Impairment of the programmed cell death-1 pathway increases atherosclerotic lesion development and inflammation

Abstract

Objective: Programmed cell death-1 (PD-1) is a member of the CD28 superfamily that delivers negative signals on interaction with its 2 ligands, PD-L1 and PD-L2. We studied the contribution of the PD-1 pathway to regulation of T cells that promote atherosclerotic lesion formation and inflammation.

Methods and results: We show that compared with Ldlr-/- control mice, Pd1-/-Ldlr-/- mice developed larger lesions with more abundant CD4+ and CD8+ T cells and macrophages, accompanied by higher levels of serum tumor necrosis factor-α. Iliac lymph node T cells from Pd1-/-Ldlr-/- mice proliferated more to αCD3 or oxidized low-density lipoprotein stimulation compared with controls. CD8+ T cells from Pd1-/-Ldlr-/- mice displayed more cytotoxic activity compared with controls in vivo and in vitro. Administration of a blocking anti-PD-1 antibody increased lesional inflammation in hypercholesterolemic Ldlr-/- mice with more lesional T cells and more activated T cells in paraaortic lymph nodes. The changes in lesional T-cell content when PD-1 was absent or blocked were also observed in bone marrow chimeric Ldlr-/- mice lacking PD-L1 and PD-L2 on hematopoietic cells.

Conclusions: PD-1 has an important role in downregulating proatherogenic T-cell responses, and blockade of this molecule for treatment of viral infections or cancer may increase risk of cardiovascular complications.

Figure 1. Effects of Programmed death–1 (PD-1) deficiency on atherosclerotic lesion development

a. Representative cross sections of the ORO-stained aortic sinuses are shown for Pd1^−/− Ldlr^−/− and Ldlr^−/− mice after 5 and 10 weeks of cholesterol diet (original magnification, ×40). b. Representative en face lesion of aortic arch and descending aortas are shown for Pd1^−/− Ldlr^−/− and Ldlr^−/− mice after 10 weeks of cholesterol diet (original magnification, ×40). c–d Quantitative analysis of lesion area in aortic sinus sections taken after 5 and 10 weeks of high-cholesterol diet (c), and on aortic arch and descending aorta en face lesions (d), after 10 weeks of high-cholesterol diet. Each data point represents the mean value obtained from multiple sections from each mouse (c) or total area for each mouse (d); horizontal bars represent the mean value for each group.

Figure 2. Effects of PD-1 deficiency on atherosclerotic lesion phenotype

a. Representative cross sections of immunohistochemistry for CD4, CD8, macrophage (F4/80) and smooth muscle cell (SMC-α actin) in aortic sinus section from Pd1^−/− Ldlr^−/− and Ldlr^−/− mice after 5 and 10 weeks of cholesterol diet (original magnification ×200). b. Quantitative analysis of immunohistochemical stains for each experimental group. Each data point represents the mean value determined for each mouse; horizontal bars represent the mean value for each group. *P < 0.05 (ANOVA with Tukey’s Multiple Comparison post test).

Figure 3. Cytotoxic effects of PD-1 deficiency on lesional cells in vivo and on mouse endothelial cells and smooth muscle cells in vitro

