HFE encodes an atypical MHC class I molecule which plays a major role in the regulation of iron homeostasis under basal conditions (Feder et al., 1996; Ludwiczek et al., 2004). HFE mutations, especially the homozygous C282Y substitution, result in type I (AKA classical) hereditary hemochromatosis (HH) (Camaschella et al., 2002; Pietrangelo, 2004; Weiss, 2010), the most frequent form of HH mainly found in people of Northern or Western European ancestry. HH is characterized by reduced serum levels of the antimicrobial peptide Hamp (hepcidin) and increased duodenal absorption of iron via divalent metal transporter 1 (Dmt1) and ferroportin 1 (Fpn1) despite progressive iron overload in parenchymal organs including the liver, pancreas, and heart (Zoller et al., 1999, 2001; Bridle et al., 2003; Pietrangelo, 2004; Bardou-Jacquet et al., 2013). The precise role of the HFE protein, however, remains incompletely understood. HFE binds to transferrin receptor 1 (TfR1) thus lowering its affinity for iron-laden transferrin (Feder et al., 1998; Lebrón et al., 1998; Bennett et al., 2000). This interaction controls cellular iron acquisition while also modifying the expression of the key iron-regulatory hormone Hamp (Ahmad et al., 2002; Nicolas et al., 2003; Ludwiczek et al., 2005; Vujic Spasic et al., 2008). The latter mechanism involves the sensing of circulating iron levels by TfR1 and TfR2, which reciprocally complex with HFE expressed on hepatocytes (Schmidt et al., 2008; Wallace et al., 2009). Mutations in HFE (or TFR2) impair this iron-sensing mechanism, resulting in the insufficient generation of Hamp and increased iron absorption (Goswami and Andrews, 2006; D'Alessio et al., 2012). Of note, macrophages lacking HFE display an iron-poor phenotype which has been attributed to enhanced iron export (Cairo et al., 1997; Drakesmith et al., 2002; Wang et al., 2003).

Systemic iron availability, erythropoietic iron demand, hypoxia, hormones, and inflammatory signals are key factors that modulate the production of the iron homeostatic regulator Hamp (Nemeth et al., 2004a; Bozzini et al., 2008; Theurl et al., 2010; Armitage et al., 2011; Kautz et al., 2014; Nairz et al., 2014; Canali et al., 2017). Hamp controls iron homeostasis upon binding Fpn1, which triggers Fpn1 internalization, degradation (Nemeth et al., 2004b) and blockade of iron efflux from duodenal enterocytes and macrophages, which recycle iron from senescent erythrocytes. Inflammation-driven Hamp induction thus causes iron sequestration within the mononuclear phagocyte system (MPS), which limits iron availability for extracellular pathogens (Bridle et al., 2003; Ludwiczek et al., 2003; Ganz, 2005; Theurl et al., 2008a).

In infections with the intracellular bacterium Salmonella enterica serovar Typhimurium, macrophages constitute an important habitat for pathogen replication and persistence (Malik-Kale et al., 2011). Because many bacteria are highly dependent on a sufficient supply of iron for their growth and pathogenicity, macrophage iron homeostasis is an important determinant of disease outcome (Nairz et al., 2014). On one hand, macrophage iron overload is associated with the inhibition of IFN-γ-driven antimicrobial immune effector pathways such as nitric oxide synthase 2 (Nos2) expression, resulting in impaired control of intracellular microbes (Weiss et al., 1994; Mencacci et al., 1997; Oexle et al., 2003). On the other hand, severe iron depletion of the host may result in reduced generation of ROS, which also impairs host defenses. In parallel, iron withholding from pathogens constitutes an efficient host defense strategy (Soares and Weiss, 2015). However, macrophages also contribute to host defense by the production of T-cell stimulatory cytokines and antimicrobial peptides (Graziadei et al., 1997). One of the latter, lipocalin 2 (Lcn2; also known as neutrophil gelatinase-associated lipocalin, siderocalin or 24p3), is secreted by neutrophils and macrophages in response to LPS, IL-1ß, IL-17, and IL-22 (Flo et al., 2004; Shen et al., 2006). In its best characterized function, Lcn2 captures iron-laden bacterial siderophores, small molecules that are enzymatically synthesized and actively secreted by many microbes to bind ferric iron with extraordinarily high affinity (Bachman et al., 2009). Lcn2-sensitive siderophores include enterobactin, carboxy-mycobactins, and bacillibactin. Upon neutralization of these siderophores, Lcn2 contributes to innate resistance against a range of pathogenic bacteria including enterobacteriaceae, mycobacteria and Bacillus anthracis by limiting their access to iron (Flo et al., 2004; Berger et al., 2006).

