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Departments of Human Nutritional Sciences and Pathology, and National Centre for Agri-food Research in Medicine, St. Boniface Hospital Research Centre and * CIHR National Training Program in Allergy and Asthma Research and Department of Immunology, The University of Manitoba, Winnipeg, Canada
4To whom correspondence should be addressed. E-mail: mmoghadasian{at}sbrc.ca.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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] production. In contrast, development of cytokine and chemokine responses to ovalbumin was as strong as or even improved in the phytosterol-treated mice relative to controls. The antiatherogenic effects of dietary phytosterols in apo E-knockout mice were associated with beneficial alterations in both lipoprotein metabolism and inflammatory pathways. Decreased capacity to mount proinflammatory cytokine and chemokine responses to inflammatory stimuli did not interfere with the global immunocompetence of such mice. Thus, the desirable suppression of proinflammatory cytokine production that was associated with inhibition of atherogenesis did not impair the capacity to mount responses to foreign antigens.
KEY WORDS: • proinflammatory cytokines • Th1/Th2 • immunocompetence • atherosclerosis • phytosterols
Coronary artery disease (CAD)5 is a multifactorial atherosclerotic vascular disease driven by an underlying inflammatory mechanism (1,2). The extent to which the immune and cardiovascular systems are intertwined in this process, and in particular the roles played by monocytes and T lymphocytes in establishing and maintaining the chronic inflammation that occurs in atherosclerosis, is an active area of investigation. Immune activation, specifically interactions between macrophages and oxidized LDL particles, is one of the earliest steps in the development of atherosclerotic lesions. Subsequent lipid deposition within the arterial wall accelerates the influx of inflammatory cells such as T lymphocytes and monocytes into subendothelial spaces in a process that leads to intimal thickening and atherogenesis. LDL oxidation products such as lysophosphatidylcholine and oxidized sterols activate macrophages and endothelial cells to generate oxygen radicals and express adhesion molecules that then recruit additional immune cells to the arterial wall, setting up a cycle of chronic inflammation (3,4). The recruitment of such inflammatory cells results in local release of a spectrum of proinflammatory mediators including cytokines, chemokines, and hydrolytic enzymes that promotes increased lipoprotein flux, oxidative stress, foam cell formation, and ongoing inflammation. Entrapment of lipoprotein particles within the arterial wall furthers the vicious cycle of inflammation and LDL oxidation, resulting in progressive development of atherosclerotic lesions.
Dietary and pharmacologic strategies that lower plasma LDL-cholesterol concentrations have been shown to cause significant reductions in the incidence of coronary events (5,6). As one approach to this goal, we found previously that dietary phytosterol treatment markedly reduced both plasma cholesterol concentrations and the extent and severity of atherosclerosis in the apolipoprotein E-knockout (apo E-KO) mouse model (7–9). Diets enriched with phytosterols reduced lesion size and yielded significant reductions in the involvement of inflammatory cells within the atherosclerotic lesion (7,8). In agreement with this observation, other laboratories subsequently reported immune modulatory effects of phytosterols or their derivatives in both humans and experimental animals (10–12).
The mechanism of action of the beneficial effects of phytosterols remains unclear. Given the increasing recognition of the strong immunologic component that underlies development of cardiovascular disease, considerable attention is now devoted to determining the effect of alterations in endogenous cytokine production on the development or progression of atherosclerosis. The concept of inflammatory background of atherosclerosis in a hypercholesterolemic milieu was reviewed recently by several laboratories (13–15). These studies suggest that there is a strong relation between the degree of hypercholesterolemia and the nature of cytokine production, specifically T helper (Th)1 vs. Th2-like balance. Modifying the balance of cytokine production can have a marked effect on clinical outcome. For example, daily administration of interleukin (IL)-12 to young apo E-KO mice accelerates atherogenesis (16). Treatment with the proinflammatory cytokine recombinant IL (rIL)-6 increases atherogenesis in apo E-KO mice without affecting plasma cholesterol concentrations (17). Transforming growth factor (TGF)-ß accelerates atherogenesis in apo E-KO mice, perhaps by reducing high HDL binding to macrophages, resulting in impaired reverse cholesterol transport (18).
