Macrophage plasticity: More than black and white

The traditional view of macrophages as ‘good’ or ‘bad’ immune cells has become outdated. Recent studies have shown that macrophage plasticity encompasses a complex spectrum of activation states, each of which plays an important role in health and disease. In this article, we discuss the latest understanding of macrophage polarization, the challenges in standardizing nomenclature, and why standardization is important for the development of new therapeutic approaches.

Macrophage plasticity refers to the remarkable ability of these innate immune cells to adapt their phenotype and function in response to environmental signals, including cytokines, pathogens, cellular debris, and metabolic signals.^1,2 Unlike other immune cells with fixed phenotypes and functions, macrophages can alter their gene expression, surface markers, and functional outputs based on the molecular cues they encounter.¹

This flexibility enables macrophage populations to serve various functions:

• Pathogen elimination: Detecting and destroying invading microorganisms through phagocytosis and secretion of antimicrobial substances.
• Tissue repair: Promoting healing and regeneration after injury by clearing debris and releasing growth factors.
• Homeostasis maintenance: Regulating normal physiological processes to maintain internal balance.
• Metabolic regulation: Controlling tissue metabolism and energy balance through interactions with other metabolic tissues, such as adipose tissue, liver, and muscle.

The plasticity of macrophages becomes particularly important during disease states. In cancer, for example, tumor progression often involves the reprogramming of macrophages from anti-tumor to pro-tumor phenotypes.³ Understanding these transitions opens new avenues for therapeutic intervention.

For decades, researchers classified macrophages into two categories: M1 (classically activated) and M2 (alternatively activated) macrophages.^4,5 This binary system provided a useful framework but oversimplified the true nature of macrophage plasticity.

The M1/M2 classification emerged in the 1990s when researchers observed distinct responses to different stimuli.^4,5

M1 macrophages are polarized in vitro by Th1 cytokines such as colony-stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α), and interferon-γ (IFN-γ) alone or together with lipopolysaccharide (LPS) from bacteria. M1 macrophages express pro-inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, IL-12, IL-23, and TNF-α.⁶ M1 macrophages play a crucial role in pathogen killing by exhibiting strong cytotoxic activity and mediating resistance against infections.^7,8 

In contrast, M2 macrophages are polarized by Th2 cytokines such as IL-4 and IL-13 and produce anti-inflammatory cytokines such as IL-10 and transforming growth factor beta (TGF-β). M2 macrophages play a crucial role in tissue repair, particularly in the later stages of wound healing.⁹ 

More recent studies have shown that macrophage plasticity extends far beyond the traditional binary classification into M1 and M2 phenotypes.¹⁰ Polarized macrophages exist along a continuous spectrum, with most cells displaying mixed characteristics.

Dr. Hagen Wieland from our development department explains: “The limitations of the M1/M2 model for defining macrophage polarization are becoming increasingly clear. These terms identify extremes of a continuum that rarely occur in living organisms.”

Several factors contribute to this complexity in macrophage phenotypes^11–14:

• Local microenvironment: Tissue-specific signals influence macrophage polarization
• Temporal dynamics: Macrophage phenotypes can change over time 
• Mixed signals: Macrophages often receive multiple, sometimes conflicting cues 
• Intermediate states: Macrophages can exhibit characteristics that are between classical M1 and M2 phenotypes

The molecular basis of macrophage plasticity involves complex networks of transcription factors, signaling pathways, and epigenetic modifications that control gene expression.¹⁵

Different stimuli activate distinct molecular cascades that promote macrophage polarization.

M1 polarization pathways¹⁶:

• NF-κB activation promotes inflammatory gene expression
• STAT1 signaling drives classical activation programs 
• IRF family transcription factors regulate interferon responses
• HIF-1α supports glycolytic metabolism

M2 polarization pathways¹⁶: 

• STAT6 activation follows IL-4/IL-13 stimulation 
• PPAR-γ promotes alternative activation 
• KLF4 regulates M2-specific gene programs 
• STAT3 supports anti-inflammatory responses

Macrophage plasticity doesn’t just involve changes in the genes they express or the signals they respond to. It also entails shifts in their metabolism, which is linked to their function.^17–20 

M1 metabolism relies heavily on glycolysis for rapid energy production, which helps them respond quickly to infection and produce large amounts of inflammatory molecules. However, glycolysis is less efficient, so it supports short bursts of activity, ideal for rapid immune responses. 

