The discovery of novel antiinflammatory targets to treat inflammation in the cystic fibrosis (CF) lung stands to benefit patient populations suffering with this disease. The Pseudomonas aeruginosa quorum sensing autoinducer N-3-oxododecanoyl homoserine lactone (3O-C12) is an important bacterial virulence factor that has been reported to induce proinflammatory cytokine production from a variety of cell types. The goal of this study was to examine the ability of 3O-C12 to induce proinflammatory cytokine production in normal and CF bronchial epithelial cells, and better understand the cellular mechanisms by which this cytokine induction occurs. 3O-C12 was found to induce higher levels of IL-6 production in the CF cell lines IB3-1 and CuFi, compared to their corresponding control cell lines C38 and NuLi. Systems biology and network analysis revealed a high predominance of over-represented innate immune pathways bridged together by calcium-dependant transcription factors governing the transcriptional responses of A549 airway cells to stimulation with 3O-C12. Using calcium-flux assays, 3O-C12 was found to induce larger and more sustained increases in intracellular calcium in IB3-1 cells compared to C38, and blocking this calcium flux with BAPTA-AM reduced the production of IL-6 by IB3-1 to the levels produced by C38. These data suggest that 3O-C12 induces proinflammatory cytokine production in airway epithelial cells in a calcium-dependent manner, and that dysregulated calcium storage or signalling in CF cells results in an increased production of proinflammatory cytokines.
Citation: Mayer ML, Sheridan JA, Blohmke CJ, Turvey SE, Hancock REW (2011) The Pseudomonas aeruginosa Autoinducer 3O-C12 Homoserine Lactone Provokes Hyperinflammatory Responses from Cystic Fibrosis Airway Epithelial Cells. PLoS ONE 6(1): e16246. https://doi.org/10.1371/journal.pone.0016246
Editor: Jacques Zimmer, Centre de Recherche Public de la Santé (CRP-Santé), Luxembourg
Received: November 11, 2010; Accepted: December 7, 2010; Published: January 31, 2011
Copyright: © 2011 Mayer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The lab of REWH receives funding from the Foundation for the National Institutes of Health, the Bill and Melinda Gates Foundation, and the Canadian Institutes for Health Research (CIHR) for two separate program grants from the Grand Challenges in Global Health Initiative and Genome BC for the Pathogenomics of Innate Immunity research program grant. M.L.M. holds a CIHR M.D./Ph.D. studentship and a Michael Smith Foundation for Health Research trainee award. C.J.B. was supported by the Canadian Cystic Fibrosis Foundation. S.E.T. holds a Career Development Award from the Canadian Child Health Clinician Scientist Program (a CIHR Strategic Training Program) and is funded by operating grants from the Canadian Cystic Fibrosis Foundation, British Columbia Lung Association, and the CIHR Team in Mutagenesis and Infectious Diseases. R.E.W.H. is a Canada Research Chair in Microbiology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Cystic fibrosis (CF) is a chronic pulmonary disease characterized by recurrent and excessive inflammation that causes the destruction of lung tissue, eventually resulting in respiratory failure. Inflammation in the CF lung is thought to be driven by both host factors such as cytokines – and bacterial factors – produced during lung colonization with pathogens such as Pseudomonas aeruginosa. P. aeruginosa relies on quorum sensing molecules such as the autoinducer N-3-oxododecanoyl homoserine lactone (3O-C12) to drive the expression of numerous genes related to virulence , biofilm formation , and antibiotic resistance  when colonizing the CF lung. Studies in mice have shown that 3O-C12 is a critical determinant for bacterial fitness and establishing chronic lung infections , , leading many to argue that the development of quorum sensing inhibitors would be a major advance in the ability to eradicate infections with P. aeruginosa –.
In addition to the role played by 3O-C12 in regulating bacterial virulence, this molecule has recently been reported to have numerous immunomodulatory and inflammatory properties. 3O-C12 has a powerful inhibitory effect on professional immune cells, inhibiting dendritic cell and T-cell activation , promoting apoptosis – and inhibiting the ability of macrophage and monocytes to respond to a range of Toll-like receptor (TLR) agonists through disruption of NF-κB signalling . Perplexingly, 3O-C12 is a powerful inducer of proinflammatory cytokines such as IL-6 and IL-8 in airway epithelial cells and lung fibroblasts, upregulates inflammatory enzymes such as cyclooxygenase-2 (COX-2) (both in vitro and in vivo) , –, and enhances neutrophil chemotaxis . Taken together, these data suggest that 3O-C12 suppresses the function of key immune networks responsible for bacterial clearance, while simultaneously enhancing inflammatory pathways that promote the pathogenesis of CF.
