Low Doses of Lipopolysaccharide and Minimally Oxidized Low-Density Lipoprotein Cooperatively Activate Macrophages via Nuclear Factor κB and Activator Protein-1
Possible Mechanism for Acceleration of Atherosclerosis by Subclinical Endotoxemia
Rationale: Oxidized low-density lipoprotein (LDL) is an important determinant of inflammation in atherosclerotic lesions. It has also been documented that certain chronic infectious diseases, such as periodontitis and chlamydial infection, exacerbate clinical manifestations of atherosclerosis. In addition, low-level but persistent metabolic endotoxemia is often found in diabetic and obese subjects and is induced in mice fed a high-fat diet.
Objective: In this study, we examined cooperative macrophage activation by low levels of bacterial lipopolysaccharide (LPS) and by minimally oxidized LDL (mmLDL), as a model for subclinical endotoxemia-complicated atherosclerosis.
Methods and Results: We found that both in vitro and in vivo, mmLDL and LPS (Kdo2-LipidA) cooperatively activated macrophages to express proinflammatory cytokines Cxcl2 (MIP-2), Ccl3 (MIP-1α), and Ccl4 (MIP-1β). Importantly, the mmLDL and LPS cooperative effects were evident at a threshold LPS concentration (1 ng/mL) at which LPS alone induced only a limited macrophage response. Analyzing microarray data with a de novo motif discovery algorithm, we found that genes transcribed by promoters containing an activator protein (AP)-1 binding site were significantly upregulated by costimulation with mmLDL and LPS. In a nuclear factor–DNA binding assay, the cooperative effect of mmLDL and LPS costimulation on c-Jun and c-Fos DNA binding, but not on p65 or p50, was dependent on mmLDL-induced activation of extracellular signal-regulated kinase (ERK) 1/2. In addition, mmLDL induced c-Jun N-terminal kinase (JNK)-dependent derepression of AP-1 by removing nuclear receptor corepressor (NCoR) from the chemokine promoters.
Conclusions: The cooperative engagement of AP-1 and nuclear factor (NF)-κB by mmLDL and LPS may constitute a mechanism of increased transcription of inflammatory cytokines within atherosclerotic lesions.
Lipoprotein oxidation plays an important role in initiation and progression of atherosclerosis, a vascular inflammatory disease, which leads to complications that cause myocardial infarction and stroke.1 Oxidized low-density lipoprotein (LDL) accumulated in the vascular wall activates macrophages and endothelial and smooth muscle cells to produce proinflammatory cytokines and chemokines, thereby enhancing inflammation in the lesion. LDL oxidation is a gradual process, progressing from lipid hydroperoxides to degradation products, such as aldehydes and ketones, which can form covalent adducts with proteins and lipids. With the changing composition of lipid and protein oxidation products, different forms of oxidized LDL interact with different cellular and soluble receptors. Thus, we have demonstrated that hydroperoxide-rich minimally oxidized LDL (mmLDL) activates macrophages via toll-like receptor (TLR)-4.2–4 OxLDL (more extensively oxidized LDL), containing aldehydes and modified/degraded apoB-100, binds to CD36 and other scavenger receptors.5
Certain chronic infectious diseases, such as periodontitis and chlamydial infection, accelerate atherosclerosis and exacerbate its clinical manifestations, leading to acute cardiovascular events.6–10 This is likely a consequence of immune responses to bacterial pathogens resulting in intensified vascular inflammation within atherosclerotic lesions. Moreover, recent studies have revealed that ingestion of high-fat meals leads to transient increases in plasma endotoxin levels,11 likely via chylomicron-mediated transport of endotoxin derived from intestinal microflora.12 Metabolic endotoxemia has been also observed in patients with type 2 diabetes mellitus13 and in mice fed a high-fat diet.14 Thus, a combinatorial activation of vascular cells by oxidized LDL and by threshold levels of endotoxin is a likely in vivo event, particularly relevant to the development of atherosclerosis in overweight individuals consuming a Western-type diet.