M2 metabolism uses oxidative phosphorylation and fatty acid oxidation, which are slower but more efficient energy sources. This metabolic setup supports sustained activity needed for tissue repair, anti-inflammatory functions, and long-term homeostasis. However, macrophages can exist in metabolic states that fall between the extremes of the classical M1 and M2 phenotypes. 

These intermediate states show mixed metabolic profiles. For example, they might use both glycolysis (typical of M1) and oxidative phosphorylation (typical of M2) simultaneously, or switch between these metabolic pathways depending on available nutrients and tissue demands. 

The plasticity of macrophages varies across different tissues, reflecting the functional demands of each anatomical location^.21 Tissue-resident macrophages develop specialized characteristics while maintaining their plasticity.

Different tissues harbor distinct macrophage populations.¹² The existence of distinct tissue-resident macrophages with specialized functions is a clear example of macrophage plasticity in action. It shows how a single cell type can adapt its identity and behavior in response to its location and the signals it receives. 

• Alveolar macrophages (lungs): Involved in particle clearance and surfactant metabolism
• Kupffer cells (liver): Specialize in detoxification and metabolic regulation
• Microglia (brain): Maintain neural homeostasis and respond to neuroinflammation
• Osteoclasts (bone): Control bone remodeling and calcium homeostasis
• Tumor-associated macrophages: Adapt to tumor microenvironments

Macrophage plasticity is profoundly shaped by the environment in which these cells reside through various mechanisms^21–23:

• Hypoxia: In low-oxygen environments (like inside tumors or inflamed tissues), macrophages often shift toward an M2-like phenotype, which is anti-inflammatory and supports tissue remodeling or tumor growth.
• Mechanical forces: Tissues under physical stress or strain (like in fibrosis, tumors, or muscle injury) can mechanically influence macrophage activation. For instance, stiffness of the surrounding tissue can push macrophages toward a pro- or anti-inflammatory state.
• Cell-cell interactions: Direct contact with nearby cells (like fibroblasts, epithelial cells, or tumor cells) can change how macrophages behave by providing surface signals or feedback.
• Metabolic factors: The availability of nutrients like glucose, lipids, or amino acids in the local tissue environment can influence whether macrophages adopt a pro-inflammatory (M1) or repair-oriented (M2) state.

Macrophage plasticity plays a pivotal role in disease development and progression. Different macrophage populations can either promote or prevent disease, depending on their activation state and the tissue context.²¹ 

Macrophage plasticity and function become dysregulated in several chronic inflammatory conditions:

• Rheumatoid arthritis: Persistent M1 activation drives joint destruction²⁴
• Atherosclerosis: Macrophage infiltration promotes plaque formation²⁵
• Inflammatory bowel disease: Imbalanced M1/M2 responses perpetuate inflammation²⁶

In these pathological contexts, excessive production of pro-inflammatory mediators by M1-polarized macrophages contributes to tissue injury. As a result, therapeutic approaches often aim to modulate macrophage activity, shifting polarization toward M2 phenotypes or dampening prolonged inflammatory responses.²⁷

Macrophages play an important role in cancer development and progression. Tumor cells reprogram the surrounding macrophages to support their growth, and the effects of macrophages on tumor cells depend on the stage of cancer^28–30:

• Early cancer: M1 macrophages may contribute to anti-tumor immunity by eliminating cancer cells
• Established tumors: M2-like macrophages promote tumor progression 
• Metastasis: Polarized macrophages facilitate cancer cell spread
• Therapy resistance: Tumor-associated macrophages can mediate resistance to cancer therapies

The tumor microenvironment promotes a shift from M1 to M2 phenotypes, creating a niche that enhances tumor growth.³⁰ Therefore, scientists are developing new immunotherapy approaches to macrophage reprogramming in the tumor microenvironment.

Unlike the dysregulated macrophage responses observed in chronic inflammatory diseases and cancer, the plasticity of macrophages plays an essential role in wound healing and tissue repair15:

• Initial response: M1 macrophages clear debris and fight infection
• Transition phase: Gradual shift toward M2 phenotypes
• Resolution: M2 macrophages promote tissue regeneration
• Homeostasis: Return to baseline surveillance functions

Disruption of macrophage plasticity can lead to chronic wounds or excessive scarring, highlighting the importance of a balanced inflammatory response.

