Tumor Necrosis Factor Receptor–Associated Factor 1 (TRAF1) Deficiency Attenuates Atherosclerosis in Mice by Impairing Monocyte Recruitment to the Vessel Wall
Background— Members of the tumor necrosis factor superfamily, such as tumor necrosis factor-α, potently promote atherogenesis in mice and humans. Tumor necrosis factor receptor–associated factors (TRAFs) are cytoplasmic adaptor proteins for this group of cytokines.
Methods and Results— This study tested the hypothesis that TRAF1 modulates atherogenesis in vivo. TRAF1−/−/LDLR−/− mice that consumed a high-cholesterol diet for 18 weeks developed significantly smaller atherosclerotic lesions than LDLR−/− (LDL receptor–deficient) control animals. As the most prominent change in histological composition, plaques of TRAF1-deficient animals contained significantly fewer macrophages. Bone marrow transplantations revealed that TRAF1 deficiency in both hematopoietic and vascular resident cells contributed to the reduction in atherogenesis observed. Mechanistic studies showed that deficiency of TRAF1 in endothelial cells and monocytes reduced adhesion of inflammatory cells to the endothelium in static and dynamic assays. Impaired adhesion coincided with reduced cell spreading, actin polymerization, and CD29 expression in macrophages, as well as decreased expression of the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in endothelial cells. Small interfering RNA studies in human cells verified these findings. Furthermore, TRAF1 messenger RNA levels were significantly elevated in the blood of patients with acute coronary syndrome.
Conclusions— TRAF1 deficiency attenuates atherogenesis in mice, most likely owing to impaired monocyte recruitment to the vessel wall. These data identify TRAF1 as a potential treatment target for atherosclerosis.
Received July 17, 2009; accepted March 16, 2010.
Atherosclerosis is a chronic inflammatory disease orchestrated by a network of inflammatory cytokines.1,2 Substantial in vitro and in vivo evidence implicates members of the tumor necrosis factor (TNF) receptor/interleukin (IL)-1/Toll-like receptor superfamily, such as TNF-α, CD40L, and IL-1β, in the development of atherosclerosis.3–5 TNF receptor–associated factors (TRAFs) function as intracellular adaptor proteins that mediate signaling for the TNF/IL-1/Toll-like receptor superfamily by upstream interaction with the respective receptors and consequent activation of downstream signaling molecules.6,7
Clinical Perspective on p 2044
TRAF1, a 46-kDa molecule, associates with several receptors, including TNFR1, TNFR2, and CD40. According to several studies, TRAF1 functions as an inhibitory protein.8,9 In contrast to other TRAFs, most resting cells lack TRAF1 but rapidly express TRAF1 on stimulation with TNF-α, CD40L, lipopolysaccharide, or lymphocyte receptor ligands.10,11 These data strongly suggest that TRAF1 participates in a negative-feedback loop. Several reports revealed that TRAF1 interferes with TRAF2-dependent nuclear factor-κB activation.12,13 Tsitsikov et al14 demonstrated enhanced TNF-α–induced signaling in TRAF1-deficient lymphocytes that coincided with hypersensitivity of TRAF1-deficient mice to skin necrosis provoked by TNF-α. Similarly, TRAF1-deficient mice proved more susceptible to TNF-α–induced liver damage.15 However, reports suggesting an opposite, proinflammatory role for TRAF1 as an activator of nuclear factor-κB and/or c-Jun N-terminal kinase (JNK)16,17 have hampered conclusive evaluation of the physiological role of TRAF1. Some of these controversies stem from differences in methodology and differential cell type–, cognate receptor–, and target gene–specific TRAF1-mediated functions, which warrants a disease-based in vivo evaluation.
Although TRAFs likely modulate atherogenesis in vivo, knowledge of the role of TRAFs in atherosclerosis remains rudimentary. Some reports identified TRAF6 as a mediator of CD40L-induced proinflammatory signals in monocytes and implicated this molecule in neointima formation in mice.18,19 Expression of TRAF2 and TRAF3 has been associated with shear stress in vitro and in vivo.20,21 Luo et al22 recently demonstrated that activation of TNF-receptor 2 (TNFR2) mediates ischemia-induced arteriogenesis by inducing TRAF2-dependent survival pathways. Our group recently demonstrated overexpression of several TRAFs, particularly TRAF1, in human and mouse atheromas.23 On the basis of these data, the present study tested the hypothesis that TRAF1 modulates mouse atherogenesis in vivo.
A detailed description of all methods is available in the online-only Data Supplement.
