Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl) homoserine lactone attenuates lipopolysaccharide-induced inflammation by activating the unfolded protein response.

N-3-oxododecanoyl homoserine lactone (3-oxo-C12-HSL), a quorum-sensing signal molecule produced by Pseudomonas aeruginosa (P. aeruginosa), is involved in the expression of bacterial virulence factors and in the modulation of host immune responses by directly disrupting nuclear factor-κB (NF-κB) signaling and inducing cell apoptosis. The unfolded protein response (UPR) triggered by endoplasmic reticulum (ER) stress may suppress inflammatory responses in the later phase by blocking NF-κB activation. It was recently demonstrated that 3-oxo-C12-HSL may induce UPR in human aortic endothelial cells (HAECs). Therefore, 3-oxo-C12-HSL may also inhibit NF-κB activation and suppress inflammatory responses by activating UPR. However, the possible underlying mechanism has not been fully elucidated. Accordingly, we investigated the effects of 3-oxo-C12-HSL on cellular viability, UPR activation, lipopolysaccharide (LPS)-induced NF-κB activation and inflammatory response in the RAW264.7 mouse macrophage cell line. Treatment with 6.25 μM 3-oxo-C12-HSL was not found to affect the viability of RAW264.7 cells. However, pretreating RAW264.7 cells with 6.25 μM 3-oxo-C12-HSL effectively triggered UPR and increased the expression of UPR target genes, such as CCAAT/enhancer-binding protein β (C/EBP β) and CCAAT/enhancer-binding protein-homologous protein (CHOP). The expression of C/EBP β and CHOP was found to be inversely correlated with LPS-induced NF-κB activation. 3-Oxo-C12-HSL pretreatment was also shown to inhibit LPS-stimulated proinflammatory cytokine production. Hence, 3-oxo-C12-HSL may attenuate LPS-induced inflammation via UPR-mediated NF-κB inhibition without affecting cell viability. This may be another mechanism through which P. aeruginosa evades the host immune system and maintains a persistent infection.

Pseudomonas aeruginosa is a ubiquitous bacterium present in the soil and water. P. aeruginosa is also an opportunistic pathogen that infects patients with the genetic disease cystic fibrosis (CF) 2 and also patients with burns or compromised immune functions. P. aeruginosa use N-(3-oxododecanoyl)-homoserine lactone (C12) and N-(3-oxobutyryl)-homoserine lactone (C4), so-called quorum-sensing molecules, to communicate with each other by activating the lasI/rhlI and lasR/rhlR regulatory gene networks (1)(2)(3). Activation of these regulatory genes leads to production of biofilms, masses of very large numbers and densities of bacteria (Ͼ10 9 cfu/ml), which are encased in a matrix of polysaccharides and DNA and other components and that are extremely difficult to clear (4 -6). Although concentrations of C12 in the airway surface liquid remain debated, there is some evidence indicating that the concentrations of the quorum-sensing molecules may reach high micromolar concentrations in or adjacent to biofilms (7)(8)(9).
C12, but not C4, also has multiple effects on host cells. Because it is lipid-soluble and membrane-permeant (10), C12 is likely capable of affecting all the cells near high densities of P. aeruginosa occur. In the lung airways this will include the airway epithelial cells, macrophages, neutrophils, fibroblasts, and endothelial cells lining nearby capillaries and smooth muscle cells in the serosa. C12 elicits increased expression (i.e. gene transcription) of proinflammatory mediators like IL8 (11,12). However, C12 also causes reduced secretion of these same proinflammatory mediators (13)(14)(15)(16). Recent work showed that these contradictory effects may be mediated through the effects of C12 to cause release of Ca 2ϩ from the ER, triggering ER stress and the ER stress-regulated kinase PERK, activation of IRE1␣, phosphorylation of eIF2a, and inhibition of protein (including cytokine and IB) synthesis and secretion (16). C12 also triggers apoptosis in multiple cell types (9,(17)(18)(19)(20)(21). That C12 activates apoptosis and not some other type of cell death has been documented from the multiple responses that are classically attributed to apoptosis: membrane blebbing, nuclear condensation and fragmentation, depolarization of mitochondrial membrane potential (⌬ mito ), release of cytochrome c from mitochondria into the cytosol, and activation of caspases 3/7, 8, and 9 and block by the pan-caspase inhibitor Z-VAD-fmk (9,22). A unique aspect of C12-triggered apoptosis was that it occurred equally well in fibroblasts from wild type mouse embryos and from Bax/Bak double knock-out (Bax Ϫ/Ϫ /Bak Ϫ/Ϫ ) mouse embryos (DKO MEF) (23). In the present paper we refer to these Bax/Bak double knock-out DKO MEF that were responsive to C12 as DKO R MEF. The molecular mechanisms involved in C12-triggered apoptosis have not been determined, but Haggie and colleagues (21) showed that activation of caspases and cell death required IRE1␣, splicing of XBP1, and production of XBP1s.
