The natural-resistance associated macrophage protein 1, Slc11a1, is a phagolysosomal transporter for protons and divalent ions including iron, that confers host protection against diverse intracellular pathogens including Salmonella. We investigated and compared the regulation of iron homeostasis and immune function in RAW264.7 murine phagocytes stably transfected with non-functional Slc11a1 and functional Slc11a1 controls in response to an infection with Salmonella enterica serovar Typhimurium (S. Typhimurium). We report that macrophages lacking functional Slc11a1 displayed an increased expression of transferrin receptor 1, resulting in enhanced acquisition of transferrin-bound iron. In contrast, cellular iron release mediated via ferroportin 1 was significantly lower in Salmonella-infected Slc11a1-negative macrophages in comparison to phagocytes bearing Slc11a1. Lack of Slc11a1 led to intracellular persistence of S. Typhimurium within macrophages which was paralleled by a reduced formation of nitric oxide, tumour necrosis factor-alpha and interleukin-6 in Slc11a1-negative macrophages following Salmonella infection, whereas interleukin-10 production was increased. Moreover, Slc11a1-negative phagocytes exhibited higher cellular iron content, resulting in increased iron acquisition by intracellular Salmonella. Our observations indicate a bifunctional role for Slc11a1 within phagocytes. Slc11a restricts iron availability, which firstly augments pro-inflammatory macrophage effector functions and secondly concomitantly limits microbial iron access.

Natural-resistance associated macrophage protein 1 (Nramp1) encodes a transmembrane phagolysosomal protein exerting resistance toward infections with intracellular pathogens by a mechanism not fully elucidated so far. We used the murine macrophage cell line RAW264.7, stably transfected with functional (RAW-37) or nonfunctional (RAW-21) Nramp1, to study for differences in the expression of NO, a central antimicrobial effector molecule of macrophages. Following stimulation with IFN-γ and LPS, Nramp1-expressing cells exhibit higher enzymatic activity of inducible NO synthase (iNOS) and increased cytoplasmic iNOS mRNA levels than RAW-21 cells. Time-course experiments showed that iNOS-mRNA levels remain increased in RAW-37 cells after prolonged cytokine stimulation while they decrease in RAW-21 cells. Reporter gene assays with iNOS-promoter luciferase constructs demonstrated an increased and prolonged promoter activity in Nramp1-resistant vs susceptible cells. This was paralleled by increased IFN regulatory factor 1 (IRF-1) expression and binding affinity to the iNOS promoter in RAW-37 cells, which may be related to enhanced STAT-1 binding affinity in these cells. A point mutation within the IRF-1 binding site of the iNOS promoter abolished the differences in iNOS transcription between RAW-21 and RAW-37 cells. Cells carrying functional Nramp1 express increased amounts of NO, which may be related to STAT-1-mediated stimulation of IRF-1 expression with subsequent prolonged activation of iNOS transcription. Enhanced NO expression may partly underlie the protection against infection with intracellular pathogens by Nramp1 functionality.

In mice, resistance to certain intracellular microbes depends on the expression of a late phagosomal protein termed natural‐resistance associated macrophage protein 1 (Nramp1, Slc11a1). Nramp1‐functionality is associated with alterations of cellular iron homeostasis and a sustained pro‐inflammatory immune response, including the formation of the antimicrobial effector molecule NO. To investigate the underlying mechanism we used RAW‐264.7 murine macrophage cells stably transfected with a functional Nramp1 allele (RAW‐37) or Nramp1 non‐functional controls (RAW‐21). We found that the production of and signalling by the anti‐inflammatory cytokine IL‐10 was significantly enhanced in macrophages lacking functional Nramp1. Upon infection of macrophages with Salmonella typhimurium pathogen survival was significantly better in RAW‐21 than in RAW‐37, which inversely correlated to NO and TNF‐α formation. Addition of a neutralising anti‐IL‐10 antibody to RAW‐21 cells led to a significantly reduced survival of S. typhimurium within these cells and enhanced formation of NO and TNF‐α reaching levels comparable to that observed in cells bearing functional Nramp1. Oppositely, supplementation of iron to RAW‐21 cells further increased IL‐10 formation. Thus, Nramp1 mediates effective host defence in part via suppression of excessive IL‐10 production which may relate to Nramp1‐mediated reduction of cellular iron pools, thus strengthening antimicrobial effector mechanisms.

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