. However, PQS stimulated chemotaxis of PMN in doses of 10-100 µM. This PQS-dependent chemotaxis could be inhibited with SB203580 which blocks MAPkinase p38, suggesting a signaling pathway similar to AHL-12 induction. Using bacterial cell culture supernantants of Pseudomonas aeruginosa wild-type cells and a PQS-deficient mutant strain support the in vivo relevance of these findings. Since PQS is produced in the early phase of biofilm formation, PMN infiltration could be timely enough to eradicate bacteria before biofilm formation is completed, which confers the bacteria with a relative resistance to host defense mechanisms. Key words: Chemotaxis, Quorum sensing molecule, Human neutrophils, Pseudomonas aeruginosa, Pseudomonas quinolone signal (PQS), Biofilm Accepted: November 12, 2013
INTRODUCTION Biofilm formation is the crucial factor in many chronic bacterial infections such as implant-associated osteomyelitis, endocarditis in patients with artificial heart valves, and vascular catheter or ventilator tube infections (1, 2). Moreover, pulmonary biofilm infections are the leading cause of morbidity and mortality in patients with cystic fibrosis due to persistent infections that eventually lead to lung damage and respiratory failure (3). Bacteria grown in biofilms are imbedded in a slimy, self-produced polymeric matrix composed of polysaccharides, nucleic acids, lipids and proteins (4-7). Once established, these sessile communities are protected from the host immune response and are difficult to eradicate due to their high inherent resistance against antibiotic agents (8-12). Bacterial cell-to-cell communication, also named quorum sensing (QS), is known to be involved in biofilm development and is mediated by a variety of different QS signaling molecules. One major group of QS molecules are the N-acylhomoserine lactones (AHL),
which are synthesized and secreted by many Gramnegative bacteria like the opportunistic human pathogen Pseudomonas aeruginosa. In P. aeruginosa, the two AHLs (3-oxododecanoyl)-L- (AHL-12) and N-(butanoyl)L-homoserine lactone (AHL-4) control the production of extracellular virulence factors and promote biofilm maturation by inducing expression of more than 300 genes (13). Moreover, it has been shown previously that AHL-12 strongly impacts mammalian cells, in particular host immune cells by inhibiting T-cell proliferation and antigen presentation or inducing apoptosis (14, 15). Recently, we and others have shown that AHL-12 also stimulates the host defense by increasing phagocytosis of bacteria by macrophages or neutrophils and induces chemotaxis of neutrophils through induction of p38 and leukocyte specific protein 1 (16-18). In addition to AHLs, P. aeruginosa uses the non-AHL signal 2-heptyl-3-hydroxy-4-quinolone (the Pseudomonas quinolone signal; PQS, Fig. 1) as an extra QS molecule. PQS is closely linked to the AHL signaling network and possesses numerous regulatory and physiological functions,
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
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PQS stimulates chemotaxis
Fig. 1 - The Pseudomonas quinolone signal (PQS).
