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Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:2677-2683
Published online before print September 27, 2007, doi: 10.1161/ATVBAHA.107.151274
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:2677.)
© 2007 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Induction of Oral Tolerance to HSP60 or an HSP60-Peptide Activates T Cell Regulation and Reduces Atherosclerosis

G.H.M. van Puijvelde; T. van Es; E.J.A. van Wanrooij; K.L.L. Habets; P. de Vos; R. van der Zee; W. van Eden; Th. J.C. van Berkel; J. Kuiper

From the Division of Biopharmaceutics (G.H.M.v.P., T.v.E., E.J.A.v.W., K.L.L.H., P.d.V., T.J.C.v.B., J.K.), Leiden University, and the Department of Infectious Diseases and Immunology (R.v.d.Z., W.v.E.), Utrecht University, the Netherlands.

Correspondence to G.H.M. van Puijvelde, LACDR, Division of Biopharmaceutics, PO Box 9502, 2300 RA Leiden, The Netherlands. E-mail puijvelde{at}chem.leidenuniv.nl


*    Abstract
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Objective— HSP60-specific T cells contribute to the development of the immune responses in atherosclerosis. This can be dampened by regulatory T cells activated via oral tolerance induction, and we explored the effect of oral tolerance induction to HSP60 and the peptide HSP60 (253 to 268) on atherosclerosis.

Methods and Results— HSP60 and HSP60 (253 to 268) were administered orally to LDLr–/– mice before induction of atherosclerosis and resulted in a significant 80% reduction in plaque size in the carotid arteries and in a 27% reduction in plaque size at the aortic root. Reduction in plaque size correlated with an increase in CD4+CD25+Foxp3+ regulatory T cells in several organs and in an increased expression of Foxp3, CD25, and CTLA-4 in atherosclerotic lesions of HSP60-treated mice. The production of interleukin (IL)-10 and transforming growth factor (TGF)-β by lymph node cells in response to HSP60 was observed after tolerance induction.

Conclusion— Oral tolerance induction to HSP60 and a small HSP60-peptide leads to an increase in the number of CD4+CD25+Foxp3+ regulatory T cells, resulting in a decrease in plaque size as a consequence of increased production of IL-10 and TGF-β. We conclude that these beneficial results of oral tolerance induction to HSP60 and HSP60 (253 to 268) may provide new therapeutic approaches for the treatment of atherosclerosis.

HSP60-specific T cells contributing to the development of atherosclerosis can be counteracted by Tregs, which can be activated by oral tolerance induction to HSP60 and may produce IL-10 and TGF-β. This results in a beneficial effect on atherosclerosis and may provide a new therapeutic approach for the treatment of atherosclerosis.


Key Words: atherosclerosis • immune system • tolerance induction • regulatory T cells • heat shock proteins


*    Introduction
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Heat shock proteins (HSPs) are a family of highly conserved proteins with various functions in normal and stressful situations. Expression of HSPs on endothelial cells and macrophages1,2 can be induced by fluid shear stress,3 oxidized lipoproteins,4 and cytokines.2 Under these circumstances, HSPs repair or prevent degradation of denaturated proteins and increase the ability of the cell to survive stressful stimuli.5,6 HSPs such as HSP60 are also involved in inflammatory diseases, probably resulting from their raised expression in cells exposed to proinflammatory mediators.7,8 In human atherosclerotic lesions,9 enhanced HSP60 expression has been detected. In addition, patients with atherosclerosis show an elevated concentration of HSP60-specific antibodies in serum,2 and T cell clones with self-HSP60 reactivity have been detected within the atherosclerotic plaques.10 This may be related to initial immune responses against bacterial HSPs which are highly homologous to HSPs in various other species including men, rats, and mice.11 HSP60-specific antibodies may contribute to endothelial damage and the inflammatory response in the vessel wall accelerating atherosclerosis.12

The autoimmune process in atherosclerosis is characterized by a T cell response to different autoantigens, eg, oxidized LDL,13 glycoproteins,14 and HSPs.15 HSP60-specific T cells are mainly of a Th1 phenotype, producing proatherogenic cytokines, such as IFN-{gamma}, IL-12, and tumor necrosis factor (TNF)-{alpha} and causing a disturbed balance between Th1 and Th2 cytokines.16,17 For a long time, this disturbed balance was regarded as the cause of the ongoing inflammation in atherosclerosis. Recent publications, however, suggest that regulatory T cells (Tregs) play an important role in prevention of Th1 mediated autoimmune diseases such as multiple sclerosis,18 diabetes mellitus,19 and atherosclerosis.20 Mallat et al hypothesized that in atherosclerosis an imbalance exists between pathogenic T cells (Th1 and Th2) and Tregs specific for "altered" self and nonself antigens (eg, oxidized phospholipids, heat shock proteins).21

