Ian Ball Terry E. Machen Department of Molecular Cellular Biology 231 LSA University of California Berkeley Berkeley, CA 94720 The interplay of arachidonic acid and docosahexaenoic acid in the regulation of cytosolic redox state and in the transcription of NF-κB in CF-mutated airway epithelial cells Abstract A series of experiments were designed and executed in an effort to help untangle the highly complex interdependencies between the key chemical constituencies responsible for inflammatory responses in CF cells. In these experiments we sought to investigate the interplay between NF-κB transcription and concomitant changes in the cytosolic redox potential of JME/CF15 cells in response to the exogenous application of arachidonic acid (AA) and docosahexaenoic acid (DHA), two fatty acids which have been previously implicated in the regulation of the host innate immune response. Changes in the cytosolic redox state of cells were measured with use of a plasmid encoding a redox sensitive green fluorescent protein (roGFP) characterized by a redoxsensitive disulfide bridge between cysteines linking two adjacent β-sheets of the protein. Changes in the redox state of the cytosol affect the labile disulfide bridge and allow the GFP probe to assume an oxidized or reduced conformation, each of which has its own distinct excitation wavelength while maintaining a common emission wavelength at 535nm. Real-time changes in redox potentials induced by AA and DHA were measured and compared to changes in the luciferase reporter levels of NF-κB across similar conditions. Cytosolically-oxidizing species such as hydrogen peroxide (H2O2), AA, and DHA were found to mitigate the level of flagellin-induced NF-κB transcription while not being observed to independently alter NF-κB reporter levels in the absence of flagellin. Flagellin-induced NF-κB transcription was also shown to have been mitigated by COX2modifying ibuprofen. Taken together, these data suggest that species capable of eliciting an oxidation of the cytosol are also capable of mitigating provoked immune responses (flagellin), possibly through some oxidative effect which works to inhibit further transcription of NF-κB. Additionally, DHA, widely implicated as an agent working towards resolution of the immune response, was found to be a cytosolically-oxidizing species. Introduction The mechanism by which dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) gives rise to the chronic inflammation that characterizes the pathology of the disease cystic fibrosis remains unclear. Functional CFTR acts as an anion channel and is primarily responsible for the transport of chloride (Cl-), bicarbonate (HCO3-), and glutathione – an important regulator of a cell’s redox state. Freedman et al. have previously reported fatty acid abnormalities in the lung, pancreas, and ileum of cftr -/- mice. This abnormality is characterized by higher ratios of AA over DHA compared to those ratios observed in WT mice1. This abnormality has also been observed in humans with dysfunctional CFTR both in plasma fatty acids as well as in nasal and rectal biopsy specimens.2 Since the presence of these fatty acid abnormalities in CFTR-deficient tissue positively correlates to chronic or acute inflammation, the fatty acids AA and DHA are likely players in the regulation of the innate host immune response. It has additionally been observed that individuals with acute airway inflammation, but functional CFTR, also exhibit disproportionate and elevated AA/DHA ratios in the lipid membranes of biopsy specimens. This seems to indicate that acute inflammation alone is capable of altering the AA/DHA balance normally present in airway cells.3 Taken together, such research seems to strongly suggest that AA and DHA play an important role in the regulation of inflammation in systems with both functional and dysfunctional CFTR. AA is the central component of the arachidonic acid cascade whereby extracellular signals trigger phospholipases A2 and C (to a lesser extent) to cleave AA from the phospholipid layer of the cell membrane. Once cleaved, arachidonic acid can then be reincorporated back into the cell membrane, exported, or metabolized by cycloxygenase-2 (COX2) to yield a largely inflammatory series of leukotrienes and prostaglandins (PGE2’s). However, modified forms of COX2, most notably the aspirinacetylated COX2, can interact with secreted phospholipase A2 (PLA2)4 in the cytosol leading to the formation of a largely anti-inflammatory series of prostaglandins –– most notably the PGD2 series – which undergo dehydration in vivo to produce the biologically active J2 prostaglandin