Although C12 causes apoptosis, ER stress, and altered inflammatory signaling and responses in many different types of cells in in vitro studies, it has been difficult to determine the physiological relevance of these effects, particularly whether P. aeruginosa secrete high enough concentrations of C12 to cause its characteristic responses. This apparent contradiction may result from the fact that airway and intestinal epithelia, both of which may be exposed to large numbers of P. aeruginosa under pathological conditions, express paraoxonase 2 (PON2) that has lactonase activity and can cleave C12 (24,25). It has been proposed that this cleavage reduces quorum sensing by the bacteria. This lactonase activity might also be expected to reduce effects of C12 on PON2-expressing cells.
During our recent study of DKO R MEF (23) we discovered that the uncloned pool of DKO MEF from which DKO R were isolated were non-responsive to C12, we called them DKO NR MEF. This paper first compares the Bax and Bak phenotypes and then caspase 3/7 responses of WT, DKO R , and DKO NR MEF. We then report results from RNAseq, Q-PCR, and Western blot analysis of the WT, DKO R , and DKO NR MEF. Although the DKO R MEF were isolated from the DKO NR MEF, RNAseq identified more than 5000 genes that were different between the two cell lines. By further comparison of WT and the two DKO lines, we identified PON2 as a gene of interest, although its expression was opposite from what might have been expected: DKO NR MEF expressed very low levels of PON2 mRNA and had protein below detection limits, whereas WT and DKO R MEF both expressed high levels of PON2 mRNA and protein.
We then tested whether adenoviral-mediated expression of human PON2 in DKO NR MEF caused them to become responsive to C12 in the apoptosis assays. We also tested the roles of PON2 versus PON2(H114Q) in C12-triggered apoptosis in human embryonic kidney (HEK293T) cells, which we found expressed only low levels of PON2 but could be transduced to stably express PON2 and PON2(H114Q) using a retrovirus infection approach.

EXPERIMENTAL PROCEDURES
Reagents-Unless otherwise specified, reagents and chemicals were obtained from Sigma. C12 was dissolved in dimethyl sulfoxide and frozen in separate vials, and then thawed for single experiments. JC1 and fura-2 were both purchased from Invitrogen and dissolved in dimethyl sulfoxide at 10 mM. Dyes were then diluted into solutions to give concentrations mentioned in the text. Anti-mouse PON2 were obtained from Antibodies-on-Line, Atlanta, GA, and anti-human PON2 from Abcam, Cambridge, MA.
Mouse Embryo Fibroblasts-Wild type (WT) and an uncloned population of Bak Ϫ/Ϫ Bax Ϫ/Ϫ (DKO) MEF immortalized by SV40 large T antigen expression have been described previously (23,42). DKO MEF cells were resistant to many proapoptotic stimuli. These DKO cells were not responsive to C12 and were termed DKO NR MEF. Single clonal cell lines of the DKO MEFs were acquired from the DKO NR by limited dilution. One DKO clone was selected for its ability to resist various proapoptotic stimuli. This line was termed DKO R MEF because these cells responded to C12, as described in our previous publication (23). WT, DKO R , and DKO NR MEF were cultured in DMEM/high glucose medium (Mediatech, Manassas, VA) with 10% (v/v) fetal bovine serum (Gemini, West Sacramento, CA), 100 units ml Ϫ1 of penicillin, and 100 g ml Ϫ1 of streptomycin (Mediatech). Cells were grown in a humidified 95/5% air/CO 2 incubator at 37°C. The cells were passaged at 1:5-1:10 dilutions and the remaining cell suspension was seeded directly onto 96-, 24-, or 12-well tissue culture plates (BD Falcon, Bedford, MA) or onto coverglasses for imaging.
