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. 2011 Dec 20;124(25):2933-43.
doi: 10.1161/CIRCULATIONAHA.111.034850. Epub 2011 Nov 21.

Immune activation resulting from NKG2D/ligand interaction promotes atherosclerosis

Mingcan Xia  1 , Nadia Guerra, Galina K Sukhova, Kangkang Yang, Carla K Miller, Guo-Ping Shi, David H Raulet, Na Xiong
Affiliations

Immune activation resulting from NKG2D/ligand interaction promotes atherosclerosis

Mingcan Xia et al. Circulation. .

Abstract

Background: The interplay between the immune system and abnormal metabolic conditions sustains and propagates a vicious feedback cycle of chronic inflammation and metabolic dysfunction that is critical for atherosclerotic progression. It is well established that abnormal metabolic conditions, such as dyslipidemia and hyperglycemia, cause various cellular stress responses that induce tissue inflammation and immune cell activation, which in turn exacerbate the metabolic dysfunction. However, molecular events linking these processes are not well understood.

Methods and results: Tissues and organs of humans and mice with hyperglycemia and hyperlipidemia were examined for expression of ligands for NKG2D, a potent immune-activating receptor expressed by several types of immune cells, and the role of NKG2D in atherosclerosis and metabolic diseases was probed with the use of mice lacking NKG2D or by blocking NKG2D with monoclonal antibodies. NKG2D ligands were upregulated in multiple organs, particularly atherosclerotic aortas and inflamed livers. Ligand upregulation was induced in vitro by abnormal metabolites associated with metabolic dysfunctions. Using apolipoprotein E-deficient mouse models, we demonstrated that preventing NKG2D functions resulted in a dramatic reduction in plaque formation, suppressed systemic and organ inflammation mediated by multiple immune cell types, and alleviated abnormal metabolic conditions.

Conclusions: The NKG2D/ligand interaction is a critical molecular link in the vicious cycle of chronic inflammation and metabolic dysfunction that promotes atherosclerosis and might be a useful target for therapeutic intervention in the disease.

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Conflict of interest statement

Disclosures DHR received grant support from Novo Nordisk. NX, MX and DHR received payments from Novo Nordisk through the Pennsylvania State University and University of California, Berkeley for the licensing of an invention based on findings described in this paper. NG, KY, G-PS, GKS and CKM declare no financial interests.

