Role of Suppressor of Cytokine Signaling-1 In Murine Atherosclerosis

Background While the impact of inflammation as the substantial driving force of atherosclerosis has been investigated in detail throughout the years, the influence of negative regulators of pro-atherogenic pathways on plaque development has remained largely unknown. Suppressor of cytokine signaling (SOCS)-1 potently restricts transduction of various inflammatory signals and, thereby modulates T-cell development, macrophage activation and dendritic cell maturation. Its role in atherogenesis, however has not been elucidated so far. Methods and Results Loss of SOCS-1 in the low-density lipoprotein receptor deficient murine model of atherosclerosis resulted in a complex, systemic and ultimately lethal inflammation with increased generation of Ly-6Chi monocytes and activated macrophages. Even short-term exposure of these mice to high-cholesterol dieting caused enhanced atherosclerotic plaque development with accumulation of M1 macrophages, Ly-6C positive cells and neutrophils. Conclusion Our data not only imply that SOCS-1 is athero-protective but also emphasize the fundamental, regulatory importance of SOCS-1 in inflammation-prone organisms.


Introduction
Atherosclerotic plaque growth and rupture are considered as the underlying cause of myocardial infarction, sudden cardiac death and stroke. While the fundamental contribution of inflammation to atherogenesis has already been extensively studied, approaches to determine the impact of inhibitors of inflammatory signaling pathways have remained few so far. [1] Suppressor of cytokine signalling (SOCS)-1 belongs to a family of 8 intracellular proteins and restricts type I Interferon and Interferon(IFN)-c receptor activation. Thereby, SOCS-1 acts as a classical negative feedback loop regulator of the Janus kinase andsignal transducer and activator of transcription (JAK-STAT) pathway. [2] Furthermore, SOCS-1 restricts toll-like receptor activation and modulates nuclear factor (NF)-kb dependent transcription by binding to and induction of degradation of the p65 subunit. Thereby, SOCS-1 controls innate and adaptive inflammatory cell behaviour, including macrophages, granulocytes, dendritic cells and T-cells in response to a diverse set of proatherogenic cytokines, e.g. interleukin-6, tumor necrosis factor (TNF)-a, and IFN-c. [3,4].
SOCS-1 as well as SOCS-3 expression has recently been demonstrated in murine and human atherosclerotic lesions. In addition, in vitro studies suggested an anti-inflammatory and potentially athero-protective effect of these molecules in vascular cells, e.g. monocytes, endothelial and smooth muscle cells. [5] While T-cell specific loss of SOCS-3 resulted in reduced plaque development, the role of SOCS-1 in atherosclerosis has not been determined yet. [5,6] Therefore, we investigated the impact of a systemic deficiency of SOCS-1 on atherogenesis in the established, murine low-density lipoprotein receptor (LDLR) model of atherosclerosis. We hypothesised that loss of SOCS-1 in this setting would result in advanced plaque development. In fact, our data confirm athero-protective features of this molecule but also underline the crucial, gate keeping function of SOCS-1 in inflammation.

Aortic Lipid Deposition
For the analysis of aortic lipid depositions, aortas were prepared en face and stained with Oil Red O solution as previously described. [7] Percentage of Oil Red O positive area was calculated via computer-assisted image quantification (Leica Qwin 500, Leica, Heidelberg, Germany).

Atherosclerotic Plaque Analysis
To analyse atherosclerotic plaque area and composition, aortic roots were embedded in Tissue-TekH O.C.T TM and kept at 280uC until sectioning. Within the aortic root, lesion area was analyzed in cross sections obtained at the level of all three leaflets of the aortic valve after staining with Oil Red O solution as previously described. [8] Serial cross sections (5 mm) were collected, 6-9 sections/animals were analysed via computerassisted image quantification (Leica Qwin 500, Leica, Heidelberg, Germany). Neutrophils were identified using a rat anti-mouse Ly-6G antibody (clone IA8, BD Bioscience, San Jose, USA). Monocytes/macrophages were detected with a rat anti-mouse MOMA-2 antibody (Acris, Herford, Germany), the biotinylated secondary antibody (rabbit anti-rat) was visualized by ABC reagent (Vector Laboratories, Burlingame, USA) and the AEC-Chromogen (DAKO, Glostrup, Denmark) according to the manufacturer's protocol. Sections were counterstained with hematoxylin (Carl Roth, Karlsruhe, Germany). For visualisation of Ly-6C/MOMA-2 double positive cells, sections were incubated with biotinylated anti-mouse Ly-6C antibody (AL-21, BD Pharmingen, Franklin Lakes, USA) and rat anti-mouse MOMA-2 followed by visualization using streptavidin-Fitc and anti-rat Alexa-549 (Invitrogen, Molecular Probes, Darmstadt, Germany). For staining of CD68/iNOS and CD68/CD206 double-positive cells, we followed a protocol published by Salagianni et al. [9] In short, sections were incubated with rat anti-mouse CD68 (clone FA-11, AbD serotec, Oxford, UK) and rabbit anti-mouse iNOS (AbCam, Cambridge, UK) or rabbit anti-mouse CD206 (clone MR5D3, AbD serotec, Oxford, UK) or respective isotype controls followed by incubation with anti-rat Alexa-549 or anti-rabbit Alexa-488 (both Invitrogen, Molecular Probes, Darmstadt, Germany). Nuclei were counterstained with DAPI (Invitrogen, Molecular Probes, Darmstadt, Germany).