including control of biofilm development, secondary metabolite and virulence factor production as well as iron acquisition, among others (19). Interestingly, PQS is present at sites of infection, e.g. in sputum samples of cystic fibrosis patients (20, 21), and is capable of modulating the host immune response by modulating activity in T-cell proliferation and cytokine (IL-2) release, suppressing interleukin 12 (IL-12) and tumor necrosis factor alpha (TNF-α) secretion by LPS-stimulated macrophages and repressing HIF-1α protein levels (19, 22-25). In this study we investigated the potential of PQS to activate human neutrophils and analyzed signaling pathways related to migration. We found an induction of chemotaxis and up-regulation of adherence proteins and cellular functions that enhance the local neutrophil response. MATERIAL AND METHODS Bacterials strains and materials Pseudomonas aeruginosa PA14 wild-type and the corresponding pqsA transposon insertion mutant ID23621 (mutant) were obtained from the P. aeruginosa PA14 mutant library (26). Cells of wild-type and mutant strain were grown for 20 h in 30 mL BHI medium at 37°C under shaking conditions. The culture medium was centrifuged at 5800×g for 10 min and the supernatant was subsequently sterilized by filtration and used for functional assays (see below). A FITC-labeled Escherichia coli was used in the phagocytosis assay. PQS, calcein AM and f-MLP were purchased from Sigma-Aldrich (Seelze, Germany) and SB 203580 from Calbiochem (La Jolla, USA). PQS measurement by liquid chromatography/tandem mass spectrometry (LC-MS/MS) Sterilized cell-free supernatants of bacterial overnight cultures were mixed with two volumes of dichloromethane and the lower organic layer containing PQS was transferred into a new reaction tube following evaporation under nitrogen gas at room temperature. The pellet was resuspended in pure methanol following quantification by LC-MS/MS using a Zorbax Eclipse XCB-C8 5 µm, 150×3.6 mm HPLC 22
column (Agilent, Santa Clara, CA, USA) and a mobile phase consisting of acetonitrile - water 80:20 (v/v) with 100 µM EDTA and 0.1 % acetic acid at a flow rate of 0.40 ml/min. Electron spray ionization (ESI)-MS was performed on an API 365 triple quadrupole mass spectrometer (PE Sciex, Toronto, Canada) using a turbo ion spray interface used in positive mode. Single MS experiments (Q1 scan), MS/MS experiments (product ion scan, PIC), and multiple reaction monitoring (MRM) were performed using nitrogen as curtain gas, nebulizer gas, heater gas and collision gas. Instrumental parameters were optimized by infusion experiments with PQS standard solution (10 µg/ml; Sigma-Aldrich, Seelze, Germany) infused into the mass spectrometer using a syringe pump (Harvard Apparatus, South Natick, MA, USA) at a flow rate of 10 µl/min. To quantify PQS with high selectivity and sensitivity, MRM experiments were performed using the transitions from precursor ion to fragment ion: 260/175 (quantifier), 260/146, 260/147 and 260/188 (qualifier). An external calibration was performed using PQS standard solutions with concentrations ranging from 10 ng/ml up to 1000 ng/ml. Isolation of PMN from peripheral blood Heparin blood from volunteers (mainly laboratory personnel and students) was layered on PolymorphPrep (Nycomed, Oslo, Norway) after receiving approval for the procedure from the local ethics committee. After centrifugation, the PMN fraction was harvested and washed repeatedly in phosphate-buffered saline (PBS, pH 7.4). For the analyses of chemotaxis, the cells were suspended in Hank’s balanced salt solution (HBSS) containing 0.5% bovine serum albumin (BSA), at a final concentration of 1 x 106 cells per milliliter HBSS. Because of the wide intraindividual variation of the cell response, all assays were performed with cells of at least three different donors. Determination of cell death and apoptosis Intercalation of propidium iodide into DNA was measured by cytofluorometry as described by Belloc et al (1994) (27). In parallel, binding of Annexin V FITC which binds to the membrane phospholipid phosphatidylserine (PS) in the presence of Ca2+ was determined. Isolated PMN were incubated for 30 min with PQS in doses ranging from 1 µM to 100 µM; formalydehyde (3%) was used as a “death” control. Phagocytosis A commercially available kit (Glycotope Biotechnology, Heidelberg, Germany) was used. In brief, FITC-labeled Escherichia coli were incubated with whole heparinized blood at either 4ºC or 37ºC for 15 min. Uptake into PMN of bacteria was quantified by cytofluorometry.
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
Hänsch et al
Fig. 2 - PQS does not induce apoptosis of PMN. PMN were incubated with (left panel) or without PQS (100 µM) for 30 min. Apoptosis and cell death were determined by staining with AnnexinV FITC and intercalation of propidium iodide, respectively. On the right panel, the “death control” is shown.
