Christa Pfeifhofer-Obermair

Nuclear receptors regulate key functions of mononuclear phagocytes and are critical components of the innate immune system, acting as regulators of organ health and disease. In healthy mice, the loss of the nuclear orphan receptor NR2F6 alters tissue‐resident macrophage populations in the liver, lung, and spleen. In response to Salmonella Typhimurium infection, Nr2f6 ‐deficient mice exhibit improved clinical outcomes, characterized by reduced weight loss, bacterial loads in the spleen and liver, and decreased plasma pro‐inflammatory cytokines. Despite unchanged basal iron metabolism in the spleen and liver, iron regulatory proteins and the interleukin (IL)‐6‐hepcidin axis are altered in Nr2f6‐ deficient mice during Salmonella infection, reducing hypoferremia. Transcriptomic analysis of splenic red pulp macrophages reveals significant alterations of phagocytosis‐related genes, including upregulation of signal‐regulatory protein alpha (Sirpa). In vitro, phagocytosis of red blood cells, regulated by the inhibitory CD47‐Sirpα axis, and Salmonella Typhimurium phagocytosis are significantly impaired in Nr2f6 ‐deficient splenic macrophages. Blocking Sirpα in vitro restores the phagocytic activity of Nr2f6 ‐deficient macrophages to wild‐type levels. In vivo , Salmonella Typhimurium loads are partially increased post‐infection in anti‐Sirpα treated Nr2f6 ‐deficient mice. These findings uncover a previously unrecognized role of NR2F6 in host‐pathogen interactions, positioning it as a potential therapeutic target for infectious diseases.

Background: Ether lipids are important constituents of biological membranes and harbor fatty alcohols attached via ether linkages to the sn-1 position of the glycerol backbone. Depending on the nature of the ether bond, they are subdivided into 1-O-alkyl (plasmanyl) and 1-O-alk-1′-enyl (plasmenyl) subclasses. They often contain polyunsaturated fatty acids at the sn-2 position, implicating them in cellular signaling and inflammatory processes including lipid mediator biosynthesis. Lipid mediators are produced by immune and non-immune cells, have diverse homeostatic and immunoregulatory functions and, together with other factors, orchestrate the initiation and resolution of inflammation. To date, alkylglycerol monooxygenase is the only known enzyme capable of cleaving alkylglycerols, one of two ether lipid subclasses. However, the exact role of alkylglycerol monooxygenase and that of its substrates in lipid mediator biosynthesis remains unclear. Results: Using a knockout mouse model, we demonstrate a sex- and cell type-dependent role of alkylglycerol monooxygenase in limiting prostanoid formation without affecting polyunsaturated fatty acid release, as revealed by metabololipidomics profiling of lipid mediators using ultra-performance liquid chromatography‒tandem mass spectrometry. This female-specific effect is driven by the suppression of prostaglandin G/H synthase 2 transcription, as deficiency in alkylglycerol monooxygenase significantly elevated prostaglandin G/H synthase 2 gene expression in female bone marrow-derived macrophages of the M1 phenotype. Furthermore, this regulatory role of alkylglycerol monooxygenase extends to visceral white adipose tissue, where elevated prostaglandin G/H synthase 2 expression and enhanced prostaglandin E₂ production were observed in female samples following alkylglycerol monooxygenase knockout. Conclusion: Our results expand the immunomodulatory functions of ether lipid metabolism and highlight the sex- and cell type-dependent role of alkylglycerol monooxygenase in controlling lipid mediator production and maintaining tissue homeostasis.