On the other hand, IL-10 gene transfer had a clear protective effect, significantly reducing the extent of macrophage infiltration and total atherosclerotic plaque area. These effects were associated with increased plasma IL-10 levels, depressed expression of Th1 cytokines, and, importantly, were independent of plasma cholesterol concentrations (19). Thus, alterations in the levels of expression of a variety of different pro- and anti-inflammatory cytokines can substantively influence atherosclerosis in this animal model via processes more complex than simple modification of cholesterol concentrations.
It has been shown that inflammation can adversely affect the quality of lipoproteins and hence contribute to atherosclerosis. For example, HDL-associated proteins such as apo AI, the major apolipoprotein of HDL, and paraoxonase-1 decrease during inflammation, limiting the antiatherogenic activities of HDL (15). The vicious cycle of LDL oxidation and inflammation is another major contributing factor in the pathogenesis of atherosclerosis. Abnormalities in immune function, lipoprotein metabolism, and antioxidant systems are included in the underlying mechanisms of accelerated atherosclerosis in apo E-KO mice. Apo E-KO SCID mice, which lack T and B cells, as well as apo E-KO mice lacking receptors for the CD40 ligand, develop smaller atherosclerotic lesions than do immunocompetent apo E-KO mice (20–22). Despite these important observations, the role of adaptive immunity in atherosclerosis remains unclear. Dietary phytosterols are among the few agents that can substantially prevent atherosclerosis in apo E-KO mice (7,8). Together with other relevant studies as explained above (20–22), this has made the apo E-KO mouse model attractive to investigate whether a substantial reduction in atherosclerosis by dietary plant sterols is associated with beneficial alterations in immune function. The aim of the present study was to test the hypothesis that the marked antiatherogenic properties of dietary plant sterols in apo E-KO mice are associated with altered immune function, specifically decreased T cell–dependent proinflammatory cytokine and chemokine production. Given the clinical potential of such strategies, we further assessed the effect of this dietary intervention on immune homeostasis and on the capacity of these mice to mount immune responses to unrelated antigens.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plasma lipids. Total cholesterol (TC), triglycerides (TG), and HDL cholesterol concentrations were measured at baseline, during, and at the end of the study using standard enzymatic methods (7–9). Non-HDL-cholesterol concentrations were calculated by subtraction of HDL-cholesterol concentrations from TC concentrations; a standard precipitation method was used to prepare the HDL fraction (24).
Histology and morphometry evaluations of atherosclerotic lesions. Sections at the aortic roots were cut and stained with hematoxylin and eosin and oil red O (ORO) for histological and morphometrical examinations (7–9). ORO-stained sections were used to estimate atherosclerotic lesion size and the lesion:lumen ratio using Image Pro-Plus software (7–9).
Spleen cell culture.
RPMI-1640 medium with 10% fetal calf serum (HyClone) was used in this experiment. Duplicate spleen cell suspensions were cultured at 7.5 x 109 cells/L in the presence of medium, 300 or 1000 g/L OVA, or the indicated polyclonal activators. Purified anti-CD4 monoclonal antibody (mAb) from YTS 191.1 tissue culture supernatant or CTLA-4 Ig at 5 mg/L was added to some cultures to block CD4-dependent cytokine production and to antagonize CD28-CD80/CD86 interactions, respectively. Supernatants were harvested for analysis of interferon (IFN)-
, IL-4, IL-13, IL-5, IL-6, IL-10, tumor necrosis factor (TNF)-, and chemokines (C-C motif) ligand 17 (CCL17) and (C-X-C motif) ligand 10 (CXCL10). The optimal time points for each assay were determined previously for polyclonally stimulated and antigen-driven cytokine/chemokine responses (25–27).
