Cardiovascular Research Advance Access published January 26, 2014
1
Natural Killer (NK) Cells Augment Atherosclerosis by Cytotoxic-Dependent Mechanisms
Ahrathy Selathurai1,2,*, Virginie Deswaerte1,2, Peter Kanellakis1, Peter Tipping2, Ban-Hock Toh2,*, Alex Bobik1,*, Tin Kyaw1,2 1
Baker IDI Heart & Diabetes Institute, Melbourne, Victoria, Australia
2
Centre for Inflammatory Diseases, Department of Medicine, Southern Clinical School, Faculty of
Medicine, Nursing and Health Sciences, Monash University, Clayton, Victoria, Australia
PO Box 6492 St Kilda Road Central, Melbourne, 8008, Australia, Fax: 61-3-8532-1100, Email: [email protected] *
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Current address: Department of Physiology, Monash University, Clayton, Victoria, Australia.
Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: [email protected].
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Correspondence: Alex Bobik, Vascular Biology and Atherosclerosis, BakerIDI Heart and Diabetes Institute,
2
Abstract Aims- Although natural killer (NK) cells, a key component of the innate immune system have been identified in human and mouse atherosclerotic lesions, their role in atherosclerosis development remains unclear. To determine their role in atherosclerosis we used both loss and gain of function experiments in ApoE-/- mice fed a high fat diet. Methods and Results- Treatment of ApoE-/- mice with anti-asialo GM1 antibodies depleted NK cells without affecting other lymphocytes, including NKT cells and greatly attenuated atherosclerosis. These
isolated from mouse spleens for adoptive transfer into lymphocyte-deficient ApoE-/-Rag2-/-IL2rg-/- mice. Transfer of NK cells from wild-type mice into ApoE-/-Rag2-/-IL2rg-/- mice doubled lesion size, confirming a proatherogenic role for NK cells. To determine whether their atherogenicity was dependent on production of interferon-gamma (IFN-γ) or cytotoxins, we compared the transfer of NK cells deficient in IFN-γ, perforin
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and granzyme B with the transfer of wild-type NK cells. Transfer of IFN-γ deficient NK cells increased lesion size in the lymphocyte-deficient ApoE-/- mice as wild-type NK cells. However, granzyme B and perforin-deficient NK cells were without affect. Also, only wild-type NK cells increased necrotic core size, whereas perforin and granzyme B-deficient NK cells did not. Plasma lipid were largely unaffected by the cell transfers. Conclusion- Our loss and gain of function findings provide definitive evidence that NK cells are atherogenic and their production of perforin and granzyme B contribute to atherosclerosis and the expansion of necrotic cores.
Key words: Atherosclerosis, ApoE-/- mice, NK cells, perforin, granzyme B
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effects were independent of plasma lipids. To confirm the atherogenicity of NK cells, these cells were
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Introduction
Atherosclerosis is a chronic inflammatory disease of elastic and large muscular arteries, characterized by lesions containing cholesterol, immune cells, smooth muscle cells and necrotic cores. Macrophages, dendritic cells and T cells are major immune cells populating developing lesions but other less abundant immune cell types such as NKT cells and NK cells are also present1. NK cells are bone marrow-derived lymphocytes that act as effectors, either through cell mediated cytotoxicity, by releasing perforin and
stressed cells via activating receptors3. These cells have been detected in human atherosclerotic lesions3, 4. In more advanced atherosclerotic lesions5, these cells are frequently localised to regions near necrotic cores deep within plaques and also in shoulder regions5. NK cells have also been detected in atherosclerotic lesions of mice6. These cells have the potential to modulate atherosclerosis via NK activating receptors
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which recognize MHC class I molecules including homologues such as MICA and MICB7. Despite such studies the role of NK cells in atherosclerosis is still unclear.
Early studies on the role of NK cells in atherosclerosis using a beige mutation in mice suggested an atheroprotective role8. Beige-mutant mice have a defect in lysosomal fusion/fission and trafficking and the deficiency causes defects in cytolytic function of NK cells9. The beige mutation in mice involves the Lyst gene implicated in lysosomal trafficking. The gene product is a homologue of the protein implicated in autosomal recessive human Chediak-Higashi syndrome and results in a complex phenotype of partial albinism, immunodeficiency, bleeding diathesis and neuropathy10. Defects in cell function are however not restricted to NK cells but also include neutrophils and other cells, making it unclear whether the increase in atherosclerosis in Lyst-beige LDLR-/- mice is a consequence of impaired NK cell function11. Subsequently Ly49A transgenic mice were used to assess NK cell function in atherosclerosis6. These mice overexpress the inhibitory receptor Ly49A under the granzyme A promoter12; consequently all splenic NK cells as well as approximately half the T cells express Ly49A. In addition, mature NK cell populations are reduced by about 70%, most likely due to insertion of the transgene into the ATF2 locus13. Transfer of bone marrow from
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granzymes and/or cytokines such as interferon-gamma (IFN-γ)2. NK cells sense pathological changes or
4 -/-
6
Ly49A transgenic mice into LDLR mice resulted in attenuated atherosclerosis . Whilst the authors concluded that impaired activity of NK cells is responsible for the attenuated atherosclerosis, the possibility that T cells known to express granzyme A and whose functions are affected by Ly49A, notably NKT cells14 and CD8+ T cells15, also contribute to the reduction in atherosclerosis was not excluded. We have previously shown that NKT and CD8+ T cells augment atherosclerosis development.14, 16
Because of the complexities in previous animal models used to define the role of NK cells in atherosclerosis,
studies. Loss of function studies were performed by depleting NK cells in high fat diet (HFD)-fed ApoEdeficient mice using anti-Asialo-GM1 antibodies. Asialo-GM1 is expressed on NK cells and anti-AsialoGM1 antibodies deplete NK cells without affecting NKT cells17. Gain of function studies were performed by transferring purified NK cells into ApoE-/-Rag2-/-IL2rg-/- mice, which are deficient in all lymphocytes
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including NK, NKT, T and B cells18. Using these two mouse models we provide novel insights as to how NK cells augment atherosclerosis.
Materials and Methods Expanded materials and methods are available at online supplementary materials.
