The role of natural killer cells in atherosclerosis.

Atherosclerosis is a progressive multifactorial disease of large elastic and muscular arteries in which lesions are characterized by the deposition of cholesterol, leukocyte influx, smooth muscle cell proliferation, cell death and collagen accumulation. Inflammatory cells of the innate and adaptive immune systems contribute to lesion development and progression. NK cells have been detected in human atherosclerotic lesions in low numbers and also detected in atherosclerotic lesions of LDLR-deficient mice fed a high fat diet. However their significance for atherosclerotic lesion development is still unclear. The studies presented in this thesis unequivocally demonstrate an important role for NK cells in the development of atherosclerosis and provide novel insights as to how these cells contribute to atherosclerosis.
Chapter 3 examines the role of NK cell in the development of atherosclerosis in ApoE-deficient mice fed a high fat diet. Despite being very minor lymphocyte population in developing atherosclerotic lesions, the study has definitely demonstrated that NK cells are proatherogenic in mice. Specific depletion of NK cells by more than 90% attenuated lesion size and macrophage accumulation. Whilst not specifically examined the reduction in macrophage accumulation could be due to lack of production of NK cell derived cytokines and chemokines in the lesions.
Chapter 4 examines whether the activation of NK cells further augments development of atherosclerosis. Activation of NK cells with Poly IC exacerbates the development of atherosclerosis. Since Poly IC can also activate T cells, the effects of Poly IC were determined in T and B cells deficient ApoE-/- Rag2-/- mice. Poly IC augmented atherosclerosis in these mice which suggest a strong dependency on NK cells. To confirm the dependency of Poly IC effects on atherosclerosis on NK cells, the effects of Poly IC in NK cell depleted mice were also determined. The depletion of NK cells reduced atherosclerotic lesion development. Therefore the study indicates a dependency of Poly IC’s proatherogenic effects on NK cells.
Chapter 5 examines the mechanisms by which NK cells are activated during development of atherosclerosis. NK cells can be activated by via NKT cells and/or via their activating receptor NKG2D. Activation of NKT cells has been shown to stimulate NK cell proliferation and cytotoxicity. This study demonstrates that NKT cells contribute to NK cell activation during development of atherosclerosis since the atherosclerotic lesions were similar in mice deficient in invariant NKT cells in ApoE-/- Jalpha18-/- mice and in NK cell depleted ApoE-/- Jalpha18-/- mice. As to the role of NKG2D receptors in activating NK cells during the development of atherosclerosis, the study indicate that these receptors are not involved despite the high expression of its activating ligands Rae-1δ, Rae-1ε and MULT-1.
Chapter 6 examines the mechanisms by which NK cells promote the development of atherosclerosis. Activated NK cells produce cytokines and cytotoxicity. This study examined whether NK cells participate in atherosclerosis via production of cytokines implicated in atherosclerosis, or through secretion of cytotoxic molecules. IL-2 activated NK cells from mice deficient in cytokines interferon-gamma (IFN-γ) were adoptively transferred to NK-, NKT-, T- and B- cell-deficient ApoE-/- Rag2-/- γ-/- mice. Interferon-gamma was found to be unnecessary for the proatherogenic actions of NK cells, as NK cells deficient in IFN-γ were capable of exacerbating atherosclerosis to the same extent as wild-type IL-2 activated NK cells. Conversely, adoptive transfer of IL-2 activated NK cells deficient in cytotoxic molecules perforin or granzyme B did not increase atherosclerosis in ApoE-/- Rag2-/- γ-/- mice. Therefore the atherosclerosis-promoting effects of NK cells appear to be dependent on their cytotoxic properties. Increased cytotoxicity in atherosclerotic lesions can lead to increased necrosis and inflammation via activation of inflammasomes. Lesions from mice that received perforin- or granzyme B-deficient NKT cells had smaller necrotic areas and lower expression of pro-IL-1β and caspase-1 mRNA.
In summary, the studies indicate that NK cell within the developing lesions augment atherosclerosis by secreting cytotoxic molecules perforin and granzyme B. The magnitude of effects of NK cells on lesion development is dependent not only on NK cell numbers but also on their activation status. Increased understanding of the mechanisms of NK cell actions during atherogenesis may lead to new strategies to develop immunotherapeutic treatments to attenuate atherosclerosis.