series which includes Δ12,14-PGJ2 and 15-deoxy- Δ12,14-PGJ2. These J2-series prostaglandins are now thought to play a role in the suppression of proinflammatory signaling such as NF-κB, IL-1β, and TNF-α.4 PGD2 has widely-varying biological roles in the body and has been implicated in sleep induction, the inhibition of platelet aggregation, and in smooth-muscle relaxation.5 However, since PGD2 seems to aggravate asthma-related inflammation,6, it appears that even largely-resolving species such as the PGD2 series have a dualistic and tissue-specific role in either the promotion or resolution of inflammation. Acetyl salicylic acid, or aspirin, can similarly acetylate COX2. Such acetylation modifies the functionality of COX2 enabling it to metabolize DHA and related eicosapentaenoic acid (EPA) to create 17R-hydroxy series docosanoids (resolvins) which have been shown to be powerful mediators in the resolution of localized hyper immune activity.7 The NF-κB pathway is an important pathway intimately connected with inflammation processes. NF-κB regulates the expression of pro-inflammatory cytokines, chemokines, and controls the expression of anti-apoptotic genes that are necessary for the maintenance of a sustained immune response.4 The presence of oxidizing reactive oxygen species in the cytosol is known to increase translocation of NF-κB from the cytosol to the nucleus where it acts as a transcription factor to upregulate a variety of pro-inflammatory genes. However, while translocation to the nucleus is increased, the DNA-binding ability of oxidized NF-κB is diminished.8 Utilization of a roGFP probe offers a more streamlined approach to measuring the cytosolic redox potential than older assays which primarily relied on delicate measurements of the glutathione/glutathione-disulfide or the reduced/oxidized thioredoxin ratios in cells. These measurements necessitated cellular destruction and precluded the possibility of observing real-time changes of intracellular redox potentials. Another advantage of using a genetic probe is the ease with which it can be modified by the addition of signal peptides which allow one to control the localization of the probe to various cellular compartments and organelles. NF-κB is often viewed as the central mediator of the immune response. NF-κB has been found to greatly promote the upregulation of key constituencies of the immune response such as inflammation-eliciting cytokines and chemokines, immune recognition receptors, and signals key to the regulation of programmed cell death, or apoptosis.9 Subsequently, by monitoring of NF-κB upregulation it is possible to ascertain the level of a cell’s immune response under an array of different conditions. Use of an NF-κBluciferase reporter-promoter construct allows the level of NF-κB upregulation in the cell to be measured via the resulting abundance of reporter – luciferase. Materials and Methods Tissue cultures Immortalized CF airway epithelial (JME/CF15) cells were selected for the both redox and luciferase studies because this cell line contains the highlycharacterized ΔF508 CF allele and because of the responsiveness of this cell line to transfection with the roGFP plasmid. The cells were grown in Corning cell culture dishes in a DMEM/F-12 culture media (CellGro) supplemented with 10% fetal bovine serum, 100U/mL ampicillin, 1% L-glutamine, 10ng/mL epidermal growth factor, 1 µM hydrocortisone, 5µg/mL insulin, 5µg/mL transferrin, 30nM triiodothyronine, 180mM adenine, 1% penstrip, and 5.5µM epinephrine. Cell cultures were maintained at 37ºC and 5% CO2 while the media was changed every 48hours. Cells were split onto 25mm glass cover slips for roGFP experiments and into standard 12-well plates for luciferase assay experiments. Cells were grown to confluence before all experiments. Calu-3 cells derived from human pulmonary adenocarcinoma cells were provided by the Widdicombe lab and were used for some experiments involving the NF-κβ-luciferase reporter construct. The calu-3 cell line, unlike the JME/CF15 cell line, expresses high levels of functional CFTR. Compared to JME/CF15 cells, calu-3 cells grew to confluence with greater uniformity and with cleaner, more defined monolayers than those observed with the JME/CF15 cell line. The slow growth of the calu-3 cells along with their resistance to transfection precluded their use in roGFP experiments. Calu-3 cells were grown under 5% CO2 and at 37ºC in DMEM/F-12 culture medium supplemented with 10% fetal bovine serum. Media was changed every 48 hours and cells were split every 12-16 days and into 12-well plates for experiments. Cells were grown to confluence before all experiments. Plasmids/Probe A variant of green florescent protein (GFP) engineered and developed by Dooley and Tsien et al. was used to measure cytosolic redox potentials in JME/CF15 cells. Two surface cysteine mutations close to the chromophore on adjacent β-sheets at positions 147 and 204 permit this modified protein to assume photochemically distinct and labile conformations corresponding to a reduced and oxidized state indicative of the cell’s redox state. This GFP construct had been subcloned into pcDNA3 using BamHI and EcoRI restriction sites. 