Proliferation assays. To assess the global effect of phytosterol treatment on immune capability, spleen cells were stimulated for 42 h with medium alone or immobilized anti-CD3 (2C11) at 50 µg/L, bacterial lipopolysaccharide (LPS) at 500 µg/L, concanavalin A (ConA) at 10 mg/L (Sigma) or anti-mouse IgM F(ab')2 at 10 mg/L (Jackson ImmunoResearch). In all cases, the 0.5 µCi [3H]thymidine pulse was for the final 18 h. Thymidine incorporation was measured using a Wallac 1450 MicroBeta® Trilux (EG&G Wallac).
Cytokine and chemokine quantification.
IL-4, IL-10, and IFN- levels in culture supernatants were determined as previously published (26,27). IL-5 (BD PharMingen), CXCL10 (Peprotech), IL-13, CCL17 (R&D Systems), IL-6, and TNF- (BioLegend) were measured by ELISA using cytokine-specific mAb pairs as indicated. Detection limits were 600 U/L IFN-, 2000 U/L IL-4, 9 ng/L IL-5, 90 ng/L IL-13, 300 U/L IL-10, 11 ng/L CXCL10, 3 ng/L CCL17, and 15.6 ng/L IL-6 and TNF-. These assays were performed in duplicate with an interassay variability of 5–10%.
Statistical analysis. Data are presented as means and SD. Two-tailed Student’s t tests were used to identify significant differences (P < 0.05) between the treated and control mice.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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2 times those seen in the control mice. Collectively, these data argue that there was no evidence of spontaneous immune activation in either group, as indicated by total cell numbers in vivo or by in vitro proliferation. However, there was a significantly higher immune proliferative response in phytosterol-treated mice compared with controls.
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25% in phytosterol-treated mice relative to controls (788 ± 69.6 vs. 538 ± 33.3 ng/L, P < 0.005). Plasma IL-6 and TNF- levels were below the limits of detection in both groups of mice. No difference was detectable in the levels of plasma IL-10 in resting mice.
In response to in vitro activation with the physiologically relevant toll-like receptor-4 ligand LPS, spleen cells from control mice responded strongly as anticipated (Fig. 1). However, mice treated with dietary phytosterols had markedly weaker responses to LPS stimulation relative to controls with minimal or no increase in the production of TNF- (Fig. 1A) and IL-6 (Fig. 1B). IL-12 p40 responses to LPS stimulation were comparable in the 2 groups.
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Despite lower cytokine responses to proinflammatory stimuli such as LPS, OVA-immunized control and phytosterol-fed mice had similar IL-6 and TNF- responses to OVA, indicating that phytosterol treatment does not lead to a generalized immunosuppression state or a deficient capacity to respond to foreign antigens. Strikingly, the production of anti-inflammatory cytokine IL-10 was elevated by 8–10 times in treated mice compared with controls (Fig. 2A).
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To extend our analysis, production of CCL17, a chemokine that plays an important role in trafficking of chemokine (C-C motif) receptor 4 positive (CCR4+) cells to sites of inflammation and promotes expression of Th2-like immunity (28–30), was also examined. OVA-stimulated CCL17 responses in apo E-KO mice are typical (<5 ng/L) of those seen in C57BL/6 mice background.
Phytosterol-fed mice also exhibited consistently elevated (6–8 times) OVA-specific IFN- responses (Fig. 2D). In contrast to the results seen with Th2 associated CCL17, CXCL10 responses, which are important in the trafficking of chemokine (C-X-C motif) receptor 3 positive (CXCR3+) cells and in regulation of Th1-associated cytokine production (31,32), were much stronger than were CCL17 responses and were indistinguishable in the 2 groups of mice (data not shown).