Production of oxygen radicals To determine the production of oxygen radicals, we used the method described by Babior et al (28). In brief, PMN (1×106) were suspended in cytochrome c solution (Sigma-Aldrich, Seelze, Germany, 1 mg/ml HBSS,) and stimulated with phorbol ester (PMA) (Sigma-Aldrich), or with PQS in the concentrations as indicated in the text. Reduction of cytochrome c was measured photometrically after 30 min and calculated as ΔOD (550 nm). Cytofluorometry To determine the expression of surface receptors on PMN, a FITC-labeled antibody to CD66b (Coulter Immunotech, Marseille, France) was used to identify PMN, and phycoerythrin (PE)-labeled antibodies to either anti-CD11b (BD Biosciences, Heidelberg, Germany) or anti-CD18 (Serotec GmbH, Düsseldorf, Germany). One hundred microliters of whole blood was incubated with anti-CD66b-FITC and the respective PE-labeled antibodies (0.1-5 µg) for comparison with mouse IgG (either PE- or FITC labelled; Beckman Coulter Marseille, France) for 20 min. Then the erythrocytes were lysed using BD Facs Lysing solution (BD Biosciences) and cells were subjected to cytofluorometry using FACSCalibur and CellQuest Pro software (Becton Dickinson, Heidelberg, Germany). Ten thousand events were counted, and expression of CD11b or CD18 on CD66b positive cells was shown as a histogram. Expression of CD11b or CD18 was quantified as mean fluorescence intensity (MFI). Chemotaxis Chemotaxis was determined using the BD Falcon HTS Fluoroblok 96-Multiwell Insert System with a polyethylene terephthalate (PET) membrane insert plate with 3 µm pore size. Isolated PMN were labeled with calcein (106 cells/ml were incubated for 15 min at 37°C with calcein (1 µg/ml), then washed three times with HBSS and seeded in a concentration of 2×105/50 µL into the insert
plate. PQS in concentrations of 1 µM, 10 µM, 100 µM, and for comparison HBSS or fMLP (10-7 M) was added to lower wells. After 2 h at 37°C the insert plate was removed and fluorescence was determined by a PerkinElmer VICTOR2™ microplate reader using Wallac Workstation software (Waltham, MA, USA). RESULTS AND DISCUSSION PQS does not induce apoptosis or bactericidal activity of PMN Polymorphonuclear neutrophils were isolated from at least three different donors and subsequently incubated with PQS to identify any toxic effect. After 30 min of incubation, PQS did not show any cytotoxicity towards PMN when used in doses up to 100 µM (Fig. 2). To answer the question whether PQS would modulate PMN functions, in a first set of experiments an impact on the phagocytosis of bacteria was studied. PMN efficiently took up opsonized, FITC-labeled E. coli demonstrating that PQS did not enhance phagocytosis when used in doses of 1 µM to 100 µM (example in Fig. 3). In this regard, PQS exhibited functional differences in comparison to the AHL-12 quorum sensing molecule, which was shown to significantly enhance phagocytosis (17, 29) and induce apoptosis (14). In a further set of experiments, the possible effect of PQS on the production of reactive oxygen species was tested. In concentrations of 1 to 100 µM, PQS did not induce reactive oxygen species and did not modulate the reactive oxygen production induced by f-MLP (107 or 108 M) (data not shown). In that respect, PQS does not directly affect the bactericidal activity of PMN. Furthermore, the effect of PQS on the constitutively expressed adhesion proteins CD11b/CD18 was analyzed. As demonstrated in the example shown in Figure 4, PQS only marginally enhanced their expression; on average an increase of 22% for CD11b and 17% for CD18 (mean
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
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PQS stimulates chemotaxis
Fig. 3 - PQS does not affect phagocytosis of bacteria by PMN. PMN in whole blood were incubated either with PQS (100 µM; thick line) or without (thin line) with opsonized, FITC-labeled E. coli at either 4ºC or 37ºC for 15 min. Thereafter, green fluorescence associated with the cells was determined by cytofluorometry.
