Background and Aims: The role of mitophagy in atherosclerosis has been extensively studied during the last few years. It was shown that mitophagy is involved in the regulation of macrophages, which are important players as immune cells in atherosclerosis development. In this study, we investigated the relationship between mitophagy and response to inflammatory stimulation of macrophage-like cells. Six cybrid cell lines with normal mitophagy, that is, increasing in response to stimulation, and 7 lines with defective mitophagy not responding to stimulation were obtained. The objective of the study was to compare the nature of the inflammatory response in normal and defective mitophagy in order to elucidate the role of mitophagy defects in inflammation. Methods: We used cytoplasmic hybrids (cybrids) as cellular models, created using mitochondrial DNA from different atherosclerosis patients. Mitophagy was stimulated by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and assessed as the degree of colocalization of mitochondria with lysosomes using confocal microscopy. Western blotting methods were used for the determination of proteins involved in the exact mechanism of mitophagy. Experiments with stimulation of mitophagy show a high correlation between these two approaches (microscopy and blotting). The pro-inflammatory response of cybrids was stimulated with bacterial lipopolysaccharide (LPS). The extent of the inflammatory response was assessed by the secretion of cytokines CCL2, IL8, IL6, IL1β, and TNF measured by ELISA. Results: Basal level of secretion of cytokines CCL2, IL8 and TNF was 1.5-2 times higher in cultures of cybrids with defective mitophagy compared to cells with normal mitophagy. This suggests a persistently elevated inflammatory response in cells with defective mitophagy, even in the absence of an inflammatory stimulus. Such cells in the tissue will constantly recruit other immune cells, which is characteristic of macrophages derived from monocytes circulating in the blood of patients with atherosclerosis. We observed significant differences in the degree and type of response to inflammatory activation in cybrids with defective mitophagy. These differences were not so much quantitative as they were dramatically qualitative. Compared with cells with normal mitophagy, in cells with defective mitophagy, the relative (to basal) secretion of IL8, IL6 and IL1b increased after the second LPS activation. This indicates a possible lack of tolerance to inflammatory activation in cells with defective mitophagy, since typically, re-activation reveals a smaller pro-inflammatory cytokine response, allowing the inflammatory process to resolve. In cells with normal mitophagy, exactly this normal (tolerant) inflammatory reaction was observed. Conclusion: Data on the involvement of mitophagy, including defective mitophagy, in disturbances of the inflammatory response in sepsis, viral infections, autoimmune diseases and other pathologies have previously been reported. In this work, we studied the role of defective mitophagy in non-infectious chronic inflammatory diseases using the example of atherosclerosis. We showed a dramatic disruption of the inflammatory response associated with defective mitophagy. Compared with cybrids with normal mitophagy, in cybrids with defective mitophagy, the secretion of all studied cytokines changed significantly both quantitatively and qualitatively. In particular, the secretion of 3 of 5 cytokines demonstrated an intolerant inflammatory response manifested by increased secretion after repeated inflammatory stimulation. Such an intolerant reaction likely indicates a significant disruption of the pro-inflammatory response of macrophages, which can contribute to the chronification of inflammation. Elucidating the mechanisms of chronification of inflammation is extremely important for the search for fundamentally new pharmacological targets and the development of drugs for the prevention and treatment of chronic inflammatory diseases, including atherosclerosis and diseases characteristic of inflammation. Such diseases account for up to 80% of morbidity and mortality.

中文翻译：

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有缺陷的线粒体自噬损害巨噬细胞样细胞炎症激活的反应

背景和目的：过去几年，线粒体自噬在动脉粥样硬化中的作用已得到广泛研究。研究表明，线粒体自噬参与巨噬细胞的调节，巨噬细胞是动脉粥样硬化发展中免疫细胞的重要参与者。在这项研究中，我们研究了线粒体自噬与巨噬细胞样细胞炎症刺激反应之间的关系。获得了6个具有正常线粒体自噬(即对刺激作出反应而增加)的细胞杂种细胞系，以及7个具有缺陷的线粒体自噬且对刺激没有反应的细胞系。该研究的目的是比较正常和有缺陷的线粒体自噬中炎症反应的性质，以阐明线粒体自噬缺陷在炎症中的作用。方法：我们使用细胞质杂交体 (cybrids) 作为细胞模型，使用来自不同动脉粥样硬化患者的线粒体 DNA 创建。通过羰基氰化物间氯苯腙 (CCCP) 刺激线粒体自噬，并使用共聚焦显微镜评估线粒体与溶酶体的共定位程度。蛋白质印迹方法用于测定参与线粒体自噬确切机制的蛋白质。刺激线粒体自噬的实验表明这两种方法（显微镜检查和印迹）之间存在高度相关性。细菌脂多糖（LPS）刺激细胞杂种的促炎反应。通过 ELISA 测量的细胞因子 CCL2、IL8、IL6、IL1β 和 TNF 的分泌来评估炎症反应的程度。结果：与具有正常线粒体自噬的细胞相比，具有缺陷线粒体自噬的细胞培养物中细胞因子CCL2、IL8和TNF的基础分泌水平高1.5-2倍。这表明即使没有炎症刺激，线粒体自噬缺陷的细胞中炎症反应也会持续升高。组织中的此类细胞会不断招募其他免疫细胞，这是动脉粥样硬化患者血液中循环的单核细胞衍生的巨噬细胞的特征。我们观察到线粒体自噬缺陷的细胞体对炎症激活的反应程度和类型存在显着差异。这些差异与其说是定量的，不如说是定性的。与具有正常线粒体自噬的细胞相比，在具有缺陷线粒体自噬的细胞中，第二次LPS激活后IL8、IL6和IL1b的相对（相对于基础）分泌增加。这表明线粒体自噬缺陷的细胞可能缺乏对炎症激活的耐受性，因为通常情况下，重新激活会显示出较小的促炎细胞因子反应，从而使炎症过程得到缓解。在具有正常线粒体自噬的细胞中，观察到了这种正常（耐受）的炎症反应。结论：关于线粒体自噬（包括有缺陷的线粒体自噬）参与败血症、病毒感染、自身免疫性疾病和其他病理学先前已有报道。在这项工作中，我们以动脉粥样硬化为例，研究了有缺陷的线粒体自噬在非传染性慢性炎症性疾病中的作用。我们发现与线粒体自噬缺陷相关的炎症反应受到显着破坏。与线粒体自噬正常的细胞杂种相比，线粒体自噬缺陷的细胞杂种中，所有研究的细胞因子的分泌在数量和质量上都发生了显着变化。特别是，5种细胞因子中的3种的分泌表现出不耐受的炎症反应，表现为反复炎症刺激后分泌增加。这种不耐受反应可能表明巨噬细胞的促炎反应受到显着破坏，这可能导致炎症慢性化。阐明炎症慢性化的机制对于寻找全新的药理学靶点以及开发预防和治疗慢性炎症性疾病（包括动脉粥样硬化和炎症特征性疾病）的药物极为重要。此类疾病占发病率和死亡率的 80%。