Because we previously demonstrated that mmLDL activates macrophages via TLR4,2–4 which is also activated by bacterial lipopolysaccharide (LPS), a “classic” TLR4 ligand,15 we hypothesized that low levels of mmLDL and LPS would exert cooperative activation of macrophages. TLR4-mediated responses involve the recruitment of a number of adaptor proteins to the TLR4 cytoplasmic domain. At the plasma membrane, the LPS-induced recruitment of MyD88 to TLR4 results in rapid expression of nuclear factor (NF)-κB–dependent genes and activation of c-Jun N-terminal kinase (JNK) and p38 kinases. In the endosomal compartment, the recruitment of TRIF to TLR4 induces IRF3-dependent gene expression. In contrast to the LPS-induced TLR4 responses, mmLDL induces moderate expression of proinflammatory genes but profound cytoskeletal rearrangements and reactive oxygen species generation in macrophages.2,3,16 These MyD88-independent effects of mmLDL are mediated by the recruitment of spleen tyrosine kinase (Syk) to the C-terminal domain of TLR4 and subsequent activation of extracellular signal-regulated kinase (ERK)1/2 and phospholipase Cγ.4,16
Because of the differences in the LPS- and mmLDL-induced activation of TLR4, we hypothesized that costimulation of macrophages by mmLDL and by low levels of bacterial LPS would result in cooperative effects leading to greater activation than achieved by either stimulus alone. Herein, we demonstrate that mmLDL and low levels of Kdo2-LipidA (KLA), the active moiety of LPS, cooperatively upregulate expression of a number of proinflammatory genes, including chemokines Cxcl2 (MIP-2), Ccl3 (MIP-1α), and Ccl4 (MIP-1β), both in vivo and in vitro. A de novo motif analysis of promoters of cooperatively activated genes suggested involvement of AP-1 transcription factors. Indeed, ERK1/2-dependent activation of AP-1 was characteristic for mmLDL and LPS costimulation. In addition, mmLDL induced phosphorylation of c-Jun and the release of nuclear receptor corepressor (NCoR) from the promoter regions of Cxcl2 and Ccl3. These molecular mechanisms suggest that the combination of minimally oxidized LDL and low levels of LPS cooperatively activate inflammatory responses in macrophages.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Cells, LDL Preparations, and Other Reagents
Murine macrophage-like cell lines RAW267.1 (abbreviated in the text as RAW) and J774A.1 (abbreviated as J774) were from American Type Culture Collection. Mouse peritoneal macrophages were isolated as described below. LDL (density, 1.019 to 1.063 g/mL) was isolated from plasma of normolipidemic donors by sequential ultracentrifugation.17 Native and modified LDL preparations were tested for possible endotoxin contamination using a Limulus Amoebocyte Lysate kit (Cambrex, Walkersville, Md). LDL preparations with LPS content higher than 50 pg/mg protein, corresponding to 2.5 pg/mL in most cell culture experiments, were discarded. To produce mmLDL, we incubated 50 μg/mL LDL in serum-free DMEM for 18 hours with murine fibroblast cells overexpressing human 15-lipoxygenase, as reported in detail.2,18,19 KLA, obtained from Avanti Polar Lipids (Alabaster, Ala), was used as a well-characterized active component of LPS and a highly specific TLR4 agonist.20
Mice, Intraperitoneal Injections, and Macrophage Isolation
All animal experiments were performed according to NIH guidelines and were approved by the Animal Subjects Committee of the University of California at San Diego. C57BL/6J mice were purchased from the Jackson Laboratory. Mice were anesthetized by a short exposure to isoflurane and injected intraperitoneally with 1 mL of 50 μg/mL mmLDL, 5 ng/mL KLA, or their combination. One hour later, mice were euthanized, peritoneum was lavaged, and peritoneal cells were isolated and prepared for real time quantitative polymerase chain reaction (qPCR) as described below. In some experiments, CD11b+ cells (predominantly macrophages) were rapidly separated at 4°C using magnetic bead reagents from Miltenyi (Germany).
Real-Time PCR–Based Quantitative Gene Expression Analysis
Total RNA was isolated using RNeasy columns (Qiagen, Valencia, Calif), treated with DNase, and reverse transcribed using oligo-dT and a First Strand Synthesis kit (Invitrogen, Carlsbad, Calif). Real-time qPCR analysis was performed using reagents from Applied Biosystems (Foster City, Calif) and a Rotor Gene Q (Qiagen).
Microarray and Motif Discovery Algorithm
RNA concentration and integrity were verified with a NanoDrop ND-1000 (Thermo Scientific, Waltham, Mass) and an Agilent 2100 BioAnalyzer (Agilent, Santa Clara, Calif), respectively. RNA was amplified, labeled, and hybridized to the Sentrix BeadChip array, MouseRef-8 (Illumina, San Diego, Calif). The slides were then processed according to the protocol of the manufacturer, stained with Cy3-streptavidin and scanned with a BeadStation scanner (Illumina). Intensities were normalized using the modified Loess procedure.21 Sorting of genes and statistical analysis of pathways and gene ontology terms were performed as described.21–23 De novo motif discovery was performed using HOMER (http://biowhat.ucsd.edu/homer/index.html), a comparative algorithm that searches for motifs that are specifically found in a set of regulated promoter sequences [−500, +100 bp] compared to an invariant background set of promoters.
