An &lt;em&gt;In vitro&lt;/em&gt; Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Erbel, Christian; Rupp, Gregor; Helmes, Christian M.; Tyka, Mirjam; Linden, Fabian; Doesch, Andreas O.; Katus, Hugo A.; Gleissner, Christian A.

doi:10.3791/50332-v

Cited by 2 publications

(2 citation statements)

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“…While it is common to generate macrophages from differentiation of monocytes obtained from peripheral blood 18 , macrophages could also be generated from induced pluripotent stem cells (iPSCs) 19 or from bone marrow-derived macrophages from mice 20 , 21 . These are feasible techniques to study primary macrophage cells obtained from monocyte/ macrophage progenitors that will proliferate and differentiate into a homogenous population of mature macrophage-like cells.…”

Section: Introductionmentioning

confidence: 99%

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

Mily

Kalsum

Loreti

et al. 2020

JoVE

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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

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Section: Introductionmentioning

confidence: 99%

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

Mily

Kalsum

Loreti

et al. 2020

JoVE

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“…Fresh blood samples were obtained from healthy donors of both sexes following approval by the Institutional Review Board at the Faculty of Medicine at Heidelberg University. Isolation of peripheral blood-derived monocytes (PBMCs) was performed according to an established protocol 10,11 . Either 100 ng/ml recombinant human M-CSF as control or 1 µM recombinant human CXCL4 were added to serum-free differentiation medium for a total of 6 days in culture.…”

Section: Pbmc Isolation and Cell Culturementioning

confidence: 99%

CXCL4-induced PBMCs Modulate Vascular Extracellular Matrix through Wnt5a-CaMKII-dependent Release of Calcific Extracellular Vesicles and Matrix Metalloproteinase-7

Krohn,

Sicklinger,

Spieler

et al. 2023

Preprint

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Background: Macrophage heterogeneity plays an increasing role in the study of vascular inflammatory responses. The CXCL4-induced monocyte/macrophage phenotype has previously been implicated with atherosclerotic plaque destabilization, a key process preceding plaque rupture. Monocyte-derived macrophages were found to exhibit a unique transcriptome in the presence of CXCL4 characterized by upregulation of S100A8 and MMP7. However, the mechanisms involved in CXCL4-induced monocyte-mediated vascular inflammation are unknown. Methods: Single-cell RNA sequencing data were examined for CXCL4-dependent gene expression signatures in plaque macrophages. Human PBMCs were differentiated with CXCL4 and subsequently characterized in terms of osteogenic gene and protein expression signatures and calcific extracellular vesicle release. Association of the CXCL4-induced phenotype with the Wnt pathway was investigated, and CXCL4-induced PBMC-derived EV were analyzed for their potential to elicit an inflammatory response in vSMC. In-vitro findings were verified histologically in calcified human carotid artery plaques. Results: In human plaque macrophages, single-cell sequencing revealed a CXCL4-susceptible subpopulation bearing a distinct proinflammatory gene expression profile. CXCL4-differentiated PBMCs exhibited a marked induction of S100A8, MMP7 and osteogenic marker transcription concomitant with augmented release of calcific EVs enriched with MMP7, S100A8 and alkaline phosphatase. Under osteogenic conditions, increased overt calcification of the extracellular matrix was observed in vitro. Analysis of inflammatory pathway activation identified the paracrine Wnt5a-CaMKII signaling axis to be causally linked to the CXCL4-induced osteogenic PBMC phenotype, S100A8 and MMP7 enrichment as well as calcific potential of secreted EV. Additionally, CXCL4-polarized PBMC-derived EV differentially stimulated osteogenic/inflammatory genotype transition in vSMC. In human carotid artery plaques, occurrence of CXCL4-induced mononuclear cells coincided with Wnt5a-CaMKII pathway activation and progressive plaque calcification. Conclusions: This study introduces a novel mechanism driving monocyte/macrophage-mediated extracellular matrix remodeling in calcific inflammatory responses through Wnt5a-CaMKII-activated secretion of MMP7+S100A8+ calcifying EV by CXCL4-induced pro-inflammatory monocytes.

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An <em>In vitro</em> Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Cited by 2 publications

References 0 publications

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

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

CXCL4-induced PBMCs Modulate Vascular Extracellular Matrix through Wnt5a-CaMKII-dependent Release of Calcific Extracellular Vesicles and Matrix Metalloproteinase-7

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