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Disease Mechanisms-Multiple Sclerosis

Mononuclear Phagocytes Specify and Adapt Phenotype in MS Model Study Suggests Phagocyte Activation as a Potential Therapeutic Target

Talan, Jamie

doi: 10.1097/01.NT.0000549647.11909.eb
Disease Mechanisms
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Researchers have shown in a mouse model of multiple sclerosis that phagocytes can both promote and inhibit inflammation in the central nervous system, sequentially adopting different phenotypes with distinct functions.

The role of mononuclear phagocytes in multiple sclerosis (MS) has been a puzzle to scientists. These immune cells are the first on the scene of the developing lesion, igniting an inflammatory response. And then they initiate a cleanup process to help resolve and repair the damage. Are they different phagocytes with opposing jobs?

A team of German scientists designed experiments to understand this vexing phenomenon — and the surprising answer may offer the possibility of a new treatment target.

“These are relatively plastic cells and have the capacity to destroy tissue and to clear up damaged tissue,” said Martin Kerschensteiner, MD, PhD, director of the institute of clinical neuroimmunology at the University Hospital and Biomedical Center at the Ludwig-Maximilians University in Munich and senior author of the paper in the September issue of Nature Neuroscience.

The scientists showed that these phagocytes are not two different cell types — one pro-inflammatory and one anti-inflammatory — but that they change roles in response to specific environmental cues. “These cells are capable of exhibiting both pro- and anti-inflammatory phenotypes,” Dr. Kerschensteiner said, adding that blocking the entry of phagocytes into the central nervous system (CNS) could slow or stop the disease process.

Many scientists have puzzled over these cells. Based on the polarization these cells can acquire in cell culture, they have even labeled two types of activity: M1 phagocytes for cells that have a pro-inflammatory polarization and M2 cells to describe the anti-inflammatory state. The M1s release toxic reactive species, while M2s send signals that are in line with classic phagocytosis — cleaning up tissue debris and repairing the cells in the surrounding area.

Scientists have further identified molecular signatures that characterize M1s and M2s. There is a pair of enzymes that compete for intracellular substrate l-arginine. M1s express induced nitric oxide synthase and M2s use arginase to catalyze urea, l-ornithine, and polyamines, and this mix supports tissue repair.

The question is what is driving this process, and are M1 and M2 distinct cell types?

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The German scientists used two complementary reporter mouse lines: one, in which the induction of the specific signature enzyme that characterizes proinflammatory phagocyte phenotypes results in red fluorescent labeling of the cells, and another that expresses yellow fluorescent protein under the control of the arginase promoter that characterizes anti-inflammatory cells. Yellow fluorescent protein is a genetic mutant of green fluorescent protein.

In these double transgenic mice, they then induced experimental autoimmune encephalitis (EAE), a classic mouse model of MS and followed the fates of individual phagocytes. Both fluorescent colors appeared in phagocytes in the spinal cord. Most of the cells were derived from invading monocytes. The cells at the earliest stages of EAE were red. The number of cells in the lesions decreased over the course of the disease, and over days they began to see the red change to yellow and eventually green.

The research team analyzed genetic transcripts and found different gene expression profiles when phagocytes were in their tissue-damaging stage and then in the tissue repair stage. The imaging allowed them to track the evolution of pro- and anti-inflammatory phagocytes as they found their way into different compartments in the CNS. The phagocytes acquired polarization once they made it into the CNS.





By tagging the phagocytes with different fluorescent proteins — red to identify pro-inflammatory markers and green to tag anti-inflammatory markers — and then using an in vivo microscopy imaging technique they could directly visualize how the phagocyte population changed its phenotype.

They considered two possibilities — either pro-inflammatory cells enter the CNS and do their damage at the site and leave the area as other anti-inflammatory cells come in, or the same cell changes its phenotype. They wanted to know which signals are responsible for that shift.

They watched as the red fluorescent phagocytes made it into the inflamed CNS to the lesion. They were pro-inflammatory. Several days later, they watched these same cells turn green and start their anti-inflammatory process of resolving the lesion. There were not two cell types but just one, and this cell was clearly getting signals about how to proceed with the task at hand.

