CD8 + T-cell deficiency protects mice from abdominal aortic aneurysm formation in response to calcium chloride 2

Abstract

Objective: Abdominal aortic aneurysm (AAA) is an aneurysm-like dilated and highly fatal cardiovascular disease. CD8 + T cells have been shown to be critical for vascular pathological processes, but the contribution of these lymphocytes to vascular diseases remains elusive.

Methods and results: Eight-week-old male wildtype (CD8 +/+ ) and Cd8a knockout (CD8 -/- ) mice were used in a calcium chloride 2 (CaCl 2 )-induced experimental AAA model. At 6 weeks after surgery, CD8 + T-cell deletion prevented the formation of AAA, accompanied by reductions of the levels of inflammatory (interferon-γ [IFN-γ], interleukin-1β, monocyte chemoattractant protein-1, intracellular adhesion molecule-1, vascular cell adhesion molecule-1, NOD-like receptor protein 3, caspase-1), oxidative stress [NADPH oxidase and gp91 phox ], and proteolysis (cathepsin S, cathepsin K, matrix metalloproteinase-2 [MMP-2] and MMP-9) proteins and/or genes in plasma and/or AAA tissues. Immunoreactivities of MMP-2 and MMP-9 were observed in macrophages. An injection of IFN-γ and adoptive transfer of CD8 + T cells of IFN-γ +/+ mice diminished CD8 -/- -mediated vasculoprotective actions in the AAA mice. In vitro, IFN-γ enhanced MMP-2 and MMP-9 gelatinolytic activities in macrophage and/or vascular smooth muscle cells.

Conclusion: The vasculoprotective effects of CD8 + T-cell deletion in a mouse CaCl 2 -induced AAA model were likely attributable to, at least in part, the attenuation of IFN-γ-dependent inflammation action, oxidative stress production, and proteolysis, suggesting a novel therapeutic target for AAA formation by regulating CD8 + T-cell-derived IFN-γ secretion.

Graphical abstract

FIGURE 1

CD8⁺ T-cell deletion mitigated the CaCl₂-induced AAA formation and the investigated protein levels in aortic tissues. A,B: Representative photographs and data of the maximal external aortic diameter in CD8^+/+ and CD8^−/− mice treated with NaCl or CaCl₂, respectively (n = 8/group). C–F: Representative western blotting images and quantitative data for the levels of NLRP3, caspase-1, gp91^phox, collagen I, and collagen III proteins in four experimental groups (n = 4/group). G,H: The ELISA results showing the serum levels of IFN-γ and IL-1β in the four groups (n = 6/group).

FIGURE 2

CD8⁺ T-cell deficiency alleviated the histopathological changes and recruitment of inflammatory cells. A: The histopathological analysis (H&E, EVG, and Masson trichrome staining) of abdominal aortas of CD8^+/+ and CD8^−/− mice treated with NaCl or CaCl₂, respectively. Scale bar: upper layer 100 μm, lower layer 50 μm. B: Quantification of the collagen deposition area of the abdominal aortas (n = 5/group). C: Quantification of the elastin degradation score of the abdominal aortas (n = 5/group). D-F: Representative images of IFN-γ⁺/CD8a⁺ T cell and MMP-2⁺/ and MMP-9⁺/CD68⁺ macrophage in the adventitia of CD8^+/+ and CD8^−/− AAA mice (n = 5/group). Scale bar: 50 μm. L, Lumen.

FIGURE 3

IFN-γ loading accelerated the CaCl₂-induced AAA formation in the CD8^−/− mice. A,B: Representative photographs and quantitative data of the maximal external aortic diameter in the PBS-injected (Vehicle) or IFN-γ-injected CD8^−/− mice after CaCl₂ aneurysm induction (n = 8/group). C,D: Representative immunoblotting images and quantitative data for the levels of NLRP3, caspase-1, gp91^phox, collagen I, and collagen III proteins in the two groups (n = 3/group). E: The ELISA results showing the serum levels of IL-1β in both groups (n = 6/group).

FIGURE 4

IFN-γ accelerated the histopathological changes and the recruitment of inflammatory cells in the CaCl₂-induced AAAs in the CD8^−/− mice. A: The histopathological analysis (H&E, EVG, and Masson trichrome staining) of abdominal aortas of PBS-injected (Vehicle) and IFN-γ-injected CD8^−/− mice after CaCl₂ aneurysm induction. Scale bar: upper layer 100 μm, lower layer 50 μm. B: Quantification of the collagen deposition area of the abdominal aortas (n = 5/group). C: Quantification of the elastin degradation score of the abdominal aortas (n = 6/group). D-F: Representative images of IFN-γ⁺/CD8a⁺ T cell and MMP-2⁺/ and MMP-9⁺/CD68⁺ macrophage in the adventitia of CD8^−/− AAA mice treated with PBS and IFN-γ (n = 5/group). Scale bar: 50 μm. L, lumen.

FIGURE 5

IFN-γ enhanced the inflammasome-related protein expressions and gelatinase activities in the macrophages and smooth muscle cells (SMCs). The macrophages and SMCs were treated with 0, 1, or 10 ng/mL of IFN-γ for 24 hr, and the lysates were then subjected to western blotting; the conditioned media were collected for the gelatin zymography assays. A,B: Representative western blotting images and quantitative data for NLRP3, caspase-1, gp91^phox, collagen I, and collagen III (n = 4/group). C,D: IFN-γ increased IL-1β levels and nitric oxide (NO) release (n = 5–6/group). E-H: Representative images and the quantitative data of the gelatin zymography assays of MMP-2 and/or MMP-9 in the condition medium of macrophages and SMCs (n = 3/group).