Ozonized Low Density Lipoprotein (ozLDL) Inhibits NF-κB and IRAK-1–Associated Signaling
Objective— Recent studies have provided strong evidence for the presence of ozone in atherosclerotic lesions. In addition, modification of LDL has been suggested to be involved in atherosclerosis. In the present study we wanted to investigate whether LDL exposed to ozone (ozLDL) is able to modulate the NF-κB system, as a paradigm for inflammatory signaling.
Methods and Results— We showed that activation of NF-κB by lipopolysaccharide (LPS), a prototypic inducer of innate immunity, was reversibly inhibited by ozLDL in monocytic THP-1 cells in a dose-dependent manner, whereas tumor necrosis factor (TNF) signaling was not affected. This was not attributable to a direct ozone effect or solely the presence of lipoprotein, and neither required direct contact to LPS nor was accompanied by a change in LPS binding. Comparable inhibitory effects of ozLDL were observed in human monocyte/macrophages and endothelial cells. The presence of ozLDL led to a decrease in LPS-induced IκΒα proteolysis and a reduction of κB-dependent transcription/target-gene expression. Furthermore, ozLDL markedly lowered stimulus-induced IκΒ kinase (IKK) activity and phosphorylation/proteolysis of interleukin (IL)-1 receptor-associated kinase-1 (IRAK-1). Finally, cholesterol ozonization products were identified as effective ozLDL inhibitory compounds.
Conclusion— Our study demonstrated that ozLDL inhibited NF-κB and IRAK-1–associated signaling which may impair immune function and promote apoptosis.
Recent experiments have revealed strong evidence that ozone is present in human atherosclerotic arteries and may be involved in other inflammatory diseases.1–3 Signature products unique to cholesterol ozonolysis have been found in atherosclerotic tissue from carotid endarterectomy, suggesting that ozone production occurred during lesion development.1 Furthermore, an increase of these ozone-specific products has been found after culture and activation of atherosclerotic plaque tissue in vitro.1 An intriguing process has been identified how ozone may be generated in vivo, ie, the antibody-catalyzed water-oxidation pathway which may be part of defense strategies and play a role in inflammation.2,4 Remarkably, all the components necessary to activate this pathway are thought to be present in the atherosclerotic microenvironment.1,2
The atherosclerotic lesion is characterized by a dramatic accumulation of lipids carried by lipoproteins such as LDL,5 which becomes susceptible to (non)enzymatic modifications when retained in the artery wall.5–7 These modifications make LDL a more potent affector of cellular functions (eg, cytokine expression, apoptosis).5,6 The formation of macrophage foam cells that take up massive amounts of modified lipoproteins is a hallmark of both early and late atherosclerosis.
The transcription factor NF-κB plays a role in gene regulation during inflammatory and immune reactions as well as apoptosis.8,9 Activated NF-κB has been identified in atherosclerotic lesions, eg, in monocyte/macrophages or endothelial cells.10 NF-κB exists as a dimer which is trapped in the cytosol in an inactive state by inhibitory proteins, including IκΒα.8 NF-κB may be modulated in the lesion by numerous agents including inflammatory cytokines, modified LDL, and microbial pathogens.9–13 The activation of NF-κB is facilitated by the IκΒ-kinase (IKK) complex, consisting of IKKα, -β, and -γ.8 Activation of IKK leads to IκΒ phosphorylation which is subsequently degraded by the proteasome, followed by nuclear translocation of NF-κB.8 κB regulatory sequences have been found in promoters/enhancers of a variety of genes involved in inflammation and immunity, eg, coding for the cytokines TNF, IL-1 and IL-2, the IL-2 receptor as well as chemokines such as IL-8 and MCP-1.8,9 In addition, the activation of NF-κB is considered mostly to be antiapoptotic leading to the induction of several antiapoptotic genes.8
The aim of our study was to investigate whether LDL that is exposed to ozone before addition to cells, which we named ozLDL, is able to affect cellular functions. As a readout we used the NF-κB system as a paradigm for inflammatory signaling.8,9 Our data demonstrated that ozLDL and cholesterol ozonization products inhibited the activation of NF-κB in monocytic and endothelial cells stimulated by LPS,14,15 a prototypical inducer of innate immunity, as well as IL-1 receptor-associated kinase-1 (IRAK-1)–mediated signaling,16 whereas the TNF pathway was not affected.