Histological Analysis
Paraffin embedded, serial sections from various organs were subjected to hematoxylin/eosin (HE) staining. In short, nuclei were stained with alum hematoxylin and differentiated with acid alcohol followed by staining with eosin solution. For Sirius Red staining, we followed a protocol published elsewhere. [10] In short, sections were stained with Sirius Red solution (0.1% Sirius Red in saturated picric acid solution). Section analysis was carried out using polarization microscopy.

Plasma Analysis
Systemic Tumor necrosis factor (TNF)-a, Interleukin (IL)-6 and MCP (monocyte chemotactic protein)-1 levels were measured using ELISA (all from R&D Systems, Minneapolis, USA) following the manufacturers protocol.

LDL-Isolation and Peroxidation
For in-vitro experiments, LDL was isolated from human plasma by sequential gradient ultracentrifugation. [12] LDL fraction was dialyzed at 4uC against phosphate buffer (140 mM NaCl, 1.9 mM NaH2HPO4, 8.1 mM). Protein concentration was determined by the Bradford method. For peroxidation, native (n)LDL (100 mg/mL) was incubated with CuSO 4 (10 mmol/L) in cell culture medium for 24 hrs. LDL-peroxidation was stopped with EDTA (5 mmol/L) and butylhydroxytoluol (20 mmol/L). LDLperoxidation was analyzed by detection of conjugated diene formation by measuring UV absorbance at 234 nm. [13] Additionally malondialdehyde as lipid peroxidation product was measured using the thiobarbituric acid-reactive assay (TBARS assay kit, Oxitech, Buffalo, USA).

In Vitro Foam Cell Formation
BMC were differentiated into bone-marrow derived macrophages (BMDM) as described above. In-vitro foam cell formation was induced by incubating BMDM with 25 mg/mL oxidized (ox)LDL and nLDL for 4 hrs. After stimulation, cells were washed twice with PBS, fixated in 3.7% paraformaldehyde followed by staining of intracellular lipids with Oil red O for 30 min. The percentage of Oil red O positive stained cells was quantified by

Cell Sorting
Cells were harvested from bone marrow, peripheral blood and spleens as described by Swirski et al. [16] BMCs from both tibias and femurs were harvested. Peripheral blood was drawn via cardiac puncture and collected into EDTA tubes. Mononuclear cells were purified by density centrifugation. Differential blood counts were obtained using the Vet abc Animal Blood Counter (scil animal care company GmbH, Viernheim, Germany). Spleens were removed and filtered through a nylon mesh (BD Bioscience).

Statistical Analysis
Data are presented as mean and standard error of mean (SEM) or standard deviation (SD). Statistical analyses were performed using SigmaStat 3.0 (SyStat, San Jose, USA). Comparisons between two groups were performed by Student's t-test assuming two-tailed distribution and equal variances. One-way ANOVA was used for the comparisons between three groups. For unequal group sizes with failed normality tests, Rank-based ANOVA was used and data presented as median with 25 th and 75 th percentile. P values ,0.05 were considered statistically significant.
Despite a significant weight gain, Socs-1 2/2 triple-KO mice remained lighter compared to the other genotypes after 4 weeks of HCD (table 1). Lipoprotein fractions did not differ between Socs-1 2/2 triple-KO and Ldlr 2/2 mice. However, Ldlr 2/ 2 ;Rag-2 2/2 mice displayed decreased total cholesterol levels caused by a lower LDL-and VLDL-cholesterol fraction (table 3). Analysis of plasma samples revealed significantly increased The extent of atherosclerotic plaque development was analysed in complete aortas prepared en face and stained with Oil Red O and demonstrated significantly enhanced lipid depositions in vessels of Socs-1 2/2 triple-KO mice ( figure 3A). Concomitantly, we also observed increased atherosclerotic lesion formation ( figure 3B) and an accumulation of macrophages (MOMA-2 positive cells) in aortic roots of Socs-1 2/2 triple-KO mice (figure 3C). Atherosclerotic plaque progression was analysed using the recommendations of the report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association ( figure 3D). [20] Overall, very early lesions types dominated after 4 weeks of HCD regardless of the respective genotype. However, plaques obtained from Socs-1 2/2 triple-KO mice proved to be the most advanced. To further characterize lesion composition we performed Sirius Red stainings but did not observe any significant extracellular matrix formation after 4 weeks of high-cholesterol dieting (figure S2A).