Animals and Treatments Male apolipoprotein E gene knockout (ApoE-/-) mice and ApoE-/-Rag2-/-IL2rg-/- mice were used in this study. All procedures performed on the mice were approved by AMREP Animal Ethics Committee, (AEC- No, E/0804/2009B) and conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No 85-23, revised 1996). In loss of function experiments, anti-Asialo-GM1 antibodies (54µg/injection; Wako Chemicals, Richmond, VA)17 was used to deplete NK cells whilst NK cells isolated from different donors were adoptively transferred into ApoE-/-Rag2-/-IL2rg-/mice. Donor and experimental mice were killed using pentobarbitone (150mg/kg intraperitoneal injection) and tissue and blood collected for NK cell isolation and analysis respectively.
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we have reinvestigated their significance for atherosclerosis by performing both loss and gain of function
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Purification of NK Cells Spleens from donor mice were crushed in PBS (without Mg2+ and Ca2+; PBS-/-). Following red blood cell lysis and washing with PBS-/-, cells were filtered and NK cells isolated using an NK cell isolation kit (Miltenyi Biotech). NK cells were cultured for 5 days in RPMI 1640 medium containing 10% (v/v) heatinactivated FCS, 5% penicillin/streptomycin, 5% Glutamax (Gibco), 50mM 2ME, 5% non-essential amino acid, 5% sodium pyruvate, 5% HEPES and 1000IU/ml recombinant human IL-2 (rhIL-2) (Biological
cells were FACS-sorted using a BD FACSAria, after staining with PE-conjugated NK1.1 and APCconjugated TCR-β antibodies.
Cholesterol analysis
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Plasma cholesterol levels were determined as described before16, 18.
Flow Cytometry and mAbs Lymphocyte analysis of CD4+ and CD8+ T cells, CD22+ B cells, NK1.1+TCRβ- NK cells and NK1.1+TCRβ+ NKT cells was carried out using Flow Cytometry as described16, 18. Total cell numbers from tissue preparations were determined using Coulter counter and using appropriate gates used for FACS analysis, absolute number of lymphocyte subsets was calculated.
Collection and Measurement of Atherosclerotic lesions Aortic sinuses collected in OCT compound were frozen, sectioned and stained with Oil Red-O (ORO) to determine lipid accumulation. Sections were stained by H&E to measure necrotic core areas defined as unstained acellular area and expressed as a percentage of total atherosclerotic lesion area16, 18.
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Resources Branch Preclinical Repository, National Cancer Institute, Frederick, MD). Then the cultured NK
6 Immunohistochemistry Aortic sinus atherosclerotic sections were stained with different antibodies to identify/quantitate different immune cells/proteins as described16, 18. Macrophages (anti-CD68), vascular smooth muscle cells (anti-αSM actin), VCAM-1 (anti-VCAM-1) and MCP-1 (anti-MCP-1) were quantified by using Optimas 6.2 software and CD4+ T-cells (anti-CD4) were counted in intimal lesions manually.
Tracking experiment
from congenic Ly5.1 donor mice two days before culling. Frozen section of aortic sinus atherosclerotic lesion were stained with fluorescence-labelled Ly5.1 (APC) and CD68 (FITC) antibodies, counterstained with DAPI and visualised under fluorescence microscope.
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mRNA extraction and realtime PCR mRNAs extracted from isolated cells (NK cells or macrophages) and aortic arches were analysed for expression of perforin, granzyme B, ApoE using realtime PCR as described16, 18.
Statistics Statistical analyses utilised Student’s t-test when data followed a normal distribution or Mann-Whitney U test when data did not follow a normal distribution. Normality was determined using the D’Agostino and Pearson omnibus normality test. One-way ANOVA was used to compare more than two groups. Analyses were performed using GraphPad Prism v5.02. P < 0.05 was statistically significant.
Results
Anti-asialo-GM1 Treatment Depletes NK cells in ApoE-/- mice Initially we examined the effectiveness of the anti-asialo-GM1 antibody treatment for depleting NK cells in peripheral blood. Five days after a single dose of anti-asialo-GM1 antibody, NK cells in blood were reduced by more than 90% (P < 0.05; Figure 1A). Identical effects on blood NK cells were observed after treating
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ApoE-/-Rag2-/-IL2rg-/- mice fed a HFD for 8 weeks were adoptively transferred with 3x106 NK cells isolated
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ApoE fed a HFD for 8 weeks (P0.05; data not shown), NK cells per in the spleen were reduced by almost 60% (P < 0.05; Figure 1C) whilst other lymphocytes, CD4+, CD8+ T-cells, CD22+ B-cells and NKT cells were unaffected (P > 0.05; Figure 1C).
NK cell depletion by anti-Asialo-GM1 attenuates atherosclerosis in hyperlipidemic ApoE-/- mice
development. Atherosclerotic lesions, determined by ORO-staining were reduced by 62% compared to mice administered with control normal rabbit serum (NRS) (P for difference < 0.05; Figure 2A). Macrophage accumulation was also reduced, by 60% compared with lesions from control mice (P < 0.05; Figure 2B). Lesion MCP-1 expression was reduced (P 0.05; Figure 2C), and the reduction in CD4+ T cells was not statistically significant (P > 0.05; Figure 2D); VCAM-1 expression was unaffected (P > 0.05; Figure 2E). Body weights were similar in the two groups, averaging 28.3±0.6 g (n=9) and 28.2±0.6g (n=8) as were blood cholesterol levels, 16.2±1.9 (n=9) and 13.7±2.5 (n=8) mmol/L (P >0.05). Thus the attenuation in lesion size in the anti-Asialo-GM1 antibody treated mice is due to reduced NK cell numbers and is independent of cholesterol levels.
Adoptive Transfer of NK Cells into Lymphocyte-Deficient ApoE-/-Rag2-/-IL2rg-/- Mice augments Atherosclerosis Development To confirm an atherogenic role for NK cells, we adoptively transferred 3x106 NK cells from wild-type (WT) mice into 6 week old ApoE-/-Rag2-/-IL2rg-/- mice deficient in all lymphocyte populations including NK cells and fed a HFD. NK cells from WT mice do not express ApoE protein (Figure S2). Four weeks later, we assessed the extent of NK cell reconstitution in the liver and administered a second dose of NK cells. In ApoE-/-Rag2-/-IL2rg-/- mice administered vehicle (PBS) no NK cells could be detected in the liver (Figure 3A). In contrast, NK cells were readily detectable in ApoE-/-Rag2-/-IL2rg-/- mice that received NK cells
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Treating ApoE-/-mice with anti-Asialo-GM1 antibodies for 8 weeks markedly attenuated atherosclerosis
8 -/-
-/-
-/-
(Figure 3A). The level of NK cell reconstitution in ApoE Rag2 IL2rg mice was 65% compared to agematched ApE-/- mice (Figure 3B). Transfer of NK cells into ApoE-/-Rag2-/-IL2rg-/- mice increased atherosclerotic lesion size (P < 0.05; Figure 4A and C); on average lesion size was doubled by NK cell transfer. Macrophage accumulation in lesions also increased to a similar extent (P < 0.05; Figure 4B and D). Plasma cholesterol levels and body weights were largely unaffected by adoptive transfer of NK cells (Table 1). Thus adoptively transferred NK cells augment atherosclerosis in ApoE-/- independently of plasma lipid levels.