Adaptive immune capability, assessed as OVA-dependent Th1- and Th2-associated cytokine production, in phytosterol-treated mice was as good as or was even improved relative to control mice. These observations imply that the desirable suppression of proinflammatory cytokine production associated with inhibition of atherogenesis does not necessarily lead to an impaired capacity to mount responses to foreign antigens.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The data demonstrate that phytosterol-enriched diets substantially reduce both proinflammatory cytokine production generated upon exposure to inflammatory stimuli such as LPS and the incidence of atherogenesis in a murine model of elevated susceptibility to atherosclerosis. No effects on the global indices of health examined after long-term compliance with this diet were detected. Importantly, the capacity of mice to mount protective Ag-specific CD4 T cell–dependent immune responses was enhanced rather than inhibited, and the responses were typical of those seen in wild-type C57BL/6 rather than in apo E-KO mice. Taken together, these results suggest that phytosterol-enriched diets could provide a readily incorporated, beneficial adjunct to current therapies for the prevention and management of cardiovascular disease.
Plant sterols reduce intestinal cholesterol absorption (33,34). Phytosterols reduce plasma TC concentrations and atherosclerosis by an average of 20–25 and 50–60%, respectively, in apo E-KO mice (7–9). We observed these effects in the presence (8,35–37) or absence of dietary cholesterol (7). The latter observation suggests that dietary phytosterols reduce the reabsorption of biliary cholesterol in the intestinal tract. The fact that the clinical benefits of phytosterols are also evident when there is no dietary or very little biliary cholesterol suggests that phytosterols reduce atherogenesis through additional mechanisms beyond their effect on intestinal cholesterol absorption.
The detailed mechanisms by which this intervention leads to altered cytokine and chemokine production require further investigation. Among anti-inflammatory mediators, the putatively protective role of IL-10 in the pathogenesis of atherosclerosis has been actively studied. Apo E/IL-10 double knockout mice develop significantly larger atherosclerotic lesions, despite plasma TC concentrations comparable to those of apo E-KO mice, suggesting an antiatherogenic effect of IL-10 independent of plasma lipid concentrations (38). In support of these findings, transplantation of T cells from IL-10 overexpressing mice into LDL-receptor-knockout mice resulted in a 47% reduction in atherosclerotic lesion size that was unrelated to plasma TC concentrations (39). Moreover, IL-10 deficiency in cholesterol-fed wild-type C57BL/6 mice results in marked reductions in plasma HDL cholesterol concentrations and increased atherosclerosis (40,41). Elevated levels of serum IL-10 are associated with a lower risk of CAD in humans (42,43). Here, we report up to 10 times elevated production of IL-10 along with reduced TNF- and IL-6 production by spleen cells in association with a 60% reduction in atherosclerosis in phytosterol-treated apo E-KO mice compared with controls. We speculate that reductions in plasma cholesterol concentrations may reduce the extent of LDL oxidation, which in turn reduces recruitment of immune cells to the arterial intima. Consequently, the secretion and production of cytokines, reactive oxygen substances, and adhesion molecule expression will be reduced. Reduced cytokine secretions in the arterial wall may signal the lymphoid organs and control their secretory function. IL-10 downregulates Th1 activation (41,44); however, it is unclear whether substantial increases in IL-10 in phytosterol-treated mice is a marker of reduced atherosclerosis or a direct consequence of reduced plasma cholesterol concentrations. It has been shown that hypercholesterolemia is associated with a Th1/Th2 switch in mice (45). It is possible that elevated plasma cholesterol concentrations alter T-cell membranes, resulting in alterations in cytokine production and signal transduction pathways. Another possible mechanism is that dietary phytosterols directly or indirectly alter the proportion of various subsets of spleen cells, changing the cytokine production profile as observed in this study.
IL-10 has multiple biological activities in addition to its capacity to reduce the synthesis and secretion of proinflammatory cytokines (46,47). Regulatory T cells are hypothesized to be distinct subset(s) of lymphocytes; one population of these cells secretes high levels of IL-10 (48). They are hypothesized to maintain immune homeostasis by preventing development of Th1-mediated autoimmune diseases or excessive Th2 induction associated with allergic disorders (49). Indeed, i.p. administration of regulatory T cell–like populations into apo E-KO mice (106 cells/mouse) significantly increased IL-10 production by cultured spleen and lymph T cells and decreased IgG2a serum levels compared with controls (50). These cell-transferred apo E-KO mice had significantly fewer atherosclerotic lesions. These findings parallel our own data, thereby raising the possibility that dietary phytosterols may stimulate differentiation of regulatory T cells in apo E-KO mice. However, further studies are required to test this speculation because a wide variety of cell types, including monocytes, macrophages, and dendritic cells are all capable of producing IL-10.