10 The plasmid was cloned in E. coli and extracted using Qiagen plasmid preps. The isolated plasmid was then transfected into JME/CF15 cells using Qiagen Effectene reagents and their associated protocol as provided by Qiagen. Transfected cells (~20-30% efficiency) constitutively expressed the encoded GFP whose expression was driven by the Cauliflower Mosaic Virus promoter. While the oxidized form of the protein favors excitation at 385nm, the reduced form of the protein favors excitation at 474nm. Both conformations share a common emission wavelength at 535nm. By measuring the emission intensity elicited by the two excitation wavelengths (oxidized and reduced), it was possible to determine the relative cytosolic abundance of the oxidized probe over the reduced probe and to equate this difference to a redox potential. Formulae outlined by Dooley and Tsien et al. could be utilized to transmute these ratios into absolute cellular redox potentials. Plasmid Transfection JME/CF15 cells were transiently transfected using Qiagen Effectene reagents following the protocol outlined by Qiagen. All cells were seeded one day prior to transfection. Cells were ranged from 40-80% confluence before transfection. Microscopy Images of the cells and their respective excitation intensities were taken using a camera interfaced to a microscope utilizing an oil-emersion lens and a FITC 535 filter. The camera signal was amplified before being routed to analytical software, Imaging Workbench 4.0, where the data were processed. The excitation wavelengths were created by lamps capable of emitting at the two different excitation wavelengths. Measuring cytosolic redox response JME/CF15 cells were immobilized and covered with 100μL of Ringer’s solution. A view containing 5-10 transfected cells was chosen as a region for analysis during each experiment. The intensity of these regions’ emission wavelengths under excitation by each of the above referenced optical wavelengths were analyzed and charted by Imaging Workbench 4.0 throughout the experiment. Emission data from the transfected cells were collected on the order of every 60 seconds. At each experiment’s onset, the readings were allowed to stabilize for about 3-5 minutes before the application of arachidonic and docosahexaenoic acid and other experimental compounds. After each experiment a standardized curve was created by fully oxidizing the probe under 10mM H2O2. Once a maximal value was achieved, the cells were washed three times and then reduced using 5mM reducing dithiothreitol (DTT). After reaching a minimal redox value the cells were again washed before reapplying 100μL Ringers to allow the cells to make a final recovery to initial baseline redox values before the experiment was ended. NF-κβ-driven luciferase reporter construct Both calu-3 cells and JME/CF15 cells were used in these experiments. The primary purpose of utilizing the calu-3 cell line was to examine whether or not there was a consistent response to similar stimuli across two different cell lines in the roGFP experiments. Cells were infected with a recombinant adenovirus containing a NF-κβluciferase reporter construct prepared by Vector Core at the University of Iowa. The luciferase reporter gene’s transcription was driven by four tandem copies of the NF-κβ consensus binding sequence. The presence of the four tandem copies of the NF-κβ binding sequence ensured that the transcription of both NF-κβ and luciferase were directly proportional to one another. Viral stocks were aliquoted and stored in a solution of 10mM Tris and 20% glycerol at -80ºC. Cells were apically infected and incubated 48 hours before experiments while they were 6080% confluent. After 48 hours, the cells were subjected to experimental treatments for an incubation time of 4 hours before luciferase was harvested as per the protocol outlined by Promega. The luciferase was quantified in relative light units with triplicate measurements using a luminometer. A Bradford assay was then used to adjust the luminometer data to account for discrepancies