mice To elucidate the mechanisms by which NK cells augment atherosclerosis, we assessed the significance of NK cell-derived interferon-γ, granzyme B and perforin. NK cells can produce copious amounts of
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interferon- γ19, which can promote atherosclerosis20. In addition, NK cells produce granzyme B and perforin21, which cooperatively induce target cell apoptosis22; accumulation of apoptotic cells in atherosclerotic lesions and subsequent necrosis leads to accelerated atherosclerosis23. Arterial mRNA expression of perforin and granzyme B was up-regulated in 8 week HFD-fed ApoE-/- mice compared to agematched chow diet-fed C57Bl/6 mice (P 0.05; Figure 4A and C). Macrophage accumulation was also similar to lesions in control mice (P > 0.05; Figure 4B and D). Plasma lipid levels and body weight were largely unaffected by the NK cell transfers (Table 1).
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Perforin and granzyme B are essential for the atherogenic effects of NK cells in ApoE-/-Rag2-/-IL2rg-/-
9 NK Cells and Necrotic Core Development Accumulation of apoptotic cells within atherosclerotic lesions due to impaired efferocytosis contributes to necrotic core development and post apoptotic necrosis, resulting in accelerated atherosclerosis23-25. Since perforin and granzyme B released by NK cells induce target cell apoptosis22, we next determined the extent to which NK cell derived perforin and granzyme B contributed to necrotic core development in lesions of ApoE-/-Rag2-/-IL2rg-/- mice that received NK cells. Adoptive transfer of WT NK cells to ApoE-/-Rag2-/IL2rg-/- mice increased necrotic core size nearly 6-fold (P< 0.05; Figure 5A-B). In contrast, necrotic core
(P for difference > 0.05; Figure 5A-B).
NK Cells home to atherosclerotic lesions NK cells have been reported in human and mouse atherosclerotic lesions5, 6. To determine whether NK cells
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home to atherosclerotic lesions, NK cells isolated from congenic Ly5.1 mice were transferred into ApoE-/Rag2-/-IL2rg-/- mice two days before completion of 8 week HFD. Immunofluorescent staining showed that Ly5.1 NK cells were identified near fibrous cap (Figure 5C) and in close proximity to macrophages in atherosclerotic lesions (Figure 5D).
Discussion Using two independent experimental approaches involving loss and gain of function we have unambiguously demonstrated that NK cells promote development of atherosclerosis. Specifically, deletion of NK cells ameliorates atherosclerosis, whilst transfer of NK cells into lymphocyte deficient ApoE-/- mice augments atherosclerosis. By adoptively transferring NK cells deficient in the cytokine interferron-γ or the cytotoxins perforin and granzyme B, we also demonstrate that NK cells can augment atherosclerosis independently of other lymphocytes, by mechanisms dependent on the cytotoxic molecules perforin and granzyme B.
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size was unaffected in lesions of mice that had received NK cells deficient in either perforin or granzyme B
10 Previous studies on the role of NK cells in atherosclerosis have led to conflicting conclusions, most likely due to effects on other cell types in addition to NK cells. Using chimeric LDLR-/- mice whose bone marrow cells overexpress the inhibitory Ly49A receptor regulated by the granzyme A promoter, it was concluded that NK cells contribute to atherosclerosis development6. However, in donor transgenic mice Ly49A inhibitory receptor is not only over expressed by NK cells but also by CD4+ and CD8+ T cells12, raising the possibility that transgenic over expression of Ly49A attenuates atherosclerosis by also affecting T cells. Engagement of transgenic Ly49A on CD4+ T cells inhibits activation by disrupting T cell receptor
effects on CD4+ T cell activation could contribute to the attenuated atherosclerosis in such mice. Also, the position of incorporation of the Ly49A transgene could also directly contribute to attenuated atherosclerosis independently of effects on lymphocyte activation. In donor Ly49A transgenic mice NK cell development is arrested due to insertion of the transgene into the ATF2 locus, resulting in expression of a truncated ATF2
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transcript13. Reductions in active ATF2 in endothelial cells suppress basal proinflammatory gene expression28 and may also affect proinflammatory cytokine secretion by T cells29. In contrast to these studies, LDLR-/- mice expressing the beige mutation, which also affects NK cell function exhibit accelerated atherosclerosis8. This effect was not attributed to interactions with B or T cells as atherosclerosis was accelerated in LDLR-/-Rag1-/- mice expressing the beige mutation. Since NK cells are not depleted, it has been suggested that the beige mutation is affecting a yet-to-be defined NK cell function to affect atherosclerosis development. However, more recent studies indicate that this atheroprotective effect is dependent on the presence of the beige mutation in bone marrow-derived cells, endothelial cells and vascular smooth muscle cells30. In the present study we avoided such complexities by directly demonstrating that NK cells promote atherosclerosis. Specific depletion of NK cells by anti-Asialo-GM1 antibody attenuated atherosclerosis consistent with NK cells augmenting development of atherosclerosis; other lymphocyte populations were unaffected including NKT cells. Conversely, adoptive transfer of NK cells into ApoE-/-Rag-/-IL2rg-/- accelerated atherosclerosis.
