A Potential Link Between Innate and Adaptive Immunity in Atherosclerosis and Adipose Tissue Inflammation
Transforming growth factor-β (TGF-β) is a major antiinflammatory mediator in atherosclerosis. Transgenic ApoE−/− mice with a dominant-negative TGFβ type II receptor (dnTGFβRII) on CD4+ and CD8+ T cells display aggravated atherosclerosis. The aim of the present study was to elucidate the mechanisms involved in this enhanced inflammatory response. Gene array analyses identified the 5-lipoxygenase–activating protein (FLAP) among the most upregulated genes in both the aorta and adipose tissue of dnTGFβRII transgenic ApoE−/− mice compared with their ApoE−/− littermates, a finding that was confirmed by real-time quantitative RT-PCR. Aortas from the former mice in addition produced increased amounts of the lipoxygenase product leukotriene B4 after ex vivo stimulation. FLAP protein expression in both the aorta and adipose tissue was detected in macrophages, but not in T cells. Four weeks of treatment with the FLAP inhibitor MK-886 (10 mg/kg in 1% tylose delivered by osmotic pumps) significantly reduced atherosclerotic lesion size and T-cell content. Finally, FLAP mRNA levels were upregulated approximately 8-fold in adipose tissue derived from obese ob/ob mice. In conclusion, the results of the present study suggest a key role for mediators of the 5-lipoxygenase pathway in inflammatory reactions of atherosclerosis and metabolic disease.
Atherosclerosis is regulated by both pro- and antiinflammatory stimuli.1 Among the latter, transforming growth factor-β (TGF-β) may play a major role in atheroprotective signaling. For example, selective expression of a dominant-negative TGFβ type II receptor on CD4+ and CD8+ T cells (CD4dnTβRII) leads to aggravated atherosclerosis and more vulnerable lesions in hyperlipidemic mice.2,3 Furthermore, the recent discovery that a CD25-depleting antibody increases lesion size in ApoE−/− but not ApoE−/− ×CD4dnTβRII mice has implicated TGF-β in the atheroprotective effects exerted by CD4+CD25+ regulatory T cells.4 Whereas those studies suggest a key role for T-cell TGF-β signaling in dampening inflammatory responses in atherosclerosis, the aim of the present study was to establish the proatherogenic mediators involved. In this process, we discovered the 5-lipoxygenase–activating protein (FLAP) as a main upregulated gene in the aorta from the ApoE−/− ×CD4dnTβRII mice, compared with their ApoE−/− littermates. Furthermore, we also found the adipose tissue to be a major site of inflammation in these mice, with FLAP also among the most upregulated genes in this tissue. The latter observation is of particular interest because evidence is emerging linking cardiovascular risk factors such as obesity and insulin resistance to infiltration of inflammatory cells in adipose tissue.
FLAP is necessary for the production of leukotrienes through the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism. Importantly, the gene encoding FLAP was recently associated with increased risk of myocardial infarction and stroke,5 and mechanistic studies have supported the notion of a major role of leukotriene signaling in atherosclerosis.6 Leukotriene B4 (LTB4), for example, is among the most potent chemotactic mediators formed within the atherosclerotic lesion, inducing migration of a number different cell types, such as monocytes,7 vascular smooth muscle cells,8 and T cells,9 suggesting LTB4 as a potential link between innate and adaptive immunological reactions. However, the latter notion has not previously been assessed in the context of atherosclerosis and adipose tissue inflammation. The beneficial effects observed in ApoE−/−×CD4dnTβRII mice after FLAP inhibition in the present study point to an important role of the FLAP/5-LO/leukotriene pathway in the regulation of T-cell-driven inflammatory responses in atherosclerosis and metabolic disease.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org. In brief, female ApoE−/− and male ApoE−/−×CD4dnTβRII mice were bred to obtain littermates that were either ApoE−/−×CD4dnTβRII or ApoE−/− mice lacking the dominant-negative transgene. For treatment protocols, either MK-886 or vehicle (1% tylose) was administered for 4 weeks by Alzet osmotic minipumps implanted into the intrascapular subcutaneous space in 8-week-old ApoE−/− ×CD4dnTβRII mice.
Results and Discussion
The expression of a dominant-negative TGF-β type II receptor under the control of the CD4 promoter in ApoE−/− mice has provided a model of atherosclerosis with a predominantly T-cell-driven inflammation.2,4 In the present study, microarray analysis revealed candidate genes for inflammatory mechanisms involved in the aggravated atherosclerosis of ApoE−/−×CD4dnTβRII mice (Table I in the online data supplement). In addition to genes reflecting increased T-cell infiltration (T-cell–specific GTPase, CD52, and T-cell receptor α chain) and interferon γ (IFN-γ) signaling (IFN-γ inducible protein, ubiquitin D, and Stat1), FLAP, being among the 20 most upregulated, should be considered a proatherogenic candidate gene. The 2- to 3-fold increase of FLAP mRNA levels in ApoE−/−×dnTGFβRII mice compared with their ApoE−/− littermates lacking the dominant-negative transgene confirmed by RT-PCR and immunohistochemistry was in contrast to the lack of aortic FLAP upregulation in ApoE−/− compared with wild-type mice (Figure 1A, 1B, and supplemental Figure I). Furthermore, ApoE−/−×dnTGFβRII mice also expressed increased FLAP mRNA levels when compared with 18-week-old ApoE−/− mice (supplemental I and Figure 1A), which have approximately the same lesion size as 12-week-old ApoE−/−×CD4dnTβRII, supporting a selective upregulation of FLAP transcription rather than a consequence of the increased atherosclerosis burden.