Priming of macrophages with interferon-gamma (IFNγ) or interleukin-4 (IL-4) leads to polarisation into pro-inflammatory or anti-inflammatory subtypes, which produce key enzymes such as inducible nitric oxide synthase (iNOS) and arginase 1 (ARG1), respectively, and in this way determine host responses to infection. Importantly, L-arginine is the substrate for both enzymes. ARG1 upregulation is associated with increased pathogen load in different infection models. However, while differentiation of macrophages with IL-4 impairs host resistance to the intracellular bacterium Salmonella enterica serovar Typhimurium (S.tm), little is known on the effects of IL-4 on unpolarised macrophages during infection. Therefore, bone-marrow-derived macrophages (BMDM) from C57BL/6N, Tie2Cre^+/−ARG1^fl/fl (KO), Tie2Cre^−/−ARG1^fl/fl (WT) mice were infected with S.tm in the undifferentiated state and then stimulated with IL-4 or IFNγ. In addition, BMDM of C57BL/6N mice were first polarised upon stimulation with IL-4 or IFNγ and then infected with S.tm. Interestingly, in contrast to polarisation of BMDM with IL-4 prior to infection, treatment of non-polarised S.tm-infected BMDM with IL-4 resulted in improved infection control whereas stimulation with IFNγ led to an increase in intracellular bacterial numbers compared to unstimulated controls. This effect of IL-4 was paralleled by decreased ARG1 levels and increased iNOS expression. Furthermore, the L-arginine pathway metabolites ornithine and polyamines were enriched in unpolarised cells infected with S.tm and stimulated with IL-4. Depletion of L-arginine reversed the protective effect of IL-4 toward infection control. Our data show that stimulation of S.tm-infected macrophages with IL-4 reduced bacterial multiplication via metabolic re-programming of L-arginine-dependent pathways.

Macrophages are at the center of innate immunity and are the main target cells of the intracellular pathogen Salmonella enterica serovar Typhi. The production of reactive oxygen and nitrogen species (ROS/RNS) is the host's early response to invading microbes, as oxidative stress is highly toxic for bacteria. Adequate ROS/RNS production in infected macrophages is critical for the clearance of intracellular pathogens; this is achieved by several enzymes, including inducible NADPH phagocyte oxidase (NOX) and nitric oxide synthase (iNOS), respectively. The proinflammatory cytokine interferon gamma (IFN gamma), primarily produced by activated natural killer cells and T-helper cells type 1, is a potent inducer of iNOS. Therefore, it is crucial for infection control through oxidative microbicidal activity. To characterize the early oxidative stress response via ROS formation, which is critical for the reduction of Salmonella proliferation within macrophages, we established an in vitro model of murine macrophages infected with Salmonella enterica serovar Typhimurium (S.tm). This serovar induces a systemic infection in mice that is frequently used as a model for typhoid fever, which, in human subjects, is caused by Salmonella Typhi. We generated bone marrow-derived macrophages (BMDM) from C57BL/6N wildtype mice using macrophage colony-stimulating factor (M-CSF) stimulation for six days. Thereafter, we infected BMDM with S.tm for one hour. Shortly before infection, cells were stained with CellROX (TM) Deep Red reagent. In its reduced form, CellROX (TM) is non-fluorescent. As a result of oxidation by ROS, this reagent exhibits strong fluorescence and persists within the cells. Subsequently, changes as a result of the oxidative stress response can be measured with a TECAN Spark microplate reader over time. We designed this protocol to measure oxidative stress in macrophages through the course of an infection with an intracellular bacterium. The protocol has several advantages over established techniques. First, it allows to continuously monitor and quantify ROS production in living cells from the very start of the infection to the final clearance of the intracellular pathogen. Second, this protocol enables efficient ROS detection without stressing the cells by detaching or staining procedures.