a. Representative imgaes of double immunofluorescent staining for annexin V (red) and SMC-α actin (green) and mounted with DAPI mounting medium (blue) in aortic sinus section from Pd1^−/− Ldlr^−/− and Ldlr^−/− mice after 10 weeks of cholesterol diet. Arrows indicate triple positive stained cells (yellow and orange) in top panels and annexin V (red) positive but SMC negative cells in bottom panels; scale bars: 20μm. b. Quantitative analysis of double positive staining for annexin V and SMC-α actin (upper panel) and of annexin positive but SMC-α actin negative staining (lower panel) for each experimental group. Each data point represents the mean value determined from three aortic sinus sections from each mouse; horizontal bars represent the mean value for all the mice in each group. *P < 0.05 (ANOVA with Tukey’s Multiple Comparison post test). c: FACS analyses for CTL killing of mouse aortic SMC (left panel) and mouse heart endothelial cells (EC) (right panel). Mouse effector CD8⁺ T cells prepared from Pd1^−/− OT-1 and Pd1^+/+ OT-1 transgenic mouse (see details in Supplemental Materials), were cocultured with mouse aortic SMC or mouse heart EC, respectively for one hour before stained for CD90 and annexin V and 7-AAD. AnnexinV and 7-AAD were characterized as being in late apoptosis, and cells that were positive for 7-AAD but not annexinV were categorized as dead. Data shown are mean ± SD, are one of representative from two different sets of experiments with similar results. Differences between two groups of mice were analyzed by the Mann-Whitney test. * p<0.05 indicates significant difference, compared to counterpart Pd1^+/+ T cell group.

Figure 4. Effects of PD-1 deficiency on T cell gene expression and immune responses of the atherosclerotic mice

a and b qRT-PCR analyses of Ifng, Tnf, Ccr5, Ccr6, and Cxcr3 were performed on unstimulated splenic CD8⁺ (a) and CD4⁺ T cell (b) RNA isolated from Pd1^−/− Ldlr^−/− and Ldlr^−/− mice after 10 weeks of cholesterol diet. n=8 from each group. Data shown are mean±SEM. c. FACS analyses for intracellular IFNγ expression by immunofluorescence staining on splenic CD8⁺ T cells in response to αCD3 for 48h. Splenic CD8⁺ T cells were purified from Pd1^−/− Ldlr^−/− and Ldlr^−/− mice after 5 weeks of cholesterol diet. d and e. Iliac lymphocyte proliferation measured by ³H-thymidine incorporation during the final 16 hours of 72 hr culture with αCD3 (d) or oxLDL (e). f and g. TNFα secretion measured by Luminex cytokine assays (see Materials and Methods) from supernatants of iliac lymphocytes cultured with αCD3 for 48h from Pd1^−/− Ldlr^−/− and Ldlr^−/− mice after 5 weeks of cholesterol diet (f) and from sera of Pd1^−/− Ldlr^−/− and Ldlr^−/− mice after 10 weeks of cholesterol diet (g). c to f, n= 6 from each group. g, n=10 from each group. Data shown are mean ± SEM. Differences between two groups of mice were analyzed by the Mann-Whitney test.

Figure 5. Effects of PD-1 blocking antibody treatment on atherosclerotic lesions in hypercholesterolemic Ldlr^−/− mice

a. Representative cross sections of ORO staining (original magnification ×40) and of immunohistochemistry for CD4, CD8, macrophage (F4/80) and SMC (SMC-α actin) on aortic sinus from Ldlr^−/− mice after 5 weeks of cholesterol diet. Starting from third week of cholesterol diet, mice were treated with either PD-1 antibody or rat IgG, 200μg/mouse, by i.p injections, twice a week, for three weeks (original magnification ×200). b. Quantitative analysis of each staining for each experimental group. Each data point represents the mean value determined for each mouse; horizontal bars represent the mean value for each group. Differences between two groups of mice were analyzed by the Mann-Whitney test. NS indicates not statistically significant.

Figure 6. Effects of PD-1 blocking antibody treatment on immune responses of the hypercholesterolemic Ldlr^−/− mice

a. Comparison of iliac lymph node cell counts from Ldlr^−/− mice treated with PD-1 antibody or rat IgG. b to g FACS analyses for numbers of total CD4⁺ (b), CD8⁺ (e) T cells and a fraction of CD44 (c and f), as well as IFNγ producing CD4⁺ and CD8⁺ T cells (d and g) in the iliac lymph nodes of Ldlr^−/− mice receiving either PD-1 antibody or rat IgG treatment after 5 week cholesterol diet. Horizontal bars represent the mean value for each group. Differences between two groups of mice were analyzed by the Mann-Whitney test.