All the achievements listed above are novel and beyond the state of the art. These technologies are having, and will have, a significant impact upon the Immunometabolism research field. We are currently working with colleagues within Trinity College Dublin to apply the multiplexed uptake assays to disease models including traumatic brain injury (focusing on microglea), sepsis, and age related macular degeneration.
The individual components of these technologies are not patentable but we are exploring protecting IP around the process and the "know how" required to successfully implement the technology.
With respect to achievement (E), this is a novel combination of the pre-existing Seahorse technology and our novel click-uptake technology. We are now exploring potential applications in the areas of mechanism discovery and diagnostics.
Results:
(1) We have now measured glutamine uptake (Slc1a5) and protein synthesis simultaneously in a large number of immunlogical contexts. Immunization of mice with Poly(I:C), a mimic of viral infection, specifically increases both measurements specifically in NK cells. In sepsis we see time dependent changes in metabolic fluxes. There are differences in these metabolic parameters in NK cells depending on what type of tumour NK cells are isolated from. Also, there are dramatic differences for NK cells from different tissues.
(2) The uptake of the 5 FAs has been optimised in NK92 cells, then applied to study thymocyte development. Arachidonic acid consistently shows the highest level of uptake in all cells tested. Apart from arachidonic acid, the FA uptake was affected by the chain length (C18>C16) but not saturation (C18:0=C18:1 and C16:0=C16:1). Within thymocytes, the double negative DN3 and DN4 subsets have the highest FA uptake and the double positive subset the lowest uptake.
(3) We measured Slc1a5 uptake using HPG in vivo, first injecting HPG locally to an inflammatory lymph node, then injecting systemically (i.v.) and measuring uptake in the spleen. The results of in vivo uptake correlate very closely to that of ex vivo uptakes.
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(4) 4 dimensions of metabolic flux analysis have been measured in single cells using flow cytometry using cells isolated directly ex vivo. 4D flux analysis involved (1) Click-puromycin (measures protein translation rate), (2) HPG (glutamine uptake), (3) click-Oleic acid uptake (Fatty acid uptake) and kynurenine (Slc7a5 cargo that is naturally fluorescent) and 3 separate click reactions. From a chemistry standpoint a triple Click approach like to study single cell metabolism this is extremely innovative.
(5) We have developed a protocol to measure glycolysis and oxidative phosphorylation (OXPHOS) in mixed cells and then single cell uptake of 2 click-nutient probes using the Seahorse extracellular flux analyser (see workflow image attached). We applied this to the inflammed and non-inflamed peritoneal cavity (i.p injection of LPS or PBS) and measured elevated glycolysis and OXPHOS in bulk/mixed cells and identified NK cells as the only cell with increased HPG uptake (contributing most to OXPHOS) and macrophages to have increased 6AzGal uptake (contributing most to the increased glycolysis.)
