Iron-deficiency causes aspartate-sensitive metabolic dysregulation in CD8+ T-cells

Megan TEH¹, Alexandra PRESTON¹, Jan REHWINKEL¹, Thomas MILNE⁴, Daniel TENNANT⁶, Susanna DUNACHIE⁵, Andrew ARMITAGE¹, Sarah DIMELOE², Hal DRAKESMITH¹, Nancy GUDGEON², Joe FROST¹, Linda SINCLAIR³, Alastair SMITH⁴, Christopher MILLINGTON⁴, Barbara KRONSTEINER⁵, Jennie ROBERTS⁶, Bryan MARZULLO⁶

¹MRC Translational Immune Discovery Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom
²Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, United Kingdom
³Cell Signalling and Immunology, University of Dundee, Dundee, UK , Dundee, United Kingdom
⁴MRC Molecular Hematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom
⁵NDM Centre for Global Health Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom
⁶Institute of Metabolism and Systems Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom

Iron is an essential micronutrient which interacts with ~400 human proteins involved in processes including mitochondrial metabolism, epigenetic regulation and DNA synthesis. Meanwhile, iron deficiency affects >1.2 billion people and is associated with anaemia and suppressed immune responses. However, the biochemical mechanisms underlying these effects remain unclear. Using CD8+ T-cells as a model of normal proliferating cells, we investigated how low iron availability influences cellular biochemistry. Iron starvation reduced cell proliferation, induced the P53 cell cycle arrest pathway and suppressed genes involved in mTORC1 and MYC signalling indicative of aberrant metabolic rewiring. The metabolic dysfunction appeared largely mitochondrial with iron starved cells featuring increased mROS generation and reduced mitochondrial membrane potential suggestive of ETC dysfunction. TCA cycle progression was impaired at the iron dependent enzymes ACO2 and SDH, depleting downstream metabolites including α-ketoglutarate. H3K27me3, a repressive histone mark removed by the iron and α-ketoglutarate dependent KDM6A/B enzymes during T-cell activation, significantly accumulated in iron deprived cells indicating that iron-deficiency impairs epigenetic reprogramming. Despite TCA cycle dysfunction, aspartate, produced downstream of the TCA cycle, was unexpectedly increased in iron restriction while nucleotide precursors downstream of aspartate incorporation were significantly depleted suggesting reduced aspartate usage. Exogenous aspartate substantially rescued the proliferation of iron deprived cells suggesting that endogenous aspartate sources are unusable, possibly due to mitochondrial aspartate trapping. Overall, iron deficiency results in a profound mitochondrial dysfunction resulting in impaired aspartate utilisation. This work provides insight as to how metabolic and iron modulatory interventions could be coupled to augment or suppress immunity.