Microglia/macrophage polarization: Fantasy or evidence of functional diversity?

Abstract

Microglia and non-parenchymal macrophages are increasingly recognized to play critical roles in the central nervous system (CNS) health and disease. Accumulating evidence suggests that these mononuclear phagocytes do not constitute stereotypical cell populations, but rather polarize into a variety of phenotypes at different stages of CNS development, stresses, and diseases. This commentary aims to discuss our current consensus and controversy on microglia/macrophage phenotypes. Collective single-cell level evidence validates the concept of microglia/macrophage polarization, while suggests multi-polarity instead of dichotomic polarization. Characterizing the functions of a specific microglia/macrophage phenotype is challenging yet essential to translate our scientific discoveries into clinical application.

Research on myeloid cells in the central nervous system (CNS), including microglia and macrophages, has flourished in the past two decades. Considerable time and effort have been invested to characterize the behavior and functions of these dynamic cells in the normal and diseased brain, and to decipher how they communicate with other CNS components to maintain or regain brain homeostasis. While scientific achievements in this field have been extensively reviewed, now is the time for us to think critically about the challenges faced in the field, the path that this line of inquiry has led us onto, and the implications on future clinical practice.

Diversity of microglia/macrophage function

Microglia/macrophage phenotypic heterogeneity is one of the most important—but also controversial—topics in neuroimmunology. It is widely appreciated that microglia are protean, assuming various morphologies and altering their gene expression profile in response to developmental/temporal, regional, and sex-specific cues in the CNS. If and how this heterogeneity leads to functionally distinct microglia phenotypes at steady state remains speculative. In the event of brain injuries or diseases, peripheral and border-associated macrophages promptly come to the rescue, further complicating research on myeloid cell heterogeneity in the compromised brain.

Early efforts to delineate the versatility of microglia/macrophages in stroke brains categorized them into two conceptual phenotypes with pro-inflammatory (M1) or anti-inflammatory (M2) functional identities. ¹ Although a strict demarcation of M1/M2 polarities is now known to be oversimplified, the concept of phenotypic diversity is nevertheless broadly accepted and investigated. Increasing numbers of functionally-specific microglia/macrophage subpopulations are being identified via expression of one or two unique signature genes.^2,3 Recent development of single-cell RNA sequencing (RNAseq) allows for unbiased categorization of cells based on an array of signature genes, and have uncovered various groups of microglia/macrophages in normal, aged, and diseased brains. We now know that distinct subgroups of microglia and macrophages exist in normal brains, and that confrontation with noxious stimuli shifts individual subgroups and induces the appearance of new subpopulations. Collectively, this molecular evidence validates our initial concept of functional microglia/macrophage polarization, while also revealing multipolarity as the norm, rather than dichotomic polarity. Further application of single-cell techniques to stroke is anticipated to portray complex immune landscapes at different stages after injury and to guide studies of microglia/macrophage multipolarization. Functional characterization, in addition to the detection of signature genes, is surely necessary for the ultimate definition of a specific phenotype. However, one might anticipate that the functional characterization of microglia/macrophages will also eventually identify multiple roles for each individual phenotype. That is, microglia/macrophage phenotypes shown to be beneficial in one disease, one type of tissue, or at one time point after injury may not prove to be similarly effective in a different disease, another tissue type, or other points in time post-injury. Future investigations on the spatially (tissue or region-specific) and temporally distinct roles of myeloid cells will test this speculation.

Technical approaches to distinguish CNS-resident microglia and bloodborne cells

More reliable methods of discriminating between microglia, border-associated macrophages (BAM), and monocyte-derived macrophages are currently being developed and tested by neuroimmunologists. CNS myeloid cells and blood-borne macrophages share many similarities in gene expression and function. The paucity of specific markers and many caveats in experimental approaches collectively hindered subtype-specific investigations of myeloid linage cells in the past. The recent advent of lineage-tracing models shines a spotlight on this long-standing issue. Cxcr4-CreER-mediated lineage tracing mice help to distinguish hematopoietic stem cell-derived monocytes from microglia and other tissue-resident macrophages, and reveal critical functions of bloodborne monocytes in acute ischemic brain injury. ³ Tmem119-CreER mice have also been generated, enabling the inspection of brain-resident microglia. ⁴ The crosstalk between microglia and monocytes was explored using CX3CR1 and CCR2 reporter mice. ⁵ Advances in high-throughput and single-cell technologies will further facilitate the exploration of various myeloid compartments. Given differences in the accessibility of bloodborne versus CNS-resident myeloid cells to noninvasive systemic treatments, unequivocal discrimination between myeloid subpopulations will hasten the identification of druggable targets for therapies and the optimization of drug delivery approaches.

Avoiding indiscriminate suppression of microglia/macrophage function

Indiscriminate inhibition or depletion of myeloid cells deprives the brain of both beneficial and dangerous microglia phenotypes, and is apparently not a prudent strategy for immunotherapies. Accordingly, there is growing interest in shifting microglia/macrophages towards beneficial phenotypes, showing that the original polarization concept has indeed been useful. The plasticity of individual microglia/macrophage phenotypes in response to microenvironmental cues during disease progression is quite remarkable. ⁶ Recent studies have identified a variety of transcription factors (e.g. STAT6, IRF4, and IRF5) as critical switches that control phenotypic shift in microglia/macrophages. Selective manipulation of these molecules adjusts microglia/macrophage phenotypes and transforms stroke outcomes.^7,8 In addition, the emerging concept of immunometabolism highlights an essential role of metabolic reprograming in regulating phenotypes of myeloid cells. Pro-inflammatory microglia/macrophages appear to prefer glycolysis, while the reparative/anti-inflammatory phenotypes are mainly supported by oxidative phosphorylation. Inducing a metabolic shift toward oxidative phosphorylation by CX3CL1 or away from glycolysis through hexokinase-2 inhibition has been shown to reduce pro-inflammatory responses in microglia/macrophages and ameliorate brain lesions after stroke. ⁹ An assiduous mechanistic approach to uncover molecules or pathways that dictate myeloid cell phenotypes will foster the development of novel immunomodulatory therapies.

Maintaining hope and focusing towards a cure

Research on microglia/macrophage polarization in brain injuries has progressed significantly. Many discoveries have resulted from the collaborative efforts of a community of multidisciplinary researchers. The generation of consensus nomenclatures is essential to establish unambiguous and effective communication in this field. The development of microglia/macrophage-targeted therapies is still in its infancy, but signs of hope abound. A recent study suggests that transient microglia depletion followed by repopulation results in unique transcriptomic changes in microglia and improved long-term recovery after traumatic brain injury. ¹⁰ These findings confirm the importance of the temporal dimension in microglia/macrophage reprogramming during brain recovery and reveal new opportunities for the treatment of neurological disorders. Continued perseverance will define the unique phenotypic signature of repopulating microglia as well as other favorable phenotypes, with the goal of translating our discoveries in microglia/macrophage polarization into clinical application.