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.”
“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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