Cells from CF patients exhibit an exaggerated inflammatory response to stimulation with P. aeruginosa –, and we have previously identified interactions between flagellin and TLR5 as contributing to this phenotype using CF airway epithelial cell lines and primary CF peripheral blood mononuclear cells in vitro  and in a recent CF patient cohort candidate SNP analysis study . One limitation of these previous studies was the use of heat-killed bacteria or laboratory strains with diminished virulence to control for motility or cytotoxicity differences between different P. aeruginosa strains and mutants, an experimental approach that precluded our ability to identify factors secreted by virulent live cells, such as 3O-C12. In this study, we report that 3O-C12 also differentially induces the increased production of the proinflammatory cytokine IL-6 in CF airway epithelial cells. Using systems biology and network analysis approaches, we demonstrated that the inflammatory response of airway epithelial cells to 3O-C12 relied on calcium-dependent transcription factors, and identify exaggerated calcium signalling in CF airway epithelial cells as the mechanism underlying their exaggerated cytokine response to 3O-C12.
Homoserine lactone 3O-C12 elicited markedly higher IL-6 production from CF airway epithelial cells
To examine P. aeruginosa quorum sensing autoinducers for their ability to induce exaggerated proinflammatory responses in CFs airway epithelial cells, two pairs of matched CF and non-CF airway epithelial cells (NuLi and CuFi; C38 and IB3-1) were stimulated with 3O-C12 and C4 homoserine lactones at concentrations between 10 and 100 µM. This range was selected to be consistent with the concentration of 3O-C12 used to elicit immunomodulatory or inflammatory effects in both in vitro cell models , , , , , , , , , , and in vivo animal models . When tested at 100 µM, 3O-C12 was found to induce the proinflammatory cytokine IL-6 in all four cell types tested, however the induction of IL-6 was between 2 and 4-fold greater in the CF cell lines CuFi and IB3-1 compared to their non-CF matched counterparts NuLi and C38 (Figure 1A and 1B). The difference in cytokine induction was not due to differences in cytotoxicity, as cell viability for all four cell lines was consistently >95% after 24 hr stimulation with concentrations of 3O-C12 between 10 and 100 µM (compared to 250 µM which induced cell death in 18–27% of cells) as determined by LDH release. Interestingly, 3O-C12 was highly specific for the induction of IL-6, as stimulation of cells with up to 100 µM of 3O-C12 did not elicit the production of the proinflammatory cytokine IL-8. To confirm that the absence of IL-8 did not arise from cell specific defects in the synthesis of this cytokine, cells were stimulated with flagellin (0.1 µg/ml) or IL-1β (10 ng/ml) for 24 hours. Flagellin and IL-1β induced the production of both IL-6 and IL-8 from all four cells with IL-8 production being on the order of 1 to 2-fold higher than IL-6 (data not shown), confirming that 3O-C12 was specific in its ability to induce IL-6. C4 HSL did not induce the production of IL-6 or IL-8 to greater than basal levels in any of the four cell lines tested.
Cytokine production was measured in supernatants from (A) CuFi and (B) IB3-1, or their matched non-CF counterparts 24 hr after treatment with 3O-C12 (100 µM). Bars show the mean of three independent experiments ± SEM; * p<0.05; ** p<0.01.
Systems biology and network analysis of airway cell inflammatory responses to 3O-C12 implicating innate immune pathways and calcium signalling
Having identified 3O-C12 as capable of inducing greater IL-6 production from CF airway cells, we sought to understand the mechanism by which this phenomenon occurred. The signalling pathways that govern cell responses to 3O-C12 are poorly elucidated; the molecule modulates the activity of peroxisome proliferator-activated receptor-gamma (PPARγ)  but no specific receptor(s) and/or signalling pathway(s) have been identified as mediators of 3O-C12 cytokine induction. To gain further insights into the mechanisms by which 3O-C12 induces proinflammatory cytokine secretion in airway epithelial cells, we employed systems biology and network based analysis of a recent microarray dataset which examined the transcriptional responses of A549 airway cells to this molecule. A total of 2,989 genes were identified by Bryan et al.  as differentially expressed (DE) after 6 hr stimulation with 3O-C12 (50 µM), and these genes were uploaded to InnateDB so that over-representation analysis could be carried out.
A total of 30 pathways were found to be significantly over-represented by the DE genes, of which a substantial number (21/30) related to the activation of cellular innate immune and inflammatory responses (Table 1). These included TNF-alpha pathway (p = 0.0007), NOD-like receptor signaling pathway (p = 0.0017), canonical NF-kappaB pathway (p = 0.0276) MAPK signaling pathway (p = 0.0416), and JNK cascade (p = 0.0427). Similarly, significantly over-represented gene ontology (GO) functional terms (Table 2, Table S1) included innate immune response (p = 0.0001), inflammatory response (p = 0.0017), cytokine production (p = 0.0175), and positive regulation of I-kappaB kinase/NF-kappaB cascade (p = 0.0303). A second theme present within the over-representation analysis results was presence of calcium sensitive transcription factors such as the ATF-2 transcription factor network (p = 0.0006), calcineurin-regulated NFAT-dependent transcription (p = 0.0027; includes NFATC1 and NFATC2), and Calcium signaling in the CD4+ TCR pathway (p = 0.0407). Similarly, additional significant GO terms included metal ion binding (p = 3.58×10-7), response to calcium ion (p = 0.0185), calcineurin complex (p = 0.0189), and cation transmembrane transporter activity (p = 0.0305).