Human Embryonic Kidney 293T Cells-HEK293T cells (ATCC, Bethesda MD) were obtained from the laboratory of Prof. E. Isacoff (University of California, Berkeley) and grown and passaged using methods as described above for MEF.
Adenoviral Expression of PON2-Adenovirus for EGFP, human CFTR, and human PON2 (hPON2) were obtained from the Gene Transfer Vector Core, University of Iowa. MEF were infected with 100 multiplicity of infection for 12 h, washed in medium, and used for experiments after 48 to 72 h post-adenoviral infection.
Cell Viability Assay-MEF cells were plated in 48-well tissue culture plates at the density of 2 ϫ 10 4 cells/well for 24 h. The indicated cell lines were treated with C12 or etoposide. Twentyfour hours later, cells were collected in the presence of 1 g/ml of propidium iodide (PI) and the percentage of live cells was measured by a PI exclusion method using flow cytometry analysis (FACScalibur, Beckon Dickinson, San Jose, CA) as described previously (23).
Retroviral Re-expression of Bak or Bax-Murine Bak cDNA or murine Bax cDNA was cloned into the retroviral expression vector pBABE-IRES-GFP with enhanced green fluorescent protein (EGFP) expressing from an internal ribosomal entry site (IRES). For retrovirus production, human embryonic kidney (HEK)-293T was transfected with the empty vector or the retroviral plasmid carrying the particular gene of interest along with helper plasmids pUVMC and pMDG2.0 using the jetPRIME transfection reagent (Polyplus-transfection, New York, NY). Retroviral supernatant was collected 48 -72 h after transfection with the addition of 10 g/ml of Polybrene (Sigma) to increase infection efficiency. Bak, Bax, Bak and Bax, or the control GFP alone was expressed in DKO NR MEF by retroviral infection. The expression of Bak and Bax was determined by Western blot.
Sample Preparation and RNA Sequencing-WT, DKO R , and DKO NR MEF were grown to confluence in 12-well plates (ϳ1 million cells/well; two wells of each cell line were used for RNAseq and Q-PCR analysis). Total RNA was isolated using TRIzol. RNA quality was assessed with the Agilent Bioanalyzer 2100 at the Functional Genomics Laboratory in University of California, Berkeley, and samples with RIN scores above 9 were used for cDNA production. One microgram of total RNA was used to isolate poly(A) purified mRNA, which was used for library construction by the Functional Genomics Laboratory. The average fragment sizes were ϳ400 bp. The library was sequenced with Ilumina HiSeq 2500 and each sample yielded 25 to 29 million 100-bp pair-end reads.
RNA Sequencing Read Alignment-RNAseq reads were mapped to the mouse reference genome (UCSC/mm9) using Tophat (43), and HTSeq (44) was used to sum the mapped reads to obtain gene expression levels. Read counts were normalized and differential expression between each sample pair was determined using DEseq (45). p value cutoff at p Ͻ 0.01 was used to define differential expression.
Retroviral Expression of PON2-The cDNA of wild type hPON2 was acquired from Origene (Rockville, MD), and the cDNA of hPON2 with the H114Q mutation was generated by gene synthesis (Biomatik, Wilmington, DE). Each cDNA was cloned into the retroviral expression vector pBabe-puro. The identity of the plasmids was confirmed by sequencing. To generate retrovirus, HEK293T cells were transfected with the empty vector or pBabe-puro carrying wild type PON2 or PON2(H114Q) as well as the helper plasmids (pUVMC and pMDG2.0) using the jetPRIME transfection reagent. Medium containing retrovirus was obtained 48 -72 h following transfection with 10 g/ml of Polybrene added to the medium to enhance infection efficiency. HEK293T cells stably expressing wild type PON2 or PON2(H114Q) were acquired by retroviral infection and culturing the cells in medium containing 1 g/ml of puromycin. PON2 expression was examined by Western blot.
Western Blots-Whole cell lysates with an equal number of cells (5 ϫ 10 4 ) were loaded on a 4 -12% BisTris gel (Bio-Rad) and transferred onto PVDF membrane (Millipore). The membrane was incubated with the appropriate primary or secondary antibodies either at 4°C overnight or at room temperature for 3 h in blotting buffer (1ϫ PBS ϩ 0.2% Tween 20) with 10% (w/v) nonfat dry milk (Bio-Rad). Protein levels were detected using the enhanced chemiluminescent detection system (Pierce). Antibodies used for Western blot analysis were anti-␤-actin mAb (Sigma), anti-Bak pAb (Enzo, Farmingdale, NY), anti-Bax pAb (Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse PON2 (Antibodies-on-line), and anti-human PON2 (Abcam). Western blots were quantitated using densitometry (ImageJ; NIH, Bethesda, MD) by measuring the intensity of PON2 bands relative to actin, and then calculating the ratio of PON2/actin in PON2-expressing MEF or HEK293T cells to PON2/actin in parental cells.
