title: Advanced Oxidation Protein Products-Modified Albumin Induces Differentiation of RAW264.7 Macrophages into Dendritic-Like Cells Which Is Modulated by Cell Surface Thiols.
abstract: 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 antioxidantN-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.
authors.affiliation: Division of Cardiology, IRCCS University Hospital San Martino, Research Centre of Cardiovascular Biology, University of Genova, Genova 16132, Italy. 700086@unige.it.
authors.forename: Silvano
authors.initials: S
authors.lastname: Garibaldi
authors.affiliation: Division of Cardiology, IRCCS University Hospital San Martino, Research Centre of Cardiovascular Biology, University of Genova, Genova 16132, Italy. 700915@unige.it.
authors.forename: Chiara
authors.initials: C
authors.lastname: Barisione
authors.affiliation: Department of Experimental Medicine, University of Genova, Genova 16132, Italy. Barbara.Marengo@unige.it.
authors.forename: Barbara
authors.initials: B
authors.lastname: Marengo
authors.affiliation: Division of Cardiology, IRCCS University Hospital San Martino, Research Centre of Cardiovascular Biology, University of Genova, Genova 16132, Italy. pietroameri@unige.it.
authors.forename: Pietro
authors.initials: P
authors.lastname: Ameri
authors.affiliation: Division of Cardiology, IRCCS University Hospital San Martino, Research Centre of Cardiovascular Biology, University of Genova, Genova 16132, Italy. bc@unige.it.
authors.forename: Claudio
authors.initials: C
authors.lastname: Brunelli
authors.affiliation: Division of Cardiology, IRCCS University Hospital San Martino, Research Centre of Cardiovascular Biology, University of Genova, Genova 16132, Italy. manrico.balbi@unige.it.
authors.forename: Manrico
authors.initials: M
authors.lastname: Balbi
authors.affiliation: Division of Cardiology, IRCCS University Hospital San Martino, Research Centre of Cardiovascular Biology, University of Genova, Genova 16132, Italy. 56182@unige.it.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Ghigliotti
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D062107:Advanced Oxidation Protein Products / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003713:Dendritic Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008264:Macrophages / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism; D012709:Serum Albumin / Q000378:metabolism* / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D062107:Advanced Oxidation Protein Products; D000975:Antioxidants; D017382:Reactive Oxygen Species; D012709:Serum Albumin; D013438:Sulfhydryl Compounds; D000111:Acetylcysteine
keywords: AOPP; NAC; dendritic cells; oxidative stress; thiols