Although the calcium dependent transcription factors ATF-2, NFATC1, and NFATC2 were identified by InnateDB's pathway ORA, the latter two genes were not themselves differentially expressed. This was not an unexpected result, as many key regulatory elements of signalling pathways in mammalian cells are regulated by non-transcriptional controls , , and so do not appear as DE genes within microarray datasets. To ensure that the NFAT transcription factors were included in our analysis using an unbiased approach, we resubmitted a gene list to InnateDB containing the 2,989 genes, and then visualized protein level interactions between genes using Cytoscape and the software plugin Cerebral. This analysis yielding a network containing 361 gene nodes with 773 unique protein-protein or protein-gene interactions (Figure S1A). In addition, InnateDB was tasked to create a first-order interaction network for NFATC1 and NFATC2 (Figure S1B), and the two resulting networks were merged into a single entity (Figure S1C) containing 396 gene nodes and 866 unique protein-protein or protein-gene interactions. To control for biases which might have arisen by intentionally introducing two non-DE genes into the dataset, jActive was used to identify the single most significant subnetwork that contributes to the manner in which A549 cells responded to HSL. Using this method, a single highly statistically significant subnetwork containing 101 nodes (including NFATC1 and NFATC2) with 198 unique interactions was found (Figure 2), although different cell types might express individual nodes to different extents.
The color of gene nodes is proportional to their relative fold-change versus untreated cells, node-node connections represent known protein-level interactions annotated within InnateDB, and the node size reflects its degree of interconnectivity (hub-like nature) within the network.
Analysis of this critical subnetwork revealed that numerous key innate immune signalling pathways, including the MAP Kinase, JAK, and canonical NF-κB pathways, converging in the upregulation of IL-6. In addition, network analysis also revealed that different innate immune pathways were linked together by calcium-activated transcription factors. The calcium-dependant TF NFATC1 provided a bridging point between the NF-κB family members C-Rel (REL) and p52 (NFKB2) and EGR1. Furthermore, the network analysis suggested the likelihood that NFATC2 integrated signalling between EGR1, CREB, and AP-1 (Fos/Jun) in the upregulation of the proinflammatory cytokine IL-6 in A549 cells. Based on these findings, we hypothesized that 3O-C12 mediated induction of proinflammatory cytokines in airway epithelial cells was largely mediated by calcium-dependent processes and transcription factors.
3O-C12 induces increased levels of [Ca2+]i in the CF cell line IB3-1 that is in turn drives increased cytokine production
We next sought to confirm these in silico findings using calcium-flux assays to determine if stimulation of IB3-1 and C38 with 3O-C12 induced differences in the levels of intracellular Ca2+ using a fluor-4 microplate based assay system to examine kinetic changes in the second. Concentrations of HSL ranging from 10 to 50 µM failed to induce substantial increases in intracellular calcium in C38 and IB3-1 (data not shown), whereas at concentrations over 100 µM, Δ[Ca2+]i rose significantly within minutes for the CFTR defective line IB3-1, but not the corrected line C38 (Figure 3A). At a higher concentration (250 µM) of 3O-C12, similar increases in Δ[Ca2+]i were observed in both cell lines, however [Ca2+]i returned to basal levels in C38 by the 20 min mark but remained elevated in IB3-1 for the duration of the time course (Figure 3B). Although 250 µM 3O-C12 was found to be cytotoxic, it is unlikely that cytotoxicity contributes to the differences in [Ca2+]i between IB3-1 and C38. Shiner et al. (2006)  reported that cell death following 3O-C12 stimulation is due to sustained increases in intracellular Ca2+, therefore the change in [Ca2+]i that occurred over the short (30 min) time frame used in this assay most certainly precede any LDH release detected after 24 hr.
CF cell line IB3-1 and the isogenic CFTR-corrected cell line C38 were stimulated with 3O-C12 at (A) 100 µM or (B) 250 µM. Data are reported as Δ[Ca2+]I, the change in [Ca2+]i in stimulated cells minus the change in vehicle treated cells. Data points show the mean of 6 replicate wells with serial measurements made every 50–60 s over 30 min. Graph is representative of one of three replicate experiments with similar results.
To investigate if the 3O-C12 induced elevations and sustained increases in [Ca2+]i in IB3 were responsible for differential induction of proinflammatory cytokines in these two cell types, cells were stimulated with increasing doses of 3O-C12, with or without a 1-hr pretreatment with membrane-permeable calcium chelator BAPTA-AM. Over a range of HSL concentrations, BAPTA-AM antagonized the exaggerated production of IL-6 by IB3-1 cells, reducing the levels to those produced by C38 (Figure 4).
Cells were pre-treated for 1 hr with vehicle or BAPTA-AM (intracellular calcium chelator). Data points are the mean of three independent experiments ± SEM. Statistical significance for comparisons between vehicle and BAPTA-AM within a cell line are indicated with *; comparisons between IB3-1 (vehicle) and C38 (vehicle) are indicated with #; */# p<0.05; **/## p<0.01.