Western Blot Analysis
SDS-PAGE and Western blot were performed according to standard protocols as we previously described.4
Chemiluminescent Assay for Transcription Factor–DNA Binding
RAW cell nuclear extracts were isolated using a Nuclear Extraction kit (Active Motif, San Diego, Calif). Transcription factor activation was measured using sensitive Active Motif’s TransAM NF-κB p65, NF-κB p50, c-Jun, and c-Fos assays in an ELISA format.
Enzyme-Linked Immunosorbent Assay
RAW cells or J774 cells were incubated for 5 hours with 50 μg/mL mmLDL, 1 ng/mL KLA, or mmLDL+KLA in a serum-free DMEM. Supernatants were harvested and spun for 5 minutes at 10 000 rpm to remove floating cells. Levels of Cxcl2, Ccl3, and Ccl4 proteins were measured in ELISA assays using reagents from R&D Systems.
The chromatin immunoprecipitation (ChIP) assay was performed as we previously reported.24 qPCR with SYBR Green (Invitrogen) was carried out with a Rotor Gene Q (Qiagen). Primer sequences for the promoter regions were: Cxcl2 forward, gggctctgtgcttcctgat; Cxcl2 reverse, cagtctggggctctgaggt25; Ccl3 forward, cctcagtccctcactgtggt; Ccl3 reverse, catggaacggaaactctcgt; Ccl4 forward, ttgtggcaggtgtgaacatt; Ccl4 reverse, tgtcatggcatcgagaaaga. Results are presented as enrichment of the precipitated target sequence compared to input DNA.
Graphs represent means±SE. Significance of differences was calculated using 1-way ANOVA, with Bonferroni correction, where indicated.
mmLDL and LPS Cooperatively Upregulate Gene Expression in RAW Cells
To assess the effect of cooperative stimulation with mmLDL and LPS on a genome-wide scale, we conducted microarray analysis and focused on early (1 hour) gene expression responses. Throughout this study, we used KLA (from Avanti Polar Lipids) as a well-characterized active component of bacterial LPS and a highly specific agonist of TLR4,20 at threshold levels of 1 ng/mL for in vitro experiments. We used 50 μg/mL mmLDL as previously reported.3,4,16 RNA isolated from RAW macrophages was analyzed using an Illumina microarray platform.
The analysis of macrophages stimulated with mmLDL alone and KLA alone demonstrated activation of both common and distinct signaling and metabolic pathways (Online Table I). Although both mmLDL and KLA activated TLR, mitogen-activated protein kinase, NF-κB, and interleukin-6 proinflammatory signaling pathways, the mmLDL stimulation was uniquely characterized by activation of PI3K/Akt, ERK1/2, and signaling pathways involved in cytoskeletal rearrangements, which agrees with the mmLDL-induced effects we previously documented with other techniques.2–4,19,26 Downregulation of the sterol biosynthetic pathway by mmLDL was indicative of mmLDL uptake and intracellular cholesterol accumulation, as we have reported recently.4
Although there was little overlap in expression of individual genes stimulated by mmLDL and KLA alone (Figure 1A), costimulation of macrophages with mmLDL and KLA resulted in additive and synergistic increases in expression of a number of genes, including transcription factors, kinases, phosphatases, and cytokines. In further studies, we focused on 3 proinflammatory chemokines important in the development of atherosclerosis, Cxcl2 (MIP-2), Ccl3 (MIP-1α), and Ccl4 (MIP-1β), which were additively/synergistically upregulated by mmLDL and KLA.
To confirm the microarray data, we conducted independent experiments and measured the expression of Cxcl2, Ccl3, Ccl4 and Gapdh mRNA in RAW macrophages induced my mmLDL, KLA, and their combination (Figure 1B). The costimulation with mmLDL and KLA resulted in 1.5 to 3 fold increases in chemokine expression (normalized to Gapdh), and ANOVA analysis demonstrated a significant trend toward the increase in gene expression in the mmLDL+KLA samples compared to mmLDL and KLA alone. These increases in RAW cells were moderately synergistic (Online Table II). The changes in the chemokine expression levels were not attributable to variations in the Gapdh mRNA levels (Online Figure I).
