Continuous Measurement of Reactive Oxygen Species Formation in Bacteria-infected Bone Marrow–derived Macrophages Using a Fluorescence Plate Reader

Background

The Gram-negative enteric bacterium Salmonella enterica serovar Typhi causes typhoid fever in humans, which is a major cause of disability and death worldwide (Stanaway et al., 2019). The closely related Salmonella enterica serovar Typhimurium (Salmonella Typhimurium, S.tm) induces self-limiting gastroenteritis in humans and systemic infection in mice—a commonly used typhoid fever model.

As an intracellular pathogen, S.tm is capable of actively invading various cell types, but its virulence is dependent on its ability to selectively infect and proliferate inside macrophages (Fields et al., 1986; Haraga et al., 2008). Upon phagocytosis, S.tm establishes a replicative niche within a membrane-bound compartment termed the Salmonella-containing vacuole. There, the pathogen competes with various bactericidal mechanisms deployed by the host cell. One factor pivotal for initial inhibition of bacterial proliferation as well as pathogen clearance is the production of reactive oxygen and nitrogen species (ROS/RNS) (Vazquez-Torres et al., 2000; Herb and Schramm, 2021). In response to S.tm infection, macrophages activate several innate immune pathways that lead to ROS/RNS production and thus induction of bactericidal oxidative stress. A crucial component of the innate immune response identified is the membrane-bound enzyme NADPH oxidase (NOX), with the isoform NOX2 being at the center of antimicrobial ROS generation (Herb and Schramm, 2021). The importance of this enzyme is highlighted by the increased susceptibility of NOX2-deficient mice to infection with normally avirulent S.tm strains (Felmy et al., 2013). Another source of macrophage antimicrobial oxidative stress is mitochondria-augmented ROS production, which is directly linked to Toll-like receptor activation (West et al., 2011). Finally, the activity of inducible nitric oxide synthase (iNOS), crucial for pathogen control, is positively regulated by inflammatory cytokines, including interferon gamma (IFNγ) (Mastroeni et al., 2000). In multiple infection models, IFNγ has thus been implicated in adequate ROS/RNS production and enhancement of intracellular bacterial clearance in macrophages (Mastroeni et al., 2000; Nairz et al., 2008; Brigo et al., 2021). This antimicrobial effect of ROS/RNS is not only attributed to direct toxicity to the pathogen, but also to indirect effects, with oxidative stress being implicated in numerous cellular signaling pathways (Tan et al., 2016). Especially for the multi-faceted role of oxidative stress in innate immune defense, iNOS activation is deemed critical for the activation of nuclear factor erythroid 2–related factor-2 (Nrf2)-dependent pathways, which lead to a nutritional immune response aimed at limiting essential nutrients to the pathogen’s compartment (Nairz et al., 2013). The importance of ROS/RNS-mediated pathogen control is also evident in patients lacking functional ROS induction due to chronic granulomatous disease, as they are at high risk for invasive or recurrent forms of salmonellosis (Burniat et al., 1980;Mouy et al., 1989;Dinauer, 2005).

Due to its central involvement in innate immunity, an accurate measurement of oxidative stress is vital to study host–pathogen interactions. Herein, we report a method that allows to continuously determine ROS formation during infections in vitro. Most of the already established methods, which include usage of ROS-sensitive probes or biosensors expressed in cells or bacteria, rely on flow cytometry or fluorescence imaging techniques to quantify ROS levels (van der Heijden et al., 2015; Grander et al., 2022; Leone et al., 2016). These methods typically allow measurements only at single time points, as samples must be fixed and/or mounted. Furthermore, extensive preparation procedures will likely stress cells and thus decrease the signal-to-noise ratio. Of note, our novel protocol enables efficient detection of oxidative stress over time without stressing the cells by detaching and/or staining procedures. Furthermore, as fluorescence is read in a plate reader, multiple controls and experimental conditions can be quantified simultaneously. The protocol presented herein allows the immediate and continuous monitoring and quantification of oxidative stress responses during infection of macrophages with intracellular microbes (Figure 1).

Figure 1. Timeline and 24-well plate layout of the experimental procedure