In Vivo Study
Six-week-old male low-density lipoprotein (LDL)-receptor–deficient (LDLR−/−) and TRAF1−/−/LDLR−/− mice consumed a high-cholesterol diet (HCD) for 18 and 8 weeks, respectively, and were prepared and analyzed histologically as described previously.24,25 Total wall area, intimal lesion area, and medial wall area, as well as the percentage of positively stained area for macrophages, lipids, T cells, collagen, smooth muscle cells, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1), were quantified by blinded investigators. For bone marrow (BM) transplantation, 8-week-old mice were subjected to total body irradiation (2×450 cGy). A total of 1.4×107 BM cells were injected in the tail vein. Four weeks later, an HCD was started for a total of 18 weeks.
Cell Isolation and Culture
Murine endothelial cells (ECs), BM-derived macrophages, and thioglycollate-elicited murine peritoneal leukocytes were isolated and cultured as described previously.23 For isolation of murine monocytes, TRAF1-deficient and control mice were anesthetized, and blood was taken by cardiac puncture, separated by Ficoll, and purified with CD11b MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Human umbilical vein ECs were obtained from PromoCell (Heidelberg, Germany).
Western blotting was performed as described previously.23 Primary antibodies included anti-ICAM-1 (mouse: Santa Cruz Biotechnology Inc, Santa Cruz, Calif; human: Cell Signaling Technology, Danvers, Mass), anti-VCAM (Santa Cruz), anti-integrin-β1 (Cell Signaling), anti-TRAF1 (Santa Cruz), and anti-lamin (Novocastra, Newcastle upon Tyne, United Kingdom).
Inflammatory Cytokine Quantification
Serum and arterial tissue lysates of mice were analyzed with a mouse inflammation kit (BD, Franklin Lakes, NJ). Serum from mice before and after HCD was analyzed with the Searchlight IR Cytokine Array (Endogen, Woburn, Mass). Mouse CXCL1 and CXCL2 were quantified in the supernatants of cell cultures by use of commercially available ELISA kits (R&D DuoSet, R&D Systems, Minneapolis, Minn).
Flow cytometry was performed as described previously.25 Antibodies included anti-CD106-APC, CD54-APC, CD49d-PE, CD40-PE (all from BD), CD62E (SouthernBiotech, Birmingham, Ala), CD29-FITC, CCR5-PE (BD), CCR7-APC (BD), CXCR3-APC (BD), CXCR2-PE (BD), and CD11b-FITC (Beckman Coulter, Fullerton, Calif). Intracellular staining for TNF-α, FoxP3, and interferon-γ was performed with the Cytofix/Cytoperm kit (BD).
Adhesion, Migration, Proliferation, and Apoptosis Assays
Adhesion and migration assays were performed as described previously.25 For spreading, murine peritoneal macrophages adhered to serum-coated coverslips were fixed with 4% paraformaldehyde and stained with Alexa Fluor 594–conjugated phalloidin (Invitrogen, Carlsbad, Calif). To identify proliferating cells, sections from the murine in vivo study were stained with an anti-Ki-67 antibody (DAKO, Glostrup, Denmark). Apoptosis was detected in parallel sections with the terminal deoxynucleotidyl transferase end-labeling technique (Roche Diagnostics, Indianapolis, Ind) and quantified by blinded investigators.
Mice were pretreated 4 hours before surgery with an intraperitoneal injection of 0.2 μg of murine TNF-α and anesthetized, and the cremaster muscle was exteriorized for intravital microscopy as described previously.26 Adhering leukocytes were quantified by blinded investigators.
Small Interfering RNA Transfections
Human umbilical vein ECs and human monocytes were transfected at 250 nmol/L final concentration of TRAF1-specific small interfering RNA (Qiagen, Hilden, Germany) with the Amaxa Nucleofector device (Lonza Cologne AG, Cologne, Germany) as described previously.23
Western blots were analyzed densitometrically with ImageJ (National Institutes of Health software). Data of at least 3 experiments were pooled and presented as mean±SEM. Student 2-tailed t test was used for paired or unpaired values (where appropriate). P<0.05 was considered statistically significant.
A total of 325 patients were included in the Tumor Necrosis Factor Receptor Associated Factors in Cardiovascular Risk Study (TRAFICS). After informed consent was received, blood was drawn from all patients, and total blood RNA was isolated with a Qiagen PAXgene blood RNA kit. All patients underwent coronary angiography and were divided into 3 groups: No coronary heart disease (no CHD), stable coronary heart disease (CHD), or acute coronary syndrome. Primers included for TRAF1 were 5′-TCC TgA gCT Tgg AgC AgA g-3′ (forward) and 5′-Agg gCC Tgg TCT TTC Tgg-3′ (reverse); for GAPDH, they were 5′-gAA ggT gAA ggT Cgg AgT c-3′ (forward) and 5′-gAA gAT ggT gAT ggg ATT TC -3′ (reverse). Differences across groups were compared by ANOVA followed by the Bonferroni post hoc test for normal variables and the Kruskal-Wallis test for nonnormal variables. Multivariate linear regression analysis was used to adjust for confounding factors, including age, sex, diabetes mellitus, hypertension, body mass index, and dyslipidemia.