in protein quantities present in the starting samples. Both luminometer and spectrometer readings were tabulated and analyzed using Microsoft Excel. Results Effects of AA and DHA on cytosolic redox potential: Arachidonic acid and docosahexaenoic acid (Sigma) were stored under N2 at -20ºC. Approximately 5 minutes before experiments, aliquots of these two acids were created in DMSO (redox inactive) prior to further dilution into Ringer’s for immediate use in experiments. roGFP experiments were then calibrated by oxidation (10mM H2O2) and subsequent reduction (5mM DTT) of the cytosol using known oxidants and reductants as outlined by Dooley and Tsien. Relative emission intensities corresponding to the two excitation wavelengths at 385 and 474nm were measured at 60s intervals using the imaging and data analysis software in Imaging Workbench 4.0. Both arachidonic and docosahexaenoic acid were observed to elicit a dosedependent increase in the oxidation of the cytosol in both CF15 and calu-3 cells. 100µM applications of AA and DHA to the apical surface of the cells changed the ratio of the emission ratio on the order of 0.1, indicating a net oxidation of the probe and cytosol. Application of PAK-derived flagellin, TNF-α and IL-8, known to be of considerable importance to the initiation and maintenance of the immune response, produced no discernable change in the redox state of the cytosol. Application of epinephrine, thought to promote cleavage of arachidonic acid from the cell membrane via stimulation of phospholipase C 11,12 and previously shown to activate phospholipases A and C in human platelets,13 also elicited no observable change in the cell’s cytosolic redox state. Each application of oxidation-inducing compound elicited an oxidation response with a similar characteristic shape – a sharp initial incline in oxidation followed by a gradual fettering out of this increase towards a flat signal eventually followed by a gradual reducing of the cytosol. Such a response is in line with observations made by Dooley and Tsien who similarly report on the natural, still largely uncharacterized reducing capabilities of the cytosol. Effects of AA, DHA, COX2 inhibitors on NF-κB-driven luciferase production A NF-κB-driven luciferase reporter construct was used to elucidate the expression of NF-κB during the redox active processes involved with the apical application of arachidonic acid and docosahexaenoic acid to JME/CF15 epithelial cells. Independently, neither AA (10-100µM) nor DHA (10-100µM) elicited an increase in NF-κB activation above control values. H2O2 also did not elicit any NF-κB activation above control values at concentrations ranging from 10-100µM. The null effect produced by AA on NF-κB transcription seems to be supported by observations made by Becuwe et al. who have similarly reported the absence of increased NF-κB reporter levels after exogenous application of arachidonic acid. However, using electrophoretic mobility shift assays to measure DNA binding, Becuwe et al. were able to observe that AA contributed to the activation, but not transcription of NF-κB.14 A concentration of 10-7g/mL flagellin caused a 4-fold increase in NF-κβ activation above baseline values. A ten-fold stronger concentration (10-6g/mL) of flagellin elicited a 12-fold increase in NF-κβ activation. Such flagellin-induced NF-κβ activations, we found, could be mitigated by incubating these cells with AA, DHA, or H2O2, in addition to flagellin. AA, DHA, and H2O2 all consistently mitigated flagellin-induced NF-κB activation. Similar mitigations of flagellininduced NF-κB transcription were observed across all tested concentrations of H2O2, AA, and DHA, all of which spanned a range from 10-100µM. ATP applied in addition to flagellin was shown to augment NF-κB activation above levels achieved for flagellin alone. ATP independently incubated with the cells also yielded higher luciferase levels compared to control levels. As flagellin is known to upregulate pro-inflammatory COX2, it seems to make sense that NF-κB activation was reduced in experiments where cells were treated with ibuprofen (a characterized COX2 modifier) in addition to flagellin. Additionally, inhibition of COX2 would slow or inhibit the largely inflammatory arachidonic acid cascade which can produce pro-inflammatory PGE2 prostaglandins in enzyme-catalyzed processes which also evolve reactive oxygen species. While redox active species, AA, DHA, and H2O2 did not independently elicit increases in NF-κB activation above control values, they did work towards mitigating the NF-κB upregulation induced by the application of flagellin. Figures 100uM DHA : Δredox = 0.1 (oxidation) 100uM DHA 0.83 0.78 385/474_ 0.73 0.68 0.63 0.58 0.53 0.48 0.43 0 1000 2000 3000 4000 5000 6000 Time (s) JME/CF15 cells ro-GFP probe Time (s) Treatment 800 100µM DHA 1900 1mM H2O2 2800 10mM H2O2 3100 wash 3x + 5mM DTT 5500 wash 3x Notes: cytosolic ro-GFP, JME Figure 1: DHA causes oxidation of the cytosol. Change in ratio of ~0.1 results after exogenous application of 100µM DHA to the apical side of JME/CF15 epithelial cells. Time is plotted against the ratio of the emissions at 535nm produced by the consecutive excitation of the roGFP probe by 385 and 474nm light at 60s intervals throughout duration of the experiment. 