The complexity of macrophage plasticity has created challenges for researchers attempting to classify and communicate the different phenotypes of macrophages. The proliferation of different naming systems has led to confusion in the scientific literature.

In a 2014 article on macrophage nomenclature, Dr. Peter J. Murray of St. Jude Children's Research Hospital compared the current state of macrophage nomenclature to the biblical Tower of Babel, where confusion of tongues prevented project completion.³¹ Without standardized terminology, researchers face challenges in comparing results across laboratories, reproducing experimental findings, and translating their research findings to clinical applications.

 To address these challenges, researchers have proposed several standardization approaches. For example, Murray and colleagues recommended the use of stimulus-based nomenclature, such as M(IL-4), M(IFN-γ), and M(LPS); the development of reproducible experimental standards; the implementation of minimal reporting requirements; and the avoidance of overly complex classification schemes.³¹

At PromoCell, we adhere to the nomenclature of Dr. Murray, as it precisely defines how macrophages are differentiated in vitro and which cytokines are used for their polarization and subsequent activation. “This allows scientists to reproduce and standardize the experimental processes,” says Wieland. “There is a noticeable increase in reviewers and scientists who have adopted Murray’s nomenclature in their works since 2014. This way, they can better present their results.” 

This approach has gained further support from other researchers in the field, with a 2023 review reinforcing the continuum view of macrophage activation and emphasizing the importance of Murray’s precise nomenclature framework for advancing the field.³²

Studying macrophage plasticity requires experimental approaches that can capture the dynamic nature of these cells. Both in vitro and in vivo methods have been developed to study macrophage plasticity.

In vitro systems

 In vitro models remain fundamental tools for dissecting macrophage plasticity and commonly employed approaches include^33–35:

• Primary macrophages: Derived from blood monocytes or bone marrow 
• Standardized culture conditions: Defined media and activation protocols 
• Polarization stimuli: Specific cytokines to induce M1 or M2 states 
• Functional assays: Tests of phagocytosis, cytokine production, and metabolism

In vitro studies of macrophage plasticity enable controlled experimental conditions, reproducible results across laboratories, cost-effective screening approaches, and detailed mechanistic investigations.

In vivo models

 Animal models provide critical insights into macrophage plasticity in physiological contexts^35–37:

• Disease-specific models: Cancer, inflammation, and infection studies 
• Genetic approaches: Conditional knockouts and reporter systems 
• Cell tracking: Following macrophage behavior over time 
• Advanced tissue analysis: Single-cell sequencing and spatial profiling

Emerging technologies

New technologies have been developed to study the plasticity of macrophages:

• Single-cell RNA sequencing: Reveals heterogeneity within populations³⁸
• Spatial transcriptomics: Maps gene expression in tissue context³⁸
• Live imaging: Tracks dynamic changes in real-time^39,40
• Computational modeling: Predicts plasticity responses⁴¹

The growing understanding of macrophage plasticity is opening new opportunities for the treatment of several diseases by targeting the mechanisms that control macrophage polarization. 

Several strategies target macrophage plasticity for therapeutic benefit^42–45:

• Repolarization therapy: Converting M2 to M1 phenotypes in cancer
• Blocking recruitment: Preventing macrophage infiltration into tumors 
• Enhancing resolution: Promoting M2 responses to resolve inflammation
• Metabolic targeting: Altering macrophage metabolism to change function

The development of macrophage-targeted therapies faces several obstacles^21,46:

• Selectivity: Avoiding effects on beneficial macrophage functions 
• Targeted delivery: Getting drugs to the right tissue locations 
• Timing: Coordinating interventions with disease progression 
• Heterogeneity: Accounting for diverse macrophage populations

To successfully translate macrophage plasticity research into clinical applications, researchers need robust standardization of experimental approaches and terminology.

Several organizations are working toward standardization:

• International consortia: Coordinating research efforts globally
• Professional societies: Developing best practice guidelines 
• Regulatory agencies: Establishing requirements for therapeutic development 
• Commercial partners: Providing standardized reagents and protocols

Adopting common standards offers multiple advantages, including easier comparison and combination of studies, smoother transition from research to clinical applications, and reduced time and resources spent on conflicting results.

References