TRAF1-Deficient Mice Develop Smaller Atherosclerotic Lesions That Contain Fewer Macrophages
To investigate the influence of TRAF1 on murine atherogenesis, TRAF1−/−/LDLR−/− and TRAF1+/+/LDLR−/− mice consumed an HCD for 8 and 18 weeks, respectively. Weights, leukocyte counts, total cholesterol, and triglyceride levels did not differ between the study groups (online-only Data Supplement Tables I and II). All mice appeared healthy and active, reproduced normally, and had no obvious abnormalities and a normal life span. TRAF1-deficient animals (n=8) had smaller intimal lesions of aortic roots (P=0.02) and arches (P=0.05) than controls (n=13) at both time points, which suggests a proatherogenic function of TRAF1 (Figure 1A and 1B). Plaques from TRAF1−/−/LDLR−/− mice contained significantly fewer macrophages (P=0.05 and 0.01) and more smooth muscle cells (P=0.04 and 0.04; Figure 1C). Lipid content tended to decrease, whereas collagen content tended to increase. T-cell counts remained unchanged in plaques and adventitia (see online-only Data Supplement Figure I for representative images). Taken together, these features represent characteristics attributed to more stable plaques in humans.27
TRAF1 Deficiency in BM-Derived and Resident Cells Attenuates Atherogenesis in Mice
To elucidate the relevance of TRAF1 in BM-derived and resident cells for atherogenesis, we performed BM transplantations between TRAF1−/−/LDLR−/− and TRAF1+/+/LDLR−/− mice (for study characteristics, see online-only Data Supplement Table III). Mice with simultaneous deficiency in TRAF1 in BM-derived and resident cells showed the greatest reduction in intimal lesion size compared with respective wild-type controls (39.9±10.6%; n=8 and 5 per group, respectively; P=0.01). TRAF1 deficiency in both BM-derived and resting cells alone sufficed to attenuate atherogenesis. Analysis of plaque composition revealed a significant reduction of macrophage content and lipids, whereas collagen and smooth muscle cell content tended to be increased in the TRAF1-deficient chimera (Figure 2).
TRAF1 Deficiency Attenuates Adhesion of Monocytes
Because we observed markedly decreased macrophage content in plaques from TRAF1−/−/LDLR−/− animals, we tested the hypothesis that TRAF1 modulates adhesion of inflammatory cells, a key step in atherosclerotic plaque formation. TRAF1 deficiency in both monocytes and ECs significantly inhibited adhesion compared with respective wild-type controls (n=3, P<0.001; Figure 3A). Similar findings emerged with peripheral blood mononuclear cells (n=3, P<0.001; Figure 3A). Under flow conditions relevant to those in human vessels, deficiency of TRAF1 in both ECs and thioglycollate-elicited peritoneal leukocytes also significantly inhibited adhesion (n=5, P=0.003; Figure 3B). TRAF1 deficiency in either ECs or leukocytes alone sufficed to attenuate adhesion of leukocytes (n=5, P=0.04 for both; Figure 3B; online-only Data Supplement Figure II). Similar results were observed with cells on an LDLR-deficient background (online-only Data Supplement Figure III). Accordingly, we identified markedly reduced adhesion of leukocytes in the venules of the cremaster muscle in TRAF1-deficient mice compared with wild-type mice in vivo as assessed by intravital microscopy (n=7 and 8, respectively; P=0.009; Figure 3C).
TRAF1 Deficiency Limits Actin Polymerization and Expression of Adhesion Molecules in ECs and Macrophages
Wild-type peritoneal macrophages quickly spread on glass coverslips, whereas the morphology of TRAF1-deficient macrophages remained largely unchanged as assessed by phalloidin staining (n=5, P<0.001; Figure 3D), which suggests that TRAF1 deficiency interferes with actin polymerization. Adhesion molecules regulate protrusion and adhesion. TRAF1 deficiency significantly decreased the expression of ICAM-1 and VCAM-1 in TNF-α–stimulated ECs by 22±4% (n=7, P=0.02) and 27±2% (n=6, P=0.01; Figure 4A), respectively. Respective experiments with TRAF1-deficient or TRAF1-competent ECs from animals also deficient in LDLR generated similar results (online-only Data Supplement Figure III). Furthermore, VCAM-1 and ICAM-1 expression was markedly reduced in both arterial tissue (n=3, P=0.01 and 0.007, respectively) and aortic sections of TRAF1−/−/LDLR−/− mice compared with TRAF1+/+/LDLR−/− mice as assessed by Western blotting and immunohistochemistry (Figure 4B and 4C). TRAF1-deficient BM-derived macrophages showed reduced integrin-β1 (CD29; n=4, P=0.004) expression, whereas no significant difference in CD11b expression was observed (n=3, P=0.88; Figure 4D). Similar findings were obtained in T cells (online-only Data Supplement Figure IV).
