The quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) enhances the host defence by activating human polymorphonuclear neutrophils (PMN)
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- Wagner, C., Zimmermann, S., Brenner-Weiss, G. et al. Anal Bioanal Chem (2007) 387: 481. doi:10.1007/s00216-006-0698-5
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The P. aeruginosa quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) interacts not only with bacteria, but also with mammalian cells, among others with those of the immune defence system. We focussed on the possible interaction of 3OC12-HSL with human polymorphonuclear neutrophils (PMN), because these cells are the first to enter an infected site. We found that 3OC12-HSL attracts PMN, and up-regulates expression of receptors known to be involved in host defence, including the adhesion proteins CD11b/CD18 and the immunoglobulin receptors CD16 and CD64. Furthermore, the uptake of bacteria (phagocytosis), which is crucial for an efficient defence against infection, was enhanced. Thus, recognising and responding to 3OC12-HSL not only attracts the PMN to the site of a developing biofilm, but also reinforces their defence mechanisms, and hence could be a means to control the infection in an early stage and to prevent biofilm formation.
KeywordsQuorum sensingPMNFunctional activityHomoserine lactoneHost defence
Bacterial biofilms are increasingly recognised as a major cause of persistent and destructive infectious diseases [1, 2]. Among those P. aeruginosa infections are especially devastating in immunocompromised patients [3–5]. Because the relative resistance of bacteria in biofilms towards antibiotics limits the therapeutic options, the question arises whether or not interference with biofilm formation could be a reasonable alternative [1, 6]. In that regard the quorum-sensing molecules could be suitable targets for an interventional therapy, because they are produced and released in the very early stages of biofilm formation when the bacteria are still more or less planctonic and susceptible to the immune defence mechanisms.
While structural and functional aspects of quorum-sensing molecules are discussed elsewhere in this journal, we like to draw the attention to the fact that quorum-sensing molecules, particularly those of P. aeruginosa, are not only efficient operators within the bacterial community, but also interact with mammalian cells, including epithelial cells, fibroblasts [7–9] and also cells of the adaptive or innate immune response [10–16].
Recognition of bacteria and bacterial products by cells of the immune defence is essential for the activation of the cellular effector functions, such as the efficient uptake, the phagocytosis, and intracellular killing, e.g. by the production of oxygen radicals. Recognition is accomplished by so-called pattern recognition receptors, which are selective for groups of bacterial molecules, including lipopolysaccharides, lipoteichoic acid, or peptidoglycans. Not only phagocytic cells, the major effector cells of the innate, non-adaptive immune response, express these receptors, but also T-lymphocytes, the protagonists of the specific, adaptive immune response. Triggering these receptors is not only essential for the effector function of the immune system (e.g. phagocytosis and killing), but most probably also contribute to the initiation of the adaptive immune response [17, 18].
The majority of studies have so far focussed on membrane-derived bacterial products such as lipopolysaccharides, peptidoglycans or on bacterial DNA and their receptors in immunocompetent cells [19, 20]. Only more recently, an immunmodulatory activity of the quorum-sensing molecules of P. aeruginosa was proposed, particularly of N-3-oxododecanoyl homoserine lactone (3OC12-HSL). This proposition is based on the observation that 3OC12-HSL inhibits the proliferation of T-lymphocytes and modulates the cytokine synthesis in various in vitro models [10–14]. Moreover, enhancement of phagocytosis by macrophages of yeast cells was shown .
In light of the facts that quorum-sensing molecules are central to the development of bacterial biofilms, and that polymorphonuclear neutrophils (PMN) represent the first line defence against bacterial infections, the interaction of PMN with developing biofilms or their products is of utmost interest.
Indeed, we observed induction of directed migration, chemotaxis, of human PMN by 3OC12-HSL . These findings are in apparent contrast to previous data by Tadeta et al. , who showed induction of apoptosis in mouse macrophages and in neutrophils. Apparently, species differences account for the discrepant findings. This assumption is supported by the work by Vikström et al. , who were also using human cells and did not observe apoptosis either.
In the present study we further examined the effect on PMN of 3OC12-HSL and of other acyl homoserine lactones (AHL). We found that in addition to induction of chemotaxis, expression of adhesion molecules was enhanced as were the expression of immunoglobulin receptors and the phagocytosis of antibody-coated (opsonised) bacteria.
Materials and methods
Acyl homoserine lactones (AHL)
The 3-deoxo-isomer of 3OC12-HSL was purchased from Sigma-Aldrich (München, Germany). C4-HSL was purchased from Sigma-Aldrich; 2-amino-4-butyrolactone was from Fluka (Buchs, Switzerland). Stock solutions (0.1 M) were prepared in dimethyl sulfoxide (DMSO); these were diluted in Hank’s balanced salt solution (HBSS) immediately before use.