One way to increase the number of antigen specific Tregs is "low dose" oral tolerance induction. This method is already used as a treatment in animal models for Th1 mediated autoimmune diseases such as multiple sclerosis,22 rheumatoid arthritis,23 and type I diabetes.24 Initial studies also show that oral tolerance induction to β2-glycoprotein I25 and HSP6526,27 results in the suppression of early atherosclerosis. However, these studies do not show the involvement of Tregs. We describe in a recent study an increase in the number of CD4+CD25+Foxp3+ cells after oral tolerance induction to oxidized LDL (oxLDL)28 and a subsequent reduction in plaque size. These CD4+CD25+Foxp3+ cells form a class of Tregs that may either be natural Tregs which act via cell-cell contact via surface-bound TGF-β29 or cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)30 or adaptive Tregs operating via the secretion of TGF-β.31

The present study shows that induction of oral tolerance to HSP60 and a highly conserved sequence of HSP60 (HSP60 [253 to 268]) attenuates atherosclerosis. The effect on atherosclerosis is explained by an increased number of CD4+CD25+Foxp3+ Tregs in both lymphoid organs and the atherosclerotic lesion. This is accompanied by an increase in HSP60-specific TGF-β and IL-10 production in mesenteric lymph node cells.


*    Methods
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To test the presence of T cells specific for HSP60, splenocytes of naive and immunized LDLr–/– mice were isolated and incubated with mycobacterial HSP60, a HSP60 peptide (253 to 268), and a HSP70 peptide (111 to 125). Proliferation of T cells was measured by incorporation of [6-3H]-thymidine.

Subsequently, LDLr–/– mice were treated orally with PBS, HSP60, HSP60 (253 to 268), and HSP70 (111 to 125) 4 times in 8 days. After treatment the mice were equipped with collars around both carotid arteries, and the mice were fed a Western type diet. The effect on atherosclerotic plaque development was analyzed after 6 weeks by sectioning and staining the carotid arteries and the aortic root.

Using fluorescence-activated-cell sorter (FACS) analysis and real-time polymerase chain reaction (PCR) assays, the effect of oral administration of the different antigens was investigated, respectively, on the number of regulatory T cells and the mRNA expression of markers for regulatory T cells. The production of cytokines by lymph node cells was determined by ELISA.

For details please see the supplemental materials, available online at http://atvb.ahajournals.org.


*    Results
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T Cells Specific for HSP60 and HSP60 (253 to 268) Epitopes Are Present in LDLr–/– Mice
Because of the important role of HSP60-specific T cells in atherosclerosis, we first investigated the presence of T cells specific for HSP60, HSP60 (253 to 268), or HSP70 (111 to 125) epitopes in the LDLr–/– mice. HSP60 is homologue in bacteria, mice, rats, and human, and therefore mycobacterial HSP60 was used in this study. The peptide (HSP60 [253 to 268]) was used because this peptide is present in all species. Splenocytes were isolated out of naive LDLr–/– mice and were incubated with several concentrations of the HSP epitopes. Incubation with 5 µg/mL HSP60 or HSP60 (253 to 268) had no effect on naive splenocytes whereas incubation with 20 µg/mL HSP60 or HSP60 (253 to 268) resulted in a 2.70±0.42- and 2.04±0.35-fold increase in proliferation, respectively (Figure 1A and B; P<0.05). HSP70 (111 to 125) did not stimulate proliferation of the splenocytes (data not shown). In all experiments 2 µg/mL ConA, a general pan T cell mitogen, was used as a positive control, which resulted in a more than 50-fold increase in proliferation (data not shown).


Figure 1
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  		Figure 1. Spleen cell proliferation in response to HSP60 and HSP60 (253 to 268). Splenocytes isolated from naïve LDLr–/– mice (A and B) and mice immunized with HSP60, HSP60 (253 to 268), or HSP70 (111 to 125) (C, D, and E) were restimulated in vitro with HSP60 (A and C), HSP60 (253 to 268) (B and D), or HSP70 (111 to 125) (E). *P<0.05.