Assay for Caspase 3/7-Caspase 3/7 activities were measured by cell-based homogeneous luminescent assays (Caspase-Glo, Promega, Madison, WI), in which a specific substrate that contains the tetrapeptide DEVD (specific for caspase 3/7) was cleaved by the activated caspases from the cells to release aminoluciferin reacting with the luciferase and resulting in the production of light (9,23). WT and DKO MEF and HEK293T cells were plated on a clear-bottom, white 96-well plate in 100 l of medium per well for 3-4 days until they were confluent. For the cells that were infected with adenovirus, the infection was done at 18 h after the cells were plated and then the cells were left to grow another 48 h. During the experiment, cells were treated with C12 in the 37°C incubator for 120 min or left untreated as controls. On the same plate, some wells without cells but 100 l of the same medium served as blanks. After treatment, 100 l of reagent was added to each well with cells (treated or controls) and their medium, or blank (media only). The plate was incubated at room temperature for 1 h on a shaker, and the end point luminescence was measured in a plate-reading luminometer (LmaxII 384, Molecular Devices, Sunnyvale, CA). Data were background (blank) subtracted and averaged.
Imaging Measurements of ⌬ mito and Ca cyto -For measurement of ⌬ mito , MEF or HEK293T cells were incubated with medium containing the probe JC-1 (10 M) for 10 min at room temperature and then washed three times with Ringer solution. The Ringer solution had the following composition (in mM): 145 NaCl, 5 KCl, 1.2 NaH 2 PO 4 , 5.6 glucose, 2.0 CaCl 2 , 1.2 MgCl 2 , and 10 HEPES, pH 7.4. Dye-loaded cells were mounted onto a chamber on the stage of a wide-field or a confocal imaging microscope and maintained at room temperature. Treatment consisted of diluting stock solutions into Ringer solution. Control experiments showed that equivalent amounts of dimethyl sulfoxide (0.1%) used to dissolve C12 did not affect the JC-1 signal. Real-time imaging measurements of ⌬ mito were performed using equipment and methods that have been reported previously (9). Briefly, a Nikon Diaphot inverted microscope with a ϫ40 Neofluar objective (1.4 numerical aperture) was used. A charge coupled device camera collected JC-1 emission images (green, 510 -540 nm; red, 580 -620 nm) during excitation at 490 Ϯ 5 nm using filter wheels (Lambda-10, Sutter Instruments, Novato, CA). Axon Imaging Workbench 4.0 (Axon Instruments, Foster City, CA) controlled the filters and collection of data. Images were corrected for background (region without cells). Under control conditions, mitochondria exhibited red and green fluorescence of JC-1. C12 and the protonophore FCCP (10 M) caused reductions of JC-1 red fluorescence and increases in JC-1 green fluorescence consistent with depolarization of mitochondria. When cells were treated with FCCP to elicit maximal depolarization of ⌬ mito , JC-1 red fluorescence decreased to very low levels, and JC-1 green fluorescence increased to maximal intensity until the dye leaked out of the cytosol into the bath solution. Quantitative data are reported as fluorescence intensities normalized to minimal JC1 ratios obtained at the start of the experiment and maximal JC1 ratios obtained after treatment with 10 M FCCP.