Figures

Figure 1
Figure 1
Upregulation of NKG2D ligands in sera and aortic plaques of human type 2 diabetes patients. (A) A high percentage of type 2 diabetes patients have elevated levels of sMICA in the sera. The dashed line indicates the detection limit. One dot represents one sample and the short flat lines indicate average concentrations. Fisher’s exact test was used to compare proportions of healthy vs. diabetic people with detectable MICA expression. *p<0.05; **p<0.01; ***p<0.001 (applied for all figures). (B-J) Detection of MICA/B proteins by IHC staining on human aortic plaques. Cryosections of the plaques were stained with anti-MICA/B antibodies (B, D, F, H) while adjacent sections were stained with anti-Mac-3 or CD31 antibodies to reveal macrophages or endothelial cells (C, E, G, I). A section of normal aorta was used as a control (panel J).
Figure 2
Figure 2
Preferential upregulation of NKG2D ligands in atherosclerotic aortae and livers of ApoE-/- mice. (A) Real-time RT-PCR analysis of Rae-1δ, Rae-ɛ and H60b transcripts in RNA samples isolated from aortic arch regions of WD-fed ApoE-/- mice. Samples of age-matched WD- and chow-fed WT mice were used as controls. The experiment included 3 mice/group and was representative of two repeated experiments of total 6 mice. (B) IHC staining of sections of atherosclerotic aortic arch regions (top and middle) and “plaque-free” thoracic regions (bottom) of the same WD-fed ApoE-/- mice with Rae-1 (left) or H60 antibodies (right). The pictures in the middle are higher amplifications (400X) of the boxed areas of the top panels (100X). (C) Flow cytometry of cell surface Rae-1 expression on macrophages (gated on the CD45+CD11b+F4/80+ population) and endothelial cells (CD45-CD31+ population) isolated from atherosclerotic aortae of the WD-fed ApoE-/- mice or normal aortae of WT mice. The gray areas indicated isotype-matched control antibody staining. (D) Real-time RT-PCR analysis of Rae-δ transcripts in indicated organs of WD-fed ApoE-/- mice and WT mice. *** indicates statistical significance compared to values of corresponding tissues of WT mice. N=4. (E) Real-time RT-PCR analysis of Rae-δ in purified macrophages of livers of WD-fed ApoE-/- mice and WT mice. N=2. (F and G) Flow cytometry of Rae-1 expression on CD11b+F4/80+ liver macrophages (F) and hepatocytes (G) of WD-fed ApoE-/- mice. The experiment included 3-4 mice/group and was performed twice with similar results. (H) Flow cytometry of Rae-1 expression on peritoneal macrophages of WT mice cultured in vitro in presence of oxLDL and AGE as in the panel C except that the dashed lines depict staining in the presence of a 10-fold excess of unlabeled anti-pan-Rae-1 antibodies. The experiment was performed 3 times, with pools of cells from 3 mice for each, with similar results. (I) Western blot analysis of Rae-1 proteins in macrophages cultured in vitro in presence of oxLDL or nLDL. The LPS treatment was included as a positive control. The blot was performed 3 times with similar results.
Figure 3
Figure 3
NKG2D plays a critical role in plaque formation in WD-fed and STZ-diabetic ApoE-/- mice. (A) Visibly different plaque sizes in aortic arch regions of representative WD-fed ApoE-/- and Klrk1-/-ApoE-/- mice. The whitish plaque areas are indicated by arrows. The pictures shown are representative of 16-18 mice of each genotype. (B) En face Oil red O staining to detect lipid-deposited plaque areas in aortae of representative ApoE-/- (n=12) and Klrk1-/-ApoE-/- (n=10) mice. Plaque areas stain red. The regions analyzed for percentage of plaque are boxed. (C and D) Quantitative comparison of percentages of average Oil red O staining positive plaque areas in the total aortic arch regions of Klrk1-/-ApoE-/- and ApoE-/- mice that were fed on a WD (C) or treated with STZ (D). (E) Quantitative comparison of percentages of average Oil red O staining positive plaque areas in STZ-induced diabetic ApoE-/- mice treated with NKG2D or control antibodies. Total numbers of mice analyzed in panels C, D and E are indicated in the graphs, which are compiled from data of 2-4 experiments for each model.
Figure 4
Figure 4
Preventing NKG2D/ligand interactions reduces serum levels of inflammation-associated cytokines in WD-fed and STZ-diabetic ApoE-/- mice. (A) Serum levels of multiple cytokines in WD-fed ApoE-/- and Klrk1-/-ApoE-/- mice. N=12 each. (B) Serum levels of multiple cytokines in STZ-diabetic ApoE-/- and Klrk1-/-ApoE-/- mice. N=4 each. (C) Serum levels of multiple cytokines of NKG2D antibody (n=9) and control antibody (n=6) treated STZ-diabetic ApoE-/- mice. Note that scales for different cytokines in the different models are not the same.
Figure 5
Figure 5
Preventing NKG2D/ligand interactions alleviates abnormal metabolic conditions by reducing liver inflammation in the WD-fed ApoE-/- model of atherosclerosis. (A) Reduced serum ALT activity in WD-fed Klrk1-/-ApoE-/- mice. The experiment included 5 mice/group and was representative of 2 repeated experiments of total 10 mice. (B) Reduced IL-6 production by ex vivo cultured liver explants of Klrk1-/-ApoE-/- mice. Experiment includes 3 mice/assay and was representative of 2 repeated experiments of total 6 mice. (C) Reduced numbers of immune cells in livers of Klrk1-/-ApoE-/- mice. The numbers are of each type of immune cells per liver, calculated based on total mononucleocytes isolated from livers and percentages of each type. The experiment included 3 mice/assay and was representative of 3 repeated experiments of total 9 mice.
Figure 6
Figure 6
Multiple immune cell types are involved in the liver inflammation mediated by NKG2D interactions. (A) Comparison of the production of IFN–γ by liver NKT cells of WD-fed Klrk1-/-ApoE-/- and ApoE-/- mice. Cells are gated on TCRβ+NK1.1+ cells. (B) Comparison of IFN–γ production by liver NK cells of WD-fed Klrk1-/-ApoE-/- and ApoE-/- mice. Cells are gated on CD3-NK1.1+ cells. (C) On a per cell basis, NKG2D deficiency did not affect IL-6 production by liver CD11b+F4/80+ macrophages of WD-fed ApoE-/- mice. The experiment in panels A-C included 3 mice/group and is representative of 3 repeated experiments of total 9 mice. The number below the gate in each graph is the mean percentage ± SE of the positive cells of total cells in the plot. The isotype-control staining was similar in WT, ApoE-/- and Klrk1-/-ApoE-/- mice. *** and * indicate statistical significance compared to the values from ApoE-/- mice.
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