Macrophages are at the center of innate pathogen control and iron recycling. Divalent metal transporter 1 (DMT1) is essential for the uptake of non-transferrin-bound iron (NTBI) into macrophages and for the transfer of transferrin-bound iron from the endosome to the cytoplasm. As the control of cellular iron trafficking is central for the control of infection with siderophilic pathogens such as Salmonella Typhimurium, a Gram-negative bacterium residing within the phagosome of macrophages, we examined the potential role of DMT1 for infection control. Bone marrow derived macrophages lacking DMT1 (DMT1fl/fl^LysMCre(+)) present with reduced NTBI uptake and reduced levels of the iron storage protein ferritin, the iron exporter ferroportin and, surprisingly, of the iron uptake protein transferrin receptor. Further, DMT1-deficient macrophages have an impaired control of Salmonella Typhimurium infection, paralleled by reduced levels of the peptide lipocalin-2 (LCN2). LCN2 exerts anti-bacterial activity upon binding of microbial siderophores but also facilitates systemic and cellular hypoferremia. Remarkably, nifedipine, a pharmacological DMT1 activator, stimulates LCN2 expression in RAW264.7 macrophages, confirming its DMT1-dependent regulation. In addition, the absence of DMT1 increases the availability of iron for Salmonella upon infection and leads to increased bacterial proliferation and persistence within macrophages. Accordingly, mice harboring a macrophage-selective DMT1 disruption demonstrate reduced survival following Salmonella infection. This study highlights the importance of DMT1 in nutritional immunity and the significance of iron delivery for the control of infection with siderophilic bacteria.

Macrophages are important for host defense against intracellular pathogens like Salmonella and can be differentiated into two major subtypes. M1 macrophages, which are pro-inflammatory and induce antimicrobial immune effector mechanisms, including the expression of inducible nitric oxide synthase (iNOS), and M2 macrophages, which exert anti-inflammatory functions and express arginase 1 (ARG1). Through the process of phagocytosis, macrophages contain, engulf, and eliminate bacteria. Therefore, they are one of the first lines of defense against Salmonella. Infection with Salmonella leads to gastrointestinal disorders and systemic infection, termed typhoid fever. For further characterization of infection pathways, we established an in vitro model where macrophages are infected with the mouse Salmonella typhi correlate Salmonella enterica serovar Typhimurium (S.tm), which additionally expresses red fluorescent protein (RFP). This allows us to clearly characterize macrophages that phagocytosed the bacteria, using multi-color flow cytometry.In this protocol, we focus on the in vitro characterization of pro- and anti-inflammatory macrophages displaying red fluorescent protein-expressing Salmonella enterica serovar Typhimurium, by multi-color flow cytometry.

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Arginase 1 (ARG1) is a cytosolic enzyme that cleaves L-arginine, the substrate of inducible nitric oxide synthase (iNOS), and thereby impairs the control of various intracellular pathogens. Herein, we investigated the role of ARG1 during infection with Salmonella enterica serovar Typhimurium (S.tm). To study the impact of ARG1 on Salmonella infections in vitro, bone marrow-derived macrophages (BMDM) from C57BL/6N wild-type, ARG1-deficient Tie2Cre^+/−ARG1^fl/fl and NRAMP^G169 C57BL/6N mice were infected with S.tm. In wild-type BMDM, ARG1 was induced by S.tm and further upregulated by the addition of interleukin (IL)-4, whereas interferon-γ had an inhibitory effect. Deletion of ARG1 did not result in a reduction in bacterial numbers. In vivo, Arg1 mRNA was upregulated in the spleen, but not in the liver of C57BL/6N mice following intraperitoneal S.tm infection. The genetic deletion of ARG1 (Tie2Cre^+/−ARG1^fl/fl) or its pharmacological inhibition with CB-1158 neither affected the numbers of S.tm in spleen, liver and blood nor the expression of host response genes such as iNOS, IL-6 or tumour necrosis factor (TNF). Furthermore, ARG1 was dispensable for pathogen control irrespective of the presence or absence of the phagolysosomal natural resistance-associated macrophage protein 1 (NRAMP1). Thus, unlike the detrimental function of ARG1 seen during infections with other intraphagosomal microorganisms, ARG1 did not support bacterial survival in systemic salmonellosis, indicating differential roles of arginine metabolism for host immune response and microbe persistence depending on the type of pathogen.