Human infectious diseases are still a major public health problem in particular because of the development of antibiotic resistance, the lack of new products, and the demise of antibacterial drug discovery by pharmaceutical companies (1). Such context leads to the emergence and reemergence of infectious diseases, and it becomes critical to develop alternative approaches to identify new antibacterial drugs and to propose new treatments. Therefore, the natural host defense mechanisms against invading microbes and the mechanisms regulating the virulence of microorganisms need to be better understood. An important host defense strategy against infection, known as “nutritional immunity,” relies on the sequestration of essential molecules, such as iron, preventing the growth of pathogens (2). Iron is essential for both the host and the invading microbes and plays a critical role in host–pathogen interactions. In response to infection, patients commonly develop hypoferremia (i.e., a decrease of iron in the circulation), a host response to limit iron availability to invading pathogens (3). However, for the host, hypoferremia also contributes to the establishment of the so-called anemia of inflammation (AI) (4), an anemia difficult to treat and that can add substantially to the morbidity of the underlying infection.

Iron sequestration in macrophages is a described hallmark of the AI and is an efficient mechanism to quickly deplete iron in the serum to limit the growth of extracellular pathogens (5). Two molecules, namely hepcidin (HAMP) and ferroportin (FPN), have been identified to play key roles in decreasing systemic iron level by promoting macrophage iron sequestration during infection (5). FPN is the only known mammalian iron exporter and is expressed at the cell surface of macrophages (6, 7). FPN is quickly downregulated through endocytosis and degradation upon interaction with HAMP (6, 8). HAMP is produced mainly by hepatocytes in case of inflammation and also by infected macrophages (9). Therefore, decreasing the expression of FPN to retain iron inside the macrophages could limit serum iron access to extracellular pathogens.

On the other hand, macrophages are a common niche for the replication of numerous intracellular pathogens including Salmonella. Increased iron level inside macrophages might therefore represent either an advantage for the growth of intracellular microorganisms or a host strategy to fight against intracellular bacteria through the generation of highly toxic reactive oxygen species via Fenton’s reaction (3). Recently, some studies have challenged these concepts and have suggested that macrophages infected with intracellular bacteria respond by decreasing their iron content via an upregulation of FPN to limit the growth of the invading microbes (10–12).

Therefore, the modulation of host iron homeostasis, in particular in macrophages, in response to infection with intracellular pathogens is currently a matter of debate, and the regulation of FPN is an important iron response to be evaluated in different intracellular bacterial infection settings. Salmonella is the most common bacterial cause of foodborne outbreaks, and many Salmonella strains are resistant to antibiotics. The main purpose of the current work was to explore the interplay between infection with Salmonella (Salmonella enterica serovar Typhimurium) and the systemic and macrophage iron homeostasis in different mouse models presenting distinct systemic and macrophages iron levels.

In this article, we characterized the interplay between iron homeostasis and intracellular Salmonella infection, using different mouse models presenting distinct systemic and macrophages iron contents. Indeed two distinct models were used presenting either macrophage iron overload (AcB61) or macrophage iron deficiency but systemic iron overload (Hamp^−/−).

Among our models, the AcB61 mice were the most susceptible to ST infection. AcB61 mice harbor a mutation in the gene Pklr leading to PK deficiency and resulting in chronic hemolytic anemia and tissue iron overload (20–22). We observed intensive EP activity in AcB61 tissue macrophages in vivo, which is consistent with in vitro studies showing that Pklr-deficient erythrocytes were more vulnerable to phagocytosis by macrophages than control erythrocytes (24). As a consequence of enhanced EP activity in AcB61 macrophages, strong heme recycling is observed with increased bilirubinemia and enhanced expression of both HMOX1 and FPN. Heme is known to be a potent inducer of HMOX1 transcription (25), and both heme and iron positively regulate macrophage FPN at both transcriptional and posttranscriptional levels (23, 26). In addition, FPN was strongly detected in vivo at the cell surface of AcB61 hepatic and splenic macrophages, suggesting some export of the iron from the cytosol to circulation.

The important iron storage and iron fluxes in AcB61 macrophages likely represent an advantage for the growth of Salmonella and contribute to the high susceptibility of AcB61. Interestingly, AcB61 mice have been challenged for their response to infection with several intracellular bacteria including Listeria monocytogenes (D. Malo, unpublished), different strains of Mycobacterium bovis (22, 27), and Legionella pneumophila (13). For all these models of infection, the Pklr mutation in AcB61 did not contribute to the clinical phenotype, and no further studies focusing on iron metabolism were performed. Other observations (not shown) indicate that the exacerbated susceptibility of AcB61 mice to Salmonella infection is not the consequence of a blunted immune response or a defect in the expression of the iron-siderophore binding protein lipocalin 2 and therefore likely reflects the accessibility to intracellular iron by the bacteria. Iron is an indispensable metal for the spread of virtually all human pathogens (3). Of note, numerous Salmonella-induced granulomas, which represent infected foci containing the bacteria, were mostly localized in iron-rich regions in AcB61 mice. Importantly, there was a significant decrease of serum iron in AcB61 when compared to A/J, suggesting that despite the fact that Salmonella is a facultative intracellular bacterium, it takes more advantage of the intracellular macrophage iron sources rather than of the extracellular sources. Accordingly, in spite of a high iron level in blood and hepatocytes, we observed no change in Salmonella load in Hamp^−/− mice when compared to WT mice. However, we previously showed that mice deficient in Hamp were significantly more susceptible to lethal infection than heterozygous or wild type littermates (28). Such observation indicates the involvement of Hamp during systemic model of ST infection. However, the exact mechanism underlying such differences remains obscure.