TRAF1 Deficiency Differentially Regulates Chemokine and Chemokine Receptor Expression but Does Not Alter Inflammatory Cell Migration
Chemotaxis presents a crucial step in the recruitment of monocytes to the intima.2 We previously reported a slight increase in monocyte chemotactic protein-1 expression in TRAF1-deficient ECs and macrophages.23 Similarly, we observed a significant increase in KC (CXCL1) in TRAF1-deficient ECs (n=8, P=0.03) but not in macrophages (n=6, P=0.34; 95% confidence interval [CI] 0.02 to 0.40 and 0.13 to 0.48, respectively). Macrophage inflammatory protein-2 (CXCL2) expression was not significantly regulated in either cell type (respectively, n=7 and 3, P=0.72 and P=0.81, 95% CI 0.19 to 0.86 and 0.17 to 0.94; Figure 5A and 5B). Western blot analysis confirmed no significant difference between TRAF1-deficient and wild-type mice in CXCR1 expression (n=3, P=0.76; Figure 5B). Further evaluation of chemokine receptor expression revealed a significant increase of CCR7 (P=0.02) and CXCR3 (P=0.008) and no significant regulation for CCR5 (P=0.2) or CXCR2 (P=0.4) expression in TRAF1-deficient monocytes compared with respective controls (n=3 each; Figure 5C). Similar results were obtained in TRAF1-deficient CD4 and CD8 T cells before and after α-CD3/α-CD28 stimulation (online-only Data Supplement Figure V). Migration of inflammatory cells not only depends on chemokines and chemokine receptors but also on the migratory capacity of inflammatory cells. TRAF1 deficiency did not significantly alter the chemotactic index of peripheral blood mononuclear cells to gradients of various chemokines compared with wild-type controls (n=3 each; Figure 5D).
IL-6 Levels Decrease in Blood and Arterial Tissue of TRAF1-Deficient Mice
Inflammatory cytokines were assayed in blood and aortic arterial tissue of TRAF1−/−/LDLR−/− and TRAF1+/+/LDLR−/− mice, respectively, at baseline (n=10 and 16) and after 18 weeks of HCD (n=10 and 18). TRAF1 deficiency provoked a significant reduction of IL-6 (P=0.04 and 0.059; Figure 6A). Furthermore, we identified reduced IL-6 concentrations in arterial tissue (10.56±3.4 versus 65.63±19 pg/mL, P=0.01) and serum (13.06±5.3 versus 36.61±7.3 pg/mL, P=0.02) of TRAF1-deficient animals 4 hours after intraperitoneal injection of 200 ng of TNF-α compared with respective controls. TRAF1-deficient animals expressed decreased levels of TNF-α after HCD in serum and arterial tissue (16.53±0.1 versus 10.63±0.37 pg/mL, P=0.004), whereas no significant regulation could be observed for IL-1β and IL-1α (95% CI 9.82 to 58.52 and 13.60 to 30.86, respectively; Figure 6A).
TRAF1 Deficiency Does Not Affect Differentiation of Monocytes/Macrophages
TRAF1-deficient macrophages showed no significant difference in FA-11 and F4/80 expression. The number of Ki-67–positive macrophages did not differ significantly in plaques of TRAF1-deficient and control mice (n=6 and 13, P=0.9, 95% CI 2.18 to 7.82 and 2.41 to 7.13, respectively; Figure 6B).
TRAF1 Deficiency Does Not Influence Apoptosis in Atherosclerotic Plaques In Vivo
Terminal deoxynucleotidyl transferase end-labeling assays performed on sections of the aortic root and arch of study animals revealed no significant difference in apoptosis rates in atherosclerotic plaques of TRAF1-deficient and -competent mice (n=5 and 13, P=0.88, 95% CI 0.76 to 9.33 and 3.66 to 6.05, respectively; Figure 6C). These data coincide with previous results from our group that demonstrated similar caspase-3/7 activation in TRAF1-deficient and -competent ECs.23
TRAF1 Deficiency Does Not Significantly Alter General Immune Responses
CD4+ T lymphocytes comprise several subsets, including Th1, Th2, and Th17 cells and Tregs. We tested whether TRAF1 modulates the ratios of these subsets, which may regulate atherogenesis. Splenic CD4, CD8 T-cell, and Treg numbers in mice that consumed an HCD did not differ significantly between TRAF1−/−/LDLR−/− and TRAF1+/+/LDLR−/− mice, nor did T-cell differentiation and activation markers differ significantly (n=6; Figure 7A). On α-CD3/α-CD28 stimulation, activation markers and intracellular interferon-γ and TNF-α did not significantly differ between splenocytes of the 2 groups. In accordance with this, supernatants showed no significant regulation of interferon-γ, IL12p70, or IL-10 (n=3; Figure 7B). Additionally, we did not observe modulation of Th1 and Th2 cytokines in blood or arterial tissue of TRAF1−/−/LDLR−/− or TRAF1+/+/LDLR−/− mice that consumed an HCD or respective mice on intraperitoneal challenge with TNF-α (Figure 7C and 7D).
