Abstract
Introduction. Monocytes/macrophages play an essential role in systemic iron homeostasis by their recycling and storage capacity of iron. Under chronic inflammatory condition, pro-inflammatory cytokines such as TNF-α and IL-1 stimulate production of hepcidin by liver cells, which in turn down-regulates iron exporter ferroportin expression on monocytes/macrophages. Thus, hepcidin/ferroportin axis contributes to the pathogenesis of anemia in chronic inflammation by restricting cellular iron export into plasma. Pro-inflammatory cytokine IL-1β is mainly produced by monocytes/macrophages througha molecular platform, called inflammasome. One of the best characterized members is NLRP3 inflammasome, which plays a central role in sterile inflammation in response to wide range of danger stimuli, including uric crystal, cholesterol crystal, silica and asbestos. Given that excess cellular iron increases the level of redox-active labile iron pool which is responsible for iron-mediated cytotoxicity, cellular labile iron might be sensed as a danger signal in monocytes/macrophages. In this study we addressed whether cellular labile iron activates NLRP3 inflammasome.
Methods. Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation from healthy donors. After 4 h priming with 10 ng/ml LPS, PBMCs were stimulated with various dose of ferric ammonium citrate (FAC) for 4 h. IL-1β production in culture supernatants was determined by ELISA. Cleaved caspase-1 and mature IL-1β was analyzed by western blot. Calcein-AM assay was used to determine cellular labile iron pool.To investigate the mechanism of NLRP3 inflammasome activation, total reactive oxygen species (ROS) detection assay and JC-10 mitochondrial membrane potential assay were performed. To evaluate lysosomal membrane permeabilization, PMA-differentiated THP-1 cells stained with Lyso Tracker were analyzed by fluorescence microscope.
Results. FAC induced the concentration-dependent increase of the labile iron pool and secretion of IL-1β in LPS-primed human monocytes. Ferrous iron chelator, bipyridine significantly inhibited the IL-1β production, highlighting importance of cellular chelatable iron pool for IL-1β production. IL-1β production induced by FAC was abrogated by pan-caspase inhibitor as well as by caspase-1 specific inhibitor, suggesting that inflammasome-mediated caspase-1 activation is required for this process. Consistently, cleaved caspase-1 and mature IL-1β was confirmed by western blot analysis. We next addressed whether NLRP3 inflammasome is activated in response to cellular labile iron. NLRP3 inhibitor, glyburide significantly inhibited IL-1β production. Furthermore, FAC treatment induced IL-1β production in THP-1 cells, but not in NLRP3-deficient THP-1 cells, indicating that cellular iron activates NLRP3 inflammasome. Next, we addressed how cellular iron activates NLRP3 inflammasome. Potassium efflux is thought to be a common trigger of NLRP3 inflammasome activation. Consistently, IL-1β production was completely abrogated in high-potassium media, indicating that potassium efflux is required for iron-mediated NLRP3 inflammasome activation. Since cellular labile iron is involved in ROS generation through Fenton reactions, we next investigated the role of ROS in iron-mediated NLRP3 inflammasome activation. FAC treatment increased ROS levels of monocytes in concentration-dependent manner. Accordingly, the percentage of monocytes with decreased mitochondorial membrane potential was increased. N-acetyl cysteine inhibited FAC-induced IL-1β production, suggesting that ROS-mediated mitochondorial damage is involved in iron-mediated NLRP3 inflammasome activation. In addition, ROS-induced lysosomal membrane permeabilization was observed in FAC-treated THP-1 cells. A cathepsin B inhibitor, CA-074 methyl ester, inhibited IL-1β production, suggesting that ROS-mediated lysosomal damage is also involved in iron-mediated NLRP3 inflammasome activation.
Conclusion. In this study, we found that cellular labile iron activates NLRP3 inflammasome through its redox activity. Because IL-1β has been reported to induce hepcidin synthesis, excess cellular iron in monocytes/macrophages might be implicated in a positive feedback loop for inflammation through NLRP3 inflammasome and hepcidin/ferroportin axis.
Disclosures
No relevant conflicts of interest to declare.
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