For measurements of cytosolic Ca 2ϩ (Ca cyto ) cells grown on cover glasses were incubated with growth medium containing 2 M fura-2/AM for 40 -60 min at room temperature and then washed three times with Ringer solution. Fura-2-loaded cells were mounted onto a chamber on the stage of the imaging microscope and maintained at room temperature. Treatments with agonists were made by diluting stock solutions into Ringer solution at the concentrations stated in the text. Fluorescence ratio imaging measurements of Ca cyto were performed using equipment as described above and methods reported previously (46,47). Emission (Ͼ510 nm) images were collected during alternate excitation at 350 Ϯ 5 and 380 Ϯ 5 nm. Images were corrected for background (region without cells), and 380:350 ratios (a measure of Ca cyto ) were normalized to the maximal value obtained in 10 mM CaCl 2 Ringer's ϩ 10 M ionomycin and minimal values obtained in Ca 2ϩ -free Ringer's ϩ 100 M EGTA ϩ 10 M ionomycin at the end of each experiment (48). (23) showed: (i) WT MEF expressed Bax and Bak, whereas DKO R MEF did not. (ii) DKO R and WT MEF were equivalently sensitive to C12 in terms of depolarization of ⌬ mito , release of cytochrome c from mitochondria, activation of caspases and cell death. It was concluded that C12 triggered Bax and Bakindependent apoptosis in DKO R MEF, a unique process that included release of cytochrome c from mitochondria.

Characterization of DKO NR MEF-Previous experiments
During the course of those previous experiments, we discovered that the original pool of DKO MEF from which the clone of DKO R MEF was isolated (23) was non-responsive to C12. We termed these DKO NR MEF. As shown from Q-PCR and Western blot analyses (Fig. 1, A and B), both DKO NR and DKO R MEF were similar in that they expressed no detectable Bax or Bak, whereas WT MEF did. As reported previously (23), DKO R MEF were relatively unaffected by the common proapoptotic stimulants staurosporine and etoposide, and retroviral-mediated expression of Bak or Bax or both Bak and Bax rendered them sensitive to staurosporine and etoposide. DKO NR MEF Ϯ Bax and/or Bak expression behaved similarly in their responses to staurosporine and etoposide (Fig. 2, A and B).
DKO NR and DKO R MEF were similar in that both showed low responses to conventional proapoptotic stimuli except following transfection with Bax or Bak. However, they behaved very differently from each other in their responses to C12. C12 increased caspase 3/7 activity and caused cell death (PI uptake) in WT and DKO R MEF (23), but C12 had no effect on DKO NR MEF (Fig. 3, A and B). C12 also had no effect on activating caspase 3/7 or cause PI uptake in DKO NR MEF that had been transduced with the retrovirus to express Bax, Bak, or Bax ϩ Bak (data not shown).
RNAseq Analysis of WT, DKO R , and DKO NR MEF-One potential explanation for the fact that C12 triggered apoptosis in DKO R but not in DKO NR MEF was that DKO NR MEF did not express key gene(s) required for the action of C12. We performed RNAseq analysis to identify differences in expression (transcription) of DKO R and DKO NR MEF, and also included WT MEF for comparison.
Results from these experiments are presented in the Venn diagram in Fig. 4 and in Table 1. Of the 14,303 expressed genes,

PON2 Required for C12-triggered Apoptosis
a total of 8407 genes were expressed above background in WT, DKO R , and DKO NR MEF (Fig. 4). 1729 were expressed selectively in WT, 805 were expressed selectively in DKO NR , and 1161 were expressed selectively in DKO R . Thus, both DKO R and DKO NR MEF had different gene expression profiles from WT MEF, and DKO R and DKO NR MEF also had different gene expression profiles from each other, even though clonal DKO R MEF were isolated from uncloned DKO NR MEF.
Because WT and DKO R MEF both responded to C12, whereas DKO NR MEF did not, we reasoned that candidate genes that were required for C12 to trigger apoptosis would be one of the 510 genes expressed in both WT and DKO R MEF but not in DKO NR MEF (Fig. 4). Many of the genes that exhibited the most significant differences between WT and DKO R MEF on the one hand and DKO NR MEF on the other (Table 1) were associated with cancer and/or growth regulation. Among these cancer-related genes, PON2 exhibited the most significantly different expression in DKO R MEF (high expression) compared with DKO NR MEF (low expression) (Table 1). PON3, which is thought to have similar antioxidant, anti-inflammatory, and anti-apoptotic functions as PON2 (31,32), ranked 10 in this category of genes that exhibited the most significantly different expression in DKO R MEF versus DKO NR MEF. PON1 was not detected in MEF (0 mapped reads in all sequenced samples). Because PON2 hydrolyzes lactones including C12 (25)(26)(27)(28)(29)(30), it was unexpected that PON2 was expressed at high levels in WT and DKO R MEF, which are sensitive to C12, but at low levels in DKO NR MEF, which are nonresponsive to C12. Although PON2 and PON3 would be expected to reduce, not enhance, proapoptotic effects of C12, there were reasons to think that previously unrecognized C12-PON2 interactions might be involved in the apparent proapoptotic effects of PON2 and therefore would be worth pursuing. For example, PON2 might cleave C12 into a potent proapoptotic molecule or C12 may inhibit the antiapoptotic activity of PON2 (49). We focused on PON2 for the rest of this study.