200uM DHA 0.8 0.75 385/474 _ 0.7 0.65 0.6 0.55 0.5 0.45 0.4 0 1000 2000 3000 4000 5000 Time (s) calu-3 cells Time (s) 550 2200 3300 4500 5200 ro-GFP probe Treatment 200µM DHA 1mM H2O2 10mM H2O2 wash 3x + 5mM DTT wash 3x Figure 2: DHA similarly causes oxidation in calu-3 cells. Change in ratio of ~0.2 results after exogenous application of 200µM DHA to the apical side of calu-3 epithelial cells. DHA oxidation of the cytosol is conserved across cell types, from JME/CF15 cells to calu-3 cells. A two-fold increase in concentration of DHA elicits a two-fold increase in oxidation compared to that produced using half this concentration. 200uM AA, 200uM DHA 0.8 0.75 385/474 _ 0.7 0.65 0.6 0.55 0.5 0 1000 2000 3000 4000 5000 6000 7000 Time (s) JME/CF15 cells Time (s) 200s 1350s 4000s 5000s 5500s ro-GFP JME Treatment 200µM AA 200µM DHA 1mM H2O2 10mM H2O2 wash 3x + 5mM DTT Figure 3: Arachidonic acid oxidizes the cytosol. Change in ratio of ~0.1 results after exogenous application of 200µM AA to the apical side of JME/CF15 epithelial cells. A change in ~0.2 also results when DHA is applied. AA appears to be a less potent oxidizer of the cytosol than DHA. After exposure to AA, the cytosol reaches a maximum oxidation and begins to again reduce on its own. This similarly happens after the application of DHA. The cytosol appears to have a natural buffering, or redox-regulatory capability which prevents prolonged periods of oxidation caused by species such as AA and DHA. NF-kB nondependence on AA and H2O2 across varying concentrations 5 4.5 Luciferase Activity _ Relative Control _ 4 3.5 3 2.5 2 1.5 1 0.5 0 CON.TREAT. H2O2 10uM H2O2 50uM H2O2 100uM AA 10uM AA 50uM AA 100uM Treatment JME/CF15 cells Figure 4: Cytosolically-oxidizing species H2O2 and AA do not promote the upregulation of NF-κB while flagellin does. Flagellin elicits an approximately 4-fold increase in NF-κB upregulation in JME/CF15 cells. Each bar represents a triplicate measurement of relative light units recorded for each treatment. Relative light units were normalized using a Bradford assay to account for possible discrepancies in the quantity of available protein that was harvested from the cells after experiments. Error bars are shown, but are small and not very easily resolved. Treatments were done in duplicate and normalized as a ratio to the control. flag 10^-7 NF-kB Driven Luciferase Activity _ 20 Luciferase Activity (RLU) 16 12 8 4 0 CON.TREAT. AA 10uM AA 50uM AA 100uM H2O2 10uM H2O2 50uM Treatment H2O2 100uM AA 100uM, flag 10^-6 AA 100uM, 100uM ibuprofen flag 10^-7, 100uM ibuprofen Figure 5: AA and ibuprofen appear to mitigate flagellin-induced NF-κB upregulation in JME/CF15 cells. Again, independent applications of AA and H2O2 in concentrations spanning from 10-100µM, do not elicit an upregulation of NF-κB. flag 10^-7 NF-kB Driven Luciferase Activity Relative Control 1.2 RLU Ratio to Control _ 1 0.8 0.6 0.4 0.2 0 flag 10^-7 flag 10^-7 + 10uM AA flag 10^-7 + 50uM AA flag 10^-7 + 10uM H2O2 flag 10^-7 + 50uM flag 10^-7 + 100uM H2O2 H2O2 Treatment Figure 6: AA and H2O2 consistently mitigate flagellin-induced NF-κB upregulation. NF-kB and AA, ATP, flagellin _ _ 7 Luciferase Activity Relative to Control 8 6 5 4 3 2 1 0 Figure 7: ATP promotes NF-κB upregulation both independently and on top of that already achieved by flagellin. AA again is seen to reduce flagellin-induced NF-κB activation. When ATP is added to flagellin in conjunction with AA, the mitigating potential of AA seems to be overcome by NF-κB transcription being promoted by ATP. AA+flag AA+flag AA+ATP+flag AA+ATP+flag flag 10^-6 flag 10^-6 ATP 100 ATP 100 AA 50 AA 50 control control Treatment Discussion: Metabolism of omega-6 arachidonic acid (AA) is a process which yields reactive oxygen species15 that contribute to the oxidation of the cytosol. Both AA and docosahexaenoic acid (DHA) are catabolized by the cell into various downstream metabolites, ones which work to both promote and resolve host