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signalling26. As CD4+ T cell activation has also been implicated in atherosclerosis development27, inhibitory
11 NK cells can exert their effects via a number of mechanisms. NK cells are capable of secreting proinflammatory cytokines including IFN-γ, TNF-α and IL-631, all of which have been implicated in atherosclerosis32-34. Adoptive transfer of NK cells deficient in interferon-γ did not attenuate the NK celldependent increase in atherosclerosis in ApoE-/-Rag2-/-IL2rg-/- mice indicating that NK cell-derived interferon-γ does not contribute to atherosclerosis in lymphopenic mice. Whether other NK cell-derived cytokines contribute to atherosclerosis remains to be determined. NK cells also exert their effects by killing cells via apoptosis utilising mechanisms that involve perforin and granzyme B23. Perforin is a cytolytic
proteoglycan serglycin35. Upon degranulation, perforin mediates cytosolic delivery of macromolecules in the granule without producing detectable plasma membrane pores35. Entry of granzyme B into the nucleus is also perforin dependent and precedes apoptosis36. Granzyme B cleaves target cell proteins to induce caspase-mediated cell death22, 37. Our findings indicate that both perforin and granzyme B secretion by NK
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cells are important for their atherogenic activity. NK cells deficient in either perforin or granzyme B did not increase atherosclerosis in ApoE-/-Rag2-/-IL2rg-/- mice. Furthermore perforin and granzyme B deficient NK cells did not increase the necrotic core of atherosclerotic lesions, contrasting with the effects of WT NK cells. Clearance of apoptotic cells is impaired in atherosclerotic lesions24, 38, leading to post apoptotic necrosis and accelerated atherosclerosis23, 39. Necrotic cells that release proinflammatory factors can activate inflammasomes, which together drive cardiovascular disease40-43. Our finding that NK cells do not affect vascular smooth muscle cell numbers in atherosclerotic lesions suggests that NK cells may be targeting macrophages in lesions to augment atherosclerosis. Macrophages in atherosclerotic lesions express Rae-1ε the ligand for the activating NKG2D receptor44, which is expressed by NK cells45. The binding of ligands to NKG2D receptors activates NK cells and promotes cytotoxicity of ligand expressing cells. Our observation that NK cells are present in close contact with macrophages supports the suggestion that NK cells may interact with macrophages. Whilst it is possible that additional mechanisms also contribute to the atherogenicity of NK cells, our studies clearly demonstrate that one mechanism by which NK cells accelerate atherosclerosis development is by inducing cell death via perforin and granzyme B-dependent mechanisms. Recently we have demonstrated that cytotoxic CD8+ T cells also accelerate atherosclerosis by
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protein that exists in cytoplasmic granules of NK cells as multimeric complexes with granzyme B and the
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perforin and granzyme B-dependent mechanisms , raising the possibility that such a mechanism may be common to other cytotoxic immune cells. Human NK cell numbers and their activity were reduced in patients with coronary artery disease. It was not clear whether medications such as statin and β1-receptor blocker could have affected NK cell function46, 47.
In conclusion, our loss and gain of function studies of NK cells in the development of atherosclerosis have definitively shown that NK cells are atherogenic. Their production of the cytotoxins perforin and granzyme
in immune-intact ApoE-/- mice NK cells appear to contribute to a greater extent to atherosclerosis than in lymphocyte-deficient Rag2-/-IL2rg-/-ApoE-/- mice suggests that additional mechanisms involving interactions between NK cells and other lymphocytes, possibly T cells, may also contribute to atherosclerosis. Recruitment of NK cells to lymph nodes can provide a source of IFN-γ for Th1 priming48; such a
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mechanism contributes to early resistance to Leishmania major in mice19. Thus it is possible that in addition to NK cell homing and exerting pathogenic effects via perforin and granzyme B, additional mechanisms involving NK cell-facilitated differentiation of CD4+ T cells to Th1 cells in lymph nodes may also be important. Clearly further studies are required to investigate this possibility. Our studies indicate that cytotoxic actions of NK cells within atherosclerotic lesions contribute significantly to their proatherogenic effects and targeting NK cells may be s useful therapeutic strategy to attenuate atherosclerosis.
Source of Funding This work was supported by a grant from the National Health and Medical Research Council of Australia (to A. B.), a PhD postgraduate scholarship (to A. S.) from BakerIDI Heart and Diabetes Institute and supported in part by the Victorian Government’s Operational Infrastructure Support Program.
Conflicts of Interest There are no conflicts of interest.
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B and expansion of necrotic cores within lesions contribute to atherosclerosis development. Our finding that
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Froelich CJ. Cytotoxic cell granule-mediated apoptosis: Perforin delivers granzyme b-serglycin complexes into target cells without plasma membrane pore formation. Immunity. 2002;16:417-428 36.
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Figure Legends Figure 1 Depletion of Natural Killer cells (NK cells) in peripheral blood and spleen of ApoE-/- mice after anti-Asialo-GM1 treatment (A) Representative results of depletion of NK cells identified as NK1.1+, TCR-β- expression in blood, assessed by flow cytometry, 5 days after commencing treatment with antiAsialo-GM1. Plots represents the percentage of NK1.1+ and TCR-β- cells; n=4, *P < 0.05. (B) Representative results of NK1.1+, TCR-β- expression by NK cells in blood, assessed by flow cytometry, 8
NK1.1+ and TCR-β- NK cells; n=4, *P < 0.05. (C) Total (NK1.1+ TCR-β-) NK cells, (NK1.1+ TCRβ+) NKT cells, CD4+ T cells, CD8+ T cells and CD22+ B cells in spleen of ApoE-/- mice treated with normal rabbit serum (NRS) or anti-Asialo-GM1 after 8 weeks of HFD; n=4 each group.
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Figure 2 NK cell depletion inhibits atherosclerotic plaque formation and macrophage accumulation but do not affect lesion smooth muscle cells, VCAM-1 and CD4+ T cells expression. (A) Photomicrographs of aortic sinus atherosclerotic lesions from ApoE-/- mice fed a HFD for 8 weeks and treated with NRS (left) or Anti-asialo-GM1 antibody; (right) showing mean area of ORO-staining (bar graphs) in the two groups of mice. (B) Immunohistochemical staining using anti-CD68 (macrophage) antibody showing mean areas of staining (bar graphs) in two groups. (C) Immunohistochemical staining with anti-α smooth muscle actin antibodies, detecting smooth muscle cells in plaque of ApoE-/- mice treated with NRS (left) or anti-Asialo GM1 antibodies (right) showingmean percentage area of staining (bar graph). (D) anti-CD4 stained atherosclerotic cross sections from NRS and anti-Asialo GM1 treated ApoE-/- mice detecting CD4+ T cells in lesion showing mean numbers of CD4+ T cells in lesions (bar graph). (E) Cross sections stained with anti-VCAM-1 (Vascular cell adhesion molecule-1) detecting VCAM-1 adhesion molecules in plaque of ApoE-/- mice treated with NRS (left) and anti-Asialo-GM1 (right) showing percentage of area stained (bar graph); n=8-9 in NRS and anti-Asialo-GM1 groups. *P
1
Natural Killer (NK) Cells Augment Atherosclerosis by Cytotoxic-Dependent Mechanisms
Ahrathy Selathurai1,2,*, Virginie Deswaerte1,2, Peter Kanellakis1, Peter Tipping2, Ban-Hock Toh2,*, Alex Bobik1,*, Tin Kyaw1,2 1
Baker IDI Heart & Diabetes Institute, Melbourne, Victoria, Australia
2
Centre for Inflammatory Diseases, Department of Medicine, Southern Clinical School, Faculty of
Medicine, Nursing and Health Sciences, Monash University, Clayton, Victoria, Australia
PO Box 6492 St Kilda Road Central, Melbourne, 8008, Australia, Fax: 61-3-8532-1100, Email: [email protected] *
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Current address: Department of Physiology, Monash University, Clayton, Victoria, Australia.
Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: [email protected].
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Correspondence: Alex Bobik, Vascular Biology and Atherosclerosis, BakerIDI Heart and Diabetes Institute,
2
Abstract Aims- Although natural killer (NK) cells, a key component of the innate immune system have been identified in human and mouse atherosclerotic lesions, their role in atherosclerosis development remains unclear. To determine their role in atherosclerosis we used both loss and gain of function experiments in ApoE-/- mice fed a high fat diet. Methods and Results- Treatment of ApoE-/- mice with anti-asialo GM1 antibodies depleted NK cells without affecting other lymphocytes, including NKT cells and greatly attenuated atherosclerosis. These
isolated from mouse spleens for adoptive transfer into lymphocyte-deficient ApoE-/-Rag2-/-IL2rg-/- mice. Transfer of NK cells from wild-type mice into ApoE-/-Rag2-/-IL2rg-/- mice doubled lesion size, confirming a proatherogenic role for NK cells. To determine whether their atherogenicity was dependent on production of interferon-gamma (IFN-γ) or cytotoxins, we compared the transfer of NK cells deficient in IFN-γ, perforin
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and granzyme B with the transfer of wild-type NK cells. Transfer of IFN-γ deficient NK cells increased lesion size in the lymphocyte-deficient ApoE-/- mice as wild-type NK cells. However, granzyme B and perforin-deficient NK cells were without affect. Also, only wild-type NK cells increased necrotic core size, whereas perforin and granzyme B-deficient NK cells did not. Plasma lipid were largely unaffected by the cell transfers. Conclusion- Our loss and gain of function findings provide definitive evidence that NK cells are atherogenic and their production of perforin and granzyme B contribute to atherosclerosis and the expansion of necrotic cores.
Key words: Atherosclerosis, ApoE-/- mice, NK cells, perforin, granzyme B
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effects were independent of plasma lipids. To confirm the atherogenicity of NK cells, these cells were
3
Introduction
Atherosclerosis is a chronic inflammatory disease of elastic and large muscular arteries, characterized by lesions containing cholesterol, immune cells, smooth muscle cells and necrotic cores. Macrophages, dendritic cells and T cells are major immune cells populating developing lesions but other less abundant immune cell types such as NKT cells and NK cells are also present1. NK cells are bone marrow-derived lymphocytes that act as effectors, either through cell mediated cytotoxicity, by releasing perforin and
stressed cells via activating receptors3. These cells have been detected in human atherosclerotic lesions3, 4. In more advanced atherosclerotic lesions5, these cells are frequently localised to regions near necrotic cores deep within plaques and also in shoulder regions5. NK cells have also been detected in atherosclerotic lesions of mice6. These cells have the potential to modulate atherosclerosis via NK activating receptors
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which recognize MHC class I molecules including homologues such as MICA and MICB7. Despite such studies the role of NK cells in atherosclerosis is still unclear.
Early studies on the role of NK cells in atherosclerosis using a beige mutation in mice suggested an atheroprotective role8. Beige-mutant mice have a defect in lysosomal fusion/fission and trafficking and the deficiency causes defects in cytolytic function of NK cells9. The beige mutation in mice involves the Lyst gene implicated in lysosomal trafficking. The gene product is a homologue of the protein implicated in autosomal recessive human Chediak-Higashi syndrome and results in a complex phenotype of partial albinism, immunodeficiency, bleeding diathesis and neuropathy10. Defects in cell function are however not restricted to NK cells but also include neutrophils and other cells, making it unclear whether the increase in atherosclerosis in Lyst-beige LDLR-/- mice is a consequence of impaired NK cell function11. Subsequently Ly49A transgenic mice were used to assess NK cell function in atherosclerosis6. These mice overexpress the inhibitory receptor Ly49A under the granzyme A promoter12; consequently all splenic NK cells as well as approximately half the T cells express Ly49A. In addition, mature NK cell populations are reduced by about 70%, most likely due to insertion of the transgene into the ATF2 locus13. Transfer of bone marrow from
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granzymes and/or cytokines such as interferon-gamma (IFN-γ)2. NK cells sense pathological changes or
4 -/-
6
Ly49A transgenic mice into LDLR mice resulted in attenuated atherosclerosis . Whilst the authors concluded that impaired activity of NK cells is responsible for the attenuated atherosclerosis, the possibility that T cells known to express granzyme A and whose functions are affected by Ly49A, notably NKT cells14 and CD8+ T cells15, also contribute to the reduction in atherosclerosis was not excluded. We have previously shown that NKT and CD8+ T cells augment atherosclerosis development.14, 16
Because of the complexities in previous animal models used to define the role of NK cells in atherosclerosis,
studies. Loss of function studies were performed by depleting NK cells in high fat diet (HFD)-fed ApoEdeficient mice using anti-Asialo-GM1 antibodies. Asialo-GM1 is expressed on NK cells and anti-AsialoGM1 antibodies deplete NK cells without affecting NKT cells17. Gain of function studies were performed by transferring purified NK cells into ApoE-/-Rag2-/-IL2rg-/- mice, which are deficient in all lymphocytes
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including NK, NKT, T and B cells18. Using these two mouse models we provide novel insights as to how NK cells augment atherosclerosis.
Materials and Methods Expanded materials and methods are available at online supplementary materials.
