Macrophage Cell Assays Help Treat Autoimmune and Inflammatory Disease

Macrophage properties play a critical role in protective immunity to pathogens. An imbalance in the M1/M2 (pro-inflammatory/anti-inflammatory) ratio may be related to the onset of inflammatory diseases such as inflammatory bowel disease (IBD), rheumatoid arthritis, and multiple sclerosis. These properties make macrophages key in understanding the pathology of autoimmune and inflammatory diseases.

We have developed a range of in vitro macrophage cell assays which enables characterization of macrophage phenotype and function to evaluate therapeutic effects in a variety of disease settings and exploits the polarization of monocyte derived macrophage.

Figure 1:

  • Classically activated (M1) macrophages detect pro-inflammatory cytokines or PAMPs (e.g., LPS). These macrophages express high levels of co-stimulatory molecules (CD80 and CD86).
  • Alternatively activated (M2) macrophages regulate the immune response, preventing excess inflammation or cytokine storm. M2 polarization can occur in the presence of stimuli, including IL-4/IL-13, and these cells are thought to inhibit the immune response by the release of IL-10.

Schema of macrophage differentiation and polarization

Figure 1. Schematic of macrophage cell assay with differentiation and polarization. CD14+ cells are isolated from PBMC and cultured in the presence of M-CSF or GM-CSF. M1 macrophages are then generated in the presence of LPS and IFNγ; M2a in the presence of IL-4 and IL-13; and M2b in the presence of IL-4 and LPS. Therapeutics can be tested for their ability to promote macrophage polarization towards a more M1 like phenotype (IO) or to inhibit macrophage polarization and enhance the M2 like phenotype (autoimmune).

Working With Macrophage Cell Assays In Vitro

Our in vitro assays utilize primary, human monocyte-derived macrophages. Specialized tissue resident macrophage subsets such as microglia can be isolated from murine or human tissue (where available) or in some instances differentiated from iPSC. These cells allow us to investigate changes to phenotype and cytokine production, as well as cell performance in functional assays such as phagocytosis and antigen presentation.

Phenotyping of Macrophage Cell Assays

Flow cytometry can be used to evaluate the ability of therapeutics to alter the differentiation profile of macrophages in mode of action studies (Figure 2).

Polarized macrophages that express high levels of CD40, CD80, and CD25 are thought to be classically activated and inflammatory (M1), while those that have high expression of CD206 and CD163 have a more anti-inflammatory function (M2).

Therapeutics which induce a change in profile towards an M1 macrophage phenotype are likely to show efficacy in an infection or immuno-oncology (IO) setting, whilst those that block the LPS and IFNγ phenotype are likely to be suitable within an autoimmune disease setting.

Figure 2 displays phenotypic changes that polarized macrophages undergo following treatment with an anti-inflammatory therapeutic, in this example with dexamethasone.

Schema of polarized macrophages following treatment with dexamethasone

Figure 2. M1, M2a, and M2b macrophage profiles following treatment with dexamethasone. Characteristic phenotypic cell surface markers are shown for M1, M2a, and M2b. M1 polarized macrophages treated with the immunosuppressant, dexamethasone, show an inhibition in CD25 and CD86 expression following treatment.

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Functional Macrophage Cell Assays

There are several ways to probe the functionality of macrophages using in vitro assays:

  • Macrophage Polarization Cytokine Profiles

    Alongside surface marker phenotyping (Figure 2), we can measure characteristic macrophage cytokine profiles to determine whether a pro-inflammatory or resolutory response is driven by the therapeutic compound.

    In the macrophage cell assay example shown in Figure 3, M1 macrophages produce pro-inflammatory cytokines such as IL12p70, TNFα, and IL-6. However, M2 macrophages produce high levels of IL-10. The absolute quantities present are likely to determine the nature of the downstream response such as T cell activation.

    Schema of cytokine profile of polarized macrophages.

    Figure 3: Cytokine profile of polarized macrophages during macrophage cell assay. M-CSF or GM-CSF derived M0 macrophages were cultured overnight with LPS and IFNγ (M1) or IL4 and LPS (M2b). The production of cytokines (IL12p70, IL10, TNFα and IL6) were then quantified from supernatants by Luminex

  • Macrophage and T Cell Co-Cultures

    T cell activation can be utilized as a readout for modulation of macrophage antigen presentation capacity (APC).

