Periodic Reporting for period 1 - Redox in macrophages (Adaptive redox regulation in inflammatory macrophages)
Reporting period: 2022-09-01 to 2024-08-31
Macrophages are an essential part of the innate immune system. Macrophages have diverse function that include clearing billions of dead cells each day, identifying and killing invading pathogens and initiating and terminating immune responses. They are first responders to pathogens, but also detect and eliminate cancer cells and maintain homeostasis by orchestrating the temporal progression of the wound healing response. We know now that macrophage functions are dictated by their underlying metabolic programs and metabolic adaptability that frequently center on electron transfer reactions (i.e. redox metabolism). For example, macrophages oxidize arginine to generate nitric oxide for pathogen defense; re-routing arginine for this purpose causes substantial changes to overall amino acid metabolism not observed in other cell types. Importantly, the cellular balance of redox reactions is particularly important to prevent cell death by ferroptosis. Ferroptosis is triggered by the accumulation of free Fe2+ iron and excessive free radicals (e.g. as needed for pathogen killing), leading to peroxidation of lipids in the cell membrane and ultimately cell death. Several antioxidant systems work in combination to clear free radicals and thus prevent ferroptosis. These include the glutathione pathway as well as glutathione independent biopterin and ubiquinone pathways and extracellular production of indoles from amino acids.
While these redox pathways were discovered in cancer cells, their function and regulation in macrophages (which are major producers of free radicals) was unknown at the start of this project. To address this knowledge gap, this MSCA project used reduced complexity in vitro models of macrophage activation states to answer a) which molecular pathways regulate redox adaptation in different macrophage activation states (i.e. inflammatory, chronic-inflammatory, homeostatic) and b) how macrophages can rewire their redox systems in activation compared to homeostasis.
While these redox pathways were discovered in cancer cells, their function and regulation in macrophages (which are major producers of free radicals) was unknown at the start of this project. To address this knowledge gap, this MSCA project used reduced complexity in vitro models of macrophage activation states to answer a) which molecular pathways regulate redox adaptation in different macrophage activation states (i.e. inflammatory, chronic-inflammatory, homeostatic) and b) how macrophages can rewire their redox systems in activation compared to homeostasis.
Using macrophage activation with either Th2 cytokines IL4 + IL13, bacterial lipopolysaccharide (LPS), the pro-inflammatory cytokine TNF or a combination of IL4 + IL13 + TNF activation (modelling chronic or a non-resolving inflammatory macrophage state), this project identified high expression of the cystine transporter SLC7A11 in chronic inflammatory macrophages, as opposed to induction of the heme degradation pathway (heme oxygenase, HMOX). Furthermore, we identified that IL4 + IL13 + TNF activated macrophages are protected from redox stress (via inhibition of glutathione peroxidase, GPX4) and ferroptosis. Expression of the L-amino acid oxidase IL4i1 and the Krebs cycle associated enzyme aconitase decarboxylase (ACOD1) was highly increased in macrophages activated with IL4 + IL13 + TNF. IL4i1 has previously been shown by this laboratory to generate metabolites (from the oxidation of tryptophan and tyrosine) that suppress ferroptosis. Importantly, we showed using macrophages derived from Il4i1-/- or Acod1-/- that ferroptosis protection was not dependent on these key redox nodes. However, since we detected tryptophan metabolites produced by IL4i1 in the macrophage supernatant following specific activation cues, I next developed an innovative supernatant transfer assay system to assess metabolite communication between macrophages and bystander cancer cells, which I term paracrine metabolite signaling. Indeed, this system showed that chronic inflammatory macrophages (IL4-IL13-TNF activated) could confer ferroptosis protection to bystander cancer cells in a paracrine manner, and this was dependent on the expression of IL4i1.
Furthermore, metabolic profiling showed that the immune modulatory metabolite itaconate was highly increased in macrophages activated with IL4 + IL13 + TNF (as well as upon activation with TNF or LPS). However, intrinsic ferroptosis protection was independent of itaconate (identified using Acod1-/- mice) and its effect on extrinsic ferroptosis protection is currently being followed up.
Furthermore, metabolic profiling showed that the immune modulatory metabolite itaconate was highly increased in macrophages activated with IL4 + IL13 + TNF (as well as upon activation with TNF or LPS). However, intrinsic ferroptosis protection was independent of itaconate (identified using Acod1-/- mice) and its effect on extrinsic ferroptosis protection is currently being followed up.
Overall, results from this project discovered paracrine communication between chronic inflammatory macrophages and bystander cancer cells through regulated metabolite signaling. This overall outcome links the fields of innate immunology and cancer biology in an unexpected way and may thus allow the identification of new targets (i.e. macrophage subsets that make IL4i1) in the tumor niche. Furthermore, the project opened avenues to explore the role of chronic inflammatory activated macrophages in other disease contexts (for example during lung fibrosis) and identified new knowledge gaps such as the identify of transporter systems that allow uptake of signaling metabolites in recipient cells.