Colonization of the CF lung by P. aeruginosa induces robust neutrophilic inflammation that is disproportionate to the bacterial load. Ongoing inflammation results in pulmonary obstruction, reduced lung function and ultimately respiratory failure and death . In the present study, we demonstrated that in addition to P. aeruginosa flagellin , , , 3O-C12 homoserine lactone induces a heightened production of the proinflammatory cytokine IL-6 from two different CF respiratory epithelial cell lines. The concentrations of 3O-C12 used in this study to elicit IL-6 production fell between 10 µM and 100 consistent with previous studies examining the immunomodulatory or inflammatory properties of this ligand , , , , , , , , , . Although 3O-C12 has only been detected in the 1–20 nM range in CF patient sputum , and in the 1–2 µM range in murine P. aeruginosa lung infection models , in vitro biofilm models predict that localized pockets of 3O-C12 production exist in which concentrations of the autoinducer readily approach 600 µM .
The signalling pathways in mammalian cells triggered by 3O-C12 are poorly characterized, and so we employed a systems biology approach to provide a further understanding of how this molecule induces inflammatory cytokine production. Using a previously published microarray dataset , we discovered direct engagement and activation of multiple immune pathways by 3O-C12, with 21/30 over-represented pathways related to immune processes involved in cell activation and cytokine production (Table 1). Network analysis of the dataset carried out to identify the most significant sub-network of genes, based on their protein-level interactions, further identified that 3O-C12 induction of proinflammatory cytokines occurs in tandem with the upregulation of the classical cytokine transcription factors NF-κB, Fos, and Jun (Figure 2). Further upstream, upregulation of the CXCR4-UBC and CXCR4-SOCS3-TRAF6 pathways was apparent (Figure 2). CXCR4, which monogamously binds CXCL12 (SDF-1), is able to recruit progenitor epithelial cells to the lungs in response to acute airway injury  suggesting that airway specific stress-response pathways are being triggered by 3O-C12.
3O-C12 has been shown to elicit the production of IL-8 from fibroblasts and corneal epithelial cells , , and the IL-8 gene was upregulated 47-fold in the microarray analysis of A549 cells stimulated with this ligand , so it was unsurprising that the gene was found to be a statistically significant node in the network analysis of this data set (Figure 2). However a previous study examining the effect of soluble Burkholderia cepacia products on A549 cells detected IL-8 production following stimulation with bacterial supernatant, but not after stimulation with 3O-C12 , consistent with the absence of detectable IL-8 protein after stimulating NuLi, CuFi, IB3-1 or C38 with the homoserine lactone, but not with other proinflammatory stimuli such as flagellin. The discrepancy between IL-8 transcriptional and protein levels could be due to the fact that IL-8 mRNA undergoes extensive post-transcriptional regulation , and the reliance of 3O-C12 on non-canonical signalling pathways  is insufficient in some cell types to induce the cellular machinery required for proper processing and secretion of this cytokine.
The results of our systems biology and network analysis suggested that intracellular Ca2+ may also play a role in proinflammatory cytokine production by airway epithelial cells in response to 3O-C12. Intracellular Ca2+ is an important second messenger that has a well appreciated role in the downstream activation cellular processes with proinflammatory sequelae. The results of our systems biology analysis suggested that intracellular Ca2+ may be contributing to cytokine induction by 3O-C12 as multiple Ca2+-influenced transcription factors involved in inflammatory responses, such as c-Jun, NF-κB, and NFATc , were significant nodes in the network graph (Figure 2). 3O-C12 has recently been shown to trigger Ca2+ release from the endoplasmic reticulum in airway epithelial cells , an event which has been linked in other cell types to the ability of this molecule to induce apoptosis , disrupt epithelial cell tight junctions , and circumvent host defence mechanisms against P. aeruginosa , but to our knowledge, has not been associated with the induction of proinflammatory cytokines by the autoinducer. In addition to validating our in silico predictions, the finding that the CFTR-modified airway epithelial cell line IB3-1 responded to 3O-C12 with increased and sustained rises in [Ca2+]i compared to the isogenic CFTR-corrected C38 cell line suggested Δ[Ca2+]i was mediating the differential IL-6 production between these cell lines. This hypothesis was subsequently shown to be correct as the membrane-permeable calcium chelator BAPTA-AM reduced 3O-C12-mediated IL-6 production in both cell lines. These findings are consistent with a growing body of literature demonstrating that CF cells undergo expansion of their endoplasmic reticulum Ca2+ stores, causing dysregulation of intracellular Ca2+ signalling pathways which in turn, can result in enhance proinflammatory cytokine secretion , , .
The lasI/lasR quorum sensing system responsible for the production of 3O-C12 HSL has previously been identified as a potential therapeutic target in CF. Antagonizing bacterial production of 3O-C12, either by immunization  or through the use of small molecule inhibitors of lasR , , results in decreased mortality in animal models of P. aeruginosa lung infection. A solid understanding of the mechanisms that propagate the inflammatory process in the CF lung is necessary to develop novel therapies that limit the magnitude, duration, or frequency of pulmonary inflammation which in turn is crucial to alleviate the burden of disease in CF and improve both the duration and quality of life in affected patients. This study sheds additional light on the etiology of inflammation in the CF lung, and provides additional rationale for the further investigation of the P. aeruginosa quorum sensing molecule 3O-C12 HSL as a novel therapeutic target for reducing lung inflammation in CF.
