Nutritional Immunity couples to Pathogen Recognition

Nutritional immunity comprises host antimicrobial defenses that aim to restrict essential metal ion availability to pathogens, in order to disturb microbial metabolic activity and pathogen fitness. Natural resistance-associated macrophage protein 1 (Nramp1) limits metal ion availability in the pathogen-containing phagosome as a mechanism to protect against intracellular pathogens. Nramp1 is a pH-dependent divalent metal transporter, which after pathogen uptake, localizes to phagolysosomes of macrophages and dendritic cells (DCs). By pumping metal ions from the vesicular lumen into the cytoplasm, Nramp1 generates a metal-deprived phagosomal environment that impairs pathogen survival. Additionally, Nramp1 has been associated with the up-regulation of proinflammatory macrophage and DC functions, including an enhanced cytokine response and respiratory burst. Inflammatory immune responses are initiated upon recognition of microbes by pattern recognition receptors, such as Toll-like receptors (TLRs), on host cells. Interestingly, endosomal TLRs, including TLR7, TLR8, and TLR9, seem to localize to similar endolysosomal compartments as Nramp1, suggesting a possible crosstalk between the metal transporter and intracellular TLRs. Indeed, Nramp1 has been shown to enhance TLR7 signal transduction in response to ssRNA ligands. However, the underlying mechanism and the relevance of this effect to pathogen infections is unknown. I hypothesize that Nramp1 regulates TLR signaling by affecting the processing and activation of endosomal TLRs. By performing an initial screen with receptor-specific ligands, I plan to identify those TLRs showing a potential crosstalk with Nramp1. The novelty and strength of the proposed project will be to decipher the underlying mechanism of Nramp1-mediated TLR regulation, to extend the findings from the individual TLR ligands to live pathogens, and finally translate the effects into an infection model. Specifically, a Candida glabrata (Cg) interaction model will be used to study the Nramp1-dependent activation of TLR7. Cg is an intracellular fungal pathogen that triggers type I interferon production through TLR7, providing a specific read-out for TLR activation. Finally, I aim to delineate whether the Nramp1 effect on TLR signaling results primarily from its function in promoting phagosome acidification, ROS production or its autonomous function as a metal transporter. The proposed project will significantly advance our understanding of metal homeostasis in host-defense. It has the potential to add an entirely new aspect to the regulation of TLR signaling by environmental cues within the phagosome and might open up new strategies to treat infectious diseases.

The main finding of this project was the identification of a novel regulatory pathway that controls Toll-like receptor trafficking in order to limit the potential harmful recognition of self-ligands and prevent autoimmune responses. Toll-like receptors (TLRs) are sensors of our immune system that evolved to detect microbial products and initiate an immune response to infection. However, some of these products are not unique to pathogens and TLRs have to successfully distinguish self-derived structures from foreign ones. Loss of this self vs. non-self discrimination leads to uncontrolled activation of the immune system promoting the development of autoimmune or inflammatory diseases. This concept is especially well described for TLR7 and TLR9, two members of the intracellular Toll-like receptors that recognize RNA and DNA ligands, respectively. The correct intracellular localization of these receptors is a crucial determinant for successful discrimination between host or pathogen-derived nucleic acids. Indeed, aberrant localization of TLR7/9 has been shown to facilitate the recognition of self-nucleic acids. Therefore, I hypothesized that dysregulated trafficking and localization of TLRs could promote the break in self-tolerance and contribute to the development of autoimmune disease, such as lupus. Trafficking of TLRs is regulated by the transmembrane protein Unc93b1, which delivers the receptors to the right places within the cell. To gain a better understanding of how this protein regulates TLR trafficking I performed a large-scale mutagenesis screen of Unc93b1. The goal was to identify novel regulatory motifs in Unc93b1 that control TLR trafficking and signaling. Through this screen I discovered a new regulatory pathway that helps maintain immunological tolerance of TLR7/9 to self-derived nucleic acids. This regulation involves post-translational modifications of Unc93b1. In the absence of this modification macrophages respond stronger to DNA and RNA ligands. In ongoing work I am testing if this regulatory pathway is relevant to the development of lupus disease. My findings could uncover fundamental aspects of the pathogenesis of autoimmune diseases.