To determine whether the T cell response to HSP-epitopes can be induced in vivo we immunized LDLr–/– mice by an intraperitoneal injection of 100 µg of HSP60, HSP60 (253 to 268), or HSP70 (111 to 125) using DDA as adjuvant. After 2 weeks mice were killed, and isolated splenocytes from HSP60-immunized mice incubated with 5 and 20 µg/mL of HSP60 showed a 7.40±1.29- (P<0.05) and 12.71±2.30- (P<0.05) fold increase in proliferation, respectively (Figure 1C). Incubation of splenocytes from HSP60 (253 to 268)-immunized mice with 5 and 20 µg/mL HSP60 (253 to 268) resulted in a 7.29±2.32- (P<0.05) and 9.26±2.58- (P<0.05) fold increase, respectively (Figure 1D). Incubation of splenocytes from HSP70 (111 to 125)-immunized mice with HSP70 (111 to 125) did not result in a significant effect on proliferation (Figure 1E).

Effect of Oral Tolerance Induction to HSP60, HSP60 (253 to 268), and HSP70 (111 to 125) on Atherosclerosis
Next we investigated the immunomodulatory effect of oral tolerance induction to these compounds on atherosclerosis. LDLr–/– mice were put on a Western-type diet for 1 week before oral administration of PBS (n=7) or 30 µg of HSP60 (n=7), HSP60 (253 to 268) (n=6), or HSP70 (111 to 125) (n=6). The oral treatment was given 4 times in total, every other day. Subsequently, mice were equipped with collars around both common carotid arteries and fed a Western-type diet. Six weeks thereafter, atherosclerotic plaque formation was analyzed. Representative hematoxylin-eosin stained cryosections of the carotid arteries of PBS, HSP70 (111 to 125), HSP60, and HSP60 (253 to 268)-treated mice are shown in Figure 2A through 2D. No significant difference in plaque size was observed in mice fed HSP70 (111 to 125) (21181±5273 µm2) compared with PBS-treated mice (20471±5273 µm2). Oral administration of HSP60 (3959±582 µm2) resulted in a significant 80.7% (P<0.05) reduction in plaque size when compared with PBS-treated mice. Oral tolerance induction to HSP60 (253 to 268) (3419±460 µm2) resulted in an 83.3% (P<0.05) reduction in plaque size (Figure 2E). Furthermore, the intima/lumen ratio was reduced significantly with 68.8% in the HSP60-treated mice (P<0.05; 0.082±0.007) and with 74.3% in the HSP60 (253 to 268)-treated mice (P<0.05; 0.067±0.010) when compared with the PBS-treated mice (0.261±0.074) (Figure 2F). During the experiment no significant differences in total plasma cholesterol levels were detected between the different groups (Figure 2G). In addition, a 27.4% reduction in plaque size at the aortic root was observed in HSP60-treated mice (377000±37200 µm2) when compared with PBS-treated mice (supplemental Figure I; 519000±44600 µm2; P<0.05). Immunohistochemical analysis of all plaques showed that oral tolerance induction to HSP60 and HSP60 (253 to 268) had no effect on the relative macrophage and smooth muscle cell content (data not shown).


Figure 2
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  		Figure 2. Oral tolerance induction to HSP60 and HSP60 (253 to 268) attenuates plaque formation. LDLr–/– mice were treated intragastrically with PBS (A), HSP70 (111 to 125) (B), HSP60 (C), or HSP60 (253 to 268) (D), and 6 weeks thereafter the carotid arteries were sectioned and lesions were quantified (E and F). During the experiment cholesterol levels were monitored (G). *P<0.05.

