Alternatively Activated (M2) Macrophage Polarization Assay

Monocytes and macrophages are critical effectors and regulators of the innate immune response. These cells play a key role by protecting the host by clearing pathogens and eliciting an immune response, while also contributing to the pathogenesis of inflammatory and degenerative diseases.

Macrophages are remarkably plastic, changing their phenotype depending on the microenvironment. Classically activated macrophages, called M1, are pro-inflammatory cells, which promote Th1 responses and tumoricidal activity. This is in contrast to alternatively activated macrophages, called M2, which have anti-inflammatory functions and elicit tissue repair responses, fibrosis, tumor growth, and progression. This has generated interest in  macrophage reprogramming as option for treating diseases like cancer and autoimmune disorders. We have developed an optimized in vitro macrophage polarization cell-based assay using human primary blood-derived cells to assess the translational potential of small molecules as novel therapies.

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M2 macrophages are involved in parasite control, tissue remodeling, immune regulation, tumor promotion, and efficient phagocytic activity. Several studies demonstrate that M2 polarization occurs in primary human monocytes upon exposure to IL-4/IL-10 cytokine cocktail. Cells that have undergone M2 polarization increase secretion of CCL18 and gain de novo expression of the mannose receptor CD206.

A validated, robust IL-4/IL-10-induced M2 polarization assay has been developed using primary human blood cells that are derived from healthy donors, to evaluate therapeutic candidates. M2 polarization of monocytes is measured by CCL18 secretion via ELISA using high content imaging. Charles River also offers a complementary M1 Macrophage Polarization Assay to support your fibrosis research.

 

M2 Macrophage Polarization Assay Principle

Blood-derived primary human CD14+ cells are seeded in presence of M-CSF to accommodate differentiation into so-called M(0) macrophages. They are then refreshed in preparation for addition of small molecule compounds and the IL-4/IL-10 cytokine cocktail to induce polarization into M2 macrophages. After three days, the culture supernatant is harvested and processed for CCL18 detection. In addition, the cells are fixed, stained for CD206 and DAPI, and imaged via HCA to assess the QC for M2 polarization.

Figure showing the process and timelines for the M2 Polarization assay.

M2 Macrophage Polarization Assay Setup

  • Cells ➔ CD14+ cells from up to 2 healthy donors
  • Seeding density ➔ 5,000 cells/well in 384-well plates
  • Differentiation ➔ M(0): M-CSF
  • Polarization ➔ IL-4 / IL-10 cytokine cocktail
  • Assay controls ➔ 0.1% DMSO (negative control) and 1 μM tofacitinib (positive control)
  • Compounds ➔ 8-point concentration response curves (in biological duplicate)
  • Harvest / Fix ➔ 72 hours post polarization
  • Readout ➔ ELISA: CCL18
  • QC ➔ HCA: CD206 and DAPI

 

Assay Performance

Representative images for IL-4/IL-10-mediated M2 polarization are shown below. In addition, representative concentration-response data shown for blood-derived M2 polarization.

Figure 1

The image shown is a data image from the M2 polarization assay. Using high content imaging we can see the IL-4/IL-10-mediated M2 polarization.

Figure 2

Data image from the M2 Polarization assay, demonstrating the M2 polarization concentration-response data for blood-derived M2 polarization.

Figure 3

Data image from the M2 Polarization assay, showing the reference compound concentration-response data for blood-derived M2 polarization.

Figure 4

Data image from the M2 Polarization assay, showing the Assay controls concentration-response data for blood-derived M2 polarization.

Figure 5

Data image from the M2 Polarization assay, showing the correlation concentration-response data for blood-derived M2 polarization.

Summary

IL-4/IL-10 cytokine cocktail consistently induced polarization of M(0) macrophages into CD206 positive cells secreting CCL18, indicative of successful M2 polarization. IL-4/IL-10-mediated M2 polarization could be confined by treatment with the JAK1/3 inhibitor tofacitinib, showing full inhibition of CCL18 secretion regardless the presence of IL-4/IL-10. IC50 values were consistent among different donors (not shown) and strong Spearman rank correlation denotes consistency between biological replicates. Using this fibrosis assay, polarization of M2 macrophages can be monitored to evaluate therapeutic candidates in multiple diseases involving alternative activated macrophages, such as fibrosis, rheumatoid arthritis, inflammatory bowel disease, and cancer.

Therapeutic candidates can be evaluated in 8-point concentration response curves, using two different blood donors in biological duplicate for their effect on CCL18 secretion. In addition, potential cytotoxic side effects of the tested therapeutic candidate will be measured by monitoring the loss of DAPI-stained nuclei as a measure for cell death. Data package will contain QC results for M2 polarization (CD206 expression), signal-to-background ratios (M-CSF vs. IL-4/IL-10), assay windows (vehicle control vs. tofacitinib), reference compound (tofacitinib) data, and effect of the therapeutic candidate(s) on the CCL18 secretion.

 

Complementary Fibrosis Assays

 

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