Role of thiocyanate in the modulation of myeloperoxidase-derived oxidant induced damage to macrophages

Abstract

Myeloperoxidase (MPO) is a vital component of the innate immune system, which produces the potent oxidant hypochlorous acid (HOCl) to kill invading pathogens. However, an overproduction of HOCl during chronic inflammatory conditions causes damage to host cells, which promotes disease, including atherosclerosis. As such, there is increasing interest in the use of thiocyanate (SCN − ) therapeutically to decrease inflammatory disease, as SCN − is the favoured substrate for MPO, and a potent competitive inhibitor of HOCl formation. Use of SCN − by MPO forms hypothiocyanous acid (HOSCN), which can be less damaging to mammalian cells. In this study, we examined the ability of SCN − to modulate damage to macrophages induced by HOCl, which is relevant to lesion formation in atherosclerosis. Addition of SCN − prevented HOCl-mediated cell death, altered the extent and nature of thiol oxidation and the phosphorylation of mitogen activated protein kinases. These changes were dependent on the concentration of SCN − and were observed in some cases, at a sub-stoichiometric ratio of SCN − : HOCl. Co-treatment with SCN − also modulated HOCl-induced perturbations in the expression of various antioxidant and inflammatory genes. In general, the data reflect the conversion of HOCl to HOSCN, which can induce reversible modifications that are repairable by cells. However, our data also highlight the ability of HOSCN to increase pro-inflammatory gene expression and cytokine/chemokine release, which may be relevant to the use of SCN − therapeutically in atherosclerosis. Overall, this study provides further insight into the cellular pathways by which SCN − could exert protective effects on supplementation to decrease the development of chronic inflammatory diseases, such as atherosclerosis.

Modulation of hypochlorous acid (HOCl) induced damage to vascular smooth muscle cells by thiocyanate and selenium analogues

“…These reactions have not been studied previously, and no data are available on the biological reactivity of these products, although decomposition of HO 2 SCN is reported to form SO 4 2− and OCN − [ 18 ]. These data agree well with other studies indicating that an excess of SCN − is required to give “clean”, conversion of HOCl to HOSCN [ 18 ], and studies performed with macrophages exposed to HOCl in the presence of SCN − [ 23 ].…”

“…It has been proposed that supplementation with SCN − could be a potential therapeutic strategy, as HOSCN is both bactericidal, and appears to be less toxic to host cells than bacteria [ 16 ]. This may be due to the selectivity and reversibility of HOSCN-mediated damage, which occurs primarily with thiols [ 19 , 20 ], and a higher capacity for repair in mammalian cells [ [21] , [22] , [23] ]. Thus, supplementation with SCN − has protective effects in vitro [ 16 , 23 , 24 ] and in vivo, in models of cystic fibrosis [ 25 ] and atherosclerosis [ 26 , 27 ].…”

Myeloperoxidase Modulates Hydrogen Peroxide Mediated Cellular Damage in Murine Macrophages

“…Treating the cells with glucose/GO/MPO resulted in a significant increase in the expression of NQO1, which was not seen in experiments with glucose/GO alone, or when SCN − was added. This is attributed to the exposure of the cells to a low, sub-lethal, flux (10 µM) of HOCl and was not observed in experiments with J774A.1 cells treated with higher concentrations (50 µM) of reagent HOCl [ 27 ]. It will be important in future studies to examine alterations in gene expression in experiments with sub-lethal bolus concentrations HOCl (<20 µM).…”

“…Quantification of cellular thiols: Cell lysates were prepared as described for the LDH assay and the intracellular thiol concentration was measured using the ThioGlo1 assay [ 30 ]. The cellular thiol concentration was assessed by the change in fluorescence measured using λ ex 384 nm and λ em 513 nm, as described previously [ 27 ]. Thiol concentrations were quantified using a standard curve constructed with GSH.…”

“…In general, the pathways responsible for the modulation of MPO-induced damage by SCN − , particularly in animal models, are not well characterized. Recent studies in murine J774A.1 macrophages indicated that SCN − could attenuate thiol oxidation and cell death, and alter the nature of pro-inflammatory signaling induced by a bolus addition of HOCl, in a manner consistent with the formation of HOSCN [ 27 ]. This led us to examine whether these pathways are also relevant on the exposure of these macrophages to an enzymatic glucose/glucose oxidase (GO)–MPO coupled system, which provides a continuous flux of H 2 O 2 , and hence HOCl, and may be a better model to simulate the pro-inflammatory environment [ 14 , 20 , 28 ].…”