    This macrophage cell assay offers a platform for testing therapeutics which modify the interaction between macrophage and T cells to either increase, decrease, or repolarize the T cell response. Mixed lymphocyte reaction (MLR) assays use allogenic CD4 T cells cocultured with therapeutically targeted macrophage and allows assessment of macrophage APC function (Figure 4 and 5).

    Schema of APC – antigen presentation assay

    Figure 4 Schematic of macrophage antigen presentation assay. During this macrophage cell assay: CD14+ monocytes isolated from PBMC are differentiated with M-CSF, then stimulated with Tet tox +/- LPS and co-cultured with T cells in an antigen presentation assay.

    Schemas of T cell proliferation measures by DNA incorporation with or without LPS stimulation

    Schemas of T cell proliferation measures by DNA incorporation with or without LPS stimulation

    Figure 5: CD4 T cells proliferation measured following MLR. For this macrophage cell assay, cells were cultured as described in Figure 4. Cellular proliferation was measured using DNA incorporation by tritiated thymidine as a readout – corrected counts per minute (cpm) are shown.

  • Macrophage Phagocytotic Assays

    Phagocytosis assays can be performed on macrophages using specialized bioparticles or labeled cells.

    Once the macrophage has been differentiated and polarized to the subset of interest, these cells can then be incubated with the bioparticles or labeled cells to initiate phagocytic activity:

    • When using the bioparticles, the macrophage has the ability to recognize them as foreign, engulfing and internalizing them. As the particles/cells are internalized, the acidic environment within the cell triggers fluorescence of the particles. This fluorescence can then be measured to determine the phagocytic activity.
    • Similarly, when investigating phagocytosis using labeled target cells, the macrophage is also able to engulf and internalize the cells, with phagocytosis assay quantified by macrophages also positive for the relevant label.

    Standard readouts for phagocytosis assay are flow cytometry and IncuCyte, real-time cell analysis
    (Figure 6).

    Schema of phagocytosis assay of macrophage

    Figure 6: Schematic for macrophage phagocytosis assay. During this macrophage cell assay: CD14+ cells are isolated from human PBMC before being stimulated with relevant polarizing conditions. Polarized macrophages are then co-cultured in the presence of fluorescent bioparticles.

    IncuCyte Based Phagocytosis Assays

    The IncuCyte technology allows quantification of phagocytic activity over time. Bioparticles or labeled cells can be co-cultured with macrophages and either the co-localization of cells, or the development of fluorescence, then measured and quantified. It can be a useful system for monitoring kinetics and can provide visualization in the form of either images or videos.

    IncuCyte imaging is a high-throughput technique, which is fully automated after initial co-culture, making it perfect for compound screens or larger studies (Figure 7).

    Schema of fluorescence and real time images over time analyzing phagocytose with bioparticles in unpolarized and polarized M2a macrophage

    Schema of fluorescence and real time images over time analyzing phagocytose with bioparticles in unpolarized and polarized M2a macrophage

    Figure 7: Real-time analysis of phagocytosis assay with bioparticles in unpolarized and polarized M2a macrophage. During this macrophage cell assay, macrophage subsets were incubated +/- bioparticles and imaged using real-time IncuCyte software. Once phagocytosed, bioparticles fluoresce in response to environmental changes and quantified using the IncuCyte analysis software. Cells which have engulfed bioparticles are indicated in blue.

    Flow Cytometry-Based Phagocytosis Assays

    Flow cytometry is a useful readout for phagocytosis assays when characterization of the functional capacity of macrophage is of interest. The bioparticles or labeled cells can simply be added into our phenotyping panels. It can also be coupled with the collection of supernatants prior to flow cytometric staining to allow for further cytokine analysis if required (Figure 8).

    Results of flow cytometry characterizing bioparticles macrophage

    Figure 8: Unpolarized and M2a macrophages were analyzed by flow cytometry for uptake of bioparticles. During this macrophage cell assay, cells were gated first for viable, single cells then CD11b. Cells were characterized to confirm M0 and M2a subsets and phagocytosis assay determined by pHrodo+ macrophages.

Functional Murine Macrophage Cell Assays

Although the presence of circulating macrophage in rodents is low, both bone marrow derived macrophages (BMDM) and thioglycolate induced peritoneal macrophages provide a sufficient source of mature macrophages for screening assays (Figure 9).

