Background

The intracellular Gram-negative bacterium Salmonella typhi can cause severe and often life-threatening disease in humans. Globally, approximately 200,000 deaths are caused by the bacterium every year. The intracellular pathogen is ingested through contaminated food, and transmitted from person to person. The mouse correlate of human Salmonella typhi is Salmonella enterica serovar Typhimurium (S.tm). The intracellular bacteria are phagocytosed by macrophages and are able to evade antimicrobial defense by inhibiting fusion of lysosome and phagosome. Therefore, S.tm is able to survive and replicate inside the host (Buchmeier and Heffron, 1991; Navarre et al., 2010; Lahiri et al., 2010; Mastroeni and Grant, 2011; Bhutta et al., 2018).

In general, the immune system is divided into cells of the innate immune system (monocytes, macrophages, dendritic cells, and natural killer cells), and the acquired immune system (B lymphocytes, and T lymphocytes). Macrophages are phagocytic cells of the innate immune system, and one of the body's first defense mechanisms, when a pathogen crosses the host’s skin barrier. They can be classified into two types: (I) M1 pro-inflammatory macrophages, which are responsible for killing bacterial and viral pathogens, and express inducible nitric oxide synthase (iNOS), and (II) M2 macrophages, which support the wound healing process, and have an anti-inflammatory effect. Importantly, M2 macrophages express the enzyme arginase 1 (ARG1) (Mosser and Edwards, 2008; Mills, 2012; Weiss and Schaible, 2015; Gordon and Martinez-Pomares, 2017; Murray, 2017; Hannemann et al., 2019).

The cytosolic enzyme ARG1 is primarily expressed in liver tissue. During infection, ARG1 upregulation promotes pathogen survival in macrophages, by hydroxylating l-arginine to urea and ornithine, which therefore lowers l-arginine levels for the synthesis of nitric oxide (NO) by iNOS. A fully functional iNOS needs an adequate l-arginine supply to produce NO that kills pathogens. Interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) are potent inducers of iNOS (Nairz et al., 2013; Bogdan, 2015; Brigo et al., 2021). ARG1 is induced by interleukin 4 (IL-4), which is produced by type 2 T helper cells. TNFα and IFNγ inhibit the transcription of ARG1, by interfering with IL-4–induced chromatin remodeling (Ostuni and Natoli, 2011; Schleicher et al., 2016; Piccolo et al., 2017). Additionally, several studies show that increased ARG1 activity is associated with an increased concentration of various pathogens, such as Streptococcus pneumoniae, Mycobacterium bovis, Mycobacterium tuberculosis, or Toxoplasma gondii (Iniesta et al., 2005; El Kasmi et al., 2008; De Muylder et al., 2013; Schleicher et al., 2016; Paduch et al., 2019). However, it has been reported that deletion or pharmacological inhibition of ARG1 does not lead to a better control of Salmonella infection in macrophages, or in mice, in a septicemia model (Brigo et al., 2021).

Current treatment against invasive salmonellosis with antibiotics has become more difficult, due to resistance against conventional antibiotics. Therefore, the identification of new mechanisms to better understand the host-pathogen interplay in Salmonella infection, and the detailed characterization of the cells involved in the defense against this infection are urgently needed. Herein, we describe a method that identifies pro- and anti-inflammatory bone marrow-derived macrophage subtypes, during an infection with Salmonella enterica serovar Typhimurium. Furthermore, Salmonella expressing red fluorescent protein allows the visualization of phagocytosed Salmonella in these subtypes. This method supports further research on the involvement of pro- and anti-inflammatory macrophages in the defense against Salmonella.