inflammatory responses. In these experiments, AA and DHA caused considerable oxidation of the cytosol as measured by a redox-sensitive GFP probe. Hydrogen peroxide (H2O2) was also a very potent elicitor of cytosolic oxidation. All three cytosolically-oxidizing species, H2O2, AA, and DHA had a negligible effect on the upregulation of NF-κB above control values in experiments utilizing the NF-κB-luciferase reporter construct. Activation of NF-κB is a well-characterized event whereby NF- κB dissociates from NF-κB-inhibiting IκB, an event ultimately resulting in the nuclear localization of NF-κB. Once in the nucleus, NF-κB can associate with a wide array of other transcription cofactors in a process which allows NF-κB to facilitate the upregulation of a diverse set of genes involved in regulating and sustaining of the immune response. NFκB activation is a fairly rapid process (30-60min) that occurs soon after a cell is exposed to a proinflammatory element. Its rapid activation and upregulation of immune specific genes coding for such pro-inflammatory signals as cytokines and interleukins offers a rapid response to perceived extracellular threats. Being a redox-sensitive transcription factor activated by oxidative stress, one would expect NF-κB activity to increase after the application of cytosolically-oxidizing species such as AA, DHA, and H2O2. We, however, observed that this did not happen. There were no heightened levels of luciferase expression to indicate that NF-κB had been upregulated in the presence of these species. It is probably important to differentiate between NF-κB activation (previously described) and the NF-κB upregulation, or transcription, of NF-κB which we measured in this experiment via the luciferase reporter. In fact, it could be the case that application of these cytosolically-oxidizing species, H2O2, AA, and DHA, do in fact cause activation, but not transcription of NF-κB. Such an interpretation would be in line with research conducted by Becuwe and colleagues. They found using electrophoretic mobility shift assays that AA contributed to the activation, but not transcription of NFκB16. We seemed to assume that both activation and upregulation NF-κB were two processes that essentially worked in concert with one another. This explanation would work to explain what we initially found to be rather anomalous data concerning the apparent non-activation of NF-κB in our experiments. One might suppose that increased NF-κB activity produced by the application of pro-oxidizing species would upregulate several pro-inflammatory genes that would in turn promote the upregulation of NF-κB in a positive feedback mechanism, but this was not found to be the case in our experiments. NF-κB upregulation, however, was elicited by the exogenous application of flagellin. 10-7g/mL concentrations of flagellin elicited 4-fold increases in NF-κB transcription above levels observed for control samples. 10-6g/mL concentrations of flagellin similarly caused a 16-fold increase in NF-κB transcription. This follows from previous work done which has shown flagellin to upregulate NF-κB transcription through the mediation of a toll-like receptor 5 in intestinal epithelial cells.16 Apical application of flagellin has also been shown to upregulate 1525 out of 12 625 genes including genes including those of proinflammatory cytokines and interleukins, TNF-α, antibacterial factors, as well as NF-κB.17 Flagellin has also been shown to upregulate COX2, the enzyme necessary for conversion of arachidonic acid into PGH2, a necessary precursor to the subsequent production of other proinflammatory products of the arachidonic acid cascade. In our experiments, flagellin worked to dramatically induce NF-κB transcription, but was not observed to elicit an oxidation of the cytosol. Such heightened NF-κB transcription was mitigated by the application of AA, DHA, and H2O2 in experiments exploring flagellin-induced NF-κB transcription. Since AA, DHA, H2O2 all cause an oxidation of the cytosol, it is conceivable that this oxidation damages, or otherwise impairs NF-κB’s ability as a transcription factor to fulfill its proper role as a key mediator of the host immune response. As mentioned earlier, oxidation of NF-κB has been found to both increase its activity while simultaneously impairing its DNA binding ability. In conditions where oxidizing AA, DHA, and H2O2 are applied in addition to flagellin, it is possible that the lower luciferase readouts we observed for these experiments were due, in part, to this sensitivity of NF-κB to oxidation. But then if this were true, we would likely observe lower