Materials and Methods
Human monocytic THP-1 cells (DSMZ, Braunschweig, Germany) were cultured as described.17 Most short-term experiments were performed in PBS (six-well plates, 3×106cells per well). Human monocytes were isolated from normal donors’ blood using Biocoll (Biochrom) followed by the monocyte-isolation-kit (Miltenyi-Biotec) and cultured in 24-well plates (2.5×106 per well) in THP-1-medium (0.5 vial/L OPI-Media-Supplement, Sigma; 1% MEM, GibcoBRL) (purity >95%, flow-cytometry) for 24 hours (adherent monocytes) or 8 days (macrophages). Human umbilical vein endothelial cells (HUVECs; Promocell, Heidelberg, Germany) were maintained in endothelial cell medium (2.5% supplement, Promocell; 7% fetal calf serum [Myoclone super-plus], 100 U/mL penicillin, 100 μg/mL streptomycin, GibcoBRL). LPS (E coli0111:B4) and TNF were purchased from Sigma. LPS binding-assays were performed as described18 (please see supplemental legends, available online at http://atvb.ahajournals.org). A potential toxicity of cellular conditions was monitored by cell morphology/count, trypan blue exclusion and the Luminescent-Cell-Viability-Assay (Promega). Endotoxin contamination was screened by the limulus-lysate-assay (BioWhittaker) and only reagents/lipoproteins with endotoxin levels of <10 pg/mL were used. Reactive oxygen species (ROS) were detected using DCFH-DA (Sigma) (please see supplemental legends).
Ozonization of LDL and Cholesterol
Human LDL was purchased from Biomedical-Technologies (Stoughton). EDTA was removed from LDL (PD-10 column, Amersham) which was finally dissolved in PBS (10 μg protein/mL). Three forms of cholesterol were applied (all from Sigma): a free cholesterol dispersion (3 mg/100 mL PBS) was stirred (7 days, 4°C) to obtain enough solubility and then filtered. The other two types (30 μg/mL) were cholesterol-PEG600 and water-soluble cholesterol. To generate ozonized LDL (ozLDL), PBS (ozPBS) or cholesterol (ozChol) ozone was insufflated in the respective solution according to the manufacturer’s instructions using an ozone generator (Ozonosan-photonik, Dr Hänsler, Iffezheim, Germany). Ozone concentrations were monitored by a photometer integrated in the processor and confirmed by an ozone-detector (GM-6000-NZL, Anseros) and a photometrical test for soluble ozone (Palintest). As a result of half-life tests (data not shown), the ozonized solution was left to stand ≥1 hour to allow the unreacted gas to volatilize. Ozone-induced LDL modifications were monitored by determining thiobarbituric acid–reactive substances (TBARS),13 apolipoprotein B (ApoB) fragmentation (Western blot), and cholesterol concentrations as well as by mass spectrometry (for additional techniques, see supplemental Methods). Furthermore, we used the cholesterol ozonization products 5α,6α-cholesterol epoxide (Sigma) and 5-oxo-5,6-secocholestan-6-al (atheronal A).1
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared and electrophoretic mobility shift assay (EMSA) was performed as published.13 The prototypic immunoglobulin κ-chain oligonucleotide was used as a probe and labeled with the Klenow fragment of DNA-polymerase I (Roche) together with [α-32P]dCTP (PerkinElmer-LifeSciences). The Sp-1 consensus-oligonucleotide (Promega) was labeled with [γ-32P]ATP (PerkinElmer-LifeSciences) and T4 polynucleotide kinase (Roche). Samples were run on non-denaturing 4% polyacrylamide gels and analyzed by autoradiography.