Cooperative Stimulation of Cytokine Expression by mmLDL and LPS In Vivo
To test the in vivo relevance of our findings with RAW macrophages, we used a model of sterile peritonitis. Mice were injected intraperitoneally with vehicle, mmLDL, KLA, or mmLDL+KLA. One hour later, peritoneal cells were isolated and mRNA levels of Cxcl2, Ccl3, and Ccl4 were determined using qPCR. Similarly to the results with RAW cells, expression of Cxcl2, Ccl3, and Ccl4 was higher in peritoneal cells costimulated with mmLDL and KLA, but the synergistic effect was more dramatic, reaching as much as 2.5- to 5-fold increases compared to the cells stimulated with KLA alone (Figure 2A and Online Table II). Peritoneal macrophages constitute ≈40% of all leukocytes in the peritoneum; other cells include lymphocytes, neutrophils, and dendritic cells. Using a rapid positive selection procedure with CD11b antibodies, we enriched the population of CD11b-positive cells (predominantly macrophages) from 40% to nearly 80% (Online Figure II) and confirmed that intraperitoneal stimulation with mmLDL and KLA resulted in cooperative stimulation of peritoneal macrophages (Figure 2B and Online Table II). These results, however, do not exclude the possibility that other peritoneal leukocytes can also respond to the mmLDL/KLA stimulation in an additive/synergistic manner.
Costimulation With mmLDL and LPS Upregulates AP-1 Transcriptional Activity
Further analysis of the microarray data, using a de novo motif discovery algorithm developed in our group,27 suggested that promoter regions of many genes upregulated in macrophages costimulated with mmLDL and LPS, including Cxcl2, Ccl3, and Ccl4, contained the common motif presented in Figure 3. This motif matched with the known c-Jun/c-Fos consensus sequence, suggesting the involvement of AP-1 transcription factor regulation in macrophages costimulated with mmLDL and LPS. The most common AP-1 components, transcription factors c-Jun and c-Fos, are regulated by mitogen-activated protein kinases JNK and ERK1/2, respectively. Indeed, microarray analysis demonstrated highly significant activation of mitogen-activated protein kinase–related pathways (Figure 3). These data agree with our earlier findings of strong ERK1/2 phosphorylation and Ccl5 (RANTES) expression in macrophages stimulated with mmLDL alone.3,4,16
Next, we compared time-dependent ERK1/2 activation in macrophages stimulated with either mmLDL or KLA. Interestingly, mmLDL induced ERK1/2 phosphorylation in RAW cells as early as 5 minutes after stimulation, but the levels of phosphorylated ERK1/2 rapidly declined, and by 60 minutes were back to the levels in unstimulated cells (Figure 4). In contrast, ERK1/2 phosphorylation by a low dose of KLA (1 ng/mL) was delayed by nearly 30 minutes but was sustained for over 1 hour. Costimulation with mmLDL and KLA resulted in a pattern of ERK1/2 phosphorylation similar to that of mmLDL, except that the length of activation was more sustained, for the entire hour of the experiment. Thus, we hypothesized that early and sustained phosphorylation of ERK1/2 increases activation of the AP-1 transcription complex during combined mmLDL/KLA stimulation.
To test this hypothesis, we measured binding of c-Jun and c-Fos to consensus AP-1 DNA binding sites. mmLDL alone and KLA alone induced only moderate c-Jun and c-Fos DNA binding, which was synergistically increased in RAW macrophages costimulated with mmLDL and KLA (Figure 5). Importantly, inhibition of ERK1/2 phosphorylation completely abolished the synergistic effect of mmLDL and KLA. NF-κB transcription factors p50 and p65 showed the same DNA binding pattern as the AP-1 transcription factors, but, in contrast, inhibition of ERK1/2/ did not have a significant effect on p50 and p65 DNA binding. These data agree with the role of ERK1/2 in regulation of AP-1, but not the NF-κB transcription program.
To confirm that ERK1/2 activation mediates cytokine expression in macrophages costimulated with mmLDL and LPS, we measured CXCL2, CCL3, and CCL4 protein secretion by RAW and by J774 cells. We found that costimulation of both macrophage cell lines with mmLDL and KLA led to additive-to-synergistic upregulation of cytokine secretion, which, importantly, was blocked with the inhibition of ERK1/2 signaling (Figure 6).
mmLDL-Induced NCoR Derepression
NCoR is an important checkpoint in the regulation of inflammatory responses to LPS via TLR4 signaling.25 LPS induces IKKε-dependent phosphorylation of c-Jun, which results in detachment of NCoR from c-Jun and recruitment of c-Fos to complete the AP-1 assembly on a promoter binding site.25 This ultimately leads to the transcription of inflammatory genes. Because mmLDL also signals via TLR4 and phosphorylates c-Jun,3,4 we tested whether mmLDL has the ability to detach NCoR from the promoters of Cxcl2, Ccl3 and Ccl4, the mmLDL-induced proinflammatory genes. First, we measured time-dependent phosphorylation of c-Jun in macrophages stimulated with mmLDL, KLA, and their combination. We found the same pattern as with phosphorylation of ERK1/2, characterized by rapid and transient c-Jun phosphorylation with mmLDL and delayed c-Jun phosphorylation with KLA (Figure 7). As with ERK1/2, the mmLDL and KLA combination induced early and sustained c-Jun phosphorylation.