Although a strong association exists between altered patterns of cytokine production and protection from atherosclerosis, as seen in this and related studies, a cause and effect relation between altered cytokine synthesis and protection remains a logical but unproven conclusion. Other mechanisms may also contribute or play an even larger role in protection against atherosclerosis. We showed previously that the antiatherogenic effects of dietary phytosterols are also associated with reduced plasma fibrinogen levels and significantly decreased platelet numbers (7,51). Other reports suggest an antithrombotic effect for dietary phytosterols (52,53). Increased plasma levels of fibrinogen and other procoagulation factors are substantially implicated in the pathogenesis of atherosclerosis (7,54,55). It is of interest that a lack of IL-10 in apo E-KO mice is associated with significant elevations in proteolytic and procoagulant activities in advanced atherosclerotic lesions (38). Although our data suggest that the anticoagulation effects of dietary phytosterols are related to increased production of IL-10, the precise role played by this cytokine is currently under study. Future studies should aim to understand the mechanisms by which dietary phytosterols and plasma cholesterol concentrations alter immune function. Among these studies, administration of plant sterols in the absence of dietary cholesterol to apo E-KO mice would highlight the relation of the extent of atherosclerosis and hypercholesterolemia (because these mice would have lower plasma cholesterol concentrations and develop less atherosclerosis compared with cholesterol-fed apo E-KO mice) and the effects of dietary phytosterol on immune function. Furthermore, identification of subsets of spleen cells, their membrane composition, and signaling pathways in relation to the production of cytokines will illustrate, at least in part, possible mechanism(s) of the immune-modulatory effects of plant sterols.
In conclusion, we demonstrated that dietary phytosterols modulated immune function in apo E-KO mice, moving these mice toward a less inflammatory phenotype. At the same time, the diet was well tolerated and the capacity of the mice to mount immune responses to other stimuli was as good as or better than that of mice fed the control diet. Given that dietary phytosterols reduce plasma cholesterol concentrations and atherosclerotic lesion size by 20 and 60% respectively, the data suggest the potential utility and safety of dietary interventions as an adjunct to current therapy. Further studies will be required to identify more precisely the mechanisms through which this class of molecules exerts its effects in vivo.
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FOOTNOTES |
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2 Supported in part by the Manitoba Medical Sciences Foundation, the Natural Engineering and Science Research Council of Canada (NSERC), the Heart and Stroke Foundation of Canada, and Canadian Institutes of Health Research (to M.H.M.) and the Canada Research Chairs Program, Canada Foundation for Innovation and Canadian Institutes of Health Research (to K.T.H.).
3 Drs. Moghadasian and HayGlass are joint senior authors.
5 Abbreviations used: Ag, antigen; apo E-KO, apolipoprotein E-Knockout; CAD, coronary artery disease; CCL17, chemokine (C-C motif) ligand 17; CCR4+, chemokine (C-C motif) receptor 4 positive cells; CXCL10, chemokine (C-X-C motif) ligand 10; CXCR3+, chemokine (C-X-C motif) receptor 3 positive cells; CD4, cluster of differentiation 4; CTLA4, cytotoxic T lymphocyte antigen 4; IL, interleukin; INF , interferon ; LPS, bacterial lipopolysaccharide; mAb, monoclonal antibody; ORO, oil red O; OVA, ovalbumin; rIL, recombinant IL; TC, total cholesterol; TG, triglycerides; TGF, transforming growth factor; Th, T helper; TNF, tumor necrosis factor.
Manuscript received 25 April 2005. Initial review completed 8 June 2005. Revision accepted 20 July 2005.
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