Polyacrylamide Gel Electrophoresis and Western Blot Analysis
Cytosolic extracts were isolated and electrophoresis was performed as described.17 After transfer, the membranes were incubated with antibodies against ApoB, IκBα, IKKγ, IRAK-1 (SantaCruzBiotechnology, Palo Alto, and BD-Biosciences), or with anti-actin (Sigma) followed by secondary peroxidase-conjugated antibodies (Dianova). Antibody binding was visualized on X-ray film using the Chemiluminescent-Reagent-Plus (PerkinElmer-LifeSciences).
For luciferase assays a firefly luciferase reporter plasmid (pGL2–3κB-Luc; 3 κB binding motifs) was transiently cotransfected with a constitutively active Renilla luciferase plasmid, pRLtk (Promega), using a DEAE-dextran-based protocol.18 Subsequent to stimulation, cells were lysed and luciferase activity was determined using the Dual-Luciferase-Reporter-Assay-System (Promega). Results were expressed as relative luciferase activity (RLA), ie, firefly relative light-units were divided by Renilla relative light-units.
Determination of IL-8
The IL-8 protein concentration in the supernatant was measured by immunoassay (R&D-Systems).
IKK Kinase Assay
Immunoprecipitation (IP) was carried out using protein A agarose (Roche) for 2 hours at 4°C together with anti-IKKγ (SantaCruzBiotechnology), followed by kinase assays.17 The kinase assay reaction was performed in kinase buffer at 30°C for 30 minutes in the presence of [γ-32P]ATP (PerkinElmer-LifeSciences) and the substrate GST-IκBα (Santa Cruz Biotechnology). Proteins were separated by polyacrylamide gel electrophoresis (PAGE) and substrate phosphorylation and loading was visualized by autoradiography and Western blot (densitometry).
IRAK-1 Kinase Assay
IRAK-1-IP was carried out at 4°C for 1 hour with cytosolic extracts, anti–IRAK-1, and protein A dynabeads (Dynal) which were previously washed with 0.1 mol/L sodium phosphate buffer (pH 8.1). After washing three times with PBS (containing 1 mmol/L dithiothreitol, 0.5 μmol/L 4-(2-aminoethyl)-benzenesulfonylfluoride; leupeptin, antipain, aprotinin, pepstatin A, chymostatin, 0.75 μg/mL each, Sigma) and three times with kinase buffer, the precipitated proteins were incubated with kinase buffer and [γ-32P]ATP (PerkinElmer-LifeSciences) at 30°C for 30 minutes and subsequently washed twice with kinase buffer. Proteins were separated by SDS-PAGE and IRAK phosphorylation and loading was visualized by autoradiography and Western blot.
Activation of NF-κB Is Inhibited by ozLDL
Initially, we investigated whether ozLDL, generated as described in Methods, was able to directly activate NF-κB. THP-1 monocytic cells were incubated with ozLDL (0.1 to 10 μg/mL) for different time intervals (15 to 90 minutes) and NF-κB activation was determined (EMSA). These experiments demonstrated that ozLDL alone did not have any effect on NF-κB (Figure 1; data not shown). Next, we tested whether preincubation with ozLDL affected NF-κB activation by other stimuli. Cells were preincubated with ozLDL for 30 minutes and then the potent NF-κB activator LPS was added for a further hour. In the absence of ozLDL we observed the expected marked activation of NF-κB by LPS (Figure 1A). This increase was completely abolished by ozLDL, whereas Sp-1 binding (constitutive transcription factor) was unchanged. The TBARS values of 7 ozLDL preparations used for EMSA ranged from 42.3 to 144.7 nmol/mg LDL protein (LDL controls: undetectable levels) and there was no correlation between TBARS concentrations and the degree of NF-κB inhibition observed (supplemental Table I). In all ozLDL preparations a significant proteolysis of ApoB was detected compared with the control (data not shown). Further experiments showed that LPS-induced activation of NF-κB was dose-dependently reduced by ozLDL (Figure 1B). Comparable inhibitory effects of ozLDL were obtained using human adherent monocytes, monocyte-derived macrophages, and HUVECs (Figure 1C). A potential toxicity of ozLDL was excluded by several approaches (Methods; supplemental Table II). In addition, we found no significant difference in ROS generation in the LPS-stimulated samples, regardless of the presence of ozLDL (supplemental Table III).