In addition to ERK1/2, mmLDL activation of macrophages also results in JNK phosphorylation.3 Thus, we hypothesized that in contrast to LPS-induced c-Jun phosphorylation, which was dependent on IKKε,25 mmLDL stimulates c-Jun phosphorylation via JNK. Indeed, a specific JNK inhibitor, SP600125, effectively inhibited mmLDL-induced c-Jun phosphorylation (Figure 8A). The specificity of SP600125 was validated by the lack of inhibition of ERK1/2 phosphorylation in the same macrophage lysates.
Next, in a ChIP experiment, we measured NCoR occupancy on the Cxcl2, Ccl3, and Ccl4 promoters. We found that as early as 15 minutes after stimulation, mmLDL induced release of approximately 40% to 60% of NCoR molecules from the promoter regions of Cxcl2 and Ccl3 (Figure 8B), but the results with the Ccl4 promoter were inconsistent. Importantly, inhibition of JNK with SP600125 abrogated mmLDL-induced NCoR release from the promoter regions of Cxcl2 and Ccl3 (Figure 8C).
Inflammation plays a major role in the pathogenesis of atherosclerosis, and chronic infections are known to exacerbate its complications. In this study, we demonstrated that 2 likely proinflammatory agonists involved in endotoxemia-complicated atherogenesis, minimally oxidized LDL and bacterial LPS, induced cooperative upregulation of proinflammatory genes in macrophages when applied together. Specifically, we found that mmLDL and threshold levels of LPS (1 ng/mL KLA) cooperatively activated RAW and J774 macrophages to express proinflammatory genes, including chemoattractant cytokines Cxcl2, Ccl3, and Ccl4 (Figures 1 and 6 and Online Table II). This cooperative effect was even more pronounced in vivo, achieving synergistic levels in peritoneal macrophages and possibly in other peritoneal cells (Figure 2). We further found that AP-1 response elements were enriched in the promoter regions of the genes responsive to costimulation with mmLDL and KLA (Figure 3) and that c-Jun and c-Fos binding to the response element DNA sequence was higher in the nuclear lysates of macrophages costimulated with mmLDL and KLA compared to mmLDL or KLA alone (Figures 4 and 5⇑). In addition, mmLDL rapidly induced AP-1 derepression by phosphorylating c-Jun and releasing corepressor NCoR from the chemokine promoters (Figures 7 and 8⇑).
The premise for our study was that low concentrations of LPS and mmLDL, although having one common signaling receptor, TLR4, nevertheless, activate separate, as well as common, signaling pathways. mmLDL stimulates TLR-dependent, but MyD88-independent, generation of reactive oxygen species and cytoskeletal rearrangements in macrophages, the latter leading to macropinocytosis and lipoprotein uptake. These mmLDL effects are mediated by the recruitment of Syk to the cytoplasmic domain of TLR4 and subsequent Syk-dependent activation of ERK1/2.4,16 In this study, we compared the time courses of ERK1/2 phosphorylation and found that mmLDL activates ERK1/2 as early as 5 minutes, but LPS (KLA) at a concentration of 1 ng/mL showed no significant ERK1/2 activation before 30 minutes. Interestingly, costimulation with mmLDL and KLA maintained the early ERK1/2 phosphorylation, characteristic for mmLDL only, which was sustained for a longer period of time because of the KLA component (Figure 4). It has been suggested that sustained ERK1/2-dependent phosphorylation of c-Fos leads to the AP-1 complex assembly and its DNA binding.28
In addition to the ERK1/2-mediated mechanism, mmLDL also activates a JNK pathway, leading to c-Jun phosphorylation and AP-1 derepression via the detachment of corepressor NCoR (Figures 7 and 8⇑). The dependence of NCoR detachment on c-Jun phosphorylation has been demonstrated in experiments with overexpressed phospho-mimic c-Jun mutant.25 However, as we suggested previously, the mechanism of LPS-induced c-Jun phosphorylation is via IKKε and is independent of JNK activity.25 In contrast, experiments of this study demonstrate that mmLDL-induced c-Jun phosphorylation and NCoR detachment do depend on JNK activity (Figure 8). These findings of separate pathways by which mmLDL and LPS regulate NCoR derepression further explain cooperative upregulation of proinflammatory cytokines induced by these 2 stimuli. Another difference in the mmLDL- and LPS-induced mechanisms is that mmLDL induces translocation of p65 to the nucleus but not its phosphorylation nor binding to DNA, whereas LPS stimulates all these steps of NF-κB activation.3 Thus, taken together, mmLDL and LPS complement each other by strengthening AP-1 and NF-κB promoter activity of target proinflammatory genes.