3OC12-HSL and C8-HSL were synthesised according to the method described by Chhabra et al. . In brief, equivalent amounts of Meldrums’s acid (Aldrich, 1 mmol) and the corresponding fatty acids (Fluka) were used, together with 1.1 equivalents of 4-(dimethylamino)pyridine (Fluka) and N,N-dicyclohexylcarbodiimide (Aldrich). Each compound was purified to homogeneity by preparative liquid chromatography.
P. aeruginosa were obtained from patients with implant-associated osteomyelitis. The bacteria were expanded in Difco Micro Inoculum Broth (purchased from Becton, Dickinson and Co, Sparks, MD, USA) diluted 1- to 10-fold in HBSS. For formation of biofilms, the bacteria were seeded into plastic culture dishes (6 well) 1×107 per mL in HBSS and cultivated at 37°C with vigorous shaking. Supernatants were taken after various times.
Isolation of PMN from peripheral blood
Heparin blood was drawn from healthy volunteers, mainly laboratory personal and students, observing the institutional guidelines. PMN were isolated using PolymorphPrep (Nycomed, Oslo, Norway), following the supplier’s recommendations. The PMN fraction was harvested, washed repeatedly in phosphate-buffered saline (PBS, pH 7.4) and was suspended in HBSS containing 0.1% bovine serum albumin (BSA), at a final concentration of 1×106 cells/mL. Cells were identified by expression of CD66b using cytofluorometry (see below). The purification method yielded about 90% PMN.
Evaluation of viability of PMN
Intercalation of propidium iodide into DNA was measured by cytofluorometry as described by Belloc et al. ; in parallel, PMN were examined by light microscopy, following staining of the cells by trypan blue or haematoxyllin, respectively.
Chemotaxis across a membrane filter
A modified Boyden chamber assay was used , equipped with a nitrocellulose filter (5-μm pore size; 200-μm thick; Schleicher and Schuell GmbH Dassel, Germany). As bona fide chemokines, recombinant human complement C5a (5 ng/mL) (Sigma-Aldrich) or interleukin 8 (IL-8) (8 ng/mL) (Immunotools, Friesoythe, Germany) were used. Random migration was assessed using HBSS. The cells (1×106 in 1 mL) were placed into the upper compartment, the chemokines in the lower. After 90 min the cells migrated into the filters were fixed with propanol and stained with haematoxylin and evaluated using an Omnicon Alpha Image Analyzer (Bausch and Lomb, Heidelberg, Germany). Chemotaxis was measured as the “leading front”, defined as the distance in μm from the top of the filter to a level where at least five cells could still be seen. Two parallel filters were prepared and 10 different areas were evaluated on each filter. Preincubation of cells with butyrolactone was done at room temperature for 10 min. SB203580 was purchased from Calbiochem (purchased through Merck Biosciences Darmstadt, Germany) and used in a final concentration of 50 μM.
For the experiments using inserts, tissue culture inserts (10 mm, polycarbonate membrane with 3μm pore size) were obtained from Nunc (Nümbrecht, Germany). The bacteria were cultured in 24 well plates (7×106 per mL in HBSS) for various times. The inserts were then placed and isolated PMN (1×106 per mL, 500μL per insert in HBSS) were added. After 24 h at 37°C, PMN having reached the lower chamber were counted.
To determine the expression of surface receptors on PMN, an FITC-labelled antibody to CD66b (Coulter Immunotech, Marseille, France) was used to identify PMN, and phycoerythrin (PE)-labelled antibodies to either anti-CD11b (BD Biosciences, Heidelberg, Germany), anti-CD18 (Serotec GmbH, Düsseldorf, Germany), anti-CD14 (Serotec), anti-CD16 (Becton Dickinson Biosciences) or anti-CD64 (Serotec). Whole blood (300 μL) was incubated with anti-CD66b-FITC and the respective PE-labelled antibodies (0.1–5 μg) or for comparison with mouse IgG (either PE- or FITC-labelled; Beckman Coulter Marseille, France ) for 20 min. The erythrocytes were then lysed using BD FacsLysing solution (BD Biosciences) and subjected to cytofluorometry using FACSCalibur and CellQuest software (Becton Dickinson, Heidelberg, Germany). Ten thousand events were counted. The percentage of double positive cells was used as a measure for up-regulation of CD64; enhancement of expression was determined by the increase in the mean fluorescence intensity (MFI).
A commercially available kit (Orpegen, Heidelberg, Germany) was used. In brief, FITC-labelled E. coli were incubated with whole heparinised blood. Uptake into PMN of bacteria was quantified by cytofluorometry.
Production of oxygen radicals
Basically, the method described by Babior et al.  was used. In brief, PMN (1×106) were suspended in cytochrome c solution (Sigma-Aldrich, Munich Germany, 1 mg/mL HBSS,) and stimulated with phorbol ester (PMA, Sigma-Aldrich) or with AHL. Reduction of cytochrome c was measured photometrically after 30 min and calculated as ΔOD (550 nm). For priming, the cells were exposed to tumour necrosis factor (TNF) α (2 ng) (Sigma-Aldrich) for 20 min, and then exposed to AHL or exposed to AHL (1 to 10 μM) and then stimulated with TNFα.