Iron deficiency, bone marrow suppression, and hemolysis are described to participate in the establishment of anemia in infectious diseases (4). Lower hematocrit levels after Salmonella infection were observed in AcB61 and Hamp^−/− mice despite important levels of plasma iron, suggesting that these mice do not develop iron-restrictive anemia during infection. Our observations are concordant with literature, suggesting that the main driving force for the decrease of hematocrit during Salmonella infection is an accelerated clearance of RBC (29, 30). Indeed, during Salmonella infection, AcB61 spleens present signs of strong EP activity with numerous RBC per macrophages. Salmonella infection via the stimulation of Toll-like receptor 4 has been shown to stimulate macrophages to hemophagocytosis, a process that lead to the phagocytosis of red and white blood cells (29, 30).

In addition, our work suggests that the Salmonella-induced anemia is, at least in part, independent of HAMP. Indeed, either no changes (AcB61 mice) or a decrease (WT mice) of hepatic Hamp expression is associated with anemia after ST infection, and even in Hamp^−/− mice, a decreased hematocrit is still observed. In vivo, the expression of Hamp in the hepatocyte is governed by stimulatory (iron overload and inflammation) and inhibitory factors (erythropoietic ERFE and hypoxia), the net effect of these factors defining the Hamp level in the organism (31). In uninfected AcB61 mice, the positive iron regulator is likely compensated by the negative erythropoietic regulator (ERFE) leading to normal but inadequately low level of Hamp for the degree of iron loading observed in these mice. Indeed, we observed extramedullary erythropoiesis and increase of Erfe mRNA levels (not shown) in the spleen of AcB61. As previously observed (28), in WT mice, Hamp expression was repressed after ST infection. In contrast, other studies have reported increased expression of Hamp during ST infection in mice (32) or Salmonella Typhi in humans (33). Interestingly, in a model of the AI using a heat-killed Gram-negative bacteria, Brucella abortus, Hamp was shown to be upregulated in an early phase associated with erythropoietic suppression but was downregulated in a second later phase in parallel with an increase in EPO and erythropoiesis (34, 35). In addition, Salmonella infection has been shown to initiate extramedullary erythropoiesis and splenomegaly with increases in RBC precursors and EPO production (36). These differences observed between studies in the regulation of Hamp during Salmonella infection may rely on differences in the degree of compensatory erythropoiesis at the infection time (negative regulation of Hamp via ERFE).

In this study, we observed a strong negative regulation of the iron exporter FPN at both the mRNA and the protein level in Hamp^−/− mice, indicating that the repression of the iron exporter by Salmonella infection is independent of HAMP action. Recently, a strong HAMP-independent, negative regulation of FPN mRNA and protein was also documented in BMDM and liver and spleen of mice in response to acute inflammatory conditions induced by TLR2/6 agonists (37). In this study, the reduced expression of FPN in macrophages was sufficient to rapidly induce hypoferremia in mice (37). Similarly, reduction of spleen Fpn mRNA level by TLR4 agonist was shown to be HAMP independent (38).

In WT mice, we also observed a concomitant decrease in both FPN expression and plasma iron with a decrease in Hamp expression. Together, such observations suggest that beside HAMP effect, other mechanisms exist to induce a pathogen-mediated hypoferremic response, contributing to the AI.

In vivo, the negative regulation of FPN protein was observed in tissue macrophages after ST infection. Similar observation was made after SE infection (Figure S4 in Supplementary Material). Of note, we did not observe a decrease in Fpn mRNA expression in infected AcB61 mice despite the strong loss of FPN protein expression at the cell surface of AcB61 macrophages. The strong positive regulation of Fpn mRNA expression by heme and iron in erythrophagocytic AcB61 macrophages likely counteracts the Salmonella-mediated negative regulation at the level of mRNA. Such observation suggests that posttranscriptional regulations may exist since FPN protein expression is diminished without any changes of Fpn and Hamp mRNA levels. Recently, iron regulatory proteins (IRPs) have been shown to play a role during Salmonella infection (39). FPN contains an iron-responsive element in its 5′ UTR, and its translation is repressed by the IRPs. Therefore, during Salmonella infection, IRPs could block the translation of Fpn and thereby contribute to the decrease of FPN protein levels despite no alteration at the levels of mRNA. However in the context of AcB61 mice, the high iron content observed in macrophages likely impairs the action of the IRPs. Since FPN protein expression decreased despite maintained level of Fpn mRNA in AcB61 mice during salmonella infection, other posttranscriptional mechanism(s) may occurred.