Schema showing how to isolate macrophage from mouse and bone marrow

Figure 9: Schematic of isolating macrophages from rodents for macrophage cell assay.

Induction of peritoneal macrophage with thioglycolate not only increases the yield of isolated macrophages but leads to a shift in populations. Post-treatment with thioglycolate, a greater proportion of the cells isolated exhibit a small peritoneal macrophage (SPM) phenotype compared to the large peritoneal macrophages (LPM) isolated from naïve mice (Figure 10).

Flow cytometry and small & large peritoneal macrophage graphs showing difference between naïve and thioglycolate macrophage culture

Figure 10: Phenotype of thioglycolate induced peritoneal macrophages used for macrophage cell assay: Peritoneal macrophages were recovered post induction with thioglycolate and stained for analysis by flow cytometry. A) From the macrophage population small (SPM, red gate) and large (LPM, blue gate) peritoneal macrophages were identified, and B) the frequency of macrophage is shown.

Both cell types can then be used in macrophage cell assays similar to that described for the human in vitro assays (Figure 11).

Graphs of in vitro macrophage assay profiling cytokines production within a culture of unstimulated or stimulated (by LPS) macrophage

Figure 11: Murine macrophage cytokine production for macrophage cell assay: Cytokine profile following culture in the presence or absence of LPS for A) Thioglycolate induced peritoneal macrophages and B) Bone marrow derived macrophages. Graphs show mean +/- SEM of triplicates.

Macrophages are a notable player in the activation and polarization of T cells and other lymphocyte responses, and in the modulation of the tissue microenvironment. With these macrophage cell assays, which can be used to measure and manipulate inflammatory responses, our expertise provides a platform for determining MOA or efficacy of macrophage targeting therapeutics.

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Frequently Asked Questions (FAQs) About Macrophage Cell Assays

  • What is the role of macrophages?

    Macrophages play an important role in the detection, phagocytosis, and destruction of foreign substances, and are present in almost all tissues. Besides phagocytosis, macrophages are critical players in initiating adaptive defense mechanisms by presenting antigens to T cells and initiation inflammation through the release of cytokines. Macrophages also play a significant role in wound healing and tissue remodeling. Understanding the impact of a therapeutic on primary human macrophage biology will help progress your program.

  • How do macrophages work during infection or inflammatory states?

    Macrophages are innate immune cells that play an essential part in host defense, inflammation, and tissue homeostasis. Upon tissue damage or infection, circulating blood monocytes enter the affected tissue and undergo phenotypic polarization into macrophages, mounting specific responses depending on microenvironment cues. Tissue resident macrophages, largely thought to derive from a different pool of progenitors during development, can also influence and drive the immune response.

    Depending on cell surface markers and functional properties, macrophages can be simplistically described as pro-inflammatory (M1) or anti-inflammatory (M2), although the reality is that macrophage phenotype is diverse and dependent on environmental cues. Macrophage cell assays will allow you to target different aspects of macrophage function.

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  • Do you think the concept of M1/M2 macrophages is a true representation of in vivo setting?

    While we appreciate that this is a bipolar approach to what may be happening in vivo, the macrophage cell assays have been designed with the concept that the polarization refers to how the macrophage has responded to environmental cues at a given point in time. We accept that this is not fixed and rather represents one point in the continuum between two functionally polarized states, and that macrophages are adept at integrating multiple signals and plastic enough to adjust to the tissue microenvironment in vivo. Our macrophage cell assays build a framework on which to assay therapeutic interventions and ultimately give the first step in understanding a MOA.

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  • What is the advantage of using a macrophage and T cell co-culture system over polyclonal stimulation of T cells?

    A model using macrophage offers a more physiologically relevant system, allowing an assessment of both T cell function and the ability of the key immune priming cells (macrophage) to stimulate a T cell response. For example, drugs targeting key receptors-ligand interactions may be ineffective in polyclonally-stimulated T cell monocultures where the receptor may be highly expressed, but the corresponding ligand, usually provided by antigen-presenting cells such as macrophages, is absent. The macrophage cell assay offers this receptor-ligand interaction for a more thorough screening of therapeutics.

  • How do I determine which macrophage cell assay is best for my program?

    We are happy to customize our macrophage cell assays to meet your requirements. Our expert immunologists can help by suggesting what parameters can be adapted to tailor the setup and readouts of the macrophage assays to suit your needs.