Materials and Methods
N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C12 HSL) and N-butanoyl-L-homoserine lactone (C4 HSL) were obtained from Cayman Chemical (Ann Arbor, MI), dissolved in DMSO, stored at −20°C and used within 3 months. Flagellin (ultrapure recombinant) and interleukin-1β were obtained from Invivogen (San Diego, CA). Ionomycin and BAPTA-AM were obtained from EMD Bioscience (Gibbstown, NJ), dissolved in ddH2O or DMSO respectively, and stored at −20°C. EGTA was obtained from Alfa Aesar (Ward Hill, MA) and was solubilized at 0.5M in ddH2O by the drop-wise addition of 10M NaOH until a pH of 8.00 was achieved.
Cell lines and growth conditions
This study utilized the well-characterized CF lung epithelial cell line IB3-1 (compound heterozygote for the ΔF508 and W1282X CFTR mutations) and CuFi-1 (homozygrous for the ΔF508 mutation) with their matched control lung epithelial cells C38 (“corrected” CF cell line derived from IB3-1) or NuLi-1 (wild type CFTR) (American Type Culture Collection) , . IB3-1 and C38 were grown in coated flasks (100 µg/ml BSA, 30 µg/ml bovine collagen I, and 10 µg/ml human fibronectin) in LHC-8 basal medium (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1% penicillin-streptomycin-amphotericin B solution. CuFi-1 and NuLi-1 were grown in Primaria flasks (BD, Mississauga, ON, Canada) in BEGM medium (Lonza, Walkersville, MD) supplemented with SingleQuot growth factors (Lonza). All cells were grown at 37°C in 5% CO2.
NuLi-1 and CuFi-1 were seeded into 96-well Primaria plates at a density of 1.5×105 cells/ml, and allowed to grow for 18–24 hr until confluent. Growth media was aspirated and replaced with fresh media containing 3O-C12 or DMSO vehicle. C38 and IB3-1 cells were seeded into 96-well plates (coated with BSA, collagen, and fibronectin as described above) at a density of 2.5×105 cells/ml, and were allowed to grow for 16–20 hr before confluent. Growth media was aspirated and replaced with fresh media containing BAPTA-AM (50 µM) or DMSO vehicle for 1 hr before the addition of 3O-C12. For all four cell lines, cell-free supernatants were collected 24 hr after the addition of 3O-C12 and either frozen at −80C for later cytokine quantification, or used immediately for LDH assays.
ELISA and LDH assays
Supernatants were assayed for IL-6 and IL-8 using Ready Set Go ELISA kits from eBioscience (San Diego, CA), according to the manufacturer's instructions. Cell cytotoxicity was analyzed through the quantification of lactate dehydrogenase (LDH) release using a commercial cytotoxicity kit (Roche Applied Science, Laval, QC, Canada) according to the manufacturer's instructions.
C38 and IB3-1 cell lines were seeded at a density of 2.5×105 cells per into 96-well view plates (Perkin-Elmer, Waltham, MA, USA) coated with BSA, collagen, and fibronectin as described above. Cells were allowed to rest for 16–20 hr, growth media was removed, and the calcium indicator dye Fluo-4 NW (Invitrogen) was used to load the cells for 1 hr according to the manufacturer's instructions. After loading the cells, two minutes of baseline fluorescence were was collected, and then cells were treated with EGTA (10 mM), ionomycin (10 µM), or 3O-C12 HSL. Fluorescence data was recorded every 50–60 sec with an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a Victor 3 (Perkin-Elmer). Intracellular concentration of calcium was calculated as described by Tabary et al.  using the formula: [Ca]i = Kd x ((F – FEGTA)/(FIonomycin – F) where Kd = 345 nM . Δ[Ca2+]i was defined as the intracellular calcium concentration in stimulated cells, less the intracellular calcium concentration in cells treated with DMSO vehicle, for a given time point. Fluorescence data from each well was normalized to its own baseline fluorescence before the addition of any treatment.
Systems biology and network analysis
Transcriptional responses of A549 cells stimulated with 3O-C12 HSL (50 µM) for 6 hr was recently examined using microarray by Bryan et al. . A list of differentially expressed genes with corresponding fold-changes and p-values was obtained from the paper's supplemental section, and utilized for in-depth systems biology analysis. Briefly, genes were uploaded to InnateDB and pathway and gene ontology (GO) term over-representation analysis was carried out as previously described . A list of protein level interactions between differentially expressed genes was obtained from InnateDB , as were first-order interactions for the transcription factors NFATC1 and NFATC2 (identified as important by the over-representation analysis). Network analysis was carried out as previously described , , by visualizing these interactions and then merging them into a single integrated network using Cytoscape (2.6.3 for Windows) and the software plugin Cerebral . The single most statistically significant sub-network was identified within the larger interaction network using the jActiveModule plugin for Cytoscape , , and the result was again visualized in Cerebral to appreciate network directionality within the system.