RNA Silencing in Human Cells Corroborates the Concept That TRAF1 Limits the Expression of Adhesion Molecules
TRAF1-silenced ECs expressed significantly reduced levels of VCAM-1 and ICAM-1 on stimulation with TNF-α (P=0.01 and 0.01) and IL-1β (P=0.01 and 0.03), as well as reduced VCAM-1 expression on CD40L stimulation (P=0.01), which confirmed our findings in murine ECs (n=4 each; Figure 8A). TRAF1-silenced human monocytes expressed significantly lower amounts of integrin-β1 than respective control-silenced monocytes (n=3, P=0.03), whereas no significant difference in CXCR1 and CD11b expression could be detected (Figure 8A).
TRAF1 Messenger RNA Expression Is Elevated in Blood of Patients With Acute Coronary Syndrome
We tested the hypothesis that TRAF1 expression in blood correlates with stable and/or acute coronary heart disease in humans.23 Given that TRAF1 constitutes an intracellular protein, we quantified TRAF1 messenger RNA (mRNA) in total blood RNA of 325 patients undergoing coronary angiography divided into 3 groups: No coronary heart disease (no CHD), stable coronary heart disease (CHD), or acute coronary syndrome. Gender and body mass index did not differ significantly among the groups, although patients were older in the CHD group, and those in the CHD and acute coronary syndrome groups presented with a significantly greater percentage of cardiovascular risk factors such as diabetes and hypertension (Table). TRAF1/GAPDH mRNA ratios were significantly elevated in patients with acute coronary syndrome compared with those with CHD or no CHD (0.034±0.0056, 0.023±0.0012, and 0.026±0.0024, respectively; P=0.04 for both; Figure 8B). After adjustment for age and sex, the association remained significant (β=0.16, P=0.039). After adjustment for other potential confounders (diabetes, hypertension, body mass index, and dyslipidemia), the association only approached the limit of statistical significance (β=0.16, P=0.054).
The present study reports the novel and surprising finding that TRAF1 deficiency attenuates atherosclerosis in mice. Our data challenge the traditional view of TRAF1 as an inhibitory, primarily antiinflammatory signaling molecule by identifying TRAF1 as a proinflammatory mediator of atherosclerosis, a chronic inflammatory disease.
Although we previously showed that TRAF1 inhibits certain proinflammatory signals in cell types involved in atheromas, the present study demonstrates that TRAF1 acts as a proinflammatory mediator of atherosclerosis, because TRAF1-deficient mice had reduced atherogenesis.23 The present data are in accord with 2 recent reports by Oyoshi et al28,29 implicating TRAF1 in the development of allergic lung inflammation in mice, which suggests that TRAF1 may also play an instrumental role in the pathogenesis of inflammatory diseases other than atherosclerosis. Notably, overexpression of TRAF1 has also occurred in hematologic disorders such as lymphoma and solid tumors.30,31 Genome-wide studies further showed a correlation between polymorphisms in the TRAF1/C5 locus and the incidence of rheumatoid arthritis and systemic lupus erythematosus32,33; however, functional analysis of TRAF1 in these diseases is still lacking.
The present study not only showed a reduction in overall lesion size in early and delayed TRAF1−/−/LDLR−/− plaques compared with respective controls but also demonstrated that TRAF1-deficient plaques showed histological features attributed to more stable plaques in humans. Plaques from TRAF1−/−/LDLR−/− animals had a marked reduction in macrophages. Intimal macrophages contribute importantly to atherogenesis; their accumulation progresses during plaque growth and is associated with thrombotic complications.2,34 Four main processes regulate macrophage content in plaques: Adhesion, migration, differentiation, and apoptosis. To gain mechanistic insight into how TRAF1 modulates intraplaque macrophage content, we systematically tested whether TRAF1 affected any of these steps. Interaction of integrins and adhesion molecules such as ICAM-1 and VCAM-1 mediates adhesion of inflammatory cells to the EC surface. We identified that a deficiency of TRAF1 in murine ECs and monocytes reduced adhesion of inflammatory cells to the endothelium in static and dynamic adhesion assays in vitro and in intravital microscopy of the cremaster muscle. This may be due to decreased expression of VCAM-1 and ICAM-1, which we observed both in lysates of ECs and in aortic arterial tissue of TRAF1-deficient mice. VCAM-1 binds to α4β1 integrin (CD49d/CD29). Interestingly, CD29 was also downregulated in its expression in TRAF1-deficient macrophages, which suggests that TRAF1 in both leukocytes and ECs contributes to atherogenesis. This notion is supported by our transplantation study, which showed that TRAF1 deficiency in both BM-derived and resident cells was sufficient to reduce lesion formation. In agreement with our findings, Oyoshi et al29 demonstrated impaired lymphocyte recruitment to inflamed lungs by reduced expression of VCAM-1 and ICAM-1 in TRAF1-deficient ECs.