Animals and Treatments Male apolipoprotein E gene knockout (ApoE-/-) mice and ApoE-/-Rag2-/-IL2rg-/- mice were used in this study. All procedures performed on the mice were approved by AMREP Animal Ethics Committee, (AEC- No, E/0804/2009B) and conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No 85-23, revised 1996). In loss of function experiments, anti-Asialo-GM1 antibodies (54µg/injection; Wako Chemicals, Richmond, VA)17 was used to deplete NK cells whilst NK cells isolated from different donors were adoptively transferred into ApoE-/-Rag2-/-IL2rg-/mice. Donor and experimental mice were killed using pentobarbitone (150mg/kg intraperitoneal injection) and tissue and blood collected for NK cell isolation and analysis respectively.
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we have reinvestigated their significance for atherosclerosis by performing both loss and gain of function
5
Purification of NK Cells Spleens from donor mice were crushed in PBS (without Mg2+ and Ca2+; PBS-/-). Following red blood cell lysis and washing with PBS-/-, cells were filtered and NK cells isolated using an NK cell isolation kit (Miltenyi Biotech). NK cells were cultured for 5 days in RPMI 1640 medium containing 10% (v/v) heatinactivated FCS, 5% penicillin/streptomycin, 5% Glutamax (Gibco), 50mM 2ME, 5% non-essential amino acid, 5% sodium pyruvate, 5% HEPES and 1000IU/ml recombinant human IL-2 (rhIL-2) (Biological
cells were FACS-sorted using a BD FACSAria, after staining with PE-conjugated NK1.1 and APCconjugated TCR-β antibodies.
Cholesterol analysis
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Plasma cholesterol levels were determined as described before16, 18.
Flow Cytometry and mAbs Lymphocyte analysis of CD4+ and CD8+ T cells, CD22+ B cells, NK1.1+TCRβ- NK cells and NK1.1+TCRβ+ NKT cells was carried out using Flow Cytometry as described16, 18. Total cell numbers from tissue preparations were determined using Coulter counter and using appropriate gates used for FACS analysis, absolute number of lymphocyte subsets was calculated.
Collection and Measurement of Atherosclerotic lesions Aortic sinuses collected in OCT compound were frozen, sectioned and stained with Oil Red-O (ORO) to determine lipid accumulation. Sections were stained by H&E to measure necrotic core areas defined as unstained acellular area and expressed as a percentage of total atherosclerotic lesion area16, 18.
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Resources Branch Preclinical Repository, National Cancer Institute, Frederick, MD). Then the cultured NK
6 Immunohistochemistry Aortic sinus atherosclerotic sections were stained with different antibodies to identify/quantitate different immune cells/proteins as described16, 18. Macrophages (anti-CD68), vascular smooth muscle cells (anti-αSM actin), VCAM-1 (anti-VCAM-1) and MCP-1 (anti-MCP-1) were quantified by using Optimas 6.2 software and CD4+ T-cells (anti-CD4) were counted in intimal lesions manually.
Tracking experiment
from congenic Ly5.1 donor mice two days before culling. Frozen section of aortic sinus atherosclerotic lesion were stained with fluorescence-labelled Ly5.1 (APC) and CD68 (FITC) antibodies, counterstained with DAPI and visualised under fluorescence microscope.
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mRNA extraction and realtime PCR mRNAs extracted from isolated cells (NK cells or macrophages) and aortic arches were analysed for expression of perforin, granzyme B, ApoE using realtime PCR as described16, 18.
Statistics Statistical analyses utilised Student’s t-test when data followed a normal distribution or Mann-Whitney U test when data did not follow a normal distribution. Normality was determined using the D’Agostino and Pearson omnibus normality test. One-way ANOVA was used to compare more than two groups. Analyses were performed using GraphPad Prism v5.02. P < 0.05 was statistically significant.
Results
Anti-asialo-GM1 Treatment Depletes NK cells in ApoE-/- mice Initially we examined the effectiveness of the anti-asialo-GM1 antibody treatment for depleting NK cells in peripheral blood. Five days after a single dose of anti-asialo-GM1 antibody, NK cells in blood were reduced by more than 90% (P < 0.05; Figure 1A). Identical effects on blood NK cells were observed after treating
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ApoE-/-Rag2-/-IL2rg-/- mice fed a HFD for 8 weeks were adoptively transferred with 3x106 NK cells isolated
7 -/-
ApoE fed a HFD for 8 weeks (P0.05; data not shown), NK cells per in the spleen were reduced by almost 60% (P < 0.05; Figure 1C) whilst other lymphocytes, CD4+, CD8+ T-cells, CD22+ B-cells and NKT cells were unaffected (P > 0.05; Figure 1C).
NK cell depletion by anti-Asialo-GM1 attenuates atherosclerosis in hyperlipidemic ApoE-/- mice
development. Atherosclerotic lesions, determined by ORO-staining were reduced by 62% compared to mice administered with control normal rabbit serum (NRS) (P for difference < 0.05; Figure 2A). Macrophage accumulation was also reduced, by 60% compared with lesions from control mice (P < 0.05; Figure 2B). Lesion MCP-1 expression was reduced (P 0.05; Figure 2C), and the reduction in CD4+ T cells was not statistically significant (P > 0.05; Figure 2D); VCAM-1 expression was unaffected (P > 0.05; Figure 2E). Body weights were similar in the two groups, averaging 28.3±0.6 g (n=9) and 28.2±0.6g (n=8) as were blood cholesterol levels, 16.2±1.9 (n=9) and 13.7±2.5 (n=8) mmol/L (P >0.05). Thus the attenuation in lesion size in the anti-Asialo-GM1 antibody treated mice is due to reduced NK cell numbers and is independent of cholesterol levels.