Effect of Oral Tolerance Induction to HSP60 on CD4+CD25+Foxp3+ Tregs
To evaluate whether oral tolerance induction to HSP60 was associated with an effect on Tregs, flow cytometry analysis was performed. HSP60-treated LDLr–/– mice were euthanized 4 and 14 days after the last oral treatment. In untreated control mice, CD4+CD25+Foxp3+ T cells are present in low numbers in Peyer’s patches (0.79±0.16%), blood (2.21±0.12%), spleen (0.80±0.07%), and mesenteric lymph nodes (3.82±0.25%). The dot-plots in supplemental Figure II represent examples of FACS analysis on CD4+CD25+ cells (left panels) and Foxp3+ cells within the CD4+CD25+ population (right panels) in Peyer’s patches, blood, spleen, and mesenteric lymph nodes, respectively. Four days after oral treatment with HSP60, the number of CD4+CD25+Foxp3+ T cells in the Peyer’s patches and blood was increased significantly to 1.73±0.30% (P<0.05) and 2.86±0.21% (P<0.01), respectively, when compared with untreated mice. No significant change was seen in the spleen (0.85±0.06%) and mesenteric lymph nodes (4.67±0.41%). Fourteen days after oral treatment, the number of CD4+CD25+Foxp3+ T cells decreased again to 1.07±0.08% in the Peyer’s patches and was not significantly different from untreated mice whereas the number of CD4+CD25+Foxp3+ T cells in blood was still enhanced (2.81±0.20%, P<0.01). In the spleen and mesenteric lymph nodes a significant increase to 1.24±0.11% (P<0.01) and 5.36±0.10% (P<0.01) was observed when compared with the situation without treatment (Figure 3).


Figure 3
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  		Figure 3. Oral tolerance induction to HSP60 leads to an increased amount of CD4+CD25+ Foxp3+ cells. LDLr–/– mice were treated intragastrically with HSP60 and killed 4 and 14 days after the last treatment. As a control, untreated animals were used. The graphs represent the amount of CD4+CD25+Foxp3+ cells in different organs (mean±SEM). *P<0.05, **P<0.01.

Regulatory T Cell Markers in Atherosclerotic Plaques
After tolerance induction to HSP60 and the induction of atherosclerosis, carotid arteries were dissected and mRNA was isolated. Subsequently, the expression of different Treg markers (CD25, CTLA-4 and Foxp3) in the atherosclerotic plaques in the carotid arteries was determined. After oral treatment with HSP60 (n=5) and 8 weeks of Western-type diet feeding, the mRNA expression of CD25, CTLA-4, and Foxp3 was significantly upregulated in the atherosclerotic plaque when compared with control mice (n=9). CD25 showed a 4.9-fold increase (P<0.05), CTLA-4 a 4.1-fold increase (P=0.068), and Foxp3 a 6.4-fold increase (P<0.05) (Figure 4).


Figure 4
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  		Figure 4. Increased expression of Treg markers is observed within lesions of HSP60-treated LDLr–/– mice. To investigate the presence of Tregs within atherosclerotic lesions, mRNA was isolated from carotid arteries of PBS (n=9) and HSP60-treated (n=5) mice and the expression of CD25, CTLA-4, and Foxp3 was quantitatively determined. *P<0.05.

Effect of Tolerance Induction on Cytokine Production
Furthermore, we investigated whether the increased number of CD4+CD25+Foxp3+ T cells also demonstrated a change in the production of cytokines in response to stimulation with HSP60. Mesenteric lymph node cells, isolated 14 days after the oral treatment with HSP60, were stimulated in vitro in presence or absence of 20 µg/mL of HSP60. Incubation with HSP60 resulted in a significant larger production of TGF-β (1.86±0.22 versus 0.93±0.15 ng/mL; P<0.05) and IL-10 (19.52±5.51 versus 6.41±1.72 pg/mL; P<0.05) when compared with mesenteric lymph node cells cultured without HSP60 (Figure 5A and 5B). Furthermore, HSP60-stimulated mesenteric lymph node cells isolated from HSP60-treated mice (14 days after treatment) produced significantly more TGF-β than HSP60-stimulated mesenteric lymph node cells isolated from untreated mice (1.86±0.22 ng/mL versus 0.96±0.22 ng/mL; P<0.05; data not shown). In all cases IFN-{gamma} levels were below the detection threshold.


Figure 5
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  		Figure 5. Oral tolerance induction to HSP60 induces antiatherogenic cytokine production. LDLr–/– mice were treated intragastrically with HSP60. After 14 days, mesenteric lymph nodes were isolated and the lymphocytes were cultured in vitro with or without HSP60 for 48 hours. The production of TGF-β (A) and IL-10 (B) was monitored (mean±SEM). *P<0.05.