Chemokines pave the way for monocyte extravasation.35 TRAF1 deficiency did not attenuate chemokine expression in ECs or macrophages. At the receptor level, CCR7, previously associated with regression of atherosclerosis, and CXCR3, known to promote atherogenesis in mice, were both upregulated in monocytes and T cells of TRAF1−/−/LDLR−/− mice36,37; however, CCR5, which is also known to promote atherosclerosis in mice,38 remained unchanged in monocytes and was decreased in T cells. Nevertheless, TRAF1 deficiency did not alter the migratory capacity of peripheral blood mononuclear cells toward chemokine gradients.
We identified reduced levels of IL-6 and TNF-α in serum and arterial tissue of TRAF1-deficient mice that consumed an HCD. Increasing evidence supports the idea that CD4 T cells play a crucial role in atherogenesis.1 Imbalance of the ratio of the different CD4 T-cell subsets (Th1, Th2, Th17 and Tregs) modulates atherogenesis and plaque destabilization. Bryce et al39 demonstrated TRAF1-dependent upregulation of the Th2 cytokines IL-4, IL-5, and IL-13 on α-CD3/α-CD28 stimulation in TRAF1-deficient cells compared with wild-type cells. In contrast, we observed no major variation in subset ratios and Th1 and Th2 cytokines in atherosclerotic mice in vitro and in vivo in the present study.
Several studies have suggested an antiapoptotic function of TRAF1.40–42 The present data showed no significant difference in apoptosis or proliferation of macrophages in vivo, which suggests that TRAF1 does not influence cell turnover in the atherosclerotic plaque. Because TRAF1 deficiency did not fully prevent atherosclerosis, other TRAFs may contribute. TRAF2 and TRAF6 are likely candidates.19,20
To investigate the hypothesis that TRAF1 mRNA expression in blood correlates with stable and/or acute coronary heart disease in humans, we performed a pilot study. TRAF1 mRNA levels were significantly elevated in patients who had an acute coronary syndrome, consistent with participation of TRAF1 in plaque instability in vivo. These data match previous findings from our group that demonstrated a strong increase of TRAF1 in carotid human atherosclerotic plaques prone to rupture.23 Further studies will be required to validate these findings.
In summary, we present the novel finding that TRAF1 deficiency attenuates atherogenesis in mice. We identified impaired monocyte recruitment to the vessel wall as the most likely underlying mechanism. Our data suggest that TRAF1 merits further testing as a therapeutic target in atherosclerosis and other chronic inflammatory diseases.
We thank Dr Michael Reth from the Max Planck Institute for his great mentorship; Dr Uwe Schünbeck from Pfizer, USA, for the important and fruitful discussions on the topic; Dr Jens Stein from the University of Bern, Switzerland, for excellent training and advice on intravital microscopy; Dr Marie Follow for her expert advice on quantitative polymerase chain reaction; and Dr Florian Willecke for critical review of the manuscript.
Sources of Funding
This work was supported by research grants from the German Research Foundation to Dr A. Zirlik (DFG ZI743/3-1 and 3-2) and from the US National Heart, Lung, and Blood Institute to Dr Libby (HL34636). Dr Libby’s laboratory also receives research funding from the Donald W. Reynolds Foundation and the Fondation Leducq. Dr Missiou was funded by the Excellence Initiative of the German Research Foundation (DFG GSC-4, Spemann Graduate School).
Branen L, Hovgaard L, Nitulescu M, Bengtsson E, Nilsson J, Jovinge S. Inhibition of tumor necrosis factor-alpha reduces atherosclerosis in apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol. 2004; 24: 2137–2142.
Chi H, Messas E, Levine RA, Graves DT, Amar S. Interleukin-1 receptor signaling mediates atherosclerosis associated with bacterial exposure and/or a high-fat diet in a murine apolipoprotein E heterozygote model: pharmacotherapeutic implications. Circulation. 2004; 110: 1678–1685.
Rothe M, Sarma V, Dixit VM, Goeddel DV. TRAF2-mediated activation of NF-kappa B by TNF receptor 2 and CD40. Science. 1995; 269: 1424–1427.