Importantly, the mmLDL and LPS cooperative effects were evident at a threshold LPS concentration (1 ng/mL) at which LPS alone induced a very limited macrophage response. Such levels of bacterial endotoxin can be found in human plasma during low-grade but sustained chronic infections, like periodontitis or infections with Chlamydia, which have been shown to accelerate the progression of atherosclerosis.6,7,9 In fact, the American Journal of Cardiology and the Journal of Periodontology jointly published an Editors’ Consensus recommending the early treatment of periodontitis to slow the progression of atherosclerosis in affected subjects.29
Recent clinical and experimental evidence suggests that low-level endotoxemia may be considerably more prevalent than previously realized,11 underscoring the importance of our findings. The gastrointestinal tract constitutes an enormous reservoir of biologically active bacterial products, including LPS, and a small proportion of gut-derived LPS can translocate into the circulation even in relatively healthy subjects.11 In the Bruneck (Italy) Study, plasma levels of endotoxin in the general population (516 men and women aged 50 to 79 years) ranged from 6 to 209 pg/mL, with a 3-fold higher risk of carotid atherosclerosis in individuals with endotoxin levels ≥50 pg/mL (90th percentile).30 A more recent study of a smaller population in England reported endotoxin levels of 3.1 EU/mL in nondiabetic and 5.5 EU/mL in patients with type 2 diabetes, which correspond to 310 pg/mL and 550 pg/mL LPS, respectively (according to the Food and Drug Administration EC-6 standard of 10 EU/ng).13 Miller et al analyzed ethnic differences and found a graded increase in endotoxin levels from black Africans to whites to South Asians, reporting significantly higher values than in other studies, as much as 14.4 EU/mL, or 1.44 ng/mL in South Asian men, and a strong correlation between plasma endotoxin and triglyceride concentrations.31 In addition, emerging evidence suggests that dietary habits alone may alter systemic exposure to endotoxin. For example, Erridge has demonstrated that a single high-fat meal markedly increases endotoxin concentrations postprandially in healthy human subjects, sufficient to activate vascular cells in vitro.11 These findings were supported by studies by Amar et al, correlating energy intake with endotoxemia in healthy subjects.32 In mice, a 4-week high-fat (72%) diet increased plasma endotoxin concentrations 2.7-fold to 4.9 EU/mL, or 490 pg/mL.14 Although absolute values vary considerably in different studies, the presence of subnanogram to nanogram per milliliter levels of endotoxin in plasma of seemingly normal subjects is well documented. To put the above numbers into perspective, the fatality rates for meningococcal patients with endotoxin plasma levels of 10 to 50, 50 to 100, 100 to 150, and >150 EU/mL were 30%, 100%, 89%, and 100%, respectively.33 Because a large portion of LPS is carried in plasma by lipoproteins,34,35 it is also plausible that LPS is present in atherosclerotic lesions, but this is yet to be determined.
In summary, our data demonstrate that cooperative engagement of AP-1 and NF-κB transcription factors by mmLDL and LPS results in additive/synergistic upregulation of proinflammatory genes in macrophages. This may constitute a mechanism of enhanced inflammatory activation within atherosclerotic lesions leading to the disease progression and increased risk of acute cardiovascular events, as suggested by human epidemiological studies of atherosclerosis complicated by chronic infections and other conditions associated with subclinical endotoxemia in apparently healthy subjects.
Sources of Funding
This work was supported by the NIH grants HL081862 (to Y.I.M.), GM069338 (to C.K.G., J.L.W., and Y.I.M.), HL088093 (J.L.W. and Y.I.M.), the American Recovery and Reconstruction Act administrative supplement HL081862-S1 (P.W. and Y.I.M.), and a grant from the Leducq Fondation (to P.W., C.K.G., J.L.W., and Y.I.M.).
Miller YI, Viriyakosol S, Binder CJ, Feramisco JR, Kirkland TN, Witztum JL. Minimally modified LDL binds to CD14, induces macrophage spreading via TLR4/MD-2, and inhibits phagocytosis of apoptotic cells. J Biol Chem. 2003; 278: 1561–1568.
Miller YI, Viriyakosol S, Worrall DS, Boullier A, Butler S, Witztum JL. Toll-like receptor 4-dependent and -independent cytokine secretion induced by minimally oxidized low-density lipoprotein in macrophages. Arterioscler Thromb Vasc Biol. 2005; 25: 1213–1219.
Choi S-H, Harkewicz R, Lee JH, Boullier A, Almazan F, Li AC, Witztum JL, Bae YS, Miller YI. Lipoprotein accumulation in macrophages via toll-like receptor-4-dependent fluid phase uptake. Circ Res. 2009; 104: 1355–1363.