The Effect of ozLDL Does Not Require Direct Contact to LPS, Is Reversible, and Is Not Accompanied by a Change in LPS Binding
To further characterize the ozLDL inhibitory condition, THP-1 monocytic cells were preincubated with ozLDL for 30 minutes before LPS was added for a further hour (standard treatment). For additional time points, cells were treated with ozLDL for 90 minutes, washed, and transferred to a new culture dish. LPS was then added at varying times for a 1-hour incubation period. Remarkably, a comparable inhibition of NF-κB was detected in the samples incubated under our standard conditions and when LPS was added 2 hours after removal of ozLDL (no direct contact with the lipoprotein; Figure 1D). When LPS was added 6 or 24 hours after ozLDL pretreatment almost no inhibitory effect was observed anymore. Binding studies with fluorescein isothiocyanate (FITC)-marked LPS showed approximately equal amounts of bound LPS in the presence of ozLDL compared with the control (supplemental Table IV).
IκBα Proteolysis Is Prevented
Next, we investigated the effect of ozLDL on the activation-induced proteolysis of IκΒα determined by Western blot analysis. As expected, stimulation of monocytic THP-1 cells with LPS led to a significant proteolysis of IκBα (Figure 2A and 2B). However, when we preincubated the cells with ozLDL this degradation of IκBα was inhibited in a dose-dependent manner, whereas actin levels were unchanged.
Inhibition by ozLDL Is Not Caused by a Direct Ozone Effect or Solely the Presence of Lipoprotein
Several control experiments were necessary to find out whether the inhibition in the presence of ozLDL was caused by a direct effect of ozone and whether this effect was attributable to presence of native lipoprotein or was only observed when the lipoprotein was modified by ozone. Therefore, THP-1 were incubated with PBS, ozPBS, native LDL, and ozLDL and then LPS was added. A comparable NF-κB activation was observed in samples treated with PBS, ozonized PBS, as well as native LDL (Figure 3A, upper panel), whereas ozLDL almost completely blocked the NF-κB activity. ozPBS as well as LDL were used as controls throughout the study. It should also be mentioned that our short-term experiments were performed in PBS to avoid interactions between ozone, medium components, and lipoproteins. In accordance with the data described above, the LPS-induced IκBα proteolysis was not inhibited by pretreatment with ozPBS or native LDL (Figure 3B).
Selective Inhibition of Signaling
Now we evaluated whether ozLDL also affected NF-κB activation induced by stimuli other than LPS. Therefore, THP-1 cells were incubated with ozLDL and afterward either TNF or LPS was added. These data showed that ozLDL only prevented NF-κB activation in samples stimulated with LPS, whereas TNF-induced signaling was not affected (Figure 3A, lower panel). This was confirmed by Western blot in which no inhibition of TNF-induced IκB proteolysis was observed in lipoprotein-pretreated cells (Figure 3B).
κB-Dependent Transcription and Target Gene Expression Are Prevented
To study whether the inhibition of NF-κB by ozLDL was associated with functional consequences, monocytic THP-1 were transfected with a κB-dependent luciferase construct and pretreated with ozLDL before LPS was added. Then PBS was replaced by medium and cells were harvested at the indicated time points (please note that LPS was present for only 1 hour for each condition). LPS significantly induced transcriptional activity, which was strongly inhibited by ozLDL when luciferase activity was measured at 2.5 hours after the time point of LPS addition and partially impaired at 5 hours (Figure 4A). No inhibition of luciferase activity was observed at 10 or 20 hours after the time point LPS was added. The same condition was used to test whether inhibition by ozLDL has a corresponding impact on IL-8 production. As expected, LPS led to an increase in IL-8 protein levels (Figure 4B). Treated with ozLDL, the cells showed a significantly inhibited IL-8 production when it was measured 2.5 and 5 hours after LPS addition, but only partial or no inhibition at 10 or 20 hours, respectively. The TNF-stimulated production of IL-8 was not impaired by ozLDL (data not shown).
ozLDL Inhibits IKK Activity
To learn more about upstream signaling pathways affected by ozLDL, we measured the IKK activity in the presence of ozLDL. THP-1 were preincubated with ozLDL before LPS was added for another 30 minutes and IKK activation was determined by kinase assays. Incubation with LPS induced a significant increase in IKK activity (Figure 5A). Consistent with above, a marked inhibition of IKK activity was detected when ozLDL was given to the cells before LPS stimulation.