Effect of 3OC12-HSL on the viability of PMN
In a first set of experiments the effect of 3OC12-HSL on the survival of PMN in vitro was tested. The cells were isolated from the peripheral blood of healthy donors and incubated with 3OC12-HSL in two concentrations. After various times, viability was assessed by propidium iodide incorporation and by light microscopy. At concentrations of 10 μM or 100 μM 3OC12-HSL did not induce cell death, nor did it affect the constitutive apoptosis of PMN occurring by 24 h or 48 h.
Induction of chemotaxis by 3OC12-HSL and supernatants of P. aeruginosa
Induction of chemotaxis by 3OC12-HSL
PMN preincubated with
Interleukin 8 (2 ng)
Up-regulation of adhesion proteins on PMN by 3OC12-HSL
Up-regulation of CD11b and CD18 on PMN following exposure of whole blood to AHL
Effect of 3OC12-HSL on the expression of immunoglobulin receptors or the lipopolysaccharide receptor CD14
Effect of 3OC12-HSL on immunoglobulin receptors, or on CD14
Without stimulus (20 min)
3OC12-HSL (10 μM, 20 min)
3-Deoxo-isomer (10 μM, 20 min)
Mean fluorescence intensitya (n=3)
% positive cells (mean of n=2)
Effect of 3OC12-HSL on phagocytosis and oxygen radical production
In the range of 0.1 to 100 μM, 3OC12-HSL did not induce the oxygen radical synthesis; it also had no priming effect on PMN, nor did it induce the oxygen radical synthesis of TNFα-primed PMN (data not shown).
P. aeruginosa produce and release quorum-sensing molecules, among others the acyl homoserine lactones (AHL), as they form biofilms. Here, we addressed the question whether or not these molecules are recognised by polymorphonuclear neutrophils (PMN), because these are the first cells of the innate immune response to enter an infected site. We found that supernatants of P. aeruginosa, collected within 2 to 24 h after cultivating the bacteria under conditions leading to biofilm formation, contained a chemotactic activity for PMN. This chemotactic activity was most probably due to 3OC12-HSL because as shown before 3OC12-HSL can induce the chemotaxis of PMN ; moreover, the chemotaxis could be inhibited by butyrolactone which selectively inhibited the 3OC12-HSL-induced chemotaxis.
Because chemotaxis and phagocytosis of bacteria is critically dependent on adhesion proteins, the effect of 3OC12-HSL on the surface expression of the ß2-integrin CD11b/CD18 on PMN was tested. An up-regulation was seen within 20 min after exposure, compatible with a transport to the membrane of preformed molecules, as it occurs also in response to bona fide stimuli such as IL-8. Furthermore, an up-regulation of the low-affinity immunoglobulin (IgG) receptor CD16 was seen, as was induction of CD64, the latter representing the high-affinity receptor for IgG. Corresponding to the enhanced expression of the immunoglobulin receptors, an increased uptake of opsonised bacteria by 3OC12-HSL-pretreated PMN was seen.
Our findings are in line with a previous report by Vickström et al.  who also described enhancement of phagocytosis in an experimental setting employing in vitro differentiated human macrophages and yeast particles. Also in line with the data by Vickström et al., we could not demonstrate induction of oxygen radical synthesis; moreover, we did not see a priming or modulation of the oxidative burst by 3OC12-HSL. Thus, although macrophages and PMN are different in many aspects, they obviously share the capacity to recognise and respond to 3OC12-HSL.
It is also noteworthy that in both macrophages and PMN, the activation by 3OC12-HSL is critically dependent on the activity of the p38 mitogen activated protein (MAP) kinase. Together with our previous data, where specific and saturable binding of 14C-3OC12-HSL to PMN was demonstrated, as was the dependency on a signalling cascade involving tyrosine kinase, phospholipase C, protein kinase C and again to MAP kinase , a receptor for 3OC12-HSL is rather likely. In keeping with a receptor for 3OC12-HSL is also the requirement of a definite isomeric conformation. Our data correspond to earlier findings by Chhabra et al. , who described that the immunmodulatory activity of the AHL, determined in a splenocyte proliferation assay, depended critically on the chemical structures, particularly on the length of the fatty acids, the lactone ring and the L-configuration .
As pointed out above, recognition of bacterial products is essential for the host defence response. Because bacteria embedded in biofilms are supposedly rather resistant to the host defence mechanisms [25, 26], only recognition at an early stage would allow interference with biofilm formation. In that regard, recognising quorum-sensing molecules would be of utmost importance. Given that 3OC12-HSL is released by the bacteria in the initial phase of biofilm formation, the sensing of a 3OC12-HSL gradient would attract PMN to the site of a developing biofilm. At that early stage phagocytosis and killing of the bacteria could still be possible, particularly since the receptors critically involved in the bactericidal activity are also up-regulated by 3OC12-HSL.
Thus, recognition of and attraction by 3OC12-HSL could be regarded as a means of PMN to control biofilm development.