Statistical significance was determined using independent sample t-tests for comparisons of two treatments for a given cell line, whereas 2-way ANOVA followed by Tukey's post-hoc testing was used for comparisons of multiple conditions across two cell lines. Statistical analysis for in vitro cell stimulations was carried out using SPSS 17.0 for Windows. Over-representation analysis was carried out using InnateDB's default methodology (hypergeometric algorithm, Benjamini-Hochberg multiple testing p-value correction) , .
Sequential construction of the network graph of A549 transcriptional responses to 3O-C12. Microarray gene expression data  was uploaded to InnateDB, and protein-protein interactions were visualized in Cytoscape/Cerebral (A). Interaction networks were also constructed for protein-protein interaction for NFATC1 and NFATC2 using InnateDB, and visualized in Cytoscape/Cerebral (B). These two networks were merged into a single network (C) which was then used for subnetwork analysis with the Cytoscape plugin jActive (the results of which are shown in Figure 2).
Conceived and designed the experiments: MLM JAS REWH. Performed the experiments: MLM JAS CJB. Analyzed the data: MLM JAS. Contributed reagents/materials/analysis tools: SET REWH. Wrote the paper: MLM.
- 1. Levy H, Murphy A, Zou F, Gerard C, Klanderman B, et al. (2009) IL1B polymorphisms modulate cystic fibrosis lung disease. Pediatr Pulmonol 44: 580–593.
- 2. Muselet-Charlier C, Roque T, Boncoeur E, Chadelat K, Clement A, et al. (2007) Enhanced IL-1beta-induced IL-8 production in cystic fibrosis lung epithelial cells is dependent of both mitogen-activated protein kinases and NF-kappaB signaling. Biochem Biophys Res Commun 357: 402–407.
- 3. Tabary O, Boncoeur E, Martin R de, Pepperkok R, Clément A, et al. (2006) Calcium-dependent regulation of NF-(kappa)B activation in cystic fibrosis airway epithelial cells. Cell Signal 18: 652–660.
- 4. Blohmke CJ, Victor RE, Hirschfeld AF, Elias IM, Hancock DG, et al. (2008) Innate immunity mediated by TLR5 as a novel antiinflammatory target for cystic fibrosis lung disease. J Immunol 180: 7764–7773.
- 5. Ierano T, Cescutti P, Leone MR, Luciani A, Rizzo R, et al. (2010) The lipid A of Burkholderia multivorans C1576 smooth-type lipopolysaccharide and its pro-inflammatory activity in a cystic fibrosis airways model. Innate Immun 16: 354–365.
- 6. Ornatowski W, Poschet JF, Perkett E, Taylor-Cousar JL, Deretic V (2007) Elevated furin levels in human cystic fibrosis cells result in hypersusceptibility to exotoxin A-induced cytotoxicity. J Clin Invest 117: 3489–3497.
- 7. Erickson DL, Endersby R, Kirkham A, Stuber K, Vollman DD, et al. (2002) Pseudomonas aeruginosa quorum-sensing systems may control virulence factor expression in the lungs of patients with cystic fibrosis. Infect Immun 70: 1783–1790.
- 8. Singh PK, Schaefer AL, Parsek MR, Moninger TO, Welsh MJ, et al. (2000) Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 407: 762–764.
- 9. Möker N, Dean CR, Tao J (2010) Pseudomonas aeruginosa increases formation of multidrug-tolerant persister cells in response to quorum-sensing signaling molecules. J Bacteriol 192: 1946–1955.
- 10. Smith RS, Harris SG, Phipps R, Iglewski B (2002) The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)homoserine lactone contributes to virulence and induces inflammation in vivo. J Bacteriol 184: 1132–1139.
- 11. Imamura Y, Yanagihara K, Tomono K, Ohno H, Higashiyama Y, et al. (2005) Role of Pseudomonas aeruginosa quorum-sensing systems in a mouse model of chronic respiratory infection. J Med Microbiol 54: 515–518.
- 12. Hartman G, Wise R (1998) Quorum sensing: potential means of treating gram-negative infections? Lancet 351: 848–849.
- 13. Hentzer M, Wu H, Andersen JB, Riedel K, Rasmussen TB, et al. (2003) Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors. EMBO J 22: 3803–3815.
- 14. Bjarnsholt T, Gennip M van, Jakobsen TH, Christensen LD, Jensen PØ, et al. (2010) In vitro screens for quorum sensing inhibitors and in vivo confirmation of their effect. Nat Protoc 5: 282–293.
- 15. Boontham P, Robins A, Chandran P, Pritchard D, Cámara M, et al. (2008) Significant immunomodulatory effects of Pseudomonas aeruginosa quorum-sensing signal molecules: possible link in human sepsis. Clin Sci (Lond) 115: 343–351.
- 16. Li H, Wang L, Ye L, Mao Y, Xie X, et al. (2009) Influence of Pseudomonas aeruginosa quorum sensing signal molecule N-(3-oxododecanoyl) homoserine lactone on mast cells. Med Microbiol Immunol 198: 113–121.
- 17. Jacobi C a, Schiffner F, Henkel M, Waibel M, Stork B, et al. (2009) Effects of bacterial N-acyl homoserine lactones on human Jurkat T lymphocytes-OdDHL induces apoptosis via the mitochondrial pathway. Int J Med Microbiol 299: 509–19.