IRAK-1 Phosphorylation Is Inhibited by ozLDL
Recent studies have shown that IRAK-1 is involved in mediating LPS-induced NF-κB activation.16 Therefore, we examined whether pretreatment with ozLDL affects IRAK-1 phosphorylation status. For this purpose, monocytic THP-1 were preincubated with ozLDL, followed by LPS for another 15 minutes and IRAK-1 autophosphorylation was determined by kinase assays. Stimulation with LPS induced a significant increase of phosphorylated IRAK-1 (Figure 5B), which was markedly inhibited in cells pretreated with ozLDL.
IRAK-1 Proteolysis Is Prevented
Activated IRAK-1 associates with downstream signaling proteins and is then proteolytically degraded.16 To visualize IRAK-1 degradation, THP-1 were pretreated with ozLDL before LPS was added and extracts were prepared at different time points for Western blot analysis. Exposure to LPS, but not TNF, was followed by a significant decrease of IRAK-1 levels (Figure 5C and 5D). Remarkably, in ozLDL pretreated cells only a slight amount of IRAK-1 degradation was found after LPS exposure.
Characterization of the Active Component
Finally, we aimed to further characterize the active component responsible for the inhibitory effect of ozLDL. Because ozone is able to modify cholesterol, as a major component of LDL,1,5,6,19 we measured cholesterol concentrations after ozonization by a cholesterol oxidase assay and performed mass spectrometry. These experiments showed a complete vanishing of the cholesterol values/peak in ozLDL and suggested the generation of cholesterol ozonization products such as 3,5-dioxo-5,6-secocholestan-6-al (ketoatheronal A) (for analysis of ozLDL, see supplemental materials), which was not detected in oxLDL and LDL. Therefore, we investigated whether preincubation with ozonized cholesterol (ozChol) could modulate LPS-induced NF-κB activation. Remarkably, these experiments using THP-1 demonstrated that ozChol also strongly inhibited this signaling pathway (Figure 6A). Essentially the same results were obtained with all three forms of ozChol (data not shown; Methods). These results were supported using two commercially available products of cholesterol ozonization, ie, 5α,6α-cholesterol epoxide and atheronal A,1,19 which also caused a strong inhibition of LPS-mediated NF-κB activation (Figure 6B).
Recent data imply that ozone is located in atherosclerotic arteries and it has been proposed that it may be important in additional human inflammatory diseases.1,2 An intriguing and somewhat imaginative scenario suggests that ozone may be the secret ingredient in plaques’ inflammatory stew.20 The present work describes a procedure to modify LDL by ozone and shows that LDL which was exposed to ozone was able to inhibit the NF-κB system in monocytic and endothelial cells. This inhibition was dose-dependent, reversible, and caused by the modified lipoprotein, because ozonized PBS or native LDL did not exert any inhibitory effect. The fact that κB-dependent transcription as well as NF-κB target-gene expression was affected showed that the inhibition of NF-κB by ozLDL had functional relevance.
Different effects of modified LDL on the NF-κB system have been described. On the one hand it has been reported that short exposure of cells to minimally as well as copper-modified LDL activates NF-κB, determined by gelshift which is accompanied by target-gene expression.11,13,21,22 On the other hand, when cells were pretreated with fully oxidized LDL or oxysterols, mostly for a longer time interval, a selective inhibition of NF-κB activity and subsequent gene expression has been shown.12,13,23,24 Furthermore, an inhibition of stimulus-dependent degradation of IκΒα in the presence of oxLDL was detected.25 In the present study ozonization of LDL did not generate products which activate NF-κB and a very short preincubation interval with ozLDL was sufficient to inhibit NF-κB without being toxic. Further analysis of ozLDL suggested the formation of cholesterol ozonization products which have a cleaved 5,6-double bond (thus significantly different from oxysterols23), and which were not detected in oxLDL and LDL. Most importantly, we demonstrated that ozChol as well as products of cholesterol ozonization1,19 also showed an inhibitory effect on NF-κB. Taken together, our data imply that cholesterol ozonization products represent effective NF-κB inhibitory components of ozLDL.