Salmonella Typhimurium, a facultative intracellular microbe, needs to gain sufficient access to host iron resources as a prerequisite for replication and virulence (Leung and Finlay, 1991; Vazquez-Torres et al., 1999). To acquire the metal from the host and within infected macrophages, Salmonella has evolved both siderophore-dependent and -independent strategies. Salmonella synthesizes catecholate-type siderophores such as enterochelin and salmochelins to capture and internalize ferric iron via siderophore receptors (Bäumler et al., 1998; Rabsch et al., 2003; Fischbach et al., 2005). Alternatively, Salmonella can incorporate non-siderophore-bound ionic iron using the Feo transport system. In addition, the SitABCD system, whose primary function is bacterial manganese import, may contribute through low-affinity uptake of iron (Zaharik et al., 2004). All three pathways of bacterial iron uptake are linked to Salmonella virulence (Tsolis et al., 1996; Janakiraman and Slauch, 2000; Boyer et al., 2002; Crouch et al., 2008; Kim et al., 2013).

Given the central importance of iron for the growth and proliferation of intracellular pathogens such as Salmonella and the important role of Hfe in the regulation of systemic iron balance, we performed experiments to assess the influence of Hfe and/or dietary iron overload on host iron homeostasis and immunity in response to S. Typhimurium infection. This is of specific interest because Hfe results in macrophage iron depletion whereas dietary iron overload leads to iron accumulation within the MPS.

Salmonella Typhimurium causes a systemic disease in mice characterized by a tropism for and replication within professional phagocytes (Richter-Dahlfors et al., 1997; Coburn et al., 2007). S. Typhimurium invades its preferred host cell type both by phagocytic uptake and active invasion (Pfeifer et al., 1999). Virulence factors such as those encoded by Salmonella Pathogenicity Island-2 are essential for both intracelullar survival and virulence (Hensel et al., 1995, 1998), suggesting that the ability to infect and replicate within macrophages provides a major benefit for the pathogen (Leung and Finlay, 1991). Nutrient availability within this host cell niche is therefore an important factor in Salmonella pathogenesis (Carver, 2014).

Iron is one of the essential nutrients that hold a central position in the interplay of host and pathogen (Weinberg, 1974; Schaible and Kaufmann, 2005; Skaar, 2009; Nairz et al., 2010; Drakesmith and Prentice, 2012; Ganz and Nemeth, 2015; Soares and Weiss, 2015). Sufficient access to this trace element is therefore a major determinant of the outcome of Salmonella infection. In general, the host response to any bacterial infection involves the restriction of serum iron levels (hypoferremia) through a combined limitation of intestinal iron absorption and macrophage iron recycling. Hamp and its receptor Fpn1 are primary mediators of the hypoferremia, and thereby influence Salmonella-host interactions (Nairz et al., 2013; Kim et al., 2014; Armitage et al., 2016). However, a range of additional genetic and environmental factors also influence bacterial iron availability during Salmonella infections.

The data presented herein suggest that both the local and systemic availability of iron within the mammalian host affect infection outcome (Nairz et al., 2015a). Hfe deficiency results in reduced Hamp production and increased serum iron levels (Pietrangelo, 2004). Enhanced bacterial replication might be anticipated in an Hfe-deficient host as a result of increased iron availability, and this has been experimentally demonstrated for pathogens such as Vibrio vulnificus, Yersinia enterocolitica, and Yersinia pestis in the setting of HH (Quenee et al., 2012; Arezes et al., 2015; Miller et al., 2016). However, the opposite is true in the context of infection with an intracellular bacterium such as S. Typhimurium because Hfe-deficient macrophages are iron-poor and thus provide an inferior niche for bacterial replication (Nairz et al., 2009). We find that Hfe-deficient macrophages restrict the availability of both non-transferrin-bound (NTBI) and transferrin-bound (TBI) iron to intracellular Salmonella more efficiently than WT macrophages. This suggests that the underlying mechanism is independent of Dmt1 and TfR1 and may be attributable to differential iron turnover or efflux. While Fpn1 constitutes the primary pathway for the cellular release of ferrous iron, iron may also be exported via alternative pathways (Devireddy et al., 2005; Keel et al., 2008; Du and Galán, 2009; Nairz et al., 2015b; Lok et al., 2016).

Slc11a1 (also known as Nramp1) has long been known to influence the course of infection with S. Typhimurium and certain species of Mycobacterium and Leishmania (Vidal et al., 1993; Atkinson et al., 1997; Blackwell et al., 2003). Although these pathogens are taxonomically unrelated, they share the features of infecting macrophages, persisting in phagolysosomes and depending on iron. Slc11a1 is incorporated into the phagolysosomal membrane and shifts iron and other divalent ions out of this compartment, thus withdrawing it from phagocytosed microbes (Vidal et al., 1995; Jabado et al., 2000; Wyllie et al., 2002; Fritsche et al., 2007; Valdez et al., 2008). We used C57BL/6 mice for our studies, which carry two dysfunctional Slc11a1 alleles. Therefore, the phenotypes observed in our studies cannot be attributed to this transporter. Moreover, our findings are unlikely to be specific for infections with S. Typhimurium but are also relevant to other iron-dependent intracellular pathogens such as Chlamydia, Legionella and Listeria (Paradkar et al., 2008; Bellmann-Weiler et al., 2010, 2013; Haschka et al., 2015), as well as Mycobacterium and Leishmania. Accordingly, we note that Hfe deficiency impairs the growth of Mycobacterium tuberculosis in human macrophages (Olakanmi et al., 2007).