RNAseq data were confirmed by measuring expression of PON2 in the MEF. Results from Q-PCR (Fig. 5A) and Western blots (Fig. 5B) confirmed RNAseq data showing that PON2 was expressed at high levels in WT and DKO R MEF and at low levels in DKO NR MEF.  (n ϭ 3). B, DKO NR MEF or DKO NR MEF retrovirally re-expressing GFP, Bak, Bax, or Bak ϩ Bax were left untreated or incubated with etoposide (10 M) for 24 h, then exposed to PI and processed by FACS to measure cell viability. *, p Ͻ 0.05 versus control.

C12 on Caspase 3/7, ⌬ mito , and Ca cyto in DKO NR Versus
PON2-expressing DKO NR MEF-We tested whether PON2 was required for C12 to activate apoptosis by comparing responses of DKO NR and DKO NR MEF that had been treated with an adenovirus expressing human PON2 or GFP (control). Q-PCR confirmed that DKO NR MEF treated with adenovirus-GFP expressed characteristically low levels of mPON2, whereas adenovirus-hPON2-treated MEF expressed high levels of PON2 (Fig. 6A). C12 had no effect on caspase 3/7 activity in DKO NR MEF expressing GFP, whereas C12 caused potent activation of caspase 3/7 in DKO NR MEF expressing hPon2 (Fig. 6B).
Previous data showed that one of the earliest responses to C12 in all cell types tested (including both WT and DKO R MEF) was a slow but persistent depolarization of ⌬ mito (9, 23). We tested for C12-induced changes in ⌬ mito in DKO NR MEF that had been treated with an adenovirus to express hCFTR (ϭ control) and DKO NR MEF that were treated with adenovirus to express hPON2. Results from these experiments (Fig. 7, A-C) showed that C12 had essentially no effect on ⌬ mito in DKO NR MEF but caused a slow, persistent and nearly complete depolarization of ⌬ mito in DKO NR MEF that expressed hPON2.
Another early indicator of responses to C12 is the release of Ca 2ϩ from the ER into the cytosol (9,19). This was tested by measuring Ca cyto . These experiments (Fig. 8, A-C) showed that C12 caused a small, slow increase in Ca cyto in DKO NR MEF that were treated with an adenovirus to express hCFTR (ϭ control), whereas subsequent addition of thapsigargin (blocks Ca 2ϩ -ATPase in the ER) (50) caused a rapid, large increase in Ca cyto . In DKO NR MEF treated with adenovirus to express hPON2, C12 caused an immediate, large increase in Ca cyto , and further addition of thapsigargin did not alter Ca cyto . Data in Fig. 8, A-C, were therefore consistent with the idea that C12 had little or no effect to release Ca 2ϩ from the thapsigargin-releasable pool in the ER of DKO NR MEF. In contrast, adenoviral-mediated expression of hPON2 caused DKO NR MEF to release Ca 2ϩ from the ER into the cytosol in response to C12. Overall, data in     Figs. 6 -8 were consistent with the idea that expression of hPON2 in DKO NR MEF caused them to respond to C12 by activating characteristic responses associated with apoptosis: activation of caspase 3/7, depolarization of ⌬ mito , and release of Ca 2ϩ stored in the ER into the cytosol. PON2-dependent Proapoptotic Effects of C12 on HEK293T Cells-Preliminary experiments indicated that C12 had little effect on the morphology of HEK293T cells. We hypothesized that these cells lacked PON2. Western blots showed that HEK293T cells indeed expressed little PON2 (Fig. 9A). These cells were transduced to express empty vector, PON2, or lactonase-deficient mutant PON2(H114Q), and Western blots confirmed that the transduced cells expressed similar levels of wild type PON2 or PON2(H114Q) (Fig. 9A).
These cells were also tested for their responses to C12. Killing assays (PI uptake) during treatments with 0 (control), 12.5, 25, and 50 M C12 showed that compared with responses of vector control HEK293T cells, C12 induced significantly larger, concentration-dependent cell killing in HEK293T-PON2 cells but not in HEK293T cells expressing the lactonase mutant PON2(H114Q) (Fig. 9B).
