SOCS3 expression in T cells significantly affects atherosclerotic lesion development
We first examined the effect of SOCS3 deletion in T cells on the development of atherosclerosis. We reconstituted low-density lipoprotein receptor–deficient (Ldlr−/−
) mice with either a WT bone marrow from SOCS3flox/flox mice (the reconstituted mice are designated SOCS3-WT) or a bone marrow from mice with T cell–specific deletion in SOCS3 (SOCS3flox/flox:Lck-Cre) generated by a conditional gene targeting approach using a Cre-loxP system (designated SOCS3-cKO; Yasukawa et al., 2003
; Kinjyo et al., 2006
). Phosphorylated (P)-STAT3 was readily detectable in atherosclerotic lesions, and we found a marked increase in P-STAT3 in spleen-derived T cells from SOCS3-cKO mice compared with SOCS-WT mice (Fig. S1
), indicating an efficient and functional deletion of SOCS3 in T cells. We therefore quantified lesion size after 6 wk on a high fat diet. We found an unexpected 50% reduction of aortic sinus lesion size in SOCS3-cKO mice compared with controls (), despite similar plasma cholesterol levels (18.4 ± 0.7 vs. 17.6 ± 0.5 g/liter; P = 0.32). We observed a similar protection against atherosclerosis at the levels of the aortic sinus and the descending thoracic aorta in a separate set of old Ldlr−/−
mice reconstituted with SOCS3-cKO bone marrow (Fig. S1 d).
Figure 1. SOCS3 deletion in T cells promotes IL-17 and IL-10 production, inhibits macrophage apoptosis, and limits atherosclerotic lesion development. (a) Atherosclerotic lesion size in the aortic root of chimeric Ldlr−/− SOCS3-WT or SOCS-cKO mice. (more ...)
We then tested the effect of SOCS3 overexpression in T cells on the development of atherosclerosis (Fig. S1 e). We reconstituted Apoe−/−
mice with purified CD4+
cells recovered from either WT or SOCS3-transgenic (Tg) mice (Seki et al., 2003
). As expected, we found reduced P-STAT3 in SOCS3-Tg T cells (unpublished data). After 6 wk of a high fat diet, spleen-derived CD4+
cells of mice transferred with CD4+
SOCS3-Tg cells showed reduced production of IL-17 and IL-10 but enhanced production of IL-4 (Fig. S1 e). This is consistent with previous studies that showed reduced Th17 and preferential Th2 cell differentiation of T cells isolated from SOCS3-Tg mice (Seki et al., 2003
; Tanaka et al., 2008
). Interestingly, we found a fourfold increase of lesion size in Apoe−/−
mice transferred with CD4+
SOCS3-Tg cells compared with controls (Fig. S1 e), despite similar plasma cholesterol levels (12.1 ± 1.6 vs. 14.1 ± 0.78 g/liter; P = 0.25). Thus, SOCS3 expression in T cells significantly affects atherosclerotic lesion development.
SOCS3 deletion in T cells enhances IL-10 and IL-17 production
The reduction of atherosclerosis in SOCS3-cKO mice was associated with a significant decrease of T cell infiltration within the lesions (), suggesting a modulation of the T cell phenotype. We found no difference in the number and suppressive function of natural CD4+
regulatory T cells between the two groups of mice (Fig. S2
). Thus, we analyzed cytokine production by purified spleen-derived CD4+
cells. We observed a significant reduction of IFN-γ production and an increase of IL-10 by cells recovered from SOCS3-cKO mice (), which was consistent with previous results showing preferential Th3- and/or Tr1-like differentiation and reduced Th1 polarization in mice lacking SOCS3 expression in T cells (Kinjyo et al., 2006
). However, we also detected a threefold increase in IL-17 production by the purified SOCS3-deficient CD4+
cells compared with controls, consistent with the critical role of STAT3 activation in Th17 development (; Chen et al. 2006
; Dong, 2008
). Flow cytometry analysis on freshly isolated cells showed no cells expressing both IL-17 and IL-10, suggesting that in vivo, IL-10 and IL-17 are produced by distinct T cells, which is in agreement with a recent study showing that RORγt+
T cells producing both IL-10 and IL-17 could not be observed in vivo (Lochner et al., 2008
). Consistent with enhanced IL-17 production, we found increased IL-6 but reduced IL-27 levels in the circulating blood of SOCS3-cKO mice (Fig. S2 c). We also found increased production of IL-6 by cultured SOCS3-cKO splenocytes. IL-6 neutralization did not alter IL-17 production, indicating that the Th17 profile of SOCS3-cKO cells was independent of IL-6 (Fig. S2 d). Thus, our results indicate that in the absence of SOCS3 expression in T cells, there is a preferential switch toward increased production of both IL-10 and IL-17, which helps explain seemingly divergent previous results on Th3, Tr1, and Th17 cells.