Adoptive Transfer of NK Cells into Lymphocyte-Deficient ApoE-/-Rag2-/-IL2rg-/- Mice augments Atherosclerosis Development To confirm an atherogenic role for NK cells, we adoptively transferred 3x106 NK cells from wild-type (WT) mice into 6 week old ApoE-/-Rag2-/-IL2rg-/- mice deficient in all lymphocyte populations including NK cells and fed a HFD. NK cells from WT mice do not express ApoE protein (Figure S2). Four weeks later, we assessed the extent of NK cell reconstitution in the liver and administered a second dose of NK cells. In ApoE-/-Rag2-/-IL2rg-/- mice administered vehicle (PBS) no NK cells could be detected in the liver (Figure 3A). In contrast, NK cells were readily detectable in ApoE-/-Rag2-/-IL2rg-/- mice that received NK cells
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Treating ApoE-/-mice with anti-Asialo-GM1 antibodies for 8 weeks markedly attenuated atherosclerosis
8 -/-
-/-
-/-
(Figure 3A). The level of NK cell reconstitution in ApoE Rag2 IL2rg mice was 65% compared to agematched ApE-/- mice (Figure 3B). Transfer of NK cells into ApoE-/-Rag2-/-IL2rg-/- mice increased atherosclerotic lesion size (P < 0.05; Figure 4A and C); on average lesion size was doubled by NK cell transfer. Macrophage accumulation in lesions also increased to a similar extent (P < 0.05; Figure 4B and D). Plasma cholesterol levels and body weights were largely unaffected by adoptive transfer of NK cells (Table 1). Thus adoptively transferred NK cells augment atherosclerosis in ApoE-/- independently of plasma lipid levels.
mice To elucidate the mechanisms by which NK cells augment atherosclerosis, we assessed the significance of NK cell-derived interferon-γ, granzyme B and perforin. NK cells can produce copious amounts of
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interferon- γ19, which can promote atherosclerosis20. In addition, NK cells produce granzyme B and perforin21, which cooperatively induce target cell apoptosis22; accumulation of apoptotic cells in atherosclerotic lesions and subsequent necrosis leads to accelerated atherosclerosis23. Arterial mRNA expression of perforin and granzyme B was up-regulated in 8 week HFD-fed ApoE-/- mice compared to agematched chow diet-fed C57Bl/6 mice (P 0.05; Figure 4A and C). Macrophage accumulation was also similar to lesions in control mice (P > 0.05; Figure 4B and D). Plasma lipid levels and body weight were largely unaffected by the NK cell transfers (Table 1).
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Perforin and granzyme B are essential for the atherogenic effects of NK cells in ApoE-/-Rag2-/-IL2rg-/-
9 NK Cells and Necrotic Core Development Accumulation of apoptotic cells within atherosclerotic lesions due to impaired efferocytosis contributes to necrotic core development and post apoptotic necrosis, resulting in accelerated atherosclerosis23-25. Since perforin and granzyme B released by NK cells induce target cell apoptosis22, we next determined the extent to which NK cell derived perforin and granzyme B contributed to necrotic core development in lesions of ApoE-/-Rag2-/-IL2rg-/- mice that received NK cells. Adoptive transfer of WT NK cells to ApoE-/-Rag2-/IL2rg-/- mice increased necrotic core size nearly 6-fold (P< 0.05; Figure 5A-B). In contrast, necrotic core
(P for difference > 0.05; Figure 5A-B).
NK Cells home to atherosclerotic lesions NK cells have been reported in human and mouse atherosclerotic lesions5, 6. To determine whether NK cells
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home to atherosclerotic lesions, NK cells isolated from congenic Ly5.1 mice were transferred into ApoE-/Rag2-/-IL2rg-/- mice two days before completion of 8 week HFD. Immunofluorescent staining showed that Ly5.1 NK cells were identified near fibrous cap (Figure 5C) and in close proximity to macrophages in atherosclerotic lesions (Figure 5D).
Discussion Using two independent experimental approaches involving loss and gain of function we have unambiguously demonstrated that NK cells promote development of atherosclerosis. Specifically, deletion of NK cells ameliorates atherosclerosis, whilst transfer of NK cells into lymphocyte deficient ApoE-/- mice augments atherosclerosis. By adoptively transferring NK cells deficient in the cytokine interferron-γ or the cytotoxins perforin and granzyme B, we also demonstrate that NK cells can augment atherosclerosis independently of other lymphocytes, by mechanisms dependent on the cytotoxic molecules perforin and granzyme B.
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size was unaffected in lesions of mice that had received NK cells deficient in either perforin or granzyme B
10 Previous studies on the role of NK cells in atherosclerosis have led to conflicting conclusions, most likely due to effects on other cell types in addition to NK cells. Using chimeric LDLR-/- mice whose bone marrow cells overexpress the inhibitory Ly49A receptor regulated by the granzyme A promoter, it was concluded that NK cells contribute to atherosclerosis development6. However, in donor transgenic mice Ly49A inhibitory receptor is not only over expressed by NK cells but also by CD4+ and CD8+ T cells12, raising the possibility that transgenic over expression of Ly49A attenuates atherosclerosis by also affecting T cells. Engagement of transgenic Ly49A on CD4+ T cells inhibits activation by disrupting T cell receptor
effects on CD4+ T cell activation could contribute to the attenuated atherosclerosis in such mice. Also, the position of incorporation of the Ly49A transgene could also directly contribute to attenuated atherosclerosis independently of effects on lymphocyte activation. In donor Ly49A transgenic mice NK cell development is arrested due to insertion of the transgene into the ATF2 locus, resulting in expression of a truncated ATF2
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transcript13. Reductions in active ATF2 in endothelial cells suppress basal proinflammatory gene expression28 and may also affect proinflammatory cytokine secretion by T cells29. In contrast to these studies, LDLR-/- mice expressing the beige mutation, which also affects NK cell function exhibit accelerated atherosclerosis8. This effect was not attributed to interactions with B or T cells as atherosclerosis was accelerated in LDLR-/-Rag1-/- mice expressing the beige mutation. Since NK cells are not depleted, it has been suggested that the beige mutation is affecting a yet-to-be defined NK cell function to affect atherosclerosis development. However, more recent studies indicate that this atheroprotective effect is dependent on the presence of the beige mutation in bone marrow-derived cells, endothelial cells and vascular smooth muscle cells30. In the present study we avoided such complexities by directly demonstrating that NK cells promote atherosclerosis. Specific depletion of NK cells by anti-Asialo-GM1 antibody attenuated atherosclerosis consistent with NK cells augmenting development of atherosclerosis; other lymphocyte populations were unaffected including NKT cells. Conversely, adoptive transfer of NK cells into ApoE-/-Rag-/-IL2rg-/- accelerated atherosclerosis.
