Garibaldi Silvano, Barisione Chiara, Marengo Barbara, Ameri Pietro, Brunelli Claudio, Balbi Manrico, Ghigliotti Giorgio
Division of Cardiology, IRCCS University Hospital San Martino, Research Centre of Cardiovascular Biology, University of Genova, Genova 16132, Italy.
Department of Experimental Medicine, University of Genova, Genova 16132, Italy.
Toxins (Basel). 2017 Jan 10;9(1):27. doi: 10.3390/toxins9010027.
Local accumulation of Advanced Oxidation Protein Products (AOPP) induces pro-inflammatory and pro-fibrotic processes in kidneys and is an independent predictor of renal fibrosis and of rapid decline of eGFR in patients with chronic kidney disease (CKD). In addition to kidney damage, circulating AOPP may be regarded as mediators of systemic oxidative stress and, in this capacity, they might play a role in the progression of atherosclerotic damage of arterial walls. Atherosclerosis is a chronic inflammatory disease that involves activation of innate and adaptive immunity. Dendritic cells (DCs) are key cells in this process, due to their role in antigen presentation, inflammation resolution and T cell activation. AOPP consist in oxidative modifications of proteins (such as albumin and fibrinogen) that mainly occur through myeloperoxidase (MPO)-derived hypochlorite (HOCl). HOCl modified proteins have been found in atherosclerotic lesions. The oxidizing environment and the shifts in cellular redox equilibrium trigger inflammation, activate immune cells and induce immune responses. Thus, surface thiol groups contribute to the regulation of immune functions. The aims of this work are: (1) to evaluate whether AOPP-proteins induce activation and differentiation of mature macrophages into dendritic cells in vitro; and (2) to define the role of cell surface thiol groups and of free radicals in this process. AOPP-proteins were prepared by in vitro incubation of human serum albumin (HSA) with HOCl. Mouse macrophage-like RAW264.7 were treated with various concentrations of AOPP-HSA with or without the antioxidant -acetyl cysteine (NAC). Following 48 h of HSA-AOPP treatment, RAW264.7 morphological changes were evaluated by microscopic observation, while markers of dendritic lineage and activation (CD40, CD86, and MHC class II) and allogeneic T cell proliferation were evaluated by flow cytometry. Cell surface thiols were measured by AlexaFluor-maleimide binding, and ROS production was assessed as DCF fluorescence by flow cytometry. HSA-AOPP induced the differentiation of RAW264.7 cells into a dendritic-like phenotype, as shown by morphological changes, by increased CD40, CD86 and MHC class II surface expression and by induction of T cell proliferation. The cell surface thiols dose dependently decreased following HSA-AOPP treatment, while ROS production increased. NAC pre-treatment enhanced the amount of cell surface thiols and prevented their reduction due to treatment with AOPP. Both ROS production and RAW264.7 differentiation into DC-like cells induced by HSA-AOPP were reduced by NAC. Our results highlight that oxidized plasma proteins modulate specific immune responses of macrophages through a process involving changes in the thiol redox equilibrium. We suggest that this mechanism may play a role in determining the rapid progression of the atherosclerotic process observed in CKD patients.
晚期氧化蛋白产物（AOPP）在局部肾脏的蓄积可诱导促炎和促纤维化过程，并且是慢性肾脏病（CKD）患者肾纤维化及估算肾小球滤过率（eGFR）快速下降的独立预测指标。除了造成肾脏损害外，循环中的AOPP可被视为全身氧化应激的介质，并且在此方面，它们可能在动脉壁动脉粥样硬化损害的进展中发挥作用。动脉粥样硬化是一种慢性炎症性疾病，涉及固有免疫和适应性免疫的激活。树突状细胞（DC）是此过程中的关键细胞，因为它们在抗原呈递、炎症消退及T细胞激活中发挥作用。AOPP是蛋白质（如白蛋白和纤维蛋白原）的氧化修饰产物，主要通过髓过氧化物酶（MPO）衍生的次氯酸盐（HOCl）形成。在动脉粥样硬化病变中已发现HOCl修饰的蛋白质。氧化环境及细胞氧化还原平衡的改变会引发炎症、激活免疫细胞并诱导免疫反应。因此，细胞表面硫醇基团有助于免疫功能的调节。本研究的目的是：（1）评估AOPP蛋白在体外是否能诱导成熟巨噬细胞激活并分化为树突状细胞；（2）确定细胞表面硫醇基团及自由基在此过程中的作用。通过将人血清白蛋白（HSA）与HOCl进行体外孵育制备AOPP蛋白。用不同浓度的AOPP-HSA处理小鼠巨噬细胞样RAW264.7细胞，同时加入或不加入抗氧化剂N-乙酰半胱氨酸（NAC）。经HSA-AOPP处理48小时后，通过显微镜观察评估RAW264.7细胞的形态变化，同时通过流式细胞术评估树突状细胞系及激活的标志物（CD40、CD86和MHC II类分子）以及异体T细胞增殖情况。通过AlexaFluor-马来酰亚胺结合法测定细胞表面硫醇，通过流式细胞术将DCF荧光作为活性氧（ROS）产生情况进行评估。HSA-AOPP诱导RAW264.7细胞分化为树突状样表型，表现为形态变化、CD40、CD86和MHC II类分子表面表达增加以及T细胞增殖诱导。HSA-AOPP处理后，细胞表面硫醇剂量依赖性降低，而ROS产生增加。NAC预处理增加了细胞表面硫醇的量，并防止了因AOPP处理导致的硫醇减少。NAC降低了HSA-AOPP诱导的ROS产生及RAW264.7细胞向DC样细胞的分化。我们的结果表明，氧化的血浆蛋白通过涉及硫醇氧化还原平衡变化的过程调节巨噬细胞的特异性免疫反应。我们认为，这种机制可能在决定CKD患者中观察到的动脉粥样硬化过程的快速进展中发挥作用。