SOCS-1 Regulates Monocyte Ly-6C hi Subset Formation in Atherosclerosis-prone Mice
Cell sorting experiments were performed in age-matched mice after 4 weeks of chow diet (CD) or high-cholesterol diet (HCD). Despite the overall leukopenia of Socs-1 2/2 triple-KO mice under CD, flow cytometric analysis of blood samples (figure S1) demonstrated a marked increase in circulating CD11b hi monocytes ( figure 4A) that was mainly due to an increase of the Ly-6C hi cell subset ( figure 4D). Similar observations were made in bonemarrow (figure 4B and E) and spleens of Socs-1 2/ triple-KO mice ( figure 4C and F). Analysis of the Ly-6C lo subset revealed no differences between triple-KO mice and Ldlr 2/2 mice, but significantly less circulating Ly-6C lo cells in Ldlr 2/2 ;Rag-2 2/2 mice (data not shown).
4 weeks of HCD resulted in a significant rise of total circulating leukocytes in Ldlr 2/ and Ldlr 2/2 ;Rag-2 2/2 mice compared to mice on CD (table 2). However, we observed a tendency toward decreased numbers of leukocytes and granulocytes in peripheral blood of Socs-1 2/2 triple-KO mice (table 2). Analysis of leukocyte subsets indicated a slight, but not statistically significant rise of CD11b hi monocytes and Ly-6C hi cells in peripheral blood of Ldlr 2/2 and Ldlr 2/2 ;Rag-2 2/2 animals. In Socs-1 2/2 triple-KO mice the proportion of circulating CD11b hi monocytes and Ly-6C hi cells decreased (figure 5A and D) while Ly-6C hi monocytosis persisted in bone-marrow (figure 5B and E) and spleens (figure 5C and F). Numbers of Ly-6C lo monocytes did not significantly change under HCD with regard to genotype or hematopoietic compartment (data not shown).
Considering the contribution of Ly-6C hi monocytes to atherosclerotic plaque formation together with the sustained generation of these cells in bone-marrow and spleens of Socs-1 2/2 triple-KO mice, we hypothesized, that the reduced number of circulating monocytes after 4 weeks of HCD might reflect an increased cell extravasation with subsequently enhanced lesion development at this particular point of time. Therefore, we performed double stainings of MOMA-2 and Ly-6C in atherosclerotic plaques obtained from Socs-1 2/2 triple-KO mice. While the overall number of double-positive cells proved to be quite low and was constricted to small, very early lesions, these stainings indicated an enhanced number of MOMA-2/Ly-6C positive cells in Socs-1 2/ 2 triple-KO mice although these observations did not reach statistical significance ( figure 6C). Given the neutrophilia in Socs-1 2/2 triple-KO mice, we also stained aortic roots for Ly-6G as a marker for neutrophils. Again, the total cell number detected was very low. However, quantitative analysis revealed increased lesion infiltration of Ly-6G positive cells in Socs-1 2/ 2 triple-KO mice ( figure 6D).

SOCS-1 Regulates Pro-atherogenic Macrophage Formation in Atherosclerosis-prone Mice
To further investigate the impact of SOCS-1 on macrophage phenotype in atherogenesis, scavenger receptor (SR)-A and CD36 expression were determined on bone-marrow derived macrophages (BMDM) after CD and HCD. While SR-A expression did not differ between the genotypes (data not shown), macrophages received from Socs-1 2/2 triple-KO mice on CD already showed a significantly enhanced expression of CD36 compared to Ldlr 2/ 2 ;Rag-2 2/2 or Ldlr 2/2 mice that further increased after HCD (figure 7A-D). Subsequently, stimulation with oxLDL resulted in a markedly higher proportion of foam cell formation in SOCS-1 deficient BMDM compared to BMDM derived from Ldlr 2/ 2 ;Rag-2 2/2 and Ldlr 2/2 mice on chow diet that did not further increase after HCD (figure 7E-F). Given these findings, we also analysed intra-plaque macrophage phenotypes. In this regard, we performed double stainings using CD68 in combination with iNOS for detection of M1 macrophages (figure 6A) versus CD68 combined with CD206 for tracking of M2 macrophages ( figure 6B). While we hardly detected any M2 macrophages regardless of the respective genotype, Socs-1 2/2 triple-KO mice showed an enhanced proportion of M1 macrophages although it did not reach statistical significance due to the low overall cell number.