luciferase readings for cells solely treated with AA, DHA, and H2O2, however this was not observed. This might indicate that the sensitivity of NF-κB to oxidation only becomes significant where there are higher concentrations of NF-κB present in the cell, or that the small decrease in NF-κB transcription could only be resolved during targeted upregulation of NF-κB by use of flagellin. A third explanation would involve the heightened levels of COX2 present in flagellin-stimulated cells. Heightened levels of COX2, coupled to increased amounts of AA and DHA (applied exogenously) could facilitate the formation of the earlier-described D and J-series resolvins which would work to resolve the immune response, however usually COX2 requires acetylation prior to the generation of pro-resolving species in the case of AA and DHA. Since acetylation is usually achieved through the application of aspirin and since we know of no known means of endogenous acetylation of COX2, it is unlikely this could have happened. In fact, un-acetylated COX2 which metabolizes AA should give rise to pro-inflammatory downstream metabolites which one would expect to augment flagellin-induced transcription of NF-κB, however there was no discernable effect observed. Furthermore, the notion that recruitment of COX2 to manufacture immuneresolving species subsequent to application of AA, DHA, and H2O2 runs into trouble when we consider the role H2O2 plays in mitigating this flagellin-induced immune response. H2O2 is not known to metabolize into immune-resolving species. Because of these inconsistencies in the explanation invoking a role for COX2 to explain the mitigation of flagellin-induced NF-κB upregulation, it seems more likely that this process of NF-κB mitigation is operating via redox sensitive mechanism independent of COX2 processes involving NF-κB. The modification of COX2 by ibuprofen was shown to mitigate flagellin-induced NF-κB transcription (Figure 7) through a mechanism most likely involving the production of D and J-series resolvins by COX2. When ATP was applied to flagellinstimulated cells, the NF-κB response was augmented, even in the presence of AA. The NF-κB response was also elevated above control levels on cells solely treated with NFκB (Figure 7). These data seem to indicate that the presence of ATP is a fairly consistent means of inducing NF-κB activation. If the ATP is cell permeable, perhaps this operates through the increased intracellular availability of ATP which can be utilized for the phosphorylation of IκB subunits which would promote the translocation of NF-κB to the nucleus where it would initiate transcription of a host of pro-inflammatory genes which might in turn generate more NF-κB and the slightly higher levels of luciferase that we observed. ATP, however, was not a central player that we investigated in these experiments. In summary we found AA, DHA, and H2O2 to all induce oxidation of the cytosol. AA, DHA, and H2O2 acting alone elicited no observable increase in luciferase levels above control values. When AA, DHA, and H2O2 were applied in addition to flagellin, these cytosolically oxidizing species were found to mitigate flagellin-induced upregulation of NF-κB, possibly by means of the sensitivity of NF-κB to oxidation when present at elevated levels in the cytosol. Future experiments may want to further discriminate between NF-κB activity and transcription as a means of more definitively measuring the specific role played by NF-κB in mediating the responses we observed in our experiments. Also, the oxidation induced by AA, DHA, and H2O2 were transient increases in oxidation as the oxidation they each elicited was resolved over time. It may be interesting to test whether shorter incubation times for the luciferase experiments (2 hours, instead of 4) would yield more illuminating data. Shortening the incubation time to 2 hours would increase the time during incubation during which cells would be exposed to significantly elevated levels of oxidation before the cytosol has reduced back to baseline redox values. Such experiments may provide clearer insight into a possible involvement of the oxidation of the cytosol in the upregulation of NF-κB. Additionally, to further rule out the involvement of COX2 as a mechanism by which AA and DHA work to mitigate flagellin-induced NF-κB transcription, we could employ the use of COX2 inhibitors and hope to observe a similar response. There are clearly complicated mechanisms at work here which have yet to be fully elucidated. References: 1 Freedman SD, Weinstein D, Blanco PG, et al. 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