Importantly, FLAP expression in ApoE−/−×CD4dnTβRII mice colocalized with the monocyte/macrophage marker CD68, but not with the T-cell marker CD3 (Figure 1D). These results indicate that abrogating the dampening effects of TGF-β selectively on T-cells induced paracrine effects on macrophages. The increased FLAP expression led to an increased LTB4 formation, measured after ex vivo stimulation of the aorta (Figure 1C). Because the mRNA levels of other enzymes involved in LTB4 biosynthesis, ie, 5-LO and LTA4 hydrolase, were not significantly altered in these mice (supplemental Figure I), the present study suggests that FLAP may be rate limiting in leukotriene biosynthesis during atherosclerosis.
Because the expression pattern suggested FLAP as a potential proatherogenic mediator, we subsequently evaluated the effect of the FLAP inhibitor MK-886 in ApoE−/− ×CD4dnTβRII and found significantly reduced aortic root atherosclerotic lesions (Figure 2A and 2B), without altered lipid levels (supplemental Table II). These findings provide the first reported successful treatment strategy capable of reducing atherosclerosis in this particular model. The latter observation also extends the somewhat conflicting results from previous studies of either genetic or pharmacological targeting of leukotriene synthesis in different strains of hyperlipidemic mice having reported both beneficial10,11 and neutral effects12 on atherosclerosis burden. The present study suggests that the FLAP/leukotriene signaling may have its predominant effects in T-cell-driven inflammatory responses. This notion is further extended by the observation that although the macrophage density in the lesions was not significantly changed (supplemental Table III), MK886 decreased aortic CD3+ T cells and reduced levels of mRNA encoding the T-cell chemokine IFN-γ (Figure 2C and 2D)
In addition to atherosclerosis, several findings in the present study also indicate a major role of FLAP/leukotriene signaling in T-cell-driven adipose tissue inflammation. For example, microarray analysis of adipose tissue again revealed FLAP as among the most upregulated genes in ApoE−/− ×CD4dnTβRII compared with both 12- and 18-week-old ApoE−/− mice (supplemental Table IV), which was confirmed by RT-PCR (Figure 3A). In comparison with age-matched C57BL/6 mice, FLAP mRNA levels were significantly increased in adipose tissue from ApoE−/−×CD4dnTβRII mice, and even higher levels (almost 8-fold increase) were detected in adipose tissue from age-matched ob/ob mice (Figure 3A). Whereas the adipose tissue levels of LTA4 hydrolase mRNA were not significantly different between the different groups of mice examined, ob/ob mice also expressed 2-fold higher levels of 5-LO compared with C57BL/6 mice (Figure 3A), hence supporting a potential link between leukotrienes and obesity.
Immunohistochemical staining revealed adipose tissue FLAP protein localization to macrophages infiltrating adipose tissue of ApoE−/−×CD4dnTβRII mice (Figure 3B). Furthermore, a local LTB4 formation was detected after ex vivo stimulation of adipose tissue from these mice (Figure 3C). This local LTB4 formation hence suggests that increased FLAP expression may be a mediator of proinflammatory circuits in adipose tissue, which is a potential cardiovascular risk factor. Furthermore, the notion of leukotriene-driven adaptive immunological circuits could be extended also to adipose tissue inflammation. For example, microarray analysis revealed increased expression of not only macrophage-associated genes in ApoE−/−×CD4dnTβRII mice but also of T-cell markers, such as CD52 and CD8. Interestingly, FLAP inhibitor treatment significantly reduced IFN-γ expression in adipose tissue (Figure 3D). Taken together, these observations suggest that T cells may be involved in the inflammatory alterations of adipose tissue and that targeting the FLAP/leukotriene pathway may inhibit this response.
In summary, the selective upregulation of FLAP in the hyperinflamed ApoE−/−×CD4dnTβRII mice points to an important role of the FLAP/leukotriene pathway in adaptive immunological circuits in vascular and adipose tissue inflammation. In the absence of the dampening effects of TGF-β on T cells, FLAP expression in macrophages represented one of the most upregulated genes with as a result, increased production of the T-cell chemoattractant LTB4. Pharmacological targeting of FLAP reduced atherosclerosis burden and vascular as well as adipose tissue T-cell inflammation. In conclusion, the results of the present study suggest that FLAP may be a key regulator of T-cell-driven inflammatory responses and could represent a possible target in the treatment of atherosclerosis and metabolic disease.
Sources of Funding
This study was supported by the Torsten and Ragnar Söderberg Foundation, Swedish Heart and Lung Foundation, European Union FP6, and Karolinska Institutet.
Original received January 26, 2007; revision received February 22, 2007; accepted March 9, 2007.
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