Interestingly, FPN- and F4/80-positive cluster of cells were detected only in the liver of Hamp KO mice after Salmonella infection. In these mice, our cellular analysis strongly suggests that such cellular aggregates correspond to the recruitment of uninfected circulating monocytes overexpressing FPN because of the lack of Hamp.

In vitro, the negative FPN regulation was directly observed at the level of mRNA in ST-infected cultured BMDM. Our data are consistent with previous observations showing decreased FPN mRNA expression in vivo (37, 40) and ex vivo in cultured murine and human macrophages treated with lipopolysaccharide (LPS) (16, 41–43). ST infection and LPS stimulation were shown to induce similar changes in macrophage gene expression (44). The molecular mechanism of Fpn mRNA repression in macrophages via LPS/TLR4 stimulation is still not known. Moreover, downregulation of Fpn expression in macrophages was also reported with TLR2/6 (37, 45) expanding the FPN response to various pathogen-associated molecules.

In the context of intracellular pathogen infection, low levels of FPN in macrophages will favor cellular iron sequestration and bacterial growth inside the infected cells. This is consistent with in vitro studies showing that degradation of FPN resulted in increased macrophage bacterial growth in Salmonella-infected J774 macrophages (46). In opposition to this cellular scenario, other authors proposed that during infection with macrophage-tropic intracellular pathogens, macrophages respond by an upregulation of FPN to limit intracellular iron content (10, 12). Increases in FPN mRNA and protein expression in mouse macrophages cell lines RAW264.7 or thioglycollate-elicited peritoneal macrophages have been reported during Salmonella infection (10). The same authors have proposed that upregulation of FPN during Salmonella infection involves NO production by NOS2 (10, 12). However, in our ST-infected BMDM as well as in the liver of infected mice, the FPN gene repression occurred with a concomitant increase of the NOS2 expression. The role of NOS2 and NO in the regulation of FPN needs further investigation. The discrepancy between studies regarding the regulation of FPN during intracellular infection is unclear and warrants continued effort to clarify this important regulation in the context of infectious diseases. The use of different antibodies against FPN, which are not all carefully characterized by appropriate controls of specificity, could contribute to the differences observed between different studies.

Despite FPN downregulation during Salmonella infection in our models, we observed a decrease in iron within infected spleen and liver. Such a decrease in tissues iron was strongly observed in Hamp^−/− liver but was not seen in AcB61 tissues, likely masked by the exacerbated EP activity and heme iron uptake by macrophages in these tissues. During infection, macrophage iron could be consumed, at least in part, by the bacteria itself, dependent on this metal for its growth and dissemination. Alternatively, FPN-independent export of iron may occur in infected macrophages. A peculiar iron distribution was observed in Salmonella infected Hamp^−/− liver, with some strong accumulation in CL hepatocytes and sinusoidal Kupffer cells. One possible explanation is the engulfment of iron-loaded apoptotic hepatocytes by liver macrophages. Indeed both iron overload and LPS/inflammation have been shown to induce apoptosis in hepatocytes (47, 48). Therefore, in Hamp^−/− mice, the iron overloaded hepatocytes in the CL zone are likely more sensitive to apoptotic processes during Salmonella infection. Since macrophage FPN expression is repressed, an increase of phagocytosis of such apoptotic cells could lead to iron overload in sinusoidal and CL macrophages.

Our observations suggest that to promote its intracellular growth, Salmonella modulates macrophage iron homeostasis to favor its access to intracellular iron with the reduction of iron export via the downregulation of FPN. Importantly, such a macrophage cellular host response, which promotes infection, anemia, and hypoferremia, is independent of macrophage iron and HAMP levels. To fight against bacterial infectious diseases and to correct the anemia during chronic infection, effort has to be made to understand whether this HAMP-independent downregulation of FPN expression exists in different macrophages populations and is a general host response observed with other intracellular pathogens infection.

This study was carried out in accordance with the recommendations of the Canadian Council on Animal Care. The protocol was approved by the McGill University Animal Care Committee.

AW, SB, ER, and AR designed protocols and performed experiments. AA, RM, and OT performed experiments. SB reviewed data and provided statistical analysis and correction of the manuscript. SV provided Hamp^−/− mice, reviewed the data, and provided comments and corrections of the manuscript. DM designed protocols, performed experiments, reviewed the data, and provided comments and corrections of the manuscript. FC-H designed protocols, performed experiments, reviewed the data, and wrote the paper.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a shared affiliation, though no other collaboration, with several of the authors (AW, AA, and FC-H), and states that the process nevertheless met the standards of a fair and objective review.