Keystone 2015: Microglia in Disease: Innocent Bystanders, or Agents of Destruction?

This article is Part III of a 3-part series from the 2015 Keystone Symposium on “Neuroinflammation in Diseases of the Central Nervous System”. To read all coverage from the meeting, click here.

Researchers have long sought to understand what makes the microglial cells residing in the brain different from macrophages that live in other tissues or cruise the blood. Even more elusive is how microglia respond to disease. At “Neuroinflammation in Diseases of the Central Nervous System,” a Keystone Symposium held January 25-30 in Taos, New Mexico, researchers took a renewed stab at classifying these cells in the healthy body. They also attempted to track how the cells change in the face of ongoing pathology.

Chris Glass of the University of California, San Diego, reported that transcription-factor binding to vast landscapes of enhancer sequences could dictate how microglia express different sets of genes than macrophages do (see Part 2). How do these enhancer landscapes affect microglial gene expression, and how does disease change this? Oleg Butovsky of Harvard University dove into these questions in a data-packed talk. Butovsky used microarray and proteomics approaches to compare the gene expression signature of microglia, specifically CD45lo, CD11b+ cells from adult mouse brains to those of other brain cells and monocytes isolated from the spleen. He found that 239 genes were uniquely expressed in microglia compared to other macrophages and immune cells; of those, 106 genes were uniquely expressed in microglia compared to other cells in the brain. Because this gene-expression pattern occurred under healthy conditions, Butovsky dubbed it a “homeostatic signature” of microglia (see Butovsky et al., 2014).

At the Heart of Microglia.

Microglia have a unique pattern of gene expression that distinguishes them from other cells in the brain (pictured above) as well as from macrophages in other parts of the body. [Image courtesy of Butovsky et al., Nature Neuroscience 2014.]

Interestingly, TGF-β was the most important upstream regulator of this homeostatic signature, said Butovsky. Adding TGF-β preserved the signature in cell culture; on the flip side, microglia were largely absent from the brains of TGF-β-deficient mice.

Butovsky next tested what would happen to this homeostatic signature in disease. In mouse models of multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer’s, microglia lost the signature and took on a more pro-inflammatory phenotype. What caused the signature to disappear when things got tough? Butovsky said that in every disease model he tested, microglia upregulated expression of ApoE as well as miR-155, and that both of these were required to erase the signature. The researchers looked for a specific inflammatory signal that boosted ApoE and miR-155. They found nothing. Finally, they discovered that something about dead neurons did the trick. “This is the one thing all of these diseases have in common,” Butovsky said. While he does not know how dead neurons eliminate the signature, he believes ApoE upregulation occurs upstream of miR-155 because microglia from ApoE knockout mice preserves the homeostatic signature. Blocking either ApoE or miR-155 also retains it.

It is unclear how the loss of the homeostatic signature changes the function of microglia in all disease scenarios. Butovsky recently published his finding that blocking miR-155 in a mouse model of ALS restored the microglial signature and ameliorated disease (see Butovsky et al., 2015). He is collaborating with MiRagen, a biotech company in Boulder, Colorado, to develop ALS treatments by blocking miR-155.

Richard Ransohoff of Biogen Idec in Cambridge, Massachusetts commented that it is important to remember that the microglial signature is based entirely on comparison with other cell types. “That doesn’t degrade the value of having a signature, but it means that the interpretation of the signature should be that it’s an operational, somewhat artificial construct to capture a subset of genes that are relatively characteristic of microglia as compared to other cells. It is not necessarily an indicator of the most important genes a microglial cell expresses, as some of those may be shared with other cell types, such as neurons or other types of macrophages,” he said.

In his presentation, Ransohoff also parsed microglial and monocyte differences. He analyzed the cells in a mouse model of multiple sclerosis—a disease in which peripheral monocytes invade the CNS. Ransohoff reported that in this scenario, brain microglia turned down expression of thousands of genes, whereas infiltrating macrophages ramped up expression overall. Chemokines, which attract other cells, were among the few genes that microglia did turn on in response to disease, suggesting they may have been beckoning macrophages from outside the brain. “Essentially, they shut down and scream for help,” Ransohoff said.

He added that in the case of MS, infiltrating macrophages seem to be the “bad guys” that cause damage, whereas microglia appear to be paralyzed bystanders. He found that the former appear to strip myelin off axons, while microglia only participate in cleaning up the resulting mess (see Yamasaki et al., 2014). These roles may be different in Alzheimer’s, where the influx of infiltrating monocytes is less robust, Ransohoff emphasized. However, based on recent data generated in his and other labs, he hypothesized that in AD, infiltrating macrophages enter the brain and surround plaques, while microglia may sit idly by, likely inhibited by Aβ (see Krabbe et al., 2013).

What makes the microglia so passive? Ransohoff teamed up with Glass to determine how CX3CR1 (aka fractalkine receptor) might tie in. Microglia are the only cells in the brain that express this receptor. CX3CR1 knockout mice have a mixture of phenotypes. When crossed to AD models, they have fewer amyloid plaques, but when crossed to tau-transgenic mice, they have more tau pathology (see Oct 2010 news). Ransohoff and Glass examined microglia from CX3CR1-deficient mice, and found that pro-inflammatory genes—such as NLRP3 and NFκb subunits—were more active than in microglia from normal mice. More extensive enhancer studies are yet to come, but Ransohoff said the preliminary data suggest that CX3CR1 may be important for keeping microglial inflammation in check.

At Keystone, microglia inhabiting the brain’s gray matter and living in close communication with neurons drew the lion’s share of attention. However, Marco Prinz of the University of Freiburg in Germany identified a novel pathway in microglia that inhabit white matter. He reported that only those microglia express USP18, a ubiquitin protease that regulates inflammatory responses. When Prinz knocked out this gene, either in a whole mouse or only in microglia, the microglia produced pro-inflammatory cytokines and destroyed the myelin encasing nearby axons. Prinz said that USP18 prevents this destruction not through the actions of its catalytic domain, but by directly interacting with Type I interferon receptors. Somehow, the interaction between the two molecules keeps the cells from responding to low levels of interferon that are consistently present in the brain, Prinz said.

The findings indicate that while microglia are often lumped together as a group of cells that differ from macrophages in other parts of the body, they are not monolithic but have diverse functions even among themselves, depending on where in the brain they are. “Prinz’s work demonstrated that microglia respond to very local signals within brain microenvironments,” said Hugh Perry of the University of Southampton, U.K. The work is an example of the ever-increasing complexity and specificity that researchers must contend with as they study the cells that mediate neuroinflammation, Perry added.—Jessica Shugart

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