This study investigates how chronic alcohol misuse affects alveolar macrophage immune function by altering mitochondrial metabolism. Overall, our research highlights the intersection of alcohol use, metabolism and immune function, making it relevant to fields like immunology, pharmacology and public health. While alcohol use has known detrimental effects, the specific metabolic mechanisms and particularly the role of glutamine oxidation in alveolar macrophages are not fully understood.
Additionally, this protocol fills a gap methodologically allowing for reproducibility in future studies. This protocol allows for real-time sensitive mitochondrial respiration measurements of multiple technical and biological replicates. It can be adjusted for other cell types and is more reliable for assaying metabolic function than other techniques that are more time consuming or require larger sample sizes.
These results clarify the impact of ethanol exposure on alveolar macrophage glutamine metabolism, which may influence therapeutic interventions in future studies. In general, we expect to see a shift toward understanding cellular glutamine metabolism and other disease models. Our work has focused on understanding how lung macrophages metabolize fuel sources for energy.
Since macrophages need a lot of energy for immune functions, like signaling to other immune cells and pathogen phagocytosis and clearance, our next steps will focus on linking shifts in energy metabolism with macrophage immune function. To begin, take cultured MHS cells treated with ethanol for 72 hours and grown to at least 50%confluency in T75 flasks. Create a plate map with five to six technical replicates for each biological replicate under control and ethanol-treated conditions, including media controls for BPTES comparison.
Rinse the cells with fresh media or PBS to remove debris and floating cells. Then add six milliliters of serum-containing media to the T75 flask to nourish the cells. Using a cell scraper, detach the cells until the bottom of the flask is clear.
Separate the detached cells by pipetting them using a serological pipette or a one-milliliter pipette. Transfer the suspended cells into a 15-milliliter conical tube and centrifuge at 200G for six minutes to pellet the cells. Then aspirate the supernatant from the centrifuge cell pellet.
Add one milliliter of media to the pellet, and re-suspend the cells thoroughly. Transfer 10 microliters of the cell suspension to a microcentrifuge tube. Add 10 microliters of trypan blue and mix the solution well.
Now, transfer 10 microliters of the cell trypan blue mixture onto one side of a hemocytometer or a cell counter, to count the cells. Calculate the dilution required based on the number of wells and cells needed using the formula shown here. Then apply 80 microliters of the re-suspended cells to the required wells in the 96-well extracellular flux microculture plate.
Incubate the cells overnight in a 37-degree-Celsius, humidified incubator with 5%carbon dioxide to allow them to adhere and equilibrate in the plate. After treatment, remove the top portion of the extracellular flux pack and place the probed portion of the flux pack face up on the bench. Apply 200 microliters of extracellular flux calibrant solution to each well in the bottom part of the extracellular flux pack cartridge.
Assemble the extracellular flux pack cartridge by reattaching the sections and wrapping it in Parafilm. Place the cartridge in a 37-degree-Celsius, non-carbon-dioxide, humidified incubator, or bead bath, to equilibrate. Finally, turn on the computer with the assay design and open the analysis software connected to the extracellular flux bioanalyzer instrument.
Begin by checking the control and ethanol-treated MHS cells to ensure they are healthy and adhered in a monolayer under a microscope. To prepare the extracellular flux base medium, aliquot 35 milliliters into a 50-milliliter tube. Add 350 microliters each of 100-millimolar sodium pyruvate, D-glucose and GlutaMAX for stability.
Warm the prepared solution to 37 degrees Celsius. Then gently aspirate the growth media from the extracellular flux microculture plate, leaving minimal residual media without disturbing the cells. Wash the wells with up to 50 microliters of extracellular flux base medium.
Next, add 180 microliters of extracellular flux base medium to each well. Incubate the extracellular microculture plate in a 37-degree Celsius, non-carbon-dioxide, humidified incubator for 30 minutes to one hour to allow equilibration. Add 700 microliters of extracellular flux base medium to the BPTES tube to make a 120-millimolar stock solution.
Pipette the solution for proper solubilization. Next, add 630 microliters of extracellular flux base medium to the oligomycin tube to prepare a 100-millimolar stock solution, and mix thoroughly. Then add 720 microliters of extracellular flux base medium to the FCCP tube to prepare a 100-millimolar stock solution.
Pipette the solution up and down 10 times to properly solubilize the compound. Add 540 microliters of extracellular flux base medium to the RA tube to prepare a 50-millimolar stock solution, and mix by pipetting. To prepare a 30-micromolar BPTES working solution, mix 1, 500 microliters of extracellular flux base medium with 500 microliters of 120-millimolar BPTES stock solution.
Mix 2, 520 microliters of extracellular flux base medium with 480 microliters of 100-millimolar oligomycin stock solution to create a 16-millimolar working solution. Then prepare the FCCP working solution by mixing 2, 865 microliters of extracellular flux supplemented base medium with 135 microliters of 100-millimolar FCCP stock solution. To prepare the RA working solution, mix 2, 700 microliters of extracellular flux supplemented base medium with 300 microliters of 50-millimolar RA stock solution.
Load the background, media control and cell-containing wells with the reagents shown here. Ethanol and BPTES-MHS cells exhibited a lower glutamine-dependent basal oxygen consumption rate cells and ATP-linked mitochondrial respiration from glutamine compared to control cells. Ethanol and BPTES cells displayed less loss in maximal respiration and reduced glutamine-dependent spare respiratory capacity relative to control cells.