Zapata JM, Reed JC. TRAF1: lord without a RING. Sci STKE. 2002; 2002: PE27.
Fotin-Mleczek M, Henkler F, Hausser A, Glauner H, Samel D, Graness A, Scheurich P, Mauri D, Wajant H. Tumor necrosis factor receptor-associated factor (TRAF) 1 regulates CD40-induced TRAF2-mediated NF-kappaB activation. J Biol Chem. 2004; 279: 677–685.
Song HY, Rothe M, Goeddel DV. The tumor necrosis factor-inducible zinc finger protein A20 interacts with TRAF1/TRAF2 and inhibits NF-kappaB activation. Proc Natl Acad Sci U S A. 1996; 93: 6721–6725.
Xie P, Hostager BS, Munroe ME, Moore CR, Bishop GA. Cooperation between TNF receptor-associated factors 1 and 2 in CD40 signaling. J Immunol. 2006; 176: 5388–5400.
Duckett CS, Gedrich RW, Gilfillan MC, Thompson CB. Induction of nuclear factor kappaB by the CD30 receptor is mediated by TRAF1 and TRAF2. Mol Cell Biol. 1997; 17: 1535–1542.
Mukundan L, Bishop GA, Head KZ, Zhang L, Wahl LM, Suttles J. TNF receptor-associated factor 6 is an essential mediator of CD40-activated proinflammatory pathways in monocytes and macrophages. J Immunol. 2005; 174: 1081–1090.
Donners MM, Beckers L, Lievens D, Munnix I, Heemskerk J, Janssen BJ, Wijnands E, Cleutjens J, Zernecke A, Weber C, Ahonen CL, Benbow U, Newby AC, Noelle RJ, Daemen MJ, Lutgens E. The CD40-TRAF6 axis is the key regulator of the CD40/CD40L system in neointima formation and arterial remodeling. Blood. 2008; 111: 4596–4604.
Sotoudeh M, Li YS, Yajima N, Chang CC, Tsou TC, Wang Y, Usami S, Ratcliffe A, Chien S, Shyy JY. Induction of apoptosis in vascular smooth muscle cells by mechanical stretch. Am J Physiol Heart Circ Physiol. 2002; 282: H1709–H1716.
Zirlik A, Bavendiek U, Libby P, MacFarlane L, Gerdes N, Jagielska J, Ernst S, Aikawa M, Nakano H, Tsitsikov E, Schonbeck U. TRAF-1, -2, -3, -5, and -6 are induced in atherosclerotic plaques and differentially mediate proinflammatory functions of CD40L in endothelial cells. Arterioscler Thromb Vasc Biol. 2007; 27: 1101–1107.
Bavendiek U, Zirlik A, LaClair S, MacFarlane L, Libby P, Schonbeck U. Atherogenesis in mice does not require CD40 ligand from bone marrow-derived cells. Arterioscler Thromb Vasc Biol. 2005; 25: 1244–1249.
Zirlik A, Maier C, Gerdes N, MacFarlane L, Soosairajah J, Bavendiek U, Ahrens I, Ernst S, Bassler N, Missiou A, Patko Z, Aikawa M, Schonbeck U, Bode C, Libby P, Peter K. CD40 ligand mediates inflammation independently of CD40 by interaction with Mac-1. Circulation. 2007; 115: 1571–1580.
Sukhova GK, Schonbeck U, Rabkin E, Schoen FJ, Poole AR, Billinghurst RC, Libby P. Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques. Circulation. 1999; 99: 2503–2509.
Oyoshi MK, Bryce P, Goya S, Pichavant M, Umetsu DT, Oettgen HC, Tsitsikov EN. TNF receptor-associated factor 1 expressed in resident lung cells is required for the development of allergic lung inflammation. J Immunol. 2008; 180: 1878–1885.
Zhang B, Wang Z, Li T, Tsitsikov EN, Ding HF. NF-kappaB2 mutation targets TRAF1 to induce lymphomagenesis. Blood. 2007; 110: 743–751.
Zapata JM, Krajewska M, Morse HC III, Choi Y, Reed JC. TNF receptor-associated factor (TRAF) domain and Bcl-2 cooperate to induce small B cell lymphoma/chronic lymphocytic leukemia in transgenic mice. Proc Natl Acad Sci U S A. 2004; 101: 16600–16605.
Plenge RM, Seielstad M, Padyukov L, Lee AT, Remmers EF, Ding B, Liew A, Khalili H, Chandrasekaran A, Davies LR, Li W, Tan AK, Bonnard C, Ong RT, Thalamuthu A, Pettersson S, Liu C, Tian C, Chen WV, Carulli JP, Beckman EM, Altshuler D, Alfredsson L, Criswell LA, Amos CI, Seldin MF, Kastner DL, Klareskog L, Gregersen PK. TRAF1–C5 as a risk locus for rheumatoid arthritis: a genomewide study. N Engl J Med. 2007; 357: 1199–1209.