SOCS3 deletion in T cells induces an antiinflammatory phenotype in macrophages
It was remarkable that the loss of SOCS3 in one cell type has produced significant effects on lesion development, where many other cell types are involved. Thus, we hypothesized that the T cell cytokine profile induced by SOCS3 deletion may modulate the inflammatory response of other cell types. In this regard, the innate immune system plays a critical role in atherogenesis (Hansson and Libby, 2006
). Therefore, we examined the impact of SOCS3 deletion in T cells on macrophage infiltration and activation. The extent of lesion macrophage accumulation was not altered in SOCS3-cKO compared with SOCS3-WT mice (35,022 ± 6,308 vs. 36,635 ± 5,223 µm2
; P = 0.84). We hypothesized that this could be related to a differential modulation of macrophage survival within the lesions. Interestingly, we found a marked reduction in the size of the necrotic core in lesions of SOCS3-cKO mice (). This was confirmed by the reduction of lesion Tdt-mediated dUTP-biotin nick-end labeling (TUNEL) staining (unpublished data) and a protection against macrophage apoptosis when bone marrow–derived macrophages were incubated in the presence of SOCS3-cKO CD4+
cells compared with SOCS3-WT CD4+
cells (). Macrophage death within the lesions is highly promoted by proinflammatory signals. We found a significant reduction of IL-12 and an increase in IL-10 production by macrophages after coincubation with SOCS3-cKO CD4+
cells compared with coincubation with SOCS3-WT CD4+
cells (). In addition, we found a significant reduction in the expression of NOS2 and TNF, and an increase of arginase-1 after coincubation of macrophages with SOCS3-cKO T cells compared with SOCS3-WT cells, consistent with a limitation of M1 phenotype and the preservation of an antiinflammatory potential (). These effects persisted when T cells and macrophages were physically separated using Transwells, suggesting the involvement of soluble factors (). Neutralization of IL-10 production and, to a lesser extent IL-17, prevented some of the antiinflammatory effects (), whereas incubation with IL-10 was associated with an antiinflammatory phenotype that persisted after supplementation with IL-17 (). Our results indicate that SOCS3 signaling in T cells not only affects the T cell phenotype but also regulates the production of pro- and antiinflammatory/atherogenic mediators by macrophages. This is consistent with a study showing that STAT3 activity in tumor cells mediates immune evasion through blockade of inflammatory signal production by the innate and adaptive immune system (Wang et al., 2004
). Further studies will be necessary to identify the soluble factors responsible for the full change in macrophage phenotype.
Figure 2. SOCS3 deletion in T cells promotes an antiinflammatory macrophage phenotype. (a) IL-12 and IL-10 production by macrophages after coincubation with CD4+ cells from SOCS3-WT or SOCS-cKO mice in the presence or absence of Transwells. Mean values ± (more ...)
