Estrogen signaling differentially alters iron metabolism in monocytes in an Interleukin 6-dependent manner

Highlights

• Estrogen signaling is involved in regulating iron metabolism in monocytes.

• Estrogen treatment modulates the expression of key iron regulatory proteins including hepcidin and ferroportin.

• Estrogen-dependent changes in cellular iron status in monocytes are dependent on the status of IL-6 expression.

Abstract

The ability of monocytes to release or sequester iron affects their role in cancer and inflammation. Previous work has shown that while IL-6 upregulates hepcidin synthesis and enhances iron sequestration, E2 reduces hepcidin synthesis and increases iron release. Given that E2 upregulates IL-6 production in monocytes, it is likely that the exact effect of E2 on iron metabolism in monocytes is shaped by its effect on IL-6 expression. To address this issue, the expression of key iron regulatory proteins was assessed in E2-treated U937, HuT-78, THP-1 and Hep-G2 cells. Iron status was also evaluated in U937 cells treated with the ERα agonist PPT, the ER antagonist ICI-182780, dexamethasone + E2, IL-6 + E2 and in IL-6-silenced U937 cells. E2 treatment reduced hepcidin synthesis in HuT-78, THP-1 and Hep-G2 cells but increased hepcidin synthesis and reduced FPN expression in U937 cells. E2-treated U937 cells also showed reduced HIF-1α and FTH expression and increased TFR1 expression, which associated with increased labile iron content as compared with similarly treated Hep-G2 cells. While treatment of U937 cells with interleukin 6 (IL-6) resulted in increased expression of hepcidin, dexamethasone treatment resulted in reduced hepcidin synthesis relative to E2- or dexamethasone + E2-treated cells; IL-6 silencing also resulted in reduced hepcidin synthesis in U937 cells. Lastly, while iron depletion resulted in increased cell death in U937 cells, E2 treatment resulted in enhanced cell survival and reduced apoptosis. These findings suggest that E2 differentially alters iron metabolism in monocytes in an IL-6 dependent manner.

Introduction

Iron is essential for cell survival, growth and metabolism; however, excess iron leads to oxidative stress and tissue damage. Therefore, intricate mechanisms have evolved to regulate iron metabolism in mammals (Anderson and Wang, 2012). Intracellular iron is exported through ferroportin (FPN) on iron-absorbing enterocytes and iron storing macrophages (Nemeth et al., 2004; Vashchenko and MacGillivray, 2013). Once oxidized by ceruloplasmin and/or hephaestin (Vashchenko and MacGillivray, 2013), ferric iron binds circulating transferrin to form di-ferric-transferrin, which then binds transferrin receptor (TFR1; CD71) on body cells. Iron reduced in endosomes is released into the cytoplasmic labile iron pool through endosomal divalent metal-ion transporter (DMT1) (Yanatori et al., 2016). Expression of FPN on iron releasing cells is negatively regulated by the peptide hormone hepcidin, which triggers FPN’s internalization and degradation (Nemeth et al., 2004). Therefore, increased availability of systemic iron triggers the hemochromatosis gene (HFE) protein to switch from TFR1 to TFR2, which initiates a signaling cascade that upregulates hepcidin synthesis and blocks intracellular iron release (Peyssonnaux et al., 2007; Schmidt et al., 2008). Inflammation (IL-6, tumor necrosis factor (TNFα), and IL-1) also increases hepcidin synthesis, decreases FPN expression and enhances iron sequestration in macrophages and hepatocytes (Lee et al., 2005; Pagani et al., 2011). However, increased demand for iron triggers the hypoxia inducible factor (HIF-1α) (Peyssonnaux et al., 2007) and the growth differentiation factor (GDF15) (Pagani et al., 2011) to block hepcidin synthesis in order to enhance cellular iron release. Several studies have suggested that the sex hormone estrogen (17-β estradiol; E2) can also reduce hepcidin expression and increase FPN expression (Bajbouj et al., 2018; Huang et al., 2013; Milman et al., 1992; Lehtihet et al., 2016; Raso et al., 2009). Binding of E2-ER complexes to estrogen-responsive elements (EREs) in the hepcidin gene was shown to reduce hepcidin synthesis in E2-treated human liver HuH7 and Hep-G2 cells (Yang et al., 2012; Hou et al., 2012). The ability of E2 to upregulate the expression of HIF-1α was also shown to associate with reduced hepcidin synthesis in ES-2 and SKOV3 cells (Hua et al., 2009; Kazi et al., 2009).

It is well established that perturbed iron metabolism compromises immunity (Cherayil, 2020; Ganz, 2006). Patients with iron overload resulting from hemochromatosis or blood transfusion are highly susceptible to microbial infections (Jason et al., 2001). Iron deficiency on the other hand often associates with impaired cell proliferation and delayed type hypersensitivity responses among other adverse consequences (Oppenheimer, 2001). Moreover, previous work has established that iron modulates the inflammatory response (Mills et al., 2016; Recalcati et al., 2019). For example, liver and peritoneal macrophages isolated from C57BL/6 mice on iron-rich diet show increased expression of M2 markers Arg1 and Ym1 while those isolated from mice on iron-deficient diet show decreased expression of Arg1 (Agoro et al., 2018). In vitro macrophage iron loading was reported to reduce the percentage of M1 cells and inhibit LPS-induced pro-inflammatory (iNOS, IL-1β, IL-6, IL-12 and TNFα) mediators by reducing NF-κB p65 nuclear translocation (Agoro et al., 2018). It was also reported to reduce the production of IL-6, IL-1β, TNF-α, and iNOS in IFN-γ-polarized M1 RAW264.7 macrophages by inhibiting STAT1 (Zhen-Shun et al., 2017). Interestingly, although iron overloading reduced M1 polarization in RAW264.7 cells, it enhanced the ability of M1-polarized RAW264.7 cells to phagocytose FITC-dextran (Zhen-Shun et al., 2017). Increased intracellular iron content was also reported to associate with reduced LPS-induced hepcidinsynthesis and increased FPN expression in macrophages (Agoro et al., 2018; Zhen-Shun et al., 2017). In contrast, Zanganeh et al. have reported that treatment with iron oxide nanoparticles induced a protective pro-inflammatory M1 polarization switch in tumor associated macrophages (Zanganeh et al., 2016). While these observations clearly show that cellular iron plays an important role in macrophage M1 and M2 polarization, the exact conditions under which it helps to switch macrophages to an M1 or an M2 phenotype remain ambiguous.

Numerous studies have established that E2 suppresses immunity and increases susceptibility to infection (Cutolo et al., 2004; Igarashi et al., 2001; Erlandsson et al., 2001). However, gender bias in autoimmunity, with most autoimmune diseases occurring at much higher rates in females, has been attributed to E2 among other factors (Whitacre, 2001). These effects of E2 on the immune response present a paradox, which is yet to be resolved. Hence, investigating the effects of E2 on iron metabolism in cells of the immune system may offer new insights into the paradoxical roles of E2 in immunity. This is particularly relevant given that major cell subsets of the immune system variably express different estrogen receptors including ER-α, ER-β and GPR30 and are hence responsive to E2 signaling (Cutolo et al., 2004; Igarashi et al., 2001; Erlandsson et al., 2001). In this study, intracellular iron status and the expression of key iron regulatory proteins were studied in monocytic- and nonmonocytic-lineage cells treated with increasing concentrations of E2. The effects of ER agonists and antagonists, inhibitors of IL-6 synthesis and IL-6 gene silencing on cellular iron status were also evaluated.