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signalling26. As CD4+ T cell activation has also been implicated in atherosclerosis development27, inhibitory
11 NK cells can exert their effects via a number of mechanisms. NK cells are capable of secreting proinflammatory cytokines including IFN-γ, TNF-α and IL-631, all of which have been implicated in atherosclerosis32-34. Adoptive transfer of NK cells deficient in interferon-γ did not attenuate the NK celldependent increase in atherosclerosis in ApoE-/-Rag2-/-IL2rg-/- mice indicating that NK cell-derived interferon-γ does not contribute to atherosclerosis in lymphopenic mice. Whether other NK cell-derived cytokines contribute to atherosclerosis remains to be determined. NK cells also exert their effects by killing cells via apoptosis utilising mechanisms that involve perforin and granzyme B23. Perforin is a cytolytic
proteoglycan serglycin35. Upon degranulation, perforin mediates cytosolic delivery of macromolecules in the granule without producing detectable plasma membrane pores35. Entry of granzyme B into the nucleus is also perforin dependent and precedes apoptosis36. Granzyme B cleaves target cell proteins to induce caspase-mediated cell death22, 37. Our findings indicate that both perforin and granzyme B secretion by NK
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cells are important for their atherogenic activity. NK cells deficient in either perforin or granzyme B did not increase atherosclerosis in ApoE-/-Rag2-/-IL2rg-/- mice. Furthermore perforin and granzyme B deficient NK cells did not increase the necrotic core of atherosclerotic lesions, contrasting with the effects of WT NK cells. Clearance of apoptotic cells is impaired in atherosclerotic lesions24, 38, leading to post apoptotic necrosis and accelerated atherosclerosis23, 39. Necrotic cells that release proinflammatory factors can activate inflammasomes, which together drive cardiovascular disease40-43. Our finding that NK cells do not affect vascular smooth muscle cell numbers in atherosclerotic lesions suggests that NK cells may be targeting macrophages in lesions to augment atherosclerosis. Macrophages in atherosclerotic lesions express Rae-1ε the ligand for the activating NKG2D receptor44, which is expressed by NK cells45. The binding of ligands to NKG2D receptors activates NK cells and promotes cytotoxicity of ligand expressing cells. Our observation that NK cells are present in close contact with macrophages supports the suggestion that NK cells may interact with macrophages. Whilst it is possible that additional mechanisms also contribute to the atherogenicity of NK cells, our studies clearly demonstrate that one mechanism by which NK cells accelerate atherosclerosis development is by inducing cell death via perforin and granzyme B-dependent mechanisms. Recently we have demonstrated that cytotoxic CD8+ T cells also accelerate atherosclerosis by
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protein that exists in cytoplasmic granules of NK cells as multimeric complexes with granzyme B and the
12 16
perforin and granzyme B-dependent mechanisms , raising the possibility that such a mechanism may be common to other cytotoxic immune cells. Human NK cell numbers and their activity were reduced in patients with coronary artery disease. It was not clear whether medications such as statin and β1-receptor blocker could have affected NK cell function46, 47.
In conclusion, our loss and gain of function studies of NK cells in the development of atherosclerosis have definitively shown that NK cells are atherogenic. Their production of the cytotoxins perforin and granzyme
in immune-intact ApoE-/- mice NK cells appear to contribute to a greater extent to atherosclerosis than in lymphocyte-deficient Rag2-/-IL2rg-/-ApoE-/- mice suggests that additional mechanisms involving interactions between NK cells and other lymphocytes, possibly T cells, may also contribute to atherosclerosis. Recruitment of NK cells to lymph nodes can provide a source of IFN-γ for Th1 priming48; such a
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mechanism contributes to early resistance to Leishmania major in mice19. Thus it is possible that in addition to NK cell homing and exerting pathogenic effects via perforin and granzyme B, additional mechanisms involving NK cell-facilitated differentiation of CD4+ T cells to Th1 cells in lymph nodes may also be important. Clearly further studies are required to investigate this possibility. Our studies indicate that cytotoxic actions of NK cells within atherosclerotic lesions contribute significantly to their proatherogenic effects and targeting NK cells may be s useful therapeutic strategy to attenuate atherosclerosis.
Source of Funding This work was supported by a grant from the National Health and Medical Research Council of Australia (to A. B.), a PhD postgraduate scholarship (to A. S.) from BakerIDI Heart and Diabetes Institute and supported in part by the Victorian Government’s Operational Infrastructure Support Program.
Conflicts of Interest There are no conflicts of interest.
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B and expansion of necrotic cores within lesions contribute to atherosclerosis development. Our finding that
13
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Figure Legends Figure 1 Depletion of Natural Killer cells (NK cells) in peripheral blood and spleen of ApoE-/- mice after anti-Asialo-GM1 treatment (A) Representative results of depletion of NK cells identified as NK1.1+, TCR-β- expression in blood, assessed by flow cytometry, 5 days after commencing treatment with antiAsialo-GM1. Plots represents the percentage of NK1.1+ and TCR-β- cells; n=4, *P < 0.05. (B) Representative results of NK1.1+, TCR-β- expression by NK cells in blood, assessed by flow cytometry, 8
NK1.1+ and TCR-β- NK cells; n=4, *P < 0.05. (C) Total (NK1.1+ TCR-β-) NK cells, (NK1.1+ TCRβ+) NKT cells, CD4+ T cells, CD8+ T cells and CD22+ B cells in spleen of ApoE-/- mice treated with normal rabbit serum (NRS) or anti-Asialo-GM1 after 8 weeks of HFD; n=4 each group.
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Figure 2 NK cell depletion inhibits atherosclerotic plaque formation and macrophage accumulation but do not affect lesion smooth muscle cells, VCAM-1 and CD4+ T cells expression. (A) Photomicrographs of aortic sinus atherosclerotic lesions from ApoE-/- mice fed a HFD for 8 weeks and treated with NRS (left) or Anti-asialo-GM1 antibody; (right) showing mean area of ORO-staining (bar graphs) in the two groups of mice. (B) Immunohistochemical staining using anti-CD68 (macrophage) antibody showing mean areas of staining (bar graphs) in two groups. (C) Immunohistochemical staining with anti-α smooth muscle actin antibodies, detecting smooth muscle cells in plaque of ApoE-/- mice treated with NRS (left) or anti-Asialo GM1 antibodies (right) showingmean percentage area of staining (bar graph). (D) anti-CD4 stained atherosclerotic cross sections from NRS and anti-Asialo GM1 treated ApoE-/- mice detecting CD4+ T cells in lesion showing mean numbers of CD4+ T cells in lesions (bar graph). (E) Cross sections stained with anti-VCAM-1 (Vascular cell adhesion molecule-1) detecting VCAM-1 adhesion molecules in plaque of ApoE-/- mice treated with NRS (left) and anti-Asialo-GM1 (right) showing percentage of area stained (bar graph); n=8-9 in NRS and anti-Asialo-GM1 groups. *P