Neutralization of IL-17 abrogates atheroprotection in mice with T cell–specific SOCS3 deletion and enhances vascular inflammation
The reduction of atherosclerosis in SOCS3-cKO mice was associated with a reduction in IFN-γ and an increase in both IL-10 and IL-17. The pro- and antiatherogenic roles of IFN-γ and IL-10, respectively, have been extensively addressed in previous studies (Tedgui and Mallat, 2006
). However, the role of IL-17 in atherosclerosis is still unexplored. Thus, we examined the direct role of IL-17 production in the atheroprotective effect of T cell–specific SOCS3 deletion. We generated additional series of chimeric Ldlr−/−
mice that were put on a high fat diet and were treated with either a mouse monoclonal anti–IL-17A neutralizing antibody (Uyttenhove and Van Snick, 2006
; Uyttenhove et al., 2007
) or an isotype-matched control for 6 wk (see Materials and methods). Interestingly, neutralization of IL-17 did not alter lesion size in SOCS3-WT mice, which are highly Th1 biased and produce low levels of IL-17, but totally abrogated the atheroprotective effect of T cell–specific SOCS3 deletion and led to a marked increase of lesion formation (). Acceleration of lesion development was associated with a marked increase in vascular inflammation, as revealed by increased vascular cell adhesion molecule (VCAM)–1 expression (), marked T cell infiltration within the lesions and the adventitia of anti–IL-17–treated SOCS3-cKO mice (), and a switch toward reduced IL-10 and increased IL-4 production by spleen-derived CD4+
cells () but no change in IFN-γ (15.4 ± 0.5 vs. 12.7 ± 1.1 ng/ml in SOCS3-WT and SOCS3-cKO, respectively). Regulatory T cell function was not altered by anti–IL-17 treatment (not depicted), and the proatherogenic effect of IL-17 neutralization in SOCS3-cKO mice was not prevented by IL-10 supplementation (Fig. S3
). These results identify an unprecedented role for SOCS3-controlled IL-17 in the control of vascular inflammation and T cell accumulation within atherosclerotic lesions, which profoundly affects lesion development. Our results are in agreement with a study showing that IL-17 neutralization was associated with enhanced T cell infiltration in a model of allergic asthma (Schnyder-Candrian et al., 2006
Figure 3. SOCS3-controlled IL-17 production protects against vascular inflammation and atherosclerotic lesion development. Representative photomicrographs (a and b) and quantitative analysis (c and d) of atherosclerotic lesion size (a and c) and lesion T cell infiltration (more ...)
In vivo administration of IL-17 to Ldlr−/− mice reduces endothelial VCAM-1 expression, vascular T cell infiltration, and atherosclerotic lesion development
Neutralization of IL-17 accelerated atherosclerosis in SOCS3-cKO mice but had no effect on lesion development in SOCS3-WT mice (). We reasoned that this apparent discrepancy might be caused by the low level of IL-17 production by SOCS3-WT T cells (highly Th1 biased on a C57BL/6 background) and that supplementation of SOCS3-WT mice with IL-17 might inhibit lesion development. Thus, we fed 17-wk-old female Ldlr−/− mice a high fat diet and treated them with either rIL-17 or control mouse serum albumin. Administration of rIL-17 led to a significant elevation of circulating IL-17 levels (Fig. S3 c) and to a moderate but significant elevation of IL-6 production (27.5 ± 18.7 vs. 1 ± 0.6 pg/ml; P = 0.008) comparable to levels detected in SOCS3-cKO mice, suggesting biological effects. We found no change in T cell cytokine profile (Fig. S3 d) or splenocyte inflammatory response (unpublished data) in mice treated with rIL-17, indicating that at this dosage, IL-17 did not promote inflammatory cell activation. Interestingly, we found a significant reduction of atherosclerotic lesion development in mice supplemented with rIL-17 (). We reproduced the results in a different set of younger 12-wk-old female Ldlr−/− mice (). Of note, atheroprotection was associated with a marked inhibition of vascular inflammation as revealed by a significant inhibition of vascular T cell infiltration () and a 60% reduction of endothelial VCAM-1 expression (). rIL-17 also reduced IL-1–induced endothelial VCAM-1 expression in vitro (). These results indicate that IL-17 inhibits high fat–induced vascular inflammation and atherosclerotic lesion development.