As shown in Fig. 10, C12 (50 M) similarly caused much larger activation of caspase 3/7 in HEK293T cells expressing wild type PON2 compared with HEK293T cells expressing vector alone or the lactonase mutant PON2(H114Q). C12 also caused a similar activation of caspase 3/7 in vector-treated versus untreated HEK293T cells (data not shown).
Measurements of Ca cyto showed C12 had only small effects to release Ca 2ϩ from the ER into the cytosol in HEK293T-vector and HEK293T-PON2(H114Q) cells but much larger effects in HEK293T-PON2 cells. As shown for individual experiments in Fig. 12, A and C, and summarized in Fig. 12D, C12 had essentially no effect on Ca cyto , and subsequent treatment with thapsigargin (1 M) caused an immediate increase in Ca cyto . In contrast, C12 caused a larger, immediate effect on Ca cyto in HEK293T-PON2 cells, and subsequent treatment with thapsigargin had no effect (Fig. 12, B and D). These results indicated that C12 released all the Ca 2ϩ from the ER in HEK293T-PON2 cells but much less in HEK293T cells expressing vector alone or PON2(H114Q).

DISCUSSION
C12 triggered pro-apoptotic events (activation of caspase 3/7, release of cytochrome c from mitochondria and cell killing) in both WT and DKO R MEF (23), both of which express high levels of PON2, but not in DKO NR MEF, which express little PON2 (Table 1 and Fig. 5B). In contrast, C12 had no effect on DKO NR MEF that expressed very low levels of PON2. In addition, expression of hPON in DKO NR MEF caused these cells to become sensitive to C12: C12 had only small or no effects on ⌬ mito , Ca 2ϩ release from the ER, and activation of caspase 3/7 in DKO NR MEF, but had large effects in DKO NR MEF that expressed PON2. C12 also triggered caspase 3/7 activation, release of Ca 2ϩ from the ER, depolarization of ⌬ mito , and cell  killing in HEK293T cells that expressed wild type PON2 but had only small or no effects in HEK293T-vector or HEK293T-PON2(H114Q) cells. These data showed that expression of hPON2 with active lactonase activity was required for both human and mouse cells to activate characteristic proapoptotic responses to N-(3-oxododecanoyl)-homoserine lactone. The present data along with previous work (23) also showed that this C12-triggered, PON2-dependent apoptosis was unique in that it occurred without the involvement of Bax and Bak. The lack of C12-triggered responses in HEK293T cells transduced with retrovirus to express vector alone and in MEF transduced with adenovirus to express CFTR alone in combination with   MARCH 13, 2015 • VOLUME 290 • NUMBER 11 other data indicated that responses to C12 were mediated through PON2 and that the mode of expression (adenovirus transient versus retrovirus stable) and CFTR expression did not alter responses.

PON2 Required for C12-triggered Apoptosis
Another conclusion from these experiments was that DKO R and DKO NR MEF expressed a large number of genes differently, even though clonal DKO R cells were isolated from uncloned DKO NR cells. DKO R MEF expressed a large number of genes that were cancer-or growth-related, including both PON2 and PON3 (32,37,38). PON2 is overexpressed in a number of cancers, including leukemia, hepatocellular, and prostate (33)(34)(35)(36). DKO NR MEF expressed a mixture of cancer-related genes and other genes related to tissue differentiation and cell-cell signaling. These data showed the danger of assuming that cell lines that have been immortalized by SV40 large T antigen are genetically identical.
The apparent role of PON2 with lactonase activity to mediate or cause apoptosis in response to C12 in host cells was counterintuitive. PON2 appears to be protective against the common proapoptotic agonists doxorubicin and staurosporine and is also up-regulated in many cancers (32,39,40). In addition, the lactonase activity of PON2 cleaves C12, so DKO NR MEF (with low PON2) should have been more sensitive to C12, whereas DKO R (with high PON2) MEF should have been less sensitive to C12. However, the opposite was observed. Previous work (41) showed that inactivation of the lactonase activity of PON2 using PON2(H114Q) did not alter the ability of PON2 to prevent cellular oxidation, and PON2(H114Q) appeared to be more effective than wild type PON2 in preventing apoptosis in response to staurosporine and tunicamycin. Taken together, the present and previous (41) data indicate that PON2 uses its lactonase activity to trigger unique Bax/Bak-independent apoptosis in response to C12, but PON2 uses its lactonase activity to inhibit traditional Bax/Bak-dependent apoptosis in response to tunicamycin and doxorubicin.
