Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon <em>Mycobacterium tuberculosis</em> Infection

Abstract

Human macrophages are primary host cells of intracellular Mycobacterium tuberculosis (Mtb) infection and thus have a central role in immune control of tuberculosis (TB). We have established an experimental protocol to follow immune polarization of myeloid-derived cells into M1 (classically activated) or M2 (alternatively activated) macrophage-like cells through assessment with a 10-color flow cytometry panel that allows visualization and deep-characterization of green-fluorescent-protein (GFP)-labeled Mtb in diverse macrophages subsets. Monocytes obtained from healthy blood donors were polarized into M1 or M2 cells using differentiation with granulocyte macrophage-colony-stimulating factor (GM-CSF) or macrophage-colony stimulating factor (M-CSF) followed by polarization with IFN-γ and lipopolysaccharide (LPS) or IL-4, respectively. Fully polarized M1 and M2 cells were infected with Mtb-GFP for 4 hours before detached Mtb-infected macrophages were stained with flow cytometry at 4-or 24-hours post-infection. Sample acquisition was performed with flow cytometry and the data was analyzed using a flow cytometry analysis software. Manual gating as well as dimensionality reduction with Uniform Manifold Approximation and Projection (UMAP) and phenograph analysis was performed. This protocol resulted in effective M1/M2 polarization characterized by elevated levels of CD64, CD86, TLR2, HLA-DR and CCR7 on uninfected M1 cells, while uninfected M2 cells exhibited a strong upregulation of the M2 phenotype markers CD163, CD200R, CD206 and CD80. M1polarized cells typically contained fewer bacteria compared to M2-polarized cells. Several M1/M2 markers were downregulated after Mtb infection, which suggests that Mtb can modulate macrophage polarization. In addition, 24 different cell clusters of different sizes were found to be uniquely distributed among the M1 and M2 uninfected

Effect of injectable calcium alginate–amelogenin hydrogel on macrophage polarization and promotion of jawbone osteogenesis

“…BMDMs and third-generation BMSCs in a good growth state were used for induction culture, and cell identification was performed using flow cytometry for cell surface-specific markers 17 and immunofluorescence. 18…”

Trichinella spiralis dipeptidyl peptidase 1 suppressed macrophage cytotoxicity by promoting M2 polarization via the STAT6/PPARγ pathway

“…RAW264.7 and peritoneal macrophages were cultivated in the presence of various stimulating factors. rTsDPP1, TRX tag protein and T. spiralis AW ESA (20 µg/mL) were added to the medium, LPS (200 ng/mL) and IL-4 (20 ng/mL) were used as macrophage M1/M2 polarization positive controls, and PBS was used as a negative control [ 35 ]. After incubation at 37 °C in 5% CO 2 for diverse times, the cells were washed with PBS and harvested, and total RNAs and soluble proteins from the above-stimulated cells were prepared for qPCR and Western blotting analysis, respectively [ 5 , 12 ].…”