of three experiments with cells of different donors) was detectable. PQS induces chemotaxis of PMN In addition, we investigated the chemotaxis of PMN in response to different PQS concentrations and observed that PQS was chemotactic for isolated PMN. In doses of 10 µM to 100 µM PQS significantly induced chemotaxis (examples and summary of data are shown in Figs. 5A and B). Compared to bona fide chemokines such as f-MLP or IL-8, the molar concentrations required for induction of chemotaxis were much higher, a phenomenon that was also observed for AHL-12, and which raises the question of the in vivo relevance. To this end, we investigated the effect of bacterial supernatants from P. aeruginosa PA14 wild-type cells as well as the corresponding PQS-deficient mutant strain ID23621 (26) which is not able to synthesize PQS due to a transposon mutation in the PQS synthesis gene pqsA. Bacteria were grown for 20 h at 37°C in BHI medium under shaking conditions and supernatants of the wild-type and mutant strain were sterilized and subsequently analyzed for PQS using LC–MS/MS. The amount of PQS in supernatants was quantified according the settings given above using the specific transitions from m/z 298 (molecule ion) to m/z 197 and m/z 102. As expected, PQS was only detectable in the wild-type supernatant (1240 ng/ml supernatant). Using these supernatants in our chemotaxis assay, we observed that supernatants of P. aeruginosa induced a chemotactic response, which was in accordance to our previous studies on AHL-12 (16). Culture supernatant of the mutant strain induced a lower response in comparison to the wild-type, which, however, could be reconstituted by PQS (Fig. 5C). 24
Fig. 4 - PQS upregulates expression of CD11b/CD18. PMN in whole blood were cultivated with (thick line) or without (thin line) PQS (100 µM) for 30 min at 37ºC. Shown is the surface expression on PMN (identified as CD66b positive cells).
These results indicate that sufficient PQS is produced by wild-type bacterial cells to induce chemotaxis of PMN. It is not the only chemokine, however, a likely candidate for an additional molecule is AHL-12. This notion is supported by our previous observation that AHL-12-deficient supernatants still induced chemotaxis of PMN, though to a lesser extent (16). PQS-induced chemotaxis was inhibited when cells were pre-incubated with SB203580, which blocks the MAPkinase p38 (Fig. 5D). This enzyme is required for the induction of cellular migration by so-called “end-point” chemokines, and suggests a signaling pathway similar to AHL-12. How PQS is recognized by PMN is not yet clear. As a lipid, it may enter the cell freely; chemotaxis, however, supposedly requires a polarization of the cells, which is accomplished by a clustering of appropriate surface receptors, a phenomenon that needs further investigation. CONCLUSIONS The results of our studies demonstrate that the Pseudomonas quinolone signal does not exhibit cytotoxic effects on PMN nor does it have an impact on apoptosis. However, we found that PQS stimulates chemotaxis of PMN, likely via a signaling pathway similar to AHL-12. Regardless of the activation mechanisms, the fact that PQS induces chemotaxis might have an implication for the local host defense. Since PQS is produced in a similar way to AHL-12, before and in the early state of biofilm formation, PMN as a first-line defense could be recruited in a phase of infection where bacteria might still be vulnerable and can be eradicated (30, 31). On the other hand, infiltration of PMN into sites of infection can also enhance the local inflammatory response, thus leading to a progression of tissue damage. A better understanding of the dynamics of host-pathogen interactions, in particular in cell signaling events is increasingly needed for the development of new
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
Hänsch et al
A
B
Fig. 5 - PQS induces chemotaxis of PMN. A. The migration of PMN toward the bona fide chemokine IL-8 and in response to PQS. On the very left, random migration is shown (data from one donor). B. The compiled data of 4 independent experiments with cells of 4 individuals (PQS 10 µM) (A, A, BB. According to the U-test, induction of chemotaxis by PQS is different from random migration). C. Supernatants of P. aeruginosa-induced chemotaxis of PMN; supernatants of the PQS mutant strain which is deficient in PQS induced less chemotaxis. Addition of PQS (10 µM) reconstituted the chemotactic activity. D. Preincubation of PMN with the MAP-kinase inhibitor SB203580 reduced the chemotaxis towards PQS (data compiled from two experiments using cells of different donors).
C
D
and more efficient antibiotic treatment options for biofilm infections.
Future” of the Karlsruhe Institute of Technology (KIT) as part of the German Excellence Initiative.
ACKNOWLEDGEMENTS
Conflict of interest: The authors declare that there are no conflicts of interest.
We thank Anke Neidig and Michael Nusser for technical assistance. Financial support: This study was in part funded by the BioInterfaces (BIF) Program of the Karlsruhe Institute of Technology (KIT) in the Helmholtz Association and by the “Concept for the
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