Discussion
Atherosclerosis is a chronic inflammatory disease of the arterial cardiovascular system. [21] As SOCS-1 has been demonstrated to restrict the activation of various pro-atherogenic pathways, [2] we postulated that loss of SOCS-1 in an atherosclerosis-prone organism might aggravate plaque development.
Ly-6C hi monocytes are defined as a short-lived Cx 3 CR lo cell subset that drives inflammation by extravasation, tissue infiltration and differentiation into phagocytes. In context with atherogenesis, they are considered as the major, pro-atherogenic monocyte population. [22] So far, data discussing the impact of SOCS-1 on monocyte generation or function has been limited to experimental settings of infection. [23] For example, human monocytes have been shown to up-regulate SOCS-1 in response to nitric oxide resulting in reduced secretion of IL-6 and IL-10. [24] In addition, increased SOCS-1 expression in monocytes infected with hepatitis C aggravates disease activity by modulating IL-12 secretion while in Ly-6C hi monocytes isolated from bone-marrow of C57BL/6 mice infected with Listeria monocytogenesis early SOCS-1 expression is associated with host outcome. [25,26].
As mentioned above, Ly-6C hi monocytes are regarded as a vital source for plaque macrophages. These phagocytes undergo foam cell formation by up-take of oxidized LDL particles via scavenger receptors -a dominating event in early lesion development. Interestingly, macrophages derived from Socs-1 2/2 triple-KO mice on chow diet already displayed significantly enhanced scavenger receptor CD36 expression. Accordingly, we also found a profoundly increased foam cell formation of these cells ex vivo.
Spontaneous atherogenesis is scarce in Ldlr 2/2 mice. Nevertheless, given the increased generation of Ly-6C hi monocytes and activated macrophages together with the enhanced circulating levels of IL-6, TNF-a and MCP-1, we speculated that atherosclerotic plaque development might already occur in SOCS-1 2/ 2 triple-KO mice on chow diet. However, we did not observe lesion formation in the absence of hypercholesterolemia regardless of the genotype investigated.
While lipoprotein fractions did not significantly differ between Ldlr 2/2 and Socs-1 2/2 deficient mice after the feeding period, they proved to be slightly lower in Ldlr 2/2 ;Rag-2 2/2 animals. These results are consistent with the observations of other groups investigating the impact of RAG-2 deficiency in the ApoE mouse model of atherosclerosis. Neither we nor previous studies have been able to elucidate the underlying mechanisms. However, regarding the importance of lymphocytes for barrier-functionsincluding the gastro-intestinal tract -one might speculate that lymphocyte depletion may disturb both, lipoprotein up-take as well as transport. [27,28].
Despite the short period of HCD and the young age of animals, we found enhanced atherosclerotic lesion formation throughout the aorta as well as in aortic roots of Socs-1 2/2 triple-KO mice. Accordingly, investigation of plaque composition revealed an increased content of macrophages with pro-atherogenic M1 features in these mice. SOCS-1 is a known modulator of macrophage activation by suppressing CD40, IL-6 and TNF-a expression. [29,30] Furthermore, as shown by Whyte et al. in context with parasite infection, SOCS-1 may confine classically activated M1 macrophage formation and foster alternatively activated -potentially anti-atherogenic -M2 macrophage generation. [3] Thus, these observations support the pro-atherogenic impact of SOCS-1 deficiency in this mouse model. The fact that we hardly detected any M2 macrophages regardless of the genotype examined may reflect the mainly pro-inflammatory sub-intimal environment at this particular time of plaque development. [31].
Overall, lesion phenotype analysis confirmed that atherogenesis proved to be the most advanced in Socs-1 2/2 triple-KO mice.
Flow cytometric analysis of peripheral blood demonstrated markedly decreased numbers of CD11b + ;Ly-6C hi monocytes in Socs-1 2/2 triple-KO mice after 4 weeks of HCD, while these cells remained increased in bone-marrow and spleen. We hypothesized, that the reduced number of circulating monocytes in Socs-1 2/ 2 triple-KO mice might have reflected an increase in vascular extravasation. [29] We resigned from performing sorting experiments with SOCS-1 deficient aortic tissue due to the short feeding period, the young age of mice and the overall low circulating CD11b + ;Ly-6C hi cell number. Instead, we stained aortic roots for MOMA-2, Ly-6C double-positive cells. Although the fate of Ly-6C hi monocytes after entering the sub-intimal space likely depends on the local cytokine/chemokine balance at the particular time of entrance and thus, may change during plaque development, we detected more MOMA-2, Ly-6C double-positive cells in developing Socs-1 2/2 triple-KO lesions.