Kurreeman FA, Goulielmos GN, Alizadeh BZ, Rueda B, Houwing-Duistermaat J, Sanchez E, Bevova M, Radstake TR, Vonk MC, Galanakis E, Ortego N, Verduyn W, Zervou MI, Consortium S, Roep BO, Dema B, Espino L, Urcelay E, Boumpas DT, van den Berg LH, Wijmenga C, Koeleman BP, Huizinga TW, Toes RE, Martin J. The TRAF1-C5 region on chromosome 9q33 is associated with multiple autoimmune diseases. Ann Rheum Dis. 2010; 69: 696–699.
Swirski FK, Pittet MJ, Kircher MF, Aikawa E, Jaffer FA, Libby P, Weissleder R. Monocyte accumulation in mouse atherogenesis is progressive and proportional to extent of disease. Proc Natl Acad Sci U S A. 2006; 103: 10340–10345.
Smith DF, Galkina E, Ley K, Huo Y. GRO family chemokines are specialized for monocyte arrest from flow. Am J Physiol Heart Circ Physiol. 2005; 289: H1976–H1984.
Trogan E, Feig JE, Dogan S, Rothblat GH, Angeli V, Tacke F, Randolph GJ, Fisher EA. Gene expression changes in foam cells and the role of chemokine receptor CCR7 during atherosclerosis regression in ApoE-deficient mice. Proc Natl Acad Sci U S A. 2006; 103: 3781–3786.
Veillard NR, Steffens S, Pelli G, Lu B, Kwak BR, Gerard C, Charo IF, Mach F. Differential influence of chemokine receptors CCR2 and CXCR3 in development of atherosclerosis in vivo. Circulation. 2005; 112: 870–878.
Combadiere C, Potteaux S, Rodero M, Simon T, Pezard A, Esposito B, Merval R, Proudfoot A, Tedgui A, Mallat Z. Combined inhibition of CCL2, CX3CR1, and CCR5 abrogates Ly6C(hi) and Ly6C(lo) monocytosis and almost abolishes atherosclerosis in hypercholesterolemic mice. Circulation. 2008; 117: 1649–1657.
Bryce PJ, Oyoshi MK, Kawamoto S, Oettgen HC, Tsitsikov EN. TRAF1 regulates Th2 differentiation, allergic inflammation and nuclear localization of the Th2 transcription factor, NIP45. Int Immunol. 2006; 18: 101–111.
Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS Jr. NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science. 1998; 281: 1680–1683.
Sabbagh L, Pulle G, Liu Y, Tsitsikov EN, Watts TH. ERK-dependent Bim modulation downstream of the 4-1BB-TRAF1 signaling axis is a critical mediator of CD8 T cell survival in vivo. J Immunol. 2008; 180: 8093–8101.
Speiser DE, Lee SY, Wong B, Arron J, Santana A, Kong YY, Ohashi PS, Choi Y. A regulatory role for TRAF1 in antigen-induced apoptosis of T cells. J Exp Med. 1997; 185: 1777–1783.
Tumor necrosis factor receptor–associated factors (TRAFs) mediate inflammatory signaling for important cytokines of the tumor necrosis factor/interleukin-1/Toll-like receptor superfamily such as CD40L, tumor necrosis factor-α, and interleukin-1β. Atherosclerosis is a chronic inflammatory disease governed by a network of such inflammatory cytokines. Although the inflammatory nature of atherosclerosis has been known for some time, cardiology still lacks a causal antiinflammatory or immunomodulatory treatment option. The potential of such therapies is clearly suggested by the pleiotropic treatment benefits of statins, most recently demonstrated in the JUPITER trial (Justification for the Use of Statins in Primary Prevention: An Intervention Trial Evaluating Rosuvastatin). Although overall inhibition of cytokines may produce a variety of undesirable side effects, the inhibition of specific signaling intermediates potentially may overcome some of these limitations. The present study presents the novel and somewhat unexpected finding that TRAF1 deficiency potently attenuates murine atherosclerosis, most likely by impairing monocyte recruitment to the vessel wall, which suggests a proatherogenic function of TRAF1. In line with this notion, we found increased expression of TRAF1 in blood of patients who had an acute coronary syndrome. Future studies will be needed to determine whether TRAF1 targeting might indeed represent a novel treatment strategy for chronic inflammatory diseases such as atherosclerosis.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.895037/DC1.
Guest Editor for this article was Aruni Bhatnagar, PhD.
Dr Missiou and N. Köstlin contributed equally to this work.