- 18. Tateda K, Ishii Y, Horikawa M, Matsumoto T, Miyairi S, et al. (2003) The Pseudomonas aeruginosa Autoinducer N-3-Oxododecanoyl Homoserine Lactone Accelerates Apoptosis in Macrophages and Neutrophils. Infect Immun 71: 5785–5793.
- 19. Kravchenko VV, Kaufmann GF, Mathison JC, Scott DA, Katz AZ, et al. (2008) Modulation of gene expression via disruption of NF-kappaB signaling by a bacterial small molecule. Science 321: 259–263.
- 20. Smith RS, Kelly R, Iglewski BH, Phipps RP (2002) The Pseudomonas autoinducer N-(3-oxododecanoyl) homoserine lactone induces cyclooxygenase-2 and prostaglandin E2 production in human lung fibroblasts: implications for inflammation. J Immunol 169: 2636–2642.
- 21. Smith RS, Fedyk ER, Springer TA, Mukaida N, Iglewski BH, et al. (2001) IL-8 production in human lung fibroblasts and epithelial cells activated by the Pseudomonas autoinducer N-3-oxododecanoyl homoserine lactone is transcriptionally regulated by NF-kappa B and activator protein-2. J Immunol 167: 366–374.
- 22. Sbarbati A, Tizzano M, Merigo F, Benati D, Nicolato E, et al. (2009) Acyl homoserine lactones induce early response in the airway. Anat Rec (Hoboken) 292: 439–448.
- 23. Zimmermann S, Wagner C, Müller W, Brenner-Weiss G, Hug F, et al. (2006) Induction of neutrophil chemotaxis by the quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone. Infect Immun 74: 5687–5692.
- 24. Bérubé J, Roussel L, Nattagh L, Rousseau S (2010) Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function enhances p38 and ERK MAPKs activation increasing IL-6 synthesis in airway epithelial cells exposed to Pseudomonas aeruginosa. J Biol Chem 285: 22299–22307.
- 25. Borgatti M, Bezzerri V, Mancini I, Nicolis E, Dechecchi MC, et al. (2007) Induction of IL-6 gene expression in a CF bronchial epithelial cell line by Pseudomonas aeruginosa is dependent on transcription factors belonging to the Sp1 superfamily. Biochem Biophys Res Commun 357: 977–983.
- 26. Delgado MA, Poschet JF, Deretic V (2006) Nonclassical pathway of Pseudomonas aeruginosa DNA-induced interleukin-8 secretion in cystic fibrosis airway epithelial cells. Infect Immun 74: 2975–2984.
- 27. Joseph T, Look D, Ferkol T (2005) NF-kappaB activation and sustained IL-8 gene expression in primary cultures of cystic fibrosis airway epithelial cells stimulated with Pseudomonas aeruginosa. Am J Physiol Lung Cell Mol Physiol 288: L471–L479.
- 28. Blohmke CJ, Park J, Hirschfeld AF, Victor RE, Schneiderman J, et al. (2010) TLR5 as an anti-inflammatory target and modifier gene in cystic fibrosis. J Immunol 185: 7731–7738.
- 29. Stoltz Da, Ozer Ea, Ng CJ, Yu JM, Reddy ST, et al. (2007) Paraoxonase-2 deficiency enhances Pseudomonas aeruginosa quorum sensing in murine tracheal epithelia. Am J Physiol Lung Cell Mol Physiol 292: L852–L860.
- 30. Zhu H, Conibear TCR, Thuruthyil SJ, Willcox MDP (2008) Pseudomonas aeruginosa quorum-sensing signal molecules induce IL-8 production by human corneal epithelial cells. Eye Contact Lens 34: 179–181.
- 31. Cooley MA, Whittall C, Rolph MS (2010) Pseudomonas signal molecule 3-oxo-C12-homoserine lactone interferes with binding of rosiglitazone to human PPARgamma. Microbes infect 12: 231–237.
- 32. Horke S, Witte I, Altenhöfer S, Wilgenbus P, Goldeck M, et al. (2010) Paraoxonase 2 is down-regulated by the Pseudomonas aeruginosa quorumsensing signal N-(3-oxododecanoyl)-L-homoserine lactone and attenuates oxidative stress induced by pyocyanin. Biochem J 426: 73–83.
- 33. Bryan A, Watters C, Koenig L, Youn E, Olmos A, et al. (2010) Human transcriptome analysis reveals a potential role for active transport in the metabolism of Pseudomonas aeruginosa autoinducers. Microbes Infect 12: 1042–1050.
- 34. Ideker T, Ozier O, Schwikowski B, Siegel AF (2002) Discovering regulatory and signalling circuits in molecular interaction networks. Bioinformatics 18: Suppl 1S233–S240.
- 35. Tan K, Tegner J, Ravasi T (2008) Integrated approaches to uncovering transcription regulatory networks in mammalian cells. Genomics 91: 219–231.
- 36. Shiner EK, Terentyev D, Bryan A, Sennoune S, Martinez-Zaguilan R, et al. (2006) Pseudomonas aeruginosa autoinducer modulates host cell responses through calcium signalling. Cell Microbiol 8: 1601–1610.
