Periodic Reporting for period 1 - 4Dplus_Metaflux (Multidimensional in vivo metabolic flux analyses: Resolving immune cells based on in vivo metabolic phenotypes)
Berichtszeitraum: 2023-06-01 bis 2024-11-30
Immunometabolism studies of the metabolism of immune cells and how this impacts immune responses in health and disease. The current challenge is to measure the metabolism of single cells directly after extraction or even within the disease environment. This is important because immune cells can change their metabolism after culture in the lab. New technological advances are needed to meet this challenge.
This project aimed to develop accurate assays of nutrient uptake, the first step in immune cell metabolism, using bioorthogonal (click) chemistry. A nutrient probe is used with a Click-handle that is transported into cells. Later, after isolated click chemistry is used to attach a click-fluorphore to the click-nutrient in the cell. Uptake is quantified as the light signal from the fluorophore measured by flow cytometry.
We aimed to multiplex our novel nutrient uptake assays using click-chemistry in different modes and with various click-handle pairs. The goal, to develop technology to measure 3-4 seperate metabolic fluxes in each individual cell from a complex immune cell mixture. The focus was on measure uptake of different types of amino acids and fatty acids, alongside an internal metabolic flux such as protein translation.
This project aimed to develop accurate assays of nutrient uptake, the first step in immune cell metabolism, using bioorthogonal (click) chemistry. A nutrient probe is used with a Click-handle that is transported into cells. Later, after isolated click chemistry is used to attach a click-fluorphore to the click-nutrient in the cell. Uptake is quantified as the light signal from the fluorophore measured by flow cytometry.
We aimed to multiplex our novel nutrient uptake assays using click-chemistry in different modes and with various click-handle pairs. The goal, to develop technology to measure 3-4 seperate metabolic fluxes in each individual cell from a complex immune cell mixture. The focus was on measure uptake of different types of amino acids and fatty acids, alongside an internal metabolic flux such as protein translation.
We have successfully developed multiple new nutrient uptake assays that measure bona fide cellular uptake using this bioorthogonal chemistry (Click) approach. This has been achieved using two separate Click chemical reactions; (1) the Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and (2) Copper independent Alkene and tetrazine inverse-demand Diels-Alder. These have been used to achieve the following:
(A) Using reaction (1) we developed an accurate assay for measuring amino acid uptake through Slc1a5, the dominant glutamine transporter in many immune cells. This work was published in Cell Reports in 2022.
(B) Reaction (1) can be used in two directions depending on where the azide and alkyne are placed. We have measured Slc1a5 uptake and the rate of protein translation (using an alkyne containing puromycin) together with single cell resolution in numerous immune contexts.
(C) Our collaborator has provided us with 5 different fatty acid (FA) molecules that contain a cylopropene click-handle that reacts with a tetrazine-fluorophore (reaction 2 above); these are click-analogues of arachidonic acid, linoleic acid, stearic acid, palmitic acid, oleic acid, and palmitoleic acid. FA acid uptake of different species has been measure/quantified using flow cytometry and confocal microscopy.
(D) Following the injection of L-Homopropargylglycine (HPG), which is a probe transported by Slc1a5, we could measure uptake that occurred in the mouse spleen that matched closely to results obtaine when we did the uptake after isolation of splenocytes. This is a major achievement and paves the way to measure nutrient uptake within a mouse at the site of disease.
(E) We have developed a process to combine click uptake assays with Seahorse extracellular flux analysis (see attached image). The Seahorse technique has been central to many of the advancements in the immunometabolism field as it allows for the combined measurement of glycolytic flux and oxidative phosphorylation (OXPHOS). One draw back of Seahorse analysis is that it requires 10^5 purified cells, and often is is not possible to get this number of cells directly ex vivo. Our modification of the Seahorse technology allows for analysis of mixed immune populations giving data on single cell nutrient uptake alongside the bulk metabolic flux measurements. Part of the Seahorse approach involves the sequential injection of inhibitors that allow the researcher to generate more information such as glycolytic capacity and spare respiratory capacity; there are 4 injection ports to allow the administration of different inhibitors/compounds. We used the 3rd and 4th injection ports to introduce our Click-Nutrients to allow for in-Seahorse nutrient uptake and then a fixative to fix the cells. The cells were then recovered, stained and processed to measure cells identity and nutrient uptake by flow cytometry.
(A) Using reaction (1) we developed an accurate assay for measuring amino acid uptake through Slc1a5, the dominant glutamine transporter in many immune cells. This work was published in Cell Reports in 2022.
(B) Reaction (1) can be used in two directions depending on where the azide and alkyne are placed. We have measured Slc1a5 uptake and the rate of protein translation (using an alkyne containing puromycin) together with single cell resolution in numerous immune contexts.
(C) Our collaborator has provided us with 5 different fatty acid (FA) molecules that contain a cylopropene click-handle that reacts with a tetrazine-fluorophore (reaction 2 above); these are click-analogues of arachidonic acid, linoleic acid, stearic acid, palmitic acid, oleic acid, and palmitoleic acid. FA acid uptake of different species has been measure/quantified using flow cytometry and confocal microscopy.
(D) Following the injection of L-Homopropargylglycine (HPG), which is a probe transported by Slc1a5, we could measure uptake that occurred in the mouse spleen that matched closely to results obtaine when we did the uptake after isolation of splenocytes. This is a major achievement and paves the way to measure nutrient uptake within a mouse at the site of disease.
(E) We have developed a process to combine click uptake assays with Seahorse extracellular flux analysis (see attached image). The Seahorse technique has been central to many of the advancements in the immunometabolism field as it allows for the combined measurement of glycolytic flux and oxidative phosphorylation (OXPHOS). One draw back of Seahorse analysis is that it requires 10^5 purified cells, and often is is not possible to get this number of cells directly ex vivo. Our modification of the Seahorse technology allows for analysis of mixed immune populations giving data on single cell nutrient uptake alongside the bulk metabolic flux measurements. Part of the Seahorse approach involves the sequential injection of inhibitors that allow the researcher to generate more information such as glycolytic capacity and spare respiratory capacity; there are 4 injection ports to allow the administration of different inhibitors/compounds. We used the 3rd and 4th injection ports to introduce our Click-Nutrients to allow for in-Seahorse nutrient uptake and then a fixative to fix the cells. The cells were then recovered, stained and processed to measure cells identity and nutrient uptake by flow cytometry.
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.
homo
(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.)
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.
homo
(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.)