This protocol offers the opportunity to follow immune polarization of human monocyte-derived cells based on visualization and deep characterization of green fluorescent protein-labeled Mtb in diverse macrophage subsets using 10 color flow cytometry. This represents an efficient and reproducible method to study responses to Mtb infection in M1 or M2 polarized macrophages, including assessment of phenotype and function before and after infection. To isolate PBMC from human donor buffy coats, use a pipette to slowly overlay 15 milliliters of buffy coat blood down the side of a 50 milliliter tube onto 15 milliliters of density gradient medium and separate the cells by centrifugation.
Use a sterile Pasteur pipette to remove the top plasma layer and carefully transfer the mononuclear cell layer into a new 50 milliliter tube. Add serum-free RPMI medium to the cells to a final volume of 50 milliliters and gently invert the tube a few times before centrifuging. Then resuspend the cells in 20 milliliters of serum-free medium for counting.
To initiate macrophage differentiation, dilute the cells to a 5 times 10 to the 6th cells per milliliter of serum-free medium concentration and seed two milliliters of cells into individual wells of a six-well plate. Place the plate in the cell culture incubator for two to three hours to allow the cells to adhere to the well bottoms. At the end of the incubation, wash the wells three times with one milliliter of serum-free medium per wash, and add two milliliters of complete RPMI medium supplemented with 50 nanograms per milliliter of GM-CSF or 50 nanograms per milliliter of M-CSF for M1 or M2 differentiation respectively to each well of adherent cells.
Three days after polarization initiation, carefully replace one milliliter of supernatant from each well with one milliliter of fresh complete medium supplemented with the appropriate growth factors. On day six of culture, add 50 nanograms per milliliter of interferon gamma and 10 nanograms per milliliter of LPS to the GM-CSF treated wells and 20 nanograms per milliliter of IL-4 to the M-CSF treated wells. Check the morphology of the monocyte-derived cell cultures regularly by light microscopy to ensure that the smaller monocytes are differentiating into larger macrophage-like cells.
To set up an Mtb culture, in a biosafety Level III facility, mix a one milliliter aliquot of thawed bacteria suspension with nine milliliters of TB complete medium in a 50 milliliter filtered cap tube for a 24-hour incubation in the tube in the cell culture incubator. The next day, collect the bacteria by centrifugation and resuspend the pellet in 15 to 20 milliliters of fresh TB complete medium in a new 50 milliliter filtered cap culture tube before returning the bacteria to the cell culture incubator. After seven to 10 days of culture, wash the bacteria two times in 35 to 40 milliliters of sterile wash buffer per wash, resuspending the bacteria in one milliliter of serum-free RPMI medium after the second wash.
Add another nine milliliters of serum-free RPMI medium to the bacteria and sonicate the bacteria in a water bath sonicator in Class II biosafety cabinet for five minutes at 37 degrees Celsius. Then measure the optical density of one milliliter of bacterial suspension at a 600 nanometer wavelength in a spectrophotometer placed inside the biosafety cabinet. To infect the monocyte-derived cells, dilute the bacteria to a 5 times 10 to the 6th colony forming units per milliliter concentration in serum-free medium and replace the supernatant in each well of the six-well cell culture plate with one milliliter of fresh serum-free medium and one milliliter of bacteria suspension per well to obtain a multiplicity of infection of five.
After a four-hour incubation in the cell culture incubator, wash each well three times with one milliliter of sterile wash buffer per wash, tilting the plate carefully to remove the entire volume of buffer from the corners of the wells after each wash. Then resuspend the Mtb-infected monocyte-derived cells in two milliliters of complete medium without antibiotics. To stain the cells for flow cytometry, incubate the infected cells with one milliliter of FACS buffer supplemented with 0.5 millimolar EDTA per well for at least 30 minutes.
At the end of the incubation, collect the detached cells with gentle pipetting and transfer the cells into individual screw capped microcentrifuge tubes for centrifugation. Resuspend the cells in approximately 50 microliters of fluorochrome-conjugated anti-human antibody cocktail and viability dye of interest and incubate the cells for 30 minutes at four degrees Celsius protected from light. After washing, fix the cells with 200 microliters of freshly prepared fixation buffer for 30 minutes at room temperature protected from light.
After the fixation and washing, resuspend the cells in 400 microliters of FACS buffer and transfer the cells to one milliliter microcentrifuge tubes. To analyze the cells, use compensation beads to compensate the fluorescent signal for each fluorochrome-conjugated antibody in the cocktail and use unstained cells to set the gate for the negative cell population. Then acquire a minimum of 50, 000 cells per sample.
Both M1 and M2 monocyte-derived cells display a vertical shift to a higher granularity and reduced cell size upon Mtb infection. An enhanced cell death is also observed in Mtb infected M1 and M2 cells at a multiplicity of infection of five compared to uninfected M0 cells. Of note, a substantially higher Mtb GFP expression is observed in M2 cells after four hours of infection compared to M1 cells.
Uninfected M1 and M2 cells can be characterized by their CD64 and CD86 or CD200 receptor and CD163 co-expression respectively. After four hours of Mtb infection, an increased frequency of CD200 receptor positive cells is observed in the Mtb GFP positive M1 polarized cells, while CD163 expression is reduced in M2 cells. Mtb GFP bacteria can also be observed in CD64 positive M1 cells and CD163 positive M2 cells by confocal microscopy.
UMAP analysis reveals that four hours of Mtb infection is not sufficient to affect macrophage polarization, while 24 hours of infection results in clearly separated clusters of uninfected and infected M1 and M2 cells that express distinct surface marker expression profiles. Further, PhenoGraph analysis shows that 24 different clusters of different sizes are uniquely distributed among the M1 and M2 uninfected and Mtb infected cells. The efficacy of M1 or M2 polarization or Mtb infectivity can vary substantially between human donors.
And therefore, inclusion of at least two donors in each experiment is recommended to reduce experimental variations. This model allows simultaneous assessment of human macrophages using flow cytometry, confocal microscopy, mRNA expression analysis, multiplex assays of soluble factors, and bacterial quantification using GFP expression or colony forming units.