The iron content of macrophages is influenced by several mechanisms including iron levels in the extracellular microenvironment, expression of iron importers, and exporters, and the rates of erythrophagocytosis and heme-iron recycling (Canonne-Hergaux et al., 1999; Mitterstiller et al., 2016; Theurl et al., 2016). Our models of genetic (i.e., Hfe-associated) and dietary iron overload had different effects on macrophage iron content. Long-term oral iron overload results in increased iron content in virtually all cell types expressing Dmt1 and/or TfR1. In contrast, Hfe deficiency spares the myeloid compartment from iron. We found that oral iron overload results in an increased bacterial load in the spleens and livers of Salmonella-infected mice and that Hfe deficiency reduces the bacterial load. Of note, oral iron overload in the setting of Hfe deficiency resulted in intermediate pathogen numbers in spleen and liver on days 2 and 4 of infection. In contrast, the survival of Hfe^−/− mice over a 10 day period was not affected by dietary iron. Hfe^−/− mice with dietary iron overload remain more resistant to Salmonella infection than WT mice receiving the same diet, with reduced organ loads and increased survival (Figure 3) despite the alleviation of bacterial iron-deprivation by dietary iron supplementation as measured by siderophore gene expression (Figure 6). Albeit somewhat unexpected, these findings suggest that in the later stages of Salmonella infection, Hfe plays an immunoregulatory function that is independent of its effect on bacterial iron-restriction. However, the expression of innate immune genes known to mediate host defense against S. Typhimurium was not different between Hfe^−/− and Hfe^+/+ mice (Vazquez-Torres et al., 2000; Vázquez-Torres et al., 2001), nor were differences in T cell-mediated pathways associated with immunity against S. Typhimurium such as IL-12, IFN-γ, IL-17, or IL-22, observed (data not shown) (Berger et al., 2006; Raffatellu et al., 2008; Saiga et al., 2008; Schulz et al., 2008; Chan et al., 2009; Godinez et al., 2009; Srinivasan et al., 2012). Given that Lcn2 was expressed at higher levels in Hfe^−/− mice, it is possible that one of the siderophore-independent effects of Lcn2 may play a role. Lcn2 is a chemoattractant for neutrophils, but this is unlikely to account for a survival difference beyond day 3 of infection (Schroll et al., 2012). Furthermore, Lcn2 promotes macrophage antibacterial effector mechanisms including TNF, IL-6, and Nos2, but these were not observed to be differentially expressed on day 2 or 4 of infection of Hfe^−/− and Hfe^+/+ mice (Nairz et al., 2015b). It is conceivable that the survival of mice in the late stages of systemic Salmonella infection is directly or indirectly influenced by the intestinal microbiome, which is modulated by Lcn2 (Raffatellu et al., 2009; Deriu et al., 2013; Moschen et al., 2016). Alternatively, the comparable survival of Hfe^−/− mice on an IE on IA diet may involve an Lcn2-independent mechanism beyond innate immunity. For instance, Hfe deficiency may have beneficial effects on apoptosis, ferroptosis, autophagy, or the oxidative stress response within or outside of the myeloid compartment that is independent of dietary iron. If a vital organ system were to be involved, an effect on host survival would be a plausible. An unbiased approach such as RNA-sequencing may be required to identify such a mechanism.

A central and novel finding of our study is that both the host and the microbe adapt their iron metabolism during infection. S. Typhimurium expressed genes required for siderophore-mediated iron uptake in vivo in the iron-poor spleens of Hfe^−/− mice. This induction was specifically abrogated by dietary iron overload. This observation raises the question whether virulence factors other than siderophore genes may have been repressed in the setting of dietary iron overload to enhance the survival of Hfe^−/− mice. This possibility is supported by the known cross-regulation of bacterial iron homeostasis and virulence gene expression (Zaharik et al., 2002). Our data on the upregulation of bacterial iron uptake genes are further in line with the specific induction of iron import mechanisms reported for Neisseria gonorrhoeae residing within human monocytes (Zughaier et al., 2014). Both studies thus support the concept that both host myeloid cells and facultatively intracellular bacteria actively compete for iron as essential nutrient.

In summary, the present study highlights the central role of macrophage iron homeostasis in the outcome of infections with iron-dependent intracellular microbes and the differential effects of genetic and dietary iron overload. We also demonstrate that Hfe is not required for the induction of hypoferremia in infected animals on an iron-replete diet. Nevertheless, the Hfe mutation alters the iron content of macrophages, which renders the host more resistant to infections with the intracellular pathogen S. Typhimurium. The selective pressure imposed by intracellular pathogens may have contributed to the evolutionary conservation of the HFE C282Y mutation, accounting for its high allelic frequency in Caucasians (Datz et al., 1998; Moalem et al., 2004).

MN planned and performed experiments, analyzed the data and wrote the manuscript. AS, DH, SD, PT, ED, and PM performed experiments. HH, FF, and IT interpreted results and edited the manuscript. GW conceived the study, analyzed the data and wrote the manuscript.