“If we looked at lesions in the formative stage of the lesion, they are filled with red cells,” explained Dr. Kerschensteiner. “In the restorative stage, they are green. By following individual cells over time we could literally see them shifting their phenotype.”

The scientists characterized the phenotypes and found that they behaved differently on many molecular levels: ATP generation, scavenging function, and their ability to generate reactive species or cytokines. It looked as if they were getting their cues from the CNS environment. The researchers believe that this phenotypic conversion is induced by signals from astrocytes in the CNS. Without external cues, the immune cells stay red. When they are guided by signals from the CNS environment, they begin to switch their role.

The discovery should enable researchers to obtain a better molecular understanding of the phenotype switch, and allow them to explore the therapeutic potential of targeted manipulation of phagocyte populations in MS patients. Other animal studies have shown that depleting phagocytes or blocking microglia activation prevents a CNS lesion, and it may be possible to see if approaches targeting phagocyte activation and actions would be safe in patients.

“Taken together, the comprehensive molecular characterization of differentially polarized CNS phagocytes indicated that they primarily differed in their metabolic states and in the way they interacted with the CNS environment,” the scientists wrote in the paper. “One of the conclusions emerging from this work is that the local microenvironment is critical in shaping the phagocyte response. Phagocytes that enter from the blood stream acquire their initial polarization only after spinal cord entry, and the nature of this polarization critically depends on the compartment they enter.”

The researchers said that the findings have “implications for the way we think about targeting phagocyte actions in inflammatory CNS disease like MS. For example, our findings suggest that the initial pro-inflammatory polarization of phagocytes needs to be prevented rather than reversed, considering that the latter happens endogenously shortly after the cells enter the CNS.”

“Furthermore, the timing of interventions, at least those that aim to block overall phagocyte infiltration or activation, is crucial. While such interventions are likely beneficial during the formation of lesions, the same strategies may have opposing effects during lesion resolution when anti-inflammatory phagocytes dominate the infiltrate. Expanding our understanding of how phagocyte phenotypes evolve in vivo can thus help to better define both the therapeutic opportunities and the limitations of targeting phagocyte actions in vivo.”

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“We know that the earliest cells that make it to the lesion are phagocytes,” said Francisco J. Quintana, PhD, associate professor of neurology at the Ann Romney Center for Neurologic Diseases at the Brigham and Women's Hospital–Harvard Medical School. “We've had pieces of the puzzle, but this was an elegant approach to study phagocytes and see how they can change in this environment.”

“They have some preliminary evidence that suggests that some of the cells controlling this change (from pro-inflammatory to anti-inflammatory) were astrocytes, and this fits into our data that shows astrocytes are very important in controlling phagocytes. Astrocytes can have profound effects on MS pathogenesis,” Dr. Quintana said. “Their finding offers a new treatment target to study: blocking monocytes and phagocytes before they get into the CNS.”



Ari Waisman, PhD, professor of immunology at the Institute for Molecular Medicine University Medical Center of the Johannes Gutenberg University of Mainz, added: “In this paper, the scientists used a new genetic system to be able to label macrophages based on their expression of either M1 type (inflammatory) or M2 type (anti-inflammatory) types.”

Using these mice and in vivo imaging, the group showed that during inflammation in the CNS, the first phagocytes that enter the CNS acquire the phenotype of M1 cells and gradually, as inflammation is decreased, becomes M2 type phagocytes.

“This is very important work,” he said. “This study suggests that one of the reasons why inflammation occurs is because the phagocytes change their phenotype and function and change into anti-inflammatory cells. Thus, instead of causing damage, which phagocytes can do, they possibly contribute to tissue repair.”

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•. Locatelli G, Theodorou D, Kendirli A, et al Mononuclear phagocytes locally specify and adapt their phenotype in a multiple sclerosis model Nat Neurosci 2018; 21(9):1196–1208.
© 2018 American Academy of Neurology