Given the systemic neutrophilia observed in SOCS-1 deficient animals, we also examined atherosclerotic plaques for neutrophil infiltration. Indeed, we found more Ly-6G positive cells in lesions derived from Socs-1 2/2 triple-KO mice. Neutrophils have recently gained attention and may particularly contribute to early atherogenesis by production of oxygen radicals and secretion of pro-inflammatory granules. [32] Therefore, neutrophils may be involved in the atherogenic phenotype of SOCS-1 deficient mice in this study.
We are also aware of the fact, that disturbed T-and B-cell development caused by loss of RAG-2 might have influenced atherogenesis in this mouse model especially since RAG-2 deficiency obviously modulated lipoprotein homeostasis. In this context Reardon et al found a close correlation between lipoprotein levels and lesion extent in Rag-2 2/2 ;ApoE 2/2 mice as well as a site-specific difference in plaque development. However, others did not detect any obvious effects of RAG-2 on lesion size. [27,28] In the study presented, we did not observe significant differences in plaque development between Ldlr 2/2 and Ldl 2/2 ;Rag-2 2/2 mice despite a tendency towards more advanced lesions in Ldlr 2/2 ;Rag-2 2/2 animals. These results surely do not negate the impact of lymphocytes on atherogenesis. Instead they may rather be a consequence of this specific study design with very young mice undergoing a short feeding period.
One might also argue that the atherogenic phenotype of Socs-1 2/2 triple-KO animals may be secondary to the ongoing systemic inflammation and therefore, of constricted biological relevance. However, we did not find atherosclerotic plaque formation in Socs-1 2/2 triple-KO mice until induction of hypercholesterolemia despite Ly-6C hi monocytosis, activation of macrophages and neutrophilia under chow diet conditions indicating that a specific, pro-atherogenic trigger was necessary for induction of plaque development. Furthermore, deletion of a single SOCS-1 allele resulted in effects similar to those observed in Socs-1 2/2 triple-KO animals ( figure S2B-D). In summary, this study demonstrates the athero-protective nature of SOCS-1 in experimental, murine atherosclerosis. Figure S1 Cell sorting of blood derived from Ldlr 2/2 , Ldlr 2/2 ;Rag-2 2/2 and Ldlr 2/2 ;Rag-2 2/2 ;Socs-1 2/2 . A and B. Representative dot plots showing cell sorting of CD11b hi (CD90, B220, CD49b, NK1.1, Ly-6G) lo cells from blood derived from all three, age-matched genotypes after (A) 4 weeks of chow diet (CD) or (B) 4 weeks of high-cholesterol diet (HCD). C and D. Representative histograms demonstrating the proportion of Ly-6C hi blood monocytes among CD11b hi (CD90, B220, CD49b, NK1.1, Ly-6G) lo cells in all three genotypes. Independent cell sorting experiments were performed in specimens derived from 8-17 animals per group. (TIF) Figure S2 Impact of SOCS-1 on extracellular matrix formation and Impact of SOCS-1 +/2 on atherosclerotic plaque development and plaque content of macrophages after 4 weeks of high-cholesterol diet (HCD). A. Representative pictures demonstrating that atherosclerotic plaque development did not involve significant collagen formation after 4 weeks of HCD regardless of the genotype investigated B. Aortas derived from Socs-1 +/2 mice showed significantly enhanced lipid depositions after en face preparation and staining with Oil Red O after 4 weeks of HCD. Results were comparable to those derived from Socs-1 2/2 triple-KO mice C and D. Aortic roots of Socs-1 +/ 2 mice also displayed increased macrophage content after staining with MOMA-2 (C) and enhanced lipid depositions after staining with Oil Red O (D) after 4 weeks of HCD. Both results were comparable to those derived from Socs-1 2/2 triple-KO mice. Horizontal bars represent mean, *p,0.05 vs Ldlr 2/2 and Ldlr 2/ 2 ;Rag-2 2/2 (C: scale bar: 500 mm, D: scale bar: 50 mm). Each dot indicates results for an individual animal. (TIF)