动脉粥样硬化是一种累及大型弹性动脉与肌性动脉的进行性多因素疾病，其病变特征为胆固醇沉积、白细胞浸润、平滑肌细胞增殖、细胞死亡及胶原堆积。固有免疫与适应性免疫系统的炎症细胞参与病变的发生与进展。自然杀伤细胞（NK cells）在人类动脉粥样硬化病变中以低数量存在，也在高脂饮食喂养的低密度脂蛋白受体缺陷（LDLR-deficient）小鼠的动脉粥样硬化病变中被检出。然而，其在动脉粥样硬化病变发生中的意义仍不明确。本论文所述研究明确证实NK细胞在动脉粥样硬化发生中具有重要作用，并为这些细胞如何参与动脉粥样硬化提供了新的见解。
第三章探讨了NK细胞在高脂饮食喂养的载脂蛋白E缺陷（ApoE-deficient）小鼠动脉粥样硬化发生中的作用。尽管NK细胞在进展中的动脉粥样硬化病变中仅占淋巴细胞群体的极小部分，但该研究明确表明NK细胞在小鼠中具有促动脉粥样硬化作用。特异性清除90%以上的NK细胞可减轻病变大小及巨噬细胞堆积。虽然未具体研究，但巨噬细胞堆积的减少可能归因于病变中NK细胞来源的细胞因子和趋化因子产生不足。
第四章探讨NK细胞激活是否会进一步加剧动脉粥样硬化的发生。用Poly IC激活NK细胞可促进动脉粥样硬化进展。由于Poly IC也可激活T细胞，研究在T细胞与B细胞缺陷的ApoE-/- Rag2-/-小鼠中测定了Poly IC的作用。Poly IC在这些小鼠中加剧了动脉粥样硬化，提示其作用强烈依赖于NK细胞。为证实Poly IC的促动脉粥样硬化作用依赖于NK细胞，研究还测定了Poly IC在NK细胞清除小鼠中的效应。清除NK细胞可减少动脉粥样硬化病变的发生。因此，该研究表明Poly IC的促动脉粥样硬化作用依赖于NK细胞。
第五章探讨动脉粥样硬化发生过程中NK细胞的激活机制。NK细胞可通过自然杀伤T细胞（NKT cells）和/或其激活受体NKG2D被激活。已有研究表明NKT细胞激活可刺激NK细胞增殖及细胞毒性。本研究证实，NKT细胞参与动脉粥样硬化发生过程中的NK细胞激活——因为在恒定NKT细胞缺陷的ApoE-/- Jalpha18-/-小鼠与NK细胞清除的ApoE-/- Jalpha18-/-小鼠中，动脉粥样硬化病变相似。关于NKG2D受体在动脉粥样硬化发生过程中激活NK细胞的作用，尽管其激活配体Rae-1δ、Rae-1ε和MULT-1高表达，研究表明这些受体并未参与该过程。
第六章探讨NK细胞促进动脉粥样硬化发生的机制。激活的NK细胞可产生细胞因子并具有细胞毒性。本研究探讨NK细胞是否通过产生与动脉粥样硬化相关的细胞因子或分泌细胞毒性分子参与动脉粥样硬化。将来自干扰素-γ（IFN-γ）缺陷小鼠的IL-2激活NK细胞过继转移至NK细胞、NKT细胞、T细胞及B细胞缺陷的ApoE-/- Rag2-/- γ-/-小鼠中。研究发现，IFN-γ并非NK细胞促动脉粥样硬化作用所必需——因为IFN-γ缺陷的NK细胞与野生型IL-2激活的NK细胞可同等程度地加剧动脉粥样硬化。相反，过继转移缺乏穿孔素（perforin）或颗粒酶B（granzyme B）等细胞毒性分子的IL-2激活NK细胞，并未增加ApoE-/- Rag2-/- γ-/-小鼠的动脉粥样硬化程度。因此，NK细胞的促动脉粥样硬化作用似乎依赖于其细胞毒性特性。动脉粥样硬化病变中细胞毒性增加可通过激活炎症小体导致坏死与炎症加剧。接受穿孔素或颗粒酶B缺陷NKT细胞的小鼠病变坏死区域更小，且pro-IL-1β与caspase-1 mRNA表达更低。
综上，研究表明病变进展中的NK细胞通过分泌穿孔素与颗粒酶B等细胞毒性分子加剧动脉粥样硬化。NK细胞对病变发生的影响程度不仅取决于NK细胞数量，还与其激活状态相关。深入理解动脉粥样硬化发生过程中NK细胞的作用机制，可能为开发免疫治疗策略以减轻动脉粥样硬化提供新方向。