Background

Human typhoid fever, a severe and often life-threatening infectious disease, is caused by the facultative intracellular Gram-negative bacterium Salmonella enterica serovar Typhi, leading to major health loss globally (Stanaway et al., 2019). For murine infection models, the strain Salmonella enterica serovar Typhimurium (Salmonella Typhimurium, S.tm), which causes a systemic disease in mice but a self-limiting gastroenteritis in humans, is frequently used.

In the case of a bacterial infection, invading pathogens are phagocytized by macrophages, the first line of innate immune defense (Weiss and Schaible, 2015). S.tm, however, thrives within macrophages. Despite its ability to invade various types of cells, virulence is dependent on intramacrophage proliferation (Fields et al., 1986; Leung and Finlay, 1991). One factor critically affecting the outcome of this host–pathogen interaction is the availability of nutrients, as intramacrophage S.tm depends on the acquisition of essential nutrients to sustain efficient proliferation. This includes amino acids or trace metals, like iron. On one hand, S.tm–driven metabolic reprogramming grants the pathogen access to intracellular nutrients (Liss et al., 2017). On the other hand, macrophages exploit this nutrient demand by withdrawing iron from the spatio-temporal localization of the pathogen in a process termed nutritional immunity (Nairz et al., 2007; Murdoch and Skaar, 2022). Appropriately, a state of systemic or cellular iron excess is associated with increased bacterial proliferation (Khan et al., 2007; Porto and De Sousa, 2007; Kao et al., 2016).

Macrophage cellular iron metabolism and its adaptation to an infection have been extensively studied. In the case of an infection with an intracellular pathogen like S.tm, regulation of the key iron metabolism proteins Ferroportin-1 (FPN1) and the Transferrin receptor-1 (TFR1) facilitates a decrease of cellular iron content and thus leads to an improved infection control of intracellular bacteria (Nairz et al., 2008; Fritsche et al., 2012; Wessling-Resnick, 2015; Abreu et al., 2020). Furthermore, transport of iron into the cytosolic lumen is accomplished by the divalent metal transporter-1 (DMT1) and by the natural resistance-associated macrophage protein-1 (NRAMP1 or SLC11A1), both of which have also been implicated in bacterial iron withdrawal (Forbes and Gros, 2003; Fritsche et al., 2012; Grander et al., 2022). DMT1 is responsible for the uptake of non-transferrin-bound iron (NTBI) from outside the macrophage and for transporting transferrin-bound iron (TBI) from the early endosome into the lumen of the phagocyte. NRAMP1 transports iron out of the late phagosome. As both are only capable of binding Fe²⁺, the function of the six-transmembrane epithelial antigen of prostate 3 (STEAP3) in the late endosome to reduce Fe³⁺ to Fe²⁺ is indispensable (Wang and Pantopoulos, 2011).

Another factor at play is the acute phase protein hepcidin (HAMP1), a liver-derived hormone regarded as the systematic master regulator of iron metabolism. During an infection, hepcidin targets the iron exporter FPN1, leading to its degradation and thus iron sequestration, causing hypoferremia (Nemeth et al., 2004). However, its role in an infection with intracellular pathogens is not yet completely understood (Chlosta et al., 2006; Lim et al., 2018).

During infection studies, cellular iron quantification is frequently relevant. Standard procedures like the usage of radioactive ⁵⁹Fe isotopes are elaborate, expensive, and might be inapplicable in certain experimental settings. Iron quantification with the help of the quenchable probe Calcein-AM is easily available and often used but has several disadvantages. Acquiring fluorescence by flow cytometry needs extensive preparation of samples that exposes cells to mechanical stress; furthermore, as Calcein does not pass into the cellular membrane compartments (e.g., lysosomes), which are rich in labile iron (chelatable), total cellular iron is most likely drastically underestimated when this method is applied (Tenopoulou et al., 2007).

Herein, we report a simple and powerful tool to accurately determine alterations in cellular iron levels upon diverse stimuli, which can be employed for cultured immune cells, here exemplified in a murine macrophage infection with intracellular bacteria. As the cellular iron trafficking machinery primarily utilizes Fe²⁺, monitoring metabolically active intracellular ferrous iron levels is most insightful (Hentze et al., 2010; Moroishi et al., 2011; Cronin et al., 2019). We apply a fluorescent probe that specifically detects Fe²⁺, based on N-oxide chemistry (RhoNox-4; commercial name: FerroOrange) (Hirayama et al., 2020). By using this approach, cellular levels of the trace metal can be quantified in a plate reader without additionally exposing cells to stressors.