Boullier A, Gillotte KL, Hörkkö S, Green SR, Friedman P, Dennis EA, Witztum JL, Steinberg D, Quehenberger O. The binding of oxidized low density lipoprotein to mouse CD36 is mediated in part by oxidized phospholipids that are associated with both the lipid and protein moieties of the lipoprotein. J Biol Chem. 2000; 275: 9163–9169.
Lehr HA, Sagban TA, Ihling C, Zahringer U, Hungerer KD, Blumrich M, Reifenberg K, Bhakdi S. Immunopathogenesis of atherosclerosis: endotoxin accelerates atherosclerosis in rabbits on hypercholesterolemic diet. Circulation. 2001; 104: 914–920.
Takaoka N, Campbell LA, Lee A, Rosenfeld ME, Kuo CC. Chlamydia pneumoniae infection increases adherence of mouse macrophages to mouse endothelial cells in vitro and to aortas ex vivo. Infect Immun. 2008; 76: 510–514.
Pussinen PJ, Tuomisto K, Jousilahti P, Havulinna AS, Sundvall J, Salomaa V. Endotoxemia, immune response to periodontal pathogens, and systemic inflammation associate with incident cardiovascular disease events. Arterioscler Thromb Vasc Biol. 2007; 27: 1433–1439.
Ghoshal S, Witta J, Zhong J, de Villiers W, Eckhardt E. Chylomicrons promote intestinal absorption of lipopolysaccharides. J Lipid Res. 2009; 50: 90–97.
Creely SJ, McTernan PG, Kusminski CM, Fisher f, Da Silva NF, Khanolkar M, Evans M, Harte AL, Kumar S. Lipopolysaccharide activates an innate immune system response in human adipose tissue in obesity and type 2 diabetes. Am J Physiol Endocrinol Metab. 2007; 292: E740–E747.
Cani PD, Amar J, Iglesias MA, Poggi M, Knauf C, Bastelica D, Neyrinck AM, Fava F, Tuohy KM, Chabo C, Waget A, Delmee E, Cousin B, Sulpice T, Chamontin B, Ferrieres J, Tanti JF, Gibson GR, Casteilla L, Delzenne NM, Alessi MC, Burcelin R. Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes. 2007; 56: 1761–1772.
Bae YS, Lee JH, Choi SH, Kim S, Almazan F, Witztum JL, Miller YI. Macrophages generate reactive oxygen species in response to minimally oxidized low-density lipoprotein: Toll-like receptor 4- and spleen tyrosine kinase-dependent activation of NADPH oxidase 2. Circ Res. 2009; 104: 210–218.
Benz DJ, Mol M, Ezaki M, Mori-Ito N, Zelaan I, Miyanohara A, Friedmann T, Parthasarathy S, Steinberg D, Witztum JL. Enhanced levels of lipoperoxides in low density lipoprotein incubated with murine fibroblast expressing high levels of human 15-lipoxygenase. J Biol Chem. 1995; 270: 5191–5197.
Harkewicz R, Hartvigsen K, Almazan F, Dennis EA, Witztum JL, Miller YI. Cholesteryl ester hydroperoxides are biologically active components of minimally oxidized LDL. J Biol Chem. 2008; 283: 10241–10251.
Raetz CRH, Garrett TA, Reynolds CM, Shaw WA, Moore JD, Smith DC Jr, Ribeiro AA, Murphy RC, Ulevitch RJ, Fearns C, Reichart D, Glass CK, Benner C, Subramaniam S, Harkewicz R, Bowers-Gentry RC, Buczynski MW, Cooper JA, Deems RA, Dennis EA. Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4. J Lipid Res. 2006; 47: 1097–1111.
Sasik R, Woelk CH, Corbeil J. Microarray truths and consequences. J Mol Endocrinol. 2004; 33: 1–9.
Sasik R, Calvo E, Corbeil J. Statistical analysis of high-density oligonucleotide arrays: a multiplicative noise model. Bioinformatics. 2002; 18: 1633–1640.
Ogawa S, Lozach J, Jepsen K, Sawka-Verhelle D, Perissi V, Sasik R, Rose DW, Johnson RS, Rosenfeld MG, Glass CK. A nuclear receptor corepressor transcriptional checkpoint controlling activator protein 1-dependent gene networks required for macrophage activation. Proc Natl Acad Sci U S A. 2004; 101: 14461–14466.
Ghisletti S, Huang W, Jepsen K, Benner C, Hardiman G, Rosenfeld MG, Glass CK. Cooperative NCoR/SMRT interactions establish a corepressor-based strategy for integration of inflammatory and anti-inflammatory signaling pathways. Genes Dev. 2009; 23: 681–693.