We showed that ozLDL selectively prevented LPS-induced NF-κB activation, whereas TNF signaling was not affected. The cytokine TNF induces a signaling pathway that is different from that of LPS and involves the formation of a specific multiprotein signaling complex at the cell membrane followed by the activation of the IKK complex.26 Therefore, the fact that ozLDL inhibits LPS-induced IKK activity and IκΒα proteolysis leads to the conclusion that the LPS pathway upstream of IKK is specifically affected by ozLDL. Here it should be mentioned that we found no significant difference in ROS generation in LPS-stimulated samples, regardless of the presence of ozLDL, indicating that the ROS system is not involved in ozLDL-mediated inhibition of NF-κB. LPS signaling initially leads to the assembly of the LPS recognition complex, which induces a cascade of association/phosphorylation events involving proteins such as IRAK-1.27 After cellular exposure to LPS the IRAK-1 protein is (auto) phosphorylated and finally inactivated by degradation.16 Activated IRAK-1 interacts with downstream signaling proteins resulting in IKK activation. Our data showed that LPS-induced phosphorylation of IRAK-1 was prevented by ozLDL. Furthermore, exposure to LPS, but not TNF, induced IRAK-1 degradation which was also inhibited by ozLDL. In addition, we found that the effect of ozLDL does not require direct contact to LPS, because a comparable inhibition of NF-κB was observed when ozLDL was removed after preincubation and LPS was added 2 hours thereafter. A recent report shows that oxidized phospholipids inhibit NF-κB activation by blocking the interaction of LPS with LPS binding protein (LBP) and CD14.28 Our studies with FITC-marked LPS showed approximately equal amounts of bound LPS in the presence of ozLDL compared with the control, suggesting that the inhibitory effect of ozLDL was not caused by a reduction in LPS binding. In summary, ozLDL appears to affect an LPS-specific pathway at the level and/or upstream of IRAK-1, but downstream of the “cell surface binding level”.27 Interestingly, it has been recently revealed that oxPAPC, a phospholipid oxidation product, inhibits LPS action by disrupting assembly of the LPS signaling complex in cell caveolae.27,29
The vasculature as the site of LDL accumulation5,6 is a compartment close to inflammatory or malignant processes. Furthermore, the immune response in the vessel wall is characterized, to a varying extent, by the production of antibodies as well as chemotaxis of leukocytes.1,14,15 Both antibodies and leukocytes are involved in the generation of ozone, because there is strong evidence for ozone production in the antibody-catalyzed water-oxidation pathway as well as during the oxidative burst of activated leukocytes.2 The ozone thus produced can react with surrounding lipoproteins to generate lipid ozonation products which could strongly affect various cellular functions such as NF-κB–associated signaling. Interestingly, there is a link between diseases with antibody accumulation in human tissue and atherosclerosis. For example, the risk of cardiovascular disease is very high in a prototypic autoimmune disease, systemic lupus erythematosus, and is also raised in other autoimmune diseases such as rheumatoid arthritis.15,30 Inhibition of NF-κB may impair the expression of genes involved in the immune response but also induce apoptotic processes.8,13 It is also of note that a link between the dysregulation of adaptive immunity and atherosclerosis is being discussed.14,15,30 Seen in a wider context, it is striking to speculate that the inhibition of innate immunity by ozLDL may be relevant not only to atherosclerosis but also to other inflammatory or malignant diseases.
Atheronal A was a kind gift from Dr Paul Wentworth (Department of Chemistry, The Scripps Research Institute, La Jolla, Calif). We thank Andreas Ertl for excellent technical support.
Sources of Funding
This work was supported by the Stiftung für Pathobiochemie und Molekulare Diagnostik (DGKL), the Wilhelm Sander-Stiftung and the Medical Faculty of the Technische Universität München.
C.C. and B.S. contributed equally to this study.
Original received November 12, 2005; final version accepted September 28, 2006.
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