Oral Tolerance Induction to HSP60 Reduces the Proliferative Response of Splenocytes to HSP60
To determine the effect of tolerance induction to HSP60 on the HSP60-specific proliferation, LDLr–/– mice were treated orally with PBS or HSP60. After oral treatment all mice were immunized with HSP60, and 2 weeks later splenocytes were isolated and cultured with or without 5 and 20 µg/mL of HSP60. Splenocytes from PBS-treated mice respond to HSP60 with an increased proliferation; a stimulation index of 4.4±0.7 and 10.4±2.5 when incubated with 5 and 20 µg/mL of HSP60, respectively. Mice orally treated with HSP60 showed a 56.8% and 68.2% reduction in the proliferative response to 5 and 20 µg/mL of HSP60, respectively (supplemental Figure III; 1.9±0.2 and 3.3±0.4; P<0.05).

Effect of Oral Tolerance to HSP60 on HSP60-Specific Antibodies
After oral treatment with HSP60 and the induction of atherosclerosis, HSP60-specific IgG1, IgG2a, and IgM levels in serum were determined. No detectable differences in HSP60-specific IgG1, IgG2a and IgM levels were observed (supplemental Figure IV).


*    Discussion
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 *Discussion
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Previous studies have demonstrated the importance of HSPs in the pathology of atherosclerosis. HSP70 is expressed in atherosclerotic lesions32 and oxLDL enhances the expression of HSP70.33,34 Autoantibodies to HSP60 causing endothelial damage12 and macrophage lysis,35 are associated with an increase in susceptibility in atherosclerosis. T cells reactive to HSP60 are found to correlate with early atherosclerotic events36 and are found in atherosclerotic plaques in rabbits37 and humans.38 Furthermore, Chlamydia pneumoniae, can induce an immune response because of its HSP60 expression, which is highly homologous to human and mouse HSP60.

We now show that LDLr–/– mice contain T cells specific for HSP60 and for the small peptide HSP60 (253 to 268) but no T cells with any reactivity against the HSP70 (111 to 125) peptide. HSP60 was derived from Mycobacterium bovis bacillus, but because of the high degree of amino acid sequence homology between different species, T cells specific for this HSP60 were found to be cross reactive against self-HSP60.39 A spleen cell proliferation assay demonstrated a 2- to 3-fold increase in T cell proliferation in response to HSP60 or HSP60 (253 to 268). Immunization of LDLr–/– mice with HSP60 or HSP60 (253 to 268) and a subsequent proliferation assay with HSP60 or HSP60 (253 to 268) resulted in a 13- and 9-fold increase in proliferation, respectively. These data confirm that HSP60 but also the small HSP60-peptide can induce a T cell response in LDLr–/– mice, whreas the small HSP70-peptide was not effective.

Intervention in the anti-HSP60 autoimmune response could be beneficial for atherosclerosis. Many strategies are used to interrupt autoimmune responses directed toward autoantigens, and one of these strategies is mucosal tolerance induction. Mucosal tolerance induction can lead to a deletion of Th1 and Th2 cells or to an activation of Tregs depending on the administered dose of the antigen. Tregs, induced by low doses of the antigen, are known for the production of TGF-β and IL-10, which both have antiatherogenic properties. Recently, Mallat et al hypothesized that in atherosclerosis an imbalance exists between pathogenic T cells (Th1 and/or Th2) and Tregs specific for "altered" self and nonself antigens.21 Tregs play an important role in controlling the development of atherosclerosis in mice,20 and a transfer of HSP60-specific Tregs to RAG1–/–LDLr–/– mice reduced the development of atherosclerotic lesions.40 Consequently, mucosal tolerance induction and the subsequent activation of Tregs may be a useful strategy to ameliorate atherosclerosis. It was already shown that oral tolerance induction to β2-glycoprotein I,25 HSP65,26,27 and oxLDL28 reduced early atherosclerosis. However, the studies on β2-glycoprotein I and HSP65 do not give a clear explanation for the observed reduction in atherosclerosis and they do not show whether Tregs are involved in this process. Oral administration of HSP60 suppresses adjuvant arthritis due to an expansion of T cells specific for HSP60 producing IL-10.41 We showed that an induction of Tregs after oral administration of oxLDL was observed and these oxLDL specific Tregs were found to be responsible for the reduction in atherosclerotic plaque formation.28