Miller YI, Worrall DS, Funk CD, Feramisco JR, Witztum JL. Actin polymerization in macrophages in response to oxidized LDL and apoptotic cells: role of 12/15-lipoxygenase and phosphoinositide 3-kinase. Mol Biol Cell. 2003; 14: 4196–4206.
Hevener AL, Olefsky JM, Reichart D, Nguyen MTA, Bandyopadyhay G, Leung HY, Watt MJ, Benner C, Febbraio MA, Nguyen AK, Folian B, Subramaniam S, Gonzalez FJ, Glass CK, Ricote M. Macrophage PPAR gamma is required for normal skeletal muscle and hepatic insulin sensitivity and full antidiabetic effects of thiazolidinediones. J Clin Invest. 2007; 117: 1658–1669.
Friedewald VE, Kornman KS, Beck JD, Genco R, Goldfine A, Libby P, Offenbacher S, Ridker PM, Van Dyke TE, Roberts WC. The American Journal of Cardiology and Journal of Periodontology Editors’ Consensus: periodontitis and atherosclerotic cardiovascular disease. Am J Cardiol. 2009; 104: 59–68.
Miller MA, McTernan PG, Harte AL, Silva NF, Strazzullo P, Alberti KG, Kumar S, Cappuccio FP. Ethnic and sex differences in circulating endotoxin levels: a novel marker of atherosclerotic and cardiovascular risk in a British multi-ethnic population. Atherosclerosis. 2009; 203: 494–502.
Amar J, Burcelin R, Ruidavets JB, Cani PD, Fauvel J, Alessi MC, Chamontin B, Ferrieres J. Energy intake is associated with endotoxemia in apparently healthy men. Am J Clin Nutr. 2008; 87: 1219–1223.
Kallio KAE, Buhlin K, Jauhiainen M, Keva R, Tuomainen AM, Klinge B, Gustafsson A, Pussinen PJ. Lipopolysaccharide associates with pro-atherogenic lipoproteins in periodontitis patients. Innate Immun. 2008; 14: 247–253.
Wurfel MM, Kunitake ST, Lichenstein H, Kane JP, Wright SD. Lipopolysaccharide (LPS)-binding protein is carried on lipoproteins and acts as a cofactor in the neutralization of LPS. J Exp Med. 1994; 180: 1025–1035.
Novelty and Significance
What Is Known?
Oxidation of low-density lipoprotein (LDL) significantly contributes to the progression of atherosclerosis, and oxidized LDL induces inflammatory responses in vascular cells.
Certain chronic infectious diseases, such as periodontitis and chlamydial infection, exacerbate clinical manifestations of atherosclerosis.
Low-level but persistent metabolic endotoxemia is often found in diabetic and obese subjects, who are at risk of cardiovascular disease, and is induced in mice fed a high-fat diet.
What New Information Does This Article Contribute?
Minimally oxidized LDL (mmLDL) and low doses of bacterial lipopolysaccharide (LPS) cooperatively activate macrophages to express higher levels of proinflammatory cytokines Cxcl2, Ccl3, and Ccl4.
The mechanism of this synergistic stimulation involves early and sustained activation of ERK1/2 (extracellular signal-regulated kinase 1/2) and JNK (c-Jun N-terminal kinase), release of NCoR (nuclear receptor corepressor) from the gene promoter regions, and derepression of the AP-1 (activator protein-1) transcription program.
Chronic inflammation in atherosclerotic lesions in coronary and cerebral arteries leads to formation of plaques vulnerable to rupture. Ruptured plaques cause intravascular thrombosis, which often results in a myocardial infarction or a stroke. In this study, we tested the hypothesis that 2 factors involved in the pathogenesis and complications of atherosclerosis (mmLDL and LPS) may synergize in inducing proinflammatory responses in macrophages. We demonstrated that mmLDL and LPS have different modes of activation of intracellular signaling pathways and that combined mmLDL and LPS stimulation of macrophages leads to enhanced transcriptional activity and elevated expression of proinflammatory cytokines. These mechanistic findings may explain why chronic infectious diseases, such as periodontitis and chlamydial infection, characterized by low-level but systemic elevations in LPS levels, accelerate atherosclerosis and exacerbate its clinical manifestations, leading to acute cardiovascular events. Our results are also relevant to the development of atherosclerosis in diabetic and overweight individuals consuming a Western-type diet, because recent studies have demonstrated that high-fat diets result in subclinical metabolic endotoxemia. Because lipoprotein oxidation is a well-documented event in the development of atherosclerosis, our data showing cooperative activation of macrophages with mmLDL and LPS suggest a possible mechanism of accelerated atherosclerosis in patients with subclinical endotoxemia.
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Original received February 5, 2010; revision received May 3, 2010; accepted May 7, 2010.