- 37. Elizur A, Cannon CL, Ferkol TW (2008) Airway inflammation in cystic fibrosis. Chest 133: 489–495.
- 38. Miyairi S, Tateda K, Fuse ET, Ueda C, Saito H, et al. (2006) Immunization with 3-oxododecanoyl-L-homoserine lactone-protein conjugate protects mice from lethal Pseudomonas aeruginosa lung infection. J Med Microbiol 55: 1381–1387.
- 39. Charlton TS, Nys R de, Netting a, Kumar N, Hentzer M, et al. (2000) A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatography-mass spectrometry: application to a model bacterial biofilm. Environ Microbiol 2: 530–541.
- 40. Gomperts BN, Belperio JA, Rao PN, Randell SH, Fishbein MC, et al. (2006) Circulating progenitor epithelial cells traffic via CXCR4/CXCL12 in response to airway injury. J Immunol 176: 1916–1927.
- 41. Palfreyman RW, Watson ML, Eden C, Smith AW (1997) Induction of biologically active interleukin-8 from lung epithelial cells by Burkholderia (Pseudomonas) cepacia products. Infect Immun 65: 617–622.
- 42. Hoffmann E, Dittrich-Breiholz O, Holtmann H, Kracht M (2002) Multiple control of interleukin-8 gene expression. J Leukoc Biol 72: 847–855.
- 43. Kravchenko VV, Kaufmann GF, Mathison JC, Scott D a, Katz AZ, et al. (2006) N-(3-oxo-acyl)homoserine lactones signal cell activation through a mechanism distinct from the canonical pathogen-associated molecular pattern recognition receptor pathways. J Biol Chem 281: 28822–28830.
- 44. Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI (1997) Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature 386: 855–858.
- 45. Schwarzer C, Wong S, Shi J, Matthes E, Illek B, et al. (2010) Pseudomonas aeruginosa homoserine lactone activates store-operated cAMP and CFTR-dependent Cl secretion by human airway epithelia. J Biol Chem 285: 34850–34863.
- 46. Vikström E, Bui L, Konradsson P, Magnusson K-E (2010) Role of calcium signalling and phosphorylations in disruption of the epithelial junctions by Pseudomonas aeruginosa quorum sensing molecule. Eur J Cell Biol 89: 584–597.
- 47. Martino MEB, Olsen JC, Fulcher NB, Wolfgang MC, O'Neal WK, et al. (2009) Airway epithelial inflammation-induced endoplasmic reticulum Ca2+ store expansion is mediated by X-box binding protein-1. J Biol Chem 284: 14904–14913.
- 48. Ribeiro CMP, Paradiso AM, Schwab U, Perez-Vilar J, Jones L, et al. (2005) Chronic airway infection/inflammation induces a Ca2+i-dependent hyperinflammatory response in human cystic fibrosis airway epithelia. J Biol Chem 280: 17798–17806.
- 49. Wu H, Song Z, Hentzer M, Andersen JB, Molin S, et al. (2004) Synthetic furanones inhibit quorum-sensing and enhance bacterial clearance in Pseudomonas aeruginosa lung infection in mice. J Antimicrob Chemother 53: 1054–1061.
- 50. Amara N, Mashiach R, Amar D, Krief P, Spieser SAH, et al. (2009) Covalent inhibition of bacterial quorum sensing. J Am Chem Soc 131: 10610–10619.
- 51. Flotte TR, Afione SA, Solow R, Drumm ML, Markakis D, et al. (1993) Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter. J Biol Chem 268: 3781–3790.
- 52. Zabner J, Karp P, Seiler M, Phillips SL, Mitchell CJ, et al. (2003) Development of cystic fibrosis and noncystic fibrosis airway cell lines. Am J Physiol Lung Cell Mol Physiol 284: L844–L854.
- 53. Gee KR, Brown K a, Chen WN, Bishop-Stewart J, Gray D, et al. (2000) Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes. Cell Calcium 27: 97–106.
- 54. Lynn DJ, Winsor GL, Chan C, Richard N, Laird MR, et al. (2008) InnateDB: facilitating systems-level analyses of the mammalian innate immune response. Mol Syst Biol 4: 218.
- 55. Lynn DJ, Chan C, Naseer M, Yau M, Lo R, et al. (2010) Curating the innate immunity interactome. BMC Syst Biol 4: 117.
- 56. Mookherjee N, Hamill P, Gardy J, Blimkie D, Falsafi R, et al. (2009) Systems biology evaluation of immune responses induced by human host defence peptide LL-37 in mononuclear cells. Mol Biosyst 5: 483–496.
- 57. Lee SMY, Gardy JL, Cheung CY, Cheung TKW, Hui KPY, et al. (2009) Systems-level comparison of host-responses elicited by avian H5N1 and seasonal H1N1 influenza viruses in primary human macrophages. PLoS One 4: e8072.
- 58. Barsky A, Gardy JL, Hancock RE, Munzner T (2007) Cerebral: a Cytoscape plugin for layout of and interaction with biological networks using subcellular localization annotation. Bioinformatics 23: 1040–1042.