Macrophage

Immune-Oncology Assays

ADCP
MDSC
M1/M2
TAM
Efferocytosis
Phagocytosis
Cytokine Release Assay (CRA)-predictive toxicity

Inflammation Assays

M1/M2 polarisation and function
Cytokine Release Assay (CRA)-predictive toxicity

M1/ M2a-d polarisation assay

Monocyte derived macrophages (moMΦ) are differentiated from monocytes isolated from healthy blood donors followed by polarisation into a range of phenotypes M1/ M2a-d and TAM phenotypes using the stimuli described below. Readouts include flow cytometric analysis of surface markers associated with different subsets, production of cytokines and chemokines and ability to phagocytose. Therapeutics can be added at the monocyte to macrophage differentiation phase or polarisation phase to prevent or drive polarisation into a distinct macrophage phenotype.

**Figure 1:** Schematic representation of human macrophage M1/ M2a-d phenotype, associated disease and polarising conditions

**Figure 2:** Effector cytokines and chemokines released by peripheral blood monocyte derived macrophages following differentiation for 6 days and repolarisation.

Figure 3: In vitro differentiation and polarization of macrophages under varying culture conditions modulates the phenotypic characteristics of the cells. Differences in morphology are observed by light microscopy (A) alongside distinct profiles in surface marker expression as determined by flow cytometry summarised in (B). Pro-inflammatory signals generate an M1 phenotype with increased expression of CD25 and CD80. In contrast, alternatively activated M2 macrophages exhibit a spectrum of anti-inflammatory phenotypes including an increase in CD206 (M2a) and CD184. CD163 remains high on most M2 and TAM phenotypes but is downregulated by M1 macrophage.

Screening assay to assess modulation of human M1/M2 macrophage function

We present a medium-throughput human macrophage assay to screen novel therapeutics that modulate pro-inflammatory M1 (IFNγ/LPS) and anti-inflammatory M2 (IL-4/IL-10) macrophage subsets. Dexamethasone and tofacitinib are used as reference drugs for benchmarking.

Dysregulated macrophage polarisation exacerbates various diseases. Therapeutics that target macrophage re-polarisation could modify the disease state by shifting macrophages to a beneficial phenotype. For example, promoting an inflammatory M1 phenotype could benefit the tumor microenvironment, while fostering an anti-inflammatory phenotype could help in autoimmune and inflammatory diseases.

Macrophages were differentiated from monocytes and polarised to M1 or M2a phenotypes following the schema to the left. Polarisation was assessed through CD80 staining and TNFα secretion for M1, and CD206 staining and CCL18 secretion for M2a; staining quantified via HCS analysis. Inhibition of M1 and M2a polarisation was demonstrated using dexamethasone and tofacitinib as reference compounds. Novel therapeutics can be benchmarked against these references.

A THP-1 macrophage cytokine release assay (CRA) can be used to explore modulators of an inflammatory response or “de-risk” any potential unwanted effects of novel therapeutic delivery systems

THP-1 cells were differentiated with PMA and then stimulated with LPS to induce cytokine release in the presence of different concentrations of the corticosteroid dexamethasone. Cytokine release was measured at 4h and 24h post LPS stimulation.

Inflammasomes are key signaling platforms within the immune system. Inflammasome complexes form in response to infection, tissue damage or metabolic imbalances. Once formed the inflammasomes activate Caspase 1 which in turn activated the pro-inflammatory cytokines IL-1β and IL-18. Targeting the inflammasome and the resulting signaling pathways is a useful tool in modulating the immune system.

Effect of MCC950 on Inflammasome activation – IL-1β and IL-18.

PBMC from three healthy donors were pretreated with MCC950, vehicle (DMSO) or media for 1 hours prior to stimulation with LPS for 4 hours. Nigericin was then added for a further 45 minutes prior to harvest of the cell culture supernatant for quantification of (A) IL-1β and (B) IL-18 by TR-FRET and ELISA respectively. Data points show mean +/- SEM of technical replicates. One-way ANOVA with Dunnett’s multiple comparisons test comparing stimulated (media) to unstimulated, LPS alone, vehicle and MCC950 to vehicle; *<0.05, **<0.01, ***<0.001.

Other formats: PBMC, macrophage subsets, microglia

THP-1 macrophage cell line phagocytosis and efferocytosis

THP-1 macrophage phagocytosis of E.Coli bioparticles and efferocytosis of Heat Shock treated A-375 melanoma tumour cells. THP-1 cells were cultured with E.Coli bioparticles 0-12.5ug/ml; at 1h phagocytosis was evaluated by pHrodo+ cells using flow cytometry. THP-1 cells were cultured with three E:T ratios of A-375 or heat shock treated A-375; at 1h efferocytosis was evaluated by pHrodo+ cells using flow cytometry.

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