PONs 1-3 all hydrolyze lactones and arylesters, although with different specificities (26,29). It has been proposed that the lactonase activity of PON2 serves an antibacterial function in the lungs and other epithelia that are exposed to bacteriaproduced quorum-sensing lactones like C12 (24,26,30,31). Indeed, increases in PON2 expression in airway epithelial cells lead to hydrolysis and inactivation of C12 in the medium bathing the cells (24,25). This hydrolysis is expected to reduce quorum-sensing by the bacteria and to reduce responses of host cells to C12. PON2 is thought to have a phospholipase-like structure (28) similar to that of PON1 (27), which may contribute to its ability to cleave a wide range of esters (26,29).
The molecular mechanism that connects the lactonase activity of PON2 to the unique suite of effects of C12 leading to Bax/Bak-independent apoptosis remains unknown. As summarized in Fig. 13, one possibility that emerges from the present data is that C12 is cleaved by PON2 in the ER (51) and mitochondria (52) into (a) secondary derivative(s) that then has wide-ranging effects. Because the C12 derivative is highly reactive, it is possible that multiple derivatives of C12 will be formed, and these may be the direct activators of multiple events in both the ER and mitochondria. PON2 is reported to localize in the ER with its lactonase and critical Ca 2ϩ -binding site, including His-114, oriented toward the lumen (51), so the C12 derivative would be expected to be produced inside the ER FIGURE 13. Proposed mechanism for C12-triggered, PON2-mediated activation of early events in ER and mitochondria of host cells. P. aeruginosa produces and releases C12 that enters host cells. PON2 use its lactonase activity to cleave the lactone ring of C12 into the shown derivative. In the ER, the active site of PON2 is located in the ER lumen, so the C12 derivative is produced inside the ER and then activates the inositol trisphosphate receptor to release Ca 2ϩ from the ER, leading to ER stress thereby stimulating apoptosis. In the mitochondria PON2 is associated with complex III and coenzyme Q on the inner mitochondrial membrane, and the C12 derivative is predicted to be formed close to and then inhibit the electron transport chain (ETC), leading to depolarization of ⌬ mito , and also cytochrome c, leading to its release across the outer mitochondrial membrane through Bax-and Bak-independent mechanisms (shown by ?) into the cytosol to trigger caspase 3 and subsequent apoptostic processes. Because the C12 derivative is highly reactive, it is possible that other derivatives of C12 will also be formed, and these may be the direct activators of events in both the ER and mitochondria. and then activate the inositol trisphosphate receptor (53) directly from the ER face or permeate the ER to act on the cytosolic side of the inositol trisphosphate receptor. Release of Ca 2ϩ from the ER will activate ER stress and changes in inflammatory signaling/response (11)(12)(13)(14)(15)(16). In mitochondria, PON2 is reported to localize on the inner mitochondrial membrane associated with complex III and coenzyme Q (52), so production of C12 derivatives would be expected to be produced near and then inhibit the electron transport chain and cytochrome c. These effects could lead to the observed depolarization of ⌬ mito and release and movement of cytochrome c from the inner mitochondrial membrane across the outer mitochondrial membrane into the cytosol to activate caspase 3 and downstream effects leading to apoptosis. It is also proposed that the C12 derivative induces pore formation in the outer mitochondrial membrane that mediates movement of cytochrome c into the cytosol without involvement of Bax or Bak.
Although speculative, the proposed model provides a useful summary of the data as well as a starting point for further tests of the role of the lactonase activity of PON2 in mediating the effects of C12 on host cells. It will be important to identify and then test the proposed C12 derivatives generated in the ER and mitochondria. Because PON3 was up-regulated in DKO NR and WT MEF compared with DKO R MEF similar to PON2 (Table 1) and PON3 may serve similar functions as PON2 (31,32), a direct test of whether PON3 is capable of mediating PON2-like responses to C12 is warranted. Because PON2 is expressed in the airways of CF patients (25,54) where C12 concentrations may reach high levels, tests of the role of PON2 in lung pathophysiology of CF patients could have important clinical implications.