Figure 4. Supplementation with IL-17 reduces vascular inflammation and limits atherosclerotic lesion development. (a) Atherosclerotic lesion size in the aortic root of 17-wk-old female chimeric Ldlr −/− SOCS3-WT mice fed a high fat diet and treated (more ...)
Vascular expression of IL-17 and P-Stat3 is associated with plaque stability
We then examined IL-17 expression in mouse atherosclerotic arteries (Ldlr−/− mice reconstituted with SOCS3-WT or SOCS3-cKO) using immunohistochemistry. As expected, we were unable to detect IL-17 expression in T cells of SOCS3-WT mice (not depicted), and only occasional IL-17+ inflammatory cells were detected in lesions of SOCS3-cKO mice (, left, a and b), which may be explained, at least in part, by the low number of activated SOCS3-deficient T cells that infiltrated the lesions. Unexpectedly, we found a marked staining of IL-17 in medial smooth muscle cells (SMCs) of plaque-free areas (which was confirmed in normal arteries; , left, e), and the staining appeared to be rapidly lost in medial SMCs underlying early lipid lesions (, left, c and d). We confirmed the detection of IL-17 in normal mouse aorta by Western blotting using a different antibody (unpublished data). These results show that C57BL/6 Ldlr−/− mice, which are Th1 biased, produce low levels of IL-17 in T cells and that vascular wall production of IL-17 decreases during the development of atherosclerosis, suggesting a relationship between SMC differentiation and IL-17 expression.
Figure 5. Expression of IL-17 in atherosclerotic vessels. (left) Mouse atherosclerosis. (a and b) Occasional staining for IL-17 (red, arrows) in inflammatory cells within the intima (i) and adventitia (arrows in b) of SOCS3-cKO mice. (c and d) Representative examples (more ...)
Finally, we studied human carotid atherosclerotic arteries retrieved from patients undergoing carotid endarterectomy. Patients characteristics are given in Table S1
. P-Stat3 and IL-17 expression were readily detectable in CD3+
cells of human plaques (), although not all CD3+
cells expressed IL-17 or P-Stat3 (). We could also detect P-Stat3 in other cell types, as expected (, and not depicted). Interestingly, we also detected IL-17 expression in vascular SMCs, and consistent with our data in mice, we found a clear decrease in its expression by medial SMCs underlying advanced lesions (, right). Interestingly, semiquantitative assessment of P-Stat3 and IL-17 expression showed increased levels of these two markers in plaques with fibrous (stable) compared with atheromatous (unstable) phenotype (). Consistent with this finding, increased levels of P-Stat3 were significantly associated with a low macrophage content, and increased levels of IL-17 were associated with a lower macrophage infiltration and a higher SMC content (). No relation was found between Stat3 or P-Stat1 levels and plaque phenotype (unpublished data).
Figure 6. Stat3 phosphorylation and IL-17 expression in human carotid atherosclerotic plaques are associated with markers of plaque stability. (a) Stat3 phosphorylation (P-Stat3) in CD3+ cells of human carotid atherosclerotic plaques (arrows). Arrowheads show a (more ...)
In conclusion, we show that endogenous expression of SOCS3 in T cells interrupts a major regulatory pathway in atherosclerosis through inhibition of IL-17 production. Interestingly, patients have been identified with defective STAT3 signaling (Minegishi et al., 2007
) and IL-17 production (Ma et al., 2008
; Milner et al., 2008
), and recent observational studies have reported the abnormal occurrence of vascular inflammation (van der Meer et al., 2006
; Ling et al., 2007
) in this setting despite the absence of classical cardiovascular risk factors. Although the reported vascular abnormalities consisted mainly of inflammatory vascular aneurysms, some cases of coronary atherosclerosis have been reported in these young individuals (Freeman et al., 2007
). Thus, our results may have important implications for the understanding of the pathophysiological mechanisms of vascular inflammation in humans and identify novel targets for disease modulation.