We now show that oral tolerance induction to HSP60 and HSP60 (253 to 268) attenuates atherosclerosis. A relatively low dose of HSP60 significantly reduced early atherosclerotic lesion formation by 80.7%. We now propose that an immunogenic peptide present in HSP60 (aa 253 to 268) can also induce regulatory T cells and reduces plaque size by 83.3%. The specificity of the response is reflected by the finding that HSP70 (111 to 125), a peptide based on a conserved sequence found in the HSP70 protein of men, rats, and mice, was not effective in reducing atherosclerosis. The experimental setup of our current study is comparable with 2 previous studies on oral tolerance induction to HSP65/HSP60.26,27 Both Harats et al26 and Maron et al27 show a decreased proliferation of splenocytes after oral treatment but no effects on Tregs were described. Maron et al27 observed a decreased IFN-{gamma} and an increased IL-10 production by lymphocytes after oral treatment with HSP65. This could indicate an activation of Tr1 cells, a subset of adaptive Tregs, particularly producing IL-10.28 In our current study low doses of HSP60 and HSP60 (253 to 268) were administered and therefore we investigated the possible activation of Tregs. Four days after the oral HSP60-treatment, the number of CD4+CD25+Foxp3+ Tregs was significantly increased in Peyer’s patches and blood. After two weeks, the number of CD4+CD25+Foxp3+ T cells was significantly increased in blood, mesenteric lymph nodes, and spleen. In the Peyer’s patches, the first site of activation, the number of Tregs decreased after 2 weeks, which may be attributed to the migration of the activated CD4+CD25+Foxp3+ T cells to peripheral lymphoid organs and the site of inflammation (atherosclerotic lesions) where they may recognize self-HSP60. We also investigated the mRNA-expression of markers for Tregs within the lesions and we observed an increased expression of CD25, CTLA-4, and Foxp3. In addition, oral treatment with HSP60 reduced the proliferative response of splenocytes to HSP60 which is in line with the studies by Maron et al and Harats et al.26,27 Furthermore, mesenteric lymph node cells of HSP60-treated mice also produced increased levels of TGF-β and IL-10 after in vitro restimulation with HSP60. All these data suggest that oral administration of HSP60 induced Tregs, which were may be able to dampen the immune response to HSP60 in atherosclerosis prone mice.

Natural Tregs, which are CD4+CD25+Foxp3+ T cells, can display their specific immunosuppressive effects via cell contact. TGF-β on their surface may bind to TβRII expressed on T cells specific for the same antigen. This TGF-β–TβRII interaction leads to the activation of a Smad-dependent pathway, resulting in a blockade of IL-2 production and a reduced proliferation of antigen-specific T cells. CTLA-4 is also important in the cell–cell interaction between Tregs and other T cells. It is, however, more likely that adaptive Tregs (Th3 and Tr1 cells) are involved in oral tolerance induction.31 Th3 cells are known for the production of antiinflammatory TGF-β and on activation they may express Foxp3.42,43 Tr1 cells, which can also be activated in the periphery, produce particularly antiinflammatory IL-10, but whether these Tregs express Foxp3 is still not clear. Therefore we assume that oral tolerance induction led to an increase in Foxp3-expressing natural Tregs or Th3 cells, producing excessive amounts of TGF-β but also IL-10. IL-10 may also be produced by activated Tr1 cells, which however do not contribute to the increase in Foxp3+ Tregs. To definitively prove that the protection against atherosclerosis after oral administration of bacterial HSP60 is attributable to the induction of Tregs specific for murine HSP60 additional experiments are required, for example by deletion of Tregs after tolerance induction or transfer of the Tregs.

In conclusion we describe that LDLr–/– mice can be tolerized to HSP60 and a HSP60 peptide (HSP60 [253 to 268]) which results in an attenuation of early atherosclerotic lesions. The mechanism underlying this effect can be attributed to the induction of CD4+CD25+Foxp3+ Tregs, which may produce TGF-β and IL-10 in atherosclerotic lesions. In this way they may downregulate the inflammatory response locally. Altogether, these beneficial results of oral tolerance induction to HSP60 and HSP60 (253 to 268) on atherosclerosis may provide new therapeutic approaches for the treatment of atherosclerosis.


*    Acknowledgments
 
Sources of Funding

Dr Kuiper is an Established Investigator of the Netherlands Heart Foundation (grant 2000T040). The authors belong to the European Vascular Genomics Network (http://www.evgn.org), a Network of Excellence supported by the European Community’s Sixth Framework Program for Research Priority 1 (Life Sciences, Genomics, and Biotechnology for Health; contract LSHM-CT-2003-503254).

Disclosures

None.


*    Footnotes
 
Original